The Interactive Fly

Zygotically transcribed genes

Genes coding for Neuromuscular Junctional proteins

Presynaptic Active Zone

Five evolutionarily conserved proteins - RIM (Drosophila Rab3 interacting molecule) , Munc13 (Drosophila unc-13), RIM-BP (Drosophila Rim-binding protein), &alpha-liprin (Drosophila Liprin-α), and ELKS (Drosophila Bruchpilot) proteins - form the core of active zones. SYD-1 is a Rho GAP that is essential for synapse assembly in invertebrates but its functional homolog is unknown in vertebrates. RIM, Munc13, and RIM-BP are multidomain proteins composed of a string of identifiable modules, whereas α-liprin and ELKS exhibit a simpler structure. The five core active zone proteins form a single large protein complex that docks and primes synaptic vesicles, recruits Ca2+ channels to the docked and primed vesicles, tethers the vesicles and Ca2+ channels to synaptic cell-adhesion molecules, and mediates synaptic plasticity (Südhof, 2013). Imaging of developing Drosophila glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent active zones by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A is positioned by Bruchpilot and Rim-binding protein complexes at maturing active zones (Bohme, 2016).

Postsynapse of the larval neuromuscular junction

(A) At the postsynaptic membrane, shown at the bottom, Discs large (Dlg) localizes to spectrin-actin complexes. Homophilic adhesion between Fasciclin 2 (Fas2) transmembrane proteins links the presynaptic and postsynaptic sides, with the intracellular C-terminal domains anchored to the first and second PDZ domains of Dlg. The adducin Hu-li tai shao (Hts) is in a complex with Dlg at the postsynaptic membrane, though the interaction may not be direct. Hts also binds to the lipid Phosphatidylinositol 4,5-bisphosphate (PIP2) via the MARCKS-homology domain. (B) Hts promotes the accumulation of par-1 and camkII transcripts in the muscle cytoplasm through an as of yet identified mechanism. PAR-1 and CaMKII phosphorylate Dlg. Phosphorylation disrupts Dlg postsynaptic targeting. (C) Phosphorylation translocates Hts away from the postsynaptic membrane and hinders Hts' ability to regulate Dlg localization, presumably through the control of PAR-1 and CaMKII at the transcriptional level. Phosphorylation of the MARCKS-homology domain also inhibits Hts' ability to bind to PIP2 (Wang, T., 2014).

Neuropilin and tolloid-like (Neto) engages the iGluR complexes extrajunctionally and together they traffic and cluster at the synapses, opposite from the active zones marked by T-bars. Neto and the essential iGluR subunits are limiting for formation of functional iGluR complexes at the NMJ and for growth of synaptic structures (Kim, 2014).





Biology of the Presynapse
  • Transmission, Development, and Plasticity of Synapses - a review by Kathryn Harris & Troy Littleton
  • Genetic analysis in Drosophila reveals a role for the mitochondrial protein p32 in synaptic transmission
  • Pathogenic Huntington alters BMP signaling and synaptic growth through local disruptions of endosomal compartments
  • Myosin VI contributes to synaptic transmission and development at the Drosophila neuromuscular junction
  • Structural and molecular properties of insect type II motor axon terminals
  • A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity
  • Live observation of two parallel membrane degradation pathways at axon terminals
  • Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton
  • Drosophila neuronal injury follows a temporal sequence of cellular events leading to degeneration at the neuromuscular junction
  • Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction
  • A pre-synaptic regulatory system acts trans-synaptically via Mon1 to regulate Glutamate receptor levels in Drosophila
  • Drosophila homolog of human KIF22 at the autism-linked 16p11.2 loci influences synaptic connectivity at larval neuromuscular junctions
  • Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction
  • Tao negatively regulates BMP signaling during neuromuscular junction development in Drosophila
  • The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila
  • Human APP gene expression alters active zone distribution and spontaneous neurotransmitter release at the Drosophila larval neuromuscular junction
  • In vivo single-molecule tracking at the Drosophila presynaptic motor nerve terminal
  • Characterization of developmental and molecular factors underlying release heterogeneity at Drosophila synapses
  • A neuropeptide signaling pathway regulates synaptic growth in Drosophila
  • Sphingolipids regulate neuromuscular synapse structure and function in Drosophila
  • Parvalbumin expression affects synaptic development and physiology at the Drosophila larval NMJ
  • GAL4 drivers specific for type Ib and type Is motor neurons in Drosophila
  • Oxidative stress induces overgrowth of the Drosophila neuromuscular junction
  • A circuit-dependent ROS feedback loop mediates glutamate excitotoxicity to sculpt the Drosophila motor system
  • Neuron-specific knockdown of Drosophila HADHB induces a shortened lifespan, deficient locomotive ability, abnormal motor neuron terminal morphology and learning disability
  • Non-enzymatic activity of the alpha-Tubulin acetyltransferase alphaTAT limits synaptic bouton growth in neurons
  • Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila]
  • POU domain motif3 (Pdm3) induces wingless (wg) transcription and is essential for development of larval neuromuscular junctions in Drosophila
  • O-GlcNAcase contributes to cognitive function in Drosophila
  • Developmental arrest of Drosophila larvae elicits presynaptic depression and enables prolonged studies of neurodegeneration
  • Structural and functional synaptic plasticity induced by convergent synapse loss in the Drosophila neuromuscular circuit
  • The conserved alternative splicing factor Caper regulates neuromuscular phenotypes during development and aging
  • Fragile X Premutation rCGG Repeats Impair Synaptic Growth and Synaptic Transmission at Drosophila larval Neuromuscular Junction

    Synaptic Proteins
  • The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization
  • Extended synaptotagmin localizes to presynaptic ER and promotes neurotransmission and synaptic growth in Drosophila
  • Presynaptic spinophilin tunes Neurexin signalling to control active zone architecture and function
  • Neurexin, neuroligin and wishful thinking coordinate synaptic cytoarchitecture and growth at neuromuscular junctions
  • Drosophila Cbp53E regulates axon growth at the meuromuscular junction
  • Distinct regulation of transmitter release at the Drosophila NMJ by different isoforms of nemy
  • The guanine exchange factor Gartenzwerg and the small GTPase Arl1 function in the same pathway with Arfaptin during synapse growth
  • σ2-adaptin facilitates basal synaptic transmission and is required for regenerating endo-exo cycling pool under high frequency nerve stimulation in Drosophila
  • Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory
  • Hsc70-4 deforms membranes to promote synaptic protein turnover by endosomal microautophagy
  • Rab3-GEF controls active zone development at the Drosophila neuromuscular junction
  • The Strip-Hippo pathway regulates synaptic terminal formation by modulating actin organization at the Drosophila neuromuscular synapses
  • Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins
  • Presynaptic CamKII regulates activity-dependent axon terminal growth
  • Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function
  • Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability
  • Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca(2+) channels
  • The Ih channel gene promotes synaptic transmission and coordinated movement in Drosophila melanogaster
  • A Ca2+ channel differentially regulates Clathrin-mediated and activity-dependent bulk endocytosis
  • Neuroligin 4 regulates synaptic growth via the Bone morphogenetic protein (BMP) signaling pathway at the Drosophila neuromuscular junction
  • Inwardly rectifying potassium (Kir) channels represent a critical ion conductance pathway in the nervous systems of insects
  • The Neurexin-NSF interaction regulates short-term synaptic depression
  • Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability
  • Kinetochore proteins have a post-mitotic function in neurodevelopment
  • Secreted C-type lectin regulation of neuromuscular junction synaptic vesicle dynamics modulates coordinated movement

    Vesicles in the Presynapse
  • The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles
  • Estimation of the readily releasable synaptic vesicle pool at the Drosophila larval neuromuscular junction
  • Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila
  • Presynaptic biogenesis requires axonal transport of lysosome-related vesicles
  • Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins
  • Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration
  • Na+ /H+ -exchange via the Drosophila vesicular glutamate transporter (DVGLUT) mediates activity-induced acid efflux from presynaptic terminals
  • Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles
  • Fife organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release
  • An improved catalogue of putative synaptic genes defined exclusively by temporal transcription profiles through an ensemble machine learning approach
  • Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner
  • Rapid regulation of vesicle priming explains synaptic facilitation despite heterogeneous vesicle:Ca(2+) channel distances
  • High-probability neurotransmitter release sites represent an energy-efficient design

    Homeostasis and plasticity at the presynapse
  • Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling
  • Phosphorylation of Complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity
  • The long 3'UTR mRNA of CaMKII is essential for translation-dependent plasticity of spontaneous release in Drosophila melanogaster
  • Reactive oxygen species regulate activity-dependent neuronal plasticity in Drosophila
  • The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity
  • The schizophrenia susceptibility gene dysbindin controls synaptic homeostasis
  • Distinct roles of Drosophila cacophony and Dmca1D Ca(2+) channels in synaptic homeostasis: genetic interactions with slowpoke Ca(2+) -activated BK channels in presynaptic excitability and postsynaptic response
  • Dysbindin links presynaptic proteasome function to homeostatic recruitment of low release probability vesicles
  • A Presynaptic ENaC Channel Drives Homeostatic Plasticity
  • Composition and control of a Deg/ENaC channel during presynaptic homeostatic plasticity
  • Transsynaptic control of presynaptic Ca(2)(+) influx achieves homeostatic potentiation of neurotransmitter release
  • RIM-binding protein links synaptic homeostasis to the stabilization and replenishment of high release probability vesicles
  • RIM-binding protein couples synaptic vesicle recruitment to release site
  • A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation
  • The auxiliary glutamate receptor subunit dSol-1 promotes presynaptic neurotransmitter release and homeostatic potentiation
  • Neto-alpha controls synapse organization and homeostasis at the Drosophila neuromuscular junction
  • The innate immune receptor PGRP-LC controls presynaptic homeostatic plasticity
  • Molecular interface of neuronal innate immunity, synaptic vesicle stabilization, and presynaptic homeostatic plasticity
  • An auxiliary subunit of the presynaptic calcium channel, α2δ-3, is required for rapid transsynaptic homeostatic signaling
  • Input-specific plasticity and homeostasis at the Drosophila larval neuromuscular junction
  • Endogenous tagging reveals differential regulation of Ca(2+) channels at single AZs during presynaptic homeostatic potentiation and depression
  • Synaptic plasticity induced by differential manipulation of tonic and phasic motoneurons in Drosophila
  • Target-wide induction and synapse type-specific robustness of presynaptic homeostasis
  • Activity-dependent global downscaling of evoked neurotransmitter release across glutamatergic inputs in Drosophila
  • FOXO Regulates Neuromuscular Junction Homeostasis During Drosophila Aging

    Biology of the Postsynapse
  • Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction
  • The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function
  • Mucin-type core 1 glycans regulate the localization of neuromuscular junctions and establishment of muscle cell architecture in Drosophila
  • Shank modulates postsynaptic wnt signaling to regulate synaptic development
  • The influence of postsynaptic structure on missing quanta at the Drosophila neuromuscular junction
  • Hebbian plasticity guides maturation of glutamate receptor fields in vivo
  • Dbo/Henji modulates synaptic dPAK to gate glutamate receptor abundance and postsynaptic response
  • Filamin, a synaptic organizer in Drosophila, determines glutamate receptor composition and membrane growth
  • TOR is required for the retrograde regulation of synaptic homeostasis at the Drosophila neuromuscular junction
  • The maintenance of synaptic homeostasis at the Drosophila neuromuscular junction is reversible and sensitive to high temperature
  • The Drosophila postsynaptic DEG/ENaC channel ppk29 contributes to excitatory neurotransmission
  • Synaptic excitation is regulated by the postsynaptic dSK channel at the Drosophila larval NMJ
  • Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila
  • Development of a tissue-specific ribosome profiling approach in Drosophila enables genome-wide evaluation of translational adaptations
  • Acute fasting regulates retrograde synaptic enhancement through a 4E-BP-dependent mechanism
  • Disparate postsynaptic induction mechanisms ultimately converge to drive the retrograde enhancement of presynaptic efficacy
  • A postsynaptic PI3K-cII dependent signaling controller for presynaptic homeostatic plasticity
  • Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction
  • Regulation of SH3PX1 by dNedd4-long at the Drosophila Neuromuscular Junction
  • Calcium-activated Calpain specifically cleaves Glutamate Receptor IIA but not IIB at the Drosophila neuromuscular junction
  • Ttm50 facilitates calpain activation by anchoring it to calcium stores and increasing its sensitivity to calcium
  • Cul3 and insomniac are required for rapid ubiquitination of postsynaptic targets and retrograde homeostatic signaling
  • Vps54 regulates Drosophila neuromuscular junction development and interacts genetically with Rab7 to control composition of the postsynaptic density
  • Vesicle clustering in a living synapse depends on a synapsin region that mediates phase separation

    Signaling between Pre- and Post-synapse
  • Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth
  • Postsynaptic Syntaxin 4 negatively regulates the efficiency of neurotransmitter release
  • Postsynaptic glutamate receptors regulate local BMP signaling at the Drosophila neuromuscular junction
  • MAN1 restricts BMP signaling during synaptic growth in Drosophila
  • Jelly belly trans-synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture
  • Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling
  • Drosophila Neuroligin3 regulates neuromuscular junction development and synaptic differentiation
  • Anterograde Activin signaling regulates postsynaptic membrane potential and GluRIIA/B abundance at the Drosophila neuromuscular junction
  • Regulation of postsynaptic retrograde signaling by presynaptic exosome release
  • A distinct perisynaptic glial cell type forms tripartite neuromuscular synapses in the Drosophila adult
  • The extracellular-regulated kinase effector Lk6 is required for Glutamate receptor localization at the Drosophila neuromuscular junction
  • Drosophila ortholog of intellectual disability-related ACSL4, inhibits synaptic growth by altered lipids
  • Activity-induced synaptic structural modifications by an activator of integrin signaling at the Drosophila neuromuscular junction
  • Two algorithms for high-throughput and multi-parametric quantification of Drosophila neuromuscular junction morphology
  • Myostatin-like proteins regulate synaptic function and neuronal morphology
  • Secreted tissue inhibitor of matrix metalloproteinase restricts trans-synaptic signaling to coordinate synaptogenesis
  • Notum coordinates synapse development via extracellular regulation of Wnt Wingless trans-synaptic signaling
  • Carrier of Wingless (Cow) regulation of Drosophila neuromuscular junction development
  • Retrograde semaphorin-plexin signalling drives homeostatic synaptic plasticity
  • Synapse-specific and compartmentalized expression of presynaptic homeostatic potentiation
  • Distinct homeostatic modulations stabilize reduced postsynaptic receptivity in response to presynaptic DLK signaling
  • A glutamate homeostat controls the presynaptic inhibition of neurotransmitter release
  • Neuronal glutamatergic synaptic clefts alkalinize rather than acidify during neurotransmission
  • Distinct Target-Specific Mechanisms Homeostatically Stabilize Transmission at Pre- and Post-synaptic Compartments

    The Extracellular Matrix
  • A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling
  • Endostatin is a trans-synaptic signal for homeostatic synaptic plasticity
  • N-glycosylation requirements in neuromuscular synaptogenesis
  • Three-dimensional imaging of Drosophila motor synapses reveals ultrastructural organizational patterns
  • Unraveling synaptic GCaMP signals: differential excitability and clearance mechanisms underlying distinct Ca(2+) dynamics in tonic and phasic excitatory, and aminergic modulatory motor terminals in Drosophila
  • Inter-relationships among physical dimensions, distal-proximal rank orders, and basal GCaMP fluorescence levels in Ca(2+) imaging of functionally distinct synaptic boutons at Drosophila neuromuscular junctions
  • Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles
  • Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction
  • The HSPG glypican regulates experience-dependent synaptic and behavioral plasticity by modulating the non-canonical BMP pathway

    Miscellaneous
  • RNAi-mediated reverse genetic screen identified Drosophila chaperones regulating eye and neuromuscular junction morphology
  • Ultrastructural comparison of the Drosophila larval and adult ventral abdominal neuromuscular junction
  • Downregulation of glutamic acid decarboxylase in Drosophila TDP-43-null brains provokes paralysis by affecting the organization of the neuromuscular synapses
  • Genes coding for neuromuscular junction proteins


    The schizophrenia susceptibility gene dysbindin controls synaptic homeostasis

    The molecular mechanisms that achieve homeostatic stabilization of neural function remain largely unknown. To better understand how neural function is stabilized during development and throughout life, an electrophysiology-based forward genetic screen was used, and the function of more than 250 neuronally expressed genes was assessed for a role in the homeostatic modulation of synaptic transmission in Drosophila. This screen ruled out the involvement of numerous synaptic proteins and identified a critical function for dysbindin, a gene linked to schizophrenia in humans. dysbindin was found to be required presynaptically for the retrograde, homeostatic modulation of neurotransmission, and functions in a dose-dependent manner downstream or independently of calcium influx. Thus, dysbindin is essential for adaptive neural plasticity and may link altered homeostatic signaling with a complex neurological disease (Dickman, 2009).

    At glutamatergic synapses of species ranging from Drosophila to human, disruption of postsynaptic neurotransmitter receptor function can be precisely offset by an increase in presynaptic neurotransmitter release to homeostatically maintain normal postsynaptic excitation. The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is used as a model for this form of homeostatic signaling in the nervous system. Efficient homeostatic modulation of presynaptic release at the Drosophila NMJ can occur in ten min following bath application of philanthotoxin-433 (PhTx; a polyamine toxin present in the venom sac of the solitary digger wasp Philanthus triangulum), which persistently and specifically inhibits postsynaptic glutamate receptors (Dickman, 2009).

    This study has systematically screened for mutations that block the rapid, PhTx-dependent induction of synaptic homeostasis. Mutations in 276 genes were screened electrophysiologically. For each mutant, an average value was calculated for the amplitude of both the spontaneous miniature excitatory junctional potential (mEJP) and evoked excitatory junctional potential (EJP) following treatment of the dissected neuromuscular preparation with PhTx for 10 min. 14 mutants were isolated with average EJP amplitudes more than two standard deviations smaller than the distribution mean. From these candidates, 7 mutants were identified that block synaptic homeostasis without an obvious effect on NMJ morphology or baseline synaptic transmission. It is concluded that the molecular mechanisms of synaptic homeostasis can be genetically separated from the mechanisms responsible for normal neuromuscular development and baseline synaptic transmission (Dickman, 2009).

    A fraction of the mutants assayed (19.5%) are previously published genetic lesions. This allows ruling out of the involvement of numerous genes and associated biochemical processes. Mutations that disrupt RNA-interference/micro-RNA processing, retrograde trans-synaptic signaling, synaptic transmission, active zone assembly, synaptic vesicle endocytosis and mitochondria all showed reliable homeostatic compensation. Therefore, synaptic homeostasis is a robust phenomenon, unperturbed by a broad spectrum of synaptic mutations. In addition, significant homeostatic compensation in synaptojanin and endophilin mutants argues against the involvement of synaptic vesicle endocytosis and indicates that the size of the recycling synaptic vesicle pool is not a limiting factor for synaptic homeostasis. These data also emphasize the importance and specificity of those identified mutations that do block synaptic homeostasis. These include four ion channels, two of which are of unknown function, and two calcium-binding proteins of unknown function. Thus, homeostatic signaling at the NMJ may include previously unexplored mechanisms of synaptic modulation (Dickman, 2009).

    One mutation that was identified with a specific defect in homeostatic compensation is a transposon insertion that resides in the Drosophila homologue of dysbindin (CG6856). The DTNBP1 (dysbindin) locus is linked with schizophrenia in humans. A transposon insertion was identified within the dysbindin locus (pBace01028, referred to as dysb1, that showed a complete absence of homeostatic compensation following application of PhTx. A similar effect was observed when dysb1 was placed in trans to a deficiency that uncovers the dysb locus, indicating that the dysb1 mutant was a strong loss of function or null mutation. No significant change in baseline synaptic transmission was observed in dysb1 mutant animals (0.5 mM extracellular calcium). Thus, under these recording conditions, this mutation disrupted synaptic homeostasis without altering baseline neurotransmission. As a control, synaptic homeostasis was normal in animals in which the pBace01028 transposon was precisely excised (Dickman, 2009).

    The dysb gene is ubiquitously expressed in Drosophila embryos. Therefore, a dysbindin transgene was generated and expressed in the dysb1 mutant. Presynaptic expression of dysb fully restored homeostatic compensation in the dysb1 mutant background, whereas muscle-specific expression of dysb did not. Thus, Dysbindin is necessary presynaptically for the rapid induction of synaptic homeostasis (Dickman, 2009).

    It was next asked whether Dysbindin is also required for the sustained expression of synaptic homeostasis. Double mutant animals were generated harboring both the dysb1 mutation and a mutation in a gene encoding a postsynaptic glutamate receptor (GluRIIA). GluRIIA mutant animals normally show robust homeostatic compensation. However, homeostatic compensation was blocked in GluRIIA; dysb1 double mutant animals. Thus, dysbindin was also necessary for the sustained expression of synaptic homeostasis over several days of larval development (Dickman, 2009).

    Synapse morphology was qualitatively normal in dysb mutants including both the shape of the presynaptic nerve terminal and the levels, localization and organization of synaptic markers including futsch-positive microtubules, synapsin and synaptotagmin. Bouton number and active zone density are also normal in dysb mutants. Thus, the disruption of synaptic homeostasis in dysb1 mutants is not a secondary consequence of altered or impaired NMJ development (Dickman, 2009).

    In the vertebrate nervous system, Dysbindin is associated with synaptic vesicles. The localization was examined of a Venus-tagged dysb transgene (ven-dysb) that rescues the dysb1 mutant. Ven-Dysb showed extensive overlap with synaptic vesicle associated proteins when expressed in neurons. Thus, Dysbindin functions presynaptically, potentially at or near the synaptic vesicle pool (Dickman, 2009).

    To further define the function of Dysbindin, baseline synaptic transmission in the dysb mutant was investigated in greater detail. At 0.5 mM extracellular calcium, synaptic transmission in dysb1 mutant animals was indistinguishable from wild type. However, when extracellular calcium was reduced, baseline synaptic transmission was significantly impaired in dysb compared to wild type and this defect was rescued by presynaptic expression of dysb. Thus, there is an alteration of the calcium dependence of synaptic transmission in the dysb mutant. Indeed, at reduced extracellular calcium, both paired-pulse facilitation and facilitation that occurs during a prolonged stimulus train were increased in dysb mutants (Dickman, 2009).

    In vertebrates, the levels of dysb expression correlate with parallel changes in extracellular glutamate concentration. Therefore, whether dysb overexpression might increase presynaptic release was tested. In wild-type animals overexpressing dysb in neurons, synaptic transmission is normal at low extracellular calcium (0.2 and 0.3 mM Ca2+) but was enhanced at relatively higher extracellular calcium (0.5 mM Ca2+). The complementary effects of dysb loss-of-function and overexpression confirm that Dysbindin has an important influence on calcium-dependent vesicle release (Dickman, 2009).

    The presynaptic CaV2.1 calcium channel, encoded by cacophony (cac), is required for synaptic vesicle release at the Drosophila NMJ. cac mutations decrease presynaptic calcium influx and also block synaptic homeostasis. Genetic interaction between dysb and cac was tested during synaptic homeostasis. Because homozygous cac and dysb mutations individually block synaptic homeostasis, analysis of double mutant combinations would not be informative. An analysis of heterozygous mutant combinations and gene overexpression were examined. Synaptic homeostasis was suppressed by a heterozygous mutation in cac. However, this suppression was not enhanced by the presence of a heterozygous mutation in dysb. In addition, neuronal overexpression of cac did not restore homeostatic compensation in dysb mutant animals and the enhancement of presynaptic release caused by neuronal dysb overexpression still occurs in a heterozygous cac mutant background. Thus, Dysbindin may function downstream or independently of Cac during synaptic homeostasis (Dickman, 2009).

    To further explore the relationship between Dysbindin and Cac, it was asked whether dysb mutations might directly influence presynaptic calcium influx. The spatially averaged calcium signal in dysb1 was indistinguishable from wild type, indicating no difference in presynaptic calcium influx. Thus, Dysbindin appears to function downstream or independently of calcium influx to control synaptic homeostasis (Dickman, 2009).

    Through a systematic electrophysiological analysis of more than 250 mutants this study could rule out the involvement of numerous synaptic proteins and biochemical processes in the mechanisms of synaptic homeostasis and demonstrate that this phenomenon is separable from the molecular mechanisms that specify structural and functional synapse development. Dysbindin is therefore identified as an essential presynaptic component within a homeostatic signaling system that regulates and stabilizes synaptic efficacy. Dysbindin functions downstream or independently of the presynaptic CaV2.1 calcium channel in the mechanisms of synaptic homeostasis (Dickman, 2009).

    Emerging lines of evidence suggest that glutamate hypofunction could be related to the etiology of schizophrenia. Likewise, reduced levels of dysbindin expression were associated with schizophrenia. The sandy mouse, which lacks Dysbindin, has a decreased rate of vesicle release (~30% decrease), a correlated decrease in vesicle pool size and an increased thickness of the postsynaptic density. This study confirms a modest, facilitatory function for Dysbindin during baseline transmission. However, numerous mutations with similar or more severe defects in baseline transmission show normal synaptic homeostasis. By contrast, loss of Dysbindin completely blocks the adaptive, homeostatic modulation of vesicle release, suggesting that the potential contribution of dysbindin mutations to schizophrenia may be derived from altered homeostatic plasticity as opposed to decreased baseline glutamatergic transmission (Dickman, 2009).

    Distinct roles of Drosophila cacophony and Dmca1D Ca(2+) channels in synaptic homeostasis: genetic interactions with slowpoke Ca(2+) -activated BK channels in presynaptic excitability and postsynaptic response

    Ca(2+) influx through voltage-activated Ca(2+) channels and its feedback regulation by Ca(2+) -activated K(+) (BK) channels is critical in Ca(2+) -dependent cellular processes, including synaptic transmission, growth and homeostasis. This study report differential roles of cacophony (CaV 2) and Dmca1D (CaV 1) Ca(2+) channels in synaptic transmission and in synaptic homeostatic regulations induced by slowpoke (slo) BK channel mutations. At Drosophila larval neuromuscular junctions (NMJs), a well-established homeostatic mechanism of transmitter release enhancement is triggered by experimentally suppressing postsynaptic receptor response. In contrast, a distinct homeostatic adjustment is induced by slo mutations. To compensate for the loss of BK channel control presynaptic Sh K(+) current is upregulated to suppress transmitter release, coupled with a reduction in quantal size. We demonstrate contrasting effects of cac and Dmca1D channels in decreasing transmitter release and muscle excitability, respectively, consistent with their predominant pre- vs. postsynaptic localization. Antibody staining indicated reduced postsynaptic GluRII receptor subunit density and altered ratio of GluRII A and B subunits in slo NMJs, leading to quantal size reduction. Such slo-triggered modifications were suppressed in cac;;slo larvae, correlated with a quantal size reversion to normal in double mutants, indicating a role of cac Ca(2+) channels in slo-triggered homeostatic processes. In Dmca1D;slo double mutants, the quantal size and quantal content were not drastically different from those of slo, although Dmca1D suppressed the slo-induced satellite bouton overgrowth. Taken together, cac and Dmca1D Ca(2+) channels differentially contribute to functional and structural aspects of slo-induced synaptic modifications (Lee, 2014).

    The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity

    Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis, and in neurons, synaptic vesicle assembly, neurotransmission, and plasticity. Protein networks, or interactomes, downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. This study consisted of a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. Components of the vesicle fusion machinery were identified as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons, among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa, and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function, the role for NSF was studied in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described, this study found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However, neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. These results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity (Gokhale, 2015).

    Dysbindin associates with seven other polypeptides to form the biogenesis of lysosome-related organelles complex 1. Null mutations in mouse dysbindin reduce the expression of other BLOC-1 subunit mRNAs and polypeptides. This suggests that dysbindin genetic downregulation could elicit multiple alterations of protein content in cells. This study identified 224 proteins whose content was modified by dysbindin/BLOC-1 partial loss of function using unbiased quantitative mass spectrometry. The screen prominently identified components of the N-ethylmaleimide-sensitive factor (NSF)-dependent vesicle fusion machinery. Focus was placed on NSF, a catalytic component of the fusion machinery, and it was asked whether NSF participates in dysbindin/BLOC-1-dependent synaptic mechanisms. Drosophila presynaptic plasticity produced by the inhibition of postsynaptic receptors was used as an assay. As previously, it was observed that mutations in fly dysbindin precluded the establishment of homeostatic synaptic plasticity, a phenotype that is rescued by presynaptic expression of dysbindin. Neuron-specific expression of dNSF1, the gene encoding Drosophila NSF, by itself does not modulate this form of plasticity, yet NSF1 expression at the synapse of dysbindin mutants rescued homeostatic synaptic plasticity defects to the same extent as dysbindin re-expression in the presynaptic compartment. These results demonstrate that partial dysbindin/BLOC-1 loss of function alters the cellular content of proteins that specifically have roles in synaptic mechanisms (Gokhale, 2015).

    Genetic polymorphisms associated with schizophrenia mostly reside in noncoding regions modifying gene and/or protein levels rather protein sequence. The question of how widespread the effects are of a single mutation or polymorphism across the proteome has been poorly explored. This study addressed this question by modeling a partial reduction in the cellular content of dysbindin/BLOC-1 using shRNAs against BLOC-1 complex subunits. 224 proteins were whose content is affected by a partial loss of function of dysbindin/BLOC-1 and focused on an interactome centered around a schizophrenia susceptibility gene, dysbindin, and NSF, a component of the membrane fusion machinery that localizes to the synapse and was previously implicated in schizophrenia mechanisms. Functional outcomes of the dysbindin/BLOC-1 and NSF association were confirmed using a Drosophila synaptic adaptive response. The results demonstrate that dysbindin/BLOC-1 expression defects induce multiple downstream quantitative protein expression traits associated with the vesicle fusion apparatus, which influence synaptic plasticity in an invertebrate model synapse (Gokhale, 2015).

    A proteomic search prominently highlights the following components of the vesicle fusion apparatus: munc18, tomosyn, NSF; and the SNAREs syntaxin 7, syntaxin 17, SNAP23, SNAP25, SNAP 29, and VAMP7. Importantly, most of the aforementioned vesicle fusion machinery components have been implicated by genomic and postmortem studies in several neurodevelopmental disorders, including schizophrenia, intellectual disability, and autism spectrum disorder. The current strategy is validated by the identification of proteins previously known to be downregulated in null alleles of BLOC-1 subunits and/or known to interact with BLOC-1. These proteins include subunits of the BLOC-1 complex and the SNARE VAMP7. This study further authenticated these fusion machinery components as part of a dysbindin/BLOC-1 network by (1) coimmunoprecipitation of a fusion machinery component with dysbindin/BLOC-1 subunits and/or (2) downregulation of a fusion machinery component after genetic or shRNA-mediated reduction of dysbindin/BLOC-1 subunits. NSF was studied since it is a hub of protein-protein interactions with components of the fusion machinery, and is a catalytic activity that is required for the resolution of fusion reaction products and other protein-protein complexes. NSF was found to associate with dysbindin and BLOC-1 subunits in neuroblastoma cells in culture. However, efforts to document the association of NSF and dysbindin-BLOC-1 by immunoprecipitation with NSF antibodies were unsuccessful in brain. This outcome occurred regardless of whether NSF was immunoprecipitated from brain homogenates or cross-linked synaptosomal lysates from adult mouse brain. This negative result is attributed to the high abundance of NSF in brain compared with dysbindin/BLOC-1. Reverse immunoprecipitations with dysbindin/BLOC-1 antibodies were not possible, as none of the available antibodies were suitable for immunoprecipitation. Since most of the associations between NSF and dysbindin/BLOC-1 are detected in the presence of the cross-linker DSP in cell lines, it is likely that the biochemical interactions between NSF and dysbindin/BLOC-1 are indirect. However, NSF cellular levels are decreased following shRNA-mediated or genomic reduction of BLOC-1 complex members, arguing in favor of a functional outcome of this association. No NSF downregulation phenotype was detected in hippocampal extracts of Bloc1s8sdy/sdy mice at days 7 or 50 of postnatal development. This suggests that NSF phenotypes may be anatomically restricted either to a region of the hippocampus or to an earlier and transient developmental stage. However, this reduced NSF trait is robustly and reversibly induced by genetic disruption of the dysbindin/BLOC-1 complex or by downregulation of dysbindin/BLOC-1 subunits in neuroblastoma and human embryonic kidney cells, neuroectodermal cells, and iPSC-derived human neurons (Gokhale, 2015).

    These studies indicate that the functional outcome of NSF reduction in BLOC-1 loss of function become evident only when the synapse is challenged. Constitutive secretion in Drosophila or mammalian non-neuronal cells is unaffected, as are spontaneous and evoked neurotransmission at the Drosophila neuromuscular junction. However, a requirement for NSF in BLOC-1 loss-of-function phenotypes can be localized to a presynaptic homeostatic mechanism, which is engaged when postsynaptic receptors are blocked with philanthotoxin. After a brief incubation with philanthotoxin, the resultant reduction in postsynaptic signal transduction rapidly induces a compensatory increase in quantal content, a response known as homeostatic synaptic plasticity. This adaptive compensatory mechanism is precluded by dysbindin mutations, and can be rescued by presynaptic expression of dysbindin. However, it was possible to rescue this phenotype in the dysbindin mutants to the exact same extent through presynaptic expression NSF. The observation that RNAi downregulation or overexpression of NSF in the neuromuscular junction does not interfere with homeostatic synaptic plasticity argues that the NSF is not an obligate component downstream of dysbindin/BLOC-1 in a linear pathway, but rather is an adaptive response to network perturbation induced by a dysbindin mutant allele. This hypothesis predicts that transheterozygotic reduction of NSF and Dysbindin should impair plasticity, a result that is at odds with the finding that plasticity is normal in dysb1-/+;UAS-NSF RNAi. It is believe that this may be a consequence of a modest reduction of dysbindin polypeptide in dysb1-/+ animals, which was predicted to be ~25% (Gokhale, 2015).

    How does the BLOC-1-NSF interaction affect synaptic mechanisms? A model integrating these findings has to consider three key elements. First, BLOC-1 subunits reside at endosomes as well as on synaptic vesicles in presynaptic terminals in neurons. Second, BLOC-1 binds monomeric SNAREs rather than tetrahelical SNARE bundles in vitro. Finally, NSF and SNAREs bind to dysbindin/BLOC-1, yet they do not seem to form a ternary complex. Thus, it is proposed that BLOC-1 bound to a single SNARE (perhaps for SNARE sorting into vesicles) is resolved by NSF, making SNAREs permissive for vesicle fusion. Therefore, when dysbindin and NSF levels are reduced by hypomorphic mutations in the fly or as a quantitative expression trait in humans, SNARE-dependent mechanisms might be impaired due to defective SNARE sorting, a consequence of the reduced levels of BLOC-1 complex and, additionally, by decreased NSF content that would impair the resolution of remaining SNARE-BLOC-1 complexes. Thus, noncoding polymorphisms in several genes and their quantitative expression traits may converge to impair synaptic mechanisms. It is proposed that unbiased identification of quantitative traits across the proteome of neurodevelopmental deficiency models is a simple approach to unravel mechanisms of complex neurodevelopmental disorders (Gokhale, 2015).

    Dysbindin links presynaptic proteasome function to homeostatic recruitment of low release probability vesicles

    This study explores the relationship between presynaptic homeostatic plasticity and proteasome function at the Drosophila neuromuscular junction. First, it was demonstrated that the induction of homeostatic plasticity is blocked after presynaptic proteasome perturbation. Proteasome inhibition potentiates release under baseline conditions but not during homeostatic plasticity, suggesting that proteasomal degradation and homeostatic plasticity modulate a common pool of vesicles. The vesicles that are regulated by proteasome function and recruited during homeostatic plasticity are highly EGTA sensitive, implying looser Ca2+ influx-release coupling. Similar to homeostatic plasticity, proteasome perturbation enhances presynaptic Ca2+ influx, readily-releasable vesicle pool size, and does not potentiate release after loss of specific homeostatic plasticity genes, including the schizophrenia-susceptibility gene dysbindin. Finally, genetic evidence is provided that Dysbindin levels regulate the access to EGTA-sensitive vesicles. Together, these data suggest that presynaptic protein degradation opposes the release of low-release probability vesicles that are potentiated during homeostatic plasticity and whose access is controlled by dysbindin (Wentzel, 2018).

    At the Drosophila NMJ, PHP can be rapidly induced within minutes in the presence of the protein synthesis inhibitor cyclohexamide, suggesting that the acute induction of PHP does not require synthesis of new proteins. In contrast, protein degradation has not been studied in the context of PHP at this synapse. The ubiquitin-proteasome system (UPS) is a major protein degradation pathway. At the Drosophila NMJ it has been shown that all components of the UPS are present at presynaptic terminals and that acute proteasome inhibition causes a rapid strengthening of neurotransmission There is accumulating evidence for links between neural activity and UPS-mediated degradation of presynaptic proteins in mice and rats. However, only two presynaptic proteins -- Rab3-interacting molecule (RIM: Jiang, 2010; Lazarevic, 2011; Yao, 2007) and Dunc-13/munc-13 (Speese, 2003) -- as well as one E3-ligase (SCRAPPER Yao, 2007) have so far been implicated in UPS-dependent control of presynaptic protein turnover and release. Thus, the molecular pathways underlying the regulation of presynaptic release through protein degradation remain enigmatic (Wentzel, 2018).

    PHP at the Drosophila NMJ requires high-release probability (pr) vesicles that are 'tightly coupled' to Ca2+ channels through rim-binding protein. The distance between Ca2+ channels and Ca2+ sensors of exocytosis, typically referred to as 'coupling distance', is a major factor determining the pr of synaptic vesicles. However, despite their implication in short-term plasticity, little is known about how vesicles that are differentially coupled to Ca2+ influx are modulated during synaptic plasticity (Wentzel, 2018).

    This study explored the relationship between PHP and presynaptic proteasome function at the Drosophila NMJ and provides evidence for links between presynaptic protein degradation and homeostatic potentiation of loosely-coupled synaptic vesicles (Wentzel, 2018).

    Rapid effects of proteasome perturbation were observed on neurotransmitter release and the abundance of ubiquitinated proteins on the minute time scale. These data suggest that the proteasomal degradation rate is relatively high under baseline conditions, consistent with previous work on local protein degradation in the presynaptic or postsynaptic compartment, and considerably faster than the average neuron-wide turnover rates of synaptic proteins (2-5 days). Such rapid degradation rates could in turn allow for potent modulation of protein abundance and/or ubiquitination through regulation of UPS function during PHP (Wentzel, 2018).

    Perturbation of proteasome function has diverse effects on cellular physiology, such as altering the levels of free ubiquitin and mono-ubiquitinated proteins, activating macroautophagy or upregulation of lysosomal enzyme levels. The observed phenotypes could thus be due to indirect effects of impaired proteasome function on synaptic physiology. Even if it is not possible to rule out this possibility, several lines of evidence argue against a major contribution of indirect effects. First, acute or prolonged proteasome perturbation does not affect release at synapses that express PHP. Second, genetic evidence is provided that proteasome perturbation-induced changes in release are blocked in two PHP mutants. Third, no major changes were detected in synaptic morphology or synaptic development upon prolonged proteasome perturbation. Fourth, interfering with proteasome function does not impair neurotransmitter release, but rather results in a net increase in release. Taken together, these data suggest that proteasome function opposes release by degrading proteins under baseline conditions, and that normal degradation of these proteins is required for PHP (Wentzel, 2018).

    The genetic data imply that not all proteins required for PHP are regulated through UPS-dependent degradation under the experimental conditions (pharmacological proteasome perturbation for 15 min) used in this study, because release can be potentiated upon brief proteasome inhibition in most PHP mutants. This observation is consistent with a recent study demonstrating that the abundance of most synaptic proteins does not change after prolonged pharmacological proteasome perturbation in cultured mouse hippocampal neurons. Based on the current observation that PHP is blocked in cut-up mutants, which were recently shown to have a defect in proteasome trafficking, it is conceivable that proteasome mobility and/or recruitment are modulated during PHP (Wentzel, 2018).

    Evidence is provided that proteasome perturbation and homeostatic signaling recruit EGTA-sensitive vesicles with a lower pr in addition to vesicles with higher pr. Previous work revealed that tightly-coupled, high-pr vesicles are required for PHP. PHP therefore likely involves vesicle pools with different pr . Homeostatic regulation of EGTA-sensitive, loosely-coupled vesicles depends on dysbindin, whereas tightly-coupled vesicles are controlled by RIM-binding protein. Together, these results imply that PHP involves two genetically separable populations of vesicles with different pr (Wentzel, 2018).

    Vesicle pools with different pr and release kinetics have been observed at various synapses, and these pools might be differentially regulated during synaptic plasticity. This study provides evidence that dysbindin is required for the recruitment of EGTA-sensitive vesicles during PHP. It will be exciting to investigate the roles of other presynaptic proteins that have been implicated in the release of EGTA-sensitive vesicles, such as Tomosyn, in the context of proteasome degradation and PHP. Interestingly, a recent study at the mouse NMJ observed accelerated release kinetics of 'slow' synaptic vesicles during PHP. Thus, homeostatic potentiation of vesicles with lower pr /slower release kinetics may be an evolutionarily conserved mechanism (Wentzel, 2018).

    Which mechanisms could potentiate the release of low-pr vesicles? This study revealed that presynaptic proteasome perturbation results in enhanced presynaptic Ca2+ influx, independent of major changes in Ca2+ buffering and/or extrusion. Therefore changes in presynaptic Ca2+ influx are considered as a possible mechanism underlying the increase in low-pr vesicle release. Proteasome perturbation also increased RRP size. Earlier work revealed that changes in presynaptic Ca2+ influx modulate release in part by altering apparent RRP size. The increase in RRP size or EGTA sensitivity upon proteasome inhibition may therefore be in part a secondary consequence of enhanced presynaptic Ca2+ influx. However, several observations, such as the increased amplitude of the fast recovery phase after pool depletion or the slowing of EPSC decay kinetics upon presynaptic proteasome perturbation, which are not seen after increasing [Ca2+]e, indicate that the increase in release is not caused by presynaptic Ca2+ influx alone. Together, these data imply that a combination of increased presynaptic Ca2+ influx and RRP size underlie the enhancement of release after proteasome or glutamate receptor perturbation. Which molecular mechanisms may link UPS function to the modulation of presynaptic Ca2+ influx or RRP size? Genetic data suggest that dysbindin functions independently of presynaptic Ca2+ influx. Interestingly, rim mutants were shown to have a defect in homeostatic RRP size modulation, but unchanged homeostatic control of presynaptic Ca2+ influx. It is therefore speculated that RIM and Dysbindin may be involved in proteasome-dependent RRP size regulation. How do these observations relate to mammalian synapses? At cultured hippocampal rat synapses, proteasome inhibition augments recycling vesicle pool size or prevents a decrease in RRP size induced by prolonged (4h) depolarization. Moreover, several studies at mammalian synapses suggest that UPS-dependent regulation of RIM abundance regulates neurotransmitter release during baseline synaptic transmission and synaptic plasticity. Finally, there is evidence that ubiquitination acutely regulates release on the minute time scale at cultured hippocampal rat synapses (Rinetti, 2010). Together, these studies suggest that rapid, UPS-dependent control of RRP size may be evolutionarily conserved. Future studies will further elucidate the molecular signaling pathways relating UPS function to neurotransmitter release during baseline synaptic transmission and homeostatic plasticity (Wentzel, 2018).

    Genetic analysis in Drosophila reveals a role for the mitochondrial protein p32 in synaptic transmission

    Mitochondria located within neuronal presynaptic terminals have been shown to play important roles in the release of chemical neurotransmitters. In the present study, a genetic screen for synaptic transmission mutants of Drosophila has identified the first mutation in a Drosophila homolog of the mitochondrial protein P32. Although P32 is highly conserved and has been studied extensively, its physiological role in mitochondria remains unknown and it has not previously been implicated in neural function. The Drosophila P32 mutant, referred to as dp32EC1, exhibited a temperature-sensitive (TS) paralytic behavioral phenotype. Moreover, electrophysiological analysis at adult neuromuscular synapses revealed a TS reduction in the amplitude of excitatory postsynaptic currents (EPSC) and indicated that dP32 functions in neurotransmitter release. These studies are the first to address P32 function in Drosophila and expand the knowledge of mitochondrial proteins contributing to synaptic transmission (Lutas, 2012).

    A genetic screen for synaptic transmission mutants in Drosophila isolated a new mutation in a Drosophila homolog of the mitochondrial protein P32, which represents the first P32 mutation in a multicellular organism. Although P32 is highly conserved and has been studied extensively, its physiological function in mitochondria remains unknown. This new mutant, referred to as dP32EC1, exhibited a temperature-sensitive (TS) paralytic behavioral phenotype. Moreover, electrophysiological analysis at adult neuromuscular synapses revealed a TS reduction in neurotransmitter release, indicating that dP32 serves an important function in synaptic transmission. Immunocytochemical analysis has shown that dP32 is located within presynaptic mitochondria, which are known to be important in ATP production and calcium signaling at synapses. Furthermore, the basic molecular and structural organization of synapses appears to be normal in the dP32 mutant, suggesting a direct role for this protein in synaptic function. At the molecular level, biochemical studies indicated conserved homomultimeric interactions of dP32 subunits. Finally, assessment of presynaptic mitochondrial function was examined in the dP32 mutant through measurement of ATP levels and imaging studies of mitochondrial membrane potential and presynaptic calcium. This work indicated that mitochondrial ATP production and membrane potential in the dP32 mutant resembled wild-type, whereas the mutant exhibited a TS increase in both resting and evoked presynaptic calcium concentration. Taken together, the preceding findings reveal a role for dP32 in synaptic transmission and mitochondrial regulation of presynaptic calcium (Lutas, 2012).

    Mitochondrial localization of P32 proteins involves an N-terminal targeting domain that is cleaved from the mature targeted protein. Comparison of Drosophila and vertebrate P32 sequences indicates conservation of the proteolytic cleavage site in dP32. In the present study, an equivalent targeting function for the N-terminal domain of dP32 was demonstrated through its ability to mediate mitochondrial targeting. When the first 71 amino acids of dP32, including the proteolytic cleavage site, was fused to GCaMP3, this fusion protein (mito-GCaMP3) was efficiently targeted to mitochondria. Although only modest sequence conservation was observed between the N-terminal domains of dP32 and vertebrate P32 proteins, previous studies suggest that mitochondrial targeting domains vary in amino acid sequence but often share an amphipathic helical structure (Lutas, 2012).

    Structural studies have established that P32 is a homotrimer in which monomers are arranged around a central pore in a donut-like structure. In the present study, homomultimerization of dP32 subunits was demonstrated in co-immunoprecipitation experiments. The trimeric structure of P32 exhibits a highly asymmetric charge distribution that creates a concentration of negatively charged residues along one side of the donut, raising the possibility that P32 may participate in calcium binding within the mitochondrial matrix. Notably, five residues that are spatially clustered to form a pocket on the negatively charged side of human P32, Glu-89, Leu-231, Asp-232, Glu-264, and Tyr-268, are identical in the Drosophila protein. Further genetic analysis may address the importance of these clustered residues in dP32 function at synapses (Lutas, 2012).

    Several possible mechanisms of dP32 function in mitochondria and synaptic transmission were considered and investigated in this paper, most notably possible roles in supporting mitochondrial membrane potential, ATP production, and presynaptic calcium signaling. Among these, the observations favor a function for dP32 in mitochondrial mechanisms regulating presynaptic calcium. Although neurotransmitter release was reduced at restrictive temperatures in dP32EC1, the presynaptic calcium concentration was increased both at rest and in response to synaptic stimulation. It is of interest to consider why the increase in presynaptic calcium in dP32EC1 is TS in what appears to be a complete loss-of-function mutant. Previous studies at Drosophila larval neuromuscular synapses at elevated temperatures have observed a TS increase in resting cytosolic calcium and associated inhibition of neurotransmitter release. This calcium increase was enhanced by pharmacological inhibition of presynaptic calcium clearance mechanisms or genetic removal of presynaptic mitochondria, but it remained dependent on temperature. The present findings may reflect a similar TS process involving calcium-dependent inhibition of neurotransmitter release and dP32-dependent mitochondrial mechanisms. Efforts to further address these mechanisms were pursued by employing a calcium indicator targeted to the mitochondrial matrix, mito-GCaMP3. Although this approach was successful for imaging mitochondrial calcium transients elicited by motor axon stimulation in both WT and dP32EC1 at 20°C, robust calcium transients could not be observed in either genotype when the temperature was increased to the restrictive temperatures of 33° or 36° (Lutas, 2012).

    The preceding observations suggest that sustained elevation of presynaptic calcium in the dP32 mutant may lead to reduced neurotransmitter release. Such a calcium-dependent mechanism has been reported previously in the squid giant synapse and attributed to calcium-dependent adaptation of the neurotransmitter release apparatus. Understanding the precise mechanism by which loss of dP32 impairs neurotransmitter release will require further investigation. One interesting question is how the absence of dP32 in the mitochondrial matrix leads to increased presynaptic calcium and whether this reflects the putative calcium binding capacity of this protein. Finally, while the present study is focused on the newly discovered role for P32 in neurotransmitter release, the resulting research materials are expected to facilitate in vivo analysis of P32 function in a broad range of biological processes (Lutas, 2012).

    The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles

    Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. Previous studies have shown that loss of the Drosophila ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca2+ channels. This paper explores additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and are important for proper T-bar formation. Basal transmission is decreased in isoform-specific mutants, attributable to a reduction in the size of the readily releasable pool (RRP) of SVs. A corresponding reduction was found in the number of SVs docked close to the remaining cytomatrix. It is proposed that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca2+ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP (Matkovic, 2013).

    An elaborate protein cytomatrix covering the AZ membrane is meant to facilitate and control the SV release process. Quantitative analysis of neurotransmitter release has provided evidence that the number of SV release sites per AZ might be fixed. Although these sites are thought to be located in close proximity to presynaptic Ca2+ channels, ultrastructural and molecular information is largely missing here. Potentially, specific interactions between SVs and certain cytomatrix components might be involved. This study provides evidence that the BRP-based cytomatrix plays a role in defining the number of readily releasable SVs, possibly by offering morphological and molecular-determined 'release slots' (Matkovic, 2013).

    Previous studies have characterized the role of BRP based on null alleles, which result in a complete absence of AZ cytomatrix (T bar), partially declustered Ca2+ channels, and likely as a direct consequence, reduced vesicular release probability. In contrast, in the analysis of BRP isoform-specific mutants, the current study neither observed any Ca2+ channel clustering deficits nor changes in vesicular release probability (Matkovic, 2013).

    Previous studies have found a binding site between the intracellular C terminus of the Cac Ca2+ channel and an N-terminal stretch of BRP, which is unique to BRP-190 (Fouquet, 2009). That solely losing BRP-190 is not sufficient to affect Ca2+ channel clustering could possibly be explained by the presence of redundant binding sites within BRP-170. Ca2+ channel clustering might well be a collective feature of the cytomatrix, and Ca2+ channels likely use multiple simultaneous interactions with several cytomatrix proteins to anchor within the AZ membrane (Matkovic, 2013).

    In fact, RIM-binding protein family proteins at rodent and Drosophila AZs bind Ca2+ channels, and loss of the only RIM-binding protein in Drosophila results in partial loss of Ca2+ channels from AZs. RIM-binding protein levels at AZ were slightly but significantly reduced in the BRP isoform mutants. Clearly, it remains a possibility that RIM-binding protein is a major scaffold determinant of the release slots and that e.g., subtle mislocalizations of RIM-binding protein might in part contribute to the BRP isoform mutant phenotype. The brp-null phenotype can now be interpreted as a 'catastrophic event' in which a complete loss of this large scaffold protein leads to a severe decrease of cytomatrix avidity (potentially mediated via a loss of RIM-binding protein) below a critical level, resulting in a 'collapse' of the normal cytomatrix architecture. Thus, functionalities associated with discrete regions of BRP and RIM-binding protein can apparently be masked when the BRP-based AZ scaffold is completely eliminated (Matkovic, 2013).

    The distal cytomatrix in brpnude is bare of SVs in EM, and SV replenishment is defective, resulting in short-term depression (and not facilitation as in brp nulls). However, no change of short-term plasticity could be detected in the brp isoform alleles with the same analyses, consistent with neither a change in Ca2+ channel clustering nor in SV clustering at the distal cytomatrix. Nevertheless, a basal release deficit was observed, which can be explained by a reduction in the size of the readily releasable vesicle pool, assigning an additional function to the BRP cytomatrix (Matkovic, 2013).

    Release-ready SVs are meant to be molecularly and positionally primed for release. Important factors are the equipment with or the attachment to the proteins of the core release machinery and the localization of the SV in proximity to the Ca2+ source. At the Drosophila NMJ, SV release is insensitive to slow Ca2+ buffers such as EGT; therefore, SVs are thought to be spatially tightly coupled to Ca2+ channels (nanodomain coupling; Eggermann, 2012). Since Ca2+ channels are found localized directly underneath the T-bar pedestal composed of the N-terminal region of BRP (Fouquet, 2009), release-ready SVs might well correspond to the SVs that were found docked at the pedestal of the T bar and thus in very close proximity to the Ca2+ channels. This in turn is in agreement with BRP itself being important for defining the number of release-ready SVs determined by electrophysiology and EM (Matkovic, 2013).

    Light microscopic inspection of an AB directed against the C terminus of BRP, common to both isoforms, with 50-nm STED resolution, typically revealed approximately five dots arranged as a circle or regular pentagon. Both isoforms were labelled individually, and it was found that (1) both isoforms seem to localize with their C termini similarly toward the distal edge of the cytomatrix and (2) both isoforms typically form an identical number of dots per AZ similar to the number of dots observed with the BRPC-Term AB recognizing both isoforms. Thus, the BRP isoforms seem to be arranged in neighboring but not overlapping clusters, forming a circular array. Consistent with both BRP isoforms not overlapping in space, there was neither efficient co-IP between them nor did elimination of one isoform substantially interfere with the AZ localization of the respective other isoform. Thus, BRP-190 and -170 seem to form discrete oligomers. The alternating pattern of BRP-190 and -170 appears to set a typical cytomatrix size, as both isoform mutants had a reduced T-bar width in EM and a reduced mean number of BRP dots per AZ. As this corresponded with a similar reduction in the number of SVs in the RRP, this AZ architecture could set a typical number of Ca2+ nanodomain-coupled RRP slots possibly located between BRP clusters. However, beyond providing a discrete morphological architecture, the two BRP isoforms described in this study might harbor additional functionalities. The brpΔ190 phenotype was more pronounced than the brpΔ170, leaving the possibility that the highly conserved N terminus of BRP-190 promotes release by further mechanisms going beyond the points analyzed in this study. Future analysis will also have to address whether localization and regulation of additional cytomatrix and release components, such as RIM-binding protein, Unc-13 family proteins, or RIM, contribute to the formation of release slots as well (Matkovic, 2013).

    Ultimately, functional differences between individual synaptic sites must be defined by variances in their molecular organization. Functional features of a synapse can be extracted electrophysiologically. Thereby, the number of Ca2+ channels was recently identified as a major determinant of the release probability of single vesicles, Pvr, in rat calyces (Sheng, 2012). Furthermore, AZ size seems to scale with the overall likelihood of release from a given AZ (Holderith, 2012). The current results suggest that the BRP-based cytomatrix should be a general determinant of the release likelihood per AZ by establishing Pvr, through Ca2+ channel clustering, as shown previously, and, as shown in this study, by determining the size of the RRP. The genetic results show that the cytomatrix can, in principle, control the RRP size independent of Ca2+ channel clustering. A coupled increase in the size of the T-bar cytomatrix together with increasing SV release was previously observed at NMJs compensating for loss of the glutamate receptor subunit glurIIA. Moreover, an increase in the number of release-ready SVs together with an increase in the amount of BRP was recently described as part of a homeostatic presynaptic response after pharmacological block of postsynaptic GluRIIA (Weyhersmuller, 2011). In line with this scenario, it was recently shown that lack of acetylation of BRP in elp3 mutants led to an increase in the complexity of the AZ cytomatrix along with an increase in RRP size (Miskiewicz, 2011). Furthermore, in vivo imaging of synaptic transmission with single synapse resolution revealed that the likelihood of release correlates with the amount of BRP present at an individual AZ (Peled, 2011). This cytomatrix size-SV release scaling might be a general principle, as a correlation between the amount of SV exocytosis, measured by an optical assay, and the amount of the AZ protein Bassoon at individual synapses of cultured rat hippocampal neurons has also been observed (Matz, 2010). The current results suggest that not only the mere size, but also the distinct architecture of the cytomatrix influence release at individual synapses through determining RRP size (Matkovic, 2013).

    Estimation of the readily releasable synaptic vesicle pool at the Drosophila larval neuromuscular junction

    Presynaptic boutons at nerve terminals are densely packed with synaptic vesicles, specialized organelles for rapid and regulated neurotransmitter secretion. Upon depolarization of the nerve terminal, synaptic vesicles fuse at specializations called active zones that are localized at discrete compartments in the plasma membrane to initiate synaptic transmission. A small proportion of synaptic vesicles are docked and primed for immediate fusion upon synaptic stimulation, which together comprise the readily releasable pool. The size of the readily releasable pool is an important property of synapses, which influences release probability and can dynamically change during various forms of plasticity. A detailed protocol is described for estimating the readily releasable pool at a model glutamatergic synapse, the Drosophila neuromuscular junction. This synapse is experimentally robust and amenable to sophisticated genetic, imaging, electrophysiological, and pharmacological approaches. The experimental design, electrophysiological recording procedure, and quantitative analysis that is necessary to determine the readily releasable pool size is described in this paper. This technique requires the use of a two-electrode voltage-clamp recording configuration in elevated external Ca(2+) with high frequency stimulation. This assay is sused to measure the readily releasable pool size and reveal that a form of homeostatic plasticity modulates this pool with synapse-specific and compartmentalized precision. This powerful approach can be utilized to illuminate the dynamics of synaptic vesicle trafficking and plasticity and determine how synaptic function adapts and deteriorates during states of altered development, stress and neuromuscular disease (Goel, 2019).

    Pathogenic Huntington alters BMP signaling and synaptic growth through local disruptions of endosomal compartments

    Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) stretch within the Huntingtin (Htt) protein. Pathogenic Htt disrupts multiple neuronal processes, including gene expression, axonal trafficking, proteasome and mitochondrial activity, and intracellular vesicle trafficking. However, the primary pathogenic mechanism and subcellular site of action for mutant Htt are still unclear. Using a Drosophila HD model, this study found that pathogenic Htt expression leads to a profound overgrowth of synaptic connections that directly correlates with the levels of Htt at nerve terminals. Branches of the same nerve containing different levels of Htt show distinct phenotypes, indicating Htt acts locally to disrupt synaptic growth. The effects of pathogenic Htt on synaptic growth arise from defective synaptic endosomal trafficking, leading to expansion of a recycling endosomal signaling compartment containing Sorting Nexin 16, and a reduction in late endosomes containing Rab11. The disruption of endosomal compartments leads to elevated BMP signaling within nerve terminals, driving excessive synaptic growth. Blocking aberrant signaling from endosomes or reducing BMP activity (see Wishful thinking) ameliorates the severity of HD pathology and improves viability. Pathogenic Htt is present largely in a non-aggregated form at synapses, indicating cytosolic forms of the protein are likely to be the toxic species that disrupt endosomal signaling. These data indicate that pathogenic Htt acts locally at nerve terminals to alter trafficking between endosomal compartments, leading to defects in synaptic structure that correlate with pathogenesis and lethality in the Drosophila HD model (Akbergenova, 2017).

    Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability

    Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. This study interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. NO was found to compromise synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, these data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release (Robinson, 2018).

    Throughout the central nervous system (CNS), the volume transmitter nitric oxide (NO) has been implicated in controlling synaptic function by multiple mechanisms, including modulation of transmitter release, plasticity, or neuronal excitability. NO-mediated posttranslational modifications (PTMs) in particular have become increasingly recognized as regulators of specific target proteins. S-nitrosylation is a nonenzymatic and reversible PTM resulting in the addition of a NO group to a cysteine (Cys) thiol/sulfhydryl group, leading to the generation of S-nitrosothiols (SNOs). In spite of the large number of SNO-proteins thus far identified, the functional outcomes and mechanisms of the underlying specificity of S-nitrosylation in terms of target proteins and Cys residues within these proteins are not clear (Robinson, 2018).

    Synaptic transmitter release is controlled by multiple signaling proteins and involves a cascade of signaling steps. This process requires the assembly of the soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) complex and associated proteins, the majority of which can be regulated to modulate synapse function. Regulatory mechanisms include phosphorylation of SNARE proteins as well as SNARE-binding proteins such as Complexin (Cpx), which have been reported at different synapses such as the Drosophila neuromuscular junction (NMJ) or in the rat CNS (Robinson, 2018).

    Several contrasting effects on transmitter release are induced by NO-mediated PTMs. Other forms of protein modification to modulate cellular signaling include prenylation, an attachment of a farnesyl or geranyl-geranyl moiety to a Cys residue in proteins harboring a C-terminal CAAX prenylation motif. This process renders proteins attached to endomembrane/endoplasmic reticulum (ER) and Golgi structures until further processing, as shown for Rab GTPases. Farnesylation also regulates mouse cpx 3/4 and Drosophila Cpx function. The Cys within CAAX motifs can also undergo S-nitrosylation, which interferes with the farnesylation signaling; however, direct evidence in a physiological environment is lacking. Cpx function has been studied in many different systems and there is controversy regarding its fusion-clamp activity. Cpx supports Ca2+-triggered exocytosis but also exhibits a clamping function. Analysis of mouse cpx double-knockout neurons lacking cpx 1 and 2 found only a facilitating function for cpx on release, and different D. melanogaster and Caenorhabditis elegans cpx mutant lines exhibit altered phenotypes in clamping or priming/fusion function, illustrating the controversial actions of Cpx (Robinson, 2018).

    This study investigated the effects of NO on synaptic transmission and found that NO reduces Ca2+-triggered release as well as the size of the functional vesicle pool, which was reversed by glutathione (GSH) signaling. At the same time, spontaneous release rates were negatively affected by NO. It was confirmed that cpx is S-nitrosylated and that NO changes the synaptic localization of cpx, as also seen following genetic and pharmacological inhibition of farnesylation. Thus, it is proposed that the function of cpx is regulated by S-nitrosylation of Cys within the CAAX motif to prevent farnesylation. This increases cpx-SNARE-protein interactions, thereby rendering cpx with a dominant clamping function, which suppresses both spontaneous and evoked release (Robinson, 2018).

    NO regulates a multitude of physiological and pathological pathways in neuronal function via generation of cGMP, thiol-nitrosylation, and 3-nitrotyrosination in health and disease. This study shows by employing biochemical and genetic tools in Drosophila, mouse, and HEK cells that NO can S-nitrosylate Cpx and modulate (in a cGMP-independent manner) neurotransmitter release at the NMJ by interfering with its prenylation status, thereby affecting the localization and function of this fusion-clamp protein. These nitrergic effects are reversed by GSH application or overexpression of GSH-liberating and de-nitrosylating enzymes (GCLm/c, GSNOR). GSH is the major endogenous scavenger for the NO moiety by the formation of S-nitrosoglutathione (GSNO) and consequently reduces protein-SNO levels via trans- and de-nitrosylation. The suppression of NOS activity facilitates synaptic function and the data support the notion that endogenous or exogenous NO enhances S-nitrosylation, reduces cpx farnesylation, and diminishes release (Robinson, 2018).

    Of the numerous synaptic molecules involved in release, Cpx in particular has been implicated in the regulation of both evoked and spontaneous release due to its fusion-clamp activity. Despite the seemingly simple structure of Cpx, its physiological function is highly controversial, as this small SNARE-complex binding protein can both facilitate but also diminish fast Ca2+-dependent and spontaneous release, depending on the system studied. In addition, there are different mammalian isoforms of Cpx (1-4), which differ in their C-terminal region, with only cpx 3/4 containing the CAAX prenylation motif. Farnesylation in general determines protein membrane association and protein-protein interactions, and some cpx isoforms, such as muscpx 3/4 and Dmcpx 7A, are regulated in this manner. However, muscpx 1/2 does not possess a CAAX motif, suggesting differential regulatory pathways to modulate cpx function. In Drosophila, there are alternative splice variants resulting from a single cpx gene, but the predominant isoform contains the CAAX motif (Dmcpx 7A), implicating the importance of this signaling molecule. The other splice isoform (Dmcpx 7B) lacks the CAAX motif and is expressed at about 1,000-fold lower levels at the larval stage [15], thus making Dmcpx 7A the dominant isoform to be regulated by farnesylation. However, the lack of Dmcpx 7B phosphorylation by PKA induces similar phenotypes as seen in the current experiments when assessed following an induction of activity-dependent potentiation of mEJC frequency, which also may involve cpx-synaptotagmin 1 interactions (Robinson, 2018).

    Interestingly, both depletion and excessive levels of cpx suppress Ca2+-dependent and -independent exocytosis. Cpx may promote SNARE complex assembly and simultaneously block completion of fusion by retaining it in a highly fusogenic state. Ca2+-dependent fusion is promoted below a concentration of 100 nM of cpx, whereas above 200 nM, it exhibits a clamping function resulting in a bell-shaped response curve. Previous work suggests that synaptotagmin 1, once bound to Ca2+, relieves the cpx block and allows fusion. Another study reported that selective competition between cpx and synaptotagmin 1 for SNARE binding allows regulation of release. The data are in agreement with the latter findings, as reduced synaptotagmin 1-Cpx interactions was observed following the block of farnesylation, indicating fewer synaptotagmin molecules binding to the SNARE complex to displace Cpx. This limited replacement of Cpx by synaptotagmin has been implicated in biochemical studies showing that local excess of Cpx inhibits release, presumably by outcompeting synaptotagmin binding. Thus, synaptotagmin-SNARE binding is strongly dependent upon the local concentration of Cpx. Alternatively, and this possibility cannot be excluded, the modulation of Cpx may simply alter its binding to the SNARE complex without directly displacing synaptotagmin, but interpretation of the data from the current assays (PLA, co-localization) would not allow distinguishing between these possibilities (Robinson, 2018).

    The data are compatible with the idea that Cpx binds to the SNARE complex, facilitates assembly, and then exerts its clamping function by preventing full fusion due to SNARE complex stabilization and subsequent increased energy barrier to allow fusion. The current model could provide an explanation of how Cpx can be regulated to signal downstream to modulate transmitter release. So far, there are no data available, apart from mutation studies, as to how Cpx function can be altered. This study provides data indicating a physiologically relevant mechanism to adjust Cpx function, possibly to the requirements of the neuron to adjust synaptic transmission. This likely occurs due to Cys S-nitrosylation and suppression of farnesylation, allowing greater amounts of hydrophilic Cpx, not bound to endomembranes, to be available for binding with the SNARE complex in an altered configuration. This cross signaling between nitrosylation/farnesylation has been proposed to act as a molecular switch to modulate Ras activity. The current data show that enhanced nitrergic activity and blocking farnesylation, either genetically (CpxΔX) or pharmacologically, alters the localization of Cpx at the Drosophila NMJ and that of GFP-CAAX in HEK cells. Furthermore, by using a nitroso-mimetic cpx mutant, this study found enhanced co-localization of Cpx with the AZ protein Brp, implying a localization-function relationship. This consequently increases the net-clamping function because of elevated local concentrations of Cpx. Dmcpx specifically exhibits a strong clamping function, as shown following overexpression in hippocampal neurons, which causes suppression of evoked and spontaneous release accompanied by a reduction of the release probability or reduced vesicle fusion efficiency in in vitro assays (Robinson, 2018).

    Two independent studies eliminating the CAAX motif in Dmcpx (cpx572 and cpx1257) investigated localization-function interactions and showed disagreeing effects on both release and cpx localization. In particular, it has also been shown that the truncated Cpx (cpx572, lacking the last 25 amino acids) does not co-localize with Brp. Interestingly, this mutant causes a strong decrease in C-terminal hydrophobicity and a modest physiological response (increased mini frequency, decreased evoked amplitudes equivalent to a loss of clamping and loss of fusion function) relative to the total knock-out (KO). In contrast, the cpx mutant with single amino acid deletion (cpx1275) causes no effect on evoked but identical effects on the frequency of spontaneous release, suggesting a lack of clamping but no lack of fusion function. In addition, this mutant now co-localizes with the AZ at the NMJ. These two studies indicate that the different mutations cause contrasting electrophysiological and morphological phenotypes, indicating that it is due to the nature of the mutation (lack of the last 25 amino acids versus 1 amino acid), which highlights the importance of a functional C-terminus. More recent studies have shown that deletions of the final amino acids (6 or 12 residues) completely abolished the membrane binding of Cpx-1, impairing its inhibitory function and confirming the requirement of an intact C-terminus for inhibition of release. This study used an endogenous Cpx with intact hydrophobic C-terminus, allowing physiological membrane binding. This is essential for inhibitory function, as the C-terminus is required for selective binding to highly curved membranes, such as those of vesicles. Thus, as different approaches are used to alter farnesylation, and a single a single amino acid mutant cpx (Dmcpx 7AC140W) was created, leaving the C-terminus intact, these studies were performed under conditions of endogenous regulation of Cpx function and thus provide new functional data on Cpx signaling. Importantly, the data show that this regulation alters cpx function, and this is the first study to provide an explanation for the differential effects observed using cpx mutants or even Cpx protein fragments in mammals, worm, and fly in various cross-species rescue experiments (Robinson, 2018).

    The data are in agreement with a model that non-farnesylated hydrophilic and soluble cytosolic Cpx binds to the vesicular membrane via its C-terminal interactions, thereby exerting its inhibitory effect. When proteins are farnesylated, they are likely tethered to endomembranes, other than vesicle membranes. It has to be distinguished between Cpx interaction with the vesicle membrane as a result of the hydrophobic C-terminus, allowing Cpx to become in close proximity to the AZ, and Cpx endomembrane binding following farnesylation, which prevents Cpx interactions with the AZ. However, in the current, SNO modification may enhance the binding to other proteins (e.g., SNAREs), thereby augmenting the effects. These additional interactions with unknown binding partners may affect proper Cpx function and explain some of the discrepancies seen in studies using other genetically altered Cpxs (Robinson, 2018).

    In summary, this study provides new data to illustrate a potential mechanism to regulate cpx function in a physiological environment, and it was shown that NO acts as an endogenous signaling molecule that regulates synaptic membrane targeting of Cpx, a pathway that may reconcile some of the controversial findings regarding Cpx function. It is suggested that increased S-nitrosylation and consequent lack of farnesylation leads to enhanced cytosolic levels of a soluble hydrophilic Cpx and less endomembrane-bound fractions, because farnesylation-incompetent proteins remain in the cytosol. These novel observations advance understanding of similar nitrergic regulation of farnesylation that may be relevant for mammalian cpx-dependent synaptic transmission at the retina ribbon synapse and other brain regions. Finally, this work has broader implications for physiological or pathological regulation of the prenylation pathway not only during neurodegeneration and aging, when enhanced S-nitrosylation might contribute to abnormal farnesylation signaling, but also in other biological systems in which nitrergic activity and prenylation have important regulatory functions such as in cardio-vasculature or cancer signaling (Robinson, 2018).

    Kinetochore proteins have a post-mitotic function in neurodevelopment

    The kinetochore is a complex of proteins, broadly conserved from yeast to man, that resides at the centromere and is essential for chromosome segregation in dividing cells. There are no known functions of the core complex outside of the centromere. This study shows that the proteins of the kinetochore have an essential post-mitotic function in neurodevelopment. At the embryonic neuromuscular junction of Drosophila melanogaster, mutation or knockdown of many kinetochore components cause neurites to overgrow and prevent formation of normal synaptic boutons. Kinetochore proteins were detected in synapses and axons in Drosophila. In post-mitotic cultured hippocampal neurons, knockdown of mis12 increased the filopodia-like protrusions in this region. It is concluded that the proteins of the kinetochore are repurposed to sculpt developing synapses and dendrites and thereby contribute to the correct development of neuronal circuits in both invertebrates and mammals (Zhao, 2019).

    Although there is no precedent for core kinetochore proteins functioning outside of chromosome mechanics, several lines of evidence argue that the observed Drosophila phenotypes in neurons are not secondary to chromosome segregation defects. First, errors of kinetochore assembly are lethal during cell division, arresting at the spindle assembly checkpoint, and this would have prevented motor neurons from forming; this study saw no loss of motor neurons in mis12 mutants. Second, were aneuploid or polyploid cells sometimes to escape that lethality, they would do so rarely, randomly, and with heterogeneity in the chromosomes lost. This study, however, detect synaptic phenotypes consistently at the NMJs of all mutant embryos examined. Third, the selective nature of the NMJ defect is difficult to reconcile with chromosomal aberrations: muscles are correctly patterned and have the correct complement; motor neurons are born and target consistently and appropriately to their muscles; and synaptic specializations form with appropriate components, despite the failure to form large boutons. Fourth, Ndc80 immunoreactivity is present at the embryonic NMJ and elsewhere in the nervous system; tagged kinetochore proteins, expressed under control of their normal promoters, were detected outside of nuclei in synaptic and axonal regions. In mammalian neurons, although only mis12 has been examined so far, the function of mis12 is clearly post-mitotic. It was detected by western blot in post-mitotic neurons, and the knockdown of mis12 in post-mitotic hippocampal neurons altered the morphology of hippocampal dendrites. A similar post-mitotic requirement for the proteins of the kinetochore has also been demonstrated in C. elegans, where the degradation of kinetochore proteins selectively in post-mitotic and differentiated sensory neurons disrupts their morphogenesis. Interestingly, there is precedent for the repurposing of other mitotic proteins, such as those of the anaphase-promoting complex, for post-mitotic functions in synaptogenesis (Zhao, 2019).

    This study was fortunate to find the synaptogenic role of mis12 in screen: had the maternal contribution been less, the motor neurons might not have formed, and had the contribution persisted into late-stage embryos, it might have been sufficient fully to support synaptogenesis. The observation that at least eight kinetochore components, including representatives of each of the kinetochore subcomplexes, give rise to the same phenotype at the NMJ suggests that they are functioning in a complex very much similar to that at the centromere. This supposition is further strengthened by the observations that, in several cases examined, the proper localization of one component was altered in a genetic background mutant for another component (Zhao, 2019).

    Although some alleles and RNAi lines did not have a phenotype, their role may have been obscured by the persistence of sufficient maternal contribution, as in the case of Cenp-C, or poor efficacy of the RNAi line. This is particularly true for Nuf2, which was detectable in larval axons although its RNAi line lacked a phenotype. It remains to be determined if the complete complement of kinetochore proteins or only mis12 function in the development of hippocampal dendrites (Zhao, 2019).

    In light of the knockdown phenotype of ndc80, the microtubule-binding subunit, at the embryonic NMJ and its presence in sparse puncta at that synapse, one parsimonious hypothesis is that the kinetochore proteins interact with neuronal microtubules akin to their function at the centromere. Microtubule dynamics are crucial to the formation of both synapses and dendrites, and this is consistent with the observation of significant overextension of both synaptic branches and sensory dendrites in Drosophila and alterations in dendritic morphology in hippocampal neurons. At the embryonic NMJ and in larval nerves, where individual puncta of kinetochore proteins could be resolved, the puncta were sparse, with just one or two per bouton. The axonal puncta in larval nerves were not as bright as those in nearby glial nuclei. Whereas multiple kinetochores and microtubule + ends are present at each centromere, the dim axonal puncta may represent individual kinetochore-like complexes at individual + ends, and this would account for the difficulty of imaging the proteins outside of the densely synaptic neuropil of the ventral nerve cord. In axons, the + ends of axonal microtubules are oriented toward growth cones and synapses, and while microtubules are splayed in growth cones, they are replaced during synaptogenesis by more stable bundles. When microtubules are not appropriately stabilized, synapse formation is perturbed, giving rise to abnormal extensions of axons and improper bouton formation. The phenotypes now reported suggest that this developmental transition requires a kinetochore-like complex. Hippocampal dendrites contain microtubules of both polarities and the increased filopodia-like protrusions that appear upon the knockdown of mis12 may arise from misorganization or overgrowth of dendritic microtubules. Future studies will need to clarify how the kinetochore influences microtubule organization. Analysis of kinetochore mutations has thus uncovered a previously unknown mechanism that appears to co-opt the fundamental mitotic functions of the ancient kinetochore complex for non-mitotic functions in both invertebrate and vertebrate neurodevelopment. A deeper understanding of these synaptogenic functions should therefore illuminate a process central to the accurate wiring of the brain (Zhao, 2019).

    Secreted C-type lectin regulation of neuromuscular junction synaptic vesicle dynamics modulates coordinated movement

    The synaptic cleft manifests enriched glycosylation, with structured glycans coordinating signaling between presynaptic and postsynaptic cells. Glycosylated signaling ligands orchestrating communication are tightly regulated by secreted glycan-binding lectins. Using the Drosophila neuromuscular junction (NMJ) as a model glutamatergic synapse, this study identified a new Ca2+-binding (C-type) lectin, Lectin-galC1 (LGC1), which modulates presynaptic function and neurotransmission strength. LGC1 is enriched in motoneuron presynaptic boutons and secreted into the NMJ extracellular synaptomatrix. LGC1 limits locomotor peristalsis and coordinated movement speed, with a specific requirement for synaptic function, but not NMJ architecture. LGC1 controls neurotransmission strength by limiting presynaptic active zone (AZ) and postsynaptic glutamate receptor (GluR) aligned synapse number, reducing both spontaneous and stimulation-evoked synaptic vesicle (SV) release, and capping SV cycling rate. During high-frequency stimulation (HFS), mutants have faster synaptic depression and impaired recovery while replenishing depleted SV pools. Although LGC1 removal increases the number of glutamatergic synapses, we find that LGC1-null mutants exhibit decreased SV density within presynaptic boutons, particularly SV pools at presynaptic active zones. Thus, LGC1 regulates NMJ neurotransmission to modulate coordinated movement (Bhimreddy, 2021).

    Activity-dependent global downscaling of evoked neurotransmitter release across glutamatergic inputs in Drosophila

    Within mammalian brain circuits, activity-dependent synaptic adaptations such as synaptic scaling stabilise neuronal activity in the face of perturbations. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. This study tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. Motoneuron-specific genetic manipulations were employed to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. An adaptation was detected which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This 'presynaptic downscaling' maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter (DVGLUT) expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. These results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side (Karunanithi, 2020).

    FOXO Regulates Neuromuscular Junction Homeostasis During Drosophila Aging

    The transcription factor Foxo is a known regulator of lifespan extension and tissue homeostasis. It has been linked to the maintenance of neuronal processes across many species and has been shown to promote youthful characteristics by regulating cytoskeletal flexibility and synaptic plasticity at the neuromuscular junction (NMJ). However, the role of foxo in aging neuromuscular junction function has yet to be determined. This study profiled adult Drosophila foxo- null mutant abdominal ventral longitudinal muscles and found that young mutants exhibited morphological profiles similar to those of aged wild-type flies, such as larger bouton areas and shorter terminal branches. Changes to the axonal cytoskeleton and an accumulation of late endosomes were also observed in foxo null mutants and motor neuron-specific foxo knockdown flies, similar to those of aged wild-types. Motor neuron-specific overexpression of foxo can delay age-dependent changes to NMJ morphology, suggesting foxo is responsible for maintaining NMJ integrity during aging. Through genetic screening, several downstream factors mediated through foxo-regulated NMJ homeostasis were identified, including genes involved in the MAPK pathway. Interestingly, the phosphorylation of p38 was increased in the motor neuron-specific foxo knockdown flies, suggesting foxo acts as a suppressor of p38/MAPK activation. This work reveals that foxo is a key regulator for NMJ homeostasis, and it may maintain NMJ integrity by repressing MAPK signaling (Birnbaum, 2020).

    Sphingolipids regulate neuromuscular synapse structure and function in Drosophila

    Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function and degeneration. Serine Palmitoyl-transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to approximately 50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig-domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. This data points to a role for sphingolipids in the regulation and fine-tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg (West, 2018).

    A prominent enrichment of wglycosphingolipids has been demonstrated within synaptic structures in the mammalian brain. To date understanding of the role of these enigmatic lipids in synapse structure and function has yet to be fully elucidated. Sphingolipids are major lipid components of the plasma and endomembrane system and have been implicated in many forms of neuropathy and neurodegeneration. Sphingolipids are proposed to generate structure in membranes due to their rigidity and association with cholesterol. They are also known to be potent signalling molecules regulating processes such as apoptosis, proliferation, migration and responses to oxidative stress (West, 2018).

    Numerous neurological and neurodegenerative conditions are directly attributable to the inability to synthesise or catabolise sphingolipids. The failure to synthesise all or particular sphingolipids gives rise to a number of neurological conditions such as infant-onset symptomatic epilepsy (loss of GM3 ganglioside synthesis, bovine spinal muscular atrophy (loss of 3- ketohydrosphingosine reductase and hereditary sensory and autonomic neuropathy type 1 (HSAN1; recessive and dominant mutations in serine palmitoyl transferase subunit 1 (SPTLC1). Conversely failure to catabolise sphingolipids in the lysosome generates a subset of lysosomal storage diseases/disorders (LSD's) known as sphingolipidoses, of which there are approximately 14 identified separate genetic conditions. Sphingolipids are now suggested to have a prominent role in the onset and progression of Alzheimer's disease while the production after bacterial infection of autoimmune antibodies to gangliosides present at the neuromuscular synapse is likely to cause the dramatic and often lethal paralysis seen in Guillain-Barrê and Miller-Fisher syndromes. The presence of sphingolipids at the synapse is further attested by the ability of tetanus and botulinal toxins to effect their entry to synapses via co-attachment to synaptic glycosphingolipids (West, 2018).

    While the presence of sphingolipids (in particular, glycosphingolipids) at the synapse is well established, little is known about their functional or structural role in the operational life of the synapse. Some in vitro studies have addressed the role of sphingolipids at synapses in the context of sphingolipid/cholesterol microdomains and indicate roles in the function and localisation of neurotransmitter receptors and synaptic exocytosis. The prominence of sphingolipids in neurological disease suggests that absence or accumulation of sphingolipids can exert an influence in synaptic function and indicates an inappropriately large gap in knowledge regarding the actions of these lipids at the synapse. In the above outlined context, roles for sphingolipids in synapse structure and function remain to be determined. To this end, this study has undertaken an analysis of sphingolipid function at a model synapse, the third instar neuromuscular junction of Drosophila. Mutations were analyzed in SPT2/SPTLC2 (Serine Palmitoyltransferase, Long Chain Base Subunit 2), which encodes an essential subunit of the Serine Palmitoyltransferase (SPT) heterodimer necessary for the initial step in sphingolipid synthesis, for defects in neuromuscular synapse structure. Evidence is presented to suggest that sphingolipids are essential for synaptic structure and function, and structural regulation may be mediated partially through function of the Ig domain adhesion protein Basigin/CD147 (Bsg) (West, 2018).

    The enrichment of sphingolipids at synapses has been long known. Assigning functions for these enigmatic lipids at the synapse has remained problematic. Ablation of gangliosides in mouse has identified subtle defects in neurotransmission while loss of G3- ganglioside synthesis results in an infantile onset epilepsy, the mechanism for which remains obscure. A specific role for sphingosine has been identified in promoting SNARE protein fusion and synaptic exocytosis (West, 2018).

    Many sphingolipid species present in the outer leaflet of the plasma membrane are found in association with cholesterol as 'lipid rafts'. Neurons receive supplementary cholesterol from glia which is essential for supporting synapse maturation and additional synaptogenesis suggesting cholesterol, and potentially lipid rafts, are rate limiting for these processes. Depletion of both cholesterol and sphingolipids together has been shown to reduce and enlarge dendritic spines with eventual loss of synapses in hippocampal neurons in culture possibly due to reduced association with lipid rafts of synapse structure promoting proteins such as Post-Synaptic Density protein 95 (PSD95). This study reduced synthesis of sphingolipids with a mutation in SPTLC2, and examined the development of neuromuscular synapses in the Drosophila larval preparation. This approach has allowed study of the genetic depletion of sphingolipids at an identified synapse in vivo and investigate a role for sphingolipids in the regulation of synaptic structure and activity. As part of this study, a potential role was identified for the Ig domain cell adhesion protein Bsg in sphingolipid dependent regulation of synaptic structure (West, 2018).

    The enrichment of sphingolipids at synapses has been long known. Assigning functions for these enigmatic lipids at the synapse has remained problematic. Ablation of gangliosides in mouse has identified subtle defects in neurotransmission while loss of G3-ganglioside synthesis results in an infantile onset epilepsy, the mechanism for which remains obscure. A specific role for sphingosine has been identified in promoting SNARE protein fusion and synaptic exocytosis. Many sphingolipid species present in the outer leaflet of the plasma membrane are found in association with cholesterol as 'lipid rafts'. Neurons receive supplementary cholesterol from glia which is essential for supporting synapse maturation and additional synaptogenesis suggesting cholesterol, and potentially lipid rafts, are rate limiting for these processes. Depletion of both cholesterol and sphingolipids together has been shown to reduce and enlarge dendritic spines with eventual loss of synapses in hippocampal neurons in culture possibly due to reduced association with lipid rafts of synapse structure promoting proteins such as Post-Synaptic Density protein 95 (PSD95). This study has reduced synthesis of sphingolipids with a mutation in SPTLC2, and examined the development of neuromuscular synapses in the Drosophila larval preparation. This approach has allowed study of the genetic depletion of sphingolipids at an identified synapse in vivo and investigate a role for sphingolipids in the regulation of synaptic structure and activity. As part of this study, a potential role has been identified for the Ig domain cell adhesion protein Bsg in sphingolipid dependent regulation of synaptic structure (West, 2018).

    On examination of sphingolipid deficient synapses, a disruption to the normal synaptic structure was observed. Synaptic boutons were enlarged and the overall numbers of boutons reduced by ~50% while the length of the neuromuscular synapse remained indistinguishable from wildtype. This phenotype is highly reminiscent of the reduction of synapse number, but increase in synapse size observed in hippocampal neurons in culture depleted for lipid rafts. Nevertheless, beyond the structural deficit of the synapse, the ultrastructure of the synapse was remarkably intact, suggesting a role in fine-tuning of synaptic properties (West, 2018).

    Synapses depleted for sphingolipids were capable of greater growth when combined with the synaptic overgrowth mutation highwire (hiw). The data suggests the mutations in lace, encoding a pyridoxal phosphate-dependent transferase, and sphingolipid depletion decouples bouton structure from normal synaptic length. Large boutons are observed in mutants of mothers against dpp (mad), thick veins (tkv), saxophone (sax) medea (med), and glass-bottom-boat (gbb), components of the TGF-β pathway that is known to regulate synaptic growth. However these mutations reduce synaptic length by ~50% and ultrastructural synaptic defects such as non-plasma membrane attached active zones (T-bars), large endosomal vesicles and ripples in pre-synaptic peri-active membranes are observed. One obvious ultrastructural defect that is present in sphingolipid depleted synapses is enlarged mitochondria. Enlarged mitochondria are observed in a number of sensory neuropathies and it is of interest that dominant mutations in SPTLC1 and SPTLC2 that generate aberrant sphingolipids give rise to Hereditary and Sensory Neuropathy Type 1 (HSAN1) where enlarged mitochondria are often observed. This may be attributable to a recognised role for sphingolipids in mitochondrial fission (West, 2018).

    To dissect the spatial requirement for sphingolipid regulation of synapse structure, the lace mutant was rescued with a rescue transgene, expressed globally, pre- or post-synaptically. It was possible to rescue synaptic bouton size and number with a global expression of the rescue transgene, but no aspects of the phenotype could be rescued with a pre-synaptic expression. Perturbed NMJ morphology could also be rescued by glial or post-synaptic expression of lace, however post-synaptic muscle expression generated a partial rescue, with an excess of 'satellite' boutons, a phenotype normally associated with integrin dysfunction or endocytic defects. Previous data feeding lace mutant larvae with sphingosine, the product of the SPT enzyme, partially rescued lace mutant associated phenotypes. Taken together with the current analysis, there is a strong suggestion that sphingolipid precursors such as sphingosine may be able to act non-cell autonomously, and traffic between cells to support synapse structure and function, but not when supplied from the nervous system (West, 2018).

    Analysis of EJP and miniEJP characteristics at the 3rd instar larval NMJ reveals mutations in lace produce, at the Ca2+/Mg2+ concentrations that were used, a small but significant increase in synaptic strength, accompanied by a change in short-term plasticity, with synaptic depression predominating over synaptic facilitation. NMJs with high-quantal content EJPs normally show synaptic depression during paired or short-train repetitive stimulation, while those with a low basal quantal content show synaptic facilitation (Lnenicka, 2000; Lnenicka, 2006). Further analysis is required, for instance using a range of Ca2+ concentrations, to establish whether this apparent change in synaptic plasticity is commensurate with a greater basal synaptic strength in the lace mutant larvae, or whether it represents a specific effect of the mutation, disrupting the normal link between mechanisms that couple basal quantal content to short-term synaptic plasticity. In vitro and in vivo analysis has suggested a role for sphingolipids in synaptic vesicle endocytosis and exocytosis in addition to a role in neurotransmitter distribution. No evident defects were observed in neurotransmitter receptor distribution. Interestingly, ablation of major subsets of gangliosides and subsequent synaptic function at the NMJ in a mouse model reveals a more pronounced run-down of neurotransmitter release upon sustained stimulation, consistent with the data presented in this study. It is not possible, however, to directly attribute the apparent deficit in synaptic facilitation observed in lace mutants to exoF or endocytosis, at this point (West, 2018).

    A phenotypic similarity at the larval neuromuscular synapse is noted between the lace mutants and mutations in the small Ig domain adhesion protein Basigin/CD147. Bsg is a glycoprotein localised in the plasma membrane that is known to genetically interact with integrins during development of the Drosophila eye. In Bsg mutants, synaptic boutons at the larval neuromuscular junction are enlarged in size and reduced in number with a modest reduction in synaptic span. Bsg has previously been localised to sphingolipid enriched lipid rafts in invading epithelial breast cells, and this study observed that Bsg is abundant in the lipid raft associated membrane fraction, co-sedimenting with syntaxin, a known component of lipid rafts. It cannot be said at this juncture if Bsg function is directly regulated by sphingolipids. Indeed, recruitment of Bsg to lipid rafts can be critical for the recruitment of other protein factors such as claudin-5 in retinal vascular epithelial cells. However, given the genetic interaction between Bsg and lace, with bsg;lace transheterozygous double mutants phenocopying both lace and bsg mutants, the data suggests Bsg and sphingolipids genetically interact to regulate synaptic structure. This interaction is interpreted as indirect; the loss of sphingolipid generated in the lace mutant affecting Bsg function to regulate synapse structure and function (West, 2018).

    Synaptic sphingolipids have previously been implicated in synaptic vesicle release, endocytosis, neurotransmitter receptor localisation and maintenance of synaptic activity. However other roles at the synapse for these enigmatic lipids remain elusive. Two potential functions for sphingolipid at the synapse are suggested by this study. Mitochondrial uptake of Ca2+ shapes Ca2+ dependent responses. The enlarged mitochondria observed in lace mutants may impinge on Ca2+ uptake to affect synaptic facilitation. A further deficit in Ca2+ handling at the synapse is suggested by the recent finding that Bsg is an obligatory subunit of plasma membrane Ca2+-ATPases (PMCAs). PMCAs extrude Ca2+ to the extracellular space, and knock-out of Bsg considerably affects Ca2+ handling by PMCAs. Sphingolipid deficient synapses in the lace mutant have deficits in Bsg function which may in turn have an effect on Ca2+ dynamics via PMCA function (West, 2018).

    Ablation of sphingolipid synthesis at a Drosophila model synapse supports a role for sphingolipids in maintenance of synaptic activity and regulation of synaptic structure. The analysis also points to sphingolipid dependent regulation of synaptic structure via function of the small Ig-domain protein Bsg. The precise regulation of synapse structure and function is a potent mechanism underlying synaptic plasticity and it is suggested that the presence of sphingolipids at synapse may partially reflect this function (West, 2018).

    Parvalbumin expression affects synaptic development and physiology at the Drosophila larval NMJ

    Presynaptic Ca(2+) appears to play multiple roles in synaptic development and physiology. This study examined the effect of buffering presynaptic Ca(2+) by expressing parvalbumin (PV) in Drosophila neurons, which do not normally express PV. The studies were performed on the identified Ib terminal that innervates muscle fiber 5. The volume-averaged, residual Ca(2+) resulting from single action potentials (APs) and AP trains was measured using the fluorescent Ca(2+) indicator, OGB-1. PV reduced the amplitude and decay time constant (tau) for single-AP Ca(2+) transients. For AP trains, there was a reduction in the rate of rise and decay of [Ca(2+)]i but the plateau [Ca(2+)]i was not affected. Electrophysiological recordings from muscle fiber 5 showed a reduction in paired-pulse facilitation, particularly the F1 component; this was likely due to the reduction in residual Ca(2+). These synapses also showed reduced synaptic enhancement during AP trains, presumably due to less buildup of synaptic facilitation. The transmitter release for single APs was increased for the PV-expressing terminals and this may have been a homeostatic response to the decrease in facilitation. Confocal microscopy was used to examine the structure of the motor terminals and PV expression resulted in smaller motor terminals with fewer synaptic boutons and active zones. This result supports earlier proposals that increased AP activity promotes motor terminal growth through increases in presynaptic [Ca(2+)]i (He, 2018).

    GAL4 drivers specific for type Ib and type Is motor neurons in Drosophila

    The Drosophila melanogaster larval neuromuscular system is extensively used by researchers to study neuronal cell biology, and Drosophila glutamatergic motor neurons have become a major model system. There are two main types of glutamatergic motor neurons, Ib and Is, with different structural and physiological properties at synaptic level at the neuromuscular junction. To generate genetic tools to identify and manipulate motor neurons of each type, GAL4 driver lines were screened for this purpose. This study describes GAL4 drivers specific for examples of neurons within each Type, Ib or Is. These drivers showed high expression levels and were expressed in only few motor neurons, making them amenable tools for specific studies of both axonal and synapse biology in identified Type I motor neurons (Perez-Moreno, 2018).

    Oxidative stress induces overgrowth of the Drosophila neuromuscular junction

    Synaptic terminals are known to expand and contract throughout an animal's life. The physiological constraints and demands that regulate appropriate synaptic growth and connectivity are currently poorly understood. Previous work has identified a Drosophila model of lysosomal storage disease (LSD), spinster (spin), with larval neuromuscular synapse overgrowth. This study identified a reactive oxygen species (ROS) burden in spin that may be attributable to previously identified lipofuscin deposition and lysosomal dysfunction, a cellular hallmark of LSD. Reducing ROS in spin mutants rescues synaptic overgrowth and electrophysiological deficits. Synapse overgrowth was also observed in mutants defective for protection from ROS and animals subjected to excessive ROS. ROS are known to stimulate JNK and fos signaling. Furthermore, JNK and fos in turn are known potent activators of synapse growth and function. Inhibiting JNK and fos activity in spin rescues synapse overgrowth and electrophysiological deficits. Similarly, inhibiting JNK, fos, and jun activity in animals with excessive oxidative stress rescues the overgrowth phenotype. These data suggest that ROS, via activation of the JNK signaling pathway, are a major regulator of synapse overgrowth. In LSD, increased autophagy contributes to lysosomal storage and, presumably, elevated levels of oxidative stress. In support of this suggestion, this study reports that impaired autophagy function reverses synaptic overgrowth in spin. These data describe a previously unexplored link between oxidative stress and synapse overgrowth via the JNK signaling pathway (Milton, 2011).

    Reactive oxygen species regulate activity-dependent neuronal plasticity in Drosophila

    Reactive oxygen species (ROS) have been extensively studied as damaging agents associated with ageing and neurodegenerative conditions. Their role in the nervous system under non-pathological conditions has remained poorly understood. Working with the Drosophila larval locomotor network, this study showed that in neurons ROS act as obligate signals required for neuronal activity-dependent structural plasticity, of both pre- and postsynaptic terminals. ROS signaling is also necessary for maintaining evoked synaptic transmission at the neuromuscular junction, and for activity-regulated homeostatic adjustment of motor network output, as measured by larval crawling behavior. The highly conserved Parkinson's disease-linked protein DJ-1β was identified as a redox sensor in neurons where it regulates structural plasticity, in part via modulation of the PTEN-PI3 Kinase pathway. This study provides a new conceptual framework of neuronal ROS as second messengers required for neuronal plasticity and for network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction (Oswald, 2018).

    This study set out to investigate potential roles for ROS in the nervous system under non-pathological conditions, which are much less well understood. The brain is arguably the most energy demanding organ and mitochondrial oxidative phosphorylation is a major source of ROS. It was therefore asked whether neurons might utilize mitochondrial metabolic ROS as feedback signals to mediate activity-regulated changes. As an experimental model the motor system was used of the fruitfly larva, Drosophila melanogaster allowing access to uniquely identifiable motoneurons in the ventral nerve cord and their specific body wall target muscles. An experimental paradigm was established for studying activity-regulated structural adjustments across an identified motoneuron, quantifying changes at both pre- and postsynaptic terminals. Thermogenetic neuronal over-activation leads to the generation of ROS at presynaptic terminals, and ROS signaling is necessary and sufficient for the activity-regulated structural adjustments. As a cellular ROS sensor the conserved redox sensitive protein DJ-1β, a homologue of vertebrate DJ-1 (PARK7), was identified and the phosphatase and tensin homolog (PTEN) and PI3kinase were identified as downstream effectors of activity-ROS-mediated structural plasticity. ROS signaling is also required for maintaining constancy of evoked transmission at the neuromuscular junction (NMJ) with a separate ROS pathway regulating the amplitude of spontaneous vesicle release events. Behaviourally, ROS signaling is required for the motor network to adjust homeostatically to return to a set crawling speed following prolonged overactivation (Oswald, 2018).

    Building on previous work that had shown oxidative stress as inducing NMJ growth (Milton, 2011), this study identified ROS as obligatory signals for activity-regulated structural plasticity. It was further shown that ROS are also sufficient to bring about structural changes at synaptic terminals that largely mimic those induced by neuronal overactivation. A mitochondrially targeted ROS reporter suggests a positive correlation between levels of neuronal activity and ROS generated in mitochondria, potentially as a byproduct of increased ATP metabolism or triggered by mitochondrial calcium influx. Although this study did not specifically investigate the nature of the active ROS in this context, three lines of evidence suggest that H2O2, generated by the dismutation of O2-, is the principal signaling species. First, under conditions of neuronal overactivation (but not control levels of activity) over-expression of the O2- to H2O2 converting enzyme SOD2 potentiated structural plasticity phenotypes. Second, over-expression of the H2O2 scavenger Catalase efficiently counter-acts all activity-induced changes that were quantified, at both postsynaptic dendritic and presynaptic NMJ terminals. Third, over-expression of the H2O2 generator Duox in motoneurons is sufficient to induce NMJ bouton phenotypes that mimic overactivation. In addition to mitochondria, other sources of ROS include several oxidases, notably NADPH oxidases. These have been implicated during nervous system development in the regulation of axon growth and synaptic plasticity. NADPH oxidases can be regulated by NMDA receptor stimulation and activity-associated pathways, including calcium, Protein kinases C and A and calcium/calmodulin-dependent kinase II (CamKII). The precise sources of activity-regulated ROS, potentially for distinct roles in plasticity, will be interesting to investigate (Oswald, 2018).

    This study demonstrated that ROS are necessary for activity-dependent structural plasticity of Drosophila motoneurons, at both their postsynaptic dendrites in the CNS and presynaptic NMJs in the periphery. The mechanisms by which ROS intersect with other known plasticity pathways now need to be investigated. Among well documented signaling pathways regulating synaptic plasticity, are Wnts, BMPs, PKA, CREB and the immediate early gene transcription factor AP-1. ROS signaling could be synergistic with other neuronal plasticity pathways, potentially integrating metabolic feedback. Indeed, ROS modulate BMP signaling in cultured sympathetic neurons and Wnt pathways in non-neuronal cells. Biochemically, ROS are well known regulators of kinase pathways via oxidation-mediated inhibition of phosphatases. Redox modifications also regulate the activity of the immediate early genes Jun and Fos, which are required for LTP in vertebrates, and in Drosophila for activity-dependent plasticity of motoneurons, both at the NMJ and central dendrites. It was therefore hypothesized that ROS may provide neuronal activity-regulated modulation of multiple canonical synaptic plasticity pathways (Oswald, 2018).

    This study focused on three aspects of synaptic terminal plasticity: dendritic arbor size in the CNS, and bouton and active zone numbers at the NMJ. These were used as phenotypic indicators for activity-regulated changes. By working with identified motoneurons adaptations could be observed across the entire neuron, relating adjustments of postsynaptic dendritic input terminals in the CNS to changes of the presynaptic output terminals at the NMJ in the periphery. For the aCC motoneuron, the degree of neuronal overactivation correlates with changes in synaptic terminal growth: notably reductions of dendritic arbor size centrally and of active zones at the NMJ. Interestingly, presynaptic active zone numbers did not show a linear response profile. Within a certain range low-level activity increases lead to more active zones, associated with potentiation; however, with stronger overactivation active zone number decrease. Reduction of active zones, as was observed at the NMJ, and of Brp levels by increased activation was previously also reported in photoreceptor terminals of the Drosophila adult visual system. At a finer level of resolution it will be interesting to determine how these activity-ROS-mediated structural changes might change active zone cytomatrix composition, which can impact on transmission properties, such as vesicle release probability (Oswald, 2018).

    Previous work found that in these motoneurons dendritic length correlates with the number of input synapses and with synaptic drive) Therefore, the negative correlation between the degree of overactivation and the reduction in central dendritic arbors is tentatively interpreted as compensatory. In agreement, it was found that blockade of activity-induced structural adjustment targeted to the motoneurons prevents behavioral adaptation normally seen after prolonged overactivation. Less clear is if and how overactivation-induced structural changes at the NMJ might be adaptive. Unlike many central synapses that facilitate graded analogue computation, the NMJ is a highly specialized synapse with a large safety factor and intricate mechanisms that ensure constancy of evoked transmission in essentially digital format. Rearing larvae at 29°C (which acutely increases motor activity) leads to more active zones at the NMJ and potentiated transmission, yet these larvae crawl at the same default speed as other larvae reared at 25°C (control) or 32°C with reduced numbers of active zones. This suggests that at least with regard to regulating crawling speed, plasticity mechanisms probably operate at the network level, rather than transmission properties of the NMJ. Indeed, recordings of transmission at the NMJ, and those reported by others, show homeostatic maintenance of eEJP amplitude irrespective of changes in bouton and active zone number. Though in this study focus was placed on anatomical changes, these structural adjustments are expected to be linked to, and probably preceded by compensatory changes in neuronal excitability that have been documented (Oswald, 2018).

    The observations of activity-regulated adjustments of both dendritic arbor size and NMJ structure give the impression of processes coordinated across the entire neuron. If this was the case, it could be mediated by transcriptional changes, potentially via immediate early genes (AP-1), which are involved in activity and ROS-induced structural changes at the NMJ and motoneuron dendrites (Oswald, 2018).

    In neurons the highly conserved protein DJ-1β; is critical for both structural and physiological changes in response to activity-generated ROS. In neurons DJ-1β might act as a redox sensor for activity-generated ROS. In agreement with this idea, DJ-1β has been shown to be oxidized by H2O2 at the conserved cysteine residue C106 (C104 in Drosophila). Oxidation of DJ-1 leads to changes in DJ-1 function, including translocation from the cytoplasm to the mitochondrial matrix, aiding protection against oxidative damage and maintenance of ATP levels. The ability of motoneurons to respond to increased activation is potently sensitive to DJ-1β dosage. It is also blocked by expression of mutant DJ-1βC104A that is non-oxidisable on the conserved Cys104. These observations suggest that DJ-1β is critical to ROS sensing in neurons. They also predict that cell type-specific DJ-1β levels, and associated DJ-1β reducing mechanisms, could contribute to setting cell type-specific sensitivity thresholds to neuronal activity (Oswald, 2018).

    The data suggest that DJ-1β could potentially be part of a signaling hub. At the NMJ, this might mediate plasticity across a range, from the addition of active zones associated with potentiation to, following stronger overactivation, the reduction of active zones. This study identified disinhibition of PI3Kinase signaling as one DJ-1β downstream pathway, a well-studied intermediate in metabolic pathways and a known regulator of synaptic terminal growth, including active zone addition. However, with stronger overactivation DJ-1β might engage additional downstream effectors that reduce active zone addition or maintenance, potentially promoting active zone disassembly. While at the presynaptic NMJ PI3Kinase disinhibition explains activity-regulated changes in bouton addition, different DJ-1β effectors likely operate in the somato-dendritic compartment, which responds to overactivation with reduced growth and possibly pruning. Thus, sub-cellular compartmentalization of the activity-ROS-DJ-1β signaling axis could produce distinct plasticity responses in pre- versus post-synaptic terminals (Oswald, 2018).

    Previous studies demonstrated a requirement for ROS for LTP and found learning defects in animal models with reduced NADPH oxidase activity, suggesting that synaptic ROS signaling might be a conserved feature of communication in the nervous system. Sharp electrode recordings from muscle DA1 revealed three interesting aspects. First, that changing ROS signaling in the presynaptic motoneuron under normal activity conditions does not obviously impact on NMJ transmission. Second, quenching of presynaptic ROS by expression of Catalase under overactivation conditions led to a significant decrease in eEJP amplitude and concomitantly reduced quantal content. This shows that upon chronic neuronal overactivation ROS signaling is critically required in the presynaptic motoneuron for maintaining eEJP amplitude by increasing vesicle release at the NMJ. This could be achieved by increasing vesicle release probability, which would counteract the reduction in active zone number following a period of neuronal overactivation. In this context it is interesting that components of the presynaptic release machinery, including SNAP25, are thought to be directly modulated by ROS, while others, such as Complexin, might be indirectly affected, for example via ROS-mediated inhibition of phosphatases leading disinhibition of kinase activity. Third, this study found that overactivation of motoneurons leads to reduced mEJP amplitude, also recently reported by others (Yeates, 2017). Curiously, mEJP amplitude, unlike eEJP amplitude, is regulated by DJ-1β, but is not impacted on by artificially increased cytoplasmic levels of the H2O2 scavenger Catalase. How it is that under conditions of neuronal overactivation eEJP and mEJP amplitudes are differentially sensitive to cytoplasmic Catalase versus DJ-1β oxidation is unclear, though it marks these two processes as distinct. One possibility is that cytoplasmic Catalase changes the local redox status, which could directly affect the properties of the presynaptic active zone cytomatrix. In contrast, mEJP amplitude regulation might be indirect and cell non-autonomous, via modulation of glutamate receptors in the postsynaptic target muscle (Oswald, 2018).

    Thus, several distinct ROS responsive pathways appear to operate at the NMJ. Structural adjustments in terms of synaptic terminal growth and synapse number are mediated by mechanisms sensitive to DJ-1β oxidation, potentially regulated via local reducing systems, including Catalase. In addition and distinct from these structural changes, at least in part, are the ROS-regulated adjustments in synaptic transmission that show different ROS sensitivities, one maintaining quantal content of evoked transmission while the other reduces mEJP amplitude when neuronal activity goes up. It is conceivable that spatially distinct sources of ROS, for example mitochondria versus membrane localized NADPH oxidases, with different temporal dynamics could potentially mediate such differences in ROS sensitivities at the NMJ (Oswald, 2018).

    Experiments exploring the potential behavioral relevance of activity-regulated structural plasticity demonstrated that network drive is regulated by ambient temperature. Acute elevation in ambient temperature produces faster crawling, while acute temperature reduction has the opposite effect. In contrast, with chronic temperature manipulations, larval crawling returns to its default speed (approx. 0.65-0.72 mm/sec). This adaptation to chronic manipulations might overall be energetically more favorable. It also allows larvae to retain a dynamic range of responses to relative changes in ambient temperature (i.e., speeding up or slowing down) (Oswald, 2018).

    Where in the locomotor network these adjustments take place remains to be worked out. It is reasonable to assume that proprioceptive sensory neurons, and potentially also central recurrent connections, provide feedback information that facilitates homeostatic adjustment of network output. This study's manipulations of the glutamatergic motoneurons show these cells are clearly important. For example, cell type-specific overactivation of the glutamatergic motoneurons (via dTrpA1) on the one hand, and blockade of activity-induced structural adjustment (by mis-expression of non-oxidizable DJ-1βC104A) on the other demonstrated that ROS-DJ-1β-mediated processes that were showed important for structural adjustment are also required for implementing homeostatic tuning of locomotor network output. The capacity of motoneurons as important elements in shaping motor network output, might be explicable in that these neurons constitute the final integrators on which all pre-motor inputs converge (Oswald, 2018).

    In conclusion, this study identified ROS in neurons as novel signals that are critical for activity-induced structural plasticity\. ROS levels regulated by neuronal activity have the potential for operating as metabolic feedback signals. The conserved redox-sensitive protein DJ-1β was further as important to neuronal ROS sensing, and the PTEN/PI3Kinase synaptic growth pathway was identified as a downstream effector pathway for NMJ growth in response to neuronal overactivation. These findings suggest that in the nervous system ROS operate as feedback signals that inform cells about their activity levels. The observation that ROS are important signals for homeostatic processes explains why ROS buffering is comparatively low in neurons. This view also shines a new light on the potential impact of ROS dysregulation with age or under neurodegenerative conditions, potentially interfering with neuronal adaptive adjustments and thereby contributing to network malfunction and synapse loss (Oswald, 2018).

    A circuit-dependent ROS feedback loop mediates glutamate excitotoxicity to sculpt the Drosophila motor system

    Overproduction of reactive oxygen species (ROS) is known to mediate glutamate excitotoxicity in neurological diseases. However, how ROS burdens can influence neural circuit integrity remains unclear. This study investigated the impact of excitotoxicity induced by depletion of Drosophila Eaat1, an astrocytic glutamate transporter, on locomotor central pattern generator (CPG) activity, neuromuscular junction architecture, and motor function. Glutamate excitotoxicity triggers a circuit-dependent ROS feedback loop to sculpt the motor system. Excitotoxicity initially elevates ROS to inactivate cholinergic interneurons, consequently changing CPG output activity to overexcite motor neurons and muscles. Remarkably, tonic motor neuron stimulation boosts muscular ROS, gradually dampening muscle contractility to feedback-enhance ROS accumulation in the CPG circuit and subsequently exacerbate circuit dysfunction. Ultimately, excess premotor excitation of motor neurons promotes ROS-activated stress signaling to alter neuromuscular junction architecture. Collectively, these results reveal that excitotoxicity-induced ROS can perturb motor system integrity by a circuit-dependent mechanism (Peng, 2019).

    Reactive oxygen species (ROS) are generated as the by-product of mitochondrial oxidative phosphorylation. In the central nervous system, under physiological conditions, high energy demand results in higher levels of ROS production relative to those in other body parts. In the past, endogenously generated ROS were recognized as signaling molecules that regulate a range of nervous system processes, including neuronal polarity, growth cone pathfinding, neuronal development, synaptic plasticity, and neural circuit tuning (Li, 2016; Oswald, 2018). By contrast, ROS overproduction and/or overwhelming the antioxidant machinery can generate ROS burdens, termed oxidative stress, in aging and diverse pathological conditions. In turn, excess ROS causes the malfunction and overactivation of ROS-regulated cell signaling pathways. Moreover, the highly oxidative properties of ROS are damaging to nucleotides, proteins, and lipids, eventually leading to neuronal dysfunction or demise. Hence, advancing understanding of the mechanisms underlying ROS-induced neurotoxicity should aid the development of potent therapeutic treatments for neurological disorders (Peng, 2019).

    Glutamate acts as the major excitatory neurotransmitter that regulates nearly all activities of the nervous system, with a tight balance between glutamate release and reuptake keeping the micromolar concentration of extracellular glutamate low. In diseases, accumulation of extrasynaptic glutamate results in glutamate-mediated excitotoxicity to the nervous system. Dysfunction of Na+/K+-dependent excitatory amino acid transporters (EAATs) is a key element of glutamate-mediated excitotoxicity. In mammals, there are five EAAT subtypes, that is EAAT1 (GLAST), EAAT2 (GLT1), EAAT3 (EAAC1), EAAT4, and EAAT5. EAAT3, EAAT4 and EAAT5 are expressed in neurons, whereas EAAT1 and EAAT2 are mainly present in astrocytes, where they are enriched in astrocyte terminal processes that form tripartite synapses with neurons and where they take up approximately 90% of released glutamate. Glutamate-mediated excitotoxicity can trigger bulk Ca2+ influx into postsynaptic neurons via NMDA receptors, which causes mitochondrial Ca2+ overload, along with other cellular responses, and which subsequently generates excess amounts of ROS. Notably, it has emerged that dysregulation of neural circuit activity can initiate subsequent disruption of the integrity of other constituents in the same network, resulting in overall circuit dysfunction and even neurodegeneration. However, it still remains unclear whether and how excitotoxicity-induced ROS can influence the integrity of neural circuits (Peng, 2019).

    Coordinated animal behaviors are linked to the activity of spinal cord central pattern generators (CPG), which are known to be specialized circuits that integrate inputs from the central brain and sensory neurons, and that subsequently generate rhythmic and patterned outputs to motor neurons. The Drosophila feed-forward locomotor circuit has served as an appropriate model for exploring the pathogenic network mechanisms that underlie neurodegenerative diseases, because it has a relatively simple neural circuitry compared to mammals yet retains conserved functions. This study explored whether glutamate-mediated excitotoxicity impacts locomotor CPG activity, neuromuscular junction (NMJ) architecture, and motor function. Interestingly, it was found that glutamate-mediated excitotoxicity due to depletion of Drosophila Eaat1, the sole Drosophila homolog of human EAAT2, can induce a circuit-dependent ROS feedback loop that impairs the proper activities of the locomotor CPG circuit and muscles, ultimately leading to motor neuron overexcitation, abnormal NMJ growth and strength, and compromised movement. Together, this work reveals a circuit-dependent mechanism for increasing ROS, which mediates glutamate excitotoxicity to sculpt the Drosophila locomotion network (Peng, 2019).

    This work utilized a fly model of glutamate excitotoxicity induced by loss of Drosophila eaat1 to explore the impact of glutamate excitotoxicity on the integrity of the motor system. A circuit-dependent feedback mechanism is described for increasing ROS that mediates excitotoxicity to alter premotor circuit activity, NMJ architecture, and motor function. Glutamate excitotoxicity initially alters locomotor CPG activity and hence prolongs CPG output bursts onto motor neurons by ROS-mediated inactivation of the cholinergic interneurons constituting the CPG circuit. Then, tonic premotor stimulation triggers activity-dependent ROS overproduction in both motor neurons and muscles. In muscles, the increased ROS level gradually dampens muscle contractility and consequent sensory input back to the locomotor CPG circuit, with this feedback strengthening ROS accumulation within the CPG circuit to exacerbate circuit dysfunction. Thus, a positive feedback loop between ROS production in the CPG circuit and muscles is established. Finally, in motor neurons, the induced ROS activate JNK stress signaling to promote abnormal NMJ bouton outgrowth and strength. Apart from genetic rescue, pharmacological treatment with the antioxidant AD4 or the K+ channel blocker 4-AP can also significantly alleviate these motor-system deficits (Peng, 2019).

    The locomotor CPG circuit for Drosophila larval feed-forward locomotion is positioned in the VNC and is activated by input from the central brain. Furthermore, acute treatment of dissected Drosophila larvae with non-competitive NMDA antagonists has been shown to reduce the initial output burst duration of the locomotor CPG and eventually abolishes all output activity (Cattaert, 2001), suggesting that glutamatergic transmission drives locomotor CPG activity and positively controls its output burst duration during larval movement. Consistent with these latter results, this study has shown that VNC-restricted expression of eaat1-venus using tsh-GAL4 could reverse the prolonged CPG output burst but not the reduced CPG output frequency in eaat1 mutants, indicating that central brain removal of eaat1 reduced burst frequency, whereas VNC removal markedly extended burst duration. The exact neuronal identity and network connections that build up the core components of the Drosophila larval locomotor CPG circuit remain unknown. Intriguingly, the phenotype of prolonged CPG output has also been reported under conditions in which the motor neuron inputs from proprioceptive sensory neurons or period-positive median segmental interneurons (PMSI) are limited. Moreover, RNAi-mediated knockdown of eaat1 extends PMSI-evoked inhibitory postsynaptic currents in motor neurons (MacNamee, 2016). An increase in extrasynaptic glutamate at the axonal synapses of PMSI was noticed when eaat1 is lost, raising an alternative possibility that excess extrasynaptic glutamate may desensitize GluClα to further diminish sensory inhibition feedback. However, this study found that reducing gluRIID but not gluClα in the eaat1 mutant background shortened prolonged CPG output. Hence, upon loss of eaat1, glutamate-mediated excitotoxicity mainly contributes to locomotor CPG circuit dysfunction. In this regard, the CPG outputs to motor neurons may be elongated and/or the potential inhibition from CPG output to PMSI or its upstream interneurons may be abrogated. Further experiments will be needed to unravel the detailed mechanism operating in the CPG circuit upon loss of eaat1 (Peng, 2019).

    Targeted relief of the increased ROS in cholinergic interneurons by genetic approaches could significantly alleviate altered CPG activity arising from either eaat1 mutation or short-term exposure to H2O2, indicating that a subset of cholinergic interneurons, which presumably constitutes the locomotor CPG circuit, is vulnerable to and influenced by the ROS increase. Temporal rescue experiments further suggest that the effect of the ROS increase on circuit activity is acute and reversible. In support of the fact that ROS is known to reduce the inactivation of voltage-gated potassium channels in neurons, long-term food-mediated feeding of (or even short-term exposure to) the K+ channel blocker 4-AP led to a restoration of CPG activity in eaat1 mutants. Thus, temporal hypoexcitability of cholinergic interneurons most probably underlies ROS-induced locomotor CPG dysfunction upon eaat1 loss. Interestingly, immediate blockade of glutamatergic transmission shortens the burst duration of the CPG output (Cattaert, 2001). Under this scenario, it is expected that shortened rather than prolonged CPG burst durations should occur upon loss of eaat1. Thus, it is likely that the induced ROS may occur in a restricted way in a certain subset of cholinergic interneurons, resulting in uneven suppression of cholinergic transmission in the locomotor CPG circuit (Peng, 2019).

    The GABA neurotransmitter has a crucial role in neuronal inhibition in the central nervous system through its actions on GABA receptors. Notably, regulation of GABAergic transmission by redox signaling is increasingly recognized. ROS, especially those derived from mitochondrial respiration, act to strengthen the neuronal inhibition mediated by GABAA receptors. Thus, it cannot be excluded that, if those ROS-vulnerable cholinergic interneurons also receive GABAergic input, the increased ROS may silence cholinergic transmission of the locomotor CPG circuit by strengthening GABA-mediated inhibition (Peng, 2019).

    The pathological roles of ROS in the regulation of skeletal muscles have been studied extensively, and most targets of redox signaling in skeletal muscle participate in muscle contraction. For instance, excess ROS can modulate SR calcium ATPase (SERCA) and ryanodine receptor (RyR) activity, both of which control the Ca2+ homeostasis of sarcoplasmic reticulum. ROS exposure can also oxidize some myofilament proteins, such as myosin heavy chain and troponin C and, in turn, can impair their functions. Consistent with these findings, the ROS increase dampens the muscle contractility of Drosophila larvae. This study found that mitochondrial and cytosolic ROS levels increase upon excess motor neuron stimulation when eaat1 is lost. Moreover, while increasing ROS by dsod1 knockdown reduced muscle contractility and movement velocity, relieving excess ROS in eaat1 mutant muscles improved locomotion. In the motor system, muscles are not only recognized as the end executive tissues for body movement, but also have a crucial role in triggering proprioceptive sensory feedback input to the central circuit. Recent studies in Drosophila have also revealed that proprioceptive sensory feedback plays a vital role in tuning locomotor circuit activity in the homeostatic adjustment of Drosophila larval crawling and in a Drosophila model of amyotrophic lateral sclerosis (ALS). Unexpectedly, this study found that, upon glutamate excitotoxicity, ROS-induced muscle weakness can cause inefficient sensory feedback input to worsen the ROS burden and can negatively impact the functioning of the central locomotor network. Therefore, under pathological conditions, impaired muscle activity can serve as a key mediator for initiating the ROS feedback loop between the CPG circuit and muscles, which may contribute to network dysfunction in excitotoxicity-associated diseases (Peng, 2019).

    Excitotoxicity-induced premotor circuit dysfunction elicits activity-dependent synaptic changes ROS are known to activate the JNK/AP-1 signaling pathway that regulates synaptic formation and strength in Drosophila. The mutation in Drosophila spinster (spin), which encodes a late endosome and lysosome protein, causes impaired lysosomal activity and a consequent ROS burden, leading to synaptic bouton outgrowth by activating the JNK signaling pathway. Intriguingly, c-FOS but not c-JUN is important for bouton outgrowth under spin loss. Similarly, it was found that the synaptic bouton phenotypes of eaat1 mutants are dependent on ROS and c-FOS activity. Interestingly, Milton (2011) have shown that 'constitutive' boosting of mitochondria-derived ROS under loss of dsod2 or after paraquat treatment can also promote bouton growth, but in that case it requires both c-FOS and c-JUN activities. By contrast, in eaat1 mutants, the altered CPG pattern possibly elicits a pulsed increase of mitochondrial ROS. Thus, it may be postulated that different resources and temporal generation of ROS may be responsible for engaging different cellular signaling processes. In support of this notion, in addition to mitochondria, NADPH oxidases provide another major source of ROS to control diverse cellular processes. Recently, DJ-1β, a Parkinson's disease-linked protein, has been identified as a redox sensor that mediates the mitochondrial ROS regulating activity-dependent synaptic plasticity at Drosophila NMJ. It will be interesting to further investigate the underlying mechanisms in detail (Peng, 2019).

    Potential relevance of ROS-induced motor-circuit dysregulation for neurodegenerative diseases Downregulation of EAAT2 has been demonstrated in patients with Alzheimer's disease or amyotrophic lateral sclerosis (ALS), as well as in ALS rodent models. ALS is a fatal adult-onset disease that predominantly causes NMJ denervation, motor neuron degeneration, and compromised motor function. Spinal removal of mouse EAAT2 is sufficient to elicit motor neuron death. Transgenic expression of EAAT2or treatment with the small compound LDN/OSU-0212320, which mainly increases translation of EAAT2 mRNA, improves the motor performance of an ALS mouse model expressing hSOD1G93A. However, a recent clinical study testing ceftriaxone, an FDA-approved β-lactam antibiotic that can transcriptionally promote EAAT2 expression, in ALS patients concluded that this drug treatment had no therapeutic effect. Therefore, it is questionable whether increasing EAAT2 expression represents a feasible therapeutic strategy for ALS. It has been argued, however, that EAAT2 downregulation largely occurs at the posttranslational and not at the transcriptional level in ALS. There was no evidence for increased EAAT2 in patients treated with ceftriaxone, and ceftriaxone treatment only slightly increases protein levels of EAAT2 in hSOD1G93A mice. In addition, the efficacy of ceftriaxone in hSOD1G93A mice is not consistent among different studies. Hence, more investigations will be needed to strengthen evidence for the pathogenic contribution of EAAT2 dysfunction in ALS (Peng, 2019).

    Oxidative stress is known as a hallmark of Alzheimer's disease, Parkinson's disease, and ALS. During aging, neurons are thought to be susceptible to excitotoxicity, and the nervous system and muscles are vulnerable to ROS accumulation because of high oxygen consumption demand. Administration of antioxidants can improve the motor function of hSOD1G93A mice and ALS patients. Nonetheless, how oxidative stress is produced in ALS and how this burden is involved in disease pathogenesis is not well understood. Interestingly, in this study, Drosophila Eaat1 depletion was shown to cause ALS-like characteristics, including motor neuron excitotoxicity, NMJ bouton abnormalities, muscle weakness, and compromised motor performance. In the future, it will be worth exploring whether ROS-induced motor circuit dysfunction might also participate in ALS progression and age-dependent motor system decline (Peng, 2019).

    It has previously been shown that reduced excitability of proprioceptive sensory neurons and cholinergic interneurons is causative of locomotor CPG circuit dysfunction and compromised locomotion in Drosophila smn mutants, which are used as a Drosophila model of spinal muscular atrophy (SMA), a motor neuron disease of juveniles. Increasing neuronal excitability by 4-AP treatment reverses these motor system defects. Intriguingly, the current data show that long-term food-mediated feeding of (or even short-term exposure to) the K+ channel blocker 4-AP also rescued altered locomotor CPG activity in eaat1 mutants. Notably, although the precise mechanisms are unknown, 4-AP has been used to treat several motor system-related disorders such as spinal cord injury, Lambert-Eaton syndrome, and hereditary canine spinal muscular atrophy, and it is an FDA-approved therapy for multiple sclerosis. Thus, as supported by the current findings, it seems plausible that neuronal hypoexcitability may be a shared mechanism underlying the motor-system defects displayed in motor-related disorders (Peng, 2019).

    Neuron-specific knockdown of Drosophila HADHB induces a shortened lifespan, deficient locomotive ability, abnormal motor neuron terminal morphology and learning disability

    Mutations in the HADHB gene induce dysfunctions in the beta-oxidation of fatty acids and result in a Mitochondrial trifunctional protein (MTP) deficiency, which is characterized by clinical heterogeneity, such as cardiomyopathy and recurrent Leigh-like encephalopathy. In contrast, milder forms of HADHB mutations cause the later onset of progressive axonal peripheral neuropathy (approximately 50-80%) and myopathy with or without episodic myoglobinuria. The mechanisms linking neuronal defects in these diseases to the loss of HADHB function currently remain unclear. Drosophila has the CG4581 (dHADHB) gene as a single human HADHB homologue. This study established pan-neuron-specific dHADHB knockdown flies and examined their phenotypes. The knockdown of dHADHB shortened the lifespan of flies, reduced locomotor ability and also limited learning abilities. These phenotypes were accompanied by an abnormal synapse morphology at neuromuscular junctions (NMJ) and reduction in both ATP and ROS levels in central nervous system (CNS). The Drosophila NMJ synapses are glutamatergic that is similar to those in the vertebrate CNS. The present results reveal a critical role for dHADHB in the morphogenesis and function of glutamatergic neurons including peripheral neurons. The dHADHB knockdown flies established herein provide a useful model for investigating the pathological mechanisms underlying neuropathies caused by a HADHB deficiency (Li, 2019).

    Non-enzymatic activity of the alpha-Tubulin acetyltransferase alphaTAT limits synaptic bouton growth in neurons

    Neuronal axons terminate as synaptic boutons that form stable yet plastic connections with their targets. Synaptic bouton development relies on an underlying network of both long-lived and dynamic microtubules that provide structural stability for the boutons while also allowing for their growth and remodeling. However, a molecular-scale mechanism that explains how neurons appropriately balance these two microtubule populations remains a mystery. It was hypothesized that alpha-tubulin acetyltransferase (alphaTAT), which both stabilizes long-lived microtubules against mechanical stress via acetylation and has been implicated in promoting microtubule dynamics, could play a role in this process. Using the Drosophila neuromuscular junction as a model, this study found that non-enzymatic αTAT activity limits the growth of synaptic boutons by affecting dynamic, but not stable, microtubules. Loss of αTAT results in the formation of ectopic boutons. These ectopic boutons can be similarly suppressed by resupplying enzyme-inactive αTAT or by treatment with a low concentration of the microtubule-targeting agent vinblastine, which acts to suppress microtubule dynamics. Biophysical reconstitution experiments revealed that non-enzymatic alphaTAT1 activity destabilizes dynamic microtubules but does not substantially impact the stability of long-lived microtubules. Further, during microtubule growth, non-enzymatic αTAT activity results in increasingly extended tip structures, consistent with an increased rate of acceleration of catastrophe frequency with microtubule age, perhaps via tip structure remodeling. Through these mechanisms, αTAT enriches for stable microtubules at the expense of dynamic ones. It is proposed that the specific suppression of dynamic microtubules by non-enzymatic αTAT activity regulates the remodeling of microtubule networks during synaptic bouton development (Coombes, 2020).

    Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila

    Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. This study reports that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila (Syed, 2019)

    POU domain motif3 (Pdm3) induces wingless (wg) transcription and is essential for development of larval neuromuscular junctions in Drosophila

    Wnt is a conserved family of secreted proteins that play diverse roles in tissue growth and differentiation. Identification of transcription factors that regulate wnt expression is pivotal for understanding tissue-specific signaling pathways regulated by Wnt. This study identified pdm3m7, a new allele of the pdm3 gene encoding a POU family transcription factor, in a lethality-based genetic screen for modifiers of Wingless (Wg) signaling in Drosophila. Interestingly, pdm3m7 larvae showed slow locomotion, implying neuromuscular defects. Analysis of larval neuromuscular junctions (NMJs) revealed decreased bouton number with enlarged bouton in pdm3 mutants. pdm3 NMJs also had fewer branches at axon terminals than wild-type NMJs. Consistent with pdm3m7 being a candidate wg modifier, NMJ phenotypes in pdm3 mutants were similar to those of wg mutants, implying a functional link between these two genes. Indeed, lethality caused by pdm3 overexpression in motor neurons was completely rescued by knockdown of wg, indicating that pdm3 acts upstream to wg. Furthermore, transient expression of pdm3 induced ectopic expression of wg-LacZ reporter and wg effector proteins in wing discs. It is proposed that pdm3 expressed in presynaptic NMJ neurons regulates wg transcription for growth and development of both presynaptic neurons and postsynaptic muscles (Kim, 2020).

    Transcription factors play essential roles by inducing genes during the formation of body plans, organ development, tissue specificity, and generation of diverse cell types. Numerous transcription factors are grouped based on similarity in their sequences and domain structures. Pituitary-specific positive transcription factor 1, Octamer transcription factor-1, Uncoordinated-86 domain (POU) transcription factors belong to a subfamily of homeodomain transcription factors, and are highly conserved in all metazoans. POU domain consists of two DNA binding domains, POU homeodomain and POU specific domain, and these two domains are linked by a flexible linker. Based on sequence homology of the POU domain and the linker, POU proteins are grouped into six classes. POU proteins are often expressed in spatiotemporally restricted patterns during development, implying that they may be specialized for differentiation of specific cells or tissues by activating required signal transduction pathways (Kim, 2020).

    The class VI Drosophila POU domain motif 3 (Pdm3) protein is reported to function in olfactory receptor neurons (ORNs) by regulating olfactory receptor gene expression and axon targeting, and in ring (R) neurons by regulating the development of ellipsoid body (EB) and axon targeting to EB in the central brain. pdm3 is also important for the axon targeting of a type of tracheal dendrite (td) neurons. In particular, td neurons that normally form synapse in the nerve cord change their target to the central brain by ectopic expression of Pdm3. Besides the neuronal functions of Pdm3, pdm3 also acts as a repressor of abdominal pigmentation in D. melanogaster, and plays a role in female-limited color dimorphism in abdomen of D. montium. Despite these studies, it is still unknown how pdm3 performs these neuronal and non-neuronal functions (Kim, 2020).

    pdm3f00828 and pdm31 homozygotes exhibit defects in axon targeting, odor perception, and locomotion. pdm3f00828 allele has insertion of a piggyback element in an intron near the 3' end of the pdm3 gene, and pdm31 has a premature stop codon in the middle of the coding region that results in the deletion of the POU domain. This study identified a new pdm3 allele, pdm3m7, as a suppressor of lethality induced by Sol narae (Sona) overexpression in a genetic screen. Sona is a fly ADAMTS (A disintegrin and metalloprotease with thrombospondin motif) whose family members are secreted metalloproteases important for cell proliferation, cell survival and development. This study has shown that Sona positively regulates Wingless (Wg) signaling and is essential for fly development, cell survival, and wg processing. wg is a prototype of Wnt family that initiates signal transduction cascade as extracellular signaling proteins, and activation of Wnt signaling leads to transcriptional induction of multiple genes for regulation of cell proliferation, cell survival, cell fate decision, and cell migration. wg is important for the development of all appendages, and the wing imaginal disc has been a great tool to study wg signaling because wg secreted from its dorsal-ventral midline is crucial for growth and development of wings (Kim, 2020).

    Wg also plays an essential role in the development of NMJ. During larval development, NMJs continue to form synaptic boutons that are specialized structures with axon terminals of motor neurons surrounded by reticular subsynaptical reticulum (SSR) formed by the plasma membrane of postsynaptic muscle19. Among multiple types of boutons such as type Ib, Is, II, and III, wg is secreted at a high level from the glutamatergic type Ib bouton known as the main localization site of wg protein and wg signaling components, and is absent or at very low levels in other types of boutons (Packard, 2002; Kim, 2020).

    Type Ib boutons also have more extensive SSR compared to other bouton types, so are easily detected by the high level of Discs-Large (Dlg) as a postsynaptic marker. Type Ib boutons in NMJs of wg mutants show reduction in bouton number but increase in bouton size. Components in wg signaling such as Arrow (Arr) that positively regulates wg signaling as a coreceptor of wg also shows its mutant phenotype similar to wg, but Shaggy (Sgg)/GSK3β that negatively regulates wg signaling as a kinase shows opposite phenotype to wg. Thus, dynamic regulation of wg signaling is essential for the development of NMJ (Kim, 2020).

    Secreted wg also signals to the presynaptic motor neuron to regulate Futsch, one of the microtubule-associated proteins (MAPs). Futsch is a homolog of mammalian MAP1B, and both Futsch and MAP1B are phosphorylated at a conserved site by Sgg/GSK3β. The phosphorylated MAP1B does not bind microtubules, which results in reduced stability of microtubules. Therefore, localization of Futsch at NMJ faithfully reflects the stability of microtubules that is dynamically regulated by wg signaling. Loss of futsch phenotype is similar to the loss of wg phenotype in NMJ (Kim, 2020).

    This study reporta that pdm3 is identified as a suppressor of Sona-induced lethality. Based on the involvement of Sona in wg signaling and the neuronal role of Pdm3, the roles of pdm3 in NMJ were specifically studied. Similar to loss of wg, loss of pdm3 in NMJ caused decrease in number but increase in size of boutons. Lethality induced by overexpressed pdm3 was completely rescued by the knockdown of wg in motor neurons but not vice versa. This indicated that pdm3 functions upstream to wg, and prompted a test whether pdm3 can induce wg transcription. Indeed, transient expression of pdm3 in wing discs induced wg transcription and wg effector proteins. Based on these data, it is propose that one of the main functions of pdm3 in NMJ is to induce wg transcription (Kim, 2020).

    This study reports that pdm3 regulates growth and development of NMJs. pdm3 mutants showed increase in bouton size and decrease in bouton number, which are similar to the phenotype of wg mutants. Lethality induced by the overexpression of pdm3 was rescued by knockdown of wg in NMJ, indicating that pdm3 functions upstream to wg. Furthermore, overexpression of pdm3 induced wg transcription in wing discs. It is proposed that a major function of pdm3 in motor neurons is to induce wg transcription, and secreted wg from motor neurons regulates growth, development, and maturation of both pre- and post-synaptic regions of NMJ (Kim, 2020).

    The mammalian homolog of pdm3 is Brain-5 (Brn-5)/POU class 6 homeobox 1 (POU6F1) mainly expressed in brain and spinal cord. Brn-5 is heavily expressed in embryonic brain but also expressed in adult brain and multiple adult organs such as kidney, lung, testis, and anterior pituitary. In developing brain, Brn-5 is expressed in postmitotic neurons after neuronal progenitor cells exit cell cycle in the early process of terminal neuronal differentiation. Therefore, both pdm3 and Brn-5 function in differentiation of neurons. Interestingly, ectopic expression of Brn-5 inhibits DNA synthesis, which is similar to cell cycle arrest phenotype by wg overexpression. Given the homology between pdm3 and Brn-5 as well as functional similarities, Brn-5 may also induce wnt transcription (Kim, 2020).

    Most of pdm3 functions identified so far are related to the maturation of neurons such as olfactory neurons, R neurons and td neurons as well as their postsynaptic partners. Ectopic expression of pdm3 induced lethality without exception, indicating that expression of pdm3 in fly tissues is generally repressed in vivo in order to express wg under the strict spatiotemporal control. An important question is whether pdm3 directly transcribe wg. This study found that wg transcription is induced only after 36 hours of transient overexpression of Pdm3. It is possible that the level of pdm3 needs to be over a threshold to induce wg transcription. Alternatively, pdm3 may need to turn on other components to indirectly induce wg transcription. DNA sequence of Brn-5 binding site has been reported, so analysis on wg and wnt regulatory regions will help understand the mechanism of wnt induction by pdm3 and Brn-5 (Kim, 2020).

    This study consistently found more significant NMJ phenotypes in A2 than A3 in both pdm3 and wg mutants. Therefore, pdm3 and wg may play more prominent roles in the A2 than the A3 segment. In fact, the level of pdm3 was higher in the anterior region than the posterior region of ventral ganglion, which suggests that more wg may be present in the NMJs of anterior abdominal segments. Consistent with this idea, the number of type Ib boutons in the A2 segment was 1.8 times more than A3 segment. One difference between pdm3 and wg mutants is the lack of certain phenotypes in the A3 segment of pdm3 NMJs: the size of boutons and the number of axon terminals in A3 were not affected in pdm3 mutant. It is possible that pdm3 turns on both common and segment-specific genes besides wg, and A3 segment-specific components may alleviate the loss of wg phenotype in the A3 segment. Similarly, other proteins induced by pdm3 may also play important roles in NMJ growth, differentiation and maintenance. In fact, multiple signaling pathways including Glass-bottom-boat (Gbb) pathway also play roles in NMJ development. Gbb is secreted from muscles and induces development of both pre- and post-synaptic structures, similar to wg signaling (Kim, 2020).

    This study identified a defective hobo element in the pdm3m7 allele. The hobo element belongs to Ac family found in maize and has short inverted terminal repeats. Laboratory and wild strains of D. melanogaster have average 28 and 22 copies of hobo elements in the genome that are either full-length or defective, respectively. Because other suppressors identified in the genetic screen using Sona overexpression did not have hobo element in the pdm3 gene, the transposition of the hobo element to the pdm3 gene may have occurred subsequent to the generation of a point mutation in the arr gene by EMS. Since both arr and pdm3 are positively involved in wg signaling, this hobo insertion may have helped the original arrm7 mutation to further decrease the activity of wg signaling under the condition of Sona overexpression (Kim, 2020).

    Besides the neuronal roles of Pdm3, all pdm3 mutants show minor but consistent defects in planar cell polarity in a restricted region of the wing as well as adhesion between the dorsal and ventral wing blades. Other phenotypes such as wing drooping and premature death were also observed in all pdm3 mutants, but these may be due to malformation of synaptic structures. pdm3 also plays a role in female-limited color dimorphism in abdomen of D. montium. The authors found in sexually dimorphic females that the first intron of the pdm3 gene has four tandem sets with predicted binding sites for the HOX gene Abdominal-B (Abd-B) and the sex determination gene doublesex (dsx). Interestingly, it has been shown that wg expression is repressed by the combinatory work of Abd-B and Dsx proteins. Taken together, it is possible that transcription of wg and pdm3 is co-repressed by Abd-B and Dsx. Such co-repression of wg and pdm3 transcription may be also required for synaptic growth and differentiation in neurons. Further studies on pdm3 will help understand how this understudied transcription factor is involved in the final differentiation of various cell types (Kim, 2020).

    O-GlcNAcase contributes to cognitive function in Drosophila

    O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, this study generated catalytically inactive and precise knock-out Oga alleles (Oga(D133N) and Oga(KO), respectively) in Drosophila melanogaster. Adult Oga(D133N) and Oga(KO) flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionary conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, this study report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila (Muha, 2020).

    Developmental arrest of Drosophila larvae elicits presynaptic depression and enables prolonged studies of neurodegeneration

    Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. This study has investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions where Drosophila larvae are terminally arrested, this study has characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). While wild-type larvae transition to pupae after 5 days, arrested third instar (ATI) larvae persist for 35 days, during which NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites, and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales (Perry, 2020).

    Structural and functional synaptic plasticity induced by convergent synapse loss in the Drosophila neuromuscular circuit

    Throughout the nervous system, the convergence of two or more presynaptic inputs on a target cell is commonly observed. The question asked in this study is to what extent converging inputs influence each other's structural and functional synaptic plasticity. In complex circuits, isolating individual inputs is difficult because postsynaptic cells can receive thousands of inputs. An ideal model to address this question is the Drosophila larval neuromuscular junction (NMJ) where each postsynaptic muscle cell receives inputs from two glutamatergic types of motor neurons (MNs), known as 1b and 1s MNs. Notably, each muscle is unique and receives input from a different combination of 1b and 1s MNs; this study surveyed multiple muscles for this reason. In this study a cell-specific promoter was identified that allows ablation of 1s MNs post-innervation and structural and functional responses of convergent 1b NMJs were measured using microscopy and electrophysiology. For all muscles examined in both sexes, ablation of 1s MNs resulted in NMJ expansion and increased spontaneous neurotransmitter release at corresponding 1b NMJs. This demonstrates that 1b NMJs can compensate for the loss of convergent 1s MNs. However, only a subset of 1b NMJs showed compensatory evoked neurotransmission, suggesting target-specific plasticity. Silencing 1s MNs led to similar plasticity at 1b NMJs, suggesting that evoked neurotransmission from 1s MNs contributes to 1b synaptic plasticity. Finally, 1s innervation was genetically blocked in male larvae and robust 1b synaptic plasticity was eliminated, raising the possibility that 1s NMJ formation is required to set up a reference for subsequent synaptic perturbations (Wang, 2021).

    The nervous system is characterized by complex wiring patterns that include different neurons converging onto the same postsynaptic cell. This wiring paradigm is found in pyramidal neurons that receive input from excitatory and inhibitory contacts, and in esophageal striated muscles that receive enteric and vagal nerve inputs. While dynamic regulation of individual synapses has been examined, interplay between convergent neurons has been predominantly studied by monitoring postsynaptic spine changes. Understanding how convergent neurons respond to such perturbations will shed light on the etiologies of neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), which display progressive neuronal cell death and devastating functional consequences (Wang, 2021).

    Analysis of individual inputs in the central nervous system is complicated by the high density of converging inputs on the same target cell. The Drosophila larval neuromuscular circuit, however, circumvents this with a simple, hard-wired connectivity map. The body plan is segmentally repeated and each hemisegment is bilaterally symmetrical and comprised of 35 motor neurons (MNs) and 30 postsynaptic muscles. Like most vertebrate central synapses, the individual glutamatergic neuromuscular contacts consist of axon terminal swellings, called boutons, and elaborate postsynaptic complexes. Each bouton houses several active zones (AZs) that enable neurotransmission. Larval muscles are innervated by two main MNs, type 1b (big) and 1s (small), which resemble tonic and phasic neurons, respectively, unlike vertebrate skeletal muscles that are monoinnervated. Most 1b MNs innervate a single muscle, whereas 1s MNs innervate functional muscle groups, highlighting important structural and functional distinctions between these classes of neurons (Wang, 2021).

    In the larval neuromuscular circuit, 1b and 1s MNs are required for normal locomotion. Each MN type has unique electrophysiological properties: 1b MNs display a higher rate of spontaneous neurotransmitter release but the quantal size is smaller compared to 1s MNs (Newman, 2017; Nguyen, 2016). In traditional neuromuscular junction (NMJ) electrophysiology experiments, excitatory postsynaptic potentials (EPSPs) in muscles represent simultaneous stimulation of both neurons. However, several studies have isolated 1b-derived and 1b+1s-derived EPSPs from different muscles and demonstrated that convergent1b and 1s MNs do not equally contribute to the EPSP amplitude. Several forms of synaptic plasticity have been observed in the larval NMJ including facilitation and depression. As each muscle is co-innervated and individual synaptic activities can be distinguished, this system provides an ideal platform to investigate structural and physiological plasticity changes that enable one MN to compensate for perturbations of a convergent MN. A recent study found that ablating the 1s MN on muscle 1 (m1) elevated evoked neurotransmission of the corresponding 1b NMJ (Aponte-Santiago, 2020). However, each muscle is innervated by a unique 1b-1s MN pair, so it is unclear whether this plasticity is consistent across all muscles (Wang, 2021).

    This study examined three separate muscles to test structural and functional plasticity. First, muscle-specific 1b activities were confirmed. Next, 1s MNs were ablated after innervation, and corresponding 1b NMJ compensatory responses were examined. All three 1b NMJs increased their bouton numbers and rate of spontaneous neurotransmitter7 release without a corresponding upregulation of active zones (AZs) and some 1b NMJs also8 partially increased their evoked neurotransmission. An important factor for 1b synaptic plasticity9 is the 1s EPSP as silencing 1s evoked activity caused similar 1b NMJ responses. Additionally,0 robust 1b synaptic plasticity does not occur if the 1s NMJ is never formed, indicating initial 1s innervation is required. These results demonstrate a novel, target-specific synaptic plasticity that2 may be a common feature of convergent neural circuits (Wang, 2021).

    This study examined the Drosophila neuromuscular circuit and demonstrate 1b synaptic plasticity upon loss of convergent 1s MNs. The muscles examined in this study, m6, m12, and m4, are co-innervated by unique 1b-1s MN pairs. First, an activity baseline was established in wild type and uncovered that 1b MNs contribute a unique percentage of the total EPSP in a muscle-specific manner. Genetic ablation of 1s MNs (vCE and dCE) after innervation led to expansion of 1b NMJs and elevation of their spontaneous release rates. Furthermore, some 1b MNs elevated evoked neurotransmission while others remained unchanged. A recent study examining m1 found similar 1b functional plasticity when m1-1s was ablated (Aponte-Santiago, 2020), but no structural compensation. This target specificity indicates heterogeneity in synaptic plasticity mechanisms. Silencing 1s MNs yielded partial m4-1b compensation, suggesting 1s evoked neurotransmission is an important factor in eliciting 1b NMJ plasticity. In mutant larvae where the m4-1s NMJs never form, no changes were observed in m4-1b bouton number or spontaneous release rate, and decreased EPSP compensation. These results suggest that initial 1s innervation may set up a reference point for robust 1b synaptic plasticity (Wang, 2021).

    A well-established form of plasticity at the larval NMJ is synaptic homeostasis, which can manifest in pre- and/or postsynaptic changes, including neurotransmitter release and neurotransmitter receptor dynamics and abundance, respectively. Presynaptic homeostatic plasticity (PHP) maintains EPSPs at baseline when postsynaptic glutamate receptor function is perturbed, and can be induced in a synapse specific manner. In this study, the loss of 1s MNs could be interpreted as a decrease in glutamate receptor function by the muscle and trigger similar PHP mechanisms to induce compensatory changes at adjacent 1b NMJs. Thus, further analysis of PHP pathways could reveal mechanisms underlying this convergent plasticity (Wang, 2021).

    1b NMJs must adapt to accommodate the increased spontaneous and evoked neurotransmission. The results do not reveal robust expansion of AZs or GluRs to support 1b synaptic plasticity, as observed in other studies where NMJ size is altered but total AZs or QCs remain the same (Aponte-Santiago, 2020; Goel, 2019a; Goel, 2019b; Goel, 2020). Therefore, it is proposed that existing AZs may alter their properties. AZs exist in two different states, active or silent, and a subset of AZs specialize in spontaneous release or evoked neurotransmission. Thus, even without an increase in AZs, 1b plasticity mechanisms may modify AZ properties to respond to the loss of 1s inputs. Overall, the data suggest silent AZs may become activated to increase the pools of spontaneous and evoked AZs as ablation of 1s MNs led to enhanced 1b spontaneous release rates, target-specific compensation of EPSPs, and increased QC. Additionally, spontaneous and evoked activities may be independently regulated. Furthermore, the readily-releasable pool (RRP) size is under dynamic control during synaptic plasticity and could modulate m4-1b evoked neurotransmitter release. Detailed examination of AZs will significantly bolster understanding of mechanism underlying 1b NMJ plasticity. Prior studies reported that spontaneous neurotransmitter release regulates synaptic development in both mammals and Drosophila. Thus, the expanded size of all 1b3 NMJs following 1s ablation may be caused by the elevated spontaneous activity. These data also suggest that all 1b MNs can detect and respond to the loss of adjacent 1s inputs. However, the ability to differentially compensate the spontaneous and evoked activity is likely due to independent mechanisms since only some 1b MNs elevate their EPSPs (Wang, 2021).

    In complex neural circuits, dissecting contributions of individual inputs to the total postsynaptic activity, also referred to as synaptic weight, remains difficult due to thousands of converging inputs on a single cell. The larval NMJ facilitates the partitioning of synaptic inputs as each muscle is innervated by few MNs. This study combined electrophysiology with calcium imaging and found that 1b synaptic weights differ on m6, m12, and m4. Taken together with the degree of EPSP compensation after ablation of 1s5 MNs, there was a direct correlation with the level of target-specific synaptic weight. Thus, robust 1b MNs that carry more synaptic drive may be endowed with certain synaptic plasticity mechanisms that respond to loss of adjacent inputs. However, regulatory roles for type II and type III MNs that are present on some muscles cannot be rule out (Wang, 2021).

    Interestingly, a similar correlation exists in Hebbian plasticity, where stronger synapses are more likely strengthened than weaker ones. This correlation is also reflected in PHP. Two studies examined input-specific PHP on different muscles: on m4, PHP can be only induced at 1b NMJs (Newman, 2017); however, on m6, PHP can be induced on both 1b and 1s NMJs (Genc, 2019). This correlates with the observation that the m4-1b has more synaptic weight than m4-1s, whereas m6-1b and m6-1s have similar synaptic weights. Taken together, homeostatic plasticity varies in target-specific and input-specific manners, suggesting heterogeneous mechanisms (Wang, 2021).

    Models of synaptic homeostasis rely on an activity set point to stabilize neurons when confronted with perturbations. Each target neuron must account for all presynaptic inputs to produce a defined output (i.e., the set point). The structural and functional properties of each input thus determine not only its contribution to the postsynaptic activity but also its ability to respond to perturbations in synaptic function. For example, transcription factors not only regulate the temporal expression of ion channels that shape neuronal excitability, but also homeostatic mechanisms. Like many activity-dependent processes, the optimal set point may be established during a narrow time-window of development. This hypothesis was tested in a Drosophila seizure mutant by inhibiting activity during embryonic development and observing suppression of seizures in postembryonic stages. Thus, manipulating activity during an embryonic critical period4 may alter the activity set point (Wang, 2021).

    In this study, one intriguing hypothesis is that 1b+1s co-innervation determines the EPSP set point during embryogenesis and is referenced by some 1b NMJs to compensate for the loss of 1s MNs. A model is proposed to describe how 1b NMJs increase their sizes and spontaneous and evoked neurotransmission due to the loss of convergent 1s MNs. The neuromuscular innervation map is formed during late embryonic development, the activity set point is defined in a target-specific manner and how neurons respond to dysfunctional neighbors (Wang, 2021).

    The conserved alternative splicing factor Caper regulates neuromuscular phenotypes during development and aging

    RNA-binding proteins play an important role in the regulation of post-transcriptional gene expression throughout the nervous system. This is underscored by the prevalence of mutations in genes encoding RNA splicing factors and other RNA-binding proteins in a number of neurodegenerative and neurodevelopmental disorders. The highly conserved alternative splicing factor Caper is widely expressed throughout the developing embryo and functions in the development of various sensory neural subtypes in the Drosophila peripheral nervous system. This study found that caper dysfunction leads to aberrant neuromuscular junction morphogenesis, as well as aberrant locomotor behavior during larval and adult stages. Despite its widespread expression, the results indicate that caper function is required to a greater extent within the nervous system, as opposed to muscle, for neuromuscular junction development and for the regulation of adult locomotor behavior. Moreover, Caper was found to interact with the RNA-binding protein Fmrp to regulate adult locomotor behavior. Finally, it was shown that caper dysfunction leads to various phenotypes that have both a sex and age bias, both of which are commonly seen in neurodegenerative disorders in humans (Titus, 2021).

    Fragile X Premutation rCGG Repeats Impair Synaptic Growth and Synaptic Transmission at Drosophila larval Neuromuscular Junction

    Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disease that develops in some premutation (PM) carriers of the FMR1 gene with alleles bearing 55-200 CGG repeats. The discovery of a broad spectrum of clinical and cell developmental abnormalities among PM carriers with or without FXTAS and in model systems suggests that neurodegeneration seen in FXTAS could be the inevitable end-result of pathophysiological processes set during early development. Hence, it is imperative to trace early PM-induced pathological abnormalities. Previous studies have shown that transgenic Drosophila carrying PM-length CGG repeats are sufficient to cause neurodegeneration. This study used the same transgenic model to understand the effect of CGG repeats on the structure and function of the developing nervous system. Presynaptic expression of CGG repeats restricts synaptic growth, reduces the number of synaptic boutons, leads to aberrant presynaptic varicosities, and impairs synaptic transmission at the larval neuromuscular junctions. The postsynaptic analysis shows that both glutamate receptors and subsynaptic reticulum proteins were normal. However, a high percentage of boutons show a reduced density of Bruchpilot protein, a key component of presynaptic active zones required for vesicle release. The electrophysiological analysis shows a significant reduction in quantal content, a measure of total synaptic vesicles released per excitation potential. Together, these findings suggest that synapse perturbation caused by rCGG repeats mediates presynaptically during larval NMJ development. It is also suggested that the stress-activated c-Jun N-terminal kinase protein Basket and CIDE-N protein Drep-2 positively mediate Bruchpilot active zone defects caused by rCGG repeats (Bhat, 2021).

    Synaptic plasticity induced by differential manipulation of tonic and phasic motoneurons in Drosophila

    Structural and functional plasticity induced by neuronal competition is a common feature of developing nervous systems. However, the rules governing how postsynaptic cells differentiate between presynaptic inputs are unclear. In this study synaptic interactions was characterized following manipulations of tonic Ib or phasic Is glutamatergic motoneurons that co-innervate postsynaptic muscles of male or female Drosophila melanogaster larvae. After identifying drivers for each neuronal subtype, ablation or genetic manipulations was performed to alter neuronal activity and examined the effects on synaptic innervation and function at neuromuscular junctions (NMJs). Ablation of either Ib or Is resulted in decreased muscle response, with some functional compensation occurring in the Ib input when Is was missing. In contrast, the Is terminal failed to show functional or structural changes following loss of the co-innervating Ib input. Decreasing the activity of the Ib or Is neuron with tetanus toxin light chain resulted in structural changes in muscle innervation. Decreased Ib activity resulted in reduced active zone (AZ) number and decreased postsynaptic subsynaptic reticulum (SSR) volume, with the emergence of filopodial-like protrusions from synaptic boutons of the Ib input. Decreased Is activity did not induce structural changes at its own synapses, but the co-innervating Ib motoneuron increased the number of synaptic boutons and AZs it formed. These findings indicate tonic Ib and phasic Is motoneurons respond independently to changes in activity, with either functional or structural alterations in the Ib neuron occurring following ablation or reduced activity of the co-innervating Is input, respectively (Aponte-Santiago, 2020).

    Functional and structural changes in neuronal circuits occur during development and in response to environmental stimuli, learning, and injury. Disruptions of these plasticity pathways contribute to neurodevelopmental diseases and impair rewiring after brain injury, highlighting the importance of the underlying mechanisms. In contrast to mammals, invertebrate nervous systems like that of Drosophila melanogaster are more stereotypical in their organization. Neuroblasts divide and differentiate in a specific order to generate fixed cellular lineages with genetically hardwired synaptic targets. Although Drosophila display stereotypical neuronal connectivity, plasticity can occur throughout development and into adulthood. Structural plasticity is most prominent during metamorphosis, when larval neurons reorganize their processes and synaptic partners form functional adult circuits. Alterations in connectivity also occur in response to changes in environmental stimuli or following acute or chronic manipulations of neuronal activity (Aponte-Santiago, 2020).

    Although plasticity occurs broadly across neuronal circuits, the motor system has played an important role in defining mechanisms for activity-dependent structural changes in connectivity. Locomotion is an essential behavior in many animals and requires coordinated output from central pattern generators to orchestrate motoneuron (MN) firing patterns that activate specific muscles. In vertebrates, individual muscle fibers receive transient innervation from many cholinergic motoneurons during early development. As many as 10 distinct motor axons can innervate a single muscle fiber before an activity-dependent competition results in retention of only a single axon. This axonal competition allows a large pool of identical motoneurons to transition from dispersed weak outputs to the muscle field to strong innervation of a smaller subset of muscles (Aponte-Santiago, 2020).

    Unlike vertebrate neuromuscular junctions (NMJs), early promiscuity in synaptic partner choice and subsequent synapse elimination does not occur in Drosophila. Instead, the larval motor system is composed of ~36 motoneurons from four subclasses that are genetically programmed by specific transcription factors and guidance molecules to form stereotypical connections to the 30 muscles in each abdominal hemisegment. Although synaptic partner choice is hardwired, activity-dependent plasticity and homeostatic mechanisms have been characterized, making Drosophila an ideal system to study synaptic interactions between motor neurons. Although individual muscles normally restrict innervation to a single neuron from each subclass, it is unclear whether motoneurons interact during innervation of the same muscle target or respond when the output of one motoneuron is altered. Therefore, a system was extablished to differentially manipulate the two primary glutamatergic inputs and characterized the subsequent effects on synaptic morphology and function. Pnly the tonic Ib motoneuron is capable of partially compensating following ablation or silencing of the phasic Is input (Aponte-Santiago, 2020).

    To characterize how changes in the presence or activity of tonic Ib versus phasic Is motoneurons alter NMJ development and function in Drosophila, GAL4 drivers specific for each class that innervate M1 were identifed and use to alter the balance of input to the muscle. The data indicate that Ib and Is motoneurons largely form independent inputs that make similar contributions to muscle excitability and contractile force. The Ib subclass was capable of structural and functional changes following manipulations that altered their output or that of the coinnervating Is motoneuron. These changes were observed only during conditions when neuronal activity was decreased or when the Is input was ablated. Functional increases in evoked release without enhanced synapse number were observed in Ib motoneurons following ablation of Is. In contrast, morphologic changes that increased AZ number without enhancing evoked release occurred when Is synaptic output was blocked with tetanus toxin. While Ib motoneurons were capable of several forms of plastic change following reduced input to the muscle, Is motoneurons were insensitive to manipulations of their own activity or that of the Ib input. Unlike the plasticity observed in Ib neurons following reduction in synaptic drive to the muscle, enhancing excitability of either the Ib or Is input was ineffective at triggering changes in either motoneuron class. These data indicate that reductions in activity from either input trigger a structural or functional change primarily from the tonic Ib motoneuron subclass (Aponte-Santiago, 2020).

    The stereotypical connectivity found in the abdominal musculature of Drosophila larvae suggest that individual muscles normally allow synaptic innervation from only a single motoneuron of each subclass. However, expanded postsynaptic target choice has been observed following muscle loss induced by laser ablation or genetic mutation, with the affected Ib motoneuron targeting inappropriate nearby muscles without altering the innervation pattern of the correctly targeted Ib neuron. Similarly, ablation of some motoneurons can result in axonal spouting from neighboring connections that target the deinnervated muscle. Misexpression of synaptic cell surface proteins can also alter target choice for some Ib and Is motoneurons. In addition, silencing neuronal activity during development has been demonstrated to induce ectopic NMJs formed primarily by type II neuromodulatory neurons. No axonal sprouting onto M1 from other motoneurons was observed that resulted in altered target choice when MN1-Ib or MNIs was ablated or silenced in these experiments. Given that M1 is the most dorsal muscle of the abdominal musculature, axons from other motoneurons are not present in the direct vicinity, so any signals released from M1 might be insufficient to attract additional innervation. Evidence was found that M1 may attempt to promote synaptic innervation when MN1-Ib was silenced with tetanus toxin. Under these conditions, the MN1-Ib terminal maintained an immature-like state with the presence of filopodial-like extensions. This effect was observed only in Ib motoneurons, highlighting differences in how Is terminals interact with or respond to signals from the muscle. Similar filopodial-like extensions at newly forming NMJ connections have been observed in late embryos and early first instar larvae. These presynaptic filopodial processes contain elevated levels of the Cacophony N-type Ca2+ channel and interact with GluRIIA-rich myopodia, with some progressing to form new synapses during early development. Because of the lack of reinforcement signals caused by the absence of synaptic activity in silenced MN1-Ib motoneurons, it was hypothesized that these processes fail to properly drive AZ assembly and new synapse formation. Indeed, a role for neuronal activity in regulating synaptogenic filopodial stabilization has been characterized in the Drosophila visual system (Aponte-Santiago, 2020).

    Many forms of plasticity, including synapse elimination at mammalian NMJs, ocular dominance plasticity, and cerebellar climbing fiber pruning, require Hebbian-like input imbalances to trigger synaptic interactions. As such, it was of interest to see whether changes in the activity of Ib or Is motoneurons that created an imbalance between the output of the two neurons could drive unique changes compared with when one input was missing. In the case of the Ib neuron, this was indeed observed. In the absence of Is input, either because of natural variation in innervation in control animals or following ablation with UAS-RPR, there was no structural response in terms of adding additional release sites. However, the loss of Is triggered a functional increase in evoked release from the Ib neuron. In contrast, when an activity imbalance was created by expressing tetanus toxin in the Is neuron, Ib displayed structural plasticity that increased the number of release sites. Although the underlying molecular pathways that mediate the two distinct responses are unknown, the results suggest that the physical presence of Is likely alters the signaling systems responsible for triggering compensation in Ib motoneurons in response to reduced muscle drive (Aponte-Santiago, 2020).

    For every manipulation made beyond increasing excitability of the neurons, a response from the tonic Ib class was detected, while the phasic Is motoneuron displayed less plasticity. Since each muscle is innervated by a single Ib motoneuron, this plasticity may allow more robust and local regulation of muscle function. Although the Is did not show plastic change in response to manipulation of its activity or the coinnervating Ib in these experiments, it cannot be ruled out that Is neurons are capable of such plasticity but display less sensitivity to putative muscle-derived retrograde signals. Given that Is neurons innervate multiple muscles compared with Ib, it is also possible that small plastic changes occurring in Is are not synapse specific and are distributed over a larger population of AZs onto multiple muscles, resulting in little effect at any single postsynaptic target. Similar differences in homeostatic plasticity in Ib versus Is motoneurons have been described following reduced postsynaptic muscle glutamate receptor function, with the Ib motoneuron showing a more robust upregulation of presynaptic release compared with Is. Although homeostatic plasticity has been observed in Is motoneurons in low extracellular Ca2+ (Genc and Davis, 2019), the elevated Pr of Is synapses may occlude further functional increases in release output in higher [Ca2+] normally found in larval hemolymph. Together, these results indicate that tonic Ib motoneurons express distinct plasticity mechanisms that can be triggered by reduced muscle function that are less robust or lacking in the phasic Is subclass. Whether the differential plasticity found in this study is linked directly to the tonic or phasic properties of Ib and Is motoneurons, or is under separate regulatory control, will require further investigation (Aponte-Santiago, 2020).

    An important question moving forward is to identify mechanisms that control structural and functional plasticity in Ib motoneurons. Similarly, defining why the Is fails to respond to many of the same manipulations is poorly understood. Whether homeostatic plasticity mechanisms triggered in response to acute or chronic reduction in glutamate receptor function are also activated following the absence or functional silencing of presynaptic inputs as described in this study is unknown. Several molecular pathways contributing to homeostatic plasticity have been described at the NMJ. Beyond Drosophila, studies in crustacean motor systems have shown that long-term alterations in activity can induce cell type-specific changes in tonic or phasic motoneuron structure or release properties. Given that tonic and phasic neurons are abundant in the nervous systems of both invertebrates and vertebrates, it will be interesting to determine whether such properties play a key role in defining their capacity for plastic change. The tools described in this study provide an opportunity to identify the distinct transcriptional profiles of each neuronal subclass in Drosophila to identify candidate mechanisms that mediate the differential plasticity responses of tonic Ib and phasic Is motoneurons (Aponte-Santiago, 2020).

    Target-wide induction and synapse type-specific robustness of presynaptic homeostasis

    Presynaptic homeostatic plasticity (PHP) is an evolutionarily conserved form of adaptive neuromodulation and is observed at both central and peripheral synapses. This work has made several fundamental advances by interrogating the synapse specificity of PHP. This study defines how PHP remains robust to acute versus long-term neurotransmitter receptor perturbation. A general PHP property is described that includes global induction and synapse-specific expression mechanisms. Finally, a novel synapse-specific PHP expression mechanism is detailed that enables the conversion from short- to long-term PHP expression. If these data can be extended to other systems, including the mammalian central nervous system, they suggest that PHP can be broadly induced and expressed to sustain the function of complex neural circuitry (Genc, 2019).

    This study has taken advantage of cell-type-specific GAL4 drivers to achieve selective, optogenetic stimulation of single motoneuron inputs to a single muscle fiber. This has enabled exploration of the synapse specificity of PHP. It is demonstrated that the global disruption of postsynaptic glutamate receptors, either pharmacologically or genetically, induces robust PHP at both tonic (Ib) and phasic (Is) presynaptic terminals contacting a single muscle target. The fact that both phasic and tonic synapses participate in presynaptic homeostasis is consistent with previously published work demonstrating that the expression of PHP does not alter the short-term dynamics of presynaptic release when both the phasic and tonic synapses are stimulated simultaneously (Genc, 2019).

    It was also demonstrated that PHP is differentially expressed at phasic and tonic synapses when external calcium concentration is decreased. Remarkably, however, the synapse-specific effects of reducing external calcium depend upon how PHP is initiated. Acute induction of PHP is robustly expressed by the phasic synapses but not as robustly at tonic synapses. However, the situation completely reverses during chronic induction of PHP, where PHP is robustly expressed by the tonic synapses but not by the phasic synapses. These data highlight a novel, fundamental difference in the mechanisms responsible for the rapid versus prolonged expression of PHP (Genc, 2019).

    It was previously argued that PHP is synapse specific in that it is expressed exclusively by the tonic nerve terminal. These prior experiments included an optical quantal analysis of synaptic transmission in the GluRIIA mutant. This study has confirmed that PHP is expressed solely by the tonic synapse under conditions of diminished release, the GluRIIA mutant background, and low external calcium (conditions that reflect those pursued in the prior optogenetic study, which utilized high concentrations of external magnesium, presumably to stabilize the optical recording conditions). However, by exploring multiple methods of PHP induction at multiple external calcium concentrations, this study has clearly demonstrated that PHP is globally induced and expressed, such that differences in PHP expression emerge only when external calcium is diminished and are dependent on how PHP is induced (acutely versus chronically) (Genc, 2019).

    The current understanding of PHP expression is based primarily on the genetic deletion of key molecular components of the presynaptic homeostatic machinery, discovered in forward genetic screens. A presynaptic ENaC channel, composed of subunits transcribed by the PPK11, PPK16, and PPK1 genes, is necessary for both the rapid induction and sustained expression of PHP, driving increased calcium influx through the presynaptic CaV2.1 calcium channel (Younger, 2013). ENaC-channel function is required for both short- and long-term PHP, and this study has now demonstrated that it functions at both phasic and tonic synapses. So, differential activity of the ENaC channel cannot account for synapse-specific PHP expression at low external calcium (Genc, 2019).

    It is proposed that the differential expression of acute versus chronic PHP might relate to the EGTA sensitivity of presynaptic release. The chronic induction of PHP is differentially sensitive to EGTA, implying that the chronic expression of PHP includes a population of synaptic vesicles that are weakly coupled to presynaptic calcium channels. This finding could explain the differential synapse-specific expression of PHP at phasic and tonic synapses. During the acute induction of PHP, low-release-probability tonic synapses release neurotransmitters less efficiently at low external calcium, and PHP expression is correspondingly impaired. During the chronic induction, it is proposed that a transformation of the release site allows the participation of weakly coupled vesicles in the release process. It is hypothesized that the different anatomical geometry of release sites (phasic and tonic synapses) causes PHP to selectively fail at phasic synapses. More specifically, phasic synapses might be organized in such a way that weakly coupled vesicles cannot be accessed when external calcium is diminished. Such an effect would only be observed after the chronic induction of PHP combined with diminished external calcium (Genc, 2019).

    The trigger that initiates PHP, whether acute or chronic, is the loss or inhibition of postsynaptic neurotransmitter receptors, whether the glutamatergic Drosophila NMJ or the cholinergic mammalian NMJ is considered. On the basis of the data presented in this study, it appears that any synapse at which postsynaptic receptors are perturbed will express PHP. However, it has yet to be determined whether selective disruption of glutamate receptors at one synapse on a given target will induce synapse-selective PHP. The tools to achieve this experiment in a clearly reproducible manner do not yet exist. If PHP is expressed at every synapse that becomes perturbed, then PHP should stabilize the flow of information through complex neural circuitry. The related question of synapse-specific induction will determine whether information transfer can be selectively sustained through the regulation of some, but not all, synapses on a given target. Finally, the data suggest that some synapses might be more or less efficient at acutely and chronically expressing PHP. It is noted that the tonic synapse always expresses PHP in a more variable manner. There is a large range of synapse types with different release probabilities throughout the complex neural circuitry in the mammalian CNS, suggesting that PHP could be more or less robustly expressed at different synapses. As such, the global induction of PHP could differentially affect neural-circuit function in previously unsuspected ways, a possibility that could be important for considering the action of chronically administered neural therapeutics or drugs of addiction (Genc, 2019).

    Activity-dependent global downscaling of evoked neurotransmitter release across glutamatergic inputs in Drosophila

    Within mammalian brain circuits, activity-dependent synaptic adaptations such as synaptic scaling stabilise neuronal activity in the face of perturbations. Stability afforded through synaptic scaling involves uniform scaling of quantal amplitudes across all synaptic inputs formed on neurons, as well as on the postsynaptic side. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. This study tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. Motoneuron-specific genetic manipulations were employed to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. An adaptation was detected which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This 'presynaptic downscaling' maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter (DVGLUT) expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. These results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side (Karunanithi, 2020).

    Changes in neuronal activity can alter synaptic strength and refine synaptic connections as part of normal developmental and behavioral plasticity. Abnormal levels of activity could disrupt normal activity-dependent synaptic modifications, leading to pathophysiological imbalances in activity within neural circuits. Neurons have however evolved the capacity to functionally adapt and maintain stable activities in the face of perturbations. The most intensely studied adaptation, which stabilizes neuronal activity within mammalian brain circuits, is synaptic scaling. It operates globally by uniformly scaling quantal amplitudes across all synaptic inputs formed on a neuron, through changes in postsynaptic receptor numbers. Synaptic scaling causes compensatory changes in excitatory synaptic drive to the neuron in a direction that enables its firing rate, which has strayed out of set point range, to return back within range. Uniform scaling is also proposed to maintain the relative differences in synaptic weights among inputs, thereby maintaining the ability of neural circuits to appropriately process and interpret divergent inputs with physiological relevance. Considering that maintenance of stable activity is imperative for the robust function of the nervous system as a whole, this study tested whether activity-dependent uniform scaling could also operate within peripheral circuits (Karunanithi, 2020).

    Peripheral circuits also exhibit compensatory adaptations and activity-dependent synaptic modifications. One such circuit is the Drosophila larval neuromuscular circuit which drives crawling. This is a converging circuit, where different glutamatergic motoneurons form discrete neuromuscular junctions (NMJs) on each muscle. The highly active 1b and the less active 1s motoneurons form NMJs on virtually all larval muscles. Compensatory adaptations observed at those NMJs are mainly those which preserve strong muscular excitation by homeostatically maintaining synaptic drive to the muscle. They include presynaptic homeostatic potentiation, presynaptic homeostatic depression (PHD), and synaptic homeostasis through structural compensations. Those adaptations are recruited through changes at synapses themselves, rather than through changes in neuronal and muscle activities (firing rates), and are therefore not regarded as activity-dependent. It is unclear whether there are activity-dependent adaptations that manifest at NMJs, involving uniform scaling, to afford robust muscle activity (Karunanithi, 2020).

    Activity-dependent synaptic modifications, which appear within the larval neuromuscular circuit following long-term increases in neuronal activity, have been associated with synapse formation and maturation, and with synaptic modifications correlated to information storage. Such work used fly strains where activity was chronically increased in all neurons or motoneurons. With current knowledge regarding activity-dependent compensatory adaptations, it was asked whether such synaptic modifications were fully or partly compensatory to afford robust muscle activity. It was also asked whether compensatory adaptations could be recruited by increasing the activity of only one of the two motoneuron types (Karunanithi, 2020).

    Using genetic manipulations, the activity of the 1s motoneuron, innervating muscle 2, was transformed from being lowly active to being highly active. Then whether that transformation recruited compensatory adaptations was assessed by testing for alterations in muscle activity and NMJ properties formed by the genetically manipulated 1s and the unmanipulated 1b motoneurons. A compensatory adaptation was uncovered that was termed 'presynaptic downscaling,' which uniformly downscaled 1b and 1s evoked neurotransmitter release to afford robust and stable muscle activity. Activity-dependent uniform scaling can therefore manifest also within the periphery and on the presynaptic side (Karunanithi, 2020).

    Activity-dependent uniform scaling of a determinant of synaptic strength has previously been reported to occur only on the postsynaptic side within central circuits. This work showed that such scaling of a determinant of synaptic strength can also occur on the presynaptic side within peripheral circuits. Presynaptic downscaling describes the uniform decreases in QCs across all inputs, maintaining their relative neurotransmitter release differences around a homeostatic set point. Presynaptic downscaling was induced by increasing the activity of only the 1s motoneuron but was manifest by decreasing release probabilities at both the manipulated 1s and the unmanipulated 1b boutons. Presynaptic downscaling decreased synaptic drive to the muscle: (1) stabilizing muscle firing rates around the downshifted set point and homeostatically maintaining the wide variation in firing rates around that set point; and (2) maintaining robust muscle activity by preventing muscle firing rates from being displaced outside of the physiological range. An activity-dependent increase in 1s DVGLUT expression resulted in presynaptic downscaling, adding support to the idea that regulation of VGLUT expression plays a role in the recruitment of presynaptic compensatory adaptations (Karunanithi, 2020).

    The findings regarding muscle activity appear to fit within the framework of biological robustness, a concept in systems biology which accounts for variations in set points to maintain robust function against perturbations. Within that framework, a robust system/component can operate stably at different set points. That framework also accounts for set point shifts, as in this study, where excessive 1s neural drive to the muscle causes the muscle firing rate set point to downshift, without firing rates overshooting the physiological range. Through such shifts, robust muscle activity is afforded to potentially generate a wide range of contractions to drive varied movements under adverse conditions (Karunanithi, 2020).

    In synaptic downscaling, which operates centrally on the postsynaptic side, the quantal amplitudes are uniformly downscaled across all inputs, decreasing synaptic drive to the postsynaptic neuron. That decrease in synaptic drive affords stability by lowering firing rates back within the narrow set point range. In presynaptic downscaling, QCs are uniformly downscaled across all glutamatergic inputs through decreases in release probabilities (although uniform downscaling of QCs could also occur through decreases in active release site numbers). The downscaling of QCs decreases synaptic drive, affording robust and stable muscle activity. Activity-dependent uniform scaling appears to therefore operate on both the presynaptic and postsynaptic sides by scaling different determinants of synaptic strength to maintain target cell firing rates within operational range. When the operational range is narrow, as in neurons, uniform downscaling stabilizes firing rates around a set point. But when the range is wide, as in larval muscle, uniform downscaling stabilizes firing rates around a different set point without displacing firing rates outside of the physiological range, maintaining robust outputs. Activity-dependent uniform downscaling appears to therefore operate in different capacities in mammalian neurons and larval muscle (Karunanithi, 2020).

    A difference between the induction of synaptic scaling centrally and of presynaptic downscaling peripherally is that the former can be produced cell-autonomously, but not the latter. Synaptic scaling can be produced by directly changing the activity of a postsynaptic neuron. Such scaling can be swift, produced in an hour, as the signals activated in response to changes in activity directly affect the synapses formed on that postsynaptic neuron. Presynaptic downscaling, on the other hand, is produced by increasing the activity of a single input neuron. Induction signals, once initiated in that input neuron, would need to travel anterograde to the target cell, and then retrograde from the target cell to the different synaptic inputs to produce uniform downscaling. Induction signals could also involve signaling between 1b and 1s inputs, possibly like those reported among mammalian glutamatergic inputs. Those signaling pathways suggest that presynaptic downscaling induction is a relatively slow (requiring more than an hour) and nonautonomous process. Some candidate molecules carrying anterograde signals being vesicular glutamate and postsynaptic receptors, and retrograde signals could include Glass bottom boat, Dystrophin, and Semaphorin. Molecules evidenced to mediate signaling among mammalian glutamatergic inputs include glutamate, calcium, protein kinase A, and neurotrophic factors (Karunanithi, 2020).

    The experiments argue for presynaptic downscaling resulting from increased 1s DVGLUT expression following increased 1s motoneuron activity. That order of process leading to presynaptic downscaling does not however imply direct causality among those steps. A number of intermediate steps may likely be involved, including retrograde signaling from the muscle (discussed above) to calibrate neurotransmitter release in order to prevent muscle activity from overshooting its physiological range. These possibilities need to be investigated in future work (Karunanithi, 2020).

    Uniform scaling is proposed to maintain the relative differences among synapses around a set point to prevent disruption of mechanisms which rely on such differences being preserved. For example, in postsynaptic neurons within brain circuits, synaptic downscaling would enable synaptic inputs which underwent Hebbian potentiation to retain their stronger synaptic weighting relative to other inputs, while concurrently preventing such strengthening from causing runaway excitation. In the periphery, recent work at the larval NMJ shows that 1b synapses primarily drive larval muscle contractions during crawling, whereas 1s synapses drive intersegmental coordination of muscle contractions (Newman, 2017). Neurotransmitter release differences between 1b and 1s synapses underlie the differential execution of these two functions. The current findings therefore suggest that uniform downscaling could preserve the relative neurotransmitter release differences to enable 1b and 1s synapses to continue driving these two functions against perturbations. Uniform downscaling on the presynaptic or postsynaptic sides could therefore afford stability within circuits by maintaining the relative differences among synaptic inputs around a homeostatic set point (Karunanithi, 2020).

    Larval NMJs display forms of compensatory adaptations, which preserve strong muscular excitation in the face of perturbations. In those forms, EJP amplitude is homeostatically maintained through compensatory changes in both QC and quantal size. PHD is one such adaptation, where the increases in quantal sizes are offset by compensatory decreases in QCs at both 1b and 1s boutons. PHD is induced by directly overexpressing DVGLUT into both 1b and 1s motoneurons, and as such, has not been shown to be induced by altered activity. By contrast, presynaptic downscaling causes a compensatory decrease in EJP amplitude to afford robust and stable muscle activity. It results following an activity-dependent increase in only 1s DVGLUT expression, causing the uniform downscaling of both 1b and 1s QCs. PHD is therefore a compensatory adaptation that preserves muscular excitation by homeostatically maintaining EJP amplitude, whereas presynaptic downscaling is a compensatory adaptation which stabilizes muscle activity by decreasing EJP amplitude. The two different adaptations are produced by two different patterns of DVGLUT expression, but both involve uniform decreases in QCs across inputs. However, in the case of presynaptic downscaling, the uniform decreases are activity-dependent. In this preparation, compensatory adaptations therefore not only regulate muscle excitation, but also muscle activity (Karunanithi, 2020).

    Uniform downscaling has been implicated in processes related to sleep and memory. Work in mammalian brain circuits proposes that uniform downscaling of quantal amplitudes on the postsynaptic side renormalizes synapses during sleep following Hebbian potentiation throughout the day, and prevents runaway excitation following Hebbian potentiation during memory formation. This finding suggests that the scope for evaluating such a process should be broadened to include any uniform downscaling on the presynaptic side, and questions whether such downscaling would involve changes in vesicular neurotransmitter transporter expression (Karunanithi, 2020).

    In future work, the molecular mechanisms underlying presynaptic downscaling will need to be elucidated, including identification of retrograde and anterograde signals, and the role of DVGLUT in such signaling. Assessments are also needed of how regulatory molecules, and changes in the activity profiles of the 1b motoneuron and both 1b and 1s motoneurons, contribute to robust and stable circuit function. Finally, detailed analysis of the frequency components within muscle bursts could provide better understanding of the mechanisms shaping muscle activity (Karunanithi, 2020).

    Endogenous tagging reveals differential regulation of Ca(2+) channels at single AZs during presynaptic homeostatic potentiation and depression

    Neurons communicate through Ca(2+)-dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca(2+) channel levels are modulated to calibrate synaptic function. This study used CRISPR to tag the Drosophila CaV2 Ca(2+) channel Cacophony (Cac) and, in males in which all endogenous Cac channels are tagged, investigated the regulation of endogenous Ca(2+) channels during homeostatic plasticity. Heterogeneously distributed Cac was found to be highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, AZ Cac levels are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca(2+) channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca(2+) influx during PHD is achieved through functional adaptions to pre-existing Ca(2+) channels. Thus, distinct mechanisms bi-directionally modulate presynaptic Ca(2+) levels to maintain stable synaptic strength in response to diverse challenges, with Ca(2+) channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales (Gratz, 2019).

    Diverse synaptic release properties enable complex communication and may broaden the capacity of circuits to communicate reliably and respond to changing inputs. This study has investigated how the regulation of Ca2+ channel accumulation at AZs contributes to the establishment and modulation of AZ-specific release properties to maintain stable communication. Endogenous tagging of Cac allowed tracking of Ca2+ channels live and in fixed tissue without the potential artifacts associated with transgene overexpression. This approach revealed differences in the regulation of endogenous and exogenous Ca2+ channels, underlining the value of developing and validating reagents for following endogenous proteins in vivo (Gratz, 2019).

    The abundance of endogenous Cac at individual AZs of single motorneurons is heterogeneous and correlates with single-AZ Pr. This is consistent with previous studies in multiple systems linking endogenous Ca2+ channel levels at individual AZs to presynaptic release probability and efficacy, and a recent investigation of transgenically expressed Cac. This strong correlation suggests Ca2+ channel levels might be regulated to tune Pr during plasticity, so this study investigated the modulation of endogenous Cac levels in several Drosophila models of presynaptic homeostatic plasticity. Previous studies have suggested that the bidirectional regulation of Ca2+ influx at synapses contributes to the modulation of presynaptic release observed during both PHP and PHD. A long-standing question is whether these changes are achieved through the regulation of channel levels, channel function, or through distinct mechanisms. Multiple mechanisms have been proposed to explain the increase in Ca2+ influx observed during the expression of PHP. For example, a presynaptic epithelial sodium channel (ENaC) and glutamate autoreceptor (DKaiR1D) have been implicated in promoting Ca2+ influx during PHP, leading to the model that modulation of presynaptic membrane potential might increase influx through Cac channels. On the other hand, the Eph‐specific RhoGEF Ephexin signals through the small GTPase Cdc42 to promote PHP in a Cac-dependent manner, raising the possibility that it does so through actin-dependent accumulation of new channels. Further, multiple AZ cytomatrix proteins, including Fife, RIM, and RIM-binding protein, are necessary to express PHP and also regulate Ca2+ channel levels during development. However, whether Ca2+ channel abundance is modulated during PHP remained an open question (Gratz, 2019).

    This study demonstrates that Cac abundance is indeed enhanced during both the acute and chronic expression of PHP. This increase occurs in conjunction with the accumulation of Brp and enhancement of the RRP, pointing to the coordinated remodeling of the entire neurotransmitter release apparatus during PHP on both timescales. Studies in mammals have found that AZ protein levels are dynamic and subject to homeostatic modification over chronic timescales, indicating that structural reorganization of AZs is a conserved mechanism for modulating release. As ENaC and DKaiR1D-dependent functional modulation occur in tandem with the structural reorganization of AZs, it is interesting to consider why redundant mechanisms may have evolved. One remarkable feature of PHP is the incredible precision with which quantal content is tuned to offset disruptions to postsynaptic neurotransmitter receptor function. It is therefore tempting to hypothesize that PHP achieves this analog scaling of release probability by simultaneously deploying distinct mechanisms to calibrate the structure and function of AZs (Gratz, 2019).

    In contrast to the many mechanisms proposed for modulating Ca2+ influx during PHP, far less is known about how Ca2+ influx is regulated during PHD. One attractive idea was a reduction in AZ Ca2+ channel levels based on studies revealing reduced levels of transgenic UAS-Cac-GFP upon vGlut overexpression. However, this study found that endogenous Cac channels do not change in conjunction with vGlut overexpression-induced PHD. Because all Cac channels are tagged in cacsfGFP-N, this observation indicates that a reduction in Cac abundance at AZs is not necessary to achieve PHD. It was determined that the source of the discrepancy is the use of the transgene to report overexpressed versus endogenous Cac levels, demonstrating that exogenous and endogenous channels are regulated differently, at least during this form of PHD. This indicates that a mechanism other than modulation of Cac abundance drives PHD expression. Levels of Brp and RRP size are also unchanged during PHD. Thus, the coordinated reorganization of the AZ appears to be specific to PHP. Interestingly, reversible downregulation of a subset of AZ proteins, but not Cac, was observed at Drosophila photoreceptor synapses following prolonged light exposure. In the future, it will be of interest to determine whether PHP and PHD share any mechanisms to control the bidirectional modulation of Ca2+ influx. PHD signaling operates independently of PHP, and was recently proposed to function as a homeostat responsive to excess glutamate, not synaptic strength, raising the possibility that mechanisms distinct from those that have been elucidated for PHP may regulate presynaptic inhibition during PHD (Gratz, 2019).

    Finally, live imaging of CacsfGFP-N during acute PHP enabled the investigation of how baseline heterogeneity in Cac levels and Pr intersects with the homeostatic reorganization of AZs. Monitoring endogenous Cac at the same AZs before and after PhTx treatment, the accumulation was observed of Ca2+ channels across AZs with diverse baseline properties. As with PHP expression over chronic timescales, the findings leave open the possibility of multiple mechanisms acting simultaneously, perhaps to ensure precise tuning and do not rule out additional modulation of channel function or indirect regulation of Ca2+ influx. In fact, a prevailing model posits rapid events that acutely modulate Pr followed by consolidation of the response for long-term homeostasis. Coincident changes in Ca2+ channel function and levels coupled with long-term restructuring of AZs provides an attractive mechanism for this model. This study also found that Cac accumulation is proportional across low- and high-Pr AZs. Therefore, baseline heterogeneity in Cac levels is maintained following the expression of PHP. At mammalian excitatory synapses, proportional scaling of postsynaptic glutamate receptor levels stabilizes activity while maintaining synaptic weights. The findings suggest an analogous phenomenon could be occurring presynaptically at the Drosophila NMJ. Notably, receptor scaling can occur globally or locally. A recent study reported that PHP can be genetically induced and expressed within individual axon branches, demonstrating a similar degree of specificity in the expression of PHP at the Drosophila NMJ. A proportional increase in Cac levels could arise through homeostatic signaling from individual postsynaptic densities responding to similar decreases in quantal size; a strategy that would allow for both the remarkable synapse specificity and precision with which homeostatic modulation of neurotransmitter release operates (Gratz, 2019).

    Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling

    Synapses and circuits rely on neuroplasticity to adjust output and meet physiological needs. Forms of homeostatic synaptic plasticity impart stability at synapses by countering destabilizing perturbations. The Drosophila melanogaster larval neuromuscular junction (NMJ) is a model synapse with robust expression of homeostatic plasticity. At the NMJ, a homeostatic system detects impaired postsynaptic sensitivity to neurotransmitter and activates a retrograde signal that restores synaptic function by adjusting neurotransmitter release. This process has been separated into temporally distinct phases, induction and maintenance. One prevailing hypothesis is that a shared mechanism governs both phases. This study shows the two phases are separable. Combining genetics, pharmacology, and electrophysiology, a signaling system consisting of PLCbeta, inositol triphosphate (IP3), IP3 receptors, and Ryanodine receptors was shown to be required only for the maintenance of homeostatic plasticity. It was also found that the NMJ is capable of inducing homeostatic signaling even when its sustained maintenance process is absent (James, 2019).

    The Drosophila melanogaster NMJ is an ideal model synapse for studying the basic question of how synapses work to counter destabilizing perturbations. At this NMJ, reduced sensitivity to single vesicles of glutamate initiates a retrograde, muscle-to-nerve signaling cascade that induces increased neurotransmitter vesicle release, or quantal content (QC). As a result, the NMJ maintains a normal postsynaptic response level. Mechanistically, this increase in QC depends upon the successful execution of discrete presynaptic events, such as increases in neuronal Ca2+ influx and an increase in the size of the readily releasable pool (RRP) of synaptic vesicles. The field has termed this compensatory signaling process as presynaptic homeostatic potentiation (PHP) (Delvendahl, 2019). Two factors that govern the expression of PHP are the nature of the NMJ synaptic challenge and the amount of time elapsed after presentation of the challenge. Acute pharmacological inhibition of postsynaptic glutamate receptors initiates a rapid induction of PHP that restores synaptic output in minutes. By contrast, genetic lesions and other long-term reductions of NMJ sensitivity to neurotransmitter induce PHP in a way that is sustained throughout life (James, 2019).

    Previously work has identified the Plc21C gene as a factor needed for PHP (Brusich, 2015). Plc21C encodes a Drosophila Phospholipase Cβ (PLCβ) homolog known to be neuronally expressed, but recent ribosomal profiling data also indicates possible muscle expression of Plc21C (Chen, 2017). In canonical signaling pathways, once PLCβ is activated by Gαq, it cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). DAG can affect synaptic function by activating Protein Kinase C (PKC), while IP3 binds its receptor (IP3R) to trigger release of calcium from intracellular stores. It is not understood which aspects of this signaling machinery are mobilized during PHP. Potential downstream consequences of PLCβ activity at the NMJ include phosphorylation of neuronal proteins, modulation of ion channel activity, and changes in localization of neurotransmission machinery (James, 2019).

    This study scrutinized PLCβ-directed signaling further. Tests were performed to see whether PLCβ-directed signaling was required solely for the maintenance of PHP or if it could also be required for induction. In addition to PLCβ, the IP3 Receptor (Drosophila Itpr, herein IP3R) and Ryanodine receptor (Drosophila RyR) were identified as being part of the same signaling process. Neither PLCβ, nor IP3R, nor RyR are required for the rapid induction of PHP. Additionally, it was found that the rapid induction of PHP is still possible in synapses already sustaining PHP. Surprisingly, it was found that NMJs are capable of rapidly inducing PHP -- even when the sustained expression of PHP is already blocked by impairments in PLCβ, IP3R, or RyR signaling. Taken together, these data show that the induction and maintenance of PHP are separable. Even though there is compelling evidence that parts of the induction and maintenance signaling mechanisms overlap (Goel, 2017), it is also true that acute PHP is possible in scenarios where long-term PHP is not (James, 2019).

    This study divides the acute induction and chronic maintenance stages of presynaptic homeostatic potentiation. The data support two core findings. The first is that the short-term induction and long-term maintenance of PHP are separable by genetic and pharmacological manipulations. The second is that an IP3-mediated signaling system is specifically required for the maintenance of PHP (see Model depicting PLCβ/IP3R/RyR signaling underling the maintenance of PHP in both muscle and neuron.) (James, 2019).

    For several years, one assumption has been that both the acute and chronic forms of PHP are executed in a similar way, and possibly by shared mechanisms. The issue has been clouded by the fact that both PhTox and a GluRIIA deletion mutant -- the primary reagents utilized to induce PHP -- have the same molecular target, that is GluRIIA-containing glutamate receptors. The process of combining these acute and chronic forms of plasticity within a single genotypic background was cumbersome due to a lack of reagents available to conduct temporally separate targeting experiments (James, 2019).

    Several groups ascertained insights into temporal requirements by targeting potential homeostatic signaling genes. The main finding has been that the majority of molecules identified are essential to both the acute and chronic forms of PHP. Neurons tightly control neurotransmitter release probability, and the core presynaptic machinery directly responsible for increasing quantal content is shared. These shared components include the CaV2-type voltage-gated calcium channel or factors gating influx through the channel. They also include factors that regulate the size of the readily releasable pool (RRP) of presynaptic vesicles or factors that control the baseline excitability or plasticity of the presynaptic motor neuron, and neurotransmitter fusion events themselves. As a result, both the acute and chronic forms of PHP signaling cause increases in readily releasable pool (RRP) size and CaV2-mediated calcium influx; and in turn, these presynaptic mechanisms underlie the increases in QC which constitute PHP (James, 2019).

    This study shows that although the acute and chronic processes might overlap, they are functionally separable. The fact that they are separable is not necessarily surprising. This finding mirrors data for discrete molecules required for long-term PHP maintenance, such as Target of Rapamycin (Tor), the Rho-type guanine exchange factor Ephexin, the transcription factor Gooseberry, C-terminal Src Kinase, innate immune signals molecules IMD, IKKβ, Relish and the kinesin adaptor Arl8. Importantly, this list contains molecules implicated both in neuron and muscle. This study has added PLCβ (Brusich, 2015) and its effectors IP3R and RyR to this list (James, 2019).

    Recent studies have augmented the idea of overlapping signaling pathways and added a degree of specificity. Both acute and chronic forms of PHP begin as instructive retrograde signals after perturbations are detected in the muscle. These forms of PHP involve a decrease in phosphorylation of muscle CaMKII levels, and converge upon the same signaling components in the presynaptic neuron. These studies suggest that Tor signaling converges on the same molecular targets as acute forms of PHP (Goel, 2017). However, the precise roles for Tor and CaMKII in either form of PHP are as yet unknown (James, 2019).

    These data appear to contradict the idea of PHP pathway convergence (Goel, 2017). Yet, the current findings are not incompatible with this idea. Multiple lines of evidence indicate discrete signaling requirements for acute forms of PHP on both sides of the synapse. A convergence point is undefined. Accounting for the separation of acute and chronic forms of PHP -- as well as their discrete signaling requirements -- long-term maintenance of PHP might integrate multiple signals between the muscle and neuron over time. For future studies, it will be important to clearly define roles of signaling systems underlying PHP and how distinct signaling systems might be linked (James, 2019).

    This work presents unexpected findings. The first is that even in the face of a chronic impairment or block of homeostatic potentiation, the NMJ is nevertheless capable of a full rapid induction of PHP. Given that most molecules required for PHP identified to date are needed for both phases, significant functional separation between them was not expected. A priori it was expected that a failure of the chronic maintenance of PHP would make core machinery unavailable for its acute induction. The second unexpected finding is how quickly the chronic maintenance of PHP can be nullified by pharmacology (10 min), resulting in a return to baseline neurotransmitter release probability after only minutes of drug exposure. This study showed that homeostatic potentiation in GluRIIA mutant larvae or GluRIIC knock-down larvae was abrogated by four different reagents previously known to block IP3R or RyR. Those findings are reminiscent of prior work showing that acute blockade of DAG/ENaC channels with the drug benzamil abolishes PHP in both a GluRIIA mutant background, as well as in the presence of PhTox (Younger, 2013). A difference between benzamil application and the pharmacological agents used in the current study is that the drugs employed in this study only abolished PHP in a chronically challenged background (James, 2019)?

    It is unclear how signaling systems that drive homeostatic plasticity transition from a state of induction to a state of maintenance. It is also not understood how interdependent short-term and long-term HSP implementation mechanisms are. A more complete understanding of the timing and perdurance of these properties could have important implications for neurological conditions where synapse stability is episodically lost (James, 2019).

    The current findings parallel recent data examining active zone protein intensities in the contexts of induction of PHP and maintenance of PHP at the NMJ. There are multiple results informative to this study. First, the expression of any form of PHP (acute or chronic) appears to correlate with an increased intensity of active zone protein levels, such as CaV2/Cacophony, UNC-13, and the Drosophila CAST/ELKS homolog Bruchpilot. Unexpectedly, however, two of these studies also reported that the rapid induction of PHP does not require this protein increase in order to be functionally executed. These are conundrums for future work. How does rapid active zone remodeling happen in minutes on a mechanistic level? In the absence of such remodeling, how is PHP able to be induced rapidly? Moreover, is the observed short-term active zone remodeling the kernel for the longer-term changes to the active zone and release probability - or is some other compensatory system triggered over long periods of developmental time (James, 2019)?

    The current findings add a new dimension to those puzzles with the data that IP3 signaling is continuously required to maintain PHP. If active zone remodeling truly is instructive for PHP maintenance, then it will be interesting to test what roles IP3 signaling and intracellular calcium release play in that process. The screen did include a UAS-RNAi line against unc-13 and an upstream GPCR-encoding gene methuselah. Moreover, a previously study of PHP used a UAS-cac[RNAi] line (Brusich, 2015). Chronic PHP maintenance was intact for all of those manipulations. Those findings are not necessarily contradictory to the recent work from other groups. For instance, knockdown of an active zone protein by RNAi is not a null condition. As such, RNAi-mediated knockdown should leave residual wild-type protein around. In theory, that residual protein could be scaled with homeostatic need (James, 2019).

    The data strongly suggest that intracellular calcium channel activation and store release fine tune neurotransmitter release that is implemented by PHP. The exact mechanism by which IP3R and RyR function to maintain PHP at the NMJ is unclear. It appears to be a shared process with IP3. If these store-release channels are acting downstream of IP3 activity, then the data suggest that this would be a coordinated activity involving both the muscle and the neuron, with loss of IP3 signaling in the neuron being more detrimental to evoked release (James, 2019).

    It remains unclear what signals are acting upstream. PLCβ is canonically activated by Gαq signaling. From prior work, evidence was garnered that a Drosophila Gq protein plays a role in the long-term maintenance of HSP (Brusich, 2015). Logically, there may exist a G-protein-coupled receptor (GPCR) that functions upstream of PLCβ/IP3 signaling. A screen did not positively identify such a GPCR. Several genes encoding GPCRs were examined, including TkR86C, mAChR-A, GABA-B-R1, PK2-R2, methuselah, AdoR, and mGluR. Genes encoding Gβ subunits or putative scaffolding molecules were also examined, including CG7611 (a WD40-repeat-encoding gene), Gβ13F, and Gβ76C, again with no positive screen hits (James, 2019).

    The data are consistent with dual pre- and post-synaptic functions of IP3. This could mean dual pre- and post-synaptic roles for calcium store release, through an undetermined combination of RyR and IP3R activities, again either pre- or post-synaptic. Both RyR and IP3R have been shown to be critical for specific aspects of neuroplasticity and neurotransmission. Activities of both RyR and IP3R can activate molecules that drive plasticity, such as Calcineurin and CaMKII. At rodent hippocampal synapses, electrophysiological measures like paired-pulse facilitation and frequency of spontaneous neurotransmitter release are modulated by RyR and/or IP3R function, as is facilitation of evoked neurotransmitter at the rat neocortex. In addition to vesicle fusion apparatus, activity of presynaptic voltage-gated calcium channels is modulated by intracellular calcium. Work from this lab at the NMJ has shown that impairing factors needed for store-operated calcium release can mollify hyperexcitability phenotypes caused by gain-of-function CaV2 amino-acid substitutions (Brusich, 2018; James, 2019 and references therein).

    Within the pre-synaptic neuron, IP3R and RyR could activate any number of calcium-dependent molecules to propagate homeostatic signaling. Some candidates were tested in a screen, but none of those tests blocked PHP. One possibility is that the reagents utilized did not sufficiently diminish the function of target molecules enough to impact PHP in this directed screen. Detection of downstream effectors specific to muscle or neuron might also be ham>pered by the fact that attenuation of IP3 signaling in a single tissue is insufficient to abrogate PHP. Another possibility is that presynaptic store calcium efflux via IP3R and RyR may directly potentiate neurotransmitter release, either by potentiating basal calcium levels or synchronously with CaV2-type voltage-gated calcium channels (James, 2019).

    Both pre- and post-synaptic voltage-gated calcium channels are critical for the expression of several forms of homeostatic synaptic plasticity. Much evidence supports the hypothesis that store-operated channels and voltage gated calcium channels interact to facilitate PHP. In various neuronal populations, both RyR and IP3R interact with L-type calcium channels physically and functionally to reciprocally impact the opening of the other channel. In presynaptic boutons, RyR calcium release follows action potential firing. Calcium imaging experiments show that both the acute expression and sustained maintenance of PHP requires an increase in presynaptic calcium following an action potential. Because IP3Rs are activated by both free calcium and IP3, elevated IP3 levels in the case of chronically expressed PHP could allow IP3Rs and RyRs to open in a way that is time-locked with CaV2-mediated calcium influx or in a way to facilitate the results of later CaV2-mediated influx (James, 2019).

    Dual separable feedback systems govern firing rate homeostasis

    Firing rate homeostasis (FRH) stabilizes neural activity. A pervasive and intuitive theory argues that a single variable, calcium, is detected and stabilized through regulatory feedback. A prediction is that ion channel gene mutations with equivalent effects on neuronal excitability should invoke the same homeostatic response. In agreement, this study demonstrates robust FRH following either elimination of Kv4/Shal protein or elimination of the Kv4/Shal conductance. However, the underlying homeostatic signaling mechanisms are distinct. Eliminating Shal protein invokes Kruppel-dependent rebalancing of ion channel gene expression including enhanced slo, Shab, and Shaker. By contrast, expression of these genes remains unchanged in animals harboring a CRISPR-engineered, Shal pore-blocking mutation where compensation is achieved by enhanced IKDR. These different homeostatic processes have distinct effects on homeostatic synaptic plasticity and animal behavior. It is proposed that FRH includes mechanisms of proteostatic feedback that act in parallel with activity-driven feedback, with implications for the pathophysiology of human channelopathies (Kulik, 2019).

    Firing Rate Homeostasis is a form of homeostatic control that stabilizes spike rate and information coding when neurons are confronted by pharmacological, genetic or environmental perturbation. FRH has been widely documented within invertebrate neurons and neural circuits as well as the vertebrate spinal cord, cortical pyramidal neurons and cardiomyocytes. In many of these examples, the genetic deletion of an ion channel is used to induce a homeostatic response. The mechanisms of FRH correct for the loss of the ion channel and precisely restore neuronal firing properties to normal, wild-type levels). To date, little is understood about the underlying molecular mechanisms (Kulik, 2019).

    FRH induced by an ion channel gene deletion is truly remarkable. The corrective response is not limited to the de novo expression of an ion channel gene with properties that are identical to the deleted channel, as might be expected for more generalized forms genetic compensation. Instead, the existing repertoire of channels expressed by a neuron can be 'rebalanced' to correct for the deletion of an ion channel. How is it possible to precisely correct for the absence of an essential voltage-gated ion channel? The complexity of the problem seems immense given that many channel types functionally cooperate to achieve the cell-type-specific voltage trajectory of an action potential (Kulik, 2019).

    Theoretical work argues that different mixtures of ion channels can achieve similar firing properties in a neuron. These observations have led to a pervasive and intuitively attractive theory that a single physiological variable, calcium, is detected and stabilized through regulatory feedback control of ion channel gene expression. Yet, many questions remain unanswered. There are powerful cell biological constraints on ion channel transcription, translation, trafficking and localization in vivo. How do these constraints impact the expression of FRH? Is calcium the only intracellular variable that is sensed and controlled by homeostatic feedback? There remain few direct tests of this hypothesis. Why are homeostatic signaling systems seemingly unable to counteract disease-relevant ion channel mutations, including those that have been linked to risk for diseases such as epilepsy and autism (Kulik, 2019)?

    This study has taken advantage of the molecular and genetic power of Drosophila to explore FRH in a single, genetically identified neuron subtype. Specifically, two different conditions are compared that each eliminate the Shal/Kv4 ion channel conductance and, therefore, are expected to have identical effects on neuronal excitability. Robust FRH is demonstrated following elimination of the Shal protein and, independently, by eliminating the Shal conductance using a pore blocking mutation that is knocked-in to the endogenous Shal locus. Thus, consistent with current theory, FRH can be induced by molecularly distinct perturbations to a single ion channel gene. However, these two different perturbations were found to induce different homeostatic responses, arguing for perturbation-specific effects downstream of a single ion channel gene (Kulik, 2019).

    Taken together, these data contribute to a revised understanding of FRH in several ways. First, altered activity cannot be the sole determinant of FRH. Two functionally identical manipulations that eliminate the Shal conductance, each predicted to have identical effects on neuronal excitability, lead to molecularly distinct homeostatic responses. Second, homeostatic signaling systems are sensitive to the type of mutation that affects an ion channel gene. This could have implications for understanding why FRH appears to fail in the context of human disease caused by ion channel mutations, including epilepsy, migraine, autism and ataxia. Finally, the data speak to experimental and theoretical studies arguing that the entire repertoire of ion channels encoded in the genome is accessible to the mechanisms of homeostatic feedback, with a very large combinatorial solution space. The data are consistent with the existence of separable proteostatic and activity-dependent homeostatic signaling systems, potentially acting in concert to achieve cell-type-specific and perturbation-specific FRH (Kulik, 2019).

    These data advance mechanistic understanding of FRH in several ways. First, it was demonstrated that FRH can be induced and fully expressed in single, genetically identified neurons. Since changes in the activity of a single motoneuron are unlikely to dramatically alter the behavior of the larvae, these data argue strongly for cell autonomous mechanisms that detect the presence of the ion channel perturbation and induce a corrective, homeostatic response. Second, this study demonstrates that FRH functions to preserve the waveform of individual action potentials. This argues for remarkable precision in the homeostatic response. Third, new evidence is provided that the transcription factor Krüppel is essential for FRH, and selectively controls the homeostatic enhancement of IKCa,the voltage-gated sodium current (INa) and the voltage-gated calcium current (ICa) without altering the baseline ion channel current. Finally, it was demonstrated that different mechanisms of FRH are induced depending upon how the Shal current is eliminated, and these differential expression mechanisms can have perturbation-specific effects on animal behavior (Kulik, 2019).

    The existence is proposed of parallel homeostatic mechanisms, responsive to differential disruption of the Shal gene. Different compensatory responses were observed depending upon whether the Shal protein is eliminated or the Shal conductance is eliminated. The following evidence supports the functional equivalence of these manipulations. First, the ShalW362F mutation completely eliminates somatically recorded fast activating and inactivating potassium current IKA. Second, a dramatic reduction in IKA was demonstrated when Shal-RNAi is driven by MN1-GAL4 in a single, identified neuron. Notably, the current-voltage relationship observed for Shal-RNAi is identical to that previously published for the ShalW362F protein null mutation, being of similar size and voltage trajectory including a + 50 mV shift in voltage activation. This remaining, voltage-shifted, IKA-like conductance is attributed to the compensatory up-regulation of the Shaker channel on axonal membranes an effect that does not occur in the ShalW362F mutant. Thus, it seems reasonable to assume that Shal protein elimination and Shal conductance blockade initially create identical effects on neuronal excitability by eliminating Shal function. Subsequently, these perturbations trigger divergent compensatory responses. But, it is acknowledged that direct information is lacking about the immediate effects of the two perturbations (Kulik, 2019).

    This study defines FRH as the restoration of neuronal firing rate in the continued presence of a perturbation. This definition is important because it necessitates that the underlying molecular mechanisms of FRH must have a quantitatively accurate ability to adjust ion channel conductances such that firing rate is precisely restored. Mechanistically, a prior example of FRH involves an evolutionarily conserved regulation of sodium channel translation by the translational repressor Pumillio. This work, originally pursued in Drosophila, was extended to mouse central neurons where it was shown that Pumilio-dependent bi-directional changes in the sodium current occur in response to altered synaptic transmission, initiated by application of either AMPA antagonist NBQX or GABA antagonist Gabazine. These data highlight the emerging diversity of molecular mechanisms that can be induced and participate in the execution of FRH (Kulik, 2019).

    It is necessary to compare the current results with prior genetic studies of the Shal channel in Drosophila. A prior report, examining the effects of partial Shal knockdown in larval motoneurons, observed a trend toward an increase in the sustained potassium current, but concluded no change. However, the small sample size for potassium current measurements in that study (n = 3 cells) and the incomplete Shal knockdown that was achieved, likely conspired to prevent documentation of the significant increase in IKDR that was observed. A second prior study examined over-expression of a pore-blocked Shal transgene in cultured Drosophila embryonic neurons, revealing elevated firing rate and a broadened action potential. This was interpreted as evidence against the existence of FRH. However, neuronal precursors were cultured from 5 hr embryos, prior to establishment of neuronal cell fate and prior to the emergence of IKA currents in vivo, which occurs ~10 hr later in development. It remains unclear whether these cultured neurons are able to achieve a clear cell identity, which may be a prerequisite for the expression of homeostatic plasticity. Another possibility concerns the time-course of FRH, which remains uncertain. Finally, over-expression of the transgene itself might interfere with the mechanisms of FRH, emphasizing the importance of the scarless, CRISPR-mediated gene knock-in approach that was employed (Kulik, 2019).

    It is clear from studies in a diversity of systems that FRH can be induced by perturbations that directly alter neuronal activity without genetic or pharmacological disruption of ion channels or neurotransmitter receptors. For example, monocular deprivation induces an immediate depression of neuronal activity in the visual cortex, followed by restoration of normal firing rates. Research on the lobster stomatogastric system ranging from experiments in isolated cell culture to de-centralized ganglia have documented the existence of FRH that is consistent with an activity-dependent mechanism. It is equally clear that FRH can be induced by the deletion of an ion channel gene, including observations in systems as diverse as invertebrate and vertebrate central and peripheral neurons and muscle. But, it has remained unknown whether FRH that is induced by changes in neural activity is governed by the same signaling process that respond to ion channel gene mutations. The current data speak to this gap in knowledge (Kulik, 2019).

    Changes in neural activity cannot be solely responsible for FRH. This study compared two different conditions that each completely eliminate the Shal ion channel conductance and, therefore, are expected to have identical effects on neuronal excitability. Robust FRH was demonstrated in both conditions. However, two separate mechanisms account for FRH. Shal-RNAi induces a transcription-dependent homeostatic signaling program. There is enhanced expression of Krüppel and a Krüppel-dependent increase in the expression of the slo channel gene and enhanced IKCA current. By contrast, the ShalW362F mutant does not induce a change in the expression of Krüppel, slo or any of five additional ion channel genes. Instead, a change was observed in the IKDR conductance, the origin of which has not yet been identified, but which appears to be independent of a change in ion channel gene transcription (Kulik, 2019).

    The existence is proposed of two independent homeostatic signaling systems, induced by separate perturbations to the Shal channel gene. First, it is proposed that Shal-RNAi and the Shal null mutation trigger a homeostatic response that is sensitive to the absence of the Shal protein. In essence, this might represent an ion channel-specific system that achieves channel proteostasis, a system that might normally be invoked in response to errors in ion channel turnover. It is speculated that many, if not all ion channels could have such proteostatic signaling systems in place. In support of this idea, the induction of Kr is specific to loss of Shal, not occurring in eight other ion channel mutant backgrounds, each of which is sufficient to alter neural activity, including eag, para, Shaker, Shab, Shawl, slo, cac and hyperkinetic. Each of these channel mutations is well established to alter neuronal activity. But, Kr responds only to loss of Shal (Kulik, 2019).

    Next, it is proposed that eliminating the Shal conductance in the ShalW362F mutant background induces a separable mechanism of FRH that is independent of ion channel transcription. While the mechanisms of this homeostatic response remain unknown, it is tempting to speculate that this mechanism is activity dependent, consistent with data from other systems. Finally, it remains possible that these homeostatic signaling systems are somehow mechanistically linked. If so, this might provide a means to achieve the precision of FRH. For example, changes in ion channel gene expression might achieve a crude re-targeting of set point firing rates, followed by engagement of activity-dependent processes that fine tune the homeostatic response. Notably, distinct, interlinked negative feedback signaling has been documented in cell biological systems, suggesting a common motif in cell biological regulation (Kulik, 2019).

    An interesting prediction of this model is that activity-dependent mechanisms of FRH could be constrained by the action of the channel-specific homeostatic system. For example, loss of Shal induces a Shal-specific gene expression program and activity-dependent homeostatic signaling would be constrained to modulate the Shal-specific response. As such, the homeostatic outcome could be unique for mutations in each different ion channel gene. Given this complexity, it quickly becomes possible to understand experimental observations in non-isogenic animal populations where many different combinations of ion channels are observed to achieve similar firing rates in a given cell. The combined influence of dedicated proteostatic and activity-dependent homeostatic signaling could achieve such complexity, but with an underlying signaling architecture that is different from current theories that focus on a single calcium and activity-dependent feedback processor (Kulik, 2019).

    Finally, although the existence is proposed of proteostatic feedback induced by the Shal null mutant and pan-neuronal RNAi, other possibilities certainly exist for activity-independent FRH, inclusive of mechanisms that are sensitive to channel mRNA. For example, the transcriptional compensation that was documented could be considered a more general form of 'genetic compensation'. Yet, the data differ in one important respect, when compared to prior reports of genetic compensation. In most examples of genetic compensation, gene knockouts induce compensatory expression of a closely related gene. For example, it was observed that knockout of β-actin triggers enhanced expression of other actin genes. The compensatory effects that were observed involve re-organization of the expression profiles for many, unrelated ion channel genes. Somehow, these divergent conductances are precisely adjusted to cover for the complete absence of the somato-dendritic A-type potassium conductance. Thus, a more complex form of genetic compensation is favored based upon homeostatic, negative feedback regulation (Kulik, 2019).

    How does Kr-dependent control of IKCa participate in FRH? IKCa is a rapid, transient potassium current. Therefore, it makes intuitive sense that elevated IKCa could simply substitute for the loss of the fast, transient IKA current mediated by Shal. If so, this might be considered an instance of simple genetic compensation. But, if this were the case, then blocking the homeostatic increase in IKCa should lead to enhanced firing rates. This is not what was observe. Instead, average firing rates decrease when Kr is eliminated in the background of Shal-RNAi. Thus, the Kr-dependent potentiation of IKCa seems to function as a form of positive feedback, accelerating firing rate in order to achieve precise FRH, rather than simply substituting for the loss of Shal. Consistent with this possibility, acute pharmacological inhibition of IKCa decreases, rather than increases, average firing rate. However, it should also be emphasized that the role of IKCa channels in any neuron are quite complex, with context-specific effects that can either increase or decrease neuronal firing rates. Indeed, it has been argued that BK channels can serve as dynamic range compressors, dampening the activity of hyperexcitable neurons and enhancing the firing of hypoexcitable neurons. This broader interpretation is also consistent with the observed Kr-dependent increase IKCa during FRH (Kulik, 2019).

    In the stomatogastic nervous system of the crab, single-cell RT-PCR has documented positive correlations between channel mRNA levels, including transcript levels for IKCa and Shal. The molecular mechanisms responsible for the observed correlations remain unknown, but it seems possible that these correlations reflect a developmental program of channel co-regulation. Upon homeostatic challenge, the steady-state positive correlations are supplanted by homeostatic compensation, notably enhanced IKCa in the presence of 4-AP. The pressing challenge is to define molecular mechanisms that cause the observed correlations and compensatory changes in ion channel expression during homeostatic plasticity. The Kr-dependent control of IKCa following loss of Shal is one such mechanism. Clearly, there is additional complexity, as highlighted by the differential response to Shal null and Shal pore blocking mutations and the pumilio-dependent control of sodium channel translation in flies and mice (Kulik, 2019).

    Why do ion channel mutations frequently cause disease? If activity-dependent homeostatic signaling is the primary mechanism of FRH, then any ion channel mutation that alters channel function should be detected by changes in neural activity and firing rates restored. One possibility is that FRH is effective for correcting for an initial perturbation, but the persistent engagement of FRH might become deleterious over extended time. Alternatively, each solution could effectively correct firing rates, but have additional maladaptive consequences related to disease pathology. While this remains to be documented in disease, this study has shown that loss of Shal protein throughout the CNS causes deficits in animal behavior that are not observed in animals harboring a pore-blocking channel mutation. Indeed, if one considers that FRH can include altered expression of a BK channel, the potential for maladaptive consequences is high. Altered BK channel function has been repeatedly linked to neurological disease including idiopathic generalized epilepsy, non-kinesigenic dyskinesia and Alzheimer's disease. Thus, there are potentially deleterious ramifications of altering BK channel expression if a homeostatic signaling process is engaged throughout the complex circuitry of the central nervous system. Although the phenotype of maladaptive compensation that was observe is clear, a block in synaptic homeostasis and impaired animal motility, there is much to be learned about the underlying cause. Ultimately, defining the rules that govern FRH could open new doors toward disease therapies that address these maladaptive effects of compensatory signaling (Kulik, 2019).

    Myosin VI contributes to synaptic transmission and development at the Drosophila neuromuscular junction

    Myosin VI, encoded by jaguar (jar) in Drosophila, is a unique member of the myosin superfamily of actin-based motor proteins. Myosin VI is the only myosin known to move towards the minus or pointed ends of actin filaments. Although Myosin VI has been implicated in numerous cellular processes as both an anchor and a transporter, little is known about the role of Myosin VI in the nervous system. Previous studies recovered jar in a screen for genes that modify neuromuscular junction (NMJ) development and this study reports on the genetic analysis of Myosin VI in synaptic development and function using loss of function jar alleles. The experiments on Drosophila third instar larvae revealed decreased locomotor activity, a decrease in NMJ length, a reduction in synaptic bouton number, and altered synaptic vesicle localization in jar mutants. Furthermore, studies of synaptic transmission revealed alterations in both basal synaptic transmission and short-term plasticity at the jar mutant neuromuscular synapse. Altogether these findings indicate that Myosin VI is important for proper synaptic function and morphology. Myosin VI may be functioning as an anchor to tether vesicles to the bouton periphery and, thereby, participating in the regulation of synaptic vesicle mobilization during synaptic transmission (Kisiel, 2011).

    Although Myosin VI function in the vesicle cycle has been implicated in mammalian cells, this report provides the first evidence that Myosin VI is important for maintaining normal peripheral vesicle localization at the bouton. In Drosophila, there are four types of boutons, which are the sites of neurotransmitter release at the NMJ, and they differ in their morphological and chemical properties. Of interest for this study were the largest synaptic boutons found at type I axon terminals, which are present at all NMJs of mature larvae. Visualization of synaptotagmin staining using confocal imaging revealed a mislocalization of synaptic vesicles in jar mutant boutons. An increasing number of jar mutant boutons, corresponding to the severity of Myosin VI loss of function, were found to exhibit diffuse staining over the entire bouton area as opposed to the doughnut-shaped staining pattern present in control boutons. Bouton centre occupancy has previously been observed at Drosophila NMJs of larvae lacking synapsin, a phosphoprotein that reversibly associates with vesicles, using FM1-43 loading under low frequencies. EM analysis confirmed that in synapsin knockouts there was a spread of vesicles into the bouton centre, accompanied by a reduction in the size of the reserve pool. Thus, synapsin is thought to function in maintaining the peripheral distribution of vesicles in Ib boutons. Likewise, the unexpected diffuse synaptotagmin staining of jar mutant boutons suggests Myosin VI participates in restricting vesicles to the bouton periphery. It is possible that Myosin VI is functioning as a regulator of the actin cytoskeleton at the synapse. Mutant studies have revealed that the presynaptic actin cytoskeleton is required for proper synaptic morphogenesis. Myosin VI has already been shown to function in regulating the actin cytoskeleton during the process of spermatid individualization, by acting either as a structural cross-linker or as an anchor at the front edge of the actin cone, and during nuclear divisions in the syncytial blastoderm. However, live imaging of actin dynamics at the synaptic boutons revealed no major defects in the actin cytoskeleton at jar loss of function mutant nerve terminals (Kisiel, 2011).

    To assess whether the morphological defects in vesicle localization observed at jar mutant synapses impact synaptic transmission, electrophysiological assays with different stimulation paradigms were used to recruit vesicles from different functional pools. The data add to the knowledge of this protein's physiological role at synapses. Myosin VI mutant mouse hippocampal neurons exhibit defects in the internalization of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, responsible for fast glutamatergic transmission, suggesting Myosin VI normally plays a role in AMPAR endocytosis. In addition, basal synaptic transmission is reduced in Myosin VI deficient mouse hippocampal slices compared to wild-type controls. Electrophysiological experiments also indicate that Myosin VI mediates glutamate release induced by brain-derived neurotrophic factor, which is known to modulate synaptic transmission and plasticity in the mammalian central and peripheral nervous system (Kisiel, 2011).

    This study is the first to show that Myosin VI's role in synaptic transmission involves mobilization of vesicles from different functional pools, indicating that Myosin VI is important for synaptic plasticity. At the Drosophila NMJ, three pools of vesicles with differential release properties have been identified using FM1-43 staining loaded by various stimulation protocols. The immediately releasable pool (IRP), representing approximately 1% of all vesicles at the NMJ, consists of vesicles docked and primed at active zones for immediate release and experiences rapid depletion within a few stimuli. The readily releasable pool (RRP), making up 14% to 19% of all vesicles at the NMJ, is mobilized by moderate stimulation of ≤3 Hz and maintains exo/endocytosis at these stimulation frequencies. The reserve pool (RP) represents the vast majority of vesicles, 80% to 90%, and is mobilized upon depletion of the RRP. Recruitment from the RP occurs with high frequency stimulation of ≥10 Hz. Spontaneous release was reduced in the most severe jar loss of function mutants. Evoked response at 1 Hz stimulation was also reduced in the jar maternal null mutant. Although less severe jar mutants exhibited a significant decrease in bouton number, they did not experience an accompanying reduction in evoked potential amplitude at low frequency stimulation, suggesting that other homeostatic mechanisms are important for maintaining synaptic strength. The impaired synaptic response in the jar maternal null mutant may be due to a reduction in the probability of RRP vesicle release or in RRP size. If Myosin VI functions to anchor synaptic vesicles, it may act on the RRP to ensure vesicles are localized in manner that makes them readily available for release. Thus, in jar maternal null mutants the reduction in EJP amplitude may occur because a significant number of vesicles were displaced from areas of higher probability release. Alternately, RRP pool size may be reduced at jar mutant synapses (Kisiel, 2011).

    Different synaptic vesicle pool properties, such as rate of recruitment of the RP in response to high frequency stimuli, may translate to changes in short-term synaptic plasticity. The increase in EJP amplitude observed at 10 Hz stimulation in 1 mM Ca2+ saline may be attributable to enhanced mobilization of the RP for jar322/Df(3R)crb87-5 NMJs. Filamentous actin has been implicated in RP mobilization as cytochalasin D, an inhibitor of actin polymerization, has been shown to reduce RP dynamics. This suggests translocation from the RP to the RRP may be mediated by an actin-based myosin motor protein. If Myosin VI functions as a synaptic vesicle tether to regulate recruitment from the RP pool, RP vesicles would be more readily mobilized and transitioned into the RRP upon high frequency stimulation in jar loss of function mutants. Consistent with the idea that RP vesicles were more rapidly incorporated into the RRP, a greater initial depression is observed at jar322/Df(3R)crb87-5 mutant synapses during high frequency stimulation in 10 mM Ca2+ saline corresponding to the depletion of vesicles at high calcium concentrations. Taken together, the data suggest that Myosin VI mediates synaptic transmission and short-term plasticity by regulating the mobilization of synaptic vesicles from different functional pools. In mammalian cells, Myosin VI has been implicated as a mediator of vesicle endocyctosis and has been shown to transport uncoated vesicles through the actin-rich periphery to the early endosome. The current experiments, however, indicate that endocytosis is not likely affected at jar mutant synapses. Typically, endocytotic mutants are unable to maintain synaptic transmission in response to high frequency stimulation, whereas the Myosin VI loss of function mutants exhibited enhanced EJP amplitude observed at 10 Hz stimulation in 1 mM Ca2+ saline. Additional experiments are required to confirm that Myosin VI is functioning as a vesicle tether. Fluorescence recovery after photobleaching analysis can be used to examine the effect of Myosin VI on synaptic vesicle mobility. If Myosin VI is functioning as a vesicle tether, synaptic vesicle mobility is expected to be increased in jar mutants compared to controls (Kisiel, 2011).

    In summary, the present work shows that Myosin VI is important for proper synaptic morphology and physiology at the Drosophila NMJ. Myosin VI function in peripheral vesicle localization at the bouton may underlie its contribution to basal synaptic transmission and expression of synaptic plasticity. Future work will address the mechanism by which Myosin VI performs its roles at the synapse, whether as a vesicle tether or by some other involvement in vesicle trafficking (Kisiel, 2011).

    Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila

    Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4 GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. This study reports that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network (Dey, 2017).

    The apical compartment of a neuronal cell extends into specific processes forming axons and synapses that are maintained by a distributed endosomal system. For example, vesicle recycling at the synapse, orchestrated by various RabGTPases and associated proteins, renews the readily releasable pool of synaptic vesicles sustaining the neurotransmission. Rab4 is thought to play an important role in balancing the vesicle traffic between the recycling and degradation pathways involved in various cellular processes such as metabolism, cell secretion, and antigen processing. Recruitment of Rab4 on sorting intermediate endosomes allows the transfer of vesicular cargoes from early endosomes to recycling endosomes in axons and at synapses. The activity of Rab4 has also been implicated in the progression of growth cone in Xenopus and the maintenance of dendritic spines in rat hippocampal neurons. Furthermore, endosomal abnormalities found in the cholinergic basal forebrain of patients with Alzheimer's disease correlate with elevated levels of Rab4. All these reports suggest that Rab4-dependent vesicle sorting is essential for the development and maintenance of the nervous system (Dey, 2017).

    Vesicular cargoes, including the RabGTPases, originate at the trans-Golgi network, and microtubule-dependent motors transport them to different destinations within a cell. Specific association with motors ensures differential and dynamic subcellular localizations of various RabGTPases according to tissue-specific activities and metabolic demands. For instance, the anterograde trafficking of early, sorting, and late endosomes is dependent on the Kinesin-3 motors KIF13, KIF16B, and KIF1A/IBβ, respectively. Similarly, in Drosophila, Rab5-positive early endosomes associate with Khc-73, which is the ortholog of mammalian KIF13, showing a conserved machinery of Kinesin-Rab interaction. The same vesicles also associate with Dynein for their retrograde movement. It is further indicated that multiple RabGTPases can be grouped together and transported to a destination within a cell where they can engage in different functions. Although the long-range transport of certain presynaptic RabGTPases has been studied in cultured neurons, it is still unclear how the bidirectional movement of Rab-associated vesicles could localize them in distant compartments such as the synapse (Dey, 2017).

    This study has shown that Rab4-associated vesicles are transported with an anterograde bias, induced by a specific interaction with the tail domain of Kinesin-2α subunit, which results in its moderate enrichment at the synapses. The rate of pre-synaptic inflow of Rab4 is positively correlated with its activation. Interestingly, this study found that a decreased flow of the Rab4-associated vesicles expanded the region occupied by synapses in the neuropil region of the larval ventral ganglion and enhanced larval motility. Altogether, these results indicate that the flow of Rab4-associated vesicles could maintain synaptic homeostasis in a neuronal network (Dey, 2017).

    Heterotrimeric Kinesin-2, implicated in the trafficking of apical endosomes and late endosomal vesicles, was initially thought to bind to its cargoes through the accessory subunit KAP3. However, recent studies have reported that motor subunits could independently interact with soluble proteins through the tail domain. This study shows that the Kinesin-2α tail can also bind to a membrane-associated protein, Rab4. A preliminary investigation in the lab suggests that the interaction in not direct. The RabGTPases are known to recruit a variety of different motors to the membrane, directing intracellular trafficking. Although a previous report indicated that Rab4 could bind to Kinesin-2, this study found that such an interaction leads to the anterograde trafficking of Rab4-associated vesicles in the axon. Rab4 associates with early and recycling endosomes carrying a diverse range of proteins. The FRB-FKBP assay indicated that a particular subset of Rab4-only vesicles associates with Kinesin-2. These vesicles predominantly move toward the synapse in axons. Although Kinesin-2 is also found to bind and transport vesicles containing Acetylcholinesterase (AChE) in the same axon, it was observed that Rab4 does not mark them. Similarly, the post-Golgi vesicles carrying N-cadherin and β-catenin, which are known to associate with Kinesin-2, exclude Rab4. Therefore, the Rab4-associated vesicles transported in the axon are likely to contain a unique set of proteins (Dey, 2017).

    An association between RabGTPases and putative transport proteins was identified using coimmunoprecipitation, pull-down, and yeast two-hybrid assays. While these assays provide information on the possible interactions, their nature in a cellular or physiological scenario is unknown. Long-range transport of the Rab27/CRMP2 complex by Kinesin-1 and the Rab3/DENN MADD complex by KIF1A/1Bβ are substantiated by genetic perturbations and imaging. The FKBP-FRB-inducible interaction system used in this study identified Kinesin-2α and KIF13A/B as the key motors for nascent Rab4-associated vesicles. The latter was also shown to associate with Rab5-marked early endosomes. The results excluded the possibility that the Rab4 effectors involved in this interaction could bind to Rab5 and Rab11. In vivo analysis of Rab4-associated vesicular traffic further confirmed that heterotrimeric Kinesin-2 plays a dominant role in the trafficking of nascent Rab4 vesicles toward the synapse (Dey, 2017).

    Anterograde axonal movement of Rab4 vesicles is mostly mediated by Kinesin-2, which transports nascent Rab4 vesicles, whereas Kinesin-3 transports sorting endosomes. In a recent study, it was shown that combined activity of Kinesin-1 and Kinesin-2 steers AChE-containing vesicles in the axon (Kulkarni, 2016). A simultaneous association between these two motors was suggested to induce longer runs of AChE-containing vesicles. A similar collaboration between Khc-73 and Kinesin-2 could propel longer runs of Rab4 vesicles. Khc-73 has been implicated in neuronal functions. Khc-73 knockdown in lch5 neurons decreased the motility of Rab4-associated vesicles to a limited extent, which suggests that the motor is only engaged in transporting a relatively small fraction of Rab4-associated vesicles in axons. Khc-73 is a processive motor with an in vitro velocity of 1.54 ± 0.46 &mi;m/s, and this study found that Khc-73 RNAi particularly affected the longer runs. Thus, Khc-73 could support the relatively longer runs (3.05 &mi;m) of Rab4 vesicles in axons. It was previously shown that Rab5-associated vesicles bind to KIF13A/B/Khc-73. Hence, it is inferred that sorting endosomes, positive for both Rab4 and Rab5, can engage both Kinesin-2 and KIF13A/Khc-73 through the cognate adaptors of these two Rabs. However, one needs suitably tagged reagents to establish this conjecture through direct observation in vivo (Dey, 2017).

    Local synaptic vesicle recycling mediated by RabGTPases is critical for the availability of fusion-ready synaptic vesicles to sustain neurotransmission. The machinery involved in the translocation of RabGTPases from cell bodies to the synapses is poorly understood. Certain RabGTPases such as Rab3 and Rab27 are known to associate with Kinesin-3 and Kinesin-1, respectively, for their anterograde axonal transport. Rab4 is a protein present on the endosomal sorting intermediates, and it allows for the transfer of cargoes from early endosomes to recycling endosomes. As Rab4 is present in both early and recycling endosomal populations, the logistics of Rab4 transport into the presynaptic terminal has been unclear. The current results show that Rab4-associated vesicles are present throughout the neuron and moderately enriched in the synapse, which is a consequence of a small anterograde bias in their transport (Dey, 2017).

    The activity of Rab4 is critical for the physiology of the neuron-like extension of the growth cone. The overexpressed levels and activity of Rab4 found in conjunction with neuropathy, such as Alzheimer's disease, suggest that overactivation of the endosomal system could influence disease progression. In Drosophila, Rab4 is expressed in both neuronal and non-neuronal tissues throughout development with the exception of optic lobe neurons of the pupal brain. These studies project the idea that the expression and activity of Rab4 are crucial for the maintenance of neuronal physiology. This study has shown that the synaptic Rab4 is maintained through axonal transport by heterotrimeric Kinesin-2 and Kinesin-3. As the synaptic localization of Rab4 is dependent on its active form, other factors like PI3K are also implicated in this trafficking. The results further suggest that the function and localization of Rab4 at axon termini are critical for the maintenance of synaptic balance at ventral ganglion. Overactivation of Rab4 in cholinergic neurons reduced the synapse-bearing region of the ventral ganglion, although levels of the marker, Brp, remained unchanged. This observation suggests that local Rab4 activity at the axon termini suppresses synapse formation. Consistent with this conjecture, this study found that the synapse-bearing zone at the neuropil expands when Rab4DN is overexpressed. Although the current data are insufficient to explain the underlying mechanism, they indicate that alteration of the trafficking logistics driven by heterotrimeric Kinesin-2 and Kinesin-3 family motors might play an essential role in the development of the defect. Further analysis of larval behavior and neuronal stability in the ventral ganglion with aging would be useful in uncovering the mechanism (Dey, 2017).

    Presynaptic biogenesis requires axonal transport of lysosome-related vesicles

    Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) supplying presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is unclear. Live imaging in Drosophila larvae and mouse hippocampal neurons provides evidence that presynaptic biogenesis depends on axonal co-transport of SV and AZ proteins in presynaptic lysosome-related vesicles (PLVs). Loss of the lysosomal kinesin adaptor Arl8 results in the accumulation of SV- and AZ-protein-containing vesicles in neuronal cell bodies and a corresponding depletion of SV and AZ components from presynaptic sites, leading to impaired neurotransmission. Conversely, presynaptic function is facilitated upon overexpression of Arl8. These data reveal an unexpected function for a lysosome-related organelle as an important building block for presynaptic biogenesis (Vukoja, 2018).

    The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization

    The kinesin-3 family member KIF1A has been shown to be important for experience dependent neuroplasticity. In Drosophila, amorphic mutations in the KIF1A homolog unc-104 disrupt the formation of mature boutons. Disease associated KIF1A mutations have been associated with motor and sensory dysfunctions as well as non-syndromic intellectual disability in humans. A hypomorphic mutation in the forkhead-associated domain of Unc-104, unc-104bris, impairs active zone maturation resulting in an increased fraction of post-synaptic glutamate receptor fields that lack the active zone scaffolding protein Bruchpilot. This study shows that the unc-104bris mutation causes defects in synaptic transmission as manifested by reduced amplitude of both evoked and miniature excitatory junctional potentials. Structural defects observed in the postsynaptic compartment of mutant NMJs include reduced glutamate receptor field size, and altered glutamate receptor composition. In addition, there is a marked loss of postsynaptic scaffolding proteins and reduced complexity of the sub-synaptic reticulum, which can be rescued by pre- but not postsynaptic expression of unc-104. These results highlight the importance of kinesin-3 based axonal transport in synaptic transmission and provide novel insights into the role of Unc-104 in synapse maturation (Y. V. Zhang, 2017).

    Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling

    Adrenergic receptors and their ligands are important regulators of synaptic plasticity and metaplasticity, but the exact mechanisms underlying their action are still poorly understood. Octopamine, the invertebrate homolog of mammalian adrenaline or noradrenaline, plays important roles in modulating behavior and synaptic functions. Previous work (Koon, 2011) uncovered an octopaminergic positive-feedback mechanism to regulate structural synaptic plasticity during development and in response to starvation. Under this mechanism, activation of Octβ2R autoreceptors by octopamine at octopaminergic neurons initiated a cAMP-dependent cascade that stimulated the development of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). However, the regulatory mechanisms that served to brake such positive feedback were not known. This study reports the presence of an alternative octopamine autoreceptor, Octβ1R, with antagonistic functions on synaptic growth. Mutations in octβ1r result in the overgrowth of both glutamatergic and octopaminergic NMJs, suggesting that Octβ1R is a negative regulator of synaptic expansion. As does Octβ2R, Octβ1R functions in a cell-autonomous manner at presynaptic motorneurons. However, unlike Octβ2R, which activates a cAMP pathway, Octβ1R likely inhibits cAMP production through inhibitory Goα. Despite its inhibitory role, Octβ1R is required for acute changes in synaptic structure in response to octopamine and for starvation-induced increase in locomotor speed. These results demonstrate the dual action of octopamine on synaptic growth and behavioral plasticity, and highlight the important role of inhibitory influences for normal responses to physiological stimuli (Koon, 2012).

    Adrenaline/noradrenaline and their receptors have emerged as important modulators of synaptic plasticity, metaplasticity, and behavior in the mammalian brain. However, the mechanisms underlying this regulation of synaptic structure are not known (Koon, 2012 and references therein).

    In insects, adrenergic signaling is accomplished through octopamine and octopamine receptors and is a powerful modulator of behaviors such as appetitive behavior and aggression. It also regulates synaptic function and synaptic structure (Koon, 2011; Koon, 2012).

    A previous study has demonstrated that at the Drosophila larval neuromuscular junction (NMJ) octopamine regulates the expansion of both modulatory and excitatory nerve terminals (Koon, 2011). Larval NMJs are innervated by glutamatergic, octopaminergic, and peptidergic motorneurons. Of these, glutamatergic nerve terminals provide classical excitatory transmission, while octopaminergic nerve endings support global modulation of excitability and synaptic growth. Larval NMJs are continuously expanding to compensate for muscle cell growth and respond to acute changes in activity by extending new synaptic boutons. By binding to the octopamine autoreceptor Octβ2R, octopamine activates a cAMP second messenger pathway that leads to CREB activation and transcription, which in turn promotes the extension of new octopaminergic nerve endings (Koon, 2011). This positive-feedback mechanism was required for an increase in locomotor activity in response to starvation. In addition, this mechanism positively regulated the growth of glutamatergic nerve endings through Octβ2R receptors present in glutamatergic neurons. An important question regards the mechanisms that serve to brake such positive feedback (Koon, 2012).

    This study demonstrates the presence of a second octopamine receptor, Octβ1R, which serves as such a brake. Octβ1R is also an autoreceptor in octopaminergic neurons that serves to inhibit synaptic growth. This inhibitory influence is excerpted through the activation of the inhibitory G-protein subunit, Goα, and thus by limiting cAMP production. Like Octβ2R receptors, Octβ1R receptors are also present at excitatory glutamatergic endings. Thus, octopamine release induces a dual excitatory (through Octβ2R) and inhibitory (through Octβ1R) function on the growth of both octopaminergic and glutamatergic endings. The presence of both the excitatory and inhibitory receptors is required for normal structural plasticity at octopaminergic terminals and for normal responses to starvation, as obliterating Octβ1R (this study) or Octβ2R (Koon, 2011) prevents the acute growth of octopaminergic ending in response to octopamine and the increase in locomotor speed in response to starvation. Thus, this study highlights the requirement of both excitatory and inhibitory influences for normal synaptic and behavioral plasticity (Koon, 2012).

    The previous study demonstrated that octopamine regulates synaptic and behavioral plasticity through an autoregulatory positive-feedback mechanism involving Octβ2R, which promotes both type I and type II outgrowth (Koon, 2011). This study has now identified an octopamine receptor, Octβ1R, which antagonizes the function of Octβ2R. It is proposed that Octβ1R may serve as a brake for the positive feedback induced by Octβ2R. Octβ1R receptors inhibit the cAMP pathway via the inhibitory G-protein Goα, as loss of Octβ1R or Goα function results in synaptic overgrowth of type I and type II endings in an octopamine cell-autonomous manner, and as octβ1r and goα interact genetically. Notably, defective Octβ1R signaling appears to saturate cAMP levels, occluding the function of Octβ2R. Thus, the loss of Octβ1R function results in insensitivity to octopamine stimulation. In turn, this abolishes starvation-induced behavioral changes that require Octβ2R signaling. While this study centered primarily on Octβ1R function at octopaminergic NMJ terminals, it is important to emphasize that octopamine neurons are also present in the larval brain. Thus, with current tools it is not possible to discern whether the defects are exclusively due to the function of octopaminergic motorneurons, or whether other central octopaminergic neurons contribute to these effects. While the phenotypes on NMJ development are most parsimoniously explained by a local function at NMJ terminals, it is likely that the behavioral effects are more complex, also involving important contribution from brain octopaminergic neurons (Koon, 2012).

    At the Drosophila larval NMJ, three type II motorneurons innervate most of the body wall muscles in each segment (Koon, 2011). This layout suggests that octopamine is likely to globally regulate plasticity, by tuning the excitability levels of multiple excitatory synapses on the body wall muscles. Together, the observations in the previous study (Koon, 2011) and this investigation identify the presence of excitatory and inhibitory octopamine receptors that are coexpressed in the same cells. This suggests that global regulation of synapses and behavior by octopamine can be tipped toward excitation or inhibition depending on receptor expression levels, affinity of the receptors for octopamine, and availability of these receptors for binding octopamine on the target cells. This dual mode of controlling excitability likely provides enhanced flexibility, allowing a broader level of control over synaptic functions (Koon, 2012).

    An important question is how can Octβ1R and Octβ2R regulate development of innervation and behavior given that they are activated by the same ligand, are localized in the same cells, and their functions are antagonistic. Several alternatives can be proposed. Octβ1R and Octβ2R might have different affinities for octopamine binding. Thus, different levels of octopamine release could differentially activate the receptors. For instance, if Octβ1R receptors have higher affinity for octopamine, and octopamine is normally released at low levels, a stable degree of innervation could be maintained by continuous inhibition of synaptic growth-promoting signals. High levels of octopamine release, as would occur during starvation, would then activate the lower affinity Octβ2R, eliciting synaptic growth. Precedence for this type of regulation has been obtained in honeybees and olive fruit flies, where low concentrations of octopamine are inhibitory while high concentrations are excitatory to cardiac contraction (Koon, 2012).

    An alternative possibility is based on the well known internalization of GPCRs upon ligand binding. It is possible that such a mechanism would maintain an appropriate ratio of Octβ1R and Octβ2R at the cell surface, actively keeping or removing octopamine receptor-mediated excitation or inhibition, depending on physiological states. A third alternative is that receptors could be posttranslationally modified upon ligand binding, which might also affect their downstream functions. For example, dimerization of β2-adrenergic receptors can inhibit its adenylate cyclase-activating activity and phosphorylation of β1-adrenergic receptor by PKA reduces its affinity for Gsα and increases its affinity for Giα/oα. Last, Octβ1R and Octβ2R receptors could be spatially separated in neurons, with one receptor being closer and the other distant to sites of octopamine release. In this scenario, the receptors would likely be exposed to different octopamine concentration (Koon, 2012).

    Simultaneous expression of excitatory and inhibitory GPCRs in the same neuron has been reported previously. For instance, mammalian dopamine receptors can couple to both stimulatory and inhibitory G-proteins, with the D1 receptor-like family being coupled to Gsα and the D2-like family being coupled to Giα/oα (Koon, 2012).

    Previous studies have investigated the effect of octopamine on synaptic transmission at the Drosophila first-instar and third-instar larval NMJ. While the studies at the third-instar larval NMJ demonstrated an excitatory effect of octopamine in neurotransmission, the study on the first-instar larval stage substantiated an inhibitory effect. A recent study now provides a potential explanation for such discrepancy between the responses to octopamine at the two larval stages). In particular, it was found that that Octβ1R is expressed at high levels in first instar and at low levels in third instar. In contrast, Octβ2R is expressed at low levels in first instar and at high levels in third instar. The current studies demonstrating an inhibitory role for Octβ1R (this study) and an excitatory role for Octβ2R (Koon, 2011) are in agreement with the idea that octopamine may play an inhibitory role during first instar, but an excitatory role during third instar (Koon, 2012).

    Octopamine receptors have been shown to elicit intracellular Ca2+ and/or cAMP increase. OAMB, the only α-adrenergic-like receptor in Drosophila, has been implicated to function via Ca2+ signaling in the Drosophila oviduct. However, OAMB is expressed in the oviduct epithelium, and not in the oviduct muscle cells. Given that octopamine induces relaxation of oviduct muscles, the presence of an alternative, inhibitory octopamine receptor in oviduct muscles was proposed. The identification of Octβ1R receptor as an inhibitory receptor raises the possibility that this is the inhibitory receptor in the oviduct (Koon, 2012 and references therein).

    In apparent contradiction to these findings, a previous study has shown that Octβ1R (also known as OA2) is capable of increasing cAMP. In that study, HEK293 cells transfected with Octβ1R were exposed to different octopamine concentrations, which resulted in an increase in cAMP levels. A potential explanation for the disparate results is that GPCR overexpression might alter its coupling to downstream pathways. For instance, mammalian β2-adrenergic receptors are known to couple to both Gsα and Giα/oα proteins. However, overexpression of β2-adrenergic receptors constitutively couples the receptor to Gsα and not to Giα or Goα. Furthermore, analysis of its binding specificity through immunoprecipitation shows that, when the receptor was overexpressed in transgenic mice, it coprecipitated with Gsα but not with Giα/oα in the absence of agonist. An additional explanation is that human embryonic kidney HEK293 cells are unlikely to express the same transduction pathways as endogenous Drosophila cells. Indeed, a recent study showed that HEK293 cells express virtually no Goα, which could also explain the lack of inhibitory response of overexpressed Octβ1R in this cell line (Koon, 2012).

    Goα is expressed in the nervous system of Drosophila and shows a marked increase in levels during the development of axonal tracts. Goα levels are altered in memory mutants including dunce and rutabaga, and Goα is necessary for associative learning. PTX overexpression in mushroom bodies of adult Drosophila severely disrupts memory, suggesting a role of Goα in synaptic plasticity. However, homozygous goα mutants are lethal due to defective development of the heart preventing the use of null mutants in studies of the NMJ or the adult brain. Moreover, overexpression of inhibitory G-proteins is known to sequester available Gβ and Gγ subunits, resulting in unspecific downregulation of other G-protein signaling. Thus, there are significant problems associated with the use of an overexpression approach to study Goα function. Fortunately, the availability of PTX and multiple Goα-RNAi strains allowed downregulation of Goα function in a cell-specific manner to examine synaptic development at the NMJ, which was found to phenocopy defects observed at the NMJ of octβ1r mutants. The presence of genetic interactions between the octβ1r and goα genes further support the notion that the two proteins act in the same signaling pathway to inhibit synaptic growth. These results provide strong evidence for the involvement of Goα in synaptic plasticity at the NMJ (Koon, 2012).

    In summary, these studies reveal that octopamine acts both as an inhibitory and excitatory transmitter to regulate synaptic growth and behavior. Thus, the inhibitory function of octopamine in global synaptic growth is as crucial as its excitatory function in maintaining plasticity in a dynamic range (Koon, 2012).

    A comparison between the axon terminals of octopaminergic efferent dorsal or ventral unpaired median neurons in either desert locusts (Schistocerca) or fruit flies (Drosophila) across skeletal muscles reveals many similarities. In both species the octopaminergic axon forms beaded fibers where the boutons or varicosities form type II terminals in contrast to the neuromuscular junction (NMJ) or type I terminals. These type II terminals are immunopositive for both tyramine and octopamine and, in contrast to the type I terminals, which possess clear synaptic vesicles, only contain dense core vesicles. These dense core vesicles contain octopamine as shown by immunogold methods. With respect to the cytomatrix and active zone peptides the type II terminals exhibit active zone-like accumulations of the scaffold protein Bruchpilot (BRP) only sparsely in contrast to the many accumulations of BRP identifying active zones of NMJ type I terminals. In the fruit fly larva marked dynamic changes of octopaminergic fibers have been reported after short starvation which not only affects the formation of new branches ('synaptopods') but also affects the type I terminals or NMJs via octopamine-signaling. Starvation experiments of Drosophila-larvae revealed a time-dependency of the formation of additional branches. Whereas after 2 h of starvation a decrease was found in 'synaptopods', the increase is significant after 6 h of starvation. In addition, evidence is provided that the release of octopamine from dendritic and/or axonal type II terminals uses a similar synaptic machinery to glutamate release from type I terminals of excitatory motor neurons. Indeed, blocking this canonical synaptic release machinery via RNAi induced downregulation of BRP in neurons with type II terminals leads to flight performance deficits similar to those observed for octopamine mutants or flies lacking this class of neurons (Stocker, 2018).

    A Presynaptic ENaC Channel Drives Homeostatic Plasticity

    An electrophysiology-based forward genetic screen has identified two genes, pickpocket11 (ppk11) and pickpocket16 (ppk16), as being necessary for the homeostatic modulation of presynaptic neurotransmitter release at the Drosophila neuromuscular junction (NMJ). Pickpocket genes encode Degenerin/Epithelial Sodium channel subunits (DEG/ENaC). This study demonstrates that ppk11 and ppk16 are necessary in presynaptic motoneurons for both the acute induction and long-term maintenance of synaptic homeostasis. ppk11 and ppk16 are cotranscribed as a single mRNA that is upregulated during homeostatic plasticity. Acute pharmacological inhibition of a PPK11- and PPK16-containing channel abolishes the expression of short- and long-term homeostatic plasticity without altering baseline presynaptic neurotransmitter release, indicating remarkable specificity for homeostatic plasticity rather than NMJ development. Finally, presynaptic calcium imaging experiments support a model in which a PPK11- and PPK16-containing DEG/ENaC channel modulates presynaptic membrane voltage and, thereby, controls calcium channel activity to homeostatically regulate neurotransmitter release (Younger, 2013).

    Homeostatic signaling systems are believed to interface with the mechanisms of learning-related plasticity to achieve stable, yet flexible, neural function and animal behavior. Experimental evidence from organisms as diverse as Drosophila, mouse, and humans demonstrates that homeostatic signaling systems stabilize neural function through the modulation of synaptic transmission, ion channel abundance, and neurotransmitter receptor trafficking. In each experiment, the cells respond to an experimental perturbation by modulating ion channel abundance or synaptic transmission to counteract the perturbation and re-establish baseline function. Altered homeostatic signaling is hypothesized to contribute to the cause or progression of neurological disease. For example, impaired or maladaptive homeostatic signaling may participate in the progression of autism-spectrum disorders, posttraumatic epilepsy, and epilepsy (Younger, 2013).

    The homeostatic modulation of presynaptic neurotransmitter release has been observed at mammalian central synapses and at neuromuscular synapses in species ranging from Drosophila to mouse and human. The Drosophila neuromuscular junction (NMJ) is a prominent model system for the study of this form of homeostatic plasticity. At the Drosophila NMJ, decreased postsynaptic neurotransmitter receptor sensitivity is precisely counteracted by a homeostatic potentiation of neurotransmitter release, thereby maintaining appropriate muscle excitation. The homeostatic enhancement of presynaptic release is due to increased vesicle release without a change in active zone number (Younger, 2013 and references therein).

    This process is referred to as 'synaptic homeostasis,' recognizing that it reflects a subset of homeostatic regulatory mechanisms that have been shown to stabilize neural function through modulation of ion channel gene expression and neurotransmitter receptor abundance (quantal scaling) (Younger, 2013 and references therein).

    An electrophysiology-based, forward genetic screen has been pioneered to identify the mechanisms of synaptic homeostasis. To date, a role has been ascribed for several genes in the mechanism of synaptic homeostasis including the Eph receptor, the schizophrenia-associated gene dysbindin, the presynaptic CaV2.1 calcium channel, presynaptic Rab3 GTPase-activating protein (Rab3-GAP), and Rab3-interacting molecule (RIM). An emerging model suggests that, in response to inhibition of postsynaptic glutamate receptor function, a retrograde signal acts upon the presynaptic nerve terminal to enhance the number of synaptic vesicles released per action potential to precisely offset the severity of glutamate receptor inhibition. Two components of the presynaptic release mechanism are necessary for the execution of synaptic homeostasis, increased calcium influx through presynaptic CaV2.1 calcium channels and a RIM-dependent increase in the readily releasable pool of synaptic vesicles. Many questions remain unanswered. In particular, how is a change in presynaptic calcium influx induced and sustained during synaptic homeostasis (Younger, 2013)?

    This study reports the identification of two genes, pickpocket16 and pickpocket11, that, when mutated, block homeostatic plasticity. Drosophila pickpocket genes encode Degenerin/Epithelial Sodium channel (DEG/ENaC) subunits. Channels in this superfamily are voltage insensitive and are assembled as either homomeric or heteromeric trimers. Each channel subunit has two transmembrane domains with short cytoplasmic N and C termini and a large extracellular loop implicated in responding to diverse extracellular stimuli (Younger, 2013).

    Little is known regarding the function of pH-insensitive DEG/ ENaC channels in the nervous system. DEG/ENaC channels have been implicated as part of the mechanotransduction machinery and in taste perception in both invertebrate and vertebrate systems. In Drosophila, PPK11 has been shown to function as an ENaC channel subunit that is required for the perception of salt taste and fluid clearance in the tracheal system, a function that may be considered analogous to ENaC channel activity in the mammalian lung (Younger, 2013 and references therein).

    This study demonstrates that ppk11 and ppk16 are coregulated during homeostatic synaptic plasticity and that homeostatic plasticity is blocked when gene is genetically deleted, when gene expression is disrupted in motoneurons, or when pickpocket channel function is pharmacologically inhibited. Advantage was taken of the fact that presynaptic homeostasis can be blocked pharmacologically to demonstrate that the persistent induction of homeostatic plasticity does not interfere with synapse growth and development. Homeostatic plasticity can be acutely and rapidly erased, leaving behind otherwise normal synaptic transmission. Finally, pharmacological inhibition of this pickpocket channel was demonstrated to abolish the homeostatic modulation of presynaptic calcium influx that was previously shown to be necessary for the homeostatic increase in neurotransmitter release (Younger, 2013).

    A model for DEG/ENaC channel function can be based on the well-established regulation of ENaC channel trafficking in the kidney during the homeostatic control of salt balance. Enhanced sodium reabsorption in the principle cells of the cortical collecting duct of the kidney is achieved by increased ENaC channel transcription and trafficking to the apical cell surface, which enhances sodium influx. Sodium is then pumped out of the basolateral. By analogy, a model is proposed for synaptic homeostasis in which the trafficking of DEG/ENaC channels to the neuronal membrane, at or near the NMJ, modulates presynaptic membrane potential to potentiate presynaptic calcium channel activity and thereby achieve precise homeostatic modulation of neurotransmitter release (Younger, 2013).

    This study provides evidence that a presynaptic DEG/ENaC channel composed of PPK11 and PPK16 is required for the rapid induction, expression, and continued maintenance of homeostatic synaptic plasticity at the Drosophila NMJ. Remarkably, ppk11 and ppk16 genes are not only required for homeostatic plasticity but are among the first homeostatic plasticity genes shown to be differentially regulated during homeostatic plasticity. Specifically, it was shown that expression of both ppk11 and ppk16 is increased 4-fold in the GluRIIA mutant background. It was also demonstrated that ppk11 and ppk16 are transcribed together in a single transcript and behave genetically as an operon-like, single genetic unit. This molecular organization suggests a model in which ppk11 and ppk16 are cotranscribed to generate DEG/ ENaC channels with an equal stoichiometric ratio of PPK11 and PPK16 subunits. This is consistent with previous models for gene regulation in Drosophila. However, the possibility cannot be ruled out that two independent DEG/ ENaC channels are upregulated, one containing PPK11 and one containing PPK16. The upregulation of ppk11 and ppk16 together with the necessity of DEG/ENaC channel function during the time when synaptic homeostasis is assayed, indicates that these genes are probably part of the homeostat and not merely necessary for the expression of synaptic homeostasis (Younger, 2013).

    DEG/ENaC channels are voltage-insensitive cation channels that are primarily permeable to sodium and can carry a sodium leak current. A model for DEG/ENaC channel function during synaptic homeostasis can be based on the well-established regulation of ENaC channel trafficking in the kidney during the homeostatic control of salt balance. Enhanced sodium reabsorption in the principle cells of the cortical collecting duct of the kidney is triggered by aldosterone binding to the mineralocorticoid receptor. This increases ENaC channel transcription and trafficking to the apical cell surface, which enhances sodium influx. Sodium is then pumped out of the basolateral side of the cell, accomplishing sodium reabsorption (Younger, 2013 and references therein).

    It is speculated that a retrograde, homeostatic signal from muscle triggers increased trafficking of a PPK11/16-containing DEG/ENaC channel to the neuronal plasma membrane, at or near the NMJ. Since the rapid induction of synaptic homeostasis is protein synthesis independent, the existence is hypothesized of a resting pool of PPK11/16 channels that are inserted in the membrane in response to postsynaptic glutamate receptor inhibition. If postsynaptic glutamate receptor inhibition is sustained, as in the GluRIIA mutant, then increased transcription of ppk11/16 supports a persistent requirement for this channel at the developing NMJ. Once on the plasma membrane, the PPK11/16 channel would induce a sodium leak and cause a moderate depolarization of the nerve terminal. This subthreshold depolarization would lead, indirectly, to an increase in action potential-induced presynaptic calcium influx through the CaV2.1 calcium channel and subsequent neurotransmitter release (Younger, 2013).

    There are two major possibilities for how ENaC-dependent depolarization of the nerve terminal could potentiate calcium influx and evoked neurotransmitter release. One possibility, based on work in the ferret prefrontal cortex and Aplysia central synapses, is that presynaptic membrane depolarization causes action potential broadening through potassium channel inactivation, thereby enhancing both calcium influx and release. A second possibility is that subthreshold depolarization of the nerve terminal causes an increase in resting calcium that leads to calcium-dependent calcium channel facilitation. Consistent with this model, it has been shown at several mammalian synapses that subthreshold depolarization of the presynaptic nerve terminal increases resting calcium and neurotransmitter release through low-voltage modulation of presynaptic P/Q-type calcium channels. However, at these mammalian synapses, the change in resting calcium does not lead to an increase in action potential-evoked calcium influx, highlighting a difference between the mechanisms of homeostatic potentiation and the type of presynaptic modulation observed at these other synapses. The mechanism by which elevated basal calcium potentiates release at these mammalian synapses remains under debate. It should be noted that small, subthreshold depolarization of the presynaptic resting potential, as small as 5 mV, are sufficient to cause a 2-fold increase in release at both neuromuscular and mammalian central synapses. This is within a reasonable range for modulation of presynaptic membrane potential by pickpocket channel insertion. Unfortunately, it is not technically feasible to record directly from the presynaptic terminal at the Drosophila NMJ. Finally, it is noted that it remains formally possible that a PPK11/16-containing DEG/ENaC channel passes calcium, based upon the ability of mammalian ASIC channels to flux calcium (Younger, 2013).

    This model might provide insight regarding how accurate tuning of presynaptic neurotransmitter release can be achieved. There is a supralinear relationship between calcium influx and release. Therefore, if changing calcium channel number is the mechanism by which synaptic homeostasis is achieved, then there must be very tight and tunable control of calcium channel number within each presynaptic active zone. By contrast, if homeostatic plasticity is achieved by ENaC-dependent modulation of membrane voltage, then variable insertion of ENaC channels could uniformly modulate calcium channel activity, simultaneously across all of the active zones of the presynaptic nerve terminal. Furthermore, if the ENaC channel sodium leak is small, and if presynaptic calcium channels are moderately influenced by small changes in resting membrane potential, then relatively coarse modulation of ENaC channel trafficking could be used to achieve precise, homeostatic control of calcium influx and neurotransmitter release. Again, these are testable hypotheses that will be addressed in the future (Younger, 2013).

    Composition and control of a Deg/ENaC channel during presynaptic homeostatic plasticity

    The homeostatic control of presynaptic neurotransmitter release stabilizes information transfer at synaptic connections in the nervous system of organisms ranging from insect to human. Presynaptic homeostatic signaling centers upon the regulated membrane insertion of an amiloride-sensitive degenerin/epithelial sodium (Deg/ENaC) channel. Elucidating the subunit composition of this channel is an essential step toward defining the underlying mechanisms of presynaptic homeostatic plasticity (PHP). This study demonstrates that the ppk1 gene (pickpocket) encodes an essential subunit of this Deg/ENaC channel, functioning in motoneurons for the rapid induction and maintenance of PHP. Genetic and biochemical evidence is provided that PPK1 functions together with PPK11 and PPK16 as a presynaptic, hetero-trimeric Deg/ENaC channel. Finally, tight control of Deg/ENaC channel expression and activity is highlighted, showing increased PPK1 protein expression during PHP and evidence for signaling mechanisms that fine tune the level of Deg/ENaC activity during PHP (Orr, 2017a).

    Homeostatic signaling systems stabilize neural function through the modulation of presynaptic transmitter release, ion channel abundance and neurotransmitter receptor trafficking. The homeostatic modulation of neurotransmitter release has been observed at mammalian central synapses and at neuromuscular synapses in species ranging from Drosophila to mouse and human. In these systems, decreased neurotransmitter receptor sensitivity is precisely counteracted by a homeostatic potentiation of neurotransmitter release, thereby maintaining appropriate muscle excitation, a process termed presynaptic homeostatic potentiation (PHP) (Orr, 2017a).

    Core molecular components of the signaling systems that achieve PHP have begun to emerge through the results of large-scale forward genetic screens at the Drosophila NMJ and additional hypothesis-driven studies. A core finding has been the demonstration that an amiloride-sensitive Deg/ENaC channel functions presynaptically to drive the induction and sustained expression of PHP (Younger, 2013). Presynaptic Deg/ENaC channel activity is not detected at baseline, but is rapidly induced upon postsynaptic neurotransmitter receptor inhibition (Younger, 2013). A current model suggests that ENaC channel insertion causes a sodium leak that depolarizes the presynaptic membrane and subsequently potentiates calcium influx through active-zone localized CaV2.1 calcium channels. This model is supported by a combination of ENaC channel pharmacology and presynaptic calcium imaging (Orr, 2017a).

    Deg/ENaC channels encompass a broad family of non-voltage activated sodium and, in some instances, calcium permeable channels that are believed to be heterotrimeric subunit assemblies. This family includes sodium leak channels, channels gated by low pH, chemoreceptors, and mechanoreceptors. Beyond this, a growing list of ligands has been shown to activate these channels including small neuropeptides and opioid peptides. Increasing evidence suggests that channel subunit composition determines the biophysical properties of the channel, as well as the intracellular trafficking and channel localization. Thus, defining the subunit composition of the ENaC channel that drives PHP is an essential step toward defining the feedback control mechanisms that govern the expression of PHP (Orr, 2017a).

    In Drosophila, there is the potential for tremendous Deg/ENaC channel heterogeneity. The ppk gene family encodes a diverse family of Deg/ENaC channel subunits and with 31 independent ppk genes encoded in the genome, the combinatorial space for unique ion channel assemblies is tremendous. To date, however, the full subunit composition of Drosophila Deg/ENaC channels with clearly defined biological activities has yet to be achieved and, therefore, very little is known about diversity and biological relevance of this large gene family. By screening a collection of ppk-RNAi transgenes, this study has defined a third essential subunit of the Deg/ENaC channel that is required for presynaptic homeostatic plasticity (PHP) at the Drosophila NMJ. This subunit is encoded by the ppk1 gene. This finding is a surprise, given that PPK1 participates in a very different type of channel in peripheral sensory neurons. In the DA sensory neurons, PPK1 functions with PPK26 as part of a highly-expressed mechanotransduction channel (Gorczyca, 2014; Guo, 2014; Mauthner, 2014). The third subunit of the DA-neuron PPK1-containing mechanotransduction channel has yet to be identified, although it remains possible that the DA-neuron channel is not heterotrimeric. By characterizing a novel PPK1 containing ENaC channel in the context of PHP, this study provides direct evidence that different PPK subunit assemblies can generate channels with very different properties and unique physiological roles in Drosophila neurons (Orr, 2017a).

    Deg/ENaC and ASIC ion channels are expressed throughout the mammalian central and peripheral nervous systems, being implicated in learning related plasticity and the response to physiological stress. But, given that ENaC channels have potent activities to control cellular physiology in tissues as diverse as the kidney, lung and colonic epithelium, it seems likely that these studies are only scratching the surface with respect to ENaC and ASIC channel function in the nervous system. Furthermore, even in those systems where ENaC channels are a focus of significant research, there remains much to learn about the regulatory mechanisms that control channel expression, trafficking and surface retention (Orr, 2017a).

    The ENaC channel that controls PHP appears to be subject to exquisite post-translational control. First, the channel is either prevented from reaching the plasma membrane or is rendered inactive under baseline conditions. Neither pharmacological inhibition of the channel nor mutations of any of the three subunits influences baseline presynaptic release. Second, ENaC channel activity is induced in seconds to minutes at the isolated NMJ, implying that the channel is either rapidly transferred to the plasma membrane or is activated at that site (Younger, 2013). Third, PHP is a rheostat-like process, adjusting release to the precise level of GluR inhibition. The number of active ENaC channels on the plasma membrane must be precisely controlled to achieve this effect. Fourth, PHP can be sustained for weeks in Drosophila and decades in human. This implies, at least in Drosophila, that the number of active ENaC channels on the plasma membrane is precisely maintained. It will be of great interest to define the regulatory mechanisms that achieve such speed and precision (Orr, 2017a).

    By analogy with other systems such as ENaC channel trafficking in the collecting duct of the kidney, and the control of Glut4 surface expression, three types of post-translational control could be employed to regulate ENaC channel levels at the NMJ during PHP. First, there appears to be potent mechanisms to sequester intracellular ENaC channels, effectively preventing them from reaching the plasma membrane. The efficiency of this system is emphasized by the fact that over-expression of a hyper-active ENaC channel in Drosophila motoneurons is without effect. Second, there must be efficient forward trafficking of ENaC channels to the plasma membrane to enable the rapid induction of PHP. Third, regulated recycling of the ENaC channels would be necessary to precisely maintain levels of the channel, given that channel residency is estimated to be as short as 20-30 minutes in other systems. If these regulatory systems exist, the induction of PHP would require dis-inhibition of internal sequestration followed by the engagement of forward trafficking mechanisms. Alternatively, there could be mechanisms that restrict forward trafficking of ENaC channels to the membrane, and these mechanisms would have to be disabled. The trigger for the induction of PHP might immediately act upon these processes. The feedback signaling system that tunes PHP to precise levels would presumably act upon the ENaC channel recycling mechanism, controlling the number of active ENaC channels on the synaptic plasma membrane. Together, these represent testable models for PHP induction and expression BThe discovery that PPK1 is an essential component of a presynaptic ENaC channel expressed in motoneurons is surprising. Prior to this, PPK1 was thought to function primarily in the dendrites of peripheral multi-dendritic sensory neurons. ppk1 is highly expressed in peripheral sensory neurons and the GFP ppk1-promoter fusion line revealed high expression in these cells. It is now apparent that ppk1 is also expressed at lower levels in muscle, trachea and motoneurons based on experiments with the GFP ppk1-promoter fusion line and the results of the genetic rescue and ppk1-RNAi experiments in motoneurons (Orr, 2017a).

    In peripheral sensory neurons, PPK1 has been shown to function as part of a mechanosensitive ion channel that is believed to be orthologous to C. elegans DEG/ENaC mechanosensors (Gorczyca, 2014; Guo, 2014). However, PPK1-containing channels that are expressed in Drosophila peripheral sensory neurons can also be gated by low pH, suggesting a parallel with acid-sensitive ion channels (ASIC) that are expressed in mammalian peripheral sensory neurons and implicated in neuropathic pain. There is, as yet, no evidence that PHP at the NMJ is induced by either mechanical or pH-dependent stimuli. PHP can be maximally induced in a relaxed NMJ preparation in the absence of action-potential induced muscle contraction. Similarly, there is no evidence for low pH participating in the induction of PHP. Acid-sensitive currents are generally transient events with rapid inactivation, including those identified by ppk1 in class IV md sensory neurons. This is in contrast to the requirement for a robust, sustained ENaC-mediated effect during the sustained expression of PHP, which can persist for weeks in adult Drosophila. Thus, the demonstration that PPK1 incorporates into DEG/ENaC channels with diverse physiological activities highlights the potential for tremendous DEG/ENaC channel diversity in Drosophila (Orr, 2017a).

    There are 31 PPK genes expressed in Drosophila that have been categorized into five sub-classes. While it has been speculated that PPK genes co-assemble within subclasses, this does not seem to be the case given that PPK1 (subclass 5) appears to function with PPK11/16 (subclass 4) in motoneurons. There are other examples that suggest heterologous assembly including evidence that PPK11 (subclass 4) functions with PPK19 (subclass 3) and that PPK23 functions with PPK29 (subclass 1) . As the subunit composition of ENaC channels in Drosophila is gradually resolved, the underlying rules may be discoverednfor channel diversity and function base of PPK subunit composition. Interestingly, there are hints that mammalian ASIC and non-acid sensing ENaC channel subunits can co-assemble, thereby providing the potential for significant channel diversity in mammals as well (Orr, 2017a).

    Structural and molecular properties of insect type II motor axon terminals

    A comparison between the axon terminals of octopaminergic efferent dorsal or ventral unpaired median neurons in either desert locusts (Schistocerca) or fruit flies (Drosophila) across skeletal muscles reveals many similarities. In both species the octopaminergic axon forms beaded fibers where the boutons or varicosities form type II terminals in contrast to the neuromuscular junction (NMJ) or type I terminals. These type II terminals are immunopositive for both tyramine and octopamine and, in contrast to the type I terminals, which possess clear synaptic vesicles, only contain dense core vesicles. These dense core vesicles contain octopamine as shown by immunogold methods. With respect to the cytomatrix and active zone peptides the type II terminals exhibit active zone-like accumulations of the scaffold protein Bruchpilot (BRP) only sparsely in contrast to the many accumulations of BRP identifying active zones of NMJ type I terminals. In the fruit fly larva marked dynamic changes of octopaminergic fibers have been reported after short starvation which not only affects the formation of new branches ('synaptopods') but also affects the type I terminals or NMJs via octopamine-signaling. Starvation experiments of Drosophila-larvae revealed a time-dependency of the formation of additional branches. Whereas after 2 h of starvation a decrease was found in 'synaptopods', the increase is significant after 6 h of starvation. In addition, evidence is provided that the release of octopamine from dendritic and/or axonal type II terminals uses a similar synaptic machinery to glutamate release from type I terminals of excitatory motor neurons. Indeed, blocking this canonical synaptic release machinery via RNAi induced downregulation of BRP in neurons with type II terminals leads to flight performance deficits similar to those observed for octopamine mutants or flies lacking this class of neurons (Stocker, 2018).

    Transsynaptic control of presynaptic Ca(2)(+) influx achieves homeostatic potentiation of neurotransmitter release

    Given the complexity of the nervous system and its capacity for change, it is remarkable that robust, reproducible neural function and animal behavior can be achieved. It is now apparent that homeostatic signaling systems have evolved to stabilize neural function. At the neuromuscular junction (NMJ) of organisms ranging from Drosophila to human, inhibition of postsynaptic neurotransmitter receptor function causes a homeostatic increase in presynaptic release that precisely restores postsynaptic excitation. This study addresses what occurs within the presynaptic terminal to achieve homeostatic potentiation of release at the Drosophila NMJ. By imaging presynaptic Ca(2+) transients evoked by single action potentials, a retrograde, transsynaptic modulation of presynaptic Ca(2+) influx was revealed that is sufficient to account for the rapid induction and sustained expression of the homeostatic change in vesicle release. The homeostatic increase in Ca(2+) influx and release is blocked by a point mutation in the presynaptic CaV2.1 channel, demonstrating that the modulation of presynaptic Ca(2+) influx through this channel is causally required for homeostatic potentiation of release. Together with additional analyses, this study establishes that retrograde, transsynaptic modulation of presynaptic Ca(2+) influx through CaV2.1 channels is a key factor underlying the homeostatic regulation of neurotransmitter release (Muller, 2012a).

    The homeostatic modulation of presynaptic neurotransmitter release has been observed in organisms ranging from Drosophila to human, at both central and neuromuscular synapses. However, the molecular mechanisms underlying this form of synaptic plasticity are poorly understood. The Drosophila neuromuscular synapse has emerged as a powerful model system to dissect the cellular and molecular basis of this phenomenon. Forward genetic screens at this synapse have begun to identify loss-of-function mutations that prevent this form of neural plasticity. Among the loss-of-function mutations that have been shown to block this process is a mutation in the presynaptic CaV2.1 Ca2+ channel (Frank, 2006). However, these prior genetic data do not inform us regarding whether this calcium channel normally participates in homeostatic plasticity or how it might do so. It remains to be shown that a change in presynaptic Ca2+ influx through the CaV2.1 Ca2+ channel occurs during homeostatic plasticity. It is equally likely that a genetic disruption of the CaV2.1 Ca2+ channel simply occludes this form of plasticity by generally impairing calcium influx or synaptic transmission. Furthermore, if a change in Ca2+ influx occurs during homeostatic plasticity, can it be shown that this change is causally required for the observed homeostatic change in presynaptic release? Finally, if a change in presynaptic Ca2+ influx occurs, can it account for both the rapid induction of homeostatic plasticity as well as the long-term maintenance of homeostatic plasticity, which has been observed to persist for several days (Muller, 2012a)?

    To address these outstanding questions, this study probed Ca2+ influx during homeostatic plasticity by imaging presynaptic Ca2+ transients at the Drosophila neuromuscular junction (NMJ). This was done by comparing wild-type controls with animals harboring a mutation in the glutamate receptor subunit IIA (GluRIIA) of the muscle-specific ionotropic GluR at the fly NMJ (GluRIIASP16). The GluRIIASP16 mutation causes a reduction in miniature excitatory postsynaptic potential (mEPSP) amplitude, and induces a homeostatic increase in presynaptic release that precisely offsets the postsynaptic perturbation thereby restoring EPSP amplitudes toward wildtype levels. The GluRIIASP16 mutation is present throughout the life of the animal, and therefore this assay reports the sustained expression of homeostatic plasticity (Muller, 2012a).

    If the homeostatic enhancement of release is solely due to a change in presynaptic Ca2+ influx without concomitant changes in the number of releasable vesicles, then repetitive stimulation of homeostatically challenged synapses is expected to result in more pronounced vesicle depletion. Indeed, there is evidence for increased synaptic depression in GluRIIA mutants and following PhTX application. However, in agreement with a recent study, this study observed that homeostatic potentiation was paralleled by an increased number of release-ready vesicles, as assayed by the method of back extrapolation of cumulative excitatory postsynaptic current amplitudes during stimulus trains. The increase in the number of release-ready vesicles detected by this assay could be a direct or an indirect consequence of the homeostatic change in presynaptic Ca2+ influx and might help the potentiated synapse to sustain release during ongoing activity (Muller, 2012a).

    Work from several laboratories has provided evidence that chronic manipulation of neural activity in cultured mammalian neurons is associated with a compensatory change in presynaptic neurotransmitter release and a change in presynaptic Ca2+ influx. However, it remains unknown whether these homeostatic changes in release are caused by altered pre- versus postsynaptic activity, and it is unclear whether a change in presynaptic calcium influx is essential for this form of homeostatic plasticity in mammalian central neurons. Ultimately, it will be exciting to determine whether the molecular mechanisms identified in Drosophila, such as those described in this study, will translate to mammalian central synapses (Muller, 2012a).

    Extended synaptotagmin localizes to presynaptic ER and promotes neurotransmission and synaptic growth in Drosophila

    The endoplasmic reticulum (ER) is an extensive organelle in neurons with important roles at synapses including the regulation of cytosolic Ca2+, neurotransmission, lipid metabolism, and membrane trafficking. Despite intriguing evidence for these crucial functions, how presynaptic ER influences synaptic physiology remains enigmatic. To gain insight into this question, mutations were generated and characterized in the single Extended Synaptotagmin (Esyt) ortholog in Drosophila melanogaster. Esyts are evolutionarily conserved ER proteins with Ca2+-sensing domains that have recently been shown to orchestrate membrane tethering and lipid exchange between the ER and plasma membrane. Esyt was shown to localize to presynaptic ER structures at the neuromuscular junction. It was shown that synaptic growth, structure, and homeostatic plasticity are surprisingly unperturbed at synapses lacking Esyt expression. However, neurotransmission is reduced in Esyt mutants, consistent with a presynaptic role in promoting neurotransmitter release. Finally, neuronal overexpression of Esyt enhances synaptic growth and the sustainment of the vesicle pool during intense activity, suggesting that increased Esyt levels may modulate the membrane trafficking and/or resting calcium pathways that control synapse extension. Thus, this study has identified Esyt as a presynaptic ER protein that can promote neurotransmission and synaptic growth, revealing the first in vivo neuronal functions of this conserved gene family (Kikuma, 2017).

    Vps54 regulates Drosophila neuromuscular junction development and interacts genetically with Rab7 to control composition of the postsynaptic density

    Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). In the wobbler mouse, a model for human motor neuron (MN) disease, reduction in the levels of Vps54 causes neurodegeneration. However, it is unclear how disruption of the GARP complex leads to MN dysfunction. To better understand the role of Vps54 in MNs, this study has disrupted expression of the Vps54 ortholog in Drosophila and examined the impact on the larval neuromuscular junction (NMJ). Surprisingly, it was shown that both null mutants and MN-specific knockdown of Vps54 leads to NMJ overgrowth. Reduction of Vps54 partially disrupts localization of the t-SNARE, Syntaxin-16, to the TGN but has no visible impact on endosomal pools. MN-specific knockdown of Vps54 in MNs combined with overexpression of the small GTPases Rab5, Rab7, or Rab11 suppresses the Vps54 NMJ phenotype. Conversely, knockdown of Vps54 combined with overexpression of dominant negative Rab7 causes NMJ and behavioral abnormalities including a decrease in postsynaptic Dlg and GluRIIB levels without any effect on GluRIIA. Taken together, these data suggest that Vps54 controls larval MN axon development and postsynaptic density composition through a mechanism that requires Rab7 (Patel, 2020).

    Vesicle clustering in a living synapse depends on a synapsin region that mediates phase separation

    Liquid-liquid phase separation is an increasingly recognized mechanism for compartmentalization in cells. Recent in vitro studies suggest that this organizational principle may apply to synaptic vesicle clusters. This study tested this possibility by performing microinjections at the living lamprey giant reticulospinal synapse. Axons are maintained at rest to examine whether reagents introduced into the cytosol enter a putative liquid phase to disrupt critical protein-protein interactions. Compounds that perturb the intrinsically disordered region of synapsin (see Drosophila Synapsin), which is critical for liquid phase organization in vitro, cause dispersion of synaptic vesicles from resting clusters. Reagents that perturb SH3 domain interactions with synapsin are ineffective at rest. These results indicate that synaptic vesicles at a living central synapse are organized as a distinct liquid phase maintained by interactions via the intrinsically disordered region of synapsin (Pechstein, 2020).

    Chemical synapses can sustain neurotransmitter release at high rates by mobilizing synaptic vesicles (SVs) from a pool clustered at the release sites. Despite the critical role of SV clusters in neural signaling, the mechanisms underlying their organization remain unclear. Two principal models have been proposed, both of which are centered at synapsins that are essential for the maintenance of the major, distal part of SV clusters. In the first model, called the scaffold model here, the clustering of SVs is suggested to depend on their anchoring via synapsins to the cytoskeleton, because synapsins can bind both SVs and cytoskeletal components like actin, spectrin, and microtubules. Key to the scaffold model is the observation that Ca2+-dependent phosphorylation of synapsin by Ca2+/calmodulin-dependent protein kinase II (CaMKII) causes its dissociation from SVs and actin, thereby enabling vesicle mobilization. A variant of the scaffold model implies that SVs are kept in place by intervesicular filaments, or tethers, that are partly composed of synapsin but also contain unidentified components. In the second model, called the liquid phase model here, SV clustering is suggested to result from phase separation (Milovanovic, 2017). Liquid-liquid phase separation is an increasingly recognized mechanism for membraneless subcellular compartmentalization, in which a distinct liquid phase forms in the cytosol because of weak multimeric interactions between proteins or between RNA and proteins (Banani, 2017; Gomes, 2019). In the case of protein-based liquid phase organization, the interactions typically involve intrinsically disordered regions (IDRs) and/or modular binding domains such as SH2 and SH3 domains. In support of the liquid phase model for SV clustering, in vitro studies have shown that recombinant synapsin, or its isolated IDR (also termed region D), can form droplets in aqueous solution (Milovanovic, 2018). Droplet formation was enhanced by adding synapsin-binding SH3 domains and disrupted by CaMKII phosphorylation. Moreover, small liposomes could be captured in synapsin droplets (Milovanovic, 2018). At present, neither of the two models of SV clustering has been rigorously tested in vivo. Moreover, with regard to liquid-liquid phase separation, in vitro results are not easily transferred to a cellular context (Pechstein, 2020).

    This study examined whether the requirements for synapsin droplet formation in vitro pertain to SV clusters at the lamprey giant reticulospinal synapse. Earlier studies in this model have demonstrated that SV clusters in stimulated synapses depend on synapsin. In the present experiments, synapses were maintained at rest to test whether reagents microinjected into the axonal cytosol would enter a putative vesicular liquid phase to disrupt critical protein-protein interactions. This study found that compounds that perturb the synapsin IDR cause dispersion of SVs from resting clusters. However, reagents that perturb SH3 domain interactions with synapsin are ineffective (Pechstein, 2020).

    Recent studies with purified components suggest that presynaptic compartments, including the SV cluster and the active zone, may be organized by liquid-liquid phase separation (Milovanovic, 2018, Wu, 2019). However, the precise biological relevance of these data has been unclear. This study used a living giant synapse to examine the conditions underlying organization of the SV cluster. The results fulfill two main criteria predicted by the liquid phase model. First, fusion proteins and antibodies microinjected into the axonal cytosol readily entered, and affected, key interaction sites within resting SV clusters containing thousands of densely packed vesicles. This finding is in line with the property of a liquid phase compartment. The low level of spontaneous vesicle turnover in the lamprey synapse underscores that the SV cluster can be regarded as near a complete resting state. If the cluster had been organized as a rigid scaffold structure, it would have been expected that the reagents penetrated less effectively, thus not giving rise to complete dispersion of clustered vesicles. Second, and most importantly, the intrinsically disordered regions (IDR) defined as critical for synapsin droplet formation in vitro was found to be equally critical for maintaining SV clusters in living synapses. Disturbance of the IDR, by binding of antibodies or the SH3A domain, led to complete dissociation of the distal part of SV clusters. In vitro experiments confirmed the efficacy of the reagents used (Pechstein, 2020).

    The SH3A domain potently inhibited droplet formation of the IDR. Its efficacy may suggest that the region within the lamprey IDR that contains the SH3A binding site plays an important role to support IDR-IDR interactions. In mammalian synapsin I, at least two SH3A binding sites have been identifiedr. Thus, it cannot be excluded that occlusion of several SH3A domain binding sites may be required to disrupt liquid droplet formation. Higher than stoichiometric amounts of SH3A domains are more efficient to disperse synapsin IDR phase separation in vitro, suggesting that SH3A acts on other proline-rich regions in synapsin IDR at high concentrations. However, the antibodies stimulated droplet formation in vitro, suggesting that the divalent antibodies may drive aberrant droplet formation with the IDR. In the living synapse, the antibodies may thus have acted in a more complex way by perturbing endogenous IDR-IDR interactions and by forcing formation of antibody-IDR condensates that disrupt recruitment of vesicles to liquid droplets. This interpretation agrees with in vivo observations of electron-dense condensates associated with SVs (Pechstein, 2020).

    Although the present results do not formally disprove the scaffold model, several arguments can be raised against it. The original version of the scaffold model implies that SVs are anchored via actin filaments. This possibility appears unlikely for two reasons. First, it would suggest that the C and E regions, which mediate actin binding, would be important, rather than the IDR. Second, actin filaments are mainly localized around SV clusters, with few filaments penetrating their interior. The version of the scaffold model suggesting a role for intervesicular tethers is more difficult to rule out, because their molecular identity is unclear, with only part consisting of synapsin. However, the estimated number of synapsin molecules per vesicle does not correlate well with the number of tethers. Moreover, intervesicular tethers can still be observed after knockout of synapsin, which argues against their primary role in SV clustering (Pechstein, 2020).

    It is important to note that the present results only concern the resting synapse, and the situation may be somewhat different during activity. Stimulation causes a fraction of the vesicular synapsin pool to disperse into the axon, at least partly because of its phosphorylation by CaMKII. It is conceivable, and consistent with in vitro data (Milovanovic, 2018), that the mobile synapsin fraction temporarily leaves the liquid phase to reenter it during rest (Pechstein, 2020).

    This study found that the antibodies directed to region E of synapsin did not effectively disrupt vesicle clusters at rest. In earlier experiments, these antibodies were shown to deplete the distal vesicle pool when the microinjection was followed by action potential stimulation. This stimulus-dependent effect can be explained by the stimulation-induced dispersal of synapsin discussed earlier. Following its dispersion in the cytosol, synapsin may have been captured by the E region-directed antibodies and thus prevented from reassociating with SVs to maintain a liquid phase. It is possible that the marked stimulus-dependent effect is enhanced by exposure of a normally hidden E region. The E region-directed antibodies produced a minor, non-significant loss of vesicles. The lack of a consistent effect suggests that the E region is not involved in maintaining the liquid phase. It is speculated that the modest effect results from mobility of some vesicles even at rest that may be linked with minute dispersion of synapsin. It is also possible that the E region-directed antibodies exerted a small influence on the adjacent IDR within a liquid phase (Pechstein, 2020).

    Another notable observation was the occurrence of numerous scattered SVs and SV aggregates in the axonal cytosol following IDR perturbation, which sheds new light on a long-standing problem. Knockout of synapsins is known to cause a general decrease in the number of SVs, which has led to the proposition that synapsins play a role in SV maintenance, possibly via its ATP-binding C region. In an alternative hypothesis, the loss of SVs was proposed to be secondary to a clustering defect, eventually resulting in SV degradation. The dispersion of SVs following acute IDR perturbation supports a primary role of synapsin in vesicle clustering in central synapses (Pechstein, 2020).

    A proximal pool of SVs remained tethered at the active zone after IDR perturbation. The mechanisms underlying the organization of this membrane-proximal pool are presently unclear. It may be organized by scaffolding, with the vesicles tethered to filaments extending from the active zone. Alternatively, these vesicles may compose a distinct liquid phase organized by other proteins than synapsin. The active zone has been proposed to form a distinct liquid phase involving RIM, RIM-BP, and calcium channels, but it is unclear how far into the cytosol this phase may extend (Pechstein, 2020).

    Intersectin, amphiphysin, endophilin, and syndapin are all localized in SV clusters at rest. Upon stimulation, they partly redistribute to the periactive zone, where they participate in different aspects of SV endocytosis. Although the endocytic functions of these proteins are well established, their possible roles within the SV cluster remain unclear. No effect on resting SV clusters was observed by disrupting intersectin or amphiphysin SH3 domain interactions with synapsin, which is consistent with results on intersectin- and amphiphysin-deficient mice. In previous studies, no effect on clusters was observed after perturbing endophilin or syndapin SH3 domain interactions. Thus, individual interactions with any one of these four presynaptic SH3 domain-containing proteins are dispensable for the maintenance of resting SV clusters in vivo. However, this study does not rule out the possibility that multivalent interactions simultaneously involving these proteins may promote liquid phase organization of the SV cluster (Pechstein, 2020).

    The accumulation of SH3 domain-containing proteins in SV clusters may merely reflect passive buffering, but they may also play active roles at this location. In the case of intersectin, a role in SV mobilization has been proposed, because deletion of intersectins I and II in mice causes enhanced synaptic depression during high-frequency stimulation. Such enhanced depression is consistent with impaired SV mobilization, but it could also be secondary to an endocytosis defect compromising the clearance of release sites. The roles of SH3 domain-containing proteins in SV clusters thus remain an important topic for future studies (Pechstein, 2020).

    RIM-binding protein links synaptic homeostasis to the stabilization and replenishment of high release probability vesicles

    This study defines activities of RIM-binding protein (RBP) that are essential for baseline neurotransmission and presynaptic homeostatic plasticity. At baseline, rbp mutants have a approximately 10-fold decrease in the apparent Ca(2+) sensitivity of release that this study attributes to (1) impaired presynaptic Ca(2+) influx, (2) looser coupling of vesicles to Ca(2+) influx, and (3) limited access to the readily releasable vesicle pool (RRP). During homeostatic plasticity, RBP is necessary for the potentiation of Ca(2+) influx and the expansion of the RRP. Remarkably, rbp mutants also reveal a rate-limiting stage required for the replenishment of high release probability (p) vesicles following vesicle depletion. This rate slows approximately 4-fold at baseline and nearly 7-fold during homeostatic signaling in rbp. These effects are independent of altered Ca(2+) influx and RRP size. It is proposed that RBP stabilizes synaptic efficacy and homeostatic plasticity through coordinated control of presynaptic Ca(2+) influx and the dynamics of a high-p vesicle pool (Muller, 2015).

    RIM-binding protein couples synaptic vesicle recruitment to release site

    At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. This study shows in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites (Petzoldt, 2020).

    Chemical synapses are the fundamental building blocks of neuronal communication, allowing for a fast and directional exchange of chemical signals between a neurotransmitter releasing presynaptic and receiving postsynaptic target cells. To couple synaptic vesicle (SV) release to electrical stimulation by action potentials, Ca2+ ions entering the cell through voltage-gated Ca2+ channels activate the Ca2+ sensor synaptotagmin that is anchored on SVs to trigger fusion events. The presynaptic site of SV fusion ('active zone' [AZ]) is covered by an electron-dense scaffold ('cytomatrix') formed by a set of large conserved multidomain proteins. How individual AZ scaffold proteins intersect mechanistically with the SV cycle still remains largely enigmatic, though such knowledge was of importance to properly model synapse function in healthy and diseased circuits (Petzoldt, 2020).

    On the level of the individual AZs, recent data from both cultivated rodent neurons and Drosophila in vivo neurons suggest that the AZ cytomatrix provides stable SV release sites or 'fusion slots' via the clustering of the critical release factor (m)Unc13. The number of such release sites seems to be determined independently of the mechanisms controlling release probability. How SV release sites might be coupled and integrated with additional processes organized by the AZ scaffold, such as the recruitment of SVs under high demand periods of resupply (e.g., high action potential frequencies), remains a largely open question (Petzoldt, 2020).

    The physically extended ELKS family protein (glutamic acid [E], leucine [L], lysine [K],and serine [S]-rich protein) Bruchpilot (BRP) not only operates as the fundamental building block of the Drosophila AZ scaffold but was also shown to promote SV recruitment depending on a binding motif at its extreme, AZ membrane-distal C terminus. Adaptor proteins physically and functionally connecting such extended scaffold proteins, which sample the cytoplasm, and the SV release sites at the AZ membrane might well be relevant in this regard. Rab3-interacting molecule (RIM)-binding proteins (RIM-BPs) are an evolutionarily conserved family of extended AZ scaffold proteins, obviously critical for SV release at Drosophila and mammalian central synapses. RIM-BP family proteins with the C-terminal SH3-II/III domains bind to Ca2+ channels, release factor Unc13A, and RIM, another critical AZ scaffold protein. At Drosophila neuromuscular junction (NMJ) synapses, rim-bp null alleles provoke a most severe functional phenotype, stronger than for rim and rim-bp single mutants at most mammalian synapses. This study exploits the severity of the synaptic Drosophila rim-bp phenotype for a detailed structure-function analysis at Drosophila larval neuromuscular (NMJ) synapses. Elimination of the individual RIM-BP SH3 domains affected transport to AZs severely; however, the low levels of properly locating RIM-BP-ΔSH3 variants were still able to rescue the 'nanoscopic' defects of rim-bp null mutants AZ scaffolds. Crystallographic analysis of the RIM-BP fibronectin III-like (FN-III) domain cluster describes an extended 'hinge' in the protein center, which likely plays conformational roles. Finally, the N-terminal region (NTR) of RIM-BP was required to properly organize the overall nanoscopic architecture of the AZ scaffold. While SV docking and numbers of (m)Unc13 clusters remained at normal levels, the NTR was specifically needed for efficient SV recruitment. Thus, it is suggested that RIM-BP family proteins evolved as adaptors physically and functionally connecting SV release sites with BRP/ELKS-dependent SV recruitment processes (Petzoldt, 2020).

    The current view of AZ scaffolds emphasizes their role in providing a 'smart catalytic surface' integrating sub-functionalities that facilitate and control the SV cycle. Providing SV release sites with a proteinaceous 'nano-environment' defining release probability, by precisely defining their spatial distance to voltage-operated Ca2+ channels, is probably one fundamental function using these highly conserved, 'ancient' protein architectures. The SV release sites probably also have to be coupled to processes retrieving SVs from more membrane-distant positions. Concerning protein architectures harvesting SVs, evolutionary solutions might have been somewhat more flexible, obviously adopted to the synapse-type specific needs in physiologically relevant fusion rates. Identifying proteins that might be involved in structurally and functionally coupling recruitment protein architectures with release sites is an emerging topic. This study provides evidence that RIM-BP with its interactions obviously plays a role in both 'nanoscopic locations.' First, through its SH3 domains, it binds RIM, the intercellular C-terminus of the voltage-operated Ca2+ channel and also Unc13A, the release factor whose nanoscale positioning has been recently identified as critical for SV release definition (Reddy-Alla, 2017). Second, this study found that it also binds conserved regions of BRP in the core of the AZ central scaffold. Deleting this interaction surface, though not reducing AZ protein levels, obviously undermines the proper bundling of BRP filaments, at least for a major fraction of AZs. Though it is not easy to prove that individual RIM-BP protein molecules span this distance physically, it is tempting to hypothesize that RIM-BP might literally connect the recruitment of SVs from more membrane distant pools, probably starting at the distal end of the BRP filaments, with their integration into SV release sites (see RIM-BP NTR localizes to the center of the AZ to stabilize the BRP scaffold and promote SV recruitment) (Petzoldt, 2020).

    At mammalian synapses, recent multi-loci genetics have demonstrated most severe deficits when eliminating combinations of AZ scaffold proteins between RIM, ELKS, and RIM-BP family members. In this study, quadruple knockouts of RIM1/2, together with RIM-BP1/2 proteins in mice, exhibit a total loss of neurotransmitter release from severe impairments in SV priming and docking, a dramatic loss of AZ scaffold density, with a trans-synaptic effect that impairs the organization of the postsynaptic density. These severe synthetic 'catastrophic' phenotypes, eliminating ultrastructural specializations and release factor targeting, demonstrate a principal functional redundancy between RIM-BPs, RIMs, and ELKS family proteins. Similar results were also retrieved from work on mice in which RIM1αβ and RIM2αβγ, together with ELKS1α and ELKS2α isoforms, have been completely eliminated. Cultured hippocampal synapses of these mutant mice consequently lose Munc13, Bassoon, Piccolo, and RIM-BP, following a mass disassembly of the AZ scaffold (Wang, 2016). Similarly, this study observed that RIM seemingly operates synergistically with RIM-BP in establishing the AZ nanoscopic architecture (Petzoldt, 2020).

    Despite the obvious importance of these findings for understanding the collective role of the AZ scaffold, the fact that the domain organization for all specific scaffold proteins has been individually conserved over hundreds of millions of years motivated a search for ways to demonstrate specific functions of these AZ core scaffold proteins. This study has molecularly isolated an additional sub-functionality for the so-far functionally nonconsidered RIM-BP NTR, and provide evidence that its protein architecture might have been conserved for the reason that it operates as an adaptor coupling SV recruitment processes with the membrane-associated SV release sites (Petzoldt, 2020).

    Mechanistic analysis of RIM-BPs so far has largely focused on SH3 domains II and III, which bind to Ca2+ channels and RIM in both mammals and Drosophila, and Unc13A in Drosophila. Analysis of RIM-BP at Drosophila NMJ synapses was the first to demonstrate a major role of the protein family in neurotransmission, characterized by a severe reduction in release probability and signs of defective Ca2+ channel clustering. This study has exploited the glutamatergic NMJ synapses for a stringent genetic analysis with multimodal readouts, also including an analysis of transport and nanoscale integration into the AZ scaffold. Deletion of individual SH3 domains majorly affected RIM-BP transport, probably directly reflecting their high affinity binding to JIP-1 homologue Aplip1, whose deletion also interferes with effective axonal transport of BRP/RIM-BP 'packages'. Somewhat surprisingly, however, moderately or even strongly reduced levels of RIM-BPΔSH3-II or RIM-BPΔSH3-III, respectively, still restored RIM-BP functionality when expressed in a null background. It obviously might be argued that only combined elimination of SH3-II together with SH3-III might uncover their function collectively. Indeed, Aplip1 binds both SH3-II and III, and AZ levels were decreased even more strongly in the RIM-BPΔSH3-II/III double-deleted variant. However, at least concerning the role of Ca2+ channel binding, the AZ scaffold can seemingly compensate for the absence of RIM-BP-SH3 mediated interactions, as Ca2+ channels with the highly conserved PXXP RIM-BP binding motif deleted still retained full activity in a genetic rescue assay (instead of resembling the rim-bp null phenotype). It is suggested that redundant interactions stabilize Ca2+ channel: AZ scaffold contacts at NMJ synapses, an idea that might be relevant for other synapses as well. Indeed, it was shown previously that the N terminus of BRP also binds the Ca2+ channel α1 subunit intracellular C terminus. Given the essential character of SH3-III for survival, however, it appears likely that slightly different rules and 'vulnerabilities' apply to other synapse types, for example, in the Drosophila brain. In fact, it was found recently that synapses in the Drosophila brain differ substantially in their BRP content, with, for example, interneuron synapses largely lacking BRP (Fulterer, 2018). This might result in different efficacy of compensation via BRP and be responsible for the essential character of SH3-III. Put differently, it still appears plausible that the C-terminal close SH3 domains might be of truly essential character dependent on synapse type given the results retrieved at mammalian synapses (Petzoldt, 2020).

    Finally, the presence of three FN-III domains, despite their invariable character, remained and remains an enigma. FN-III modules are common in extracellular but rarer in intracellular proteins, such as RIM-BP, here found chiefly as the main components of a group of intracellular proteins associated with the contractile apparatus of muscles. The first x-ray structure of the RIM-BPs, which is presented in this study, depicts a typical FN-III-type organization; however, it is oriented in an extended hinge-like arrangement. It is tempting to speculate that the hinge-like architecture might support RIM-BP conformations or conformational change when connecting release sites with recruitment processes (Petzoldt, 2020).

    Physiological analysis points toward a discrete function of the RIM-BP N-terminal domain. Analysis of SV recruitment based on the rim-bp null allele is complicated due to the severe release probability deficits dominating the physiological scenario. Nonetheless, a careful analysis, using high Ca2+ concentrations to milden the influence of release probability differences, indeed identified recruitment defects. Remarkably, similar to the NTR-specific deletions, a previous study analyzing the rim-bp null mutant for RIM-BP suggested a rate-limiting function for the replenishment of high release probability SVs following vesicle depletion at NMJ synapses. Moreover, in mouse rim-bp2 knockouts, recruitment/replenishment deficits at auditory hair cells were reported. In rim-bp null mutants, UNC13A levels are strongly reduced, while for the dNTR-2 construct, UNC13A levels and consequently release site number were not reduced (instead rather slightly increased). Thus, the data apparently uncouple the role of RIM-BP for release site organization (via Unc13A clustering) from its role in overall scaffold organization where the NTR plays an important role (Petzoldt, 2020).

    The extended BRP filaments in Drosophila probably operate as 'antennae' to harvest SVs from the reserve pool, a process facilitated by the C-terminal amino acids of BRP, made evident by the defects of the brp nude allele lacking only the last 17 amino acids. BRP in its first half is highly homologous to ELKS/CAZ-associated structural proteins (CAST) AZ proteins; however, its C-terminal half is specific for insects and obviously evolved for harvesting SVs. The large vertebrate-specific AZ protein Bassoon was shown to help SV replenishment at the central synapse under conditions of heavy stimulation. Notably, Bassoon binds to the first SH3 domain of RIM-BP, suggestive of convergent evolution where different molecular antennae were used to finally target RIM-BP, the generic 'old' adaptor coupling to the membrane close release sites (Petzoldt, 2020).

    In its current form, static STED microscopy as used here only samples average epitope distributions, while the relevant proteins likely are dynamically switched in the course of the SV cycle. As an indication of this, the RIM-BP C-terminal SH3 domains were found to bind both the Ca2+ channel C-terminus and Unc13A N terminus. As these binding events can hardly be accomplished by a single Unc13A molecule at a single time point, future analysis will have to address the underlying conformational dynamics likely involved here (Petzoldt, 2020).

    Taken together, RIM-BPs might take a generic role of SV replenishment, apart from their obvious role in coclustering of release machinery and Ca2+ channels at the AZ membrane. Indeed, the highly conserved domain architecture might have evolved for exactly this reason, to guide SVs into the proper release environment (Petzoldt, 2020).

    A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation

    Homeostatic signaling systems are thought to interface with other forms of plasticity to ensure flexible yet stable levels of neurotransmission. The role of neurotransmitter receptors in this process, beyond mediating neurotransmission itself, is not known. Through a forward genetic screen, the Drosophila kainate-type ionotropic glutamate receptor subunit DKaiR1D was identified as being required for the retrograde, homeostatic potentiation of synaptic strength. DKaiR1D is necessary in presynaptic motor neurons, localized near active zones, and confers robustness to the calcium sensitivity of baseline synaptic transmission. Acute pharmacological blockade of DKaiR1D disrupts homeostatic plasticity, indicating that this receptor is required for the expression of this process, distinct from developmental roles. Finally, this study demonstrates that calcium permeability through DKaiR1D is necessary for baseline synaptic transmission, but not for homeostatic signaling. It is proposed that DKaiR1D is a glutamate autoreceptor that promotes robustness to synaptic strength and plasticity with active zone specificity (Kiragasi, 2017).

    The nervous system is endowed with potent and adaptive homeostatic signaling systems that maintain stable functionality despite the myriad changes that occur during neural development and maturation. The importance of homeostatic regulation in the nervous system is underscored by associations with a variety of neurological diseases, yet the genes and mechanisms involved remain enigmatic. A powerful model of presynaptic homeostatic plasticity has been established at the Drosophila neuromuscular junction (NMJ), a model glutamatergic synapse with molecular machinery that parallels central synapses in mammals. Here, genetic and pharmacological manipulations that reduce postsynaptic (muscle) glutamate receptor function trigger a trans-synaptic, retrograde feedback signal to the neuron that increases presynaptic release to precisely compensate for this perturbation. This process is referred to as 'presynaptic homeostatic potentiation' (PHP), because the expression mechanism requires a presynaptic increase in neurotransmitter release (Kiragasi, 2017).

    In recent years, forward and candidate genetic approaches have revealed several new and unanticipated genes necessary for PHP expression. While perturbations to the glutamate receptors in muscle are crucial events in the induction of PHP, whether other ionotropic glutamate receptors (iGluRs) function in PHP or are even expressed at the Drosophila NMJ is unknown. Finally, although evidence has emerged that homeostatic modulation is synapse specific, no roles for neurotransmitter receptors or other factors have been found to enable the presynaptic tuning of release efficacy at individual synapses (Kiragasi, 2017).

    This study has identified the kainate-type iGluR subunit DKaiR1D to be necessary for PHP expression at the Drosophila NMJ. DKaiR1D is necessary for the calcium sensitivity of baseline synaptic transmission, as well as for the acute and chronic expression of homeostatic potentiation. Recently, the functional reconstitution of DKaiR1D was achieved in heterologous cells, revealing that these receptors form homomeric calcium-permeable channels with atypical pharmacological properties compared to their vertebrate homologs (Li, 2016). This study has found that DKaiR1D is expressed in the nervous system and not the muscle, is present near presynaptic active zones, and is required specifically in motor neurons to enable the robustness of baseline neurotransmission and homeostatic plasticity. It is proposd that glutamate activates DKaiR1D at presynaptic release sites to translate autocrine activity into the robust stabilization of synaptic strength with active zone specificity (Kiragasi, 2017).

    This unexpected role for iGluRs in sensing glutamate at presynaptic terminals indicates an autocrine mechanism that responds to glutamate release to adaptively modulate presynaptic activity at individual active zones (Kiragasi, 2017).

    Glutamate receptors have diverse functions in modulating presynaptic excitability and short-term plasticity in addition to their established roles in postsynaptic excitation. Similar to what was observed with DKaiR1D at the Drosophila NMJ, rodent iGluRs also localize to presynaptic active zone, are activated by high concentrations of glutamate, and can modulate release during single action potentials. This suggests conserved autocrine modulatory mechanisms shared between these systems (Kiragasi, 2017).

    Rodent autoreceptors are known to modulate presynaptic activity on rapid timescales. In these cases, most of the impact on release is likely to derive from a calcium store-dependent mechanism, or from modulation of the action potential during the repolarization phase, when most of the calcium influx that drives vesicle release occurs. In a similar fashion, activation of presynaptic DKaiR1D during a single action potential could lead to a rapid additional source of presynaptic calcium influx from DKaiR1D itself and/or through modulation of presynaptic membrane potential to drive increased vesicle release. Voltage imaging at the Drosophila NMJ has found the half width of the action potential waveform to be ~5 ms (Ford, 2014), which is sufficient time to be modulated through such a mechanism. Therefore, dynamic changes in voltage or calcium influx at or near active zones could, in principle, drive additional vesicle release during a single action potential. This modulation may be restricted to nearby active zones and compartments relative to the site of glutamate release. Indeed, presynaptic kainate autoreceptors have the capacity to confer short-range, synapse-specific modulation to synaptic transmission (Scott, 2008), while presynaptic ligand-gated ion channels in C. elegans can also rapidly modulate synaptic transmission (Takayanagi-Kiya, 2016). Local activation of DKaiR1D could, therefore, subserve a powerful and flexible means of tuning presynaptic efficacy at or near individual release sites (Kiragasi, 2017).

    How does DKaiR1D promote the expression of presynaptic homeostatic plasticity? In contrast to the role of DKaiR1D in baseline release, the data indicate that the DKaiR1D-dependent mechanism that drives PHP is calcium independent. This implies two changes to DKaiR1D functionality that are unique to homeostatic adaptation compared to baseline transmission. First, because presynaptic release is acutely potentiated following application of PhTx, the activity, levels, and/or localization of DKaiR1D receptors must change to acquire a novel influence on neurotransmitter release following PHP induction. The activity of synaptic glutamate receptors can change through associations with additional subunits and auxiliary factors such as Neto. Furthermore, various forms of plasticity are expressed through dynamic changes in the levels and localization of glutamate receptors trafficking between active zones and endosome pools or extra-synaptic membrane. Indeed, when DKaiR1D is overexpressed in motor neurons, it rescues baseline transmission and PHP expression while localizing to heterogeneous puncta of varying distances relative to active zones. Notably, there is evidence that DKaiR1D interacts with other glutamate receptor subunits in vivo (Karuppudurai, 2014), which may contribute to the pharmacological differences observed in this study compared with the in vitro characterization (Li, 2016) and may also be targets of modulation during PHP (Kiragasi, 2017).

    Second, calcium signaling through DKaiR1D differentially drives baseline release and homeostatic plasticity. Therefore, mechanisms distinct from calcium permeability of the channel must contribute to PHP expression. One possibility is that DKaiR1D signals through an undefined metabotropic mechanism during PHP, which might contribute to the ability of the calcium impermeable DKaiR1DR transgene, with reduced conductance (Li, 2016), to rescue PHP expression. Alternatively, an attractive possibility is that following PHP induction, glutamate released from nearby active zones may dynamically modulate the presynaptic membrane potential and/or action potential waveform to promote additional synaptic vesicle release. Indeed, small, sub-threshold depolarizations of the presynaptic resting potential, as small as 5 mV, are sufficient to induce a 2-fold increase in presynaptic release. The timescale of this activity could occur within a few milliseconds as discussed above, and studies at the Drosophila NMJ have revealed that glutamate is released from single synaptic vesicles over timescales of milliseconds. Interestingly, epithelial sodium channels (ENaCs) have been proposed to enable PHP expression through changes in the presynaptic membrane potential, and such a mechanism could be shared by DKaiR1D but gated by glutamate release at individual active zones. Thus, DKaiR1D may serve to homeostatically modulate presynaptic release through modulation of presynaptic voltage and, intriguingly, with active zone specificity (Kiragasi, 2017).

    This characterization of DKaiR1D has revealed a role for presynaptic glutamate signaling in homeostatic plasticity. In the mammalian CNS, glutamate signaling drives the adaptive regulation of postsynaptic AMPA receptor insertion and removal, known as homeostatic scaling. Further, kainate receptors were recently demonstrated to regulate postsynaptic homeostatic scaling. Together with the present study, these results demonstrate that glutamatergic signaling through kainate receptors orchestrate the potent and adaptive homeostatic control of synaptic strength on both sides of the synapse. Future studies will reveal the integration between synaptic glutamate signaling and other forces that modulate synaptic strength to enable robust, flexible, and stable neurotransmission (Kiragasi, 2017).

    The auxiliary glutamate receptor subunit dSol-1 promotes presynaptic neurotransmitter release and homeostatic potentiation

    Presynaptic glutamate receptors (GluRs) modulate neurotransmitter release and are physiological targets for regulation during various forms of plasticity. Although much is known about the auxiliary subunits associated with postsynaptic GluRs, far less is understood about presynaptic auxiliary GluR subunits and their functions. At the Drosophila neuromuscular junction, a presynaptic GluR, DKaiR1D, localizes near active zones and operates as an autoreceptor to tune baseline transmission and enhance presynaptic neurotransmitter release in response to diminished postsynaptic GluR functionality, a process referred to as presynaptic homeostatic potentiation (PHP). This study identified an auxiliary subunit that collaborates with DKaiR1D to promote these synaptic functions. This subunit, dSol-1, is the homolog of the Caenorhabditis elegans CUB (Complement C1r/C1s, Uegf, Bmp1) domain protein Sol-1. dSol-1 functions in neurons to facilitate baseline neurotransmission and to enable PHP expression, properties shared with DKaiR1D. Intriguingly, presynaptic overexpression of dSol-1 is sufficient to enhance neurotransmitter release through a DKaiR1D-dependent mechanism. Furthermore, dSol-1 is necessary to rapidly increase the abundance of DKaiR1D receptors near active zones during homeostatic signaling. Together with recent work showing the CUB domain protein Neto2 (see Drosophila Neto) is necessary for the homeostatic modulation of postsynaptic GluRs in mammals, these data demonstrate that dSol-1 is required for the homeostatic regulation of presynaptic GluRs. Thus, it is proposed that CUB domain proteins are fundamental homeostatic modulators of GluRs on both sides of the synapse (Kiragasi, 2020).

    Synaptic strength is dynamically tuned during both Hebbian and homeostatic forms of plasticity. One major mechanism that achieves this modulation targets the abundance, localization, and/or functionality of ionotropic glutamate receptors (GluRs). For instance, the expression of long-term potentiation and depression requires bidirectional changes in the abundance of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to adjust synaptic strength. Furthermore, some forms of homeostatic plasticity also tune the abundance of N-methyl-D-aspartate (NMDA), AMPA, and kainate receptors at postsynaptic densities to stabilize neuronal activity . Auxiliary subunits associated with GluRs are key factors that control GluR trafficking and dynamics during plasticity, where transmembrane AMPA receptor regulatory protein (TARP), Cornichon, and Neto subunits orchestrate AMPA and kainate receptor function and plasticity. Although much is known about the GluR subtypes and associated auxiliary subunits that regulate GluR trafficking, abundance, and functionality at postsynaptic densities during both Hebbian and homeostatic plasticity, far less is understood about these mechanisms at presynaptic release sites (Kiragasi, 2020).

    Presynaptic autoreceptors have emerged as important regulators of neurotransmitter release at glutamatergic synapses. For example, presynaptic kainate receptors are present in hippocampal mossy fibers, where autocrine feedback facilitates neurotransmission during trains of activity. In addition, presynaptic NMDA receptors in the hippocampus mediate presynaptic inhibition in response to excess glutamate release as well as presynaptic facilitation following the induction of long-term potentiation. Furthermore, presynaptic metabotropic receptors play critical roles in various forms of plasticity and can bidirectionally tune presynaptic neurotransmitter release. Finally, ionotropic neurotransmitter receptors at presynaptic terminals can modulate release at neuromuscular junctions (NMJs) in Caenorhabditis elegans and Drosophila. While it is now clear that presynaptic autoreceptors are important bidirectional modulators of neurotransmitter release, how the levels, activity, and localization of these receptors are controlled to establish baseline function, and to what extent they are further modified during plasticity, remains unclear (Kiragasi, 2020).

    A kainate-type ionotropic GluR, DKaiR1D, was previously shown to be necessary at the Drosophila NMJ for the expression of presynaptic homeostatic potentiation (PHP). PHP is a fundamental form of synaptic plasticity in which pharmacological and genetic challenges that diminish postsynaptic neurotransmitter receptor functionality trigger a transsynaptic retrograde signal that enhances presynaptic neurotransmitter release to precisely compensate for reduced postsynaptic excitability (19, 20). PHP has been observed at NMJs of Drosophila, rodents, and humans and was recently demonstrated to be rapidly expressed in the mammalian central nervous system. DKaiR1D was identified in a forward genetic screen to be required for the rapid expression of PHP at the fly NMJ. DKaiR1D receptors form homomers that are permeable to both sodium and calcium, localized near presynaptic release sites, and proposed to homeostatically regulate presynaptic voltage following autocrine activation by glutamate. This DKaiR1D-dependent enhancement in neurotransmitter output implies a rapid modulation in the abundance, functionality, and/or localization of these receptors must occur in the course of PHP induction. This regulation could, in principle, be achieved through interactions with auxiliary GluR subunits. However, the precise mechanisms that control DKaiR1D and enable robust and stable neurotransmission at baseline and during plasticity, and whether auxiliary factors are involved, are unknown (Kiragasi, 2020).

    A candidate screen of Drosophila GluR modulators and auxiliary subunits was performed to identify potential functions in PHP expression. This effort has discovered an uncharacterized auxiliary GluR subunit that functions in neurons to promote neurotransmitter release and enable homeostatic potentiation. This factor, a homolog of the C. elegans auxiliary GluR subunit Sol-1 (Walker, 2006), contains multiple CUB domains and is structurally similar to the Neto/Sol-2 family of auxiliary GluR subunits. dSol-1 mutants essentially phenocopy DKaiR1D mutants in neurotransmission and PHP. Further experiments demonstrate that dSol-1 functions to homeostatically modulate presynaptic glutamate release by promoting the rapid accumulation of DKaiR1D receptors near active zones. Together, these data indicate that the interactions between CUB domain auxiliary subunits and their associated GluRs are fundamental physiological targets of homeostatic signaling (Kiragasi, 2020).

    In the context of baseline neurotransmission, dSol-1 promotes release without measurably changing the abundance or localization of DKaiR1D receptors, indicating a functional role in modulating DKaiR1D activity. However, dSol-1 is necessary during PHP signaling to drive a rapid increase in DKaiR1D receptor abundance at presynaptic terminals. These findings define a CUB domain auxiliary GluR subunit as a central target for the presynaptic modulation of synaptic efficacy and homeostatic plasticity (Kiragasi, 2020).

    Several lines of evidence suggest that dSol-1 enhances baseline neurotransmitter release by targeting DKaiR1D receptor functionality. First, dSol-1 promotes baseline neurotransmission in low extracellular Ca2+, a function shared with DKaiR1D. In addition, neurotransmitter release is reduced in dSol-1 mutants and enhanced by neuronal overexpression of dSol-1, indicating a capacity for dSol-1 expression levels to bidirectionally tune release. However, this potentiation in baseline transmission occurs without a significant increase in DKaiR1D receptor abundance, at least when both dSol-1 and DKaiR1D are overexpressed in motor neurons, suggesting a change in DKaiR1D functionality. Interestingly, in C. elegans, Sol-1 regulates GLR1 functionality by modulating channel gating, promoting the open state, and slowing sensitization, without an apparent change in glr1 expression. In heterologous cells, both Sol-1 and dSol-1 promote GLR1 function without altering expression levels. This indicates a potentially conserved function between sol-1 and dSol-1 to confer similar modulations to GluR functionality. In mammals, Neto auxiliary subunits selectively associate with kainate-subtype GluRs, while TARPs such as Stargazin associate with AMPA-type receptors. In Drosophila, Neto is an important auxiliary subunit for the postsynaptic GluRs at the NMJ, which, like DKaiR1D, are generally characterized as non-NMDA, kainate-type GluRs. However, in C. elegans, both Sol-1 and Sol-2/Neto form a complex together with the AMPA receptor subtype GLR1, suggesting some level of promiscuity, at least in invertebrates, between AMPA and kainate GluRs and their auxiliary subunits. One possibility is that at baseline states, a substantial proportion of DKaiR1D receptors do not interact with dSol-1. By increasing levels of dSol-1, more DKaiR1D receptors may become associated with dSol-1, perhaps leading to changes in gating properties that enhance DKaiR1D receptor function. However, the possibility cannot be ruled out that dSol-1 somehow regulates DKaiR1D through a more indirect mechanism. While DKaiR1D receptors can form homomers and traffic to the cell surface when expressed alone in heterologous cells, future in vitro studies will be needed to determine the precise role dSol-1 has in DKaiR1D receptor trafficking and/or functionality (Kiragasi, 2020).

    dSol-1 enables PHP expression through a mechanism that is distinct from its role in baseline transmission, although both functions converge on DKaiR1D. The data suggest that PHP signaling leads to a rapid accumulation of DKaiR1D receptors near presynaptic release sites that requires dSol-1. This dSol-1-dependent increase in DKaiR1D levels may be a unique feature of homeostatic signaling in Drosophila, as there is no evidence for worm sol-1 to promote surface levels or changes in GLR1 localization in vivo or in vitro. Although the rapid increase in DKaiR1D receptor levels at synaptic terminals during PHP signaling is surprising, it is not unprecedented. DKaiR1D receptors are present near presynaptic release sites, and many other active zone components rapidly accumulate and/or remodel following PhTx application. An attractive possibility is that DKaiR1D receptors participate in this process of rapid active zone remodeling during PHP signaling. Mechanistically, new protein synthesis of DKaiR1D is unlikely to be involved, as PHP expression and active zone remodeling can occur without new translation. Recently, the lysosomal kinesin adaptor arl-8 and other axonal transport factors were identified to be necessary for the rapid increase in active zone components during PHP signaling. Thus, it is tempting to speculate that DKaiR1D receptors might be cotransported during PHP as part of this pathway. In vertebrates, auxiliary subunits traffic GluRs during synaptic plasticity, so dSol-1 may function similarly in delivering DKaiR1D receptors to the plasma membrane and/or to release sites during PHP signaling. Finally, it is possible that DKaiR1D receptors are constitutively degraded under baseline conditions, and that PHP signaling through dSol-1 inhibits this degradation. The role of protein degradation in PHP signaling has been recently studied in both pre- and postsynaptic compartments at the Drosophila NMJ. Interestingly, inhibition of proteasomal degradation in presynaptic compartments is capable of rapidly enhancing neurotransmission to levels similar to what is observed after overexpression of dSol-1. In both cases, no further increase in neurotransmitter release is observed after PhTx application. While there are apparently distinct roles for dSol-1 in baseline function and homeostatic plasticity, a common point of convergence is DKaiR1D (Kiragasi, 2020).

    CUB domains define a structural motif in a large family of extracellular and plasma membrane-associated proteins present in invertebrates to humans. While the specific four extracellular CUB domains that define sol-1/dSol-1 are unique to invertebrates, genes containing multiple CUB domains (between two and eight) are present throughout vertebrate species and function in diverse processes including intercellular signaling, developmental patterning, inflammation, and tumor suppression. CUB domains mediate dimerization and binding to collagen-like regions; this interaction may be relevant to its role in promoting PHP, as the Drosophila collagen member Multiplexin is present in the extracellular matrix and has been proposed to be part of the homeostatic retrograde signaling system. This characterization of dSol-1 contrasts with what is known about another CUB domain auxiliary glutamate receptor in Drosophila, Neto-β. neto-β is highly expressed in the larval muscle, where it is clearly involved in the trafficking and/or stabilization of postsynaptic GluRs at the NMJ. In contrast, dSol-1 is exclusively expressed in the nervous system. Another interesting distinction is that while both dSol-1 and Neto contain multiple extracellular CUB domains and a single transmembrane domain, dSol-1 lacks any intracellular domain while two isoforms of Neto are expressed with one of two alternative intracellular C-terminal cytosolic domains, Neto-α or Neto-β. Neto-β is clearly the major isoform and performs the key functions in controlling postsynaptic GluR levels and composition, while neto-α was recently proposed to function in motor neurons with DKaiR1D and to be necessary for PHP. Interestingly, the C. elegans receptor GLR1 requires the auxiliary subunits Sol-1, Stargazin, and Neto/Sol-2 for functionality in vivo and in vitro. It is therefore possible that Drosophila Neto-α and/or Stargazin-like interact with both dSol-1 and DKaiR1D. In mammals, there is a large body of evidence demonstrating that presynaptic GluRs, including kainate receptors, modulate presynaptic function. While postsynaptic kainate receptors in mammals associate with the CUB domain auxiliary GluR subunit Neto2 to regulate synaptic function and homeostatic plasticity, to what extent Neto2 or other auxiliary subunits function with presynaptic kainate receptors remains enigmatic. This study indicates that CUB domain proteins may be fundamental modulators of GluRs in synaptic function and plasticity on both sides of the synapse (Kiragasi, 2020).

    Neto-alpha controls synapse organization and homeostasis at the Drosophila neuromuscular junction

    Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), this study shows that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, this study demonstrates that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR (KaiR1D)-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. It is proposed that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities (Han, 2020).

    Formation of functional synapses during development and their fine-tuning during plasticity and homeostasis relies on ion channels and their accessory proteins, which control where, when, and how the channels function. Auxiliary proteins are diverse transmembrane proteins that associate with channel complexes and mediate their properties, subcellular distribution, surface expression, synaptic recruitment, and associations with various synaptic scaffolds. Channel subunits have expanded and diversified during evolution to impart different channel biophysical properties, but whether auxiliary proteins have evolved to match channel diversity remains unclear (Han, 2020).

    Ionotropic glutamate receptors (iGluRs) mediate neurotransmission at most excitatory synapses in the vertebrate CNS and at the neuromuscular junction (NMJ) of insects and crustaceans and include α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), N-methyl-D-aspartic acid receptors (NMDARs), and kainate receptors (KARs). Sequence analysis of the Drosophila genome identified 14 iGluRs genes that resemble vertebrate AMPARs, NMDARs, and KARs. The fly receptors have strikingly different ligand binding profiles; nonetheless, phylogenetic analysis indicates that two of the Drosophila genes code for AMPARs, two code for NMDARs, and 10 code for subunits of the KAR family, which is highly expanded in insects. In flies and vertebrates, AMPARs and KARs have conserved, dedicated auxiliary proteins. For example, AMPARs rely on Stargazin and its relatives to selectively modulate receptors' gating properties, trafficking, and interactions with scaffolds such as PSD-95-like membrane-associated guanylate kinases. Stargazin is also required for the functional reconstitution of invertebrate AMPARs. KARs are modulated by the Neto (Neuropilin and Tolloid-like) family of proteins, including vertebrate Neto1 and Neto2, C. elegans SOL-2/Neto (Wang et al., 2012), and Drosophila Neto. Neto proteins differentially modulate the gating properties of vertebrate KARs. A role for Neto in the biology of KARs in vivo has been more difficult to assess because of the low levels of KARs and Neto proteins. Nevertheless, vertebrate Netos modulate synaptic recruitment of selective KARs by association with synaptic scaffolds such as GRIP and PSD-95, and the PDZ binding domains of vertebrate KAR/Neto complexes are essential for basal synaptic transmission and long-term potentiation (LTP). Post-translational modifications regulate Neto activities in vitro, but the in vivo relevance of many of these observations remains unknown (Han, 2020).

    Drosophila NMJ is an excellent genetic system to probe the repertoire of Neto functions. This glutamatergic synapse appears to rely exclusively on KARs, with one presynaptic and five postsynaptic subunits. Previous work has shown that Drosophila Neto is an obligatory auxiliary subunit of the postsynaptic KAR complexes: in the absence of Neto, postsynaptic KARs fail to cluster at synaptic sites and the animals die as paralyzed embryos. Heterologous reconstitution of postsynaptic KARs in Xenopus oocytes revealed that Neto is required for functional receptors. The fly NMJ contains two glutamate receptor (GluR) complexes (types A and B) with different subunit compositions (either GluRIIA or GluRIIB, plus GluRIIC, GluRIID, and GluRIIE) and distinct properties, regulation, and localization patterns. The postsynaptic response to the fusion of single synaptic vesicles (quantal size) is reduced for NMJs with type B receptors only, and the dose of GluRIIA and GluRIIB is a key determinant of quantal size. The fly NMJ is also a powerful model system to study homeostatic plasticity. Manipulations that decrease the responsiveness of postsynaptic GluR (leading to a decrease in quantal size) trigger a robust compensatory increase in presynaptic neurotransmitter release or quantal content (QC). This increase in QC restores evoked muscle responses to normal levels. A presynaptic KAR, KaiRID, has recently been implicated in basal neurotransmission and presynaptic homeostatic potentiation (PHP) at the larval NMJ (Kiragasi, 2017; Li, 2016). The role of KaiRID in modulation of basal neurotransmission resembles GluK2/GluK3 function as autoreceptors (Pinheiro, 2007). The role of KaiRID in PHP must be indirect, because a mutation that renders this receptor Ca2+ impermeable has no effect on the expression of presynaptic homeostasis (Kiragasi, 2017) (Han, 2020).

    The fly NMJ reliance on KARs raises the possibility that Drosophila diversified and maximized its use of Neto proteins. Drosophila Neto encodes two isoforms (Neto-α and Neto-β) with distinct intracellular domains generated by alternative splicing. Both cytoplasmic domains are rich in phosphorylation sites and docking motifs, suggesting rich modulation of Neto/KAR distribution and function. Neto-β, the predominant isoform at the larval NMJ, mediates intracellular interactions that recruit PSD components and enables synaptic stabilization of selective receptor subtypes. Neto-α can rescue viability and receptor clustering defects of Neto null. However, the endogenous functions of Neto-α remain unknown (Han, 2020).

    This study shows that Neto-α is key to synapse development and homeostasis and fulfills functions distinct from those of Neto-β. Using isoform-specific mutants and tissue-specific manipulations, it was found that loss of Neto-α in the postsynaptic muscle disrupts GluR fields and produces enlarged PSDs. Loss of presynaptic Neto-α disrupts basal neurotransmission and renders these NMJs unable to express PHP. The different functions of Neto-α were mapped to distinct protein domains and Neto-α was shown to be both required and sufficient for PHP, functioning as a bona fide effector for PHP. It is proposed that Drosophila ensured NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities (Han, 2020).

    This study showed that Neto-α is required in both pre- and postsynaptic compartments for the proper organization and function of the Drosophila NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field; the PSDs are significantly enlarged in muscle where Neto-α has been perturbed. In MNs, Neto-α is required for two distinct activities: (1) modulation of basal neurotransmission in a KaiRID-dependent manner and (2) effector of presynaptic homeostasis response. This is an extremely rare example of a GluR auxiliary protein that modulates receptors on both sides of a particular synapse and plays a distinct role in homeostatic plasticity (Han, 2020).

    Vertebrate KARs depend on Neto proteins for their distribution and function (Copits and Swanson, 2012). Because of their reliance on KARs, Drosophila Netonull mutants have no functional NMJs (no postsynaptic KARs) and consequently die as paralyzed embryos. Previous work has shown that muscle expression of Neto-ΔCTD, or minimal Neto, at least partly rescues the recruitment and function of KARs at synaptic locations. This study reports that neuronal Neto-ΔCTD also rescues the KaiRID-dependent basal neurotransmission. Thus, Neto-ΔCTD, the part of Neto conserved from worms to humans, seems to represent the Neto core required for KAR modulatory activities (Han, 2020).

    The intracellular parts of Neto proteins are highly divergent, likely reflecting the microenvironments in which different Neto proteins operate. Similar to mammalian Neto1 and Neto2, Drosophila Neto-α and Neto-β are differentially expressed in the CNS and have different intracellular domains that mediate distinct functions. These large intracellular domains are rich in putative phosphorylation sites and docking motifs and could further modulate the distribution and function of KARs or serve as signaling hubs and protein scaffolds. Post-translational modifications regulate vertebrate Neto activities in vitro, although the in vivo relevance of these changes remains unknown. The current data demonstrate that Neto-α and Neto-β could not substitute for each other. For example, Neto-β, but not Neto-α, controls the recruitment of PAK, a PSD component that stabilizes selective KAR subtypes at the NMJ, and ensures proper postsynaptic differentiation. Conversely, postsynaptic Neto-β alone cannot maintain a compact PSD size; muscle Neto-α is required for this function. Neto-β cannot fulfill presynaptic functions of Neto-α, presumably because is confined to the somato-dendritic compartment and cannot reach the synaptic terminals. Histology and western blot analyses indicate that Neto-α constitutes less than 1/10th of the net Neto at the Drosophila NMJ. These low levels impaired direct visualization of endogenous Neto-α. Several isoform-specific antibodies have been generated, but they could only detect Neto-α when overexpressed. Similar challenges have been encountered in the vertebrate Neto field (Han, 2020).

    The two Neto isoforms are limiting in different synaptic compartments. Neto-β limits the recruitment and synaptic stabilization of postsynaptic KARs. In contrast, several lines of evidence indicate that Neto-α is limiting in MNs. First, overexpression of KaiRID cannot increase basal neurotransmission (Kiragasi, 2017); however, neuronal overexpression of Neto-ΔCTD increases basal neurotransmission, indicating that Neto, but not KaiRID, is limiting in the MNs. Second, neuronal overexpression of Neto-α exacerbates the PHP response to PhTx exposure and even rescues this response in KaiRIDnull. These findings suggest that KaiRID's function during PHP is to help traffic and stabilize Neto-α, a low-abundance PHP effector. Similarly, studies in mammals reported that KARs trafficking in the CNS do not require Neto proteins; instead, KARs regulate the surface expression and stabilization of Neto1 and Neto2. Nonetheless, the KAR-mediated stabilization of Neto proteins at CNS synapses supports KAR distribution and function. In flies, KaiRID-dependent Neto-α stabilization at synaptic terminals ensures KAR-dependent function, normal basal neurotransmission, and Neto-α-specific activity as an effector of PHP (Han, 2020).

    Previous studies showed that presynaptic KARs regulate neurotransmitter release; however, the site and mechanism of action of presynaptic KARs have been difficult to pin down. This study provides strong evidence for Neto activities at presynaptic terminals. First, Neto-α is both required and sufficient for PHP. It has been shown that the PhTx-induced expression of PHP occurs even when the MN axon is severed. In addition, the signaling necessary for PHP expression is restricted to postsynaptic densities and presynaptic boutons. Second, Neto-ΔCTD, but not Neto-β, rescued basal neurotransmission defects in Neto-αnull. Both variants contain the minimal Neto required for KAR modulation, but only Neto-ΔCTD can reach the presynaptic terminal, whereas Neto-β is restricted to the somato-dendritic compartment. This suggests that Neto-ΔCTD (or Neto-α), together with KaiRID, localizes at presynaptic terminals, where KaiRID could function as an autoreceptor. Finally, upon PhTx exposure, Neto-α enabled fast recruitment of Brp at the active zone. Multiple homeostasis paradigms trigger Brp mobilization, followed by remodeling of presynaptic cytomatrix. These localized activities support Neto-α functioning at presynaptic terminals (Han, 2020).

    Rapid application of glutamate to outside-out patches from HEK cells transfected with KaiRID indicated that KaiRID forms rapidly desensitizing channels; addition of Neto increases the desensitization rates and open probability for this channel. Neto-α has a large intracellular domain (250 residues) rich in post-translational modification sites and docking motifs, including putative phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and protein kinase A (PKA). This intracellular domain may engage in finely tuned interactions that allow Neto-α to (1) further modulate the KaiRID properties and distribution in response to cellular signals and (2) function as an effector of presynaptic homeostasis in response to low postsynaptic GluR activity. Mammalian Neto1 and Neto2 are phosphorylated by multiple kinases in vitro (Lomash, 2017); CaMKII- and PKA-dependent phosphorylation of Neto2 restrict GluK1 targeting to synapses in vivo and in vitro. Similarly, Neto-α may function in a kinase-dependent manner to stabilize KaiRID and/or other presynaptic components. Second, Neto-α may recruit Brp or other presynaptic molecules that mediate activity-related changes in glutamate release at the fly NMJ. Besides Brp, several presynaptic components have been implicated in the control of PHP. They include (1) Cacophony (Cac), the α1 subunit of CaV2-type calcium channels and its auxiliary protein α2Δ-3, that control the presynaptic Ca2+ influx; (2) the signaling molecules Eph, Ephexin, and Cdc42 upstream of Cac; and (3) the BMP pathway components, Wit and Mad, required for retrograde BMP signaling. In addition, expression of PHP requires molecules that regulate vesicle release and the RRP size, such as RIM, Rab3-GAP, Dysbindin, and SNAP25 and Snapin. Recent studies demonstrated that trans-synaptic Semaphorin/Plexin interactions control synaptic scaling in cortical neurons in vertebrates and drive PHP at the fly NMJ (Orr, 2017a). Neto-α may interact with one or several such presynaptic molecules and function as an effector of PHP. Future studies on what the Neto-α cytoplasmic domain binds to and how is it modulated by post-translational modifications should provide key insights into the understanding of molecular mechanisms of homeostatic plasticity (Han, 2020).

    On the muscle side, Neto-α activities may include (1) engaging scaffolds that limit the PSD size and (2) modulating postsynaptic KAR distribution and function. For example, Neto-α may recruit trans-synaptic complexes such as Ten-a/Ten-m or Nrx/Nlgs that have been implicated in limiting the postsynaptic fields (Banovic, 2010; Mosca, 2012). In particular, DNlg3, like Neto-α, is present in both pre- and postsynaptic compartments and has similar loss-of function phenotypes, including smaller boutons with larger individual PSDs, and reduced EJP amplitudes (Xing, 2014). Neto-α may also indirectly interact with the Drosophila PSD-95 and Dlg and help establish the PSD boundaries. Fly Netos do not have PDZ binding domains, but the postsynaptic Neto/KAR complexes contain GluRIIC, a subunit with a class II PDZ binding domain. It has been reported that mutations that change the NMJ receptors' gating behavior alter their synaptic trafficking and distribution (Petzoldt, 2014). Neto-α could be key to these observations, because it may influence both receptor gating properties and ability to interact with synapse organizers (Han, 2020).

    Phylogenetic analyses indicate that Neto-β is the ancestral Neto. In insects, Neto-β is predicted to control NMJ development and function, including recruitment of iGluRs and PSD components, and postsynaptic differentiation. Neto-α appears to be a rapidly evolving isoform present in higher Diptera. This large order of insects is characterized by a rapid expansion of the KAR branch to ten distinct subunits. Insect KARs have unique ligand binding profiles, strikingly different from vertebrate KARs. However, like vertebrate KARs, they all seem to be modulated by Neto proteins. It is speculated that the rapid expansion of KARs forced the diversification of the relevant accessory protein, Neto, and the extension of its repertoire. In flies, the Neto locus acquired an additional exon and consequently an alternative isoform with distinct expression profiles, subcellular distributions, and isoform-specific functions. It will be interesting to investigate how flies differentially regulate the expression and distribution of the two Neto isoforms and control their tissue- and synapse-specific functions. Mammals have five KAR subunits, three of which have multiple splice variants that confer rich regulation. In addition, mammalian Neto proteins have fairly divergent intracellular parts that presumably further integrate cell-specific signals and fine-tune KAR localization and function. In Diptera, KARs have relatively short C tails and thus limited signaling input, whereas Netos have long cytoplasmic domains that could function as scaffolds and signaling hubs. Consequently, most information critical for NMJ assembly and postsynaptic differentiation has been outsourced to the intracellular part of Neto-β. Neto-α-mediated intracellular interactions may also hold key insights into the mechanisms of homeostatic plasticity. This study reveals that Neto functions as a bona fide effector of presynaptic homeostasis (Han, 2020).

    Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function

    Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. This study reports that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1 (RhoGAP100F). Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. It is concluded that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis (Muhammad, 2015).

    Chemical synapses release synaptic vesicles (SVs) at specialized presynaptic membranes, so-called active zones (AZs), which are characterized by electron-dense structures, reflecting the presence of extended molecular protein scaffolds. These AZ scaffolds confer stability and facilitate SV release. Importantly, at individual AZs, scaffold size is found to scale with the propensity to engage in action potential-evoked release. An evolutionarily conserved set of large multi-domain proteins operating as major building blocks for these scaffolds has been identified over the last years: Syd-2/Liprin-α, RIM, RIM-binding-protein (RBP) and ELKS family proteins (of which the the Drosophila homologue is called Bruchpilot (BRP)). However, how presynaptic scaffold assembly and maturation are controlled and coupled spatiotemporally to the postsynaptic assembly of neurotransmitter receptors remains largely unknown, although trans-synaptic signalling via Neurexin-1 (Nrx-1)-Neuroligin-1 (Nlg1) adhesion molecules is a strong candidate for a conserved 'master module' in this context, based on Nrx-Nlg signalling promoting synaptogenesis in vitro, synapses of rodents, Caenorhabditis elegans and Drosophila (Muhammad, 2015).

    With respect to scaffolding proteins, Syd-1 was found to promote synapse assembly in C. elegans, Drosophila and rodents. In Drosophila, the Syd-1-PDZ domain binds the Nrx-1 C terminus and couples pre- with postsynaptic maturation at nascent synapses of glutamatergic neuromuscular junctions (NMJs) in Drosophila larvae. Syd-1 cooperates with Nrx-1/Nlg1 to stabilize newly formed AZ scaffolds, allowing them to overcome a 'threshold' for synapse formation. Additional factors tuning scaffold assembly, however, remain to be identified. This study shows that the conserved scaffold protein spinophilin (Spn) is able to fine-tune Nrx-1 function by binding the Nrx-1 C terminus with micromolar affinity via its PDZ domain. In the absence of presynaptic Spn, 'excessive seeding' of new AZs occurred over the entire NMJ due to elevated Nrx-1/Nlg1 signalling. Apart from structural changes, this study shows that Spn plays an important role in neurotransmission since it is essential to establish proper SV release probability, resulting in a changed ratio of spontaneous versus evoked release at Spn NMJ terminals. The trans-synaptic dialogue between Nrx-1 and Nlg1 aids in the initial assembly, specification and maturation of synapses, and is a key component in the modification of neuronal networks. Regulatory factors and processes that fine-tune and coordinate Nrx-1/Nlg1 signalling during synapse assembly process are currently under investigation. These data indicate that Drosophila Spn-like protein acts presynaptically to attenuate Nrx-1/Nlg1 signalling and protects from excessive seeding of new AZ scaffolds at the NMJ. In Spn mutants, excessive AZs suffered from insufficient evoked release, which may be partly explained by their reduced size, and partly by a genuine functional role of Spn (potentially mediated via Nrx-1 binding). In mice, loss of Spn (Neurabin II), one of the two Neurabin protein families present in mammals, was reported to provoke a developmental increase in synapse numbers (Feng, 2000). While Spinophilin was found to be expressed both pre- and post-synaptically (Muly, 2004; Muly, 2004a; Muly, 2004b), its function, so far, has only been analysed in the context of postsynaptic spines (Feng, 2000; Terry-Lorenzo 2005; Allen, 2006; Sarrouilhe, 2005). Given the conserved Spn/Nrx-1 interaction reported in this study, Spn family proteins might execute a generic function in controlling Nrx-1/Nlg1-dependent signalling during synapse assembly (Muhammad, 2015).

    This study consistently found that Spn counteracts another multi-domain synaptic regulator, Syd-1, in the control of Nrx-1/Nlg1 signalling. Previous genetic work in C. elegans identified roles of Syd-1 epistatic to Syd-2/Liprin-α in synaptogenesis. Syd-1 also operates epistatic to Syd-2/Liprin-α at Drosophila NMJs. Syd-1 immobilizes Nrx-1, positioning Nlg1 at juxtaposed postsynaptic sites, where it is needed for efficient incorporation of GluR complexes. Intravital imaging suggested an early checkpoint for synapse assembly, involving Syd-1, Nrx-1/Nlg1 signalling and oligomerization of Liprin-α in the formation of an early nucleation lattice, which is followed later by ELKS/BRP-dependent scaffolding events. As Spn promotes the diffusional motility of Nrx-1 over the terminal surface and limits Nrx-1/Nlg1 signalling, and as its phenotype is reversed by loss of a single gene copy of nrx-1, nlg1 or syd-1, Spn displays all the features of a 'negative' element mounting, which effectively sets the threshold for AZ assembly. As suggested by FRAP experiments, Spn might withdraw a population of Nrx-1 from the early assembly process, establishing an assembly threshold that ensures a 'typical' AZ design and associated postsynaptic compartments. As a negative regulatory element, Spn might allow tuning of presynaptic AZ scaffold size and function (Muhammad, 2015).

    The C. elegans Spn homologue NAB-1 (NeurABin1) was previously shown to bind Syd-1 in cell culture recruitment assays (Chia, 2012). This study found consistent evidence for Syd-1/Nrx-1/Spn tripartite complexes in salivary gland experiments. Moreover, the PDZ domain containing regions of Spn and Syd-1 interacted in Y2H experiments. It would be interesting to dissect whether the interaction of Spn/Syd-1 plays a role in controlling the access of Nrx-1 to one or both factors. For C. elegans HSN synapses, a previous study showed that loss of NAB-1 results in a deficit of synaptic markers, such as Syd-1 and Syd-2/Liprin-α, while NAB-1 binding to F-actin was also found to be important for synapse assembly. Though at first glance rather contradictory to the results described in this study, differences might result from Chia (2012) studying synapse assembly executed over a short time window, when partner cells meet for the first time. In contrast, this study used a model (Drosophila larval NMJs) where an already functional neuronal terminal adds novel AZs. Despite the efforts of this study, no role of F-actin in the assembly of AZs of late larval Drosophila NMJs was demonstrated. F-actin patches might be particularly important to establish the first synaptic contacts between partner cells. Both the study by Chia et al. and this study, however, point clearly towards important regulatory roles of Spn family members in the presynaptic control of synapse assembly. Further, this study described a novel interaction between the Spn-PDZ domain and the intracellular C-term of Nrx-1 at the atomic level. Interestingly, it was found that all functions of Spn reported in this study, structural as well as functional, were strictly dependent on the ligand-binding integrity of this PDZ domain. It is noteworthy that the Spn-PDZ domain binds other ligands as well, for example, Kalirin-7 and p70S6K , and further elucidation of its role as a signal 'integrator' in synapse plasticity should be interesting. The fact that Nrx-1 levels were increased at Spn NMJs and, most importantly, that genetic removal of a single nrx-1 gene copy effectively suppressed the Spn AZ phenotype, indicates an important role of the Spn/Nrx-1 interaction in this context. Affinity of Spn-PDZ for the Nrx-1 C-term was somewhat lower than that of the Syd-1-PDZ, both in ITC and Y2H experiments. Nonetheless, overexpression of Spn was successful in reducing the targeting effect of Syd-1 on overexpressed Nrx-1GFP. It will be interesting to see whether this interaction can be differentially regulated, for example, by (de)phosphorylation. It is worth noting that apart from Syd-1 and Spn, several other proteins containing PDZ domains, including CASK, Mint1/X11, CIPP and Syntenin, were found to bind to the Nrxs C-termini. CASK was previously shown to interact genetically with Nrx-1, controlling endocytic function at Drosophila NMJs. However, when this study tested for an influence of CASK on Nrx-1GFP motility using FRAP, genetic ablation of CASK had no effect (Muhammad, 2015).

    Thus, CASK function seemingly resembles neither Syd-1 nor Spn. Clearly, future work will have to address and integrate the role of other synaptic regulators converging on the Nrx-1 C-term. In particular, CASK (which displays a kinase function that phosphorylates certain motifs within the Nrx-1 C-term) might alternately control Spn- and Syd-1-dependent functions. Presynaptic Nrx-1, through binding to postsynaptic Nlg1 at developing Drosophila NMJ terminals, is important for the proper assembly of new synaptic sites. It is of note, however, that while mammalian Nrxs display robust synaptogenetic activity in cellular in vitro systems, direct genetic evidence for synaptogenetic activity of Nrxs in the mammalian CNS remained rather scarce. Triple knockout mice lacking all α-Nrxs display no gross synaptic defects at the ultrastructural level. Future analysis will have to investigate whether differences here might be explained by specific compensation mechanisms in mammals; for example, by β-Nrxs, or other parallel trans-synaptic communication modules. Genuine functional deficits in neurotransmitter release were also observed after the elimination of presynaptic Spn. Elimination of ligand binding to the PDZ domain rendered the protein completely nonfunctional, without affecting its synaptic targeting. Thus, the Spn functional defects are likely to be mediated via a lack of Nrx-1 binding. Notably, ample evidence connects Nrx-1 function with both the functional and structural maturation of Drosophila presynaptic AZs. This work now promotes the possibility that binding of Spn to Nrx-1 is important for establishing correct release probability, independent of absolute AZ scaffold size. It is noteworthy that Nrx-1 function was previously shown to be important for proper Ca2+ channel function and, as a result, properly evoked SV release. Thus, it will be interesting to investigate whether the specific functional contributions of Spn are mediated via deficits in the AZ organization of voltage-gated Ca2+ channels or Ca2+ sensors, such as synaptotagmin. Taken together, this study found an unexpected function for Spn in addition of AZs at Drosophila glutamatergic terminals, through the integration of signals from both the pre- and postsynaptic compartment. Given that the Spn/Nrx-1 interaction is found to be conserved from Drosophila to rodents, addressing similar roles of presynaptic Spn in mammalian brain physiology and pathophysiology might be informative (Muhammad, 2015).

    Neurexin, neuroligin and wishful thinking coordinate synaptic cytoarchitecture and growth at neuromuscular junctions

    Using full length and truncated forms of Neurexin (Dnrx) and Neuroligins (Dnlg) together with cell biological analyses and genetic interactions, this study reports novel functions of dnrx and dnlg1 in clustering of pre- and postsynaptic proteins, coordination of synaptic growth and ultrastructural organization. dnrx and dnlg1 extracellular and intracellular regions are required for proper synaptic growth and localization of dnlg1 and Dnrx, respectively. dnrx and dnlg1 single and double mutants display altered subcellular distribution of Discs large (Dlg), which is the homolog of mammalian post-synaptic density protein, PSD95. dnrx and dnlg1 mutants also display ultrastructural defects ranging from abnormal active zones, misformed pre- and post-synaptic areas with underdeveloped subsynaptic reticulum. Interestingly, dnrx and dnlg1 mutants have reduced levels of the BMP receptor Wishful thinking (Wit), and dnrx and dnlg1 are required for proper localization and stability of Wit. In addition, the synaptic overgrowth phenotype resulting from the overexpression of dnrx fails to manifest in wit mutants. Phenotypic analyses of dnrx/wit and dnlg1/wit mutants indicate that Dnrx/Dnlg1/Wit coordinate synaptic growth and architecture at the NMJ. These findings also demonstrate that loss of dnrx and dnlg1 leads to decreased levels of the BMP co-receptor, Thickveins and the downstream effector phosphorylated Mad at the NMJ synapses indicating that Dnrx/Dnlg1 regulate components of the BMP signaling pathway. Together these findings reveal that Dnrx/Dnlg are at the core of a highly orchestrated process that combines adhesive and signaling mechanisms to ensure proper synaptic organization and growth during NMJ development (Banerjee, 2016).

    Neurexins and Neuroligins have emerged as major players in the organization of excitatory and inhibitory synapses across species. Mutational analyses in Drosophila uncovered specific functions of dnrx and dnlg1 in NMJ synapse organization and growth, where dnrx is expressed pre-synaptically and dnlg1 post-synaptically. Deletion of the extracellular and intracellular regions of dnrx or dnlg1 revealed that both regions are necessary for dnrx and dnlg1 clustering and function at the synapse. Most importantly, the data presented here suggest that dnrx and dnlg1 genetically interact with wit as well as the downstream effector of BMP signaling, Mad to allow both the organization and growth of NMJ synapses. The surprising finding that loss of dnrx and dnlg1 leads to decreased wit stability and that dnrx and dnlg1 are required for proper wit localization raises the possibility that these proteins function to coordinate trans-synaptic adhesion and synaptic growth. This is further strengthened from the observations that, in the absence of Wit, the synaptogenic activity from overexpression of dnrx did not manifest into an increased synaptic growth as is seen in the presence of wit. Loss of dnrx and dnlg1 also led to a reduction in the levels of other components of the BMP pathway, namely the Tkv and pMad. Together these findings are the first to demonstrate a functional coordination between trans-synaptic adhesion proteins dnrx and dnlg1 with wit receptor and BMP pathway members to allow precise synaptic organization and growth at NMJ. It would be of immense interest to investigate whether similar mechanisms might be operating in vertebrate systems (Banerjee, 2016).

    The trans-synaptic cell adhesion complex formed by heterophilic binding of pre-synaptic Neurexins (Nrxs) and post-synaptic Neuroligins (Nlgs), displayed synaptogenic function in cell culture experiments. In vertebrates and invertebrates alike, Nrxs and Nlgs belong to one of the most extensively studied synaptic adhesion molecules with a specific role in synapse organization and function. In Drosophila, dnrx and dnlg1 mutations cause reductions in bouton number, perturbation in active zone organization and severe reduction in synaptic transmission. These phenotypes are essentially phenocopied in both dnrx and dnlg1 mutants, and double mutants do not cause any significant enhancement in the single mutant phenotypes illustrating that they likely function in the same pathway. From immunohistochemical and biochemical analyses, it is evident that lack of dnrx or dnlg1 causes their diffuse localization and protein instability in each other's mutant backgrounds. These data suggest that trans-synaptic interaction between dnrx and dnlg1 is required for their proper localization and stability, and that these proteins have a broader function in the context of general synaptic machinery involving Nrx-Nlg across phyla. It also raises the possibility that the trans-synaptic molecular complex involving Nrx-Nlg may alter stability of other synaptic proteins and lead to impairments in synaptic function without completely abolishing synaptic structure and neurotransmission. It is therefore not surprising that Nrx and Nlg have been recently reported to be associated with many non-lethal cognitive and neurological disorders, such as schizophrenia, ASD, and learning disability (Banerjee, 2016).

    The rescue studies of dnrx and dnlg1 localization using their respective N- and C-terminal domain truncations emphasize a requirement of the full-length protein for proper synaptic clustering. Given that dnrx and dnlg1 likely interact via their extracellular domains, it is somewhat expected that an N-terminal deletion as seen in genotypic combinations of elav-Gal4/UAS-DnrxΔ N;dnrx-/- and mef2-Gal4/UAS-dnlg1Δ N;dnlg1-/- would fail to rescue the synaptic clustering of Dnlg1 and Dnrx, respectively. However, the inability of the C-terminal deletions of these proteins as seen in elav-Gal4/UAS-DnrxΔ c;dnrx-/- and mef2-Gal4/UAS-dnlg1Δ Cdnlg1-/- to cluster Dnlg1 and Dnrx, respectively, to wild type localization and/or levels suggest that the lack of the cytoplasmic domains of these proteins may render the remainder portion of the protein unstable, thereby leading to its inability to be either recruited or held at the synaptic apparatus (Banerjee, 2016).

    Finally, the subcellular localization of Dlg at the SSR and its levels in the dnrx and dnlg1 single and double mutants as well as in the rescue genotypes provides key insights into the stoichiometry of Dnrx-Dnlg1 interactions, and how Dnrx-Dnlg1 might be functioning with other synaptic proteins in their vicinity to organize the synaptic machinery. A significant reduction in Dlg levels in dnrx mutants raises the question of whether Dlg localization/levels are controlled presynaptically? Alternatively, it is possible that Dnrx-Dnlg1 is not mutually exclusive for all their synaptic functions and there might be other Neuroligins that might function with Dnrx. One attractive candidate could be Dnlg2, which is both pre- and postsynaptic. dnrx could also function through the recently identified Neuroligins 3 and 4. Although dnlg1 mutants did not show any significant difference in Dlg levels, a diffuse subcellular localization nevertheless raised the possibility of a structural disorganization of the postsynaptic terminal and defects in SSR morphology, which were confirmed by ultrastructural studies. The rescue analysis of the subcellular localization of Dlg demonstrates that Dlg localization and levels could be restored fully in a presynaptic rescue of dnrx mutants by expression of full length Dnrx, however the localization of Dlg could not be restored by expression of DnrxΔNT. These observations suggest that the extracellular domain of dnrx is essential for the localization and also the levels of Dlg. Whether the extracellular domain influences Dlg via dnlg1 or any of the other three Dnlgs (2, 3 and 4) at the NMJ remains to be elucidated (Banerjee, 2016).

    Most studies on Nrx/Nlg across species offer clues as to how these proteins assemble synapses and how they might function in the brain to establish and modify neuronal network properties and cognition, however, very little is known on the signaling pathways that these proteins may potentially function in. It has been previously reported that Neurexin 1 is induced by BMP growth factors in vitro and in vivo and that could possibly allow regulation of synaptic growth and development. In Drosophila, both dnrx and dnlg1 mutants showed reduced synaptic growth similar to wit pathway mutants. Reduced wit levels both from immunolocalization studies in dnrx and dnlg1 mutant backgrounds and biochemical studies from immunoblot and sucrose density gradient sedimentation analyses present compelling evidence towards the requirement of dnrx and dnlg1 for wit stability. Reduction of synaptic dnrx levels in wit mutants argue for interdependence in the localization of these presynaptic proteins at the NMJ synaptic boutons. The findings that synaptic boutons of dnrx and dnlg1 mutants also show reduction in the levels of the co-receptor Tkv suggest that this effect may not be exclusively Wit-specific, and possibly due to the overall integrity of trans-synaptic adhesion complex that ensures Wit and Tkv stability at the NMJ (Banerjee, 2016).

    Genetic interaction studies displayed a significant reduction in bouton numbers resulting from trans-heterozygous combinations of wit/+,dnrx/+ and wit/+,dnlg1/+ compared to the single heterozygotes strongly favoring the likelihood of these genes functioning together in the same pathway. Although double mutants of wit,dnrx and wit,dnlg1 are somewhat severe than dnrx and dnlg1 single mutants, they did not reveal any significant differences in their bouton counts compared to wit single mutants. Moreover, genetic interactions between Mad;dnrx and Mad;dnlg1 together with decreased levels of pMad in dnrx and dnlg1 mutants provided further evidence that dnrx and dnlg1 regulate components of the BMP pathway. Interestingly, reduced levels of pMad were observed in dnrx and dnlg1 mutants, in contrast to a recent study that reported higher level of synaptic pMad in dnrx and dnlg1 mutants. The differences in pMad levels encountered in these two studies could be attributed to differences in rearing conditions, nature of the food and genetic backgrounds, as these factors have been invoked to affect synaptic pMad levels. These findings strongly support that dnrx, dnlg1 and wit function cooperatively to coordinate synaptic growth and signaling at the NMJ (Banerjee, 2016).

    Loss of either dnrx or dnlg1 does not completely abolish the apposition of pre- and post-synaptic membrane at the NMJ synapses but detachments that occur at multiple sites along the synaptic zone. These observations point to either unique clustering of the Dnlg1/Dnrx molecular complexes or preservation of trans-synaptic adhesion by other adhesion molecules at the NMJ synapses. Indeed, recent studies show nanoscale organization of synaptic adhesion molecules Neurexin 1Β, NLG1 and LRRTM2 to form trans-synaptic adhesive structures (Chamma, 2016). In addition to dnrx and dnlg1 mutants, synaptic ultrastructural analysis showed similarity in presynaptic membrane detachments with a characteristic ruffling morphology in wit mutants as well suggesting that these proteins are required for maintaining trans-synaptic adhesion. Interestingly, double mutants from any combinations of these three genes such as dnlg1,dnrx or wit,dnrx and wit,dnlg1 did not show any severity in the detachment/ruffling of the presynaptic membrane suggesting that there might be a phenotypic threshold that cannot be surpassed as part of an intrinsic synaptic machinery to preserve its very structure and function. It would be interesting to test this possibility if more than two genes are lost simultaneously as in a triple mutant combination. Alternatively, there could be presence of other distinct adhesive complexes that remain intact and function outside the realm of Dnrx, dnlg1 and wit proteins thus preventing a complete disintegration of the synaptic apparatus (Banerjee, 2016).

    The same holds true for most of the ultrastructural pre- and postsynaptic differentiation defects observed in the single and double mutants of dnrx, dnlg1 and wit. Barring dnrx,dnlg1 double mutants in which the SSR width showed a significant reduction compared to the individual single mutants, all other phenotypes documented from the ultrastructural analysis showed no difference in severity between single and double mutants. Another common theme that emerged from the ultrastructural analysis was that loss of either pre- or postsynaptic proteins or any combinations thereof, showed a mixture of defects that spanned both sides of the synaptic terminal. For example, presynaptic phenotypes such as increased number of active zones or abnormally long active zones as well as postsynaptic phenotypes such as reduction in width and density of the SSR were observed when presynaptic proteins such as dnrx and wit and postsynaptic dnlg1 were lost individually or in combination. These studies suggest that pre- and postsynaptic differentiation is tightly regulated and not mutually exclusive where loss of presynaptic proteins would result only in presynaptic deficits and vice-versa (Banerjee, 2016).

    The postsynaptic SSR phenotypes seen in the single and double mutants of dnrx, dnlg1 and wit might be due to their interaction/association with postsynaptic or perisynaptic protein complexes such as Dlg and Fasciclin II. Alternatively, the postsynaptic differentiation or maturation deficits in these mutants could also be due to a failure of postsynaptic GluRs to be localized properly or their levels maintained sufficiently. It has been shown previously that lack of GluR complexes interferes with the formation of SSR. Deficits in postsynaptic density assembly have been previously reported for dnlg1 mutants, including a misalignment of the postsynaptic GluR fields with the presynaptic transmitter release sites. GluR distribution also showed profound abnormalities in dnrx mutants. These observations suggest that trans-synaptic adhesion and synapse organization and growth is highly coordinated during development, and that multiple molecular complexes may engage in ensuring proper synaptic development. Some of these questions need to be addressed in future investigations (Banerjee, 2016).

    In Drosophila, the postsynaptic muscle-derived BMP ligand, Glass bottom boat (Gbb), binds to type II receptor Wit, and type I receptors Tkv, and Saxophone (Sax) at the NMJ. Receptor activation by Gbb leads to the recruitment and phosphorylation of Mad at the NMJ terminals followed by nuclear translocation of pMad with the co-Smad, Medea, and transcriptional initiation of other downstream targets. It is interesting to note that previously published studies revealed that a postsynaptic signaling event occurs during larval development mediated by Type I receptor Tkv and Mad. Based on findings from this study, it is speculated that Dnrx and Dnlg1-mediated trans-synaptic adhesive complex allows recruitment and stabilization of wit and associated components to assemble a larger BMP signaling complex to ensure proper downstream signaling. Loss of dnrx and/or dnlg1 results in loss of adhesion and a decrease in the levels of Wit/Tkv receptors as well as decreased phosphorylation of Mad. Thus a combination of trans-synaptic adhesion and signaling mediated by Dnrx, Dnlg1 and components of the BMP pathway orchestrate the assembly of the NMJ and coordinate proper synaptic growth and architecture (Banerjee, 2016).

    The data presented in this study address fundamental questions of how the interplay of pre- and postsynaptic proteins contributes towards the trans-synaptic adhesion, synapse differentiation and growth during organismal development. dnrx and dnlg1 establish trans-synaptic adhesion and functionally associate with the presynaptic signaling receptor wit to engage as a molecular machinery to coordinate synaptic growth, cytoarchitecture and signaling. dnrx and dnlg1 also function in regulating BMP receptor levels (Wit and Tkv) as well as the downstream effector, Mad, at the NMJ. It is thus conceivable that at the molecular level setting up of a Dnrx-Dnlg1 mediated trans-synaptic adhesion is a critical component for molecules such as Wit and Tkv to perform signaling function. It would be of immense interest to investigate how mammalian Neurexins and Neuroligins are engaged with signaling pathways that not only are involved in synapse formation but also their functional modulation, as the respective genetic loci show strong associations with cognitive and neurodevelopmental disorders (Banerjee, 2016).

    A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity

    Evidence has begun to emerge for microRNAs as regulators of synaptic signaling, specifically acting to control postsynaptic responsiveness during synaptic transmission. This report provides evidence that Drosophila melanogaster miR-1000 acts presynaptically to regulate glutamate release at the synapse by controlling expression of the vesicular glutamate transporter (VGlut). Genetic deletion of miR-1000 led to elevated apoptosis in the brain as a result of glutamatergic excitotoxicity. The seed-similar miR-137 regulates VGluT2 expression in mouse neurons. These conserved miRNAs share a neuroprotective function in the brains of flies and mice. Drosophila miR-1000 showed activity-dependent expression, which might serve as a mechanism to allow neuronal activity to fine-tune the strength of excitatory synaptic transmission (Verma, 2015).

    miRNAs have emerged in recent years as important regulators of homeostatic mechanisms. Changes in miRNA expression and activity have been linked to neurodegenerative disorders. A growing body of evidence suggests that miRNAs are neuroprotective in the aging brain, as well as in the control of synaptic function and plasticity. Mouse miR-134 acts postsynaptically to regulate synapse strength, and miR-181 and miR-223 regulate glutamate receptors, thereby affecting postsynaptic responsiveness to glutamate. miR-1, a muscle-specific miRNA, acts in a retrograde fashion at the neuromuscular junction to regulate the kinetics of synaptic vesicle exocytosis. However, there are few examples of miRNAs acting directly in the presynaptic terminal to control synaptic strength. miR-485, which is found presynaptically, has been shown to control the expression of synaptic vesicle protein SV2A, thereby affecting synapse density and GluR2 receptor levels (Verma, 2015).

    This paper reports that Drosophila miR-1000 regulates neurotransmitter release from presynaptic terminals. miR-1000 regulates expression of the VGlut, which loads glutamate into synaptic vesicles. Genetic ablation of miR-1000 leads to glutamate excitotoxicity, resulting in early-onset neuronal death. Presynaptic regulation of miR-1000 is activity dependent and may serve as a mechanism for tuning synaptic transmission. Evidence is presented that this regulatory relationship is conserved in the mammalian CNS, with a seed-similar miRNA, miR-137, conferring neuroprotection through regulation of VGluT2. The consequences of misregulation of glutamatergic signaling can be severe: excitotoxicity due to excessive glutamate release has been implicated in ischemia and traumatic brain injury, as well as in neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis (Verma, 2015).

    Although postsynaptic regulation of glutamate receptor activity has been well studied, much less is known about presynaptic regulation of glutamatergic signaling. These findings suggest that miR-1000 acts presynaptically to regulate VGlut expression and thereby control synaptic glutamate release. It is tempting to speculate that this could provide a mechanism for tuning synaptic output and locally modulating synaptic strength. Such a mechanism would be most useful if the miRNA itself could be regulated in an activity-dependent manner. Evidence is provided that miR-1000 expression is regulated by light in vivo, presumably reflecting photoreceptor activity in the eye. This in turn leads to light-regulated regulation of VGlut reporter levels. These findings lend support to the notion of activity-dependent regulation of miR-1000 activity. An in depth exploration of these issues awaits the development of methods to monitor changes in presynaptic miRNA levels in real time (Verma, 2015).

    Failure of miR-1000-mediated regulation of VGlut led to excess glutamate release and resulted in excitotoxicity. Consistent with these findings, Gal4-directed overexpression of VGlut has been reported to cause neurodegeneration. Notably, elevated levels of vertebrate VGluTs have been associated with excitotoxicity in animal models of epilepsy and traumatic brain injury. The GAERS rat epilepsy model shows elevated levels of VGluT2 but not of VGluT1. Similarly, in a model of stroke, ischemic injury was found to result in elevated expression of VGluT1 but not of VGluT2. VGluT1 levels are regulated by methamphetamine treatment, likely contributing to excitotoxic consequences of methamphetamine abuse. VGluT1 levels have also been reported to increase in rat brains following antidepressant treatment (Verma, 2015).

    In the mouse, miR-223 acts on postsynaptic glutamate receptors and has a neuroprotective role in vivo. These findings provide evidence that miR-1000 has a neuroprotective role mediated through regulation of presynaptic glutamate release and that this regulatory mechanism is conserved for miR-137 and VGluT2 in the mouse. Together, these studies show that miRNA-mediated regulation of glutamatergic activity acts pre- and post-synaptically to modulate synaptic transmission and to protect against excitotoxicity. In this context, it is noteworthy that miR-137 is reported to be enriched at synapses. miR-137 levels were found to be low in a subset of Alzheimer's patients with elevated serine palmitoyltransferase 1 expression leading to increased ceramide production. Single nucleotide polymorphisms affecting miR-137 target sites could lead to low-level constitutive overexpression of its targets, even when the SNP is present in a single copy. A single nucleotide polymorphism affecting miR-137 has also been identified as a risk factor for schizophrenia. It will be of interest to learn whether misregulation of VGluT2 expression contributes to these neurological conditions. The current findings raise the possibility that miRNA mediated regulation makes VGluT and other miRNA targets possible risk factors in neurodegenerative disease (Verma, 2015).

    Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins

    Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. This work presents evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release (Uytterhoeven, 2011).

    Synaptic vesicles recycle locally at the synapse, and this study now provides genetic evidence that the synapse holds the capacity to regulate the sorting of synaptic vesicle proteins at 2xFYVE and Rab5 positive endosomes. 2xFYVE-GFP positive endosomes involved in signaling pathways exist at nerve endings. Likewise, membrane invaginations and endosomal-like cisternae form as a result of bulk membrane uptake during intense nerve stimulation, and in various endocytic mutants. However, the current data indicate that endosomes that accumulate in sky mutants are fundamentally different from these 'endocytic cisternae.' First, endocytic cisternae are not enriched for the endosomal markers 2xFYVE and Rab5. Second, endosomal structures in sky mutants do not appear to directly form at the plasma membrane. Third, endocytic cisternae may directly fuse with the membrane, however, based on mEJC and TEM analyses, sky induced endosomes appear to function as an intermediate station. Note that FM 1-43 dye can enter and leave endosomes in sky mutants, suggesting small synaptic vesicles can form at these structures. Together with time-course experiments in sky mutants, endosomes specifically accumulate upon stimulation and dissipate during rest. Therefore, the data suggest that endosomes in sky mutants constitute an intermediate step in the synaptic vesicle cycle. Such a compartment may also be operational in wild-type synapses because a 2xFYVE-GFP endosomal compartment can be largely depleted from synaptic vesicles upon endocytic blockade and because synaptic vesicles harbor proteins that are also commonly found on endosomes (Uytterhoeven, 2011).

    Rab GTPases are involved in numerous intracellular trafficking events. In particular in synaptic vesicle traffic, Rab3, and its close isoform Rab27 have been implicated in vesicle tethering and/or priming, while Rab5 has been also implicated in endocytosis. However, an involvement of Rab proteins in other aspects of the synaptic vesicle cycle, including recycling, remains enigmatic. This systematic analysis of CA Rabs now implicates several Rabs in synaptic recycling. First, consistent with previous results, the data suggest that Rab5 mediates transport to synaptic endosomes. Second, Rab7CA and Rab11CA appear to inhibit new vesicle formation, likely by controlling post endosome trafficking. Finally, this study found that Rab23CA and Rab35CA mediate transport to or retention of synaptic vesicles at sub-boutonic structures. Neither of these Rabs had yet been implicated in synaptic vesicle traffic and hence, these in vivo studies identified different Rab proteins involved in aspects of the synaptic vesicle cycle (Uytterhoeven, 2011).

    The expression of Rab5CA and Rab35CA results in increased trafficking of vesicles to -or retention of vesicles at- sub-boutonic structures, and unlike expression of Rab23CA, Rab5CA and Rab35CA also result in a facilitation of neurotransmitter release similar to sky mutants. Rab35 has been found to localize to the plasma membrane (Sato, 2008) and this study shows enrichment at NMJ boutons close to the membrane as well. Rab35 has been implicated in endosomal, clathrin-dependent traffic, phagocytic membrane uptake in non-neuronal cells and actin filament assembly during Drosophila bristle development (Allaire, 2010, Sato, 2008, Shim, 2010, Zhang, 2009). Given these roles, Rab35 is ideally posed to also play a role in the endosomal traffic of synaptic vesicles, and the current in vitro and in vivo genetic interaction studies indicate Sky to be a Rab35 GAP in synaptic vesicle trafficking. Although Rab5 mediates endosomal traffic in different cell types, the data indicate that Sky is not a GAP for Rab5 in vivo at the synapse. While this study does not exclude Sky activating the GTPase activity of alternative Rabs in different contexts, the results suggest a Sky-Rab35 partnership that restricts endosomal trafficking of synaptic vesicles (Uytterhoeven, 2011).

    In most cell types the ESCRT complex mediates sorting of ubiquitinated proteins into multivesicular bodies; however, such a function has not been characterized for synaptic vesicle components. This study provide evidence that the ESCRT complex mediates synaptic vesicle protein sorting in sky mutants where synaptic vesicles cycle excessively via endosomes. This study found genetic interactions with ESCRT genes. In addition, more efficient clearance of an artificially ubiquitinated synaptic vesicle protein, Ub-nSybHA, was also found in sky mutants, and this effect is ESCRT dependent. Combined, the data indicate endosomal sorting to control the quality of proteins in the synaptic vesicle cycle in sky mutants (Uytterhoeven, 2011).

    Increased transmitter release as a result of endosomal sorting e.g. in sky mutants, may appear beneficial in some instances, however in inhibitory neurons for example, such an effect could result in reduced neuronal signaling while in excitatory neurons this feature could over activate postsynaptic cells. Clearly, fine-tuned neurotransmission in neuronal populations may ensure optimal information flow, and misregulation of Sky activity in neuronal populations may cause systemic defects including larval paralysis and death. Further underscoring this notion, in humans, mutations in the Sky orthologue TBC1D24 are causative of focal epilepsy (Uytterhoeven, 2011).

    The sorting mechanism uncovered by loss of Sky function or upon expression of Rab35CA may address the use-dependent decline in protein- or lipid-function at synapses, and the continuous need to rejuvenate the synaptic vesicle protein pool. In addition, it may also constitute a mechanism to remove inappropriately endocytosed membrane components from the vesicle pool. The trafficking pathway inhibited by Sky may also yield the ability of synapses to adapt the functional properties of their synaptic vesicles, thus controlling the nature or abundance of proteins involved in vesicle fusion and neuronal signaling (Uytterhoeven, 2011).

    Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration

    Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. This paper studied Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome; endosome-to-lysosome trafficking was identified as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, synaptic vesicle-associated proteins were shown to be younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, it was found that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. These findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans (Fernandes, 2014).

    Live observation of two parallel membrane degradation pathways at axon terminals

    Neurons are highly polarized cells that require continuous turnover of membrane proteins at axon terminals to develop, function, and survive. Yet, it is still unclear whether membrane protein degradation requires transport back to the cell body or whether degradation also occurs locally at the axon terminal, where live observation of sorting and degradation has remained a challenge. This study reports direct observation of two cargo-specific membrane protein degradation mechanisms at axon terminals based on a live-imaging approach in intact Drosophila brains. Different acidification-sensing cargo probes are sorted into distinct classes of degradative 'hub' compartments for synaptic vesicle proteins and plasma membrane proteins at axon terminals. Sorting and degradation of the two cargoes in the separate hubs are molecularly distinct. Local sorting of synaptic vesicle proteins for degradation at the axon terminal is, surprisingly, Rab7 independent, whereas sorting of plasma membrane proteins is Rab7 dependent. The cathepsin-like protease CP1 is specific to synaptic vesicle hubs, and its delivery requires the vesicle SNARE neuronal synaptobrevin. Cargo separation only occurs at the axon terminal, whereas degradative compartments at the cell body are mixed. These data show that at least two local, molecularly distinct pathways sort membrane cargo for degradation specifically at the axon terminal, whereas degradation can occur both at the terminal and en route to the cell body (Jin, 2018).

    Neurons must regulate the turnover of membrane proteins in axons, dendrites, and the cell body to ensure normal development and function. Defects in membrane protein degradation are hallmarks of neurodegenerative diseases. Recent progress has identified several mechanisms that are required at axon terminals to prevent dysfunction and degeneration, including the local generation of autophagosomes and endolysosomes. However, it is unclear whether these degradative organelles are principally transported back to the cell body for degradation or whether degradation can occur locally. In addition, the cargo specificity of membrane degradation mechanisms at the axon terminals has remained largely unknown, i.e., it is unclear which membrane proteins are degraded by what mechanisms (Jin, 2018).

    Several mechanisms have been directly linked to synapse function or degeneration and have raised questions about cargo specificity and the ultimate locale for degradation. These include (1) local generation of autophagosomes at axon terminals, (2) maturation of autophagosomes and endosomes that depends on the ubiquitous small guanosine triphosphatase (GTPase) Rab7, (3) endosomal sorting that depends on the GTPase Rab35 and RabGAP Skywalker, and (4) endosomal sorting that depends on the neuron-specific synaptic vesicle (SV) proteins neuronal synaptobrevin (n-Syb) and V100. These mechanisms may overlap, and defects in any of them cause neurodegeneration in a variety of neurons. In the case of (macro-) autophagy, the formation of autophagosomes occurs at axon terminals, whereas degradation is thought to occur during and after retrograde transport back to the cell body. As with both the canonical and neuron-specific endolysosomal mechanisms, it remains largely unknown what cargoes are sorted into autophagosomes at axon terminals. The Rab35/Skywalker-dependent endosomal sorting mechanism was recently reported to selectively sort different SV proteins in an activity-dependent manner. Lysosomes have also been shown to localize to dendritic spines in an activity-dependent manner. In both cases, it remains unknown whether degradation occurs locally at synapses and what cargo proteins are affected. Finally, previous work has described a 'neuronal sort-and-degrade' (NSD) mechanism based on the function of the two neuron-specific synaptic genes n-syb (Haberman, 2012) and v100 (Wang, 2014). Similar to the other mechanisms, neither cargo specificity nor the locale of degradation for NSD is known. For all mechanisms, it has remained a challenge to directly observe their local roles in the context of normal development and function in an intact brain (Jin, 2018).

    This study reports the direct observation of cargo-specific endolysosomal sorting and degradation at axon terminals in vivo using live imaging in intact Drosophila brains. Axon terminal 'hub' compartments are defined based on their local dynamics, maturation, degradation, continuous mixing through fusion and fission, and budding of retrograde transport vesicles. In addition, two distinct pools of hubs were identified (see Model: Two Parallel Membrane Degradation Mechanisms at Axon Terminals) that function locally in two separate endolysosomal pathways based on different cargo specificities, different molecular sorting, and different maturation mechanisms (Jin, 2018).

    Genetically encoded fusions with pHluorin, a pH-sensitive GFP, and mCherry, a particularly pH-resistant red fluorescent protein (RFP), report cargo incorporation into degradative membrane compartments. It is surprising that the only clearly discernible compartments at axon terminals are red live, large, acidified, and spatiotemporally relatively stable endolysosomal compartments. These compartments were named hubs because of their continuous fission, fusion, and budding of smaller retrograde trafficking vesicles. Appearance or disappearance of an entire hub is never reported, but only the formation of hubs by fusion of several smaller vesicles and splitting into multiple smaller compartments that undergo renewed cycles of fusion. These dynamics are reflected in hub composition and maturation: at any point in time, some hubs are marked by early endosomal markers and contain undegraded probes, whereas others are marked by lysosomal markers and contain partially degraded probes (Jin, 2018).

    How does this 'hub flux' contribute to the sorting and degradation of dysfunctional membrane proteins? A key insight comes from the characterization of retrograde trafficking vesicles: the axonal vesicles exhibit the same composition and a mix of early and late markers and degraded and undegraded probes. These observations are most straightforwardly explained with random mixing of hubs and random budding of axonal vesicles, irrespective of their maturation stage. In this model, sorting into hubs carries a probabilistic chance of degradation that increases with time (either in hubs or in retrograde trafficking vesicles). Sorting into hubs ensures degradation if membrane proteins cannot be recycled back into the axon terminal; alternatively, degradation and recycling may both be probabilistic. The latter would imply that not only dysfunctional proteins are sorted into hubs. Both mechanisms could ensure a pool of functional synaptic proteins that increases with the amount of endolysosomal flux, as previously observed for the Skywalker/Rab35 mechanism (Jin, 2018).

    How SV membrane proteins are specifically sorted into SV hubs is unclear. Because sorting of Syt1-DF into SV hubs is Rab7 independent, SV hub maturation bypasses the requirement for Rab5-to-Rab7 conversion. Neither n-Syb (a vesicle SNARE and membrane fusion factor) nor V100 (part of a proton pump) are required for the continuous fusion and fission of SV hubs. However, the reduced axon terminal numbers of SV hubs are consistent with roles in the sorting of SVs to SV hubs. Different SV retrieval mechanisms, including ultrafast endocytosis, clathrin-mediated endocytosis, and bulk endocytosis, may account for different mechanisms and routes to local degradative hubs. Preassembled plasma membrane cargo complexes may play a role in promoting the different endocytic routes. Colocalization measurements revealed a distinction between two membrane degradation mechanisms with enrichments of SV proteins versus plasma membrane proteins between 3- and 8-fold, but not a 100% separation. Hence, if protein complexes facilitate sorting into a specific endocytic pathway or hub compartments, they may do so in a probabilistic fashion (Jin, 2018).

    Continuous flux is a hallmark of endolysosomal compartments and results in low colocalization ratios with dynamically changing molecular markers and difficulties to unambiguously identify a specific compartment at any point in time. Live observation of dynamic hubs as sorting and degradation stations at axon terminals was only made possible by their integrity over time and may provide an inroad to the study of distinct, cargo-specific mechanisms that keep neurons and their synaptic terminals functional (Jin, 2018).

    Constitutive turnover of SV hubs was observed prior to synaptogenesis and neuronal activity. In contrast, previous work on SV 'rejuvenation' focused on turnover that increases in response to neuronal activity. Colocalization of axon terminal hubs with the Rab35 GAP Skywalker (Sky) revealed equal overlap with both hub types. This could indicate an intersection of different endolysosomal pathways; alternatively, Sky may only temporarily localize to hubs depending on their maturation stage. The second possibility is favored, because the early endosomal Rab5 and the lysosomal marker Spin exhibit similar colocalization ratios and all known endolysosomal markers depend in some way on the maturation stage Colocalization results implicates Sky in both the canonical and SV pathway. Consistent with this, rab7 affects Sky-dependent rejuvenation and the sky mutant affects the turnover of an n-Syb imaging probe (Jin, 2018).

    Autophagy similarly intersects with axon terminal hub compartments based on colocalization with Atg8, albeit this colocalization is significantly higher for the rab7-dependent general PM hubs than for the SV hubs. However, the hubs and axonal trafficking vesicles are distinct from autophagosomes based on their dynamics: Atg8-positive compartments are not part of the 'hub flux,' emerge de novo, and directly enter the axon without prior fission. It is possible that autophagosomes can engulf hub compartments and thus provide an alternative degradative exit to budding of retrograde trafficking vesicles. A Rab26-dependent mechanism was recently proposed for the sorting of SVs to pre-autophagosomal compartments prior to Atg8 recruitment, which may represent a similar hub compartment (Jin, 2018).

    This study showed that cathepsin-L-like protease CP1 has specificity for the SV hubs at axon terminals. This finding is consistent with several cystein cathepsins that have been characterized for their tissue-specific expression. In mammalian systems, cathepsin L selectively degrades polyglutamine (polyQ)-containing proteins, but not other types of aggregation-prone proteins lacking polyQ. In HeLa and Huh-7 cells, cathepsin L was reported to degrade autophagosomal membrane markers, but not proteins in the lumen of autophagosomes. In contrast, the major histocompatibility complex (MHC) class-II-associated invariant chain is specifically degraded by cathepsin S, but not cathepsin L, in CD4+ T cells. The current characterization of cargo-specific membrane degradation machinery with a specific protease raises the question to what extent different membrane degradation mechanisms are characterized and may require specific proteases (Jin, 2018).

    Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles

    Synaptic neuropeptide and neurotrophin stores are maintained by constitutive bidirectional capture of dense-core vesicles (DCVs) as they circulate in and out of the nerve terminal. Activity increases DCV capture to rapidly replenish synaptic neuropeptide stores following release. However, it is not known whether this is due to enhanced bidirectional capture. Experiments at the Drosophila neuromuscular junction, where DCVs contain neuropeptides and a bone morphogenic protein, show that activity-dependent replenishment of synaptic neuropeptides following release is evident after inhibiting the retrograde transport with the dynactin disruptor mycalolide B or photobleaching DCVs entering a synaptic bouton by retrograde transport. In contrast, photobleaching anterograde transport vesicles entering a bouton inhibits neuropeptide replenishment after activity. Furthermore, tracking of individual DCVs moving through boutons shows that activity selectively increases capture of DCVs undergoing anterograde transport. Finally, upregulating fragile X mental retardation 1 protein (Fmr1, also called FMRP) acts independently of futsch/MAP-1B to abolish activity-dependent, but not constitutive, capture. Fmr1 also reduces presynaptic neuropeptide stores without affecting activity-independent delivery and evoked release. Therefore, presynaptic motoneuron neuropeptide storage is increased by a vesicle capture mechanism that is distinguished from constitutive bidirectional capture by activity dependence, anterograde selectivity, and Fmr1 sensitivity. These results show that activity recruits a separate mechanism than used at rest to stimulate additional synaptic capture of DCVs for future release of neuropeptides and neurotrophins (Cavolo, 2016).

    Synapses are supplied by anterograde axonal transport from the soma, the site of synthesis of synaptic vesicle proteins and dense-core vesicles (DCVs) that contain neuropeptides and neurotrophins. Delivery to synapses was thought to be based on a one-way anterograde trip until it was discovered that DCVs are subject to sporadic capture while traveling bidirectionally through en passant boutons as part of long-distance vesicle circulation (Wong, 2012). Interestingly, constitutive DCV capture occurs both during fast anterograde and retrograde transport, which are mediated by different motors (i.e., the kinesin 3 family member unc-104/Kif1A and the dynein/dynactin complex, respectively). Balanced capture in both directions is advantageous because DCVs are distributed equally among en passant boutons (Wong, 2012). In principle, bidirectional capture could occur by parallel regulation of anterograde and retrograde motors or by modification of the microtubules that both anterograde and retrograde DCV motors travel on (Cavolo, 2016).

    Before the discovery of bidirectional capture of circulating vesicles, activity was shown to replenish the presynaptic neuropeptide pool following release by inducing Ca2+-dependent capture of DCVs being transported through boutons. This result and subsequent experiments (Bulgari, 2014) established that capture, rather than delivery or DCV turnover, limits synaptic neuropeptide stores. Activity-dependent capture was first described with a GFP-tagged neuropeptide in the Drosophila neuromuscular junction (NMJ), but also occurs with neurotrypsin, wnt/wingless, and brain-derived neurotrophic factor. Mechanistically, activity-dependent capture was characterized in terms of the rebound in presynaptic GFP-tagged peptide content following release and correlated with decreased retrograde transport. However, it is now evident that the reduction in retrograde flux could be caused by enhanced bidirectional capture as DCVs travel back and forth through the terminal as part of vesicle circulation (Wong, 2012). Therefore, prior studies support the hypothesis that there is only one synaptic capture mechanism, which is bidirectional and facilitated by activity (Cavolo, 2016).

    This study tested the above hypothesis by investigating the directionality of activity-dependent capture. Experiments were performed with multiple approaches, including inhibiting retrograde transport, particle tracking, and simultaneous photobleaching and imaging (SPAIM; Wong, 2012). Furthermore, the effect of fragile X retardation protein (Fmr1, also called FMRP) was examined because it is known to affect bouton size and neuropeptide release. Together, these studies establish that different mechanisms mediate synaptic capture at rest and in response to activity (Cavolo, 2016).

    Until recently, it was thought that presynaptic neuropeptide stores were set by controlling synthesis and delivery by fast one-way axonal transport of DCVs. However, studies of the Drosophila NMJ have shown that there is an excess of DCVs delivered to type-I boutons by long-distance vesicle circulation. Therefore, because DCV delivery is not limiting, the presynaptic neuropeptide pool is determined by capture, which was found to be bidirectional (Wong, 2012). However, in addition to constitutive capture, activity induces Ca2+-dependent capture. This is advantageous because tapping into the circulating vesicle pool removes delays associated with synthesis and transport, which can take days in humans, to rapidly replace released peptides. Surprisingly, experiments presented in this study demonstrate that activity-dependent capture is unidirectional and selectively sensitive to a genetic perturbation (i.e., Fmr1 overexpression). Therefore, activity does not simply enhance constitutive bidirectional capture that operates at rest, but instead stimulates an independent synaptic capture mechanism (Cavolo, 2016).

    Previously, it was not possible to genetically block activity-dependent capture to determine its contribution to steady-state presynaptic stores. However, this study documented inhibition of activity-dependent capture by Fmr1 overexpression. As this was accompanied by a dramatic decrease in presynaptic DCV number, it is concluded that activity-dependent capture makes a large contribution to steady-state presynaptic peptide stores and hence the capacity for future release. At the Drosophila NMJ, DCVs contain a bone morphogenic protein and neuropeptides. Thus, it is possible that activity-dependent capture affects development and acute synaptic function (Cavolo, 2016).

    Capture efficiency measurements revealed that the previously detected decrease in retrograde traffic following activity was an indirect effect of vesicle circulation; activity-induced capture of only anterograde DCVs at each en passant bouton simply leaves fewer DCVs for the retrograde trip back into the axon without changing retrograde capture. Of interest, anterograde selectivity for activity-induced capture rules out mechanisms that would perturb transport in both directions (e.g., microtubule breaks). DCV anterograde transport is mediated by the unc-104/Kif1A motor, which also transports SSV proteins and is required for formation of boutons. Therefore, activity-dependent capture may regulate unc-104/Kif1A to affect synaptic release of both small-molecule transmitters and peptides. However, alternative targets could be involved, including proteins that mediate DCV interaction with this anterograde motor or alter the DCV itself (e.g., its phosphoinositides, which may bind to the unc-104/Kif1A pleckstrin homology domain) (Cavolo, 2016).

    Phosphorylation of Complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity

    Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca(2+) entry. This study shows that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity (Cho, 2015).

    These findings indicate that the SNARE-binding protein Cpx is a key PKA target that regulates spontaneous fusion rates and presynaptic plasticity at Drosophila NMJs. Cpx's function can be modified to regulate activity-dependent functional and structural plasticity. In vivo experiments using Cpx phosphomimetic rescues demonstrate that Cpx phosphorylation at residue S126 selectively alters its ability to act as a synaptic vesicle fusion clamp. In addition, S126 is critical for the expression of HFMR, an activity-dependent form of acute functional plasticity that modulates mini frequency at Drosophila synapses. These data indicate a Syt 4-dependent retrograde signaling pathway converges on Cpx to regulate its synaptic function. Additionally, it was found that elevated spontaneous fusion rates correlate with enhanced synaptic growth. This pathway requires Syt 4 retrograde signaling to enhance spontaneous release and to trigger synaptic growth. Moreover, the Cpx S126 PKA phosphorylation site is required for activity-dependent synaptic growth, suggesting acute regulation of minis via Cpx phosphorylation is likely to contribute to structural synaptic plasticity. Together, these data identify a novel mechanism of acute synaptic plasticity that impinges directly on the presynaptic release machinery to regulate spontaneous release rates and synaptic maturation (Cho, 2015).

    How does acute phosphorylation of S126 alter Cpx's function? The Cpx C-terminus associates with lipid membranes through a prenylation domain (CAAX motif) and/or the presence of an amphipathic helix. The Drosophila Cpx isoform used in this study (Cpx 7B) lacks a CAAX-motif, but contains a C-terminal amphipathic helix flanked by the S126 phosphorylation site. S126 phosphorylation does not alter synaptic targeting of Cpx or its ability to associate with SNARE complexes in vitro. As such, phosphorylation may instead alter interactions of the amphipathic helix region with lipid membranes and/or alter Cpx interactions with other proteins that modulate synaptic release. Given the well-established role of the Cpx C-terminus in regulating membrane binding and synaptic vesicle tethering of Cpx, phosphorylation at this site would be predicted to alter the subcellular localization of the protein and its accessibility to the SNARE complex. However, no large differences between WT Cpx and CpxS126D were observed in liposome binding. This assay is unlikely to reveal subtle changes in lipid interactions by Cpx, as this study found that C-terminal deletions (CTD) maintained its ability to bind membranes. The ability of the CTD versions of Drosophila Cpx to associate with liposomes is unlike that observed with C. elegans Cpx, and indicate domains outside of Drosophila Cpx's C-terminus contribute to lipid membrane association as well, potentially masking effects from S126 phosphorylation that might occur in vivo. Alternatively, phosphorylation of the Cpx C-terminus could alter its association with other SNARE complex modulators such as Syt 1 (Cho, 2015).

    The data indicate that enhanced minis regulate synaptic growth through several previously identified NMJ maturation pathways. The Wit signaling pathway is required for synaptic growth in the background of enhanced minis. The wit gene encodes a presynaptic type II BMP receptor that receives retrograde, transsynaptic BMP signals from postsynaptic muscles. Consistent with these data, other studies have demonstrated that downstream signaling components of the BMP pathway are necessary and sufficient for mini-dependent synaptic growth at the Drosophila NMJ. Additionally, it was found that the postsynaptic Ca++ sensor, Syt 4, is required for enhancing spontaneous release and increasing synaptic growth. The data does not currently distinguish the interdependence of the BMP and Syt 4 retrograde signaling pathways, and other retrograde signaling pathways might contribute to mini-dependent synaptic growth as well. Recently, several retrograde pathways have been identified that regulate functional homeostatic plasticity at the Drosophila NMJ. Future work will be required to fully define the retrograde signaling pathways necessary to mediate mini-dependent synaptic growth (Cho, 2015).

    How might elevated spontaneous release through Cpx phosphorylation regulate synaptic growth? It is hypothesized that the switch in synaptic vesicle release mode to a constitutive fusion pathway that occurs over several minutes following stimulation serves as a synaptic tagging mechanism. By continuing to activate postsynaptic glutamate receptors in the absence of incoming action potentials, the elevation in mini frequency would serve to enhance postsynaptic calcium levels by prolonging glutamate receptor stimulation. This would prolong retrograde signaling that initiates downstream cascades to directly alter cytoskeletal architecture required for synaptic bouton budding. Given that elevated rates of spontaneous fusion still occur in cpx and syx3-69 in the absence of Syt 4 and BMP signaling, yet synaptic overgrowth is suppressed in these conditions, it is unlikely that spontaneous fusion itself directly drives synaptic growth. Rather, the transient enhancement in spontaneous release may serve to prolong postsynaptic calcium signals that engage distinct effectors for structural remodeling that fail to be activated in the absence of elevated spontaneous release. Results from mammalian studies indicate spontaneous release can uniquely regulate postsynaptic protein translation and activate distinct populations of NMDA receptors compared to evoked release, so it is possible that spontaneous fusion may engage unique postsynaptic effectors at Drosophila NMJs as well (Cho, 2015).

    In the last few decades, intense research efforts have elucidated several molecular mechanisms of classic Hebbian forms of synaptic plasticity that include long-term potentiation (LTP) and long-term depression (LTD), alterations in synaptic function that lasts last minutes to hours. The most widely studied expression mechanism for these forms of synaptic plasticity involve modulation of postsynaptic AMPA-type glutamate receptor (AMPAR) function and membrane trafficking. In contrast, molecular mechanisms of short-term synaptic plasticity remain poorly understood. Several forms of short-term plasticity have been described, such as post-tetanic potentiation (PTP), which involves stimulation-dependent increases in synaptic strength, including changes in mini frequency. Short-term plasticity expression is likely to impinge on the alterations to the presynaptic release machinery downstream of activated effector molecules. For example, Munc 18, a presynaptic protein involved in the priming step of vesicle exocytosis via its ability to associate with members of the SNARE machinery, is dynamically regulated by Ca++-dependent protein kinase C (PKC), and its regulation is required to express PTP at the Calyx of Held. This study demonstrates that the presynaptic vesicle fusion machinery can also be directly modified to alter spontaneous neurotransmission via activity-dependent modification of Cpx function by PKA. Protein kinase CK2 and PKC phosphorylation sites within the C-terminus of mammalian and C. elegans Cpx have been identified. Therefore, activity-dependent regulation of Cpx function via C-terminal phosphorylation may be an evolutionarily conserved mechanism to regulate synaptic plasticity. Moreover, Cpx may lie downstream of multiple effector pathways to modulate various forms of short-term plasticity, including PTP, in a synapse-specific manner. Interestingly, Cpx is expressed both pre- and postsynaptically in mammalian hippocampal neurons and is required to express LTP via regulation of AMPAR delivery to the synapse, suggesting Cpx-mediated synaptic plasticity expression mechanisms may also occur postsynaptically (ACho, 2015 and references therein).

    In summary, these results indicate minis serve as an independent and regulated neuronal signaling pathway that contributes to activity-dependent synaptic growth. Previous studies found Cpx’s function as a facilitator and clamp for synaptic vesicle fusion is genetically separable, demonstrating distinct molecular mechanisms regulate evoked and spontaneous release. Evoked and spontaneous release are also separable beyond Cpx regulation, as other studies have demonstrated that minis can utilize distinct components of the SNARE machinery, distinct vesicle pools, and distinct individual synaptic release sites . These findings suggest diverse regulatory mechanisms for spontaneous release that might be selectively modulated at specific synapses (Cho, 2015).

    Fife organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release

    The strength of synaptic connections varies significantly and is a key determinant of communication within neural circuits. Mechanistic insight into presynaptic factors that establish and modulate neurotransmitter release properties is crucial to understanding synapse strength, circuit function, and neural plasticity. Drosophila Fife , a Piccolo-RIM homolog. has been shown to regulate neurotransmission and motor behavior through an unknown mechanism. This study demonstrates that Fife localizes and interacts with RIM (Rab3 interacting molecule) at the active zone cytomatrix to promote neurotransmitter release. Loss of Fife results in the severe disruption of active zone cytomatrix architecture and molecular organization. Through electron tomographic and electrophysiological studies, a decrease was found in the accumulation of release-ready synaptic vesicles and their release probability caused by impaired coupling to Ca2+ channels. Finally, Fife was found to be essential for the homeostatic modulation of neurotransmission. It is proposed that Fife organizes active zones to create synaptic vesicle release sites within nanometer distance of Ca2+ channel clusters for reliable and modifiable neurotransmitter release (Bruckner 2016).

    The strength of synaptic transmission is a critical determinant of information processing in neural circuits. Evoked neurotransmission depends on localized Ca2+ influx triggering neurotransmitter release from synaptic vesicles at specialized domains of presynaptic terminals called active zones. At the active zone membrane, synaptic vesicles are docked and molecularly primed to respond to a rise in Ca2+ concentration by fusing with the membrane to release neurotransmitter. A conserved complex of active zone-associated proteins makes up the active zone cytomatrix. In Drosophila melanogaster, these proteins include the ELKS family protein Bruchpilot, Rab3-interacting molecule (RIM), RIM-binding protein, Unc13, and Fife. Active zone cytomatrix proteins contain many lipid- and protein-binding domains that mediate diverse interactions with key players in synaptic transmission, leading to the model that the active zone cytomatrix spatially organizes presynaptic terminals for the millisecond coupling of neurotransmitter release to action potentials (Bruckner, 2016).

    The specific neurotransmitter release properties of an active zone are determined by two key parameters acting in concert: (1) the number of synaptic vesicles docked at the membrane and molecularly primed for Ca2+-triggered release, termed the readily releasable pool, and (2) the release probability of these vesicles. Vesicle release probability is established by multiple parameters, including Ca2+ channel levels, localization and function at active zones, the spatial coupling of Ca2+ channels and release-ready vesicles, and the intrinsic Ca2+ sensitivity of individual vesicles. The observation that the presynaptic parameters of synaptic strength vary significantly even between the synapses of an individual neuron indicates that neurotransmitter release properties are determined locally at active zones and raises the question of how this complex regulation is achieved. Genetic studies in Drosophila, Caenorhabditis elegans, and mice are revealing a key role for the active zone cytomatrix in determining the functional parameters underlying synaptic strength. A mechanistic understanding of how the active zone cytomatrix achieves local control of synaptic release properties will yield fundamental insights into neural circuit function (Bruckner, 2016).

    Fife, a Piccolo-RIM-related protein is required for proper neurotransmitter release and motor behavior. This study demonstrates that Fife localizes to the active zone cytomatrix, where it interacts with RIM to promote neurotransmitter release. The active zone cytomatrix is diminished and molecularly disorganized at Fife mutant synapses, and Fife is critical for vesicle docking at the active zone membrane. Not only are the number of release-ready vesicles reduced in the absence of Fife, but their probability of release is also significantly impaired because of disrupted coupling to calcium channels. These results suggest that Fife promotes high-probability neurotransmitter release by organizing the active zone cytomatrix to create vesicle release sites in nanometer proximity to clustered Ca2+ channels. Finally, it was found that in addition to its role in determining baseline synaptic strength, Fife plays an essential role in presynaptic homeostatic plasticity. Together, these findings provide mechanistic insight into how synaptic strength is established and modified to tune communication in neural circuits (Bruckner, 2016).

    This study demonstrates that Fife plays a key role in organizing presynaptic terminals to determine synaptic release properties. Fife functions with RIM at the active zone cytomatrix to promote neurotransmitter release. Functional and ultrastructural imaging studies demonstrate that Fife regulates the docking of release-ready synaptic vesicles and, through nanodomain coupling to Ca2+ channels, their high probability of release. It was further found that Fife is required for the homeostatic increase in neurotransmitter release that maintains circuit function when postsynaptic receptors are disrupted. These findings uncover Fife's role as a local determinant of synaptic strength and add to understanding of how precise communication in neural circuits is established and modulated (Bruckner, 2016).

    The finding that Fife interacts with RIM provides insight into how Fife functions within the network of cytomatrix proteins. RIM is a central active zone protein that was recently shown to facilitate vesicle priming at mammalian synapses by relieving autoinhibition of the priming factor Munc13. Like Fife, Drosophila RIM promotes Ca2+ channel accumulation at active zones and exhibits EGTA-sensitive neurotransmitter release at the Drosophila NMJ. This suggests that Fife and RIM may promote high-probability neurotransmitter release by acting together to dock and prime synaptic vesicles in close proximity to Ca2+ channels clustered at the cytomatrix. The findings are consistent with previous work in pancreatic β cells, where it was found that Piccolo and RIM2α form a complex that promotes insulin secretion through an unknown mechanism. To date, functional studies at mammalian synapses have focused on investigating interactions between Piccolo and Bassoon, which bind through their common coiled-coil regions. Thus, it will be of interest to investigate the functional relationship between Piccolo and RIM in promoting neurotransmitter release in mouse models. Piccolo also binds CAST1 through coiled-coil domain interactions. Although the Piccolo coiled-coil region is not present in Fife, future studies to determine whether this interaction is preserved through distinct interacting domains will be important, as Fife and Drosophila CAST-related Bruchpilot carry out overlapping functions. Similarly, although neither Fife nor Piccolo contains the conserved SH3-binding domain that mediates the interaction between RIM and RIM-binding protein, the overlap between Fife and RIM-binding protein phenotypes raises the possibility of functional interactions that will also be important to investigate in future experiments (Bruckner, 2016).

    Significant alterations to active zone cytomatrix size and structure were observed in Fife mutants, whereas none have been detected in RIM mutants, indicating that Fife carries out this function independently of RIM. Previous ultrastructural analysis in aldehyde-fixed samples revealed occasional free-floating electron-dense structures that resemble active zone cytomatrix material and cluster synaptic vesicles. These unanchored electron-dense structures have also been observed at low frequency in Drosophila RIM-binding protein mutants, which, like Fife mutants, exhibit smaller active zone cytomatrices, and at higher frequency in rodent ribbon synapses lacking Bassoon. These structures were not visible in HPF/FS-prepared electron microscopy samples, likely because protein components of the synapse are not cross-linked upon fixation. That these structures are not observed in control synapses but have been found in multiple active zone cytomatrix mutants argues that the extensive cross-linking of proteins in chemically fixed preparations may enable the visualization of biologically relevant complexes missed with cryofixation. This supports the idea that the two fixation methods may offer different advantages for ultrastructural studies of synapses. In any case, the active zone cytomatrix is significantly reduced in size at Fife active zones in both HPF/FS- and aldehyde-fixed electron micrographs (Bruckner, 2016).

    Although diminished or absent cytomatrices have been observed in electron micrographs of RIM-binding protein and Bruchpilot mutants, this phenotype has not been observed in electron micrographs of other active zone cytomatrix mutants, suggesting it represents more than the loss of a single component protein. Rather, the reduced complexity visible in electron micrographs likely reflects a broader underlying molecular disorganization. Further support for this model comes from superresolution imaging, which reveals molecular disorganization at Fife active zones as indicated by the loss of the characteristic ring-shaped localization pattern of Bruchpilot's C terminus. Similar disorganization of Bruchpilot was observed at active zones lacking RIM-binding protein. Superresolution imaging was used to investigate the localization of active zone proteins Cacophony and RIM-binding protein and, although the levels of Cacophony are reduced at Fife active zones, no apparent differences were observed in the patterns of these proteins. Cacophony and RIM-binding protein both localize in smaller puncta than Bruchpilot, so future studies with higher resolution imaging modalities such as stimulated emission depletion microscopy may reveal more subtle abnormalities. Correlations between active zone molecular composition and release probability have been observed at diverse synapses. At the Drosophila NMJ, functional imaging with genetically encoded Ca2+ indicators has demonstrated that active zones display a wide range of release probabilities. Active zones with high release probability contain higher levels of Bruchpilot, which may in turn correlate with higher Ca2+ channel levels. At mouse hippocampal synapses, Bassoon and RIM levels directly correlate with neurotransmitter release probability. Consistently, synaptic probability of release is significantly decreased in Fife mutants (Bruckner, 2016)

    Through a combination of morphological and functional studies, this study found that Fife acts to promote the active zone docking of synaptic vesicles and regulates their probability of release. Because the number of readily releasable vesicles appears to scale with active zone cytomatrix size and molecular composition at diverse synapses, a conserved function of the active zone cytomatrix may be to establish release sites for synaptic vesicles. Consistent with this view, the number of release-ready vesicles is also reduced in Drosophila RIM-binding protein-null mutants and isoform-specific bruchpilotΔ170 and bruchpilotΔ190 mutants, which share similar active zone structural abnormalities with Fife. By combining rapid preservation of intact Drosophila larvae by HPF/FS fixation, electron tomography, and extensive segmentation of active zone structures, this study obtained a detailed view of the 3D organization of active zones in near-native state that allowed further dissection of Fife's role in determining the size of the readily releasable vesicle pool. Membrane-docked vesicles are significantly decreased in Fife mutants, whereas more distant vesicles attached to the membrane by long tethers appear unaffected. Correlating physiological and morphological parameters of neurotransmission is an ongoing challenge in the field. It has been proposed that docking and priming are not separable events in the establishment of the readily releasable vesicle pool, but rather the morphological and physiological manifestations of a single process. Although approximately one third of docked vesicles in these preparations lack obvious short connections to the membrane, which are thought to represent priming factors, the possibility cannot be excluded that these filaments are present but obscured, perhaps because the vesicles are more tightly linked to the membrane. As this proportion is unchanged in Fife mutants, it is concluded that Fife acts to promote vesicle docking and may simultaneously facilitate molecular priming-possibly through its interactions with RIM (Bruckner, 2016).

    The data indicate that neurotransmitter release at Fife synapses is highly sensitive to EGTA, a slow Ca2+ chelator that has been used to investigate the coupling of Ca2+ influx at voltage-gated Ca2+ channels and Ca2+ sensors on synaptic vesicles. At synapses with high release probability, including inhibitory synapses in the mammalian hippocampus and cerebellum, excitatory synapses of the mature Calyx of held, and the Drosophila NMJ, molecularly primed synaptic vesicles and Ca2+ channel clusters are thought to be positioned within ~100 nm of one another to ensure the tight coupling of Ca2+ influx and Ca2+ sensors that explains observed release properties. The EGTA sensitivity of release at Fife, but not wild-type, NMJs indicates that Fife likely regulates the probability that a docked vesicle is released by positionally coupling release-ready vesicles to Ca2+ channels clustered beneath the active zone cytomatrix. The trend toward fewer docked vesicles associated with the active zone cytomatrix in tomograms of Fife synapses provides morphological support for this model. Building on detailed tomographic studies of the Drosophila NMJ to visualize how Ca2+ channels and vesicles are spatially organized at active zones in different genetic backgrounds will be an important step in advancing understanding of the geometry of release probability and how it is established (Bruckner, 2016).

    Finally, this study found that Fife is required for presynaptic homeostasis. In response to decreases in glutamate receptor levels or function, Drosophila motoneurons rapidly increase synaptic vesicle release to maintain postsynaptic excitation. This homeostatic increase in presynaptic neurotransmission is accompanied by an increase in the number of dense projections per active zone and Bruchpilot levels. Cytomatrix proteins RIM, RIM-binding protein, and now Fife have all been shown to function in presynaptic homeostasis, indicating a critical role for the active zone cytomatrix as a substrate for synaptic plasticity. These studies provide insight into the molecular mechanisms through which the active zone cytomatrix determines neurotransmitter release parameters to modulate how information flows in neural circuits (Bruckner, 2016).

    An improved catalogue of putative synaptic genes defined exclusively by temporal transcription profiles through an ensemble machine learning approach

    Assembly and function of neuronal synapses require the coordinated expression of a yet undetermined set of genes. Previously, an ensemble machine learning model was trained to assign a probability of having synaptic function to every protein-coding gene in Drosophila melanogaster. This approach resulted in the publication of a catalogue of 893 genes which was postulated to be very enriched in genes with a still undocumented synaptic function. Since then, the scientific community has experimentally identified 79 new synaptic genes. This study used these new empirical data to evaluate the original prediction. A series of changes were implemented to the training scheme of this model, and using the new data it was demonstrated that this improves its predictive power. Finally, the new synaptic genes were added to the training set and a new model was trained, obtaining a new, enhanced catalogue of putative synaptic genes. This study presents this new catalogue and announces that a regularly updated version will be available online. This study shows that training an ensemble of machine learning classifiers solely with the whole-body temporal transcription profiles of known synaptic genes resulted in a catalogue with a significant enrichment in undiscovered synaptic genes. Using new empirical data provided by the scientific community, the original approach was validated, improving the model to obtained an arguably more precise prediction. This approach reduces the number of genes to be tested through hypothesis-driven experimentation and will facilitate understanding of neuronal function (Pazos Obregon, 2019).

    Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner

    Synchronous neurotransmitter release is triggered by Ca(2+) binding to the synaptic vesicle protein Synaptotagmin 1, while asynchronous fusion and short-term facilitation is hypothesized to be mediated by plasma membrane-localized Synaptotagmin 7 (SYT7). This study generated mutations in Drosophila Syt7 to determine if it plays a conserved role as the Ca(2+) sensor for these processes. Electrophysiology and quantal imaging revealed evoked release was elevated 2-fold. Syt7 mutants also had a larger pool of readily-releasable vesicles, faster recovery following stimulation, and intact facilitation. Syt1/Syt7 double mutants displayed more release than Syt1 mutants alone, indicating SYT7 does not mediate the residual asynchronous release remaining in the absence of SYT1. SYT7 localizes to an internal membrane tubular network within the peri-active zone, but does not enrich at active zones. These findings indicate the two Ca(2+) sensor model of SYT1 and SYT7 mediating all phases of neurotransmitter release and facilitation is not applicable at Drosophila synapses (Guan, 2020).

    Rapid regulation of vesicle priming explains synaptic facilitation despite heterogeneous vesicle:Ca(2+) channel distances

    Chemical synaptic transmission relies on the Ca(2+)-induced fusion of transmitter-laden vesicles whose coupling distance to Ca(2+)-channels determines synaptic release probability and short-term plasticity, the facilitation or depression of repetitive responses. Using electron- and super-resolution microscopy at the Drosophila neuromuscular junction this study quantitatively mapped vesicle:Ca(2+)-channel coupling distances. These are very heterogeneous, resulting in a broad spectrum of vesicular release probabilities within synapses. Stochastic simulations of transmitter release from vesicles placed according to this distribution revealed strong constraints on short-term plasticity; particularly facilitation was difficult to achieve. It was shown that postulated facilitation mechanisms operating via activity-dependent changes of vesicular release probability (e.g. by a facilitation fusion sensor) generate too little facilitation and too much variance. In contrast, Ca(2+)-dependent mechanisms rapidly increasing the number of releasable vesicles reliably reproduce short-term plasticity and variance of synaptic responses. Activity-dependent inhibition of vesicle un-priming or release site activation are proposed as novel facilitation mechanisms (Kobbersmed, 2020).

    This study has described a broad distribution of SV release site:Ca2+ channel coupling distances in the Drosophila NMJ and compared physiological measurements with stochastic simulations of four different release models (single-sensor, dual fusion-sensor, Ca2+-dependent unpriming and site activation model). The two first models (single-sensor and dual fusion-sensor), where residual Ca2+ acts on the energy barrier for fusion and results in an increase in probability of vesicle release (pVr), failed to reproduce facilitation. The two latter models involve a Ca2+-dependent regulation of participating release sites and reproduced release amplitudes, variances and paired-pulse ratios (PPRs). Therefore, the Ca2+-dependent accumulation of releasable SVs is a plausible mechanism for paired-pulse facilitation at the Drosophila NMJ, and possibly in central synapses as well (Kobbersmed, 2020).

    Two models can explain paired-pulse facilitation and variance-mean behaviour at the Drosophila NMJ. Both models include a Ca2+-dependent increase in the number of participating (occupied/activated) release sites. In the Ca2+-dependent unpriming model, forward priming happens at a constant rate, but unpriming is inversely Ca2+-dependent, such that increases in residual Ca2+ lead to inhibition of unpriming, thereby increasing release site occupation between stimuli. Ca2+-dependent replenishment has been observed in multiple systems. This has traditionally been implemented in various release models as a Ca2+-dependent forward priming rate. In a previous secretion model in chromaffin cells, a catalytic function of Ca2+ upstream of vesicle fusion was proposed. However, in the context of short term facilitation such models would favour accelerated priming during the AP, which would counteract this facilitation mechanism and might cause asynchronous release, similar to the problem with the dual fusion-sensor model. In the model presented in this study this is prevented by including the Ca2+ dependency on the unpriming rate. Consistent with this idea, recent data in cells and in biochemical experiments showed that the Ca2+-dependent priming protein (M)Unc13 reduces unpriming. Another model that reproduced the electrophysiological data was the site activation model, where sites are activated Ca2+-dependently under docked (but initially unprimed) SVs. In this case, it was necessary to prevent rapid activation-and-fusion during the AP by including an extra, Ca2+-independent transition, which introduces a delay before sites are activated. The two models are conceptually similar in that they either recruit new SVs to (always active) sites, or activate sites underneath dormant SVs. Those two possibilities are almost equivalent when measuring with electrophysiology, but they might be distinguished in the future using flash-and-freeze electron microscopy . Interestingly, Unc13 has recently been shown to form release sites at the Drosophila NMJ. Therefore, the two models also correspond to two alternative interpretations of Unc13 action (to prevent unpriming, or form release sites) (Kobbersmed, 2020).

    In this model, all primed vesicles have identical properties, and only deviate in their distance to the Ca2+ channel cluster (positional priming). Alternatively, several vesicle pools with different properties (molecular priming) could be considered, which might involve either vesicles with alternative priming machineries, or vesicles being in different transient states along the same (slow) priming pathway. In principle, if different primed SV states are distributed heterogenously such that more distant vesicles are more primed/releasable, such an arrangement might counteract the effects of a broad distance distribution, although this is speculative. Without such a peripheral distribution, the existence of vesicles in a highly primed/releasable state (such as the 'super-primed' vesicles reported at the Calyx of Held synapse), would result in pronounced STD, and counteract STF, which indeed has been observed (Kobbersmed, 2020).

    In this study electrophysiological recordings were performed on muscle 6 of the Drosophila larva which receives input from morphologically distinct NMJs containing big (Ib) and small (Is) synaptic boutons, which have been shown to differ in their physiological properties. This could add another layer of functional heterogeneity in the postsynaptic responses analysed in thus study (the EM and STED analyses shown in this study were focused on Ib inputs). Because the model does not distinguish between Is and Ib inputs, the estimated parameters represent a compound behaviour of all types of synaptic input to this muscle. Future investigations to isolate the contribution of the different input types (e.g. by genetically targeting Is/Ib-specific motoneurons using recently described GAL4 lines) could help distinguish between inputs and possibly further refine the model to identify parameter differences between these input types (Kobbersmed, 2020).

    A cartoon summarizes the results for the single-sensor, dual fusion-sensor and unpriming models (see Cartoon illustrations of the single-sensor, the dual fusion-sensor, and the unpriming models during a paired-pulse simulation at 0.75 mM extracellular Ca2+). Facilitation in single and dual fusion-sensor models depend on the increase in release probability from the first AP to the next (compare colored rings representing 25% release probability between row 2 and 4). However, the increase is very small, even for the dual fusion-sensor model, and to nevertheless produce some facilitation, optimisation finds a small Ca2+ influx, which leads to an ineffective use of the broad vesicle distribution (and a too-high estimate of nsites). In the unpriming model a higher fitted Ca2+ influx (QMax) leads to a more effective use of the entire SV distribution, and facilitation results from the combination of incomplete occupancy of release sites before the first AP (row 1), combined with 'overshooting' priming into empty sites between APs (Kobbersmed, 2020).

    Molecularly, syt-7 was linked to STF behaviour in mice (Jackman, 2016), and the data does not rule out that syt-7 is essential for STF at the Drosophila NMJ (see Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner). However, this study shows clearly that a pVr-based facilitation mechanism (dual fusion-sensor model) cannot account for STF in synapses with heterogeneous distances between release sites and Ca2+ channels. Interestingly, syt-7 was also reported to function in vesicle priming and RRP replenishment. Thus, future work will be necessary to investigate whether the function of syt-7 in STF might take place by Ca2+-dependent inhibition of vesicle unpriming or release site activation (Kobbersmed, 2020).

    Similar suggestions that facilitation results from a build-up of primed SVs during stimulus trains were made for the crayfish NMJ and mammalian synapses. This is in line with the results, with facilitation arising from modulation of the number of primed SVs rather than pVr. The models are conceptually simple (e.g. all SVs are equally primed and distinguished only by distance to Ca2+ channels, sometimes referred to as 'positional priming'), and this study has improved conceptually on previous work by using estimated SV release site:Ca2+ channel distributions, stochastic simulations and comparison to variance-mean relationships and a systematic comparison was performed of pVr- and priming-based models. It has not been clear whether increases in primed SVs are also required for paired-pulse facilitation, or only become relevant in the case of 'tonic' synapses that build up release during longer stimulus trains. Paired-pulse facilitation is a more wide-spread phenomenon in synapses than frequency facilitation, and this study shows for the case of Drosophila NMJ that it also seems to require priming-based mechanisms. Thus, Ca2+-dependent increases of the RRP during STP might be a general feature of chemical synapses (Kobbersmed, 2020).

    High-probability neurotransmitter release sites represent an energy-efficient design

    Nerve terminals contain multiple sites specialized for the release of neurotransmitters. This study tested the hypothesis that high-probability release sites represent an energy-efficient design. Release site probabilities and energy efficiency were examined at the terminals of two glutamatergic motor neurons synapsing on the same muscle fiber in Drosophila larvae. Through electrophysiological and ultrastructural measurements, calculated release site probabilities were found to differ considerably between terminals (0.33 versus 0.11). The energy required to release and recycle glutamate were calculated from the same measurements. The energy required to remove calcium and sodium ions subsequent to nerve excitation was estimated through microfluorimetric and morphological measurements. Energy efficiency was calculated as the number of glutamate molecules released per ATP molecule hydrolyzed, and high-probability release site terminals were found to be more efficient (0.13 versus 0.06). This analytical model indicates that energy efficiency is optimal (~ 0.15) at high release site probabilities (~0.76). As limitations in energy supply constrain neural function, high-probability release sites might ameliorate such constraints by demanding less energy. Energy efficiency can be viewed as one aspect of nerve terminal function, in balance with others, because high-efficiency terminals depress significantly during episodic bursts of activity (Lu, 2016).

    This study reports the relationship between the average probability of release from each AZ (PAZ) and energy efficiency in terminals of two glutamatergic motor neurons (MNs) innervating the same target muscle fiber. PAZ was three times higher in one of the terminals, and this terminal was also twice as efficient, indicating that a high-probability release site is more energy efficient. Given that the brain's energy demands are high, selection away from low PAZ synapses because of their low efficiency might be expected to favor the adoption of a uniform high PAZ design. However, this study found that PAZ values in situ fell short of the high PAZ values predicted for optimal energy efficiency. Terminals with the lowest PAZ and lowest energy efficiency depressed the least at endogenous release rates and performed most of the work during fictive locomotion. The interpretation of these data is that selection away from energy inefficiency has favored high PAZ but that increased PAZ is held in check by selection away from an inability to sustain release (Lu, 2016).

    Selection away from energy inefficiency is thought to have influenced the size of neurons and properties of their synapses and, in turn, limited neural computational power. This study suggests that selection away from energy inefficiency has selected for high PAZ synapses, and this study has demonstrated a positive correlation between PAZ and energy efficiency under physiological conditions. High PAZ can result from high Ca2+ entry/AZ, sensitivity (S) or cooperativity (nH) of release. At first glance, these parameters seem to have qualitatively different influences on energy efficiency. High S or nH lead to higher PAZ and, in turn, higher PAZ leads to higher energy efficiency. More Ca2+ entry/AZ also results in higher PAZ, but simulation of how changes in Ca2+ entry/AZ affect energy efficiency produced a curve with a distinct optimum. Further analysis revealed that energy efficiency would be optimized if PAZ were elevated to 0.76 by an increase in Ca2+ entry/AZ. Taken together, these simulation results show that a nerve terminal needs a relatively high PAZ for optimal energy efficiency but that efficiency diminishes at the highest PAZ values because the purchase of more Ca2+ yields no more neurotransmitter release in return (Lu, 2016).

    Trade-offs between energy efficiency and function have been observed previously. For relay synapses such as the NMJ, the trade-off appears to be between presynaptic energy efficiency and the capacity to sustain neurotransmitter release. Some mammalian central synapses show a capacity for sustained release and are exclusively low PAZ synapses. This study found that low PAZ terminals depressed less than high PAZ terminals, consistent with previous studies showing that low PAZ synapses are likely to facilitate, whereas high PAZ synapses are likely to depress. Type-Ib terminals with an endogenous firing rate of about 20 Hz offset the risk of depletion with a low PAZ, but this measure is attended by low efficiency. The selection potential for low PAZ becomes apparent when, in combination with large NAZ, it confers a capacity to sustain high levels of release, which may translate to sustaining organismal locomotion without fatigue (Lu, 2016).

    While there is a clear rationale for selection against presynaptic design unable to sustain release at a NMJ, the rationale for selection against presynaptic energy inefficiency at a NMJ assumes that the presynaptic terminal consumes (or once consumed) a non-negligible proportion of the NMJ energy budget. Postsynaptic energy consumption can be estimated from EJC measurements that allow calculation of the amount of charge crossing the postsynaptic plasma membrane in response to neurotransmitter released during a single presynaptic AP and the amount of ATP then needed to remove those ions (see the Supplemental Information). Several assumptions that are difficult to defend have to be made to assess postsynaptic energy consumption (Is: 8.16 × 108, and, Ib: 5.18 × 108 ATP molecules). Yet, even if correct only in order of magnitude, it wouls be concluded that the presynaptic terminal consumes ∼1% of the NMJ energy budget. This proportion contrasts starkly with estimates at mammalian central synapses, at which presynaptic energy demands are ∼30% of the total synaptic signaling cost. If presynaptic terminals only consume 1% of the NMJ energy budget, it is suggested that the ability to locomote without fatigue ultimately conferred a greater selection advantage than the energy saved by an efficient terminal and that as a result low PAZ release sites persisted at the expense of high PAZ sites (Lu, 2016).

    Input-specific plasticity and homeostasis at the Drosophila larval neuromuscular junction

    Synaptic connections undergo activity-dependent plasticity during development and learning, as well as homeostatic re-adjustment to ensure stability. Little is known about the relationship between these processes, particularly in vivo. This was addressed with novel quantal resolution imaging of transmission during locomotive behavior at glutamatergic synapses of the Drosophila larval neuromuscular junction. Two motor input types, Ib and Is, were found to provide distinct forms of excitatory drive during crawling and differ in key transmission properties. Although both inputs vary in transmission probability, active Is synapses are more reliable. High-frequency firing 'wakes up' silent Ib synapses and depresses Is synapses. Strikingly, homeostatic compensation in presynaptic strength only occurs at Ib synapses. This specialization is associated with distinct regulation of postsynaptic CaMKII. Thus, basal synaptic strength, short-term plasticity, and homeostasis are determined input-specifically, generating a functional diversity that sculpts excitatory transmission and behavioral function (Newman, 2017).

    The transfer of information between neurons throughout the nervous system relies on communication across inherently unreliable chemical synapses. Synaptic communication is further complicated by the fact that individual neurons can receive inputs from many functionally diverse neurons. Even synapses formed by one presynaptic neuron and one or more postsynaptic target neurons can vary greatly in neurotransmitter release, postsynaptic sensitivity, and plasticity. Additionally, postsynaptic cells are not passive receivers of information but can produce retrograde signals, including homeostatic signals that modulate synaptic release. The mechanisms that regulate diversity in transmission at individual synapses are not well understood, nor are their relationships to the plasticity and homeostatic mechanisms that adjust synaptic strength. In particular, it is unclear whether retrograde signaling is input or synapse specific and able to maintain input context among diverse convergent synapses (Newman, 2017).

    The Drosophila larval neuromuscular junction (NMJ) is a model system for studying glutamatergic transmission, with pre- and postsynaptic molecular machinery similar to that of central excitatory synapses in vertebrates, while also possessing activity-dependent adjustments in synaptic strength, including short- and long-term plasticity, as well as homeostatic plasticity. Two morphologically distinct glutamatergic motor neurons, larger type Ib and smaller type Is, converge onto most of the larval body wall muscles used for locomotion. There is evidence that the transmission properties of these inputs differ (Kurdyak, 1994; Lnenicka, 2000; Lu, 2016), thus providing a powerful system for investigating the role of input and synapse specificity in the regulation of basal synaptic strength, plasticity, and homeostasis. In addition, there is great heterogeneity, in that both the basal release probability (Pr) of evoked release and the frequency of spontaneous release differ greatly between synapses of the same Ib axon (Peled, 2011; Peled, 2014). This study set out to understand how plasticity and homeostasis function in a diverse pool of synapses and to determine whether synaptic homeostasis operates globally or has input specificity (Newman, 2017).

    To address these questions requires high-resolution, high- speed analysis of function at many synapses simultaneously. Quantal resolution measurements of excitatory transmission through Ca2+-permeant glutamate receptors (GluRs) has been achieved by imaging chemical or genetically encoded Ca2+ indicators (GECIs) in the postsynaptic cell (Cho, 2015; Guerrero, 2005; Lin, 2016; Melom, 2013; Muhammad, 2015; Peled, 2011; Peled, 2014; Reese, 2015, Reese, 2016; Siegel, 2013). This study has generated a vastly improved postsynaptically targeted GECI based on GCaMP6f. When expressed in Drosophila larval muscle, 'SynapGCaMP6f' enables quantal imaging without voltage clamping. SynapGCaMP6f was combined with an optical platform that immobilizes larvae without anesthetics to measure synaptic transmission simultaneously at hundreds of Ib and Is synapses in the intact, behaving animal (Newman, 2017).

    The quantal imaging makes it possible to connect the elementary properties of transmission at single synapses, to the synaptic drive that is generated by convergent synaptic inputs, to the operation of muscle groups in large parts of the animal, and finally to behavior. These observations show that basal synaptic strength, short-term plasticity, and, most strikingly, synaptic homeostasis are input specific, diversifying excitatory transmission and behavioral output (Newman, 2017).

    The Ib and Is inputs to the Drosophila larval muscle respectively resemble tonic (low release probability, facilitating) and phasic (high release probability, depressing) inputs seen at neuro-muscular and neuro-neuronal connections in other organisms. Given these differences, the Is input has been proposed to initiate strong, phasic muscle contractions (Schaefer, 2010). However, this study found that the primary excitatory drive and main cause of muscle contraction in larval Drosophila is actually the Ib input. This is despite the fact that Is synapses have a higher basal release probability (Pr) and larger quantal sizes. These functional differences likely reflect the relatively short duration of the Is bursts of activity and the relatively small number of active zones of the Is terminal. Whereas elimination of transmission from Is inputs had little or no effect on muscle contraction during restrained locomotion, just as it had earlier been shown to have negligible effect on general behavior, this study found that the Is input is needed for normal intersegmental coordination of contraction waves. This is consistent with the broad, multi-muscle Is innervation pattern (Newman, 2017).

    Ib synaptic activity begins before and ends after Is activity, consistent with their common presynaptic inputs and different intrinsic excitabilities. Recent work has shown that excitatory and inhibitory interneurons regulate the differential recruitment of MNs between hemi- segments and within hemisegments, respectively (Heckscher, 2015; Zwart, 2016). While Ib inputs ramp up their synaptic drive during locomotor bursts, Is inputs abruptly reach a maximum, which they often sustain for the duration of the shorter burst. Given their substantial depression during high-frequency stimulation, the ability of the Is input to sustain a fixed output level during locomotion suggests that it increases the firing frequency during the bursts. The common interneuron drive within the VNC suggests that the progressive increase in synaptic drive during the Ib burst may also be partly due to an increase in frequency during the burst, in addition to the recruitment of silent synapses and increase in Pr of active synapses, which is observed during facilitating high-frequency trains. Thus, a combination of circuit-level and cell-autonomous differences combine to generate the complex behavioral output of the larva from a limited number MNs and muscles (Newman, 2017).

    Spontaneous glutamate release has been shown to play a role in synapse development (Choi, 2014). However, this study found that spontaneous release in vivo represents only ~1% of total release, suggesting that evoked release would drown out the influence of spontaneous release. One possible explanation is that in late third-instar larvae, spontaneous release may represent a larger fraction of total synaptic transmission than earlier in development when the developmental influences are exerted. An intriguing alternative explanation derives from the finding that, at both inputs, synapses preferentially participate in either evoked or spontaneous release. This suggests that developmental signals could occur at a subset synapses that are dominated by spontaneous release (Newman, 2017).

    Synapses of both inputs were found to differ in key properties, with Is synapses having larger quantal sizes, less total spontaneous release, higher Pr, and short-term depression (as opposed to facilitation in Ib synapses) during high-frequency trains. The difference in short-term plasticity between the inputs is consistent with electrophysiological analysis of responses to separate stimulation of Is or Ib axons (Lnenicka, 2000; Lu, 2016), as well as modeling. These resemble input-specific differences seen in the mammalian brain, such as between parallel and climbing fibers converging on Purkinje cells (Dittman, 2000; Mapelli, 2015) or interneurons in the cortex. The tendency to facilitate or depress is shaped by basal Pr, where high Pr results in greater depletion of immediately releasable vesicles leading to depression. Although overall this agrees with the observation that Is synapses have higher Pr and depress, it was found that despite overlapping distributions of single-synapse Pr, overall release tended to depress in Is and facilitate in Ib, suggesting additional differences in regulation. This greater complexity comes into stark relief when the behavior of hundreds of individual synapses is examined during high-frequency trains of presynaptic firing. This study found that the heterogeneity of short-term plasticity within an input is much greater than was previously appreciated. Local plasticity dynamics were catagorized by how transmission changed during a stimulus train, and it was found that the inputs differ only in two categories, Is inputs having a larger number of active sites that depress and Ib having a larger number of silent sites that are recruited to a releasing state. These observations reveal a previously unknown level of specialization in basal release and plasticity between neighboring synapses of a common input (Newman, 2017).

    The Drosophila larval NMJ has proven to be a powerful system for studying synaptic homeostasis. The mechanism of this homeostasis is that reduction in quantal size (because of reduced GluR conductance that results from either mutation of a GluR subunit or partial pharmacological block) triggers a retrograde signal that leads to increased transmitter release, restoring the normal the normal level of excitatory drive (Davis, 2015). The current results reveal a new aspect to homeostatic plasticity by showing that it is specific to input and acts primarily at synapses with certain properties of basal transmission, short-term plasticity and physiological function. Only glutamate release from the Ib input is boosted during homeostatic compensation. This is attributed to the fact that despite the smaller quantal size and unitary Ca2+ influx at Ib synapses, Ib inputs have longer bouts of activity, resulting in much larger aggregate Ca2+ elevation in the Ib postsynapse during locomotion. The larger Ca2+ influx drives a higher activation of CaMKII in the Ib postsynapse. Critically, mutation of the GluR subunit to reduce quantal size at both Ib and Is synapses exclusively reduces activated CaMKII at the Ib postsynapse. Why there is no reduction in activated CaMKII at the Is postsynapse is not clear, though nonlinearity in the relationship between the Ca2+ concentration profile and CaMKII activation (Stratton, 2013) and differences in GluR subunit composition that influence Ca2+ influx may contribute (Newman, 2017).

    A second mechanistic difference ensures exclusivity for homeostatic signaling to the Ib input: even when CaMKII activity is inhibited throughout the muscle, only Ib presynapses are boosted in Pr. This could mean that Is presynapses are not responsive to the homeostatic signal. Alternatively, it could mean that Is postsynapses are incapable of generating the homeostatic signal, an idea that would be consistent with the finding that elimination of release from Is axons does not induce homeostatic compensation at Ib synapses. However, this would also require that the retrograde homeostatic signal act very locally so that it could not travel even a few microns from signaling-capable Ib postsynapses to Is boutons that are often located very close by. Another possibility is that basal Pr is high at Is synapses partly due to lower levels of activity in vivo resulting in greater homeostatic enhancement in presynaptic release, which in turn could occlude further increases in synaptic reliability following changes in GluR composition or global postsynaptic CaMKII inhibition (Newman, 2017).

    Evidence for signaling compartmentalization can be seen in the structural differences between Ib and Is NMJs, particularly on the postsynaptic side. Ib axons are surrounded by a significantly thicker and more elaborate SSR. There are also substantial differences in the localization of key organizing postsynaptic proteins such as Dlg which is present at higher levels in Ib postsynapses. CaMKII activity has also been shown to affect SSR structure and Dlg localization. Thus, the different activity patterns during native behaviors may also directly regulate postsynaptic properties other than presynaptic release to maintain functional diversity (Newman, 2017).

    The presynaptic changes that mediate homeostatic compensation in Pr are only partly known. Homeostasis has been shown to be accompanied by an increase in presynaptic AP-evoked Ca2+ elevation in Ib boutons. Although a number of molecules have been demonstrated to play a role in mediating presynaptic homeostatic compensation (Davis 2015), it remains to be determined how these mechanisms interact with input-specific differences in strength and plasticity (Newman, 2017).

    Although homeostasis has been generally thought to be a global mechanism to regulate synaptic strength, evidence has emerged that homeostasis can be regulated with variable degrees of specificity. This study has now demonstrated that homeostatic changes can also propagate retrogradely to the presynaptic neuron in an input-specific and synapse-autonomous manner. Given the divergent innervation of multiple muscles by Is inputs and unique innervation by Ib inputs, a Ib-specific homeostatic mechanism can provide greater functional flexibility (Newman, 2017).

    By combining in vivo, physiological measurements of glutamate release with high-resolution quantal analysis at single synapses in the semi-dissected preparation, this study has clarified the relative properties of convergent glutamatergic inputs in the larva during native behaviors. Importantly, this study has demonstrated that the regulation of basal synaptic strength, short-term plasticity, and homeostasis are shaped in a precise input-specific manner. The Ib input has higher levels of in vivo activity, lower Pr synapses, and a propensity for facilitation by recruiting silent synapses during spike trains. Furthermore, the Ib input is the primary determinant for contraction dynamics in the behaving animal. Consistent with its dominant role, when postsynaptic sensitivity to glutamate is altered at both inputs, there is a selective homeostatic adjustment in the amount of neurotransmitter released from Ib input. CaMKII, localized to the postsynaptic density, is ideally placed to detect local changes in postsynaptic activity at both inputs, and postsynaptic inhibition of CaMKII activity is sufficient to enhance release at the Ib input, demonstrating a high degree of signaling compartmentalization within a single muscle cell. Together, these results demonstrate how synaptic activity at the Drosophila larval NMJ is precisely regulated to ensure both functional diversity and stability (Newman, 2017).

    The long 3'UTR mRNA of CaMKII is essential for translation-dependent plasticity of spontaneous release in Drosophila melanogaster

    A null mutation of the Drosophila calcium/calmodulin-dependent protein kinase II gene (CaMKII) was generated using homologous recombination. Null animals survive to larval and pupal stages due to a large maternal contribution of CaMKII mRNA, which consists of a short 3'-UTR form lacking regulatory elements that guide local translation. The selective loss of the long 3'UTR mRNA in CaMKII null larvae allows testing its role in plasticity. Development and evoked function of the larval neuromuscular junction are surprisingly normal, but the resting rate of miniature excitatory junctional potentials (mEJPs) is significantly lower in CaMKII mutants. Mutants also lack the ability to increase mEJP rate in response to spaced depolarization, a type of activity-dependent plasticity shown to require both transcription and translation. Consistent with this, overexpression of miR-289 in wild-type animals blocks plasticity of spontaneous release. In addition to the defects in regulation of mEJP rate, CaMKII protein is largely lost from synapses in the mutant. All phenotypes are non-sex-specific and rescued by a fosmid containing the entire wild-type CaMKII locus, but only viability and CaMKII localization are rescued by genomic fosmids lacking the long 3'UTR. This suggests that synaptic CaMKII accumulates by two distinct mechanisms: local synthesis requiring the long 3'UTR form of CaMKII mRNA and a process which requires zygotic transcription of CaMKII mRNA. The origin of synaptic CaMKII also dictates its functionality. Locally translated CaMKII has a privileged role in regulation of spontaneous release which cannot be fulfilled by synaptic CaMKII from the other pool (Kuklin, 2017).

    CaMKII is both ubiquitous and abundant. In mammals, CaMKII constitutes around 1% of 96 total brain protein and it is also highly expressed in fly heads. Unsurprisingly, CaMKII has been shown to have a plethora of important functions in the nervous system including roles in multiple stages and forms of learning and memory. At the Drosophila neuromuscular junction (NMJ) the roles of CaMKII encompass both development of the synapse and its activity-dependent plasticity. To date, these functions have been revealed using transgenes encoding CaMK II inhibitors or RNAi to decrease kinase activity or activated forms of the kinase to increase activity (Kuklin, 2017).

    Early studies at the larval NMJ showed that global inhibition of CaMKII with a heat shock-inducible inhibitor peptide transgene (hs-ala lines) increased branching and bouton number. This same manipulation also increased the amplitude of evoked currents and blocked paired pulse facilitation. Global inhibition of CaMKII was also associatedwith increased presynaptic excitability while expression of constitutively active CaMKII in motor neurons suppressed excitability. Subsequent studies looking at postsynaptic inhibition of CaMKII with both ala peptide and another inhibitor (CaMKIINtide) showed that muscle CaMKII activity could stimulate a retrograde signaling pathway which increased quantal content without changes in mEJP amplitude. The larger quantal content correlated with an increase in morphologically identified release sites and, for high levels of CaMKIINtide expression, an increase in mini frequency (Kuklin, 2017).

    More recently, presynaptic expression of either ala peptide or CaMKII RNAi was shown to block activity-dependent bouton sprouting. The abundance of roles is consistent with the presence of the kinase at high levels on both sides of the NMJ, and in multiple cellular compartments (Kuklin, 2017).

    The use of genetics to investigate the role of signal transduction molecules in neuronal function has been standard practice for many years in Drosophila. Although Drosophila CaMKII was cloned over 20 years ago, the location of the gene on heterochromatin-rich chromosome complicated standard mutational approaches. To begin genetic analysis of CaMKII, a null mutation was generated in the CaMKII gene by homologous recombination, inserting two stop codons into the N-terminal coding sequence. This study shows that this mutation is comp letely lethal before adulthood in the homozygous state (Kuklin, 2017).

    Homozygous mutant animals survive into late larval and pupal stages, due to a large amount of maternally contributed CaMKII mRNA which has a short 3'UTR lacking regulatory information, including binding site for miR-289 which are present in the long form. The fact that null animals survive to pupate, and show essentially normal morphological development of the neuromuscular junction (NMJ), implies that maternally-derived short 3'UTR CaMKII is able to support the majority of basic processes. Indeed transgenic expression of the short 3'UTR form can partially rescue viability indicating that lack of CaMKII protein is the cause of lethality rather than some critical role of the long 3'UTR form of the mRNA. Third instar null animals, however, lack the synaptic enrichment of CaMKII seen in wild-type animals. They also show very specific defects in miniature excitatory junctional potentials (mEJPs) and do not exhibit transcription/translation-dependent plasticity of mEJP frequency. CaMKII derived from newly transcribed mRNA can rescue synaptic localization and viability independent of the 3'UTR, but plasticity of mEJPs requires the long 3'UTR mRNA. Consistent with this, suppression of CaMKII translation in wild-type animals by overexpression of miR-289 also blocks mEJP plasticity. These results argue that synaptic localization of CaMKII can occur via multiple mechanisms, and that locally translated kinase has a special role in plasticity (Kuklin, 2017).

    Maternal CaMKII mRNA allows initiation of normal development in null larvae but cannot support metamorphosis. Given the numerous and important functions of CaMKII, it is not unexpected that loss of CaMKII is lethal by adulthood. Surprisingly, however, these mutants appear to be fairly normal with respect to the structure and function of the nervous system in larval stages. This is likely because Drosophila embryos receive large amounts of mRNA from maternally-derived support cells in the ovary. Because the maternal genotype is CaMKII w+/+, this means that even genetically null oocytes will contain mRNA encoding CaMKII. This mRNA is able to provide normal initial levels of the protein, and accordingly there is no lethality during embryonic development. By late larval stages the amount of CaMKII falls, reflecting either degradation or dilution of maternally encoded kinase. By the time animals reach third instar, functional problems can beseen at the NMJ and there is significant lethality. The severely reduced CaMKII level in pupal stages blocks the ability to complete metamorphosis. Production of new CaMKII mRNA is clearly necessary for continued viability as the animal enters this stage (Kuklin, 2017).

    One major difference between the maternal and zygotic mRNAs is that the maternal message has a truncated 3'UTR and lacks sequences known to confer post-transcriptional regulation. Interestingly, viability does not seem to require the long 3'UTR . Animals containing either a rescue fosmid lacking long UTR sequences or expressing a neuronal transgene with a truncated UTR are able to reach adulthood and reproduce. This suggests that producing new protein is sufficient for survival through metamorphosis and that the long UTR form has a specialized role. This underscores the need for future studies to utilize cell-specific and temporally-controlled genetic manipulations of kinase protein and mRNA structure (Kuklin, 2017).

    The CaMKII null NMJ phenotype differs from that of animals expressing CaMKII inhibitors. The ability to obtain CaMKII null third instar larvae allowed characterization both the structure of the NMJ and its function in the absence of zygotic transcription. What was immediately obvious was that the phenotypes of the null animals did not resemble the phenotypes reported for animals expressing CaMK II inhibitors or RNAi, manipulations that should affect CaMKII activity levels regardless of the mRNA template. Null animals had no obvious morphological defects or changes in excitability or evoked release, only a decrease in the rate of spontaneous release. In contrast, in animals with strong postsynaptic inhibition of CaMKII an increase in mini rate was reported, likely due to an increase in the number of presynaptic release sites. In the CaMKII null, the number of release sites, as assessed by staining for Brp, is unchanged indicating that the decrease in minis is due to an alteration in release probability (Kuklin, 2017).

    These qualitatively distinct phenotypes suggest that inhibition of CaMKII enzymatic activity is not the same as loss of zygotic transcription on a background of maternally-provided kinase. On the face of it, these differences are surprising since both types of manipulation, transgenic inhibition of CaMKII and loss of new transcription of the gene, should produce animals with reduced CaMKII enzyme activity. What might account for these differences? One possibility is that the absolute levels of CaMKII activity might be different. This would imply that different magnitudes of activity loss have qualitatively distinct effects. This possibility seems unlikely, however, given previous findings with the heat shock driven ala lines where different levels of peptide inhibitor were tested: high and low levels of ala expression did not differ qualitatively, only in severity. A second possibility is that the time window in which CaMKII activity is lost is the key difference between the two manipulations. Expression of inhibitors using GAL4 lines that turn on early in development could reduce activity earlier than slow depletion of maternal mRNA does. This would imply that early loss of CaMKII activity has qualitatively different effects than later loss. A third possibility is that RNAi and inhibitor peptides have off-target effects, perhaps on CaMKI, a poorly studied enzyme in the fly. A fourth possibility, and one that is favored, is that inhibition and mutation might be different because they disrupt distinct pools of CaMKII which are specified by both transcriptional and translational mechanisms (Kuklin, 2017).

    How is CaMKII from newly transcribed mRNA distinct from that encoded by maternal mRNA? Maternal CaMKII mRNA differs in two ways from zygotic mRNA. First, it differs in structure. Drosophila CaMKII has multiple polyadenylation sites and can have either a short or long 3'UTR. Based on publicly available RNA seq data sets and 3' RACE PCR, mRNA from 0-2 h embryos (which reflects maternal contribution) contains exclusively the short 3'UTR. The RNA seq data also suggest it originates from a distinct transcription start site and differs in its 3'UTR. The second difference is that zygotic mRNA has a different history. Maternal mRNA is synthesized in the nuclei of ovarian nurse cells and never sees the inside of a neuronal nucleus. For CaMK II and other mRNAs the association with mRNA transport machinery occurs in the nucleus. Newly transcribed nuclear mRNAs therefore have preferential access to the machinery that mediates RNA localization. This machinery can be cell type-specific and change over development, meaning that maternal mRNA, even if it has the correct regulatory sequences, may not be competent to localize correctly. Thus while maternal and zygotic CaMKII mRNAs encode the same protein, they do not contain the same regulatory information and may not have the same access to localization or processing factors (Kuklin, 2017).

    How do these two differences influence neuronal structure and function? CaMKII null mutants have two obvious defects: a decrease in basal and stimulated mEJP rate and a lack of synaptically localized CaMKII. These two deficits appear to be mechanistically distinct. Rescue of synaptic localization was seen with both the WT gene and a fosmid lacking long UTR sequences. Localization is therefore 3'UTR -independent but appears to require newly transcribed mRNA. Whether this is due to an mRNA-based mechanism (transport of mRNA to synaptic sites and local translation), or whether it is due to preferential transport or diffusion of protein synthesized in the soma from new mRNA templates, will require further investigation (Kuklin, 2017).

    In contrast to synaptic CaMKII localization, the presence of the long 3'UTR is absolutely required for establishing a normal basal level of spontaneous release, and for activity-dependent increases in mEJP rate. This plasticity is translation-dependent and is suppressed by miR-289 which has been previously shown to regulate activity-dependent presynaptic synthesis of CaMKII at the NMJr. The partial suppression seen with pre synaptic miR-289 could be due to relative expression levels of the mRNA and miR or to a requirement for other regulators. The postsynaptic suppression of plasticity points to involvement of CaMKII- dependent retrograde signaling in spontaneous release. Taken together, however, these data imply that there is a population of synaptic long 3'UTR CaMKII mRNA that is locally translated and acts to increase the probability of release. Why newly translated CaMKII is required is unknown, but in rodent neurons, synaptically synthesized CaMKII has preferential access to certain binding partners. These results also revealed differences be tween the NMJ and adult olfactory system synapses. In adult projection neurons, the 3'UTR was required for both localization and activity- dependent regulation of CaMKII translation in dendrites. Further investigation of the mechanisms of RNA and protein localization will be required to resolve these differences, but it is likely that there will be multiple mechanisms for regulation of synaptic CaMKII levels (Kuklin, 2017).

    Activity-dependent synthesis of CaMKII is clearly a critical feature of the enzyme and is conserved across species and developmental stages. Previous work in the adult fly brain has shown that CaMKII mRNA contains sequences that regulate activity-dependent translation. Importantly, this conserved in mammals where 3'UTR sequences in the CAMK2A gene have been shown to drive localization and activity-dependent translation, though it has been suggested that there may also be a role for 3'UTR sequences. The fly will provide a powerful model system for understanding how and why CaMKII is targeted to multiple subcellular compartments. The discovery that local translation of CaMKII is a key driver of plasticity of mini rate also provides a foothold for obtaining an understanding of this process. Spontaneous release is increasingly being recognized as mechanistically and functionally distinct from evoked release (Kuklin, 2017).

    The regulation of spontaneous release, and even the sites at which it occurs, are separate from action potential evoked activity. These miniature events can regulate nuclear gene expression, local translation and participate in developmental processes such as circuit wiring. The dependence of activity-dependent plasticity of spontaneous release on local translation of CaMKII on both sides of the synapse suggests that there are complex mechanisms for fine tuning this important type of synaptic activity (Kuklin, 2017).

    Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca(2+) channels

    The DLG-MAGUK subfamily of proteins plays a role on the recycling and clustering of glutamate receptors (GLUR) at the postsynaptic density. discs-large1 (dlg) is the only DLG-MAGUK gene in Drosophila and originates two main products, DLGA and DLGS97 which differ by the presence of an L27 domain. Combining electrophysiology, immunostaining and genetic manipulation at the pre and postsynaptic compartments, this study examined the DLG contribution to the basal synaptic-function at the Drosophila larval neuromuscular junction. The results reveal a specific function of DLGS97 in the regulation of the size of GLUR fields and their subunit composition. Strikingly the absence of any of DLG proteins at the presynaptic terminal disrupts the clustering and localization of the calcium channel DmCa1A subunit (Cacophony), decreases the action potential-evoked release probability and alters short-term plasticity. These results show for the first time a crucial role of DLG proteins in the presynaptic function in vivo (Astorga, 2016).

    dlg1 is the only gene of the DLG-MAGUK subfamily in Drosophila. Similar to vertebrate genes, two forms of the gene are expressed as the result of two transcription start sites. DLGA (α form) and DLGS97 (β form) are distinguished by the inclusion of an L27 domain in beta forms located in the amino terminus of the protein. In vertebrates DLG4/PSD95 is predominantly expressed as α form while DLG1/SAP97 is mainly expressed as β form. DLGA is expressed in epithelial tissues, somatic muscle and neurons; in turn, DLGS97 is not expressed in the epithelial tissue. In the larval neuromuscular junction (NMJ), a glutamatergic synapse, both dlg products are expressed pre and postsynaptically. Hypomorphic dlg alleles display underdeveloped subsynaptic reticulum, bigger glutamate receptors fields and an increased size of synaptic boutons, active zones and vesicles. Additionally to these morphological defects, altered synaptic responses such as increased excitatory junction currents (EJC) and increased amplitude of miniature excitatory junction potentials have been observed. The strong morphological defects make difficult to distinguish developmental defects from the role of DLGs in the basal function of the mature synapse. Previously studies have reported form-specific null mutant strains for DLGA (dlgA40.2) and DLGS97, (dlgS975). These mutants do not show the gross morphological defects observed in hypomorphic mutants, although still show functional synaptic defects, supporting a role of DLG proteins in the mature synaptic function (Astorga, 2016).

    Combining genetic, electrophysiology and immunostaining techniques this study dissected the role of DLG proteins at the pre and postsynaptic compartments. The results show the specific requirement of postsynaptic DLGS97 for normal glutamate receptor (GLUR) distribution. In turn, both DLG proteins increase the release probability by promoting voltage-dependent Ca2+ channel localization. The results demonstrate for the first time a relevant role to DLG proteins in the presynaptic function contributing to Ca2+ mediated short-term plasticity and probability of release (Astorga, 2016).

    Flies carrying the severe hypomorph dlg1 mutant allele, dlgXI-2 and the isoform specific dlgS97 null mutant displayed increased amplitude of the spontaneous excitatory postsynaptic (junctional) potential (mEJP) without changes in frequency. In addition all mutants displayed a decreased quantal content as measured by evoked post-synaptic potentials. The specific defects behind these results were explored. To characterize the synaptic transmission in WT and dlg mutants, post synaptic currents were recorded in HL3.1 solution on muscles 6 or 7 of third instar male larvae of the various genotypes. Recordings of spontaneous excitatory postsynaptic currents (mEJC) were obtained after blocking the voltage activated sodium channels. Thereafter, histogram distributions were constructed with the amplitudes of the miniature events and the quantal size was estimated by the peak value obtained adjusting a Log-Normal distribution in each genotype. It is worth to emphasize that finding a phenotype on dlgA or dlgS97 mutants means that DLGA or DLGS97 proteins by themselves cannot replace DLG function (Astorga, 2016).

    The average amplitude of spontaneous postsynaptic potentials were compared and, supporting previous results, it was found that the average amplitude of the mEJC of the mutants dlgXI-2 (0.99 ± 0.05 nA) and dlgS97 (0.98 ± 0.03 nA) were significantly larger compared to the average amplitudes of the mEJC of Canton-S strain used as WT control (0.81 ± 0.04 nA) and of dlgA (0.78 ± 0.02 nA) specific mutant. The same result was obtained comparing the quantal size. None of the mutants showed a significant change compared to the WT in the frequency of the mEJC. As an additional control, all mutants were recorded over a deficiency covering the dlg gene, finding similar results. These findings are in accordance with the idea that DLGS97 protein and not DLGA is necessary for the quantal size determination (Astorga, 2016).

    Bigger quantal size could be of pre or postsynaptic origin as the result of increased neurotransmitter (NT) content in vesicles or increased glutamate receptor field's size respectively. First, to determine the pre or post-synaptic origin of this phenotype, a UAS-dsRNA construct that targets all dlg products, was expressed under the control of the motoneuron promoter OK6-GAL4 or the muscle promoter C57-GAL4. As expected for a post-synaptic defect, the increased quantal size observed in dlgS97 mutants was mimicked only by the decrease of DLG in the muscle. The specific role of DLGS97 in the muscle is supported by the rescue of the dlgS97 mutant phenotype only by the expression of DLGS97 in the muscle and not in the motor neuron. The effect of GAL4 expression was examined in the mutant background in all experiments; neither of the GAL4 lines without the specific UAS constructs changed the phenotype of the mutants. Again, none of the genotypes studied displayed differences with the WT in the frequency of the minis (Astorga, 2016).

    Changes in quantal size of postsynaptic origin could be due to higher number of post-synaptic receptors and/or a different composition of the postsynaptic receptors. An increase in the size of glutamate receptors fields has been described in dlg hypomorphic alleles including dlgXI-2 mutants. Therefore, the size of the glutamate receptor fields was compared among the mutants and with WT, and also the active zones were measured using antibodies for the active zone protein Bruchpilot. Consistently with previous results bigger glutamate receptors fields were found compared to WT only in dlgXI-2 and dlgS97 mutants but not in dlgA mutants. Surprisingly, an increased number of active zones per bouton was also found in all mutants, a phenotype usually associated with an increase in the frequency of minis that were not observe. In addition, an increased active zone area was found in dlgA and dlgXI-2 mutants (Astorga, 2016).

    As expected for a postsynaptic defect, the bigger size of the glutamate fields in dlgS97 mutants was rescued by the expression of DLGS97 in the muscle but not by its expression in the motor neuron. These results confirm that DLGS97, but not DLGA is responsible for the regulation of the size of the receptors fields in the muscle (Astorga, 2016).

    The strict requirement of DLGS97 in the regulation of the size of GLUR fields supports results that have involved other DLGS97 interacting proteins in the regulation of the size of the glutamate receptors fields. METRO, an MPP-like MAGUK protein, has been shown to form a complex with DLGS97 and LIN-7 through the L27 domains present in each of the three proteins. metro mutants display decreased DLGS97 at the synapse and larger GLUR receptors fields than WT, even bigger than dlgS97 mutants. METRO and DLGS97 depend on each other for their stability on the synapse, thus, in dlgS97 mutants, METRO and dLIN-7 are highly reduced at the synapse. The similar post-synaptic phenotype of metro and dlgS97 and the reported interaction between these two proteins suggests the proposal that the increase size of GLUR fields is consequence of the loss of METRO due to the loss of DLGS97 protein (Astorga, 2016).

    As stated before, changes in quantal size of postsynaptic origin can also reflect a different composition of the receptors. Drosophila NMJ GLUR receptors are tetramers composed by obligatory subunits and two alternative subunits, GLURIIA and GLURIIB. Receptors composed by one of these two subunits differ in their kinetics; GLURIIB receptors desensitizes faster than GLURIIA receptors. Thus, the kinetic of the spontaneous currents (mEJCs), is associated to the relative abundance of these two types of receptors in the GLUR fields. It has been shown that the abundance of GLURIIB but not of GLURIIA in the synapse is associated with the expression of dlg. To analyze if dlg mutants display a change in the composition of the subunits abundance relative to the control, the kinetics of the mEJCs were studied. Kinetics analyses of the mEJCs revealed that only dlgS97 and the double mutant display a slower kinetic in the off response, which is compatible with a different composition of the glutamate receptors fields regarding the proportion between GLURIIA and B receptors. The value of tau also increased in larvae expressing dsRNA-dlg in the muscle, but not by its expression in the motor neuron. Finally tau-off values recovered the WT value only with the expression of DLGS97 in the muscle. As slower mEJCs were observed, the results suggest an increase in the ratio of GLURIIA/GLURIIB. It is known that receptors containing the GLURIIA subunit display bigger conductance and slower inactivation kinetics than receptors containing the GLURIIB subunit. Thus, synapses with post-synaptic receptors fields containing proportionally less GLURIIB subunits would display bigger and slower mEJCs similar to the phenotype observed in dlgS97 mutants. To confirm this hypothesis, the abundance of GLURIIA and GLURIIB receptors was evaluated by immunofluorescence in the NMJ of WT and dlg mutant larvae. The immunofluorescence that allowed the detection and quantification of GLURIII and GLURIIB fields was performed with paraformaldehyde (PFA) fixative. However, the immunofluorescence to detect GLURIIA receptors only works fixating the tissue with Bouin reagent. Thus, in order to be able to compare between these two fixations, the size of the GLUR fields was normalized by the HRP staining that labels the whole presynaptic bouton. First, as a control, GLURIIA and GLURIII were double stained in the same larvae. The results show that using PFA fixative, GLURIII fields display bigger size only in dlgS97 mutants and not in dlgA mutants. Even more, as predicted from the kinetic data, only dlgS97 and not dlgA mutants display bigger GLURIIA fields while there are not difference in the size of GLURIIB fields between WT and the mutants. Additionally the results show no difference in the number of GLURIIA or GLURIIB clusters between WT and dlg mutants. Immunohistochemical results confirm the prediction from the electrophysiological data revealing that in dlgS97 mutants, GLURIIA subunits are proportionally more abundant in GLUR fields than in control larvae. In conclusion, the results show that dlgS97 mutants display larger quanta and mEJCs with slower kinetic establishing its participation in the regulation of the size of GLUR fields where the increased size is obtained mainly through the recruitment of receptors containing GLURIIA subunits. As a similar result was obtained in another study that observed that the loss of GLURIIB receptors in the NMJ of dlgXI-2 mutant embryos, these observation suggest that either of the two DLG proteins are necessary for the localization of GLURIIB in the synapse but only DLGS97 is actively limiting the size of the clusters by regulating the number of GLURIIA receptors (Astorga, 2016).

    Taking into account previous reports that show the regulation of the synaptic localization of DLG by CAMKII, the regulation of the subunit composition by CAMKII and these results, a mechanism is proposed by which, after a strong activation of CAMKII, the phosphorylation of DLGS97 would detach it from the synapse allowing the increase of the size of the GLUR fields by the recruitment of GLURIIA over GLURIIB. These changes should increase the synaptic response by two different mechanisms (Astorga, 2016).

    To determine if DLG proteins modulate the presynaptic release probability, excitatory junction currents (EJC) were recorded in the muscle by stimulating the nerve at 0.5 Hz in low extracellular Ca2+ (0.2 mM), both conditions to avoid synaptic depression. For all mutant genotypes the average peak amplitude and quantal content (EJC amplitude/quantal size) of the evoked responses were significantly smaller than WT. In congruence with previous results, the lower amplitude of the current response is accompanied by a decrease in the quantal content. Taking into account the results on the size of the GLUR fields in the mutant's muscles, these results are compatible with a reduction of the neurotransmitter release in dlg mutants. A decreased neurotransmitter release could be associated with a decreased number of release sites in the boutons. However, the number of active zones per bouton is increased in all dlg mutants with bigger active zones in dlgXI-2 and dlgA mutants (Astorga, 2016).

    The decrease in the evoked response could be a consequence of the absence of the specific form of DLG in the postsynaptic side, transmitted by unknown mechanisms or, alternatively, it could be the result of an effect of DLG on the probability of release. In order to explore where this phenotype originates (pre or post-synaptically) DLG levels were downregulated by expressing dsRNA against all forms of dlg. Compatible with a presynaptic defect, the expression of UAS-dsRNA-dlg presynaptically decreases the amplitude of the evoked response while the same construct expressed postsynaptically using C57 promoter did not changed the amplitude of the EJCs. The presynaptic expression of the dsRNA-dlg also mimics the reduction in quantal content of the mutants, displaying a severe reduction in this parameter. On the other hand, the postsynaptic expression of the dsRNA-dlg associates to a moderate but significant decrease on the quantal content, as expected from the effect already reported of the postsynaptic dsRNA-dlg on the quantal size and the lack of effect on the amplitude of the EJCs. The presynaptic effect of DLG is supported further by the rescue experiments. Thus, the amplitude of the evoked response and the quantal content in dlgA40.2 mutant is completely rescued by the selective expression of DLGA in the presynaptic compartment but not by its expression in the postsynaptic compartment. The pre-synaptic expression of DLGS97 improves the synaptic function increasing the average size of the EJCs such that the difference between the WT and the presynaptic-rescue is not significant, suggesting a complete rescue. However, the average EJC in the presynaptic rescue is not different either from the control mutant animal, which is interpreted as the rescue not being complete and thus the term partial rescue is used. DLGS97 does not, however rescued at al the quantal content. This is explained because although the amplitude of the current increased, the quantal size remains unchanged by the presynaptic expression of DLGS97. In consequence the quantal content does not increase as much as the current. On the other hand, the postsynaptic expression did not increase the amplitude of the evoked current. However, since it does rescue the quantal size the quantal content augmented enough to be different from the mutant control. Notably, DLGA expressed presynaptically in dlgA40.2mutants not only rescued the EJC amplitude but also the number of active zones per bouton and the size of the active zones. On the other hand DLGS97 expression only partially rescued the increased number of active zones in dlgS975 mutants. These results support a role of DLG proteins in the presynaptic function where DLGA seems to regulate more aspects than DLGS97. Despite the fact that both forms of DLG share most of their protein domains, neither of the two-forms is able to fully rescue the absence of the other, suggesting that both of them participate in a complex. The binding between the SH3 and GUK domains of MAGUK proteins has been described; this interaction (at least in vitro) is able to form intra or intermolecular associations and offers a mechanism by which DLGA and DLGS97 proteins could be associated to recruit proteins to a complex (Astorga, 2016).

    Changes in the overall quantal content at these synapses may reveal presynaptic defects. However, genetic background and other independent modification could alter apparent release. To independently scrutinize alteration in the presynaptic release probability two presynaptic properties were examined, the short-term plasticity and the calcium dependency of quantal release (Astorga, 2016).

    To explore the EJC phenotype observed in dlg mutants, stimulation paradigms were carried out that allow characterization of aspects of the short term plasticity that are known to depend on presynaptic functionality and give clues about the mechanisms involved in the observed defects. First, the response were studied of the mutants to high frequency stimulation, 150 stimuli at 20 Hz. WT responses at high frequency stimulation show a fast increase in the amplitude of the response that then slows down. The fast initial increase is called facilitation and the second phase with smaller slope is called augmentation. The time constant of the facilitation is believed to reflex the calcium dynamics in the terminal and its slope to be the product of the accumulation of calcium and the consequent calcium dependent increase in the probability of release. The fractional increment in the mutants' responses showed an increased facilitation in all mutants, while an increased augmentation was only significant in dlgA mutants compared to WT. Additionally all mutants showed a trend toward steeper slopes than WT, but only the augmentation slope in dlgA mutants reached statistical significance. These results support that the mutants display a lower probability of release than WT, which could reflect defects in the calcium dynamics or in the response to calcium (Astorga, 2016).

    Previous work in dlg mutants did not report defects in short-term plasticity. These works differ from the current one in methodological aspects, mainly that they were carried out in a media with high concentration of magnesium (20 mM) and calcium (1.5 mM). This work was carried out in a media containing low magnesium (4 mM) and calcium (0.2 mM) concentration. It is known that magnesium reduces neurotransmitter release, probably due to partial blockade of VGCC. Additionally, magnesium permeates more than sodium and potassium through GLURs (Astorga, 2016).

    To better evaluate the calcium dynamics in the terminal pair pulse (PP) experiments, a well-known paradigm to evaluate presynaptic calcium dynamics, were carried out. In PP, a second depolarization shortly after the first one carried out in low extracellular calcium concentration elicits an increased release of neurotransmitter thought to reflect the increased calcium concentration in the terminal reached after the first stimulus. According to this, and posing as the working hypothesis that DLG affects presynaptic calcium dynamics, a second pulse would be expected to increase the release in a bigger proportion, since the first stimulus did not release much of the ready releasable pool. Conversely, a second stimulus given at high calcium concentration produces a decrease in the release of neurotransmitter, which is considered to originate in the partial depletion of the ready releasable pool at the release sites. Thus, a second pulse at high calcium concentration should elicit a smaller decrease of the release since an inferior entrance of calcium should produce less depletion of the ready releasable pool of vesicles (Astorga, 2016).

    Consistently with a decreased calcium entrance, all mutants displayed increased pair pulse facilitation at low calcium concentration and decreased pair pulse depression at high calcium concentration. These results support a defect in the calcium entrance to the terminal as the underlying defect in dlg mutants causing the evoked stimuli defects. To characterize the calcium dependency of the release in the mutants the evoked responses were measured at different calcium concentrations. It can be observed that for all the mutants and at most calcium concentrations, the quantal content of the evoked response is lower than the control. The only exception is seen at 2 mM calcium where the quantal content of the dlgA mutants and the control are not different to each other. However, even at this calcium concentration the quantal content of dlgS97 and the double mutant dlgXI-2 are significantly lower than the control. To get insight about the release process the responses were fit to a Hill equation. This type of fitting better estimate the maximum responses and the EC50, which is masked in the overall release of different backgrounds. This is observed in the graph with the normalized responses by the maximal quantal predicted. The adjusted curves show that mutants reach the theoretical maximal quantal content at higher calcium concentration than the WT and that the EC50 for the mutants is diminished respect to the WT. To confirm the presynaptic origin of the defect in the calcium dependency, the calcium dependency was carried out in the mutant genotypes expressing DLGA or DLGS97 pre or postsynaptically. The quantal content analysis shows that only the presynaptic expression of DLGA in dlgA mutants completely rescued the calcium dependency, in line with previous results that show the importance of DLGA in the presynaptic compartment. On the other hand the presynaptic expression of DLGS97, although it rescued the calcium dependency, failed to rescue the maximal quantal content. Observing the graph with the normalized responses, DLGA as well as DLGS97 both are able to restore the WT calcium dependency. The inability to rescue the maximal quantal content could be explained by the existence of synaptic compensatory mechanisms that allow to counterweigh the bigger quantal size in dlgS97 mutants, which were shown before not to be rescued by the presynaptic expression of DLGS97 (Astorga, 2016).

    Facilitation is thought to depend on the resultant of the calcium entrance, calcium release from intracellular stores and the clearance of cytosolic calcium. So, the defects in facilitation observed in the mutants could be due to a decreased calcium entrance but also they could be due to a defect on the clearance of calcium. In a preliminary experiment, the relative changes were measured of the total intracellular calcium concentration in the bouton using the genetically encoded calcium indicator GCamp6f. GCamp6f expressed in control flies (OK6-GAL4/UAS-GCamp6f) respond with a fast and transitory change in the cytoplasmic calcium of the boutons when they are exposed to a local pulse of potassium. The same experimental approach in dlgS97 mutant larvae reveals that the rise of the calcium response is significantly slower than the control; additionally the recovery of the response is also significantly slower. These preliminary experiments suggest a defect in calcium entrance in the mutants but they also support a defect in the extrusion that hint to additional defects. Further experiments are needed to clarify the calcium kinetics involved since these experiments were measuring the bulk of calcium change and in doing this approximation, the nanodomain changes that are known to be the ones that regulate the neurotransmitter release are being lost (Astorga, 2016).

    Since the results described above including the calcium dependency of the release as well as the parameters of the short-term plasticity suggest that the calcium entrance to the terminal is impaired, a view that is supported by the preliminary data measuring the cytosolic calcium, the next experiments focused on the calcium entrance. The main calcium entry to the terminal is the voltage gated calcium channel (VGCC) encoded by the Drosophila gene cacophony. Advantage was taken of a UAS-cacophony1-EGFP transgenic fly (CAC-GFP) to study the distribution of the channel in WT and mutant genotypes. CAC-GFP overexpressed in WT background localizes in the synapse in a strictly plasma membrane-associated manner in big clusters closely associated with release sites. However, CAC-GFP overexpressed in dlgS97 or dlgA mutant background displays a significant decrease in the expression accompanied by a more disperse localization with significantly smaller clusters, suggesting that the Cacophony protein might not be properly delivered or anchored to the plasma membrane in dlg mutants (Astorga, 2016).

    It was reasoned that if dlg mutants had a defect on calcium entrance, the over expression of calcium channels should rescue at least partially the phenotype. Advantage was taken of the fact that CAC-GFP construct encodes a functional channel, and recordings were taken from control and dlg mutants overexpressing CAC. As expected and supporting a decreased calcium entrance in the mutants, dlgS97 and dlgA mutants that overexpress CAC-GFP display significantly bigger evoked EJCs compared to dlg mutants, without a change of phenotype in the spontaneous currents. Additionally, the over expression of CAC-GFP partially rescued the pair pulse facilitation and the pair pulse depression as well as the calcium curve (Astorga, 2016).

    The disrupted localization of CAC could result from the disturbance of a normal direct association to DLG or it could be affected indirectly. To test an immunoprecipitation assay was carried out using flies that express CAC-EGFP in all neurons. Antibodies against GFP were able to precipitate DLG together with Cacophony-GFP, supporting that Cacophony channel is part of the DLG complex in the boutons (Astorga, 2016).

    A possible interaction between DLG and voltage-gated calcium channels (VGCCs is) the VGCC auxiliary subunits. The α2δ auxiliary subunit (Straightjacket in Drosophila) increases calcium channel activity and plasma membrane expression of CaV2 α1 subunits and Cacophony. The β auxiliary subunit increases plasma membrane expression of several mammalian VGCC classes. Intriguing β subunits are also MAGUK proteins and they are able to release the VGCC α subunit from the endoplasmic reticulum retention. It may be speculated that DLG through their SH3-GUK domain might be playing the role of the β subunit (Astorga, 2016).

    On the other hand, in mammalian cultured neurons it has been proposed that a complex formed by the scaffold proteins LIN-2/CASK, LIN-10/MINT and LIN-7 is involved in the localization of VGCCs at the synapse and that SAP97 forms a complex with CASK. The association of DLGS97 with LIN7 has been reported in the postsynaptic compartment in the Drosophila NMJ. Furthermore, an association between the L27 domain of DLGS97 and the L27 domain of Drosophila CASK has been shown in vitro, however there are no reports of this type of association with DLGA. Another protein involved in the localization of calcium channels in the active zone is RIM. Drosophila rim has been involved in synaptic homeostasis and the modulation of vesicle pools. Surprisingly rim mutants, display low probability of release and altered responses to different calcium concentrations. Recently it was shown that spinophilin mutants display a phenotype with bigger quantal size and GLUR fields size with a higher proportion of GLURIIA subtype of receptors as well as decreased EJCs and decreased pair pulse facilitation. This is a phenotype very similar to the one described here for dlg mutants. The authors in this report did not explore the calcium channels abundance or distribution and the current study did not explore the link of DLG to Neuroligins, Neurexins and Syd. It would be interesting to determine if there is a link between Spinophilin and DLG (Astorga, 2016).

    Taken together these results show that dlgS97 is the main isoform responsible for the postsynaptic defects in the dlgXI-2 mutants; which comprise the increase in the size of the receptors fields and the change in the ratio of GLURIIA/GLURIIB. The results as well support a model in which DLG forms a presynaptic complex that includes Cacophony where the absence of either form of DLG leads to defects in the localization of the voltage dependent calcium channel and to a decrease in the entrance of calcium to the bouton; which in turn affect the probability of release and the short-term plasticity in the mutants. The results described in this study highlight the specificity of the function of DLGS97 and DLGA proteins and describe for the first time an in vivo presynaptic role of DLG proteins (Astorga, 2016).

    The Ih channel gene promotes synaptic transmission and coordinated movement in Drosophila melanogaster

    Hyperpolarization-activated cyclic nucleotide-gated "HCN" channels, which underlie the hyperpolarization-activated current (Ih), have been proposed to play diverse roles in neurons. The presynaptic HCN channel is thought to both promote and inhibit neurotransmitter release from synapses, depending upon its interactions with other presynaptic ion channels. In larvae of Drosophila melanogaster, inhibition of the presynaptic HCN channel by the drug ZD7288 reduces the enhancement of neurotransmitter release at motor terminals by serotonin but this drug has no effect on basal neurotransmitter release, implying that the channel does not contribute to firing under basal conditions. This study shows that genetic disruption of the sole HCN gene (Ih) reduces the amplitude of the evoked response at the neuromuscular junction (NMJ) of third instar larvae by decreasing the number of released vesicles. The anatomy of the (NMJ) is not notably affected by disruption of the Ih gene. It is proposed that the presynaptic HCN channel is active under basal conditions and promotes neurotransmission at larval motor terminals. Finally, it was demonstrated that Ih partial loss-of-function mutant adult flies have impaired locomotion, and, thus, it is hypothesized that the presynaptic HCN channel at the (NMJ) may contribute to coordinated movement (Hegle, 2017).

    A Ca2+ channel differentially regulates Clathrin-mediated and activity-dependent bulk endocytosis

    Clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE) are two predominant forms of synaptic vesicle (SV) endocytosis, elicited by moderate and strong stimuli, respectively. They are tightly coupled with exocytosis for sustained neurotransmission. However, the underlying mechanisms are ill defined. Previous work has shown that the Flower (Fwe) Ca2+ channel present in SVs is incorporated into the periactive zone upon SV fusion, where it triggers CME, thus coupling exocytosis to CME. This study shows that Fwe also promotes ADBE. Intriguingly, the effects of Fwe on CME and ADBE depend on the strength of the stimulus. Upon mild stimulation, Fwe controls CME independently of Ca2+ channeling. However, upon strong stimulation, Fwe triggers a Ca2+ influx that initiates ADBE. Moreover, knockout of rodent fwe in cultured rat hippocampal neurons impairs but does not completely abolish CME, similar to the loss of Drosophila fwe at the neuromuscular junction, suggesting that Fwe plays a regulatory role in regulating CME across species. In addition, the function of Fwe in ADBE is conserved at mammalian central synapses. Hence, Fwe exerts different effects in response to different stimulus strengths to control two major modes of endocytosis (Yao, 2017).

    A tight coupling of exocytosis and endocytosis is critical for supporting continuous exocytosis of neurotransmitters. CME and ADBE are well-characterized forms of SV endocytosis triggered by moderate and strong nerve stimuli, respectively. However, how they are coupled with exocytosis under distinct stimulation paradigms remains less explored. A model is proposed based on the present data. When presynaptic terminals are mildly stimulated, SV release leads to neurotransmitter release and the transfer of Fwe channel from SVs to the periactive zone where CME and ADBE occur actively. The data suggest that this channel does not supply Ca2+ for CME to proceed. However, intense activity promotes Fwe to elevate presynaptic Ca2+ levels near endocytic zones where ADBE is subsequently triggered. Thus, Fwe exerts different activities and properties in response to different stimuli to couple exocytosis to different modes of endocytosis (Yao, 2017).

    It has been previously concluded that Fwe-dependent Ca2+ influx triggers CME (Yao, 2009). However, the current results suggest alternative explanations. First, the presynaptic Ca2+ concentrations elicited by moderate activity conditions, i.e., 1-min 90 mM K+/0.5 mM Ca2+ or 20-s 10-20 Hz electric stimulation, are not dependent on Fwe. Second, expression of 4% FweE79Q, a condition that abolishes Ca2+ influx via Fwe, rescues the CME defects associated with fwe mutants, including decreased FM1-43 dye uptake, a reduced number of SVs, and enlarged SVs. Third, raising the presynaptic Ca2+ level has no beneficial impact on the reduced number of SVs observed in fwe mutants. These data are consistent with the observations that a Ca2+ influx dependent on VGCCs triggers CME at a mammalian synapse. Hence, Fwe acts in parallel with or downstream to VGCC-mediated Ca2+ influx during CME (Yao, 2017).

    ADBE is triggered by intracellular Ca2+ elevation, which has been assumed to be driven by VGCCs that are located at the active zones. However, the data strongly support a role for Fwe as an important Ca2+ channel for ADBE. First, following exocytosis, Fwe is enriched at the periactive zone where ADBE predominates. Second, Fwe selectively supplies Ca2+ to the presynaptic compartment during intense activity stimulation, which is highly correlated with the rapid formation of ADBE upon stimulation. Third, 4% FweE79Q expression, which induces very subtle or no Ca2+ upon strong stimulation, fails to rescue the ADBE defect associated with loss of fwe. Fourth, treatment with a low concentration of La3+ solution that specifically blocks the Ca2+ conductance of Fwe significantly abolishes ADBE. Lastly, the role of Fwe-derived Ca2+ influx in the initiation of ADBE mimics the effect of Ca2+ on ADBE at the rat Calyx of Held. As loss of fwe does not completely eliminate ADBE, the results do not exclude the possibility that VGCC may function in parallel with Fwe to promote ADBE following intense stimulation (Yao, 2017).

    Interestingly, Ca2+ influx via Fwe does not control SV exocytosis during mild and intense stimulations. How do VGCC and Fwe selectively regulate SV exocytosis and ADBE, respectively? One potential mechanism is that VGCC triggers a high, transient Ca2+ influx around the active zone that elicits SV exocytosis. In contrast, Fwe is activated at the periactive zone to create a spatially and temporally distinct Ca2+ microdomain. A selective failure to increase the presynaptic Ca2+ level during strong stimulation is evident upon loss of fwe. This pinpoints to an activity-dependent gating of the Fwe channel. Consistent with this finding, an increase in the level of Fwe in the plasma membrane does not lead to presynaptic Ca2+ elevation at the Calyx of Held when the presynaptic terminals are at rest or subject to mild stimulation. However, previous studies showed that, in shits terminals, blocking CME results in the accumulation of the Fwe channel in the plasma membrane, elevating Ca2+ levels. It is possible that Dynamin is also involved in regulating the channel activity of Fwe or that the effects other than Fwe accumulation associated with shits mutants may affect intracellular Ca2+ handling. Further investigation of how neuronal activity gates the channel function of Fwe should advance knowledge on the activity-dependent exo-endo coupling (Yao, 2017).

    Although a proteomic analysis did not identify ratFwe2 in SVs purified from rat brain, biochemical analyses show that ratFwe2 is indeed associated with the membrane of SVs. The data show that 4% of the total endogenous Fwe channels efficiently promotes CME and ADBE at the Drosophila NMJ. If a single SV needs at least one functional Fwe channel complex during exo-endo coupling, and one functional Fwe complex comprises at least four monomers, similar to VGCCs, transient receptor potential cation channel subfamily V members (TRPV) 5 and 6, and calcium release-activated channel (CRAC)/Orai1, then it is anticipated that each SV contains ~100 Fwe proteins (4 monomers x 25). This suggests that Fwe is highly abundant on the SVs. It is unlikely that many SVs do not have the Fwe, as a 25-fold reduction of the protein is enough to ensure functional integrity during repetitive neurotransmission. Finally, the results for the SypHy and dextran uptake assays at mammalian central synapses indicate the functional conservation of the Fwe channel in promoting different modes of SV retrieval. In summary, the Fwe-mediated exo-endo coupling seems to be of broad importance for sustained synaptic transmission across species. (Yao, 2017).

    Neuroligin 4 regulates synaptic growth via the Bone morphogenetic protein (BMP) signaling pathway at the Drosophila neuromuscular junction

    The neuroligin (Nlg) family of neural cell adhesion molecules is thought to be required for synapse formation and development, and has been linked to the development of autism spectrum disorders in humans. In Drosophila melanogaster, mutations in the neuroligin 1-3 genes have been reported to induce synapse developmental defects at neuromuscular junctions (NMJs), but the role of neuroligin 4 (dnlg4) in synapse development has not been determined. This study reports that the Drosophila Neuroligin 4 (DNlg4) is different from DNlg1-3 in that it presynaptically regulates NMJ synapse development. Loss of dnlg4 results in reduced growth of NMJs with fewer synaptic boutons. The morphological defects caused by dnlg4 mutant are associated with a corresponding decrease in synaptic transmission efficacy. All of these defects could only be rescued when DNlg4 was expressed in the presynapse of NMJs. To understand the basis of DNlg4 function, genetic interactions were sought, and connections were found with the components of the bone morphogenetic protein (BMP) signaling pathway. Immunostaining and western blot analyses demonstrated that the regulation of NMJ growth by DNlg4 was due to the positive modulation of BMP signaling by DNlg4. Specifically, BMP type I receptor Tkv abundance was reduced in dnlg4 mutants, and immunoprecipitation assays showed that DNlg4 and Tkv physically interacted in vivo. This study demonstrates that DNlg4 presynaptically regulates neuromuscular synaptic growth via the BMP signaling pathway by modulating Tkv (X. Zhang, 2017).

    The formation, development, and plasticity of synapses are critical for the construction of neural circuits, and the Drosophila larval neuromuscular junction (NMJ) is an ideal model system to dissect these processes. In the past few decades, several subcellular events and signaling pathways have been reported to be involved in regulating synaptic growth at Drosophila NMJs, such as local actin assembly, endocytosis, ubiquitin-mediated protein degradation, the Wingless pathway, and the bone morphogenetic protein (BMP) pathway. Among these, BMP signaling is thought to be a major retrograde pathway that promotes the synaptic growth of NMJs (X. Zhang, 2017).

    At the Drosophila NMJ, the BMP homolog Glass bottom boat (Gbb) is released by muscle cells and binds to the presynaptic type II BMP receptor Wishful thinking (Wit). Wit is a constitutively active serine/threonine kinase and, upon binding to Gbb, forms a complex with the type I BMP receptor Thickvein (Tkv) or saxophone (Sax), which results in their activation by phosphorylation. The activated type I receptor subsequently phosphorylates the downstream R-Smad protein Mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) then binds to the co-Smad Medea (Med). This complex translocates to the nucleus of motoneurons to activate or repress the transcription of target genes required for NMJ growth. Mutation of any component in the BMP signaling pathway results in a striking deficiency of NMJ growth. In addition, many molecules are reported to affect NMJ growth by negatively regulating BMP signaling at different points in the pathway. This study report that Drosophila Neuroligin 4 (DNlg4), a trans-synaptic adhesion protein, acts as a positive regulator of BMP signaling to regulate NMJ growth (X. Zhang, 2017).

    Neuroligins (Nlgs) were initially reported to be the postsynaptic ligands of the presynaptic adhesion proteins neurexins (Nrxs), and loss of function of Nlgs in humans is thought to be associated with several mental disorders, including autism and schizophrenia. Nlgs are an evolutionarily conserved family of proteins encoded by four independent genes in rodents and five independent genes in humans. Nlgs have been reported to induce synapse assembly by co-cultured neurons when expressed in nonneuronal cells, and overexpression of Nlgs in neurons increases synapse density. These in vitro cell culture studies suggest a role of Nlgs in inducing the formation of synaptic contacts. However, an in vivo study showed, despite severe defects in synaptic transmission, that there was no alteration of synapse number in neurons from nlg1-3 triple knock-out mice. Similarly, loss of Nlg1 specifically in the hippocampus or amygdala did not alter the synapse number, suggesting that the role of Nlgs is not to trigger the initial synapse formation. Rather, it is more likely that upon binding to Nrx, Nlg functions in maturation of nascent synapses, including differentiation and stabilization by recruiting scaffolding proteins, postsynaptic receptors, and signaling proteins (X. Zhang, 2017).

    In Drosophila, four Nlgs have been identified. Mutations in dnlg1-3 result in defective synapse differentiation that is primarily characterized by abnormal protein levels or the ectopic postsynaptic localization of glutamate receptors in larval NMJs. In addition, loss of dnlg1-3 separately leads to impairment in NMJ synapse development, as indicated by abnormal synaptic bouton number, but the precise underlying mechanism is poorly understood. A recent study showed that flies with a dnlg4 mutation exhibit an autism-related phenotype of behavioral inflexibility, as indicated by impaired reversal learning. DNlg4 also regulates sleep by recruiting the GABA receptor to clock neurons and thus modulating GABA transmission, which suggests a role of DNlg4 in synapse differentiation. However, potential molecular mechanisms underlying the behavioral defects caused by dnlg4 mutation and the role of DNlg4 in synapse development have not been reported (X. Zhang, 2017).

    This paper reports the generation of an independent null allele of dnlg4 and characterized the role of DNlg4 in neuromuscular synaptic growth. Loss of DNlg4 led to impaired NMJ synapse growth, as indicated by decreased synaptic bouton numbers and increased bouton size. Presynaptic knockdown of DNlg4 mimicked these phenotypes. These morphological abnormalities in dnlg4 mutants led to corresponding impairment in synaptic transmission efficacy. Unexpectedly, all of these defects were only rescued when DNlg4 was expressed in presynaptic, instead of postsynaptic, areas of NMJs.DNlg4 genetically interacted with components of the BMP pathway and that the presynaptic BMP signaling at NMJs was decreased in dnlg4 mutants. A reduction of the BMP type I receptor Tkv was observed in dnlg4 mutants and DNlg4 physically interacted with Tkv in vivo. Altogether, this study revealed that DNlg4 regulated neuromuscular synaptic growth by positively modulating BMP signaling though Tkv (X. Zhang, 2017).

    Sequence analyses showed that there are four nlg genes in the Drosophila genome, and all four DNlgs share significant amino acid sequence homology and protein structures with vertebrate Nlgs. DNlg1 and DNlg2 have a positive effect on synaptic growth of NMJs, as indicated by an obvious reduction in synaptic boutons in the dnlg1 and dnlg2 mutants. Conversely, loss of DNlg3 led to increased numbers of synaptic boutons at NMJs. In the present study, a dnlg4 null mutant was generated by gene targeting, and this mutant exhibited significant defects in NMJ morphology, including fewer synaptic boutons and increased bouton size. However, neuronal overexpression of two copies of the dnlg4 transgene induced a pronounced increase in bouton number. These results demonstrated a positive role of DNlg4 in regulating synapse development. Interestingly, neuronal overexpression of one copy of dnlg4 in the WT background did not induce an increase in bouton number, but it did when expressed in a dnlg4 mutant background. These results suggested a homeostatic adjustment during synapse development in Drosophila, which could somewhat counteract the effect caused by increased DNlg4. As a result, a moderate increase of DNlg4 in WT flies did not lead to increased synaptic growth of NMJs (X. Zhang, 2017).

    In Drosophila, loss of DNlgs in the dnlg1-3 mutants also induced synaptic differentiation defects that were characterized by decreased protein levels or impaired distribution of glutamate receptors and other postsynaptic proteins at NMJs. In contrast to other dnlg mutants, statistical alteration in the distribution or protein level of glutamate receptors at NMJs in was not observed dnlg4 mutants. In addition, the distribution and protein level of some presynaptic proteins, such as BRP, CSP, and SYT, were normal in the dnlg4 mutants. However, the ultrastructural analyses of the NMJs showed that there were still some defects in synaptic ultrastructure in the dnlg4 mutants, including the increased bouton area per active zone, longer single PSD, and reduced postsynaptic SSR regions. One striking ultrastructural defect in the dnlg4 mutants was the partial detachment of presynaptic membranes from postsynaptic membranes within the active zone, which was rarely observed in WT flies, suggesting the adhesion function of DNlg4 during synaptogenesis. It was interesting that this defect also appeared in dnlg1 mutants and dnrx mutants, suggesting that the DNlg4 might affect the synaptic architecture by a mechanism similar to that of DNlg1 and DNrx (X. Zhang, 2017).

    Functionally, dnlg4 mutants showed a mild impairment in transmitter release at NMJs, as characterized by slightly reduced evoked EJP amplitude and quantal contents. This phenotype was consistent with the morphological impairments and the ultrastructural defects of NMJs, suggesting a probable reduction in the number of total synapses or functional synapses at NMJs. The amplitude of mEJP was not changed in the dnlg4 mutants, which was consistent with the normal protein levels of postsynaptic glutamate receptors. Interestingly, the frequency of mEJPs in the dnlg4 mutants was dramatically increased, indicating that DNlg4 affects spontaneous transmitter release. The detailed mechanism underlying this should be addressed in future work (X. Zhang, 2017).

    In mammals, Nlgs are generally considered to function as postsynaptic adhesion molecules and to help form trans-synaptic complexes with presynaptic Nrxs. In Drosophila, DNlg1 and DNlg3 are reported to be located in postsynaptic membranes. However, there are some exceptions to the postsynaptic localization of Nlgs. For example, an Nlg in Caenorhabditis elegans is reported to be present in both presynaptic and postsynaptic regions. DNlg2 is also required both presynaptically and postsynaptically for regulating neuromuscular synaptic growth. These studies support a more complex mechanism of Nlgs in synapse modulation and function. These data add to this complexity by suggesting a presynaptic role of DNlg4 in larval neuromuscular synaptic growth and synaptic functions (X. Zhang, 2017).

    First, in the VNC of third-instar larvae, DNlg4 was concentrated in the neuropil region where the synapses aggregated. In NMJs, although endogenous DNlg4 was not detected by anti-DNlg4 antibodies, the exogenous DNlg4 that was expressed in motoneurons was located in type I synaptic boutons of NMJs, suggesting a reasonable presynaptic location of DNlg4 at NMJs. In another assay, DNlg4 was expressed using a DNlg4-Gal4 driver to mimic the endogenous expression pattern of the dnlg4 gene. The DNlg4 promoted by DNlg4-Gal4 was distributed in type I boutons of NMJs and was located in presynaptic areas of boutons, which also supported the presynaptic location of endogenous DNlg4 at NMJs, although a simultaneous postsynaptic localization of DNlg4 at NMJs could not be excluded. Second, knockdown of DNlg4 presynaptically led to morphological defects in NMJs similar to those observed in the dnlg4 mutants, indicated by decreased bouton numbers and increased bouton size. However, knockdown of DNlg4 postsynaptically did not cause the same phenotypes. These morphological defects of NMJs in the dnlg4 mutants could be completely rescued when DNlg4 was expressed in presynaptic neurons, but not when it was expressed in postsynaptic muscles. Third, dnlg4 mutants had a significant increase in spontaneous transmitter release frequency, which was usually interpreted as a presynaptic defect. In addition, all of the functional defects in the dnlg4 mutants, including decreased amplitude of EJPs, reduced quantal contents, and increased mEJP frequency, could be rescued by presynaptic expression of DNlg4. These results indicated that presynaptic DNlg4 was essential for proper proliferation and function of synapses at NMJs. Finally, the number of synaptic boutons at NMJs was significantly increased when two copies of UAS-dnlg4 were overexpressed in presynaptic neurons, whereas this phenomenon was not observed when two copies of UAS-dnlg4 were overexpressed in muscles, suggesting that presynaptic DNlg4 alone was sufficient to promote synaptic growth. Altogether, these data provided convincing evidence that DNlg4 functions as a presynaptic molecule in regulating synaptic growth and transmitter release at NMJs (X. Zhang, 2017).

    The BMPs are major retrograde trans-synaptic signals that affect presynaptic growth and neurotransmission both in the CNS and at NMJs. This study presents immunohistochemical and genetic data showing that DNlg4 regulates NMJ growth via the BMP signaling pathway (X. Zhang, 2017).

    First, the dnlg4 mutants shared similar phenotypes with the components of the BMP signaling pathway in synapse development, synapse architecture, and synapse functions of NMJs, including reduced synaptic bouton number, increased presynaptic membrane ruffles, and decreased synaptic transmission efficacy. Second, several genetic crosses followed by neuromuscular bouton number analyses showed a definite dosage-sensitive genetic interaction between dnlg4 and the components of the BMP signaling pathway, such as tkv, wit, mad, and dad, suggesting that DNlg4 is required for BMP signaling in regulating NMJ growth. Third, both immunohistochemical and Western blot analyses showed that the pMad, which serves as an indicator of BMP signaling, is decreased in both the synaptic boutons of NMJs and motoneuronal nuclei in the dnlg4 mutants, whereas it is increased in dnlg4-overexpressing flies. Finally, the expression of the BMP signaling target gene trio, is significantly reduced in the dnlg4 mutants, but it was increased in dnlg4-overexpressing flies. Together, these results supported the hypothesis that DNlg4 promotes synaptic growth by positively regulating BMP signaling (X. Zhang, 2017).

    The critical question to be addressed, therefore, is the mechanism underlying this regulation. Through Western blot analyses of larval brain homogenates, it is found that the protein level of Tkv (as assessed by ectopically expressed Tkv-GFP) in the dnlg4 mutants is significantly decreased, whereas Wit and Mad, the other two components of the BMP pathway, were not altered. The immunohistochemical assay also showed that the Tkv protein in the dnlg4 mutants is reduced in both the VNC and the synaptic boutons of NMJs. These data indicated the specific positive regulation of Tkv by DNlg4. Previous studies reported that tkv mutants had decreased synaptic bouton number, increased presynaptic membrane ruffles, reduced amplitude of EJP, and unchanged mEJP amplitude. In addition, neuronal overexpression of one copy of transgenic tkv did not induce NMJ overgrowth, but it induced such overgrowth when two copies of transgenic tkv were expressed in neurons. These phenotypes were similar to what was observed in the dnlg4 mutants and dnlg4-overexpressing flies. All of these results strongly demonstrated that DNlg4 regulates BMP signaling by modulating Tkv protein levels (X. Zhang, 2017).

    Because the dnlg4 mutants had a level of tkv mRNA comparable with that of the WT controls, DNlg4 did not affect the transcription of tkv. The possibility of Tkv trafficking defects from the cell body to the axon terminal could also be excluded because no retention of Tkv in the soma of motoneurons was observed. Thus, it seems reasonable to speculate that DNlg4 affects the recruitment or stability of Tkvs at the presynapse. In support of this hypothesis, co-localization of Tkv and DNlg4 at presynaptic regions of NMJs was observed by immunostaining. As further confirmation, it was shown that Tkv could be co-immunoprecipitated with an antibody against DNlg4, and a reverse co-immunoprecipitation assay using anti-GFP antibodies also resulted in the precipitation of DNlg4 by Tkv-GFP, suggesting a physical interaction between DNlg4 and Tkv in vivo. Furthermore, using pull-down assay, the acetylcholinesterase-like domain of the N terminus was shown to be essential for DNlg4 interacting with Tkv (X. Zhang, 2017).

    If the recruitment of Tkv to the presynaptic membrane entirely depends on DNlg4, the existence of Tkv in the presynaptic membrane would not be detected, and accumulation of Tkv proteins in the presynaptic areas of NMJs would probably be observed in the dnlg4 mutants. However, minor Tkv protein level was detected at the presynaptic membrane, and no accumulation of Tkv was observed at NMJs in the dnlg4 mutants. Thus, a more reasonable hypothesis is that DNlg4 affected the stability of Tkv at the presynaptic membranes. The protein level of Tkv in the presynapses of NMJs was reported to be regulated by several pathways, including direct proteasomal degradation, ubiquitin-mediated degradation, and endocytosis. The simplest model is that the DNlg4 might stabilize Tkv via inhibiting its degradation. Several protein kinases, such as the ribosomal protein S6 kinase-like protein (S6KL) and the serine/threonine kinase Fused, have been reported to interact physically with Tkv in vitro and to facilitate its proteasomal degradation. In particular, S6KL has been demonstrated to degrade Tkv at NMJs. DNlg4 might stabilize Tkv by inhibiting these protein kinases, but the detailed mechanism of such activity still needs to be addressed (X. Zhang, 2017).

    In summary, this study demonstrated that DNlg4 positively regulated neuromuscular synaptic growth by modulating BMP signaling through the maintenance of Tkv protein levels in the presynapse of NMJs. To accomplish this function, DNlg4 acted as a presynaptic molecule instead of a postsynaptic molecule. This study further suggested a relationship between Nlgs and BMP signaling and provided a new understanding of the exact role of Nlgs during synapse formation and development (X. Zhang, 2017).

    Human APP gene expression alters active zone distribution and spontaneous neurotransmitter release at the Drosophila larval neuromuscular junction

    This study provides further insight into the molecular mechanisms that control neurotransmitter release. Experiments were performed on larval neuromuscular junctions of transgenic Drosophila melanogaster lines with different levels of human amyloid precursor protein (APP) production. To express human genes in motor neurons of Drosophila, the UAS-GAL4 system was used. Human APP gene expression increased the number of synaptic boutons per neuromuscular junction. The total number of active zones, detected by Bruchpilot protein puncta distribution, remained unchanged; however, the average number of active zones per bouton decreased. These disturbances were accompanied by a decrease in frequency of miniature excitatory junction potentials without alteration in random nature of spontaneous quantal release. Similar structural and functional changes were observed with co-overexpression of human APP and beta-secretase genes. In Drosophila line with expression of human amyloid-beta42 peptide itself, parameters analyzed did not differ from controls, suggesting the specificity of APP effects. These results confirm the involvement of APP in synaptogenesis and provide evidence to suggest that human APP overexpression specifically disturbs the structural and functional organization of active zone and results in altered Bruchpilot distribution and lowered probability of spontaneous neurotransmitter release (Saburova, 2017).

    In vivo single-molecule tracking at the Drosophila presynaptic motor nerve terminal

    An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. This study describes a technique to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. This study describes how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva (Bademosi, 2018).

    Inwardly rectifying potassium (Kir) channels represent a critical ion conductance pathway in the nervous systems of insects

    A complete understanding of the physiological pathways critical for proper function of the insect nervous system is still lacking. The recent development of potent and selective small-molecule modulators of insect inward rectifier potassium (Kir) channels, Kir1, Kir2, or Kir3 in Drosophila, has enabled the interrogation of the physiological role and toxicological potential of Kir channels within various insect tissue systems. Therefore, this study aimed to highlight the physiological and functional role of neural Kir channels the central nervous system, muscular system, and neuromuscular system through pharmacological and genetic manipulations. The data provide significant evidence that Drosophila neural systems rely on the inward conductance of K(+) ions for proper function, since pharmacological inhibition and genetic ablation of neural Kir channels yielded dramatic alterations of the CNS spike discharge frequency and broadening and reduced amplitude of the evoked EPSP at the neuromuscular junction. Based on these data, it is concluded that neural Kir channels in insects (1) are critical for proper function of the insect nervous system, (2) represents an unexplored physiological pathway that is likely to shape the understanding of neuronal signaling, maintenance of membrane potentials, and maintenance of the ionic balance of insects, and (3) are capable of inducing acute toxicity to insects through neurological poisoning (Chen, 2018).

    The establishment of insecticide resistance within multiple arthropod vectors of human pathogens has been, at least in part, the driving force behind the prolific advancement of the fields of insecticide science and insect molecular physiology. The goal of mitigating the various resistance mechanisms has been a multidisciplinary and transdisciplinary approach that has resulted in a detailed understanding of molecular genetics, transcriptomics, biochemistry, cellular physiology, and neuroendocrinology of non-model insects, such as mosquitoes. In addition to these fields, the reduced efficacy of currently approved classes of insecticides has dramatically increased interest of identifying novel molecular targets for insecticide design and/or development of novel chemical scaffolds targeting previously exploited proteins. A variety of new target sites and chemical scaffolds have been identified and characterized in the past decade that include transient receptor proteins, G-protein coupled receptors, dopaminergic pathways, and K+ ion channels (Chen, 2018).

    Inward rectifier potassium (Kir) channels belong to a large 'superfamily' of K+ ion channels that includes the voltage-gated, two-pore, calcium-gated, and cyclic nucleotide-gated channels. Kir channels function as biological diodes due to their unique ability to mediate the inward flow of K+ ions at hyperpolarizing membrane voltages more readily than the outward flow of K+ at depolarizing voltages. On a molecular level, Kir channel are structurally simple ion channels that consists of 4 subunits assembled around a central, water-filled pore, through which K+ ions move down their electrochemical gradient to traverse the plasma membrane. Each subunit consists of a central transmembrane domain, a re-entrant pore-forming loop, and a cytoplasmic domain comprised of amino and carboxyl termini (Chen, 2018).

    Recent genetic and pharmacological evidence suggests that Kir channels could represent viable targets for new insecticides. In Drosophila melanogaster, embryonic depletion of Kir1, Kir2, or Kir3 mRNA leads to death or defects in wing development. Reduction of Kir1 and Kir2 mRNA expression in the Malpighian (renal) tubules of Drosophila or inhibition of Kir channels in isolated mosquito Malpighian tubules with barium chloride (BaCl2) dramatically reduces the transepithelial secretion of fluid and K+ (Wu, 2015; Scott, 2004), indicating Kir channels expressed in the Malpighian tubules may be an exploitable insecticide target site. Considering this, high-throughput screens (HTS) of chemical libraries were performed to identify small-molecule modulators of mosquito Kir1 channels, which is the principal conductance pathway in mosquito Malpighian tubules. Structurally distinct small molecules were identified (i.e., VU573, VU590, or VU625) and pharmacological inhibition of Aedes aegypti Kir1 was shown to disrupt the secretion of fluid and K+ in isolated Malpighian tubules, urine production, and K+ homeostasis in intact females. Similarly, a Kir1 inhibitor, termed VU041, was identified in a subsequent HTS campaign and was shown to (1) be highly potent against the Anopheles gambiae Kir1 (ca. 500 nanomolar), (2) exhibit topical toxicity (ca. 1 μg/mosquito) to insecticide-susceptible and carbamate/pyrethroid-resistant strains of mosquitoes, (3) and display high selectivity for mosquito Kir channels over mammalian Kir channel orthologs (Chen, 2018).

    Previous work indicates that VU041-mediated toxicity stems from inhibition of the Kir1 channel within the Malpighian tubules to induce tubule failure and an inability to maintain K+ homeostatsis after blood feeding. However, after exposure to lethal doses of VU041, An. gambiae and A. aegypti were found to display both hyperexcitatory and lethargic tendencies that were complexed with uncoordinated movements, which is reminiscent of neurological poisoning. Furthermore, acute toxicity (ca. 1-3 hours) was observed after exposure to VU041, similar to other insecticides that poison the nervous system. Lastly, previous studies have shown that select Kir channel inhibitors were capable of inducing a flightless behavior where mosquitoes were ambulatory, yet were not able to fly, presumably due to failure of the nervous or muscular systems. Although it is possible that the mortality is due to complete systems failure stemming from ubiquitous expression of Kir channels or due to accumulated waste that remains due to impaired Malpighian tubule function, it is also reasonable to predict that VU041 is directly altering the functional capacity of Kir channels expressed in the nervous system to yield toxicity. Unfortunately, there have been no studies to characterize the physiological role of Kir channels in the insect nervous systems, which limits the ability to infer the toxicological potential of these neural proteins. Studies using RT-PCR have shown that the head of A. aegypti is enriched with Kir2B' (vector base accession number: AEL013373) mRNA (Dr. Peter Piermarini, The Ohio State University, personal communication to R. Chen, 2018), suggesting that poisoning of the mosquito central nervous system (CNS) through Kir inhibition is indeed possible. Unfortunately, electrophysiological recordings of mosquito CNS activity have yet to be achieved, which limits the ability to infer the physiological role or toxicological potential of neural Kir channels of mosquitoes. However, electrophysiological recordings from an excised CNS of D. melanogaster is possible and further, the gene encoding Kir2, termed irk2, is highly concentrated in the adult head, CNS, and the thoracic-abdominal ganglia. This suggests that D. melanogaster may represent a suitable substitute for mosquitoes and will enable the characterization of the physiological role Kir channels have in the insect nervous system (Chen, 2018).

    Considering (1) the foundational role of Kir channels in mammalian and insect cellular physiology, (2) deletion of irk2 gene in Drosophila is homozygous lethal, (3) the signs of intoxication after exposure to Kir channel modulators being reminiscent of neurological poisoning, and (4) the overexpression of Kir mRNA in mosquito and Drosophila neural tissues, it was hypothesized that Kir channels regulate neuronal signaling and excitability of insect nervous systems and are a critical conductance pathway for proper functioning of the insect nervous system. Therefore, the goals of the present study were to employ electrophysiological methods combined with genetic and pharmacological techniques to determine the physiological importance of Kir channels in insect CNS, neuromuscular junction, and muscular systems that will provide insight into targeting neural Kir channels as a novel insecticide target site. Additionally, data collected in this study begin to bridge the fundamental knowledge gap regarding unexplored physiological pathways in the insect nervous system that will provide a more holistic understanding to neuronal excitability and neurotransmission of insects (Chen, 2018).

    Currently, there have been no efforts to characterize the physiological role or toxicological potential of insect neural Kir channels. However, the current findings demonstrate that the recently discovered Kir-directed insecticide, VU0413, is capable of dramatically altering the neural activity of flies and, in a more general sense, that Kir channels constitute a critical K+ ion conductance pathway in the insect nervous system. Despite the nervous system being the target tissue of the extreme majority of deployed insecticides, a complete understanding of the physiological pathways critical for proper function of the insect nervous system is still lacking. This represents a critical gap in knowledge of the complex relationship between the dozens of functionally coupled ion channels, transporters, and enzyme systems that require tight regulation for proper neuronal function. This fundamental gap pertaining to the foundational neural physiology must be filled to develop a holistic understanding of insect nervous system function that will lead to the development of new insecticides (Chen, 2018).

    Knowledge of the physiological role and toxicological potential of insect Kir channels is growing rapidly with studies suggesting these channels serve a critical role in Malpighian tubule function of mosquitoes and Drosophila, insect salivary gland function, honey bee dorsal vessel function, and insect antiviral immune pathways. Furthermore, these channels represent a critical K+ conductance pathway in the mammalian nervous system as Kir knockouts in glial cells leads to membrane depolarization, enhanced synaptic potentiation, and reduced spontaneous neural activity. Considering the importance of Kir channels in the function of various insect tissues and the established role of Kir channels in mammalian neuronal tissue, it is hypothesized that Kir channels also serve a critical role in insect neural tissue and aimed to highlight the general influence of Kir channel modulation to the insect nervous system through pharmacological and genetic manipulations of the Kir channel (Chen, 2018).

    To begin testing the physiological role of insect neural Kir channels, neurophysiological recordings of the Drosophila CNS were performed using the voltage dependent Kir blocker, BaCl2. BaCl2 is useful pharmacological tool to test the physiological role of Kir channels since, at physiological membrane potentials, Kir channels are up to 1000-fold more sensitive to BaCl2 than other K+ ion channels. This enhanced potency to Kir channels when compared to other K+ ion channels enables selective inhibition of Kir channels at low- to mid-micromolar concentrations of BaCl2. An increase was observed in the spike discharge frequency followed by cessation of firing after exposure of the CNS to mid-micromolar concentrations of BaCl2, providing the first insight that Kir channels constitute a critical conductance pathway in insect CNS. However, the potential for BaCl2 to precipitate out of some saline solutions and the potential of BaCl2 to modulate non-target proteins limits the conclusions that can be drawn from these data. Fortunately, the recent identification of selective and potent small molecules designed to target insect Kir channels has facilitated the characterization of the physiological role of these channels in various insect tissue systems with more certainty than BaCl2 and other divalent cations (Chen, 2018).

    This study used the recently discovered insect Kir channel modulator (VU041) and its inactive analog (VU937) to characterize the influence these channels have in insect nervous system function. Exposure of the Drosophila CNS to VU041 dramatically altered the spike discharge frequency in a biphasic manner with low concentrations yielding neuroexcitation and higher concentrations having a depressant effect on CNS activity. A biphasic response is oftentimes observed when multiple pathways are inhibited and it is plausible that VU041 is directly or indirectly altering the functional capacity of other ion channels or transporters, such as delayed rectifier K+ channels or calcium-activated K+ channels. Although off-target effects are possible, they are unlikely since VU937 had no influence to CNS activity, suggesting the observed phenotype is through Kir inhibition. To ensure the observed effect to CNS activity was directly due to Kir2 channel modulation, CNS-specific RNAi-mediated knockdown was performed of the Kir2 encoding gene, irk2. Results from this genetic depletion of irk2 show a dramatic increase in CNS spike discharge frequency that was also substantiated through hyperactive larval behavior. These observed responses to VU041 and irk2 genetic depletion is likely due to the physiological role of only Kir2 since no mRNA reduction was observed in other Kir-encoding genes that are expressed in the CNS or any irk gene within the whole body or carcass. Previous reports have documented compensatory functions of Kir channels that arise after genetic depletion of one Kir channel, which prevents the manifestation of an observable change in phenotype (Wu, 2015). Yet, it does not appear that a compensatory mechanism arose to account for the genetic depletion of irk2 since a direct physiological response was observed and irk1 and irk3 mRNA levels remained unchanged. The influence to expression of other K+ ion transport pathways, such as Na+-K+-2Cl- cotransporter and Na+-K+-ATPase pumps, remains unknown and should be studied prior to drawing absolute conclusions regarding the physiological basis for neural Kir channels. Furthermore, exposure of the neuromuscular junction to VU041 altered the evoked EPSP waveform and muscle excitability. These data indicate that Drosophila, and likely mosquito, central and muscular nervous systems rely on the inward conductance of K+ ions through Kir channels for proper function (Chen, 2018).

    The Drosophila genome encodes three Kir channel proteins, termed ir, irk2 and irk3, and all three contain the structural features and biophysical properties that are found in mammalian Kir channel subunits. Although ir and irk3 mRNA has been found to be expressed at low levels in the fly head, the irk2 gene is highly expressed in the adult fly head where it is concentrated in the brain and eye, suggesting that, of the Kir channels, irk2 is the principal inward conductance pathway for K+ ions. The sequence of irk2 is similar to that of ir, and both are highly related to human Kir 2, 3, and 6 proteins, which are constitutively active, GIRK, and ATP-gated Kir channels, respectively. Interestingly, irk2 channels have been shown to be constitutively active in S2 cells, associate with sulphonylurea receptors (SUR) as is seen with KATP channels, and the presence of an Asn223 residue suggests similarity to the GPCR-gated Kirs (Kir3.x; mammalian nomenclature). The variable functional associations have led to the speculation that irk2 may have different mechanisms of gating and regulation based on the cell type the gene is expressed in. Due to this, pharmacological modulators of mammalian GIRK and KATP channels were used to determine the mechanisms of irk2 gating in the Drosophila CNS. The GIRK activator, ML297, is highly selective for mammalian GIRK1/2 subunit combination over other Kir channels and was found to induce neuroexcitation to the Drosophila CNS. The sustained increase in Drosophila CNS activity after ML297 exposure was unexpected since GIRK2 knockouts in mice revealed an epileptic phenotype, suggesting GIRK is responsible for depressing neuronal excitability and thus, an activator of GIRK should reduce CNS spike discharge frequency. It is important to note that ML297 was shown to have moderate activity on the mammalian serotonin (5-Ht2b) receptor, which is expressed in the Drosophila CNS and may be the cause for observed neuroexcitation to the Drosophila CNS. Unfortunately, the severely underdeveloped pharmacological library of GIRK inhibitors prevents further interrogation at this time. To determine if Irk2 is gated by ATP, four structurally distinct activators and inhibitors of mammalian KATP channels were used. No change in CNS spike discharge frequency was observed after exposure to these molecules at concentrations ranging into the upper micromolar range. Since other studies have shown clear effects to various insect systems with mammalian KATP modulators, the lack of response to the Drosophila CNS is likely to be due to the absence of ATP-gated Kir channels and not due to incompatibility of the structural scaffolds with the Drosophila KATP channel. These findings have led to the speculation that (1) irk2 is not likely to be expressed as a KATP channel in the CNS, (2) constitutively active Kir channels are present in the Drosophila CNS, and (3) GIRK-like channels may be present in the CNS yet further studies are required to interrogate this claim (Chen, 2018).

    The data presented in this study raise the question as to what the physiological role Kir channels have in nervous system function of insects at the cellular level. In mammals, astrocyte function has received significant interest for their roles in the regulation of synaptic levels of neurotransmitters, in particular glutamate, buffering of extracellular K+, and release of neurotransmitters, all of which have been shown to directly modulate neuronal excitability and transmission. In particular, Kir4.1 channels expressed in astrocytes have been directly linked to K+ influx across neural membranes where cells take up excess extracellular potassium ions, distribute them via gap junctions, and extrude the ions at sites in which extracellular K+ concentrations ([K+]out) is low, which is termed K+ spatial buffering. It is reasonable to predict that the insect nervous system employs this method of K+ transport during neuronal activity since [K+]out is dramatically increased and must be rapidly reversed to prevent membrane depolarization of neurons. Therefore, inhibition of this process through pharmacological blockage of neural Kir channels will lead to depolarization of the nervous system and induce CNS excitation, which was observed in this study at low concentrations of VU041 and after genetic knockdown of Kirs. In mammals, a complete knockout of Kir4.1 yielded a reduction of spontaneous EPSC in pyramidal neurons, similar to what was observed after CNS exposure to concentrations of VU041 greater than 10 µM (Chen, 2018).

    It is hypothesized that Kir channels provide a pathway for K+ spatial buffering during neuronal activity of Drosophila and this pathway is critical for proper CNS activity. Excitability and synaptic transmission of insect and mammalian nervous systems are dependent upon [K+]out and alteration of the K+ ion gradient directly affects excitatory neurotransmission. In accordance to this, changes were observed in the CNS spike frequency and complete cessation of evoked EPSP's at the NMJ, which is classically attributed to changes in presynaptic function that may be resultant of altered neurotransmitter release. Similarly, reduced amplitude and broadening of the evoked EPSP waveform at the neuromuscular junction were observed after pharmacological inhibition of Kirs, which may be a result of modification of postsynaptic terminal responsiveness to neurotransmitters. The influence of Kir channel inhibition to pre- and post-synaptic function can be due to changes in either extracellular ion or transmitter levels. This is evidenced by the response of the Drosophila CNS after exposure to BaCl2 and 25 μM VU041. Exposure to these pharmacological agents yielded near maximal spike discharge frequency that culminated in a relatively abrupt termination of this activity. This reduction of CNS spike frequency may be due to depolarization-induced inactivation of Na+ channels due to prolonged exposure to elevated [K+]out, thereby lowering the probability of transmitter release that will reduce neuronal firing. Therefore, it appears as though Kir channels are responsible for regulating the K+ ion gradient that ultimately controls synaptic activity and neurotransmitter release, which is essential for proper neural signaling and activity (Chen, 2018).

    Kir channels represent a critical K+ ion conductance pathway within the Drosophila, and likely mosquito, central and neuromuscular nervous systems. Considering this, it is reasonable to suggest that the recently identified Kir-directed mosquitocide, VU041, is capable of inducing toxicity through neurological poisoning in addition to inducing Malpighian tubule failure that leads to toxicity by an inability to perform osmoregulatory actions. These data provides a proof-of-concept that novel chemical scaffolds targeting neural Kir channels in insects represent a novel mechanism of action with insecticide resistance mitigating potential. Based on the data collected in this study, it is hypothesized that the function of Kir channels in the insect nervous system is responsible for reducing [K+]out during neuronal activity by the process known as K+ spatial buffering, similar to that described in mammals. It is important to note that this hypothesis cannot be fully validated until whole-cell electrophysiological recordings are performed to determine the role of Kirs in (1) glutamate and K+ uptake during neural activity, (2) maintenance of neural membrane properties (e.g., Vm, Rm, etc.), and 3) synaptic transmission and plasticity (Chen, 2018).

    The Neurexin-NSF interaction regulates short-term synaptic depression

    Although Neurexins, which are cell adhesion molecules localized predominately to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their role in the regulation of synaptic vesicle release during repetitive nerve stimulation is unknown. This study shows that Drosophila neurexin mutant synapses exhibit rapid short-term synaptic depression upon tetanic nerve stimulation. Moreover, the intracellular region of Neurexin was demonstrated to be essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, it was found that the intracellular region of Neurexin interacts with N-ethylmaleimide sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. The binding sites of each molecule were mapped, and it was demonstrated that the Neurexin-NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. These results reveal a previously unknown role of Neurexin in the regulation of short-term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of Neurexin in synaptic vesicle release (Li, 2015).

    Previous studies have shown that the α-NRXs functionally couple Ca2+ channels to the presynaptic machinery to mediate synaptic vesicle exocytosis and that defective synaptic vesicle release can be restored with 1 mm Ca2+ (Li, 2007). In the current experiments, 1.8 mm Ca2+ was used to eliminate the effects of abnormal Ca2+ sensitivity in neurotransmitter release. Under this condition, it was shown that nrx mutant synapses exhibit rapid short term synaptic depression and reduced quantal content of nerve-evoked synaptic currents at the steady state during tetanic stimulation. These observations suggest that NRX regulates activity-dependent synaptic plasticity. Similar observations have been reported in the fast-twitch diaphragm muscle of the α-NRX double knock-out mouse, which indicates that presynaptic efficacy but not presynaptic homeostatic plasticity is normal under basal conditions (Li, 2015).

    Deficits in both the presynaptic neurotransmitter release machinery and the postsynaptic neurotransmitter receptors may cause rapid short term synaptic depression. As a presynaptic adhesion molecule, NRX interacts with the postsynaptic adhesion molecule, Neuroligin, and bridges the synaptic cleft that aligns the presynaptic neurotransmitter release machinery with the postsynaptic neurotransmitter receptors. It has been suggested that the activity-dependent regulation of the NRX/Neuroligin interaction mediates learning-related synaptic remodeling and long term facilitation. A recent study reveals that the alternative splicing of presynaptic NRX-3 controls postsynaptic AMPA receptor trafficking and long term plasticity in mice (Aoto, 2013). This study showed that the kinetics of the individual synaptic currents were stable during tetanic stimulation in the nrx mutant synapses. Moreover, NRX was shown to mediate synaptic plasticity through the presynaptic machinery, which was supported by rescue experiments (Li, 2015).

    The intracellular sequence of NRX, including the C-terminal PDZ-binding motif, is identical across several vertebrate and invertebrate species. Mammalian NRXs only possesses a short cytoplasmic tail (55 amino acids), including a PDZ-binding motif. To identify the potential binding partners, several laboratories have conducted yeast two-hybrid screening by using the cytosolic tail of mammalian NRX as a bait. Two PDZ-containing proteins, CASK and syntenin, have been identified in the previous screening, and further studies show that the binding between CASK and NRX is abolished by deletion of the last three amino acids of the intracellular C-terminal region of NRX. However, it seems that the previous screenings are not saturated, as the NRX-interacted protein Mints was not identified in these screenings (Li, 2015).

    Drosophila NRX contains a long cytoplasmic tail (122 amino acids), including a functional PDZ-binding motif. Thus, it is possible that the long cytoplasmic tail may associate with some partners in a PDZ-independent manner. In this study, normalized Drosophila cDNA libraries were used in the screening, which helps to identify the binding partner with low abundance. In the screening, 23 cDNAs clones that encode the fragments of 18 proteins were recovered. Previous study has demonstrated that the cytoplasmic tail of NRX associates with RFABG and facilitates the retinol transport. This study identified the intracellular region of NRX binds with NSF through the amino acid sequence 1788-1813 but not the PDZ-binding motif of NRX. This NRX/NSF interaction was found to be essential for the NSF recruitment to the presynaptic terminals and plays an important role in synaptic vesicle release. In addition, multiple lines of evidence that NRX associates with NSF at the presynaptic terminals. An alignment analysis of the NSF-binding site of NRX revealed that this sequence is conserved across different species. Moreover, this sequence is present across many proteins that serve different functions, which implies that this sequence may be essential for protein interactions (Li, 2015).

    Each NSF molecule contains an N-terminal domain that is responsible for the interaction with α-SNAP and the SNARE complex, a low affinity ATP-binding domain (D1 domain) whose hydrolytic activity is associated with NSF-driven SNARE complex disassembly and a C terminal high affinity ATP-binding domain (D2 domain). This study mapped the NRX-binding sites of NSF to the D2 domain, which mediates the ATP-dependent oligomerization of NSF. An alignment analysis showed that both the D2 domain in NSF and the NSF-binding site in NRX are highly conserved. This result suggests that the NRX/NSF interaction may occur in other species, a possibility that needs to be investigated further (Li, 2015).

    It has been documented that the D2 domain of NSF is essential for its hexamerization. This study shows that NRX exhibits a declined NSF binding capacity in high Ca2+ concentration. These observations suggest that NSF can be released from NRX under stimulation. Although NRX and NSF do not bind with Ca2+ directly, it is possible that they undergo Ca2+ signaling-dependent modifications or bind with some Ca2+-binding proteins. In the yeast two-hybrid screening, one potential Ca2+-binding protein (CG33978) has been identified. However, the mechanism of how NRX releases NSF under high Ca2+ concentration need to be further investigated (Li, 2015).

    Previous studies have shown that the PDZ-binding motif of NRX associates with several molecules involved in the synaptic vesicle exocytosis machinery, including synaptotagmin and the PDZ domain-containing proteins CASK and Mints. Synaptotagmin functions as a Ca2+ sensor and controls synaptic membrane fusion machinery. Adaptor protein Mints regulates presynaptic vesicle release (Ho, 2006), whereas the other adaptor protein CASK is not essential for the Ca2+-triggered presynaptic release. In contrast, CASK interacts with NRX and protein 4.1 to form a trimeric complex and regulates synapse formation. The process of synaptic vesicle release includes several consecutive steps, docking, priming, and fusion. Depending on the stimulation given to synapses, different synaptic vesicle trafficking steps become rate-limiting for synaptic vesicle release. This causes short term changes in synaptic transmission that determine many higher brain functions such as sound localization, sensory adaptation, or even working memory. Thus, the PDZ-binding motif-linked synaptic vesicle exocytotic machinery might regulate the different steps during synaptic vesicle release. Indeed, nrx mutant synapses that express NRXΔ4 exhibit a rapid current decline over the first several dozen stimulations. In contrast, the expression of NRXΔ4 largely restores the reduced steady-state mean EJCs and quantal content in neurexin mutant synapses. Together with the C-terminally truncated NRX rescue experiments, these data suggest that intracellular regions of NRX other than the PDZ-binding motif also regulate short term synaptic depression. The existing literature extensively documents the roles of NSF in SNARE complex disassembly and short term synaptic depression. This study extended these findings to show that the NRX/NSF interaction facilitates the recruitment of NSF to the presynaptic terminals and promotes the subsequent SNARE complex disassembly (Li, 2015).

    Previous studies have established that the NSF hexamer serves as the only active form for SNARE complex disassembly. In this study, the binding assay showed that purified NRX binds to NSF in a concentration-dependent and saturable manner. NRXs appear to serve as scaffold proteins to recruit NSF, and the NRX/NSF interaction may promote SNARE complex disassembly in vivo. Live image studies have shown that NSF mutant (i.e. comt) synapses exhibit defective NSF re-distribution during tetanic nerve stimulation. In this study, immunocytochemical and sedimentation analyses revealed that a lack of the NRX/NSF interaction results in an altered distribution of NSF and an accumulation of 7S SNARE complexes. These results imply that NRX/NSF interaction serves as a potential mechanism to restrict the mobilization of NSF (Li, 2015).

    Electrophysiological recordings revealed that synaptic depression was comparable between nrx mutant synapses and wild-type synapses in response to low frequency stimulations. The long intervals between low frequency stimulations may allow the remaining NSF to disassemble the SNARE complexes and generate enough free t-SNARE for the subsequent synaptic vesicle fusion events. Another possibility that needs to be investigated further is that tetanic stimulation may have other effects on the re-distribution of NSF (Li, 2015).

    In summary, this study provides evidence that the NRX/NSF interaction recruits NSF to the presynaptic terminals and promotes SNARE complex disassembly. These findings have revealed a previously unknown role of NRX in the regulation of neurotransmitter release and provide a linkage between the presynaptic adhesion molecules and the presynaptic plasticity machinery (Li, 2015).

    Characterization of developmental and molecular factors underlying release heterogeneity at Drosophila synapses

    Neurons communicate through neurotransmitter release at specialized synaptic regions known as active zones (AZs). Using biosensors to visualize single synaptic vesicle fusion events at Drosophila neuromuscular junctions, this study analyzed the developmental and molecular determinants of release probability (Pr) for a defined connection with ~300 AZs. Pr was heterogeneous but represented a stable feature of each AZ. Pr remained stable during high frequency stimulation and retained heterogeneity in mutants lacking the Ca(2+) sensor Synaptotagmin 1. Pr correlated with both presynaptic Ca(2+) channel abundance and Ca(2+) influx at individual release sites. Pr heterogeneity also correlated with glutamate receptor abundance, with high Pr connections developing receptor subtype segregation. Intravital imaging throughout development revealed that AZs acquire high Pr during a multi-day maturation period, with Pr heterogeneity largely reflecting AZ age. The rate of synapse maturation was activity-dependent, as both increases and decreases in neuronal activity modulated glutamate receptor field size and segregation (Akbergenova, 2018).

    This study used quantal imaging, super resolution SIM, and intravital imaging to examine the development of heterogeneity in evoked Pr across the AZ population at Drosophila NMJs. It was first confirmed that release heterogeneity was not caused by summation of fusion events from multiple unresolvable AZs. Indeed, high Pr sites corresponded to single AZs with enhanced levels of BRP. These findings are consistent with previous observations using conventional light microscopy that indicate Pr correlates with BRP levels. By monitoring release over intervals of extensive vesicle fusion during strong stimulation, it was also observed that Pr is a stable feature of each AZ. In addition, loss of the synaptic vesicle Ca2+ sensor Syt1 globally reduced Pr without altering the heterogeneous distribution of Pr across AZs, indicating that AZ-local synaptic vesicle pools with differential Ca2+ sensitivity are not likely to account for Pr heterogeneity. Since voltage gated calcium channel (VGCC) abundance, gating, and organization within the AZ are well established regulators of Pr across synapses, heterogeneity in presynaptic Ca2+ channel abundance was a clear candidate for the generation of Pr heterogeneity at Drosophila NMJs. Indeed, the Cac Ca2+ channel responsible for neurotransmitter release is heterogeneously distributed across the NMJ . Using transgenically labeled Cac lines, it was observed that Cac density at AZs is indeed strongly correlated with Pr. To directly visualize presynaptic Ca2+ influx at single AZs, GCaMP fusions were generated to the core AZ component BRP. Ca2+ influx at single AZs was highly correlated with both Cac density and Pr. The cacNT27 mutant with decreased conductance also resulted in a global reduction in Pr without disrupting heterogeneity, further confirming that Ca2+ influx regulates Pr across the range of release heterogeneity (Akbergenova, 2018).

    Postsynaptically, high Pr AZs were enriched in GluRIIA-containing receptors and displayed a distinct pattern of glutamate receptor clustering. While most synapses showed GluRIIA and GluRIIB spread over the entire PSD, high Pr AZs were apposed by PSDs where GluRIIA concentrated at the center of the AZ, with GluRIIB forming a ring at the PSD periphery. Indeed, anti-glutamate receptor antibody staining of wildtype larvae lacking tagged glutamate receptors had previously identified a GluRIIB ring around the GluRIIA core in some mature third instar NMJ AZs (Marrus , 2004). In addition, activity-dependent segregation of GluRIIA and a GluRIIA gating mutant has been observed at individual AZs in Drosophila (Petzoldt, 2014). The correlation of Pr with GluRIIA accumulation is especially intriguing considering that this subunit has been implicated in homeostatic and activity-dependent plasticity (Davis, 2006; Frank, 2014; Petersen et al., 1997; Sigrist et al., 2003). By following the developmental acquisition of this postsynaptic property as a proxy for Pr from the first through third instar larval stages via intravital imaging in control and mutant backgrounds, it was observed that the earliest formed AZs are the first to acquire this high Pr signature over a time course of ~3 days, and that PSD maturation rate can be modulated by changes in presynaptic activity (Akbergenova, 2018).

    Similar to prior observations, this study found that most AZs at the Drosophila NMJ have a low Pr. For the current study, the AZ pool was artificially segregated into low and high release sites, with high releasing sites defined based on having a release rate greater than two standard deviations above the mean. Given that birthdate is a key predictor of glutamate receptor segregation, and by proxy Pr, the AZ pool is expected to actually reflect a continuum of Pr values based on their developmental history. However, using the two standard deviation criteria, 9.9% of AZs fell into the high Pr category, with an average Pr of 0.28. It was also observed that 9.7% of the AZs analyzed displayed only spontaneous release. No fusion events could be detected for either evoked or spontaneous release for another 14.6% of AZs that were defined by a GluRIIA-positive PSD in live imaging. Future investigation will be required to determine whether these cases represent immature AZs with extremely low evoked Pr, or distinct categories reflective of differences in AZ content. The remaining AZs that participated in evoked release had an average Pr of 0.05. Ca2+ channel density and Ca2+ influx at individual AZs was a key determinant of evoked Pr heterogeneity, as Pr and the intensity of Cac channels tagged with either TdTomato or GFP displayed a strong positive correlation. Spontaneous fusion showed a much weaker correlation with both Cac density and Ca2+ influx at individual AZs, consistent with prior studies indicating spontaneous release rates are poorly correlated with external Ca2+ levels at this synapse. With synaptic vesicle fusion showing a steep non-linear dependence upon external Ca2+ with a slope of ~3-4, a robust change in Pr could occur secondary to a relatively modest increase in Ca2+ channel abundance over development. Although the number of VGCCs at a Drosophila NMJ AZ is unknown, estimates of Cac-GFP fluorescence during quantal imaging indicate a ~ 2 fold increase in channel number would be necessary to move a low Pr AZ into the high Pr category. Similar correlations between evoked Pr and Ca2+ channel abundance have been found at mammalian synapses, suggesting this represents a common evolutionarily conserved mechanism for determining release strength at synapses (Akbergenova, 2018).

    This study did not test the correlation of Pr with other AZ proteins besides Cac and BRP, but it would not be surprising to see a positive correlation with the abundance of many AZ proteins based on the observation that maturation time is a key determinant for Pr. Indeed, recent studies have begun to correlate Pr with specific AZ proteins at Drosophila NMJs. It was also observed that PSD size was robustly increased by 1.6-fold over a 24 hr period of AZ development during the early larval period in control animals. AZ maturation is likely to promote increased synaptic vesicle docking and availability, consistent with observations that correlate AZ size with either Pr or the readily releasable pool (Akbergenova, 2018).

    Several models are considered for how AZs acquire this heterogeneous nature of Pr distribution during a developmental period lasting several days. One possibility is that unique AZs gain high Pr status through a mechanism that would result in preferential accumulation of key AZ components compared to their neighbors. Given that retrograde signaling from the muscle is known to drive synaptic development at Drosophila NMJs, certain AZ populations might have preferential access to specific signaling factors that would alter their Pr state. Another model is that AZs compete for key presynaptic Pr regulators through an activity-dependent process. High Pr AZs might also be more mature than their low Pr neighbors, having a longer timeframe to accumulate AZ components. Given the Drosophila NMJ is constantly forming new AZs at a rapid pace during development, newly formed AZs would be less mature compared to a smaller population of 'older' high Pr AZs (Akbergenova, 2018).

    To examine if the release heterogeneity observed at the third instar stage reflects AZ birth order over several days of development, the intravital imaging was extended through a longer time period beginning in the first instar larval stage. GCaMP imaging indicated high Pr sites segregate GluRIIA and GluRIIB differently from low Pr sites, with the IIA isoform preferentially localizing at the center of PSDs apposing high Pr AZs. As such, developmental acquisition of this property was used as an indicator of high Pr sites. Although segregation of glutamate receptors may not perfectly replicate the timing of Pr acquisition during development, it is currently the best tool for estimating Pr during sequential live imaging. Based on the acquisition of GluRIIA/B segregation, the data support the hypothesis that increases in Pr reflect a time-dependent maturation process at the NMJ. The continuous addition of new AZs, which double in number during each day of development, ensures that the overall ratio of high to low Pr sites represents a low percentage as the NMJ grows. Confidence in the age-dependency model of Pr was further established by mapping release after following NMJs intravitally for 24 hr; using this approach, it was found that newly formed AZs are consistently very low Pr. Finally, Pr was mapped in the second instar stage, and it was observed that the heterogeneity at this stage is shifted towards higher releasing sites, with a reduction in the fraction of low-releasing sites (Akbergenova, 2018).

    These data indicate AZs are born with low Pr and gain pre- and postsynaptic material over 3 days on an upward trajectory toward higher Pr status. Is AZ age a static determinant of Pr, or can growth rate be regulated to allow faster or slower acquisition of high Pr AZs? To answer this question, whether mutants that alter presynaptic activity influence the rate of PSD growth and GluRIIA/B segregation was assayed. In BRP69/def, syt1null, and napTS mutants with decreased presynaptic release, a significant reduction in postsynaptic maturation rate and in the percentage of PSDs with GluRIIA/IIB rings was observed. Conversely, in the shaker, eag double mutant with increased presynaptic excitability, a significantly increased rate of GluRIIB field size and a significant increase in GluRIIA/IIB rings was found compared to controls. To investigate whether differences in synapse growth and maturation rate could be seen between AZs enriched in BRP and Cac versus neighboring AZs that are deficient in these components, development was imaged in the rab3 null mutant. At the first instar stage when AZs are roughly age matched, release sites enriched in presynaptic components had developed large and mature PSDs in stark contrast with non-enriched AZs, whose PSDs appeared immature. These results indicate that PSD maturation can be influenced by presynaptic activity at the resolution of single AZs (Akbergenova, 2018).

    Although how AZs are assembled during development is still being established, the current data do not support a model where AZs are fully preassembled during transport and then deposited as a single 'quantal' entity onto the presynaptic membrane. Rather, these data support a model of seeding of AZ material that increases developmentally over time as AZs matures, consistent with previous studies of AZ development in Drosophila. Although no evidence for rapid changes in Pr were detected in the steady-state conditions used in the current study, homeostatic plasticity is known to alter Pr over a rapid time frame (~10 min) at the NMJ. It will be interesting to determine if Cac abundance can change over such a rapid window, or whether the enhanced release is mediated solely through changes in Cac function and Ca2+ influx. Changes in the temporal order of Pr development could also occur secondary to altered transport or capture of AZ material. For example, the large NMJ on muscle fibers 6 and 7 displays a gradient in synaptic transmission, with terminal branch boutons often showing a larger population of higher Pr AZs. If AZ material is not captured by earlier synapses along the arbor, it would be predicted to accumulate in terminal boutons, potentially allowing these AZs greater access to key components, and subsequently increasing their rate of Pr acquisition. Alternatively, the gradient of Pr along the axon could be due to terminal boutons being slightly older than the rest of the arbor (Akbergenova, 2018).

    In summary, the current data indicate that heterogeneity in release correlates highly with Ca2+ channel abundance and Ca2+ influx at AZs. Postsynaptically, PSDs apposed to high-releasing AZs display increased GluRIIA abundance and form segregated receptor fields, with GluRIIB forming a ring around a central core of GluRIIA. Release sites accumulate these high Pr markers during a synapse maturation process in which newly formed AZs are consistently low Pr, with AZs gaining signatures of high releasing sites over several days. Finally, mutations that increase or decrease presynaptic activity result in faster or slower rates of PSD maturation, respectively. These data add to the understanding of the molecular and developmental features associated with high versus low Pr AZs (Akbergenova, 2018).

    A neuropeptide signaling pathway regulates synaptic growth in Drosophila

    Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. This study demonstrates that cholecystokinin-like receptor (CCK-like receptor at 17D1; CCKLR), and Drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR (CCKLR-17D1 but not CCKLR-17D3) or dsk produce severe NMJ undergrowth, whereas overexpression of CCKLR causes overgrowth. Presynaptic expression of CCKLR is necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduce synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. These results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

    Proper synaptic growth is essential for normal development of the nervous system and its function in mediating complex behaviors such as learning and memory. The Drosophila larval neuromuscular junction (NMJ) has become a powerful system for studying the molecular mechanisms underlying synaptic development and plasticity, and many of the key synaptic proteins are evolutionarily conserved (Chen, 2012).

    Genetic and molecular analysis in Drosophila has uncovered numerous molecules and pathways that regulate NMJ growth, including proteins required for cell adhesion, endocytosis, cytoskeletal organization, and signal transduction via TGF-β, Wingless, JNK, cAMP, and other signaling molecules. For example, previous studies revealed that increased cAMP levels led to a down-regulation of the cell adhesion protein FasII at synapses, and the activation of the cAMP response element binding protein (CREB) transcription factor to achieve long-lasting changes in synaptic structure and function. Despite the identification and characterization of these various positive and negative regulators, understanding of the networks that govern synaptic growth is still incomplete, with many of the key components and mechanisms yet to be uncovered and analyzed (Chen, 2012).

    To search for new regulators of synaptic growth, a forward genetic screen was conducted for mutants exhibiting altered NMJ morphology. In this screen, a new mutation was discovered that exhibits strikingly undergrown NMJs, which indicates disruption of a positive regulator of NMJ growth. The affected protein was identified as cholecystokinin (CCK)-like receptor (CCKLR), a putative neuropeptide receptor that belongs to the family of G-protein coupled receptors (GPCRs) sharing a uniform topology with seven transmembrane domains. When activated by their ligands, neuropeptide GPCRs affect levels of second messengers such as cAMP, diacylglycerol, inositol trisphosphate, and intracellular calcium. Through activation of their cognate receptors, secreted neuropeptides mediate communication among various sets of neurons as well as other cell types to regulate several physiological activities, including feeding and growth, molting, cuticle tanning, circadian rhythm, sleep, and learning and memory. In general, neuropeptides act by modulating neuronal activity through both short-term and long-term effects. Short-term effects include modifications of ion channel activity and alterations in release of or response to neurotransmitters. Long-term effects include changes in gene expression through activation of transcription factors and protein synthesis. In contrast with the well-known effects of neuropeptide signaling on neuronal activity and the strength of synaptic transmission, regulation of synaptic growth and development by neuropeptides has not previously been clearly established (Chen, 2012).

    This study demonstrates that CCKLR is required presynaptically to promote NMJ growth. Moreover, mutations of drosulfakinin (dsk), which encode the predicted ligand of CCKLR, cause similar NMJ undergrowth and interact genetically with CCKLR mutations, indicating that DSK and CCKLR are components of a common signaling mechanism that regulates NMJ growth. In addition to the morphological phenotypes caused by mutations of CCKLR and dsk, mutant larvae also exhibit deficits in synaptic function. Through phenotypic analysis of double mutant combinations, it was shown that DSK/CCKLR signals through the cAMP-PKA-CREB pathway to regulate NMJ growth. The results suggest a novel role for neuropeptide signaling in regulation of synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural plasticity of synapses in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

    Neuropeptides, whose effects have been extensively studied at NMJs, are usually described as neuromodulators because they modify the strength of synaptic transmission. For example, proctolin can potentiate the action of glutamate at certain NMJs in insects. However, involvement of neuropeptides in regulating neural development has not been well characterized. Recently, a C-type natriuretic peptide acting through a cGMP signaling cascade was found to be required for sensory axon bifurcation in mice, which suggests that neuropeptides may have a broader role in development than previously appreciated. The current studies demonstrate that DSK and its receptor, CCKLR, are strong positive regulators of NMJ growth in Drosophila (Chen, 2012).

    DSK belongs to the family of FMRFamide-related peptides (FaRPs), which is very broadly distributed across invertebrate and vertebrate phyla. Originally identified in clams, FaRPs affect heart rate, blood pressure, gut motility, feeding behavior, and reproduction in invertebrates. They have been shown to enhance synaptic efficacy at NMJs in locust and to modulate presynaptic Ca2+ channel activity in crustaceans. In Drosophila, various neuropeptides derived from the FMRFa gene can modulate the strength of muscle contraction when perfused onto standard larval nerve-muscle preparations. To these previously described functions of FaRPs, this study adds a new role as a positive regulator of NMJ development (Chen, 2012).

    Transgenic rescue experiments, RNAi expression, and overexpression of WT CCKLR demonstrate that CCKLR functions presynaptically in motor neurons to promote NMJ growth. Downstream components of this pathway were identified on the basis of known biochemistry of GPCRs and phenotypic interactions in double mutant combinations. GPCRs typically function by activating second messenger pathways via G proteins. Because loss-of-function mutations in dgs (which encodes the Gsα subunit in Drosophila) cause NMJ undergrowth, it is hypothesized that CCKLR signals through Gsα. Consistent with this idea, it was found that presynaptic constitutively active dgs overexpression rescues the NMJ undergrowth phenotype of CCKLR mutants. Conversely, dominant dose-dependent interactions were observed between CCKLR-null mutations and mutations of rut, which encodes an AC; or PKA-C1, which encodes a cAMP-dependent protein kinase, resulting in significant reductions in NMJ growth. These data place CCKLR together with the other genes in a common cAMP-dependent signaling pathway that regulates NMJ growth (Chen, 2012).

    It is known that the AC encoded by rut is activated by Gsα, and on the basis of the results, it is proposed that Gsα is downstream of CCKLR signaling. However, the NMJ undergrowth in rut1, which is a presumptive null mutant, is not as severe as that of a CCKLR-null mutant. This is likely due to the fact that the Drosophila genome contains up to seven different AC-encoding genes, all of which are stimulated by Gsα. Presumably, one or more additional AC-encoding genes share some functional overlap with rut in regulation of NMJ growth. This idea is in good agreement with the results of Wolfgang (2004), who found that the NMJ undergrowth phenotype of rut1 is weaker than that of dgs mutants, which they also interpreted as an indication that multiple ACs are activated by the Gsα encoded by dgs (Chen, 2012).

    The primary effector of this pathway is CREB2, a transcriptional regulatory protein that is activated upon phosphorylation by PKA. Consistent with the idea that CCKLR ultimately acts via activation of CREB, loss-of-function mutations of dCreb2 or neuronal overexpression of a dominant-negative dCreb2 transgene cause NMJ undergrowth similar to that of CCKLR-null mutants. Additionally, loss of one copy of dCreb2 in a CCKLR heterozygous background also causes NMJ undergrowth, and overexpression of WT dCreb2 fully rescues the NMJ undergrowth phenotype of CCKLR null, even leading to NMJ overgrowth. Thus, regulation of NMJ growth through the CCKLR signaling pathway is clearly mediated by dCreb2, whose activity is itself necessary and sufficient for regulating NMJ growth. This conclusion differs from another study that suggested that dCreb2 is required for NMJ function but not NMJ growth. One possible explanation for this discrepancy is that a weaker, inducible heat shock-driven transgene was used to express dCreb2 in the earlier work, whereas strong constitutive neuronal drivers were used this study. In any case, the current results demonstrate that in addition to its known role in NMJ function, CREB2 is also a strong positive regulator of NMJ growth and is likely to play a greater role in structural plasticity of synapses in learning and memory in Drosophila than previously suggested. This conclusion is consistent with a recent study indicating that sprouting of type II larval NMJs in response to starvation is stimulated by a cAMP/CREB-dependent pathway via activation of an octopamine GPCR (Chen, 2012).

    In addition to being undergrown, NMJs in CCKLR mutant larvae also exhibit a functional deficit. This is perhaps less straightforward than it might seem. Previous analyses of mutations affecting growth of the larval NMJ in Drosophila have shown that there is no simple correlation between the size and complexity of the NMJ and the amplitude of EJPs or amount of neurotransmitter release. These discrepancies arise because of various homeostatic compensatory mechanisms and because some of the affected signaling pathways alter synaptic growth and synaptic function in different ways via distinct downstream targets. For example, a mutation in highwire, which has the most extreme NMJ overgrowth phenotype described, is associated with a decrease in synaptic transmission. Wallenda mutations have been shown to fully suppress the overgrowth phenotype, but have no effect on the deficit in synaptic transmission (Chen, 2012).

    In the case of CCKLR mutants, however, there appears to be a very good correspondence between the morphological phenotype and the electrophysiological phenotype: the reduction in the total number of active zones in CCKLR larvae as measured morphologically correlates very well with the reduction in quantal content that was observe. In addition, no difference in CCKLR mutants was detected in calcium sensitivity of transmitter release or in the size or frequency of spontaneous release events. Thus, the synaptic growth phenotype of CCKLR mutant NMJs is sufficient to account for the functional phenotype. However, the possibility cannot be ruled out that DSK/CCKLR signaling also exerts some modulatory effect on NMJ function that is distinct from its effect on NMJ development (Chen, 2012).

    DSK is identified as the Drosophila orthologue of CCK, the ligand of CCKLR in vertebrates, on the basis of sequence analysis. The genetic analysis strongly supports the conclusion that DSK is the ligand of CCKLR at the larval NMJ. First, mutations of dsk and expression of dsk RNAi result in NMJ undergrowth phenotypes similar to that of CCKLR mutants. Second, loss of one copy of both dsk and CCKLR in double heterozygotes results in NMJ undergrowth. Third, heterozygosity for dsk does not further enhance the phenotype of a CCKLR-null mutant as expected if DSK regulates NMJ growth through its action on CCKLR. Fourth, overexpression of UAS-dsk does not rescue the undergrowth phenotype of a CCKLR-null mutant, but CCKLR overexpression can rescue NMJ undergrowth of a dsk hypomorphic mutant (Chen, 2012).

    The discovery of an entirely novel role for neuropeptide signaling in NMJ growth raises several questions about how this signaling is regulated and the biological significance of this mechanism. Although answers to these questions will require much additional work, an immediate question is whether a paracrine or autocrine mechanism is involved. In the case of octopamine-mediated synaptic sprouting in response to starvation, both autocrine and paracrine signaling are involved in the sprouting of type II and type I NMJs, respectively. In an early immunohistochemical investigation, it was reported that DSK was detected in medial neurosecretory cells in the larval CNS that extended projections anteriorly into the brain and posteriorly to the ventral ganglion. As it was not possible to obtain the original DSK antiserum and raising a new antiserum was not successful, the previous report has not been extended or confirmed. Instead, tissue-specific RNAi experiments were performed to examine the spatial requirement for DSK. Pan-neuronal dsk RNAi expression indicates that DSK expression in neurons is required to promote NMJ growth. In addition, C739-Gal4-driven dsk RNAi also causes NMJ undergrowth, whereas OK-Gal4-driven dsk RNAi in motor neurons does not. The expression pattern of C739-Gal4 overlaps with the DSK-positive cells previously identified by immunohistochemistry, which suggests that DSK produced by those neurosecretory cells is required for normal NMJ growth. Thus, from available data, it seems most likely that DSK is acting in paracrine fashion to regulate NMJ growth. However, further investigation will be necessary to determine the exact source of the DSK that promotes NMJ growth to fully understand how this neuropeptide regulates NMJ development (Chen, 2012).

    Studies have demonstrated a role for CREB in long-term synaptic plasticity-structural changes in synaptic morphology that underlie the formation of long-term memories. This study shows that in addition to CREB's role in structural modification of synapses in response to experience after development is complete, it is also a key regulator of growth and morphology during development of the larval NMJ. Moreover, although CREB is the transcriptional effector for many GPCRs, the fact that NMJs in CCKLR mutants are as undergrown as those of CREB mutants suggests that DSK/CCKLR signaling is a major input to CREB during NMJ growth. Many of the genes encoding intermediate components of the pathway such as dnc, rut, and PKA also have effects on NMJ growth and development as well as on synaptic plasticity and learning and memory, further emphasizing an overlap between the mechanisms that regulate synaptic growth during development and those that regulate postdevelopmental structural synaptic plasticity. These results raise the possibility that DSK/CCKLR signaling also plays a role in long-term synaptic plasticity and learning as well as in synaptic development (Chen, 2012).

    Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton

    Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. This study demonstrates that the heterotrimeric G protein Go functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. Ankyrin 2 was identified as a target of Go signaling required for NMJ formation. Moreover, the Go-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the Go-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. This work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function (Luchtenborg, 2014).

    Ankyrins (Ank) are highly abundant modular proteins that mediate protein-protein interactions, mainly serving as adaptors for linking the cytoskeleton to the plasma membrane. Mammalian genomes encode three Ank genes [AnkR (Ank1), AnkB (Ank2) and AnkG (Ank3)], whereas Drosophila has two [Ank1 (also known as Ank - FlyBase) and Ank2]. Ank2 is expressed exclusively in neurons and exists in several splicing variants. The larger isoforms (Ank2M, Ank2L and Ank2XL) are localized to axons and play important roles in NMJ formation and function. The C-terminal part of Ank2L can bind to microtubules. Despite the well-established role of Ank2 in NMJ formation, its function has been considered somewhat passive and its mode of regulation has not been clarified. This study shows that Gαo binds to Ank2 and that these proteins and the Wg pathway components Wg, Fz2, and Sgg jointly coordinate the formation of the NMJ. The functional Gαo-Ank interaction is conserved from insects to mammals (Luchtenborg, 2014).

    Synaptic plasticity underlies learning and memory. Both in invertebrates and vertebrates, activation of Wnt signaling is involved in several aspects of synapse formation and remodeling, and defects in this pathway may be causative of synaptic loss and neurodegeneration. Thus, understanding the molecular mechanisms of synaptic Wnt signaling is of fundamental as well as medical importance. The Drosophila NMJ is a powerful model system with which to study glutamatergic synapses, and the Wnt pathway has been widely identified as one of the key regulators of NMJ formation.

    This study provides important mechanistic insights into Wnt signal transduction in the NMJ, identifying the heterotrimeric Go protein as a crucial downstream transducer of the Wg-Fz2 pathway in the presynapse. It was further demonstrated that Ank2, a known player in the NMJ, is a target of Gαo in this signaling (Luchtenborg, 2014).

    This study found that the α subunit of Go is strongly expressed in the presynaptic cell, and that under- or overactivation of this G protein leads to neurotransmission and behavioral defects. At the level of NMJ morphology, presynaptic downregulation or Ptx-mediated inactivation of Gαo was found to recapitulate the phenotypes obtained by similar silencing of wg and fz2. These data confirm that presynaptic Wg signaling, in addition to the Wg pathway active in the muscle, is crucial for proper NMJ formation, and that Go is required for this process. Furthermore, neuronal Gαo overexpression can rescue the wg and fz2 loss-of-function phenotypes, demonstrating that, as in other contexts of Wnt/Fz signaling, Go acts as a transducer of Wg/Fz2 in NMJ formation. In contrast to its evident function and clear localization in the presynapse, Gαo localization on the muscle side of the synapse is much less pronounced or absent. Unlike Gαo, the main Drosophila Gβ subunit is strongly expressed in both the pre- and postsynapse. Thus, a heterotrimeric G protein other than Go might be involved in the postsynaptic Fz2 transduction, as has been implicated in Fz signaling in some other contexts (Luchtenborg, 2014).

    A recent study proposed a role for Gαo downstream of the octopamine receptor Octβ1R. This signaling was proposed to regulate the acute behavioral response to starvation both on type II NMJs (octapaminergic) and on the type I NMJs (glutamatergic) analyzed in this study. In contrast to the current observations, downregulation of Gαo in these NMJs was proposed to increase, rather than decrease, type I bouton numbers. It is suspected that the main reason for the discrepancy lies in the Gal4 lines used. The BG439-Gal4 and C380-Gal4 lines of Koon are poorly characterized and, unlike the well-analyzed pan-neuronal elav-Gal4 and motoneuron-specific OK371-Gal4 and D42-Gal4 driver lines used in the current study, might mediate a more acute expression. In this case, this study reflects the positive role of Gαo in the developmental formation of glutamatergic boutons, as opposed to a role in acute fine-tuning in response to environmental factors as studied by Koon (Luchtenborg, 2014).

    Postsynaptic expression of fz2 was found to fully rescue fz2 null NMJs. This study found that presynaptic knockdown of Fz2 (and other components of Wg-Fz2-Gαo signaling) recapitulates fz2 null phenotypes, whereas presynaptic overactivation of this pathway increases bouton numbers; furthermore, presynaptic overexpression of fz2 or Gαo rescues the fz2 nulls, just as postsynaptic overexpression of fz2 does. The current data thus support a crucial role for presynaptic Wg-Fz2-Gαo signaling in NMJ formation. Interestingly, both pre- and postsynaptic re-introduction of Arrow, an Fz2 co-receptor that is normally present both pre- and postsynaptically, as is Fz2 itself, can rescue arrow mutant NMJs. Thus, it appears that the pre- and postsynaptic branches of Fz2 signaling are both involved in NMJ development. A certain degree of redundancy between these branches must exist. Indeed, wild-type levels of Fz2 in the muscle are not sufficient to rescue the bouton defects induced by presynaptic expression of RNAi-fz2, yet overexpression of fz2 in the muscle can restore the bouton integrity of fz2 nulls. One might hypothesize that postsynaptic Fz2 overexpression activates a compensatory pathway - such as that mediated by reduction in laminin A signaling - that leads to restoration in bouton numbers in fz2 mutants. The current data showing that the targeted downregulation of Fz2 in the presynapse is sufficient to recapitulate the fz2 null phenotype underpin the crucial function of presynaptic Fz2 signaling in NMJ formation (Luchtenborg, 2014).

    This study found that downregulation of Ank2 produces NMJ defects similar to those of wg, fz2 or Gαo silencing. However, Ank2 mutant phenotypes appear more pronounced, indicating that Wg-Fz2-Gαo signaling might control a subset of Ank2-mediated activities in the NMJ. Ank2 was proposed to play a structural role in NMJ formation, binding to microtubules through its C-terminal region. However, since the C-terminal region was insufficient to rescue Ank2L mutant phenotypes, additional domains are likely to mediate Ank2 function through binding to other proteins. This study demonstrates in the yeast two-hybrid system and in pull-down experiments that the ankyrin repeat region of Ank2 physically binds Gαo, suggesting that the function of Ank2 in NMJ formation might be regulated by Wg-Fz2-Gαo signaling. Indeed, epistasis experiments place Ank2 downstream of Gαo in NMJ formation (Luchtenborg, 2014).

    Upon dissociation of the heterotrimeric Go protein by activated GPCRs such as Fz2, the liberated Gαo subunit can signal to its downstream targets both in the GTP- and GDP-bound state (the latter after hydrolysis of GTP and before re-association with Gβγ). The free signaling Gαo-GDP form is predicted to be relatively long lived, and a number of Gαo target proteins have been identified that interact equally well with both of the nucleotide forms of this G protein. In the context of NMJ formation, this study found that Gαo-GTP and -GDP are efficient in the activation of downstream signaling, and identifies Ank2 as a binding partner of Gαo that interacts with both nucleotide forms. The importance of signaling by Gα-GDP released from a heterotrimeric complex by the action of GPCRs has also been demonstrated in recent studies of mammalian chemotaxis, planar cell polarity and cancer (Luchtenborg, 2014).

    Gαo[G203T], which largely resides in the GDP-binding state owing to its reduced affinity for GTP, might be expected to act as a dominant-negative. However, in canonical Wnt signaling, regulation of asymmetric cell division as well as in planar cell polarity (PCP) signaling in the wing, Gαo[G203T] displays no dominant-negative activity but is simply silent, whereas in eye PCP signaling this form acts positively but is weaker than other Gαo forms. Biochemical characterization of the mammalian Gαi2[G203T] mutant revealed that it can still bind Gβγ and GTP, but upon nucleotide exchange Gαi2[G203T] fails to adopt the activated confirmation and can further lose GTP. The current biochemical characterization confirms that Gαo[G203T] still binds GTP. Interestingly, Gαi2[G203T] inhibited only a fraction of Gαi2-mediated signaling, suggesting that the dominant-negative effects of the mutant are effector specific. Thus, it is inferred that a portion of Gαo[G203T] can form a competent Fz2-transducing complex, and a portion of overexpressed Gαo[G203T] resides in a free GDP-loaded form that is also competent to activate downstream targets - Ank2 in the context of NMJ formation (Luchtenborg, 2014).

    These experiments place Ank2 downstream of Gαo and also of Sgg (GSK3β). It remains to be investigated whether Ank2 can directly interact with and/or be phosphorylated by Sgg. Meanwhile, it is proposed that the microtubule-binding protein Futsch might be a linker between Sgg and Ank2. Futsch is involved in NMJ formation and is placed downstream of Wg-Sgg signaling, being the target of phosphorylation and negative regulation by Sgg as the alternative target to β-catenin, which is dispensable in Wg NMJ signaling. Abnormal Futsch localization has been observed in Ank2 mutants. In Drosophila wing and mammalian cells in culture, Gαo acts upstream of Sgg/GSK3β. Cumulatively, these data might suggest that the Wg-Fz2-Gαo cascade sends a signal to Futsch through Sgg, parallel to that mediated by Ank2 (Luchtenborg, 2014).

    The importance of the Gαo-Ank2 interaction for Drosophila NMJ development is corroborated by findings in mammalian neuronal cells, where it was demonstrated that the ability of Gαo to induce neurite outgrowth is critically dependent on AnkB and AnkG. Knockdown of either or both ankyrin reduces neurite production. Remarkably, upon AnkB/G downregulation, Gαo switches its activity from the induction of microtubule-dependent processes (neurites) to actin-dependent protrusions (lamellopodia). Furthermore, Gαo recruits AnkB to the growing neurite tips. These data demonstrate that the Gαo-ankyrin mechanistic interactions are conserved from insects to mammals and are important for control over the neuronal tubulin cytoskeleton in the context of neurite growth and synapse formation. The novel signaling mechanism that were uncovered might thus be of general applicability in animal nervous system development and function (Luchtenborg, 2014).

    Drosophila Cbp53E regulates axon growth at the meuromuscular junction

    Calcium is a primary second messenger in all cells that functions in processes ranging from cellular proliferation to synaptic transmission. Proper regulation of calcium is achieved through numerous mechanisms involving channels, sensors, and buffers notably containing one or more EF-hand calcium binding domains. The Drosophila genome encodes only a single 6 EF-hand domain containing protein, Cbp53E, which is likely the prototypic member of a small family of related mammalian proteins that act as calcium buffers and calcium sensors. Like the mammalian homologs, Cbp53E is broadly though discretely expressed throughout the nervous system. Despite the importance of calcium in neuronal function and growth, nothing is known about Cbp53E's function in neuronal development. To address this deficiency, novel null alleles of Drosophila Cbp53E were generated, and neuronal development was examined at the well-characterized larval neuromuscular junction. Loss of Cbp53E resulted in increases in axonal branching at both peptidergic and glutamatergic neuronal terminals. This overgrowth could be completely rescued by expression of exogenous Cbp53E. Overexpression of Cbp53E, however, only affected the growth of peptidergic neuronal processes. These findings indicate that Cbp53E plays a significant role in neuronal growth and suggest that it may function in both local synaptic and global cellular mechanisms (Hagel, 2015).

    Distinct regulation of transmitter release at the Drosophila NMJ by different isoforms of nemy

    Synaptic transmission is highly plastic and subject to regulation by a wide variety of neuromodulators and neuropeptides. The present study examined the role of isoforms of the cytochrome b561 homologue called no extended memory (nemy) in regulation of synaptic strength and plasticity at the neuromuscular junction (NMJ) of third instar larvae in Drosophila. Specifically, two independent excisions of nemy were generated that differentially affect the expression of nemy isoforms. The nemy45 excision, which specifically reduces the expression of the longest splice form of nemy, leads to an increase in stimulus evoked transmitter release and altered synaptic plasticity at the NMJ. Conversely, the nemy26.2 excision, which appears to reduce the expression of all splice forms except the longest splice isoform, shows a reduction in stimulus evoked transmitter release, and enhanced synaptic plasticity. It was further shown that nemy45 mutants have reduced levels of amidated peptides similar to that observed in peptidyl-glycine hydryoxylating mono-oxygenase (PHM) mutants. In contrast, nemy26.2 mutants show no defects in peptide amidation but rather display a decrease in Tyramine beta hydroxylase activity (TbetaH). Taken together, these results show non-redundant roles for the different nemy isoforms and shed light on the complex regulation of neuromodulators (Knight, 2015).

    The guanine exchange factor Gartenzwerg and the small GTPase Arl1 function in the same pathway with Arfaptin during synapse growth

    The generation of neuronal morphology requires transport vesicles originating from the Golgi apparatus (GA) to deliver specialized components to the axon and dendrites. Drosophila Arfaptin is a membrane-binding protein localized to the GA that is required for the growth of the presynaptic nerve terminal. This study provides biochemical, cellular and genetic evidence that the small GTPase Arl1 and the guanine-nucleotide exchange factor (GEF) Gartenzwerg are required for Arfaptin function at the Golgi during synapse growth. These data define a new signaling pathway composed of Arfaptin, Arl1, and Garz, required for the generation of normal synapse morphology (Chang, 2015).

    Drosophila neuronal injury follows a temporal sequence of cellular events leading to degeneration at the neuromuscular junction

    There is a critical need to improve understanding of the molecular and cellular mechanisms that drive neurodegeneration. At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components. This study used an in vivo motor neuron injury assay to acutely induce neurodegeneration in order to follow the temporal order of events that occur following injury in Drosophila. Sites of injury can be rapidly identified based on structural defects to the neuronal cytoskeleton that result in disrupted axonal transport. Additionally, the neuromuscular junction accumulates ubiquitinated proteins prior to the neurodegenerative events, occurring at 24 hours post injury. These data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism. Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans. Thus, these studies will facilitate the identification of biomedically relevant targets for future treatments (Lincoln, 2015).

    Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction

    This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment (Wong, 2015).

    Mucolipidosis type IV (MLIV) and Batten disease are untreatable lysosomal storage diseases (LSDs) that cause childhood neurodegeneration. MLIV arises from loss-of-function mutations in the gene encoding TRPML1, an endolysosomal cation channel belonging to the TRP superfamily. The absence of TRPML1 leads to defective lysosomal storage and autophagy, mitochondrial damage, and macromolecular aggregation, which together initiate the protracted neurodegeneration observed in MLIV). Batten disease arises from the absence of a lysosomal protein, CLN3), and results in psychomotor retardation. Both diseases cause early alterations in neuronal function. For instance, brain imaging studies revealed that MLIV and Batten patients display diminished axonal development in the cortex and corpus callosum, the causes of which remain unknown (Wong, 2015).

    To better understand the etiology of MLIV in a genetically tractable model, flies were generated lacking the TRPML1 ortholog. The trpml-deficient (trpml1) flies have led to insight into the mechanisms of neurodegeneration and lysosomal storage (Wong, 2015).

    This study reports that trpml1 larvae exhibit diminished synaptic growth at the NMJ, a well-studied model synapse. Lysosomal function supports Rag GTPases and MTORC1 activation, and this is essential for JNK phosphorylation and synapse development (Wong, 2015).

    Drosophila larvae and mice lacking CLN3 also exhibit diminished Rag/ MTORC1 and JNK activation, suggesting that alterations in neuronal signaling are similar in different LSDs and are evolution- arily conserved. Interestingly, the NMJ defects in the two fly LSD models were suppressed by the administration of a high-protein diet and a drug that is currently in clinical trials to treat certain forms of cancer. These findings inform a pharmacotherapeutic strategy that may suppress the neurodevelopmental defects observed in LSD patients (Wong, 2015).

    This study shows that lysosomal dysfunction in Drosophila MNs results in diminished bouton numbers at the larval NMJ. Evidence is presented that lysosomal dysfunction results in decreased activation of the amino-acid-responsive cascade involving Rag/MTORC1, which are critical for normal NMJ development (Wong, 2015).

    Despite the requirement for MTORC1 in NMJ synapse development, previous studies and the current findings show that bouton numbers are independent of S6K and 4E-BP1. Rather, MTORC1 promotes NMJ growth via a MAP kinase cascade culminating in JNK activation. Therefore, decreasing lysosomal function or Rag/MTORC1 activation in hiwND8 suppressed the associated synaptic overgrowth. However, the 'small-bouton' phenotype of hiwND8 was independent of MTORC1. Thus, MTORC1 is required for JNK-dependent regulation of bouton numbers, whereas bouton morphology is independent of MTORC1. Furthermore, although both rheb expression and hiw loss result in Wallenda-dependent elevation in bouton numbers, the supernumerary boutons in each case show distinct morphological features. Additional studies are needed for deciphering the complex interplay between MTORC1-JNK in regulating the NMJ morphology (Wong, 2015).

    Biochemical analyses revealed that both JNK phosphorylation and its transcriptional output correlated with the activity of MTORC1, which are consistent with prior observations that cln3 overexpression promotes JNK activation and that tsc1/tsc2 deletion in flies result in increased JNK-dependent transcription. These findings point to the remarkable versatility of MTORC1 in controlling both protein trans lation and gene transcription (Wong, 2015).

    Using an in vitro kinase assay, this study demonstrates that Wallenda (Wnd) is a target of MTORC1. Because axonal injury activates both MTORC1 and DLK/JNK, these findings imply a functional connection between these two pathways. Interestingly, the data also suggest that MTORC1 contains additional kinases besides MTOR that can phosphorylate Wnd. One possibility is that ULK1/Atg1, which associates with MTORC1, could be the kinase that phosphorylates Wnd. Consistent with this notion, overexpression of Atg1 in the Drosophila neurons has been shown to promote JNK signaling and NMJ synapse overgrowth via Wnd) (Wong, 2015).

    This study also found that developmental JNK activation in axonal tracts of the CC and pJNK levels in cortical neurons were compromised in a mouse model of Batten disease. Thus, the signaling deficits identified in Drosophila are also conserved in mammals. The activity of DLK (the mouse homolog of Wnd) and JNK signaling are critical for axonal development in the mouse CNS. Therefore, decreased neuronal JNK activation during development might underlie the thinning of the axonal tracts observed in many LSDs (Wong, 2015).

    Although the findings of this study demonstrate a role for an amino-acid- responsive cascade in the synaptic defects associated with lysosomal dysfunction, simply elevating the dietary protein content was not sufficient to rescue these defects. These findings were reminiscent of an elegant study that showed that the growth of Drosophila neuroblasts is uncoupled from dietary amino acids owing to the function of ALK, which suppresses the uptake of amino acids into the neuroblasts (Cheng, 2011). Indeed, simultaneous administration of an ALK inhibitor and a high-protein diet partially rescued the synaptic growth defects associated with the lysosomal dysfunction, and improved the rescue of pupal lethality associated with trpml1. Although these studies do not causally link the defects in synapse development with pupal lethality, they do raise the intriguing possibility that multiple phenotypes associated with LSDs could be targeted using ALK inhibitors along with a protein-rich diet (Wong, 2015).

    Although LSDs result in lysosomal dysfunction throughout the body, neurons are exceptionally sensitive to these alterations. The cause for this sensitivity remains incompletely understood. Given the findings of this study that mature neurons do not efficiently take up amino acids from the extracellular medium, lysosomal degradation of proteins serves as a major source of free amino acids in these cells. Therefore, disruption of lysosomal degradation leads to severe shortage of free amino acids in neurons, regardless of the quantity of dietary proteins, thus explaining the exquisite sensitivity of neurons to lysosomal dysfunction (Wong, 2015).

    σ2-adaptin facilitates basal synaptic transmission and is required for regenerating endo-exo cycling pool under high frequency nerve stimulation in Drosophila

    The functional requirement of AP2 complex (see AP-2α) in synaptic membrane retrieval by clathrin mediated endocytosis (CME) is not fully understood. This study isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction synapse, this mutation was named angur (a Hindi dialect meaning grapes). Loss-of-function alleles of angur show more than two-fold overgrowth in bouton numbers and dramatic decrease in bouton size. angur mutation was mapped to σ2-adaptin, the smallest subunit of the adapter complex 2. Reducing neuronal level of any of the subunits of AP2 complex or disrupting AP2 complex assembly in neurons phenocopied σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature sensitive paralysis at 38 degrees °C. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic level of subunits of AP2 complex and clathrin but not other endocytic proteins were reduced in the mutants. Moreover, the BMP/TGFβ signalling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, these data suggest that - 1) while σ2-adaptin facilitates SV recycling for basal synaptic transmission, its activity is also required for regenerating SV during high frequency nerve stimulation; and 2) σ2-adaptin regulates NMJ morphology by attenuating TGFβ signalling (Choudhury, 2016).

    Synaptic transmission requires fusion of synaptic vesicles (SVs) at the active zones followed by their efficient retrieval and recycling through endocytic mechanisms. Retrieval and sorting of membrane lipids and vesicular proteins at the synapse are mediated by a well-orchestrated and coordinated action of several adapter and endocytic proteins. Clathrin-mediated endocytosis (CME) is the primary pathway operative at the synapses for membrane retrieval. Genetic analysis of the components of the CME pathway in Caenorhabditis elegans and Drosophila has revealed that this pathway is required for SV re-formation, and in many cases, blocking CME at synapses results in temperature-sensitive paralysis. Additionally, CME plays a crucial role in regulating synaptic morphology. At Drosophila NMJs, blocking CME results in enhanced bone morphogenetic protein (BMP) signaling and affects synaptic growth (Choudhury, 2016).

    The heterotetrameric adapter protein 2 (AP2) complex is a major effector of the CME pathway. AP2 serves as a major hub for a large number of molecular interactions and links plasma membrane, cargo/signaling molecules, clathrin, and accessory proteins in the CME pathway and hence can directly influence synaptic signaling. The AP2 complex is pseudo-asymmetric and contains four subunits-one each of large α and β2 subunits, one medium μ2 subunit, and a small σ2 subunit. Depletion of clathrin or its major adapter, AP2, in either Drosophila or mammalian central synapses results in accumulation of endosome-like vacuoles and reduction of SVs, suggesting that CME may not be essential for membrane retrieval. Similarly, genetic perturbation of μ2-adaptin or α-adaptin shows only mild defects in vesicle biogenesis at C. elegans synapses, but simultaneous loss of both adaptins leads to severely compromised SV biogenesis and accumulation of large vacuoles at nerve terminals. While Drosophila loss-of-function mutations in α-adaptin are embryonic lethal, hypomorphic mutants exhibit reduced FM1-43 uptake, suggesting a compromised endocytosis in these mutants. Whether reduced endocytosis reflects a defect in membrane retrieval or a defect in SV biogenesis remains unclear. Moreover, the consequences of AP2 reduction on synaptic morphology and physiology remain unknown (Choudhury, 2016).

    This study presents an analysis of Drosophila σ2-adaptin in the context of regulating NMJ morphological plasticity and physiology. A mutation was identified that dramatically altered NMJ morphology. Next, this mutation was mapped to σ2-adaptin by deficiency mapping. This study shows that AP2-dependent vesicle endocytosis regulates both synaptic growth and transmitter release. The AP2 complex is a heterotetramer, and these studies in Drosophila show that the four subunits are obligate partners of each other and are required for a functional AP2 complex. This finding is in contrast to the hemicomplex model in C. elegans, in which α/σ2 and β22 can sustain the function, if any one of the subunits is mutated. Loss of AP2 disrupts stable microtubule loops of the presynaptic cytoskeleton and exacerbates growth signaling through the phosphorylated Mothers Against Decapentaplegic (pMAD) pathway, suggesting that normal AP2 constrains the TGFβ signaling module. Reducing σ2-adaptin level results in synaptic fatigue at the larval NMJ synapses during high-frequency stimulation and causes temperature-sensitive paralysis in adults. Based on these results, it is suggestd that AP2 is essential for attenuating synaptic growth signaling mediated by the TGFβ pathway in addition to its requirement in regenerating SVs under high-frequency nerve firing (Choudhury, 2016).

    AP2/CME has been proposed to play an essential role in SV endocytosis. Moreover, mutation in the proteins affecting CME also results in altered NMJ development in Drosophila, suggesting regulation of synaptic signaling by CME. This study analyzed the role of AP2, one of the major adapters for clathrin at synapses, in the context of its physiological relevance and synaptic signaling. The results demonstrate that AP2 facilitates basal synaptic transmission and SV endocytosis but also is essential during high-frequency nerve firing to regenerate SVs. Moreover, evidence is provided that AP2 regulates morphological plasticity at the Drosophila NMJ by stabilizing the microtubule loops and attenuating the BMP signaling pathway (Choudhury, 2016).

    The biogenesis of rhabdomeres is regulated by endocytosis and intracellular trafficking. Components of the endocytic machinery, when perturbed, lead to defects in rhabdomere formation. One such example is the disruption of the rhabdomere base when shits flies are grown at restrictive temperature for 4 hr. Rh1-Gal4-driven shits1 flies raised at 19° led to an enlargement of photoreceptor cell bodies with the inter-rhabdomeric space being reduced. Similarly, when α-adaptin was knocked down using GMR-Gal4, rhabdomere biogenesis was disrupted, with smaller rhabdomeres. In accordance with these observations, the TEM analysis of angur mutant eye clones showed no detectable rhabdomeres. Consistent with the defect in rhabdomere formation, no membrane depolarization was observed in angur mutant eye clones. The visual cascade in Drosophila begins with rhodopsin being converted to meta-rhodopsin by light, followed by a subsequent phosphorylation by GPRK1. The phosphorylated meta-rhodopsin binds to arrestins, where its activity is quenched. The rhodopsin-arrestin complex is endocytosed, and rhodopsin is degraded in the lysosomes. Thus, endocytosis regulates rhodopsin turnover in the photoreceptor cells, and blocking of the endocytic pathway components by mutations in Arr1, Arr2, or AP2 leads to retinal degeneration and photoreceptor cell death. This further establishes a strong link between endocytosis and retinal degeneration. Thus, it is likely that defective endocytosis in angur mutant eye clones affects meta-rhodopsin turnover, resulting into defective rhabdomeres and disorganized ommatidia. Moreover, endocytosis of several signaling receptors also has been shown to regulate morphogenesis during Drosophila eye development. The Notch signaling pathway has been studied extensively, and it has been shown that the Notch extracellular domain is transendocytosed during this process into the Delta-expressing cells. The localization of Notch and Delta is disrupted when endocytosis is acutely blocked, and this prevents internalization of Notch in the Delta-expressing cells. It is thus speculated that the endocytic defect in angur mutant clones affects turnover and internalization of signaling molecules that may lead to defective rhabdomere development (Choudhury, 2016).

    The functional analysis of the σ2-adaptin mutants as well as of other subunits of AP2 reveals its requirement in maintaining basal synaptic transmission and regeneration of synaptic vesicles under high-frequency nerve firing at Drosophila NMJ synapses. The data are consistent with previous reports showing the requirement for Drosophila α-adaptin in SV recycling at NMJ synapses. However, in contrast to its role at mammalian central synapses, this study found that loss of AP2 at Drosophila NMJ synapses affects basal synaptic transmission and SV endocytosis, albeit not very strongly. Moreover, the kinetics of SV re-formation in σ2-adaptin mutants was only mildly affected. Consistent with the recent observation that loss of the AP2 complex at mammalian central synapses or its subunits in C. elegans affects synaptic vesicle biogenesis, compromised synaptic transmission and SV re-formation was observed after high-frequency stimulation, suggesting that at Drosophila NMJ synapses, AP2 function is required not only for SV trafficking but also for SV regeneration under high-frequency nerve stimulation. A direct estimation of vesicle pool size in σ2-adaptin mutants further supports its requirement in the regulation of SV pool size and is consistent with a recent report. Because loss of AP2 in Drosophila does not completely abrogate SV re-formation, it remains possible that other known synaptic clathrin adapters such as AP180, Eps15, and Epsin might partially compensate for loss of the AP2 complex. This is corroborated by the observation that even a 96% reduction in synaptic α-adaptin level caused only an ~50% reduction in clathrin, suggesting that other clathrin adapters might contribute to retention and/or stability of synaptic clathrin (Choudhury, 2016).

    The striking physiological defect in σ2-adaptin mutants or neuronally reduced α-adaptin was their inability to recover from synaptic depression even after 4 min of cessation of high-frequency stimulation. Consistent with these observations, the endo-exo cycling pool did not recover to its initial value even after 4 min of rest following high-frequency nerve stimulation. This suggests that in addition to its requirement in synaptic vesicle endocytosis, AP2 complex functions at a relatively slower step downstream of membrane retrieval, possibly during membrane sorting from endosomes to regenerate fusion-competent synaptic vesicles (Gu, 2013; Kononenko, 2014; Choudhury, 2016 and references therein).

    Several studies in cell culture support a model that indicates that the AP2 complex is an obligate heterotetramer for subunit stability and function. However, studies of AP2 function in C. elegans suggest that α/σ2 subunits and β22 subunits may constitute two hemicomplexes that can carry out the minimal function of the AP2 complex independent of one another. Does AP2 in Drosophila form hemicomplexes and contribute to vesicle trafficking? The results do not support this model for Drosophila. First, unlike C. elegans, in which individual mutants for subunits of the AP2 complex are viable, the Drosophila mutant for α-adaptin is early larval lethal. Consistent with these findings, neuronal knockdown of α-adaptin or β2-adaptin resulted in third instar lethality. Second, if the hemicomplexes were functional in Drosophila, one would observe significant levels of synaptic β2-adaptin in α-adaptin knockdown or σ2-adaptin mutants. However, it was observed that removing any of the subunits of the Drosophila AP2 complex in neurons resulted into an unstable AP2 complex and degradation of α- and β2-adaptins. Third, inhibiting AP2 complex assembly by reducing synaptic PI(4,5)P2 levels also resulted into an unstable AP2 complex. These observations strongly suggest that in contrast to C. elegans, all the subunits of AP2 in Drosophila are obligate partners for a stable AP2 complex for clathrin-dependent SV trafficking (Choudhury, 2016).

    Mutations in genes implicated in regulating CME, BMP signaling, or actin cytoskeleton dynamics all show abnormal NMJ development in Drosophila, characterized by either supernumerary boutons or an increased number but smaller boutons. The NMJ phenotype due to loss of σ2-adaptin is consistent with that of other known endocytic mutants implicated in CME. The contrasting synaptic overgrowth observed in σ2-adaptin mutants suggests a role for AP2 in mediating presynaptic growth signaling. Such synaptic overgrowth also was observed when any of the subunits of AP2 were downregulated or its assembly was interfered with by downregulating neuronal PI(4,5)P2. This suggests a pathway in which PI(4,5)P2 and the AP2 complex interact obligatorily to regulate synapse growth. The NMJ phenotype in angur mutants is strikingly more severe from that of the other synaptic mutants and points toward multiple growth signaling pathways that are possibly affected by an AP2-dependent endocytic deficit (Choudhury, 2016).

    The morphology of the synapse is a consequence of the neuronal cytoskeleton network that shapes the growing synapse. Futsch, a protein with MAP1B homology, regulates the synaptic microtubule cytoskeleton, thereby controlling synaptic growth at the Drosophila NMJ. The hypothesis that the microtubule organization could be altered was strengthened by the dramatic decrease in the number of Futsch-positive loops in the σ2-adaptin mutants. Futsch-positive loops have long been known to be associated with stable synaptic boutons, while the absence or disruption of these loops is indicative of boutons undergoing division or sprouting. The dramatic increase in the number of boutons in these mutants with a corresponding decrease in the number of loops correlates well with the fact that these boutons might be undergoing division. The phenotype associated with futsch mutants is fewer and larger boutons with impaired microtubule organization, which is an expected phenotype when bouton division is impaired. The σ2-adaptin mutants, however, show a larger number but smaller-sized boutons, indicating that bouton division is enhanced. One of the signaling events that has been shown to dictate this process is BMP signaling. Further, BMP signaling is also required during developmental synaptic growth. Consistent with this, elevated pMAD levels were found in σ2-adaptin mutants, indicating that BMP signaling is upregulated in these mutants. It has also been demonstrated that BMP signaling plays a role in maintenance of the presynaptic microtubule network. Drosophila spichthyin and spartin mutants have upregulated BMP signaling with significantly increased microtubule loops. It has been proposed that Futsch acts downstream of BMP signaling to regulate synaptic growth. The σ2-adaptin mutants also show upregulated BMP signaling, but in contrast to spichthyin and spartin mutants, σ2-adaptin mutants have fewer Futsch loops, which also appeared fragmented. This suggests that deregulated BMP signaling, whether upregulation or downregulation, impairs microtubule stability. dap160 mutants also show supernumerary boutons, with fragmented Futsch staining suggesting that the microtubule dynamics are misregulated in these mutants. Interestingly, Nwk levels are drastically altered in dap160 mutants, suggesting that Nwk levels are important for maintaining microtubule stability. Because σ2-adaptin mutants have normal Nwk levels, the observed synaptic overgrowth is independent of Nwk and suggests regulation of Futsch through Nwk-independent pathways (Choudhury, 2016).

    Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory

    Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The tomosyn-SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting tomosyn could be an important effector in PKA-dependent synaptic plasticity. This hypothesis was tested in Drosophila, in which the role of the PKA pathway in associative learning has been well established. It was first determined that panneuronal tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. Next memory performance was assayed 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that Tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that Tomosyn is involved in a specific component of late associative memory (Chen, 2011).

    Synaptic transmission is dependent on the formation of SNARE complexes between the vesicle SNARE synaptobrevin and the plasma membrane SNAREs syntaxin and SNAP-25. SNARE complex assembly produces fusion-competent (primed) vesicles, docked at the plasma membrane. Originally identified as a syntaxin-binding protein, tomosyn has emerged as a negative regulator of secretion by directly competing with synaptobrevin to form nonfusogenic tomosyn SNARE complexes (Fujita, 1998; Hatsuzawa, 2003; Pobbati, 2004). The N terminus of tomosyn also promotes SNARE complex oligomerization, sequestering SNARE monomers required for priming, and impedes the function of the calcium-sensor synaptotagmin. By these means tomosyn is involved in regulating SNARE complex assembly and controlling the size of the readily releasable pool of synaptic vesicles. Evidence suggests that the interaction between tomosyn and the SNARE machinery can be modulated by cAMP-dependent protein kinase A (PKA) phosphorylation of tomosyn, a second messenger cascade previously implicated in several forms of behavioral and synaptic plasticity. This study characterized the synaptic function of Drosophila tomosyn and probed the functional relevance of tomosyn in the regulation of behavioral plasticity (Chen, 2011).

    The results demonstrate that tomosyn suppresses synaptic function and is necessary in mushroom body intrinsic neurons specifically for a long-term cAMP-dependent component of associative olfactory learning in Drosophila. The prominent biophysical change observed at the fly NMJ after tomosyn RNAi is a prolonged EJC resulting in an increased total charge transfer, similar to that observed at C. elegans tomosyn mutant synapses. Although the underlying cause of the altered EJC duration in either C. elegans or Drosophila has yet to be determined, one potential explanation is the occurrence of late fusion events possibly resulting from ectopically primed vesicles distal to release sites. This hypothesis is supported by ultrastructural data from C. elegans tomosyn mutants, in which a twofold increase in the number of morphologically docked vesicles was observed, the additional vesicles positioned further from the presynaptic density (Gracheva, 2006). Analyses of priming defective unc-13 and syntaxin C. elegans mutants, in which docked vesicles were found to be greatly reduced, suggest that vesicle docking is a morphological correlate of priming. On the basis of these data, it has been proposed that distally primed vesicles in C. elegans tomosyn mutants exhibit delayed release relative to those close to the presynaptic density, the presumed site of calcium entry, leading to a broadened evoked response (Chen, 2011).

    The observed increase in synaptic release at the fly NMJ after tomosyn RNAi adds to a growing body of evidence that tomosyn suppresses synaptic strength. For example, the twofold increase in docked vesicles observed at C. elegans tomosyn mutant synapses correlates with a doubling of the readily releasable pool assessed by applying hyperosmotic saline and corresponds to the enhanced evoked EJC charge integral. The increase in quantal content and reduction in PPD at the fly NMJ after tomosyn RNAi is also consistent with an enhanced primed vesicle pool. Similar conclusions were reached for changes in paired-pulse facilitation at central synapses of mouse tomosyn mutants (Chen, 2011).

    The observation that tomosyn knockdown at the fly NMJ results in the addition of mEJCs with slower decay kinetics could also be a manifestation of ectopic vesicle priming. Alternatively, this change in fly mEJCs could reflect a change in fusion pore dynamics in the absence of tomosyn, a possibility given previously observed genetic and/or physical interactions between tomosyn and several key components of the exocytic machinery, including the SNARE proteins synaptotagmin, Munc-13, and Munc-18. Because the presynaptic density is responsible for localizing elements of the vesicle fusion machinery such as UNC-13 and calcium channels, spatial misregulation of vesicle docking in the absence of tomosyn may be related to these changes in fusion properties. However, definitive evidence for ectopically primed vesicles at the fly NMJ after tomosyn RNAi is not yet available, and therefore the cause of the altered evoked response kinetics remains speculative (Chen, 2011).

    Evidence indicates that vertebrate tomosyn is a PKA target. Although it cannot yet be definitively establish that tomosyn function is down-regulated by PKA phosphorylation at fly synapses pending the availability of a tomosyn null mutant, the fact that tomosyn RNAi phenocopies the NMJ response to cAMP activation and that these two treatments show nonadditivity supports this notion (Chen, 2011).

    On the basis of this analysis of NMJ function, it is predicted that, as in mouse tomosyn mutants, synapses within the fly CNS will experience similar increases in synaptic strength after tomosyn RNAi or cAMP activation. This prediction is supported by the specific ASM aversive odor learning deficit observed in the fly olfactory CNS after tomosyn knockdown in mushroom body Kenyon cells, the site of cAMP-mediated associative memory formation (Chen, 2011).

    Drosophila aversive odor learning has long been used to investigate the molecular and cellular mechanisms underlying associative memory formation. In the Drosophila mushroom bodies, neuronal signals representing odor cues and electric shock converge onto type I adenylyl cyclase encoded by rutabaga (rut-AC I) to initiate cAMP signals necessary and sufficient to form engrams underlying associative odor memory. These instructive cAMP signals localize to the Kenyon cells in which presynaptic changes are thought to represent a particular odor memory. Stabilization of aversive odor memory over time requires signals from dorsal-paired medial (DPM) neurons, the putative release sites of amnesiac peptide — the fly homolog to mammalian pituitary adenylate cyclase-activating polypeptide (PACAP) — onto mushroom body Kenyon cells. Amnesiac neuropeptides are known to stimulate cAMP production, whereas the major fly PKA is required from 30 min to 3 h after acquisition to sustain late odor memory. The two distinct components of late odor memory, ASM and ARM, differ in both temporal dynamics and molecular mechanisms. Whereas ARM requires the active zone protein Bruchpilot, ASM is not only tomosyn-dependent, according to the current observations, but also requires synapsin, a conserved PKA phosphorylation target associated with synaptic vesicles. Adult synapsin mutant flies exhibit impaired ASM in aversive odor learning. Furthermore, PKA-dependent phosphorylation of synapsin within Kenyon cells is necessary to support larval appetitive odor learning. Thus, both tomosyn and synapsin are required for the cAMP-dependent ASM phase of associate learning (Chen, 2011).

    How might synapsin and tomosyn function together in ASM? Mechanistically, PKA phosphorylation of synapsin is implicated in the mobilization and supply of synaptic vesicles from the reserve pool to the active zone, whereas PKA phosphorylation of tomosyn promotes SNARE complex assembly (Baba, 2005). This suggests that enhanced vesicle delivery and increased priming capacity through PKA regulation of synapsin and tomosyn, respectively, may act in concert to maintain synaptic strength in support of ASM. Because knockdown of either protein disrupts ASM, it seems that both vesicle mobilization and enhanced priming capacity are required for this phase of synaptic plasticity (Chen, 2011).

    Several lines of evidence suggest that PKA-dependent modulation of tomosyn function provides a possible molecular mechanism for the transduction of cAMP signaling into synaptic plasticity within the olfactory system underlying ASM. First, at the level of the NMJ, it was shown that tomosyn regulates synaptic strength and that this regulation occludes cAMP-dependent synaptic enhancement. Second, within the olfactory neuronal network, it was demonstrated that tomosyn function is likely required within Kenyon cells to support ASM: the cells that receive instructive signals to initiate cAMP-dependent synaptic changes underlying appropriate behavioral plasticity. Third, synaptic output from Kenyon cells is necessary to support both early and late odor memories. Fourth, DPM signaling onto Kenyon cells is required for late aversive odor memory, and DPM neurons stain positive for the amnesiac peptide, which is known to stimulate cAMP production. Fifth, enhanced synaptic transmission in cultured neurons induced by the vertebrate amnesiac homolog PACAP requires PKA-dependent tomosyn phosphorylation (Baba, 2005). Sixth, postacquisitional PKA activity is necessary for late aversive odor memory and is likely mediated by an A kinase-anchoring protein (AKAP)-bound pool of PKA holoenzymes within Kenyon cells. Finally, biochemical evidence demonstrating that PKA-dependent phosphorylation of tomosyn reduces its affinity for the SNARE machinery suggests a potential mechanistic link between cAMP signaling within Kenyon cells and the up-regulation of synaptic strength (Chen, 2011).

    On the basis of this evidence, it is postulated that loss of tomosyn inhibitory function leads to a generalized up-regulation of synaptic strength at Drosophila synapses. In Kenyon cells enhanced synaptic transmission resulting from loss of tomosyn likely occludes cAMP-dependent plasticity. This speculation is supported by the observed occlusion of forskolin-dependent synaptic enhancement at the NMJ after tomosyn RNAi. Similarly, PACAP-induced synaptic plasticity in cultured neurons is occluded when tomosyn is no longer phosphorylatable by PKA (Baba, 2005). It is further speculated that within Kenyon cells the phosphorylation of Drosophila tomosyn is due to postacquisitional, Amnesiac-induced PKA activity. This hypothesis would fit with the observed requirement of AKAP-bound PKA for late but not early ASM. It is thus tempting to speculate that tomosyn phosphorylation is dependent on localized signaling via AKAPs at specific subdomains of Kenyon cells that occur after early ASM is established (Chen, 2011).

    Hsc70-4 deforms membranes to promote synaptic protein turnover by endosomal microautophagy

    Synapses are often far from their cell bodies and must largely independently cope with dysfunctional proteins resulting from synaptic activity and stress. To identify membrane-associated machines that can engulf synaptic targets destined for degradation, a large-scale in vitro liposome-based screen was performed followed by functional studies. A presynaptically enriched chaperone Hsc70-4 was identified that bends membranes based on its ability to oligomerize. This activity promotes endosomal microautophagy and the turnover of specific synaptic proteins. Loss of microautophagy slows down neurotransmission while gain of microautophagy increases neurotransmission. Interestingly, Sgt, a cochaperone of Hsc70-4, is able to switch the activity of Hsc70-4 from synaptic endosomal microautophagy toward chaperone activity. Hence, Hsc70-4 controls rejuvenation of the synaptic protein pool in a dual way: either by refolding proteins together with Sgt, or by targeting them for degradation by facilitating endosomal microautophagy based on its membrane deforming activity (Uytterhoeven, 2016).

    Rab3-GEF controls active zone development at the Drosophila neuromuscular junction

    Synaptic signaling involves the release of neurotransmitter from presynaptic active zones (AZs). Proteins that regulate vesicle exocytosis cluster at AZs, composing the cytomatrix at the active zone (CAZ). At the Drosophila neuromuscular junction (NMJ), the small GTPase Rab3 controls the distribution of CAZ proteins across release sites, thereby regulating the efficacy of individual AZs. This study identifies Rab3-GEF as a second protein that acts in conjunction with Rab3 to control AZ protein composition. At rab3-GEF mutant NMJs, Bruchpilot (Brp) and Ca(2+) channels are enriched at a subset of AZs, leaving the remaining sites devoid of key CAZ components in a manner that is indistinguishable from rab3 mutant NMJs. As the Drosophila homologue of mammalian DENN/MADD and Caenorhabditis elegans AEX-3, Rab3-GEF is a guanine nucleotide exchange factor (GEF) for Rab3 that stimulates GDP to GTP exchange. Mechanistic studies reveal that although Rab3 and Rab3-GEF act within the same mechanism to control AZ development, Rab3-GEF is involved in multiple roles. It was shown that Rab3-GEF is required for transport of Rab3. However, the synaptic phenotype in the rab3-GEF mutant cannot be fully explained by defective transport and loss of GEF activity. A transgenically expressed GTP-locked variant of Rab3 accumulates at the NMJ at wild-type levels and fully rescues the rab3 mutant but is unable to rescue the rab3-GEF mutant. These results suggest that although Rab3-GEF acts upstream of Rab3 to control Rab3 localization and likely GTP-binding, it also acts downstream to regulate CAZ development, potentially as a Rab3 effector at the synapse (Bae, 2016).

    A pre-synaptic regulatory system acts trans-synaptically via Mon1 to regulate Glutamate receptor levels in Drosophila

    Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. This study has identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. Mutants were generated in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels suggesting its involvement in a pre-synaptic mechanism that regulates post-synaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants suggesting that homeostatic mechanisms are not affected in these mutants. It is proposed that DMon1 is part of a pre-synaptically regulated trans-synaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction (Deivasigamani, 2015).

    Neurotransmitter release at the synapse is modulated by factors that control synaptic growth, synaptic vesicle recycling, and receptor turnover at postsynaptic sites. Endolysosomal trafficking modulates the function of these factors and therefore plays an important role in regulating synaptic development and function. Intracellular trafficking is regulated by Rabs, which are small GTPases. These proteins control specific steps in the trafficking process. A clear understanding of the role of Rabs at the synapse is still nascent. Drosophila has 31 Rabs, and most of these are expressed in the nervous system. Rab5 and Rab7, present on early and late endosomes, respectively, are critical regulators of endolysosomal trafficking and loss of this regulation affects neuronal viability underscored by the fact that mutations in Rab7 are associated with neurodegeneration. Rab5 along with Rab3 is present on synaptic vesicles, and both play a role in regulating neurotransmitter release. In Drosophila, Rab3 is involved in the assembly of active zones by controlling the level of both Bruchpilot-a core active zone protein-and the calcium channels surrounding the active zone. In hippocampal and cortex neurons, Rab5 facilitates LTD through removal of AMPA receptors from the synapse. In Drosophila, Rab5 regulates neurotransmission; it also functions to maintain synaptic vesicle size by preventing homotypic fusion. Compared to Rab5 or Rab3, less is known about the roles of Rab7 at the synapse. In spinal cord motor neurons, Rab7 mediates sorting and retrograde transport of neurotrophin-carrying vesicles. In Drosophila, tbc1D17-a known GAP for Rab7-affects GluRIIA levels; the effect of this on neurotransmission has not been evaluated. Excessive trafficking via the endolysosomal pathway also affects neurotransmission. This has been observed in mutants for tbc1D24-a GAP for Rab35. A high rate of turnover of synaptic vesicle proteins in these mutants is seen to increase neurotransmitter release (Deivasigamani, 2015 and references therein).

    This study has examined the synaptic role of DMon1-a key regulator of endosomal maturation. Multiple synaptic phenotypes are found associated with Dmon1 loss of function, and one of these is altered synaptic morphology. Boutons in Dmon1 mutants are larger with more satellite or supernumerary boutons-a phenotype strongly associated with endocytic mutants. Formation of satellite boutons is thought to occur due to loss of bouton maturation, with the initial step of bouton budding being controlled postsynaptically and the maturation step being regulated presynaptically. Supporting this, a recent study shows that miniature neurotransmission is required for bouton maturation. The presence of excess satellite boutons in Dmon1 mutants suggests that the number of 'miniature' events is likely to be affected in these mutants. The fact that this phenotype can be rescued upon expression of vGlut supports this possibility. However, this does not fit with the observed decrease in size and intensity of Brp positive puncta in these mutants. Active zones with low or nonfunctional Brp are known to be more strongly associated with increased spontaneous neurotransmission. Considering the involvement of postsynaptic signaling in initiating satellite bouton formation, it is thought that altered neurotransmission possibly together with impaired postsynaptic or retrograde signaling, contributes to the altered synaptic morphology in Dmon1 mutants. This may also explain why no satellite boutons are observed in neuronal RNAi animals (Deivasigamani, 2015).

    A striking phenotype associated with loss of Dmon1 is the increase in GluRIIA levels. This phenotype seems presynaptic in origin since neuronal loss of Dmon1 is sufficient to increase GluRIIA levels. Is the increase in GluRIIA due to trafficking defects in the neuron? This seems unlikely for the following reasons: First, it has been shown that although neuronal overexpression of wild-type and dominant negative Rab5 alters evoked response in a reciprocal manner, there is no change in synaptic morphology, glutamate receptor localization and density, or change in synaptic vesicle size. The role of Rab7 at the synapse is less clear. In a recent study, loss of tbc1D15-17, which functions as a GAP for Rab7, was shown to increase GluRIIA levels at the synapse. Selective knockdown of the gene in muscles, and not neurons, was seen to increase GluRIIA levels, indicating that the function of the gene is primarily postsynaptic. These data are not consistent with the current results from neuronal knockdown of Dmon1, suggesting that the presynaptic role of Dmon1 in regulating GluRIIA levels is likely to be independent of Rab5 and Rab7 and therefore novel (Deivasigamani, 2015).

    The current experiments to evaluate the postsynaptic role of Dmon1 have been less clear. Although a modest increase in GluRIIA levels are seen upon knockdown in muscles, the increase is not always significant when compared to controls. However, the fact that muscle expression of Dmon1 can rescue the GluRIIA phenotype in the mutant suggests that it is likely to be one of the players in regulating GluRIIA postsynaptically. Further, it is to be noted, that while overexpression of vGlut leads to down-regulation of the receptor at the synapse, the receptors do not seem to get trapped in the muscle, suggesting that multiple pathways are likely to be involved in regulating receptor turnover in the muscle, and the DMon1-Rab7-mediated pathway may be just one of them (Deivasigamani, 2015).

    How might neuronal Dmon1 regulate receptor expression? One possibility is that the increase in receptor levels is a postsynaptic homeostatic response to defects in neurotransmission, given that Dmon1Δ181 mutants have smaller synaptic vesicles. However, in dvglut mutants, presence of smaller synaptic vesicles does not lead to any change in GluRIIA levels, given that receptors at the synapse are generally expressed at saturating levels. Therefore, it seems unlikely that the increase in GluRIIA is part of a homeostatic response, although one cannot rule this out completely. The other possibility is that DMon1 is part of a transsynaptic signaling mechanism that regulates GluRIIA levels in a post-transcriptional manner. The observation that presynaptically expressed DMon1 localizes to postsynaptic regions and the results from neuronal RNAi and rescue experiments support this possibility. The involvement of transsynaptic signaling in regulating synaptic growth and function has been demonstrated in the case of signaling molecules such as Ephrins, Wingless, and Syt4. In Drosophila, both Wingless and Syt4 are released by the presynaptic terminal via exosomes to mediate their effects in the postsynaptic compartment. It was hypothesized that DMon1 released from the boutons either directly regulates GluRIIA levels or facilitates the release of an unknown factor required to maintain receptor levels. The function of DMon1 in the muscle is likely to be more consistent with its role in cellular trafficking and may mediate one of the pathways regulating GluRIIA turnover. These possibilities will need to be tested to gain a mechanistic understanding of receptor regulation by Dmon1 (Deivasigamani, 2015).

    Drosophila homolog of human KIF22 at the autism-linked 16p11.2 loci influences synaptic connectivity at larval neuromuscular junctions

    Copy number variations at multiple chromosomal loci, including 16p11.2, have recently been implicated in the pathogenesis of autism spectrum disorder (ASD), a neurodevelopmental disease that affects ~1%-3% of children worldwide. The aim of this study was to investigate the roles of human genes at the 16p11.2 loci in synaptic development using Drosophila larval neuromuscular junctions (NMJ), a well-established model synapse with stereotypic innervation patterns. A preliminary genetic screen based on RNA interference was conducted in combination with the GAL4-UAS system, followed by mutational analyses. The result indicated that disruption of klp68D, a gene closely related to human KIF22, caused ectopic innervations of axon branches forming type III boutons in muscle 13, along with less frequent re-routing of other axon branches. In addition, mutations in klp68D, of which gene product forms Kinesin-2 complex with KLP68D, led to similar targeting errors of type III axons. Mutant phenotypes were at least partially reproduced by knockdown of each gene via RNA interference. Taken together, these data suggest the roles of Kinesin-2 proteins, including KLP68D and KLP64D, in ensuring proper synaptic wiring (Park, 2016).

    Recent clinical studies on ASD have revealed gross alterations in the structure of nervous system. For instance, total brain size and the rate of neuronal proliferation in the prefrontal cortex are significantly increased in ASD patients. Such structural changes may reflect altered neuronal connectivity between specific brain regions. Moreover, the proposed candidate genes responsible for ASD include various synaptic proteins that play important roles in neurite outgrowth, axonal guidance, axonal targeting and synaptogenesis, suggesting structural abnormalities at a synaptic level responsible for expression of ASD phenotypes. Based on these findings, it was hypothesized that genetic perturbation at the 16p11.2 loci would lead to aberrant synaptic connectivity, thus underlying functional disturbances that lead to ASD. Results demonstrate significant axon targeting errors caused by defects in KLP68D, a Drosophila Kinesin-2 protein closely related to human KIF22 at the autism-linked 16p11.2 loci (Park, 2016).

    Hetero-trimeric Kinesin-2 complex in Drosophila, consisting of KLP68D, KLP64D and DmKAP, has been implicated in microtubule organization and axonal transport of synaptic proteins such as choline acetyltransferase. However, experimental evidence is missing to support the idea that Kinesin-2 complex may participate in delivering molecules important for axon targeting. Potential cargos of KLP68D and KLP64D motors have been estimated to include Unc-51/ATG1, Fasciclin II, EB1, Armadillo, Bazooka, and DE-cadherin, most of whom have been well characterized for their roles in synaptogenesis. It will be important to investigate whether disruptions of any of these potential cargos lead to aberrant axon targeting phenotypes observed in Klp68D and Klp64D mutants (Park, 2016).

    It should be noted that Drosophila Nod ("no distributive disjunction"), mostly involved in chromosomal segregation has been recognized as a homolog for human KIF22. However, similar levels of sequence homology to KIF22 were found in both Nod and KLP68D. In fact, a blast analysis results in higher sequence identity between KIF22 and KLP68D than Nod (41% vs. 33%). The specificity of motor protein cargos is often predicted to depend on the amino acid composition of motor proteins outside their core motor domain. Therefore, relatively lower level of homology between human KIF22 and Drosophila KLP68D may correspond to their distinct molecular functions. In contrast to KLP68D, the role of KIF22 in the mammalian nervous system has not been extensively investigated, but only limited to chromosomal segregation and genomic stability. Whether Drosophila KLP68D can be functionally replaced by human KIF22 in transgenic animals awaits further investigations (Park, 2016).

    Na+ /H+ -exchange via the Drosophila vesicular glutamate transporter (DVGLUT) mediates activity-induced acid efflux from presynaptic terminals

    Neuronal activity can result in transient acidification of presynaptic terminals and such shifts in cytosolic pH (pHcyto) likely influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals it was hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation this study investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically-encoded pH-indicator (GEpHI), named 'pHerry', in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity-induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H+ /Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid extruding protein when deposited on the plasma membrane (Rossano, 2016).

    Neuronal activity generates large cytosolic pH (pHcyto) transients in presynaptic terminals and such transients are likely to influence mechanisms underlying neurotransmitter release. Minute changes in pHcyto can influence presynaptic processes such as voltage-gated Ca2+ channel gating, endocytosis, synaptic vesicle (SV) filling, cytosolic Ca2+ buffering, and both Ca2+/calmodulin-dependent kinase II and cyclic-AMP-based forms of synaptic plasticity. pHcyto in quiescent neurons is set by the equilibrium between standing acid influx and efflux. Neuronal activity, however, results in enhanced acid influx, primarily due to electroneutral H+/Ca2+ exchange across the plasma membrane Ca2+-ATPase (PMCA) as it clears cytosolic Ca2+. pH homeostasis would be well served if nerve activity triggered acid efflux to offset acid influx (Rossano, 2016).

    Nerve activity may enhance acid efflux in many ways. Standing efflux mechanisms might be directly enhanced by decreasing pHcyto or through intracellular signalling linked to Ca2+ entry. For example, anion exchangers (AEs) are known to be modulated by Ca2+/calmodulin. Also, various mammalian Na+/H+ exchangers (NHEs) are modified by Ca2+-dependent kinases and protein kinase A and C pathways. Finally, but perhaps most significantly, activity-induced acid efflux could be mediated by translocation of acid extruders, such as the vesicular H+ ATPase (vATPase), from intracellular compartments to the plasmamembrane (PM), as described for cholinergic mouse motorneuron (MN) terminals. While mammalian NHE5, NHE6 and NHE9 are present on intracellular membranes in neurons, there are no reports of NHE translocation to the PM during nerve activity (Rossano, 2016).

    Whether activity-induced acid efflux occurs at glutamatergic nerve terminals is unknown, but Drosophila glutamatergic MN terminals represent a tractable system for investigation. vATPases are present in these terminals and may extrude acid when deposited on the PM. Drosophila NHE and AE expression patterns have only been grossly characterized. Vesicular glutamate transporters (VGLUTs) have not been previously implicated in pHcyto homeostasis but a recent report of VGLUT1 cation/H+ exchange activity in mammalian SVs (Preobraschenski, 2014) led to the speculation that VGLUTs contribute to acid efflux (Rossano, 2016).

    VGLUTs transport glutamate into vesicles using the electrical portion (ΔΨH+) rather than the chemical portion (ΔpH) of the electrochemical proton gradient (ΔμH+), and most models of VGLUT bioenergetics suggest VGLUTs are coupled to cation/H+ exchangers (Goh, 2011; Preobraschenski, 2014) or a Cl shunt. While these mechanisms have never been validated beyond in vitro preparations, H+/cation exchange by VGLUT may provide a novel acid efflux mechanism when VGLUTs are deposited on the PM during SV exocytosis and exposed to the strong PM electrochemical Na+ gradient (ΔμNa+) (Rossano, 2016).

    In this study, enhanced activity-induced acid transients under exocytotic blockade suggest that Drosophila MN terminals usually extrude acid through the translocation of vesicular acid extruders to the PM. While translocation of the vATPase accounts for some acid efflux, pharmacological and genetic tools revealed a portion of the efflux is mediated directly by the Drosophila vesicular glutamate transporter (DVGLUT). This is the first report of pHcyto modulation in situ by a VGLUT in any cell. Expression of DVGLUT in Xenopus oocytes revealed intrinsic Na+/H+ exchange, which marks the first description of ion transport by DVGLUT. Modulation of activity-induced pHcyto transients by DVGLUT though Na+/H+ exchange at the PM demonstrates novel integration of pHcyto, vesicular recycling, glutamate loading and [Ca2+]cyto in presynaptic terminals (Rossano, 2016).

    This study examined the extent to which SV exocytosis shapes activity-induced pHcyto transients in glutamatergic MN terminals through translocation of acid transporters to the PM. Cytosolic expression of the pseudo-ratiometric fluorescent GEpHI pHerry permitted measurement of acid dynamics within individual MN terminals. While intrinsic acid extrusion from MN terminals accelerates when pHcyto falls, acid extrusion following action potentials trains is much faster than predicted by activation of acid extruders by low pHcyto. Activity-induced acid extrusion partially offsets Ca2+-dependent acidification during nerve activity and continues for seconds after nerve activity. Complementary genetic and pharmacological approaches revealed this activity-induced acid efflux to be mediated by NHEs, the vATPase and Na+/H+ exchange through DVGLUT (Rossano, 2016).

    Previous work has shown depolarization produces significant acid loading in soma, axons and presynaptic terminals of many neuronal preparations. While less common, there are several reports of depolarization-induced acid efflux from neurons. This report expands upon previous studies by characterizing mechanisms which shape rapid pHcyto transients in the context of established models of vesicular trafficking and [Ca2+]cyto dynamics. The latter is particularly important as [Ca2+]cyto is tightly regulated in presynaptic terminals and [Ca2+]cyto levels drive rapid acid influx and efflux mechanisms through direct Ca2+/H+ exchange via the PMCA and Ca2+-dependent trafficking of vesicular H+ transporters, respectively (Rossano, 2016).

    Careful analysis of the relationship between pHcyto and [Ca2+]cyto was necessary to determine if alterations in activity-induced pHcyto transients were directly due to changes in the location and activity of acid transporters or secondary to changes in [Ca2+]cyto levels. To this end the relationships between [Ca2+]cyto, JH+ and acid extrusion (τrec) as well as resting pHcyto and [Ca2+]e were quantified. Many manipulations produced changes in resting pHcyto which cannot be explained by alterations in Ca2+ handling as resting pHcyto is independent of Ca2+ in Drosophila MN terminals. Similarly, manipulations which altered τrec probably altered acid efflux directly as τrec is independent of Ca2+ loading during stimulation. Interpreting manipulations which only altered JH+ during stimulation proved the most challenging as JH+ is proportional to Ca2+ loading during stimulation and thus changes in JH+ may represent changes in acid influx due to changes in Ca2+ handling or direct changes in acid efflux. MN terminals with altered expression of DVGLUT only differed from their controls with respect to JH+. As there were no differences in bulk [Ca2+]cyto at rest or during stimulation between genotypes with different DVGLUT expression levels, differences in JH+ between genotypes are probably not due to changes in Ca2+ handling, with the caveat that potential alterations in the Ca2+ and pH near-membrane micro-environments are not addressed in this analysis (Rossano, 2016).

    The data in this study indicate that activity-induced acid efflux is mediated by multiple PM and vesicular acid extruders, namely NHEs, the vATPase and DVGLUT. The contribution of NHEs is unsurprising as NHE gene products are highly expressed in Drosophila larvae (Giannakou, 2001) and spontaneous vesicular fusion at the larval NMJ is sensitive to the NHE inhibitor amiloride (Caldwell, 2013). These results are corroborated in this study as application of the amiloride derivative EIPA decreased resting pHcyto and delayed acid extrusion in MN terminals. The contributions of the vATPase and DVGLUT to pHcyto transients are probably attributable to selective trafficking of these transporters to the PM during exocytosis as they are primarily vesicular proteins, although BafA1-sensitive vATPases are constitutively present on the PM of many cells as well. Furthermore, genetic manipulations to impair vesicular endocytosis and exocytosis revealed that stopping vesicular fusion impaired acid extrusion while locking vesicular membrane at the PM enhanced acid clearance following both NH4+ withdrawal and activity-induced acid loading. The conclusion that both the vATPase and DVGLUT are functional components of the recycling pool of vesicular proteins which shape activity-induced pHcyto transients is further supported by the observation that application of both the glutamate transporter inhibitor EB and BafA1, an established vATPase inhibitor, decrease acid clearance following activity-induced acid loading. These results agree with a previous report that trafficking of the vATPase to the PM can alkalinize the cytosol of mouse cholinergic MN terminals (Zhang, 2010; Rossano, 2016 and references therein).

    The notion that the DVGLUT can function at the PM as an acid extruder requires careful consideration as exact transport mechanisms of VGLUTs are unclear and no previous studies have been conducted to elucidate the transport mechanisms of DVGLUT, although it is reasonable to assume its transport modalities are generally similar to those of mammalian VGLUT1 (Preobraschenski, 2014). Here, a combined pharmacological and genetic approach provided the most compelling evidence for acid efflux mediated by DVGLUT. This conclusion is further supported by the observation that EB can inhibit DVGLUT-mediated Na+/H+ exchange in oocytes. Experiments in which EB has been used to inhibit VGLUTs have been historically interpreted by assuming a primary effect on glutamate transport, not H+ dynamics. If intrinsic Na+/H+ exchange is a shared property of mammalian VGLUTs it is possible that prior work with EB has erroneously ascribed changes in neurotransmitter loading to direct inhibition of glutamate transport rather than secondary effects of inhibited cation/H+ exchange (Rossano, 2016).

    The bioenergetics of vesicular glutamate transporters are undoubtedly vital to modulation of neurotransmitter release. Studies of mammalian VGLUTs in heterologous expression systems suggest that VGLUTs are primarily driven by ΔΨH+ of ΔμH+ across the vesicular membrane, which is established by the vATPase. Maintenance of ΔΨH+ requires dissipation of ΔpH via H+ exchange with another cation to enable continuous proton pumping and glutamate transport (Goh, 2011). It is unclear if H+/cation exchange is due to co-transport with another ion transporter, as has been described in insect midgut where co-expression of vATPase and Na+-coupled nutrient amino acid transporters form a functional NHE (Harvey, 2009), or is an intrinsic property VGLUTs, as has been described in mammalian VGLUT1 (Preobraschenski, 2014). The data presented in this study provide the first direct evidence that intrinsic EB-sensitive Na+/H+ exchange is a property of DVGLUT. Taken with the observation that the in situ effects of EB are only additive with those of BafA1 in the presence of significant DVGLUT expression it is very likely that Na+/H+ exchange via DVGLUT contributes to activity-enhanced acid efflux across the PM of MN terminals. The electroneutrality of ion exchange by DVGLUT requires further investigation as changes in Vm upon removal of Na+ from oocytes expressing DVGLUT is probably attributable to decrease in the PM Na+ gradient rather than electrogenic Na+/H+ exchange by DVGLUT (Rossano, 2016).

    The following model of pHcyto regulation by DVGLUT reconciles the observation that DVGLUT is a mediator of activity-induced acid extrusion with the available data from mammalian VGLUT ion transport mechanisms. In quiescent nerve terminals NHEs mediate a standing acid efflux and DVGLUT is primarily on the vesicular membrane where it loads glutamate into the vesicular lumen via ΔΨH+ generated by the vATPase. The growing ΔpH across the vesicular membrane is dissipated by DVGLUT through cation/H+ exchange. Under these conditions K+/H+ exchange is most likely as K+ is much more abundant than Na+ in the cytosol and previous work has demonstrated functional K+/H+ exchange across organellar membranes in rat synaptosomes (Goh, 2011) and amphibian vestibular hair cells. Upon exocytosis to the PM the directionality of DVGLUT reverses and acid efflux is mediated by Na+/H+ exchange driven by the strong ΔμNa+ at the PM. Upon cessation of neuronal activity, DVGLUT contributes to an early phase of accelerated acid efflux until it is retrieved from the PM by endocytosis. In recently endocytosed vesicles the elevated Cl concentration of the vesicular lumen drives glutamate into the vesicle by a Cl shunt mechanism (Schenck, 2009; Preobraschenski, 2014). As the vesicular ΔΨH+ generated by the vATPase is re-established the cycle completes. The model presented above describes a mechanism by which acid efflux can scale to effectively clear the overall net acid load through enhanced trafficking of acid-extruding proteins, including DVGLUT, to the PM, thus maintaining tight control of pHcyto in the face of large PMCA-medicated acid loads during nerve activity (Rossano, 2016).

    Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction

    Retrograde signaling is essential for neuronal growth, function and survival; however, little is known about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. This study identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but a significant decrease in the number of presynaptic release sites was found. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Overexpression of the type II TGFβ receptor Wit enhanced presynaptic pMad levels. In Khc-73 mutants, this enhancement was significantly suppressed. Similarly, muscle overexpression of the ligand Gbb enhanced pMAD levels in presynaptic boutons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, loss of Khc-73 was shown to curb the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. These findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons (Liao, 2018).

    Khc-73 function plays a supporting role in retrograde BMP signaling under basal conditions. However under conditions of enhanced BMP signaling, this endosomal coordination by Khc-73 becomes critical to transmit the retrograde signal from the synapse to the neuronal cell body (Liao, 2018).

    Efficient retrograde signaling from synaptic terminals back to the neuronal soma is critical for appropriate neuronal function and survival. Nevertheless, little is known about the molecular steps that facilitate the routing of synaptic endosomes destined for retrograde axonal pathways. This study describes several lines of evidence for a potential role for Khc-73 in this process. Khc-73 mutant larvae develop grossly normal synaptic structure and function at the Drosophila larval neuromuscular junction (NMJ), but this study finds a reduction in the number of presynaptic release sites. Through genetic interaction experiments, this defect was shown to most likely be the result of abnormal BMP signaling in motoneurons: transheterozygous combinations of Khc-73 and Medea or wit mutants show a significant loss of presynaptic release sites compared to control. Khc-73 becomes even more critical, when higher demand is put on the motoneuron by activating BMP signaling: loss of Khc-73 largely blocks the retrograde enhancement in synaptic release in response to activation of BMP pathway in motor neurons. Consistently it has been shown previously that transgenic knock down of Khc-73 in motoneurons blocks the ability of the NMJ to undergo retrograde synaptic homeostatic compensation (Tsurudome, 2010). The current findings show that when BMP signaling is activated, loss of Khc-73 reduces the accumulation of pMad in motoneuron nuclei, suggesting a role for Khc-73 in the regulation of retrograde signaling. Immunohistochemical assessment and live imaging analysis of Khc-73 mutant larvae provide evidence for involvement of Khc-73 in at least two steps in endosomal dynamics in motoneurons. On the one hand, Khc-73 is required for normal dynamics of internalized endosomes through late endosomal and multivesicular stages, and on the other Khc-73 plays a role in facilitating the routing of endosomes onto the retrograde pathway. These defects have two main consequences: first, an accumulation of BMP receptors was found at the NMJ (possibly in multivesicular bodies) without increased local signaling, suggesting that these receptor containing endosomes might be trapped in a state between late endosomal and lysosomal stage. Second, a dampening was seen of the ability of retrograde bound Rab7:GFP tagged endosomes to join the retrograde pathway, illustrating a defect in retrograde movement of vesicles and possibly providing an underlying explanation for the reduction in pMAD when retrograde BMP signaling is activated in Khc-73 mutants. These results together present Khc-73, a plus-end microtubule motor, in the unexpected role of regulation of endosomal traffic from synapse to the soma in motoneurons with a role for ensuring the efficiency of retrograde BMP signaling (Liao, 2018).

    The findings point to a model in which Khc-73 facilitates the routing of retrograde bound vesicles onto the retrograde axonal pathway. This model predicts coordination between endosomes, dynein motors and kinesin Khc-73. The coordinated involvement of dynein and kinesin motor proteins in the transport and sorting of endosomes has been previously proposed and examples supporting this model are mounting. Previously published data for Khc-73 and KIF13B have provided evidence that interaction between early endosomes, dynein motors and microtubules are possible. Khc-73/KIF13B is capable of binding to the GTPase Rab5 (found on early endosomes), thus allowing Khc-73 to localize directly to Rab5 endosomes. As a kinesin motor protein, Khc-73 could then transport these endosomes to the retrograde pathway by moving along the microtubule network in the synapse (Liao, 2018).

    Compelling evidence for a dynein interaction with Khc-73 has been previously demonstrated during mitotic spindle formation. The Khc-73/KIF13B stalk domain is phosphorylated by Par1b and this creates a 14-3-3 adapter protein binding motif. It has been proposed that physical interaction between Khc-73 stalk domain and the dynein interacting protein NudE via 14-3-3 ε/ζ might underlie the interaction between Khc-73 and dynein that is necessary for appropriate mitotic spindle formation. Interestingly, transgenic knock down of NudE in Drosophila larval motoneurons leads to a reduction in the number of presynaptic release sites, a phenotype reminiscent of Khc-73 loss of function. Thus, Khc-73 contains domains and protein-protein interactions that are capable of coordinating endosomes, microtubules and dynein. It is proposed that Khc-73 is necessary for the normal endosomal sorting and exit of endosomes from the NMJ to support efficient retrograde BMP signaling (Liao, 2018).

    Tao negatively regulates BMP signaling during neuromuscular junction development in Drosophila

    The coordinated growth and development of synapses is critical for all aspects of neural circuit function and mutations that disrupt these processes can result in various neurological defects. Several anterograde and retrograde signaling pathways, including the canonical Bone Morphogenic Protein (BMP) pathway, regulate synaptic development in vertebrates and invertebrates. At the Drosophila larval neuromuscular junction (NMJ), the retrograde BMP pathway is part of the machinery that controls NMJ expansion concurrent with larval growth. This study sought to determine whether the conserved Hippo pathway, critical for proportional growth in other tissues, also functions in NMJ development. Neuronal loss of the serine-threonine protein kinase Tao, a regulator of the Hippo signaling pathway, results in supernumerary boutons, each of which contain a normal number of active zones. Tao is also required for proper synaptic function, as reduction of Tao results in NMJs with decreased evoked excitatory junctional potentials. Surprisingly, Tao function in NMJ growth is independent of the Hippo pathway. Instead, the experiments suggest that Tao negatively regulates BMP signaling as reduction of Tao leads to an increase in pMad levels in motor neuron nuclei and an increase in BMP target gene expression. Taken together, these results support a role for Tao as a novel inhibitor of BMP signaling in motor neurons during synaptic development and function (Politano, 2019).

    The Strip-Hippo pathway regulates synaptic terminal formation by modulating actin organization at the Drosophila neuromuscular synapses

    Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. This study demonstrates that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. This study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization (Sakuma, 2016).

    Since the Hippo (Hpo) pathway was discovered as the key regulator to ensure the appropriate final tissue size by coordinating cell proliferation and cell death, large-scale genetics studies have identified numerous regulators of the Hpo pathway. While most pathway components identified thus far are positive regulators of Hpo, some negative regulators were recently reported. One such negative regulator is the STRIPAK (STRiatin-Interacting Phosphatase And Kinase) complex, which is evolutionarily conserved and regulates various cellular processes including cell cycle control and cell polarity. The core component of the STRIPAK complex is the striatin family of proteins: striatins serve as B‴ subunits (one of the subfamily of regulatory B subunits) of the protein phosphatase 2A (PP2A) complex. Beyond this, the A and C subunits of PP2A, Mob3, Mst3, Mst4, Ysk1, Ccm3, Strip1, and Strip2 form the core mammalian STRIPAK complex. It has been previously reported that Strip, the Drosophila homolog of mammalian Strip1 and 2, is involved in early endosome formation, which is essential for axon elongation. Building on these findings, it was hypothesized that the Strip-Hpo pathway may also be involved in neuronal synaptic development (Sakuma, 2016).

    The Drosophila larval neuromuscular junction (NMJ) is an ideal model for studying synaptic development because of its identifiable, stereotyped morphology, accessibility, broad complement of available reagents, and suitability for a wide range of experimental approaches. Furthermore, the Drosophila NMJ, like vertebrate central synapses, is glutamatergic, suggesting that the molecular mechanisms that regulate synaptic development in Drosophila NMJ might be applicable to vertebrates. Motor neuron axons are genetically hardwired to target specific muscles by the end of the embryonic stage. There, axonal growth cones subsequently differentiate into presynaptic termini, called boutons, each of which contains multiple active zones). During the larval stage, muscle size increases nearly 100-fold and boutons are continuously and proportionately added to maintain constant innervation strength. Various molecules can negatively or positively regulate the growth of synaptic termini. Amongst the many factors, elements of the actin cytoskeleton are key effectors of morphological change, functioning downstream of several cell surface receptors and signaling pathways. Of the two types of actin filaments (branched and linear), the activity of Arp2/3 complex, responsible for nucleation of branched F-actin, the first step of actin polymerization, should be strictly regulated. Arp2/3 hyperactivation results in synaptic terminal overgrowth characterized by excess small boutons emanating from the main branch that are termed satellite boutons (Sakuma, 2016).

    This study shows that Strip negatively regulates the synapse terminal development through tuning the activity of the core Hpo kinase cassette. Loss or reduction of strip function in motor neurons increased the number of satellite boutons, which could be suppressed by reducing the genetic dosage of hpo. Similarly, activation of the core Hpo kinase cassette also increased satellite boutons. In this context, the presumptive downstream target of the core Hpo kinase cassette is Enabled (Ena), a regulator of F-actin assembly and elongation that was reported to antagonize the activity of Arp2/3. The canonical downstream transcriptional co-activator, Yorkie (Yki), appears dispensable for Hpo-mediated synaptic terminal development. It is proposed that the evolutionarily conserved Strip-Hpo pathway regulates local actin organization by modulating Ena activity during synaptic development (Sakuma, 2016).

    This study has identified Strip and components of the Hpo pathway as regulators of synaptic morphology. In addition to the intensely investigated function of Hpo in growth control in mitotic cells, a few postmitotic roles of the Hpo pathway have recently been uncovered, such as dendrite tiling in Drosophila sensory neurons and cell fate specification of photoreceptor cells in Drosophila retina. This study now finds an additional postmitotic role for Hpo in synaptic terminal development. The results indicate that Strip and the core Hpo kinase cassette regulate satellite bouton formation by regulating the activity of Ena, an actin regulator that is involved in the initiation, extension, and maintenance of linear actin filaments at the barbed end. Although it cannot be excluded that there might be other targets of Yki in motor neurons than diap1 or bantam whose transcriptional activations were not observed in this study, Yki, a well-known downstream target of the core Hpo kinase cassette, was dispensable for proper synaptic morphology. Ena phosphorylation causes its inactivation; therefore, it was reasoned that Strip can act as a positive regulator of Ena by inactivating the Hpo pathway. A model is proposed for the regulation of satellite bouton formation by Strip and Hpo pathway components (see The Strip-Hpo pathway regulates satellite bouton formation with Ena, a regulator of F-actin organization). As the presynaptic localization of endogenous Strip was punctate and non-uniform, it is expected that Strip localization could be critical for regulating the phosphorylation status of Hpo, Wts, and Ena, which locally alters actin organization and eventually specifies the position of satellite bouton formation that could be a marker for new bouton outgrowth. When Strip is present, the core Hpo kinase cassette is inactivated, which in turn locally increases the expression of the active (unphosphorylated) form of Ena. However, the core Hpo kinase cassette can be activated in the absence of Strip, which subsequently phosphorylates and inactivates Ena. Ena prevents Arp2/3-induced branching, suggesting that Ena inactivation activates Arp2/3 and results in satellite bouton formation, similar to Rac activation. It is reported that Arp2/3 is involved in bouton formation and axon terminal branching downstream of WAVE/SCAR complex in NMJ. Indeed, the cureent findings support this hypothesis. F-actin organization was altered by strip knockdown in motor neurons. When the GFP-moe reporter was expressed in motor neuron, the GFP fused to the C-terminal actin-binding domain of Moesin, which is widely used as an F-actin reporter. The intensity of actin puncta became higher and puncta were unevenly distributed when strip was knocked down. This data suggests that Strip function is important for the proper organization of F-actin (Sakuma, 2016).

    There are many indications that Strip and other STRIPAK components (Mst3, Mst4, and Ccm3) regulate the actin network. For example, Strip1, Strip2, Mst3, and Mst4 regulate the actomyosin contractions which regulate cell migration in cancer cells. In addition to regulating the actin network, STRIPAK has been suggested to function in microtubule organization. Mutants of Drosophila Mob4, a member of the core STRIPAK complex and homolog of mammalian Mob3, show abnormal microtubule morphology at NMJs and muscles. Furthermore, it has been reported that Strip forms a complex with Glued, the homolog of mammalian p150Glued, a component of the dynactin complex required for dynein motor-mediated retrograde transport along microtubules. Strip also affects microtubule stability. As previously mentioned, microtubules are also key effectors of synaptic development downstream of several receptors and signaling pathways. Taken together, the STRIPAK complex can act as a regulatory hub for multiple cellular signals including Hpo pathway-mediated actin organization, endocytic pathway-dependent BMP signals, and microtubule stability for proper synaptic development (Sakuma, 2016).

    The Hpo pathway has been reported to act as a sensor of the local cellular microenvironment, such as mechanical cues, apico-basal polarity and actin architecture to balance cell proliferation and cell death. Although synaptic morphogenesis is a postmitotic process, it is very plastic and depends on a dynamically changing extracellular environment, as exemplified by the nearly 100-fold expansion of muscle size during larval development. Thus, this study demonstrates an intriguing function for the Strip-Hippo pathway in the homeostatic control of neuronal synaptic morphology and function (Sakuma, 2016).

    The innate immune receptor PGRP-LC controls presynaptic homeostatic plasticity

    It is now appreciated that the brain is immunologically active. Highly conserved innate immune signaling responds to pathogen invasion and injury and promotes structural refinement of neural circuitry. However, it remains generally unknown whether innate immune signaling has a function during the day-to-day regulation of neural function in the absence of pathogens and irrespective of cellular damage or developmental change. This study shows that an innate immune receptor, a member of the peptidoglycan pattern recognition receptor family (PGRP-LC), is required for the induction and sustained expression of homeostatic synaptic plasticity. This receptor functions presynaptically, controlling the homeostatic modulation of the readily releasable pool of synaptic vesicles following inhibition of postsynaptic glutamate receptor function. Thus, PGRP-LC is a candidate receptor for retrograde, trans-synaptic signaling, a novel activity for innate immune signaling and the first known function of a PGRP-type receptor in the nervous system of any organism (Harris, 2015).

    The homeostatic modulation of presynaptic neurotransmitter release has been observed at mammalian central synapses and at neuromuscular synapses in species ranging from Drosophila to mouse and human. The homeostatic enhancement of presynaptic release following inhibition of postsynaptic glutamate receptors is achieved by an increase in presynaptic calcium influx through presynaptic CaV2.1 calcium channels and the simultaneous expansion of the readily releasable pool (RRP) of synaptic vesicles. To date, the retrograde, trans-synaptic signaling system that initiates these presynaptic changes following disruption of postsynaptic glutamate receptors remains largely unknown (Harris, 2015).

    A large-scale forward genetic screen for homeostatic plasticity genes, using synaptic electrophysiology at the neuromuscular junction (NMJ) as the primary assay, identified mutations in the PGRP-LC locus that block presynaptic homeostasis. In Drosophila, PGRP-LC is the primary receptor that initiates an innate immune response through the immune deficiency (IMD) pathway. In mammals, there are four PGRPs including a 'long' isoform (PGRP-L or PGlyRP2) that appears to have both secreted and membrane-associated activity. In mice, the PGlyRP2 protein has two functions in the innate immune response: a well-documented extracellular enzymatic (amidase) activity and a pro-inflammatory signaling function that is independent of its extracellular enzymatic activity. As yet, there are no known functions for PGRPs in the nervous system of any organism (Harris, 2015).

    Innate immune signaling has been found to participate in neural development and disease including a role for the C1q component of the complement cascade, Toll-like receptor signaling, and tumor necrosis factor signaling. In these examples, however, the innate immune response is induced within microglia or astrocytes. Far less clear is the role of innate immune signaling within neurons, either centrally or peripherally, although there are clear examples of downstream signaling components such as Rel and NFκB having important functions during learning related neural plasticity. This study places the innate immune receptor, PGRP-LC at the presynaptic terminal of Drosophila motoneurons and demonstrate that this receptor is essential for robust presynaptic homeostatic plasticity. It is speculated, based on the current data, that PGRP-LC could function as a receptor for the long-sought retrograde signal that mediates homeostatic signaling from muscle to nerve (Harris, 2015).

    A distinguishing feature of presynaptic homeostatic plasticity is that it can be both rapidly induced and sustained for prolonged periods. It is possible that innate immune signaling activity is adjusted to fit the requirements of presynaptic homeostasis in motoneurons. This would be consistent with the established diversity of innate immune signaling functions during development such as dorso-ventral patterning. Alternatively, the induction of innate immune signaling might serve a unique function during presynaptic homeostasis. Many homeostatic signaling systems incorporate feed-forward signaling elements. By analogy, PGRP-LCx could function as a feed-forward signaling receptor that acts more like a 'switch' to enable presynaptic homeostasis in the nerve terminal. The accuracy of the homeostatic response would be determined by other signaling elements. In favor of this idea, the induction of innate immune signaling is rapid, occurring in seconds to minutes, and can be maintained for the duration of an inducing stimulus, consistent with recent evidence that presynaptic homeostasis is rapidly and continually induced at synapses in the presence of a persistent postsynaptic perturbation. Finally, it remains formally possible that PGRP-dependent signaling is a permissive signal that allows expression presynaptic homeostasis (Harris, 2015).

    Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins

    A network of interacting Drosophila cell surface proteins has been defined in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. These findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity (Carrillo, 2015).

    This study has defined a network of interacting Drosophila IgSF CSPs in which 21 Dpr proteins bind to 9 DIPs. The structure of the Dpr-DIP complex resembles that of neural and immune cell adhesion complexes. Each of the six dpr and DIP genes examined in this study is expressed by a different subset of neurons in the larval VNC and pupal OL. In the larval neuromuscular system, Dpr11 and its binding partner DIP-γ regulate presynaptic terminal development and neurotransmission. In the pupal OL, they are required for normal formation of synapses between a Dpr11-expressing sensory neuron and a DIP-γ expressing interneuron (Carrillo, 2015).

    The crystal structure shows that Dprs and DIPs belong to a group of IgSF CSPs that interact via their N-terminal Ig domains. These include immune cell receptors such as CD2, CD58, JAML, CAR, B7-1, and CTLA-4, and Nectin/Nectin-like (Necl) proteins. The nine Nectin/Necls interact with each other, forming a small network. Although DIPs resemble Nectins/Necls, their closest vertebrate counterpart is the five-member IgLON subfamily, which is also expressed in neurons. Dprs have no clear mammalian orthologs. DIPs and Dprs are distinguished from IgLONs and Nectins in that their interactions are across subfamilies, not within a subfamily. The closest structural homolog of the Dpr-DIP complex is the SYG-1-SYG-2 complex, known to be involved in synapse specification (Carrillo, 2015).

    The Dpr-DIP complex has an interface involving no charge pairs, suggesting that binding specificity is encoded through shape complementarity. The Dpr-DIP interaction code may be created by substitution of larger or smaller residues within the binding interface in order to create more or less complementary surfaces between individual interacting Dpr-DIP pairs. This differs substantially from the electrostatic complementarity model, in which receptor-ligand specificity is created primarily through hydrogen bonding interactions and salt bridges. Interestingly, for Dscam homophilic interactions, where each of the many thousands of possible variants binds primarily to itself, both electrostatic and shape complementarity play crucial roles. Each Dscam variant has to find a single binding solution, which is a task that can be solved in many ways. By contrast, the complex cross-reactivity observed for Dpr-DIP interactions may impose restrictions on encoding of specificity that mandate the selection of shape complementarity as the primary mechanism (Carrillo, 2015).

    The larval neuromuscular system is a genetic model system for glutamatergic synapses in mammals. In mutants lacking either Dpr11 or DIP-γ, NMJs contain many small clustered boutons called satellites. The satellite bouton phenotypes are rescued by either pre- or postsynaptic expression of the proteins. mEPSP amplitude and frequency are increased to similar extents in dpr11 and DIP-γ mutants. These data, together with the fact that the two loci genetically interact, indicate that the two proteins have linked functions, and suggest that the phenotypes are due to loss of Dpr11-DIP-γ adhesion complexes (Carrillo, 2015).

    BMPs are trophic factors for mammalian neurons, and retrograde BMP signaling controls NMJ arbor growth in Drosophila. Satellites are observed in mutants in which BMP signaling is upregulated. Consistent with this, presynaptic pMad staining, which reports on the magnitude of the BMP signal, is increased in dpr11 mutants, and dpr11 and DIP-γ interact with genes encoding BMP signaling components (Carrillo, 2015).

    Each dpr and DIP examined is expressed in a unique subset of neurons that project to specific layers in the OL neuropils. Identifying these neurons can define relationships between dpr/DIP expression and synaptic connectivity, because detailed synaptic maps for units of the first two areas of the OL, the La and Me, have been created using electron microscopic reconstruction (Carrillo, 2015).

    Axons of UV-sensitive R7 photoreceptors synapse in layer M6 of the Me onto Dm8, Tm5a, Tm5b, and other targets. dpr11 is selectively expressed by yR7s, which express Rh4 opsin and are in ~70% of ommatidia. Dpr11 is the first cell surface protein to be associated with a subclass of R7s. DIP-γ is expressed by Dm8s, which arborize in M6 and receive more R7 synapses than any other neurons (Carrillo, 2015).

    To examine whether formation of synapses between yR7s and Dm8s involves interactions between Dpr11 and DIP-γ, a marker for existing active zones, Brp-shortmCherry, was expressed in yR7s. In control animals, yR7 terminals are bulb-shaped and regularly arranged in M6. In dpr11 and DIP-γ mutants, the main bodies of yR7 terminals have altered shapes, and active zone and membrane markers are found in extensions projecting into deeper Me layers. These data suggest that synapses between yR7 and its M6 targets do not form normally in the absence of Dpr11 or DIP-γ. Because most M6-projecting DIP-γ-positive cells seen in the FLP-out analysis are Dm8s, and because Dpr11's other partner, DIP-β, does not label M6, it is infered that the loss of Dpr11 or DIP-γ is likely to primarily affect yR7-Dm8 synapses in M6 (Carrillo, 2015).

    In DIP-γ mutants, there are large gaps in M6 labeling by DIP-γ or Dm8 reporters. The number of OrtC2b+, DIP-γ+ cells is reduced by >3-fold, suggesting that most DIP-γ-expressing Dm8s die. Alternatively, they might turn off expression of the OrtC2b-GAL4 driver, although this seems less likely. This effect on cell fate suggests that DIP-γ is required for reception of a neurotrophic signal. Since dpr11 mutants have no DIP-γMiMIC M6 gaps, implying that they have normal numbers of OrtC2b+, DIP-γ+ cells, this signal might be communicated through Dprs 15, 16, and/or 17, the other Dprs that bind to DIP-γ. Other OL neurons are also dependent on trophic factors for survival. R cell growth cones secrete the Jelly Belly (Jeb) ligand, which binds to its receptor Alk on L3 neurons, and L3s die in the absence of Jeb or Alk. The functions of DIP-γ in mediating normal development of yR7-Dm8 connectivity, as assayed by displacement of the active zone marker in yR7s, may be distinct from its roles in Dm8 survival, because about half of the overshoots in DIP-γ mutants appear to grow through a Dm8 arbor labeled by the DIP-γ reporter (Carrillo, 2015).

    dpr11 is expressed by subsets of direction-selective T4 and T5 neurons that arborize in the Lop layers activated by front-to-back and back-to-front motion, and DIP-γ is expressed by three LPTCs, which receive synaptic input from T4s and T5s. These data suggest that Dpr11 and DIP-γ expression patterns might have evolved to facilitate assembly of synaptic circuits for specific sensory responses: near-UV vision for yR7-Dm8 connections and movement along the anterior-posterior axis for T4/T5 subset-LPTC connections. In a conceptually similar manner, a specific type of vertebrate amacrine neuron, VG3-AC, forms synapses on W3B retinal ganglion cells, which are specialized for detecting object motion. Both VG3-ACs and W3B-RGCs selectively express the IgSF protein Sidekick2 (Sdk2), and Sdk2-mediated homophilic adhesion is required for their connectivity (Carrillo, 2015).

    An accompanying paper on gene expression in La neurons (Tan, 2015) presents ten instances in which a La neuron expressing a Dpr is synaptically connected to a Me neuron expressing a DIP to which that Dpr binds in vitro. In nine of these, as well as in the two cases described in this study (yR7 -> Dm8 and T4/T5 -> LPTC), the Dpr is in the presynaptic neuron and the DIP in the postsynaptic neuron. Each dpr and DIP gene examined in the two papers is expressed in a different subset of OL neurons, each of which projects to a distinct set of neuropil layers, and neurons can express multiple Dprs or DIPs or a combination of the two (Tan, 2015). This means that there are hundreds of different synaptic matches in the OL that could potentially be programmed by the Dpr-ome network. Dprs and DIPs are also expressed by subsets of neurons in other areas of the larval and pupal brain. These expression patterns, together with the phenotypic data presented here for one Dpr-DIP binding pair, suggest that Dpr-DIP interactions are likely to be important determinants of synaptic connectivity during brain development (Carrillo, 2015).

    An auxiliary subunit of the presynaptic calcium channel, α2δ-3, is required for rapid transsynaptic homeostatic signaling

    The homeostatic modulation of neurotransmitter release, termed presynaptic homeostatic potentiation (PHP), is a fundamental type of neuromodulation, conserved from Drosophila to humans, that stabilizes information transfer at synaptic connections throughout the nervous system. This study demonstrates that α2δ-3 (straitjacket), an auxiliary subunit of the presynaptic calcium channel, Cacophony, is required for PHP. The α2δ gene family has been linked to chronic pain, epilepsy, autism, and the action of two psychiatric drugs: gabapentin and pregabalin. Loss of α2δ-3 blocks both the rapid induction and sustained expression of PHP due to a failure to potentiate presynaptic calcium influx and the RIM-dependent readily releasable vesicle pool. These deficits are independent of α2δ-3-mediated regulation of baseline calcium influx and presynaptic action potential waveform. α2δ proteins reside at the extracellular face of presynaptic release sites throughout the nervous system, a site ideal for mediating rapid, transsynaptic homeostatic signaling in health and disease (Wang, T., 2016).

    Presynaptic homeostatic potentiation (PHP) can be initiated by disruption of postsynaptic neurotransmitter receptors and is expressed as a change in presynaptic vesicle release. As such, PHP requires retrograde, transsynaptic signaling. The homeostatic potentiation of presynaptic release is mediated by increased presynaptic calcium influx without a change in the presynaptic action potential waveform. A remarkable property of presynaptic homeostatic plasticity is that it can be induced in a time frame of seconds to minutes and can be stably maintained throughout the life of an organism -- months in Drosophila and decades in humans. Equally remarkable, presynaptic homeostasis can precisely offset the magnitude of postsynaptic perturbations that vary widely in severity. This implies the existence of profoundly stable and remarkably precise homeostatic modifications to the presynaptic release apparatus. Transsynaptic signaling systems that are capable of achieving the rapid, accurate, and persistent control of presynaptic vesicle release are generally unknown (Wang, T., 2016).

    In a large-scale forward genetic screen for homeostatic plasticity genes, mutations were identified in the α2δ-3 auxiliary subunit of the CaV2.1 calcium channel. α2δ genes encode a family of proteins that are post-translationally processed into a large glycosylated extracellular α2 domain that is linked through disulfide bonding to a short, membrane-associated δ domain. Existing loss-of-function data are consistent with the primary function of α2δ being the trafficking and synaptic stabilization of pore-forming α1 calcium channel subunits, with which they associate in the ER. There is also evidence that α2δ subunits control calcium channel kinetics in a channel-type- and cell-type-specific manner. However, the function of the α2δ gene family extends beyond calcium channel trafficking and membrane stabilization, including activities related to synapse formation and stability. As such, the large, glycosylated extracellular domain in α2δ may have additional, potent signaling activities at the active zone (Wang, T., 2016).

    Importantly, the α2δ gene family is associated with a wide range of neurological diseases, including autism spectrum disorders (ASDs), neuropathic pain, and epilepsy. The α2δ-1 and α2δ-2 proteins are the primary targets of gabapentin and pregabalin, two major drugs used to treat neuropathic pain and epilepsy. This study demonstrates that α2δ-3 is essential for PHP. Thus, while α2δ-3 is an extracellular component of the extended presynaptic calcium channel complex (Davies, 2010), it nonetheless has a profound ability to modulate the intracellular neurotransmitter release mechanism. It is proposed that α2δ-3 relays signaling information from the synaptic cleft to the cytoplasmic face of the presynaptic active zone during PHP, an activity that could reasonably be related to the function of α2δ-3 during neurological disease (Wang, T., 2016).

    This study demonstrates that α2δ-3 is essential for the rapid induction and sustained expression of presynaptic homeostatic potentiation (PHP). α2δ-3 encodes a glycosylated extracellular protein known to interact with matrix proteins that reside within the synaptic cleft. As such, it is proposed that α2δ-3 mediates homeostatic, retrograde signaling by connecting signaling within synaptic cleft to effector proteins within the presynaptic terminal, such as RIM. Since α2δ-3 associates with the pore-forming α1 subunit of calcium channels, it is ideally positioned to relay signaling to the site of high-release probability vesicle fusion adjacent to the presynaptic calcium channels (Wang, T., 2016).

    It was previously demonstrated that PHP requires not only potentiation of presynaptic calcium influx but also a parallel homeostatic expansion of the readily releasable pool (RRP). Several lines of evidence argue against the possibility that the homeostatic potentiation of presynaptic calcium influx fully accounts for the observed potentiation of the RRP. First, it is well established in mammalian systems and the Drosophila NMJ (Müller, 2015) that the calcium-dependence of the RRP is sub-linear. Therefore, the relatively small change in presynaptic calcium influx that occurs during PHP (12%-25%) would not be sufficient to account for the observed doubling of the RRP, an effect that has been quantified across a wide range of extracellular calcium (0.3-15 mM [Ca2+]e) (Müller, 2015). Second, the homeostatic increase of presynaptic calcium influx and the homeostatic expansion of RRP are genetically separable processes (Harris, 2015). Since loss of α2δ-3 completely blocks the homeostatic expansion of the RRP, it appears that α2δ-3 has an additional activity that is directed at the homeostatic modulation of the RRP (Wang, T., 2016).

    Collectively, these data argue that α2δ-3 functions with Rab3 interacting molecule (rim), either directly or indirectly, to achieve a homeostatic potentiation of the RRP. First, the loss of function phenotype of α2δ-3 is strikingly similar to that observed in rim mutants. Both mutations cause a deficit in presynaptic release that is associated with diminished baseline presynaptic calcium influx, diminished size of the baseline RRP, and enhanced sensitivity to application of EGTA-AM. Second, this study demonstrates a strong trans-heterozygous interaction between rim/+ and α2δ-3/+, suggesting that both genes function to control the same presynaptic processes during PHP. Since the rim mutation selectively disrupts the homeostatic modulation of the RRP, this genetic interaction could reflect a failure to homeostatically modulate the RRP (Wang, T., 2016).

    Both RIM and α2δ-3 bind the pore-forming α1 subunit of the CaV2.1 calcium channel. As such, signaling could be relayed from α2δ-3 to RIM through molecular interactions within the extended CaV2.1 calcium channel complex. However, not all evidence is consistent with this possibility. For example, RNAi-mediated depletion of CaV2.1 channels, sufficient to decrease release by ∼80%, does not prevent presynaptic homeostasis. Thus, loss of α2δ-3 blocks PHP, whereas loss of the CaV2.1 α1 subunit does not. In addition, the double-heterozygous mutant of rim/+ and α2δ-3/+ blocks PHP but does not disrupt baseline vesicle release, arguing that this genetic interaction is not due to a decrease in the number or organization of presynaptic α1 calcium channel subunits. Thus, it is speculated that α2δ-3 conveys signaling through a co-receptor on the plasma membrane to participate in the homeostatic modulation of the RRP. There are very few extracellular proteins known to establish baseline levels of primed, fusion-competent synaptic vesicles. Since α2δ proteins should reside at chemical synapses throughout the nervous system, this signaling could reasonably be related to the neurological and psychiatric diseases associated with α2δ genes (Wang, T., 2016).

    Presynaptic CamKII regulates activity-dependent axon terminal growth

    Spaced synaptic depolarization induces rapid axon terminal growth and the formation of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). This study identified a novel presynaptic function for the Calcium/Calmodulin-dependent Kinase II (CamKII) protein in the control of activity-dependent synaptic growth. Consistent with this function, both total and phosphorylated CamKII (p-CamKII) are were found to be enriched in axon terminals. Interestingly, p-CamKII appears to be enriched at the presynaptic axon terminal membrane. Moreover, levels of total CamKII protein within presynaptic boutons globally increase within one hour following stimulation. These effects correlate with the activity-dependent formation of new presynaptic boutons. The increase in presynaptic CamKII levels is inhibited by treatment with cyclohexamide suggesting a protein-synthesis dependent mechanism. Previous work has found that acute spaced stimulation rapidly downregulates levels of neuronal microRNAs (miRNAs) that are required for the control of activity-dependent axon terminal growth at this synapse. The rapid activity-dependent accumulation of CamKII protein within axon terminals is inhibited by overexpression of activity-regulated miR-289 in motor neurons. Experiments in vitro using a CamKII translational reporter show that miR-289 can directly repress the translation of CamKII via a sequence motif found within the CamKII 3' untranslated region (UTR). Collectively, these studies support the idea that presynaptic CamKII acts downstream of synaptic stimulation and the miRNA pathway to control rapid activity-dependent changes in synapse structure (Nesler, 2016).

    Acute spaced synaptic depolarization rapidly induces the formation of new synaptic boutons at the larval NMJ. These immature presynaptic outgrowths, also known as "ghost boutons", are characterized by the presence of synaptic vesicles but by a lack of active zones and postsynaptic specializations. IA wild-type third instar larval NMJ will typically have about 2 ghost boutons. Using an established synaptic growth protocol, a robust increase in the number of ghost boutons was observed following 5 x K+ spaced stimulation. It has been shown that activity-dependent ghost bouton formation involves both new gene transcription and protein synthesis. Furthermore, new presynaptic expansions can form within 30 min of stimulation even after the axon innervating the NMJ has been severed. These findings suggest that a local mechanism (i.e. local signaling and/or translation) is required for the budding and outgrowth of new axon terminals. As expected, application of the translational inhibitor cyclohexamide during the recovery phase prevented the formation of new ghost boutons (Nesler, 2016).

    It has been shown previously that the outgrowth of new synaptic boutons in response to spaced depolarization requires the function of activity-regulated neuronal miRNAs including miR-8, miR-289, and miR-958 (Nesler, 2013). This implies that mRNAs encoding for synaptic proteins might be targets for regulation by these miRNAs. Focused was placed on CamKII for three reasons. (1) CamKII has been shown to have a conserved role in the control of long-term synaptic plasticity and its expression at synapses requires components of the miRNA pathway. Furthermore, the fly CamKII mRNA contains two predicted binding sites for activity-regulated miR-289. (2) CamKII and PKA both phosphorylate and actives synapsin. At the fly NMJ, a synapsin-dependent mechanism is required for a transient increase in neurotransmitter release in response to tetanic stimulation. Synapsin also redistributes to sites of activity-dependent axon terminal growth and regulates outgrowth via a PKA-dependent pathway. (3) Presynaptic CamKII has been shown to function in axon pathfinding in cultured Xenopus neurons. It seemed likely that activity-dependent ghost bouton formation and axon guidance might share similar molecular machinery (Nesler, 2016).

    It was postulated that presynaptic CamKII was required to control activity-dependent axon terminal growth at the larval NMJ. To address this question, CamKII expression was disrupted in motor neurons using two transgenic RNAi constructs. Depletion of presynaptic CamKII with both transgenes prevented the formation of new ghost boutons in response to spaced stimulation. Thus, presynaptic CamKII is necessary to control the formation of new synaptic boutons (Nesler, 2016).

    To further confirm that presynaptic CamKII function was required for activity-dependent growth, a transgenic line was used that inducibly expressed an inhibitory peptide (UAS-CamKIIAla). As in mammals, the activation of Drosophila CamKII by exposure to calcium leads to the autophosphorylation of a conserved threonine residue within the autoinhibitory domain (T287 in Drosophila). Activation of CamKII then confers an independence to calcium levels that persists until threonine-287 is dephosphorylated. The synthetic Ala peptide mimics the autoinhibitory domain and its transgenic expression is sufficient to substantially inhibit endogenous CamKII activity. Expression of the Ala inhibitory peptide in larval motor neurons disrupted the formation of new ghost boutons following spaced synaptic depolarization. These observations are consistent with results from CamKII RNAi (Nesler, 2016).

    Together, these data suggest that presynaptic CamKII function is required to control new ghost bouton formation in response to acute synaptic activity. Similarly, presynaptic CamKII has been implicated in controlling both bouton number and morphology during development of the larval NMJ. Reducing neuronal CamKII levels by RNAi has recently been shown to significantly reduce the number of type 1b boutons at the larval NMJ suggesting that presynaptic CamKII is required to control normal synapse development (Gillespie, 2013). In contrast, presynaptic expression of the Ala inhibitory peptide has no effect on the total number of type 1 synaptic boutons. Given that the Ala peptide does not completely inhibit CamKII autophosphorylation, it is suggested that the activation of CamKII in response to acute spaced synaptic depolarization is likely to be more sensitive to disruption then during NMJ development (Nesler, 2016).

    It was next asked if presynaptic CamKII could induce activity-dependent axon terminal growth at the NMJ. The overexpression of genes that are necessary for the control of ghost bouton formation generally does not cause an increase in the overall number of new synaptic boutons following 5 x high K+ spaced. Instead, overexpression often leads to an increased sensitization of the synapse to subsequent stimuli (for example, significant growth is observed after 3 x instead of 5 x high K+). The overexpression of a wild-type CamKII transgene in motor neurons caused an increase of 71% in ghost bouton numbers in 3 x K+ spaced stimulation larvae compared to 0 x K+ controls. While this is trending towards an increase, it did not reach statistical significance, even though expression levels were substantially higher than endogenous CamKII. Thus, increased CamKII is not sufficient to stimulate activity-dependent axon terminal growth (Nesler, 2016).

    To further investigate the role of presynaptic CamKII in activity-dependent axon terminal growth, the effect of transgenic neuronal overexpression of either an overactive form of CamKII (CamKIIT287D) or a form that is incapable of remaining active in the absence of elevated calcium (CamKIIT287A). Much like C380-Gal4/+ controls, presynaptic expression of either transgene had no significant effect on the number of ghost boutons in 3 x high K+ stimulation. Again, levels of CamKII protein in axon terminals in both transgenic lines were elevated relative to controls. Collectively, these data suggest that constitutive activation of CamKII is not sufficient to sensitize the NMJ to stimulation (Nesler, 2016).

    The results suggest that the temporal and/or spatial regulation of CamKII expression or activation is likely required to control activity-dependent growth. In support, the Drosophila CamKII protein has been shown to phosphorylate and regulate the activity of the Ether-a-go-go (Eag) potassium channel in motor neuron axon terminals. In turn, CamKII is bound and locally activated by phosphorylated Eag. This local activation can persist even after calcium levels have been reduced. CamKII autophosphorylation and Eag localization to synapses requires the activity of the membrane-associated Calcium/Calmodulin-associated Serine Kinase, CASK. The presynaptic coexpression of CASK with CamKIIT287D reverses (to wild-type levels) the increase in type 1b boutons observed when CamKIIT287D is overexpressed alone. Thus, a mechanism exists at the larval NMJ that allows for the persistence of local CamKII activation in the absence of additional stimul (Nesler, 2016).

    After establishing that CamKII has a novel presynaptic function in activity-dependent ghost bouton formation, the distribution of CamKII protein at the larval NMJ was examined closely. It has previously been reported that CamKII strongly colocalizes with postsynaptic Discs large (DLG), the Drosophila ortholog of mammalian PSD-95, around the borders of type 1 synaptic boutons. In support, an anti-CamKII antibody coimmunoprecipitates DLG from larval body wall extracts. Interestingly, while DLG is pre-dominantly postsynaptic at the developing NMJ it is also initially expressed in the presynaptic cell and at least partially overlaps with presynaptic membrane markers in axon terminals. It has been demonstrated that while fly CamKII colocalizes with DLG within dendrites of adult olfactory projection neurons (PNs), it also localizes to presynaptic boutons within those same neurons. Consistent with the latter observations (using a different CamKII antibody), it has been shown that CamKII is substantially enriched in presynaptic terminals of type 1b boutons. To resolve these inconsistent results, both antibodies against CamKII were used to more closely analyze the localization of CamKII at the third instar larval NMJ. First, double labeling of wild-type NMJs with a monoclonal CamKII antibody and anti-horseradish peroxidase (HRP), a marker for Drosophila neurons, confirmed that CamKII was enriched in presynaptic boutons in a pattern very similar to that of HRP. A closer examination of confocal optical sections revealed that almost all CamKII localized to the presynaptic terminal and was not significantly enriched either (1) at sites surrounding presynaptic boutons, or (2) in the axons innervating synaptic arbors (Nesler, 2016).

    Within boutons, CamKII appeared to be predominantly cytoplasmic but was sometimes localized to discrete puncta that were reminiscent of antibody staining for active zones. Prolonged depolarization of hippocampal neurons with K+ leads to mobilization of CamKII from the cytoplasm to sites near active zones. Moreover, using a fluorescent reporter for CamKII activity, high frequency stimulation causes the very rapid (on the order of minutes) activation of presynaptic CamKII and promotes its translocation from the cytoplasm to sites near active zones. To address this possibility, larval NMJs were double labeled with antibodies targeting both CamKII and DVGLUT, the Drosophila vesicular glutamate transporter, in order to visualize active zones. As predicted, it was found that some presynaptic CamKII colocalized with DVGLUT in type 1b and 1s boutons. Thus, in some type 1 synaptic boutons, CamKII protein is enriched in or near active zones (Nesler, 2016).

    To confirm that CamKII was enriched in presynaptic boutons, wild-type NMJs were double labelled with a polyclonal CamKII antibody and anti-DLG. CamKII did partially colocalize with DLG at the border of type 1 synaptic boutons. However, in this study, CamKII was primarily localized to the presynaptic side of the synapse. Collectively, this study provides strong evidence that CamKII is expressed on both the pre- and postsynaptic side of the synapse but that it is clearly enriched within presynaptic boutons at the larval NMJ. This localization is analogous to CamKII distribution in mammalian axons (Nesler, 2016).

    After demonstrating that total CamKII was enriched in presynaptic axon terminals, it was next asked if any of this protein was active by assessing phosphorylation of threonine-287 using a phospho-specific polyclonal antibody. It was found that pT287 CamKII staining intensity was strong and fairly uniform in presynaptic boutons and weakly stained axons innervating synaptic arbor. Closer examination of confocal optical sections revealed that almost all p-CamKII colocalized with HRP in the presynaptic terminal and only sparsely stained the body wall muscle (Fig. 3A′). Presynaptic CamKII RNAi almost completely disrupted p-CamKII in axon terminals leaving some residual staining in the presynaptic bouton and surrounding muscle suggesting that the antibody is specific. To further demonstrate this presynaptic localization, it was found that p-CamKII staining clearly does not overlap with postsynaptic DLG but does colocalize strongly with immunostaining using the monoclonal total CamKII antibody (Nesler, 2016).

    Collectively, three different antibodies were used to show that CamKII enriched in presynaptic axon terminals. Next, it was asked as to how this enrichment was occurring. In Drosophila and mammalian neurons, the CamKII mRNA is transported to dendritic compartments and locally translated in response to synaptic stimulation. This spatial and temporal regulation requires sequence motifs found within the 5' and 3' UTRs of the CamKII transcript. In contrast, the localization of CamKII to axon terminals of Drosophila PNs does not strictly require the CamKII 3'UTR suggesting that enrichment in presynaptic boutons occurs through a mechanism that does not strictly require local translation. In mammalian neurons, CamKII is enriched in axon terminals where it can associate with synaptic vesicles and synapsin I. Recently, it has been shown that mammalian CamKII and the synapsin proteins are both conveyed to distal axons at rates consistent with slow axonal transport, with a small fraction of synapsin cotransported with vesicles via fast transport (Nesler, 2016).

    Because activity-dependent growth at the larval NMJ requires the miRNA pathway and new protein synthesis, it was asked if the localization of CamKII protein to axon terminals might require the CamKII 3'UTR. As expected, when expression was specifically driven in larval motor neurons, a transgenic CamKII:EYFP fusion protein regulated by the CamKII 3'UTR localized strongly to presynaptic boutons at the larval NMJ. However, very similar results were observed using the same CamKII:EYFP fusion protein regulated by a heterologous 3'UTR. Taken together, these data suggest that localization of CamKII protein to presynaptic boutons at the NMJ does not require mRNA transport and local translation. Thus, it is concluded that most of the Drosophila CamKII protein found in motoneuron axon terminals is likely there due to the transport of cytosolic CamKII from the cell body to synapses via a mechanism involving axonal transport. (Nesler, 2016).

    It was of interest to determining how CamKII might be regulating activity-dependent axon terminal growth, and it was speculated that either the levels or distribution of CamKII protein might be altered in response to spaced depolarization. It first asked if high K+ stimulation resulted in an increase in CamKII protein within motoneuron axon terminals. Larval preparations were stimulated, and changes in the levels of CamKII protein in presynaptic boutons was examined by immunohistochemistry and quantitative confocal microscopy. Following spaced stimulation, CamKII staining within boutons rapidly increased (in ~ 1 h) by an average of 26%. This increase in immunofluorescence was global and did not appear to be localized to particular regions of the NMJ (i.e., near obvious presynaptic outgrowths). CamKII has been reported to very rapidly translocate to regions near active zones in response to high frequency stimulation. However, when compared to DVGLUT levels in unstimulated and stimulated larvae, no significant increase in CamKII immunofluorescence was observed indicating that translocation does not occur or does not persist in the current assay. To determine if this increase in CamKII enrichment required new protein synthesis, larval preparations were incubated with the translational inhibitor cyclohexamide during the recovery phase. Surprisingly, this treatment completely blocked the activity-dependent affects on presynaptic CamKII enrichment within axon terminals. Thus, spaced high K+ stimulation results in a rapid increase in CamKII levels in presynaptic boutons via some mechanism that requires activity-dependent protein synthesis (Nesler, 2016).

    Next, it was asked if the levels or distribution of p-CamKII changed in response to spaced stimulation. Larval preparations were stimulated exactly as described above and analyzed by confocal microscopy. Interestingly, p-CamKII staining was enriched at the presynaptic membrane of many axon terminals following spaced depolarization (Nesler, 2016).

    Given the requirement for new protein synthesis, it was speculated that the additional CamKII protein in axon terminals could be derived from a pool of CamKII mRNA that is rapidly transcribed and translated in the soma in response to spaced depolarization. This newly translated CamKII would then be actively transported out to axon terminals via standard mechanisms. If this were true, it would be expected that elevated CamKII levels could be detected in the larval ventral ganglion. To examine this process more closely, global total CamKII expression levels within the larval ventral ganglion were assayed by Western blot analysis. It was found that two distinct isoforms of CamKII are expressed in explanted larval ventral ganglia, Surprisingly, no increase was observed in CamKII protein levels in the ventral ganglion (Nesler, 2016).

    What is the source of this new presynaptic CamKII protein? Three possible explanations are proposed. First, new CamKII protein might be transcribed and translated in the motor neuron cell body. However, this new protein would be rapidly transported away to axon terminals in response to spaced depolarization. Second, some CamKII protein is found in the axons innervating the NMJ (seen using the p-CamKII antibody). It is possible that activity stimulates the rapid transport of an existing pool of CamKII protein from distal axons into axon terminals. This process would be sensitive to translational inhibitors. Finally, a pool of CamKII mRNA might be actively transported into axon terminals and then locally translated in response to spaced depolarization. This would account for the both the dependence on translation and for increased CamKII enrichment in presynaptic boutons (Nesler, 2016).

    Thus far, this study has shown that activity-dependent ghost bouton formation correlates with a protein synthesis-dependent increase in CamKII levels within presynaptic boutons at the larval NMJ. The activity-dependent translation of CamKII in olfactory neuron dendrites in the adult Drosophila brain requires components of the miRNA pathway. Within the CamKII 3'UTR, there are two putative binding sites for activity-regulated miR-289. These two binding sites were of particular interest. It was previously shown that levels of mature miR-289 are rapidly downregulated in the larval brain in response to 5 x high K+ spaced training (Nesler, 2013). Moreover, presynaptic overexpression of miR-289 significantly inhibits activity-dependent ghost bouton formation at the larval NMJ (Nesler, 2013). Based on these data, it was speculated that CamKII might be a target for regulation by miR-289 (Nesler, 2016).

    To determine if CamKII is a target for repression by miR-289 in vivo, a transgenic construct containing the primary miR-289 transcript was overexpressed in motor neurons and CamKII enrichment was examined by anti-CamKII immunostaining and quantitative confocal microscopy. Relative to controls, the presynaptic overexpression of miR-289 completely abolished the observed activity-dependent increase in CamKII immunofluorescence. When analyzing global CamKII levels within axon terminals during NMJ development, presynaptic miR-289 expression led to a slight decrease in CamKII immunofluorescence. This trend is similar to results observed following treatment with cyclohexamide during the recover period. The lack of full repression by miR-289 is not surprising given that one miRNA alone is often not sufficient to completely repress target gene expression (Nesler, 2016).

    To directly test the ability of miR-289 to repress translation of CamKII, a reporter was developed where the coding sequence for firefly luciferase (FLuc) was fused to the regulatory CamKII 3'UTR (FLuc-CamKII 3'UTR). When this wild-type reporter was coexpressed with miR-289 in Drosophila S2 cells, expression of FLuc was significantly reduced. In contrast, when this reporter was coexpressed with miR-279a, a miRNA not predicted to bind to the CamKII 3'UTR, no repression was observed. To confirm that repression of the FLuc-CamKII reporter by miR-289 was via a specific interaction, the second of two predicted miR-289 binding sites was mutagenized. Binding site 2 (BS2) was a stronger candidate for regulation because it is flanked by AU-rich elements (AREs) and miR-289 has been shown to promote ARE-mediated mRNA instability through these sequences. Moreover, it is well established that the stabilization and destabilization of neuronal mRNAs via interactions between AREs and ARE-binding factors plays a significant role in the establishment and maintenance of long-term synaptic plasticity in both vertebrates and invertebrates. Altering three nucleotides within BS2 in the required seed region binding site was sufficient to significantly disrupt repression of the reporter by miR-289. The minimal predicted BS2 sequence was cloned into an unrelated 3'UTR and it was asked if miR-289 could repress translation. Coexpression of the FLuc-SV-mBS2 reporter with miR-289 led to significant repression. Taken together, these results indicate that the BS2 sequence is both necessary and sufficient for miR-289 regulation via the CamKII 3′UTR (Nesler, 2016).

    The most important conclusion of this study is that presynaptic CamKII is required to control activity-dependent axon terminal growth at the Drosophila larval NMJ. First, it was shown that CamKII is necessary to control ghost bouton formation in response to spaced synaptic depolarization. Next, it was demonstrated that spaced stimulation correlates with a rapid protein synthesis dependent increase in CamKII immunofluorescence in presynaptic boutons. This increase is suppressed by presynaptic overexpression of activity-regulated miR-289. Previous work has shown that overexpression of miR-289 in larval motor neurons can suppress activity-dependent axon terminal growth (Nesler, 2013). This study demonstrated that miR-289 can repress the translation of a FLuc-CamKII 3'UTR reporter via a specific interaction with a binding site within the CamKII 3'UTR ( Fig. 6C-E). Collectively, this experimental evidence suggests that CamKII functions downstream of the miRNA pathway to control activity-dependent changes in synapse structure. (Nesler, 2016).

    Thus, CamKII protein is expressed in the right place to regulate rapid events that are occurring within presynaptic boutons. Several questions remain regarding CamKII function in the control of activity-dependent axon terminal growth. First, it is unclear what the significance might be of a rapid increase of total CamKII in presynaptic terminals. Why is the pool of CamKII protein that is already present not sufficient to control these processes? Similar questions have been asked regarding activity-dependent processes occurring within dendrites. It is postulated that the CamKII mRNA might be locally translated in axon terminals. It has been proposed that local mRNA translation might be (1) required for efficient targeting of some synaptic proteins to specific sites, or (2) local translation may in and of itself be required to control activity-dependent processes at the synapse. Second, the impact of spaced depolarization on CamKII function needs to be assessed and downstream targets of CamKII phosphorylation involved in these processes need to be identified. One very strong candidate is synapsin which, at the Drosophila NMJ, has been shown to rapidly redistribute to sites of new ghost bouton outgrowth in response to spaced stimulation. Finally, the idea that CamKII might work through a Eag/CASK-dependent mechanism to control activity-dependent axon terminal growth needs to be examined (Nesler, 2016).

    The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila

    Using the Drosophila larval neuromuscular junction, this study shows a PI3K-dependent pathway for synaptogenesis which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, the functional hierarchy within the pathway was determined. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, an antagonistic pathway was identified that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways (Jordan-Alvarez, 2017).

    The epistasis assays have determined the in vivo functional links between PI3K and other previously known pro-synaptogenesis factors. Epistasis assays are based on the combined expression of two or more UAS constructs. Several double combinations in this study have produced a phenotype in spite of the apparent ineffectiveness of the single constructs. This type of results underscores the necessity to use epistasis assays in order to reveal functional interactions in vivo, hence, biologically relevant. In addition to the pro-synaptogenesis signaling, the study has revealed an anti-synaptogenesis pathway that composes a signaling equilibrium to determine the actual number of synapses. The magnitude of the synapse number changes elicited by the factors tested here are mostly within the range of 20%-50%. Are these values significant to cause behavioral changes? Reductions in the order of 30% of excitatory or inhibitory synapses in adult Drosophila local olfactory interneurons transform perception of certain odorants from attraction to repulsion and vice versa. In schizophrenia patients, a 16% loss of inhibitory synapses in the brain cortex has been reported. In Rhesus monkeys, the pyramidal neurons in layer III of area 46 in dorsolateral prefrontal cortex show a 33% spine loss, and a significant reduction in learning task performance during normal aging. Thus, it seems that behavior is rather sensitive to small changes in synapse number irrespective of the total brain mass (Jordan-Alvarez, 2017).

    The signaling interactions analyzed here were chosen because they were reported in other cellular systems and species previously. Some of these interactions have been confirmed (e.g., Gbb/Wit), while others have proven ineffective in the context of synaptogenesis (e.g., Ras85D/PI3K binding). Likely, the two signaling pathways, pro- and anti-synaptogenesis, are not the only ones relevant for synapse formation. For example, in spite of the null condition of the gbb and wit mutant alleles used here, the resulting synaptic phenotypes are far less extreme than expected if these two factors would be the only source of signaling for synaptogenesis. Although it could be argued that the incomplete absence of synapses in the mutant phenotypes could result from maternal perdurance, Wit is not part of the oocyte endowment while Gbb is. Three alternative possibilities may be considered, additional ligands for Wit, additional receptors for Gbb, and a combination of the previous two. Beyond the identity of these putative additional ligands and receptors, the stoichiometry between ligands and receptors may certainly be relevant. Actually, Gbb levels are titrated by Crimpy. An equivalent quantitative regulation could operate on Wit. The reported data on Wit illustrate already the diversity of the functional repertoire of this receptor. Wit can form heteromeric complexes with Thick veins (Tkv) or Saxophone (Sax) receptors to receive Dpp/BMP4 or Gbb/BMP7 as ligands. However, the same study also showed that Wit could dimerize with another receptor, Baboon, upon binding of Myoglianin to activate a different and antagonistic signaling pathway, TGFβ/activin-like (Jordan-Alvarez, 2017).

    The Gbb/Wit/PI3K signaling analyzed in this study is likely not the only pro-synaptogenesis pathway in flies and vertebrates. The ligand Wingless (Wg), member of the Wnt family, and the receptors Frizzled have been widely documented as relevant in neuromuscular junction development, albeit data on synapse number are scant. Interestingly, however, the downstream intermediaries can be as diverse as those mentioned above for Wit. Although generally depicted as linear pathways, a more realistic image would be a network of cross-interacting signaling events whose in situ regulation and cellular compartmentalization remains fully unexplored (Jordan-Alvarez, 2017).

    The quantitative regulation of receptors is most relevant to understand their biological effects. In that context, is worth noting that Tkv levels are distinctly regulated from those of Wit and Sax through ubiquitination in the context of neurite growth. On the other hand, although the receptor Wit is considered a RSTK type, the functional link with PI3K is a feature usually associated to the RTK type instead. The link of Wit with a kinase has a precedent with LIMK1 that binds to, and is functionally downstream from, Wit in the context of synapse stabilization. Thus, Wit should be considered a wide spectrum receptor in terms of its ligands, co-receptor partners and, consequently, signaling pathways elicited. Actually, the Wit amino acid sequence shows both, Tyr and Ser/Thr motifs justifying its initial classification as a 'dual' type of receptor. In this report this study did not determine if Wit heterodimerizes with other receptors, as canonical RSTKs do, or if it forms homodimers, as canonical RTKs do. However, the lack of synaptogenesis effects by the putative co-receptors, Tkv and Sax, and the phenotypic similarity with the manipulation of the standard RTK signaling effector Cic, leaves open the possibility that Wit could play RTK-like functions, at least in the context of synaptogenesis (Jordan-Alvarez, 2017).

    Consistent with the proposal of a dual mechanism for Wit, its activation seems to be a requirement to elicit two independent signaling steps, PI3K and Ras85D, that could reflect RTK and RSTK mechanisms, respectively. Both steps are independent because the mutated form of PI3K unable to bind Ras85D, PI3KΔRBD, is as effective as the normal PI3K to elicit synaptogenesis. PI3K and Ras85D signaling, however, seem to converge on Bsk revealing a novel feature of this crossroad point. The activity level of Bsk is known to be critical in many signaling processes. The peculiarities of Bsk/JNK activity include its coordinated regulation by p38a and Slpr in the context of stress heat response without interference on the developmental context. Another modulator, Puc, was described as a negative feed-back loop in the context of oxidative stress. The Puc mediated loop is operative also for synaptogenesis, while that of p38a/Slpr is relevant for p38a only, as shown here. Further, Ras85D represents an additional regulator in the neural scenario. The triple regulation of Bsk/JNK by Ras85D, Puc and the MAPKs seems to stablish a narrow range of activity thresholds within which normal number of synapses is determined (Jordan-Alvarez, 2017).

    The concept of signaling thresholds is also unveiled in this study by the identification of another signaling pathway that opposes synapse formation. The pro- and anti-synaptogenesis pathways have similar constituents, including small GTPases, MAPKs and transcriptional effectors, Mad/Smad, which are canonical for RSTK receptors. The RSTK type II receptor Put, which can mediate diverse signaling pathways according to the co-receptor bound can be discarded in either the pro- or the anti-synaptogenesis pathways. Thus, the main receptor for the anti-synaptogenesis pathway remains to be identified (Jordan-Alvarez, 2017).

    Concerning small GTPases, the pro-synaptogenesis pathway uses Ras85D while its counterpart uses the poorly studied Ras64B. The anti-synaptogenesis pathway includes an additional member of this family of enzymes, Rala. This small GTPase plays a role in the exocyst-mediated growth of the muscle membrane specialization that surrounds the synaptic bouton as a consequence of synapse activity. That is, Rala can influence synapse physiology acting from the postsynaptic side. The experimental expression of a constitutively active form of Rala in the neuron does not seem to affect the overall synaptic terminal branching. However, the null ral mutant shows reduced synapse branching and its vertebrate homolog is expressed in the central nervous system. This study found that Rala under-expression in neurons yields an elevated number of synapses. Thus, it is likely that this small GTPase acts as a break to synaptogenesis, hence its inclusion in the antagonistic pathway (Jordan-Alvarez, 2017).

    Synaptogenesis and neuritogenesis are distinct processes since each one can be differentially affected by the same mutant (e.g.: Hiw). Both features, however, share some signals (e.g., Wnd, Hep). This signaling overlap is akin to the case of axon specification versus spine formation for constituents of the apico/basal polarity complex Par3-6/aPKC. These and other examples illustrated in this study underscore the need to discriminate between synapses and boutons. This study is focused on the cell autonomous signaling that takes place in the neuron. Non-cell autonomous signals (e.g., originated in the glia or hemolymph circulating) have not been considered. The active role of glia in axon pruning and bouton number has been the subject of other studies. Considering the reported role of Hiw through the midline glia in the remodeling of the giant fiber interneuron it is not unlikely that the glia-to-neuron signaling may share components with the neuron autonomous signaling addressed here (Jordan-Alvarez, 2017).

    The summary scheme (see Summary diagram of antagonistic signaling pathways for synaptogenesis and their interactions) describes the scenario where two signaling pathways mutually regulate each other. Epistasis assays are the only experimental approach for in vivo studies of more than one signaling component, albeit this type of assay is only feasible in Drosophila Thus, it is plausible that vertebrate synaptogenesis will be regulated by a similar antagonistic signaling (Jordan-Alvarez, 2017).

    The regulatory equilibrium as a mechanism to determine a biological parameter is the most relevant feature in this scenario for several reasons. First, because this type of mechanism can respond very fast to changes in the physiological status of the cell, and, second because it provides remarkable precision to the trait to be regulated, synapse number in this case. Although bi-stable regulatory mechanisms are known in other contexts, the case of synapse number may seem unexpected because the highly dynamic nature of synapse number has been recognized only recently. Consequently, a molecular signaling mechanism endowed with proper precision and time resolution must sustain this dynamic process. The balanced equilibrium uncovered in this study, although most likely still incomplete in terms of its components, offers such a mechanism (Jordan-Alvarez, 2017).

    Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function

    Mutations in TBC1D24 cause severe epilepsy and DOORS syndrome, but the molecular mechanisms underlying these pathologies are unresolved. This study solved the crystal structure of the TBC domain of the Drosophila ortholog Skywalker, revealing an unanticipated cationic pocket conserved among TBC1D24 homologs. Cocrystallization and biochemistry showed that this pocket binds phosphoinositides phosphorylated at the 4 and 5 positions. The most prevalent patient mutations affect the phosphoinositide-binding pocket and inhibit lipid binding. Using in vivo photobleaching of Skywalker-GFP mutants, including pathogenic mutants, it was shown that membrane binding via this pocket restricts Skywalker diffusion in presynaptic terminals. Additionally, the pathogenic mutations cause severe neurological defects in flies, including impaired synaptic-vesicle trafficking and seizures, and these defects are reversed by genetically increasing synaptic PI(4,5)P2 concentrations through synaptojanin mutations. Hence, this study has discovered that a TBC domain affected by clinical mutations directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function (Fischer, 2016).

    Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

    Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1 (RhoGAP100F), neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function (McDermott, 2014).

    Localized translation is a widespread and evolutionarily ancient strategy used to temporally and spatially restrict specific proteins to their site of function and has been extensively studied during early development and in polarized cells in a variety of model systems. It is thought to be of particular importance in the regulation of neuronal development and in the plastic changes at neuronal synapses that underlie memory and learning, allowing rapid local changes in gene expression to occur independently of new transcriptional programs. The Drosophila neuromuscular junction (NMJ) is an excellent model system for studying the general molecular principles of the regulation of synaptic development and plasticity. Genetic or activity-based manipulations of synaptic translation at the NMJ has previously been shown to affect the morphological and electrophysiological plasticity of NMJ synapses. However, neither the mRNA targets nor the molecular mechanism by which such translational regulation occurs are fully understood (McDermott, 2014).

    Previous work identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, which was named Syncrip (Syp). Mammalian SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that contain CamKIIα, Arc, and IP3R1 mRNAs and is thought to regulate translation via an interaction with the noncoding RNA BC200/BC1, itself a translational repressor. Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro and regulates dendritic morphology via association with, and localization of, mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. It was previously shown that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation. Furthermore, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott, 2014).

    This study shows that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 (nrx-1), futsch, highwire (hiw), discs large 1 (dlg1), and α-spectrin (α-spec). The protein levels of a number of these mRNA targets, including msp-300 and dlg1, are significantly affected in syp null mutants. Furthermore, in addition to regulating MSP-300 protein levels, Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphology that resemble those observed in msp-300 mutants. It is proposed that Syp coordinates the protein levels from a number of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Drosophila NMJ (McDermott, 2014).

    The results demonstrate that Syp is required for the appropriate branching of the motoneurons and the number of synapses they make at the muscle. These observations are potentially explained by the finding that Syp is also required for the correct level of expression of msp-300, dlg1 and other mRNA targets. Given that it was previously shown that Syp regulates mRNA localization and localized translation in the Drosophila oocyte, and studies by others have shown that mammalian SYNCRIP/hnRNPQ inhibits translation initiation by competitively binding poly(A) sequences, these functions of Syp as occurring at the level of translational regulation of the mRNAs to which Syp binds. The data are also consistent with other work in mammals showing that SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that can regulate dendritic morphology via the localized expression of mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex (McDermott, 2014 and references therein).

    Translation at the Drosophila NMJ is thought to provide a mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in the surface area of body wall muscles or in response to changes in larval locomotion. The phenotypes observed in this study resemble, and are comparable to, those seen when subsynaptic translation is altered genetically or by increased locomotor activity. In syp null mutants, NMJ synaptic terminals are overgrown, containing more branches and synaptic boutons. Similarly, bouton numbers are increased by knocking down Syp in the muscle using RNAi. In contrast, overexpression of Syp in the muscle has the opposite phenotype, resulting in an inhibition of synaptic growth and branching. Furthermore, expressing RNAi against syp in motoneurons alone does not result in a change in NMJ morphology, indicating that Syp acts postsynaptically in muscle, but not presynaptically at the NMJ to regulate morphology. Interestingly, pan-neuronal syp knockdown or overexpression using Elav-GAL4 also results in NMJ growth defects, revealing that some of the defects observed in the syp null mutant may be attributed to Syp function in neuronal cell types other than the motoneurons, such as glial cells, which are known to influence NMJ morphology. Finally, while Syp is not required in the motoneuron to regulate synapse growth and is not detected in the motoneuron, the possibility cannot be excluded that Syp is present at low levels in the presynapse and regulates processes independent of synapse morphology. A further detailed characterization of the cell types and developmental stages in which Syp is expressed and functions is required to better understand the complex phenotypes that were observe (McDermott, 2014).

    RNA binding proteins have emerged as critical regulators of both neuronal morphology and synaptic transmision, suggesting that protein production modulates synapse efficacy. Consistent with this, it has been shown in a parallel study that Syp is required for proper synaptic transmission and vesicle release and regulates the presynapse through expression of retrograde Bone Morphogenesis Protein (BMP) signals in the postsynapse. In this role, Syp may coordinate postsynaptic translation with presynaptic neurotransmitter release. These observations provide a good explanation for how Syp influences the presynapse despite being only detectable in the postsynapse. This study has shown that Syp associates with a large number of mRNAs within third instar larvae, many of which encode proteins with key roles in synaptic growth and function. Syp mRNA targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein levels in the larval body wall but positively regulates MSP-300 and Nrx-1 protein levels. Dysregulation of these multiple mRNA targets likely accounts for the phenotypes that were observed. Postsynaptically expressed targets with key synaptic roles that could explain the synaptic phenotypes that were observed in syp alleles include MSP-300, α-Spec, and DLG. For example, mutants in dlg1 and mutants where postsynaptic DLG is destabilized or delocalized have NMJ morphology phenotypes similar to those observed upon overexpression of Syp in the muscle. Presynaptically expressed targets include syd-1, nrx-1, and hiw. However, this study has shown that syp knockdown in presynaptic motoneurons does not result in any defects in NMJ morphology. The RIP-Seq experiments were carried out using whole larvae and will, therefore, identify Syp targets in a range of different tissues and cells, the regulation of which may or may not contribute to the phenotype that were observed in syp mutants. It is, therefore, possible that Syp associates with these presynaptic targets in other neuronal cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are expressed and required postsynaptically in the muscle, but this has not been definitively determined. syp alleles may provide useful tools to examine where key synaptic genes are expressed and how they are regulated (McDermott, 2014).

    The identity of localized mRNAs and the mechanism of localized translation at the NMJ are major outstanding questions in the field. To date, studies have shown that GluRIIA mRNA aggregates are distributed throughout the muscle. The Syp targets identified in this study, such as msp-300, hiw, nrx-1, α-spec, and dlg1, are now excellent candidates for localized expression at the NMJ. Ultimately, conclusive demonstration of localized translation will involve the visualization of new protein synthesis of targets during activity-dependent synaptic plasticity. Biochemical experiments will also be required to establish the precise mode of binding of Syp to its downstream mRNA targets, the basis for interaction specificity, and the molecular mechanism by which Syp differentially regulates the protein levels of its mRNA targets at the Drosophila NMJ. Despite the fact that mammalian SYNCRIP is known to associate with poly(A) tails, this study and other published work have revealed that Syp can associate with specific transcripts. How Syp associates with specific mRNAs is unknown, and future studies are needed to uncover whether the interaction of Syp with specific transcripts is dictated by direct binding of the three Syp RRM RNA binding domains or by binding to other specific mRNA binding proteins. It is also possible that specific mRNA stem–loops, similar to the gurken localization signal, are required for Syp to bind to its mRNA targets (McDermott, 2014).

    This study shows that msp-300 is the most significant mRNA target of Syp. MSP-300 is the Drosophila ortholog of human Nesprin proteins. These proteins have been genetically implicated in various human myopathies. For example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated with Emery-Dreifuss muscular dystrophy (EDMD) as well as severe cardiomyopathies. Moreover, Syp itself is increasingly linked with factors and targets that can cause human neurodegenerative disorders. Recent work has revealed that SYNCRIP/hnRNPQ and Fragile X mental retardation protein (FMRP) are present in the same mRNP granule, and loss of expression of FMRP or the ability of FMRP to interact with mRNA and polysomes can cause cases of Fragile X syndrome. Separate studies have also shown that SYNCRIP interacts with wild-type survival of motor neuron (SMN) protein but not the truncated or mutant forms found to cause spinal muscular atrophy, and Syp genetically interacts with Smn mutations in vivo. Understanding Syp function in the regulation of such diverse and complex targets may, therefore, provide new avenues for understanding the molecular basis of complex disease phenotypes and potentially lead to future therapeutic approaches (McDermott, 2014).

    The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function

    Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these structures are shaped remain poorly understood. This study shows that the conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for the normal assembly of the subsynaptic muscle membrane reticulum (SSR) at the Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced glutamate receptor levels, and a deficit in synaptic homeostasis. The membrane-remodeling proteins Amphiphysin and Syndapin colocalize with Past1 in distinct SSR subdomains, and collapse into Amphiphysin-dependent membrane nodules in the SSR of past1 mutants. These results suggest a mechanism by which the coordinated actions of multiple lipid-binding proteins lead to the elaboration of increasing layers of the SSR, and uncover new roles for an EHD protein at synapses (Koles, 2015).

    Dozens of lipid-binding proteins dynamically remodel membranes, generating diverse cell shapes, sculpting organelles, and promoting traffic between subcellular compartments. Although the activities of many of these membrane-remodeling proteins have been studied individually, what is lacking is an understanding of how membrane-remodeling factors work together to generate specialized membranes in vivo (Koles, 2015).

    C-terminal Eps15 Homology Domain (EHD)-family proteins encode large membrane-binding ATPases with structural similarity to dynamin and function at a variety of steps of membrane transport (Naslavsky, 2011). These proteins contain an ATPase domain, a helical lipid-binding domain, and a carboxy-terminal EH domain that interacts with Asn-Pro-Phe (NPF)-containing binding partners (Naslavsky, 2011). Although their mechanism of action is not fully understood, it is postulated that C-terminal EHD proteins bind and oligomerize in an ATP-dependent manner on membrane compartments, where they are involved in the trafficking of cargo. The mouse and human genomes each contain four highly similar EHD proteins (EHD1-4), which have both unique and overlapping functions. EHD proteins interact with several members of the Bin/Amphiphysin/Rvs167 (BAR) and Fes/Cip4 homology-BAR (F-BAR) protein families, which themselves can remodel membranes via their crescent-shaped dimeric BAR domains. In mammals, EHD proteins associate with the NPF motifs of the F-BAR proteins Syndapin I and II, and these interactions are critical for recycling of cargo from endosomes to the plasma membrane in cultured cells. In Caenorhabditis elegans, the sole EHD protein Rme-1 colocalizes and functions with the BAR protein Amphiphysin and the F-BAR protein Syndapin, also via their NPF motifs. Further, EHD1 has been suggested to drive the scission of endosomal recycling tubules generated by the membrane-deforming activities of Syndapin 2 and another NPF-containing protein, MICAL-L1. However, the combined membrane-remodeling activities that might arise in vivo from the shared functions of C-terminal EHD and NPF-containing proteins remain unclear (Koles, 2015).

    The Drosophila neuromuscular junction (NMJ) is a powerful system in which to study membrane remodeling. On the postsynaptic side of the NMJ, a highly convoluted array of muscle membrane infoldings called the subsynaptic reticulum (SSR) incorporates neurotransmitter receptors, ion channels, and cell adhesion molecules. Assembly of the SSR during larval growth involves activity-dependent targeted exocytosis mediated by the small GTPase Ral and its effector, the exocyst complex, as well as the t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) receptor gtaxin/Syx18 and scaffolding proteins such as Discs Large (Dlg). Many proteins with predicted membrane-remodeling activities, including Drosophila homologues of Syndapin (Synd) and Amphiphysin (Amph), localize extensively to SSR membranes, making them prime candidates to facilitate SSR elaboration. Amph regulates the postsynaptic turnover of the trans-synaptic cell adhesion molecule FasII, but its role in organizing the SSR is unknown (Koles, 2015).

    The Drosophila melanogaster genome encodes a single C-terminal EHD protein called Putative achaete/scute target (Past1). Past1 mutants exhibit defects in endocytic recycling in larval nephrocytes, sterility and aberrant development of the germline, and short lifespan (Olswang-Kutz, 2009), but the functions of Past1 at the NMJ have not been explored. Mammalian EHD1 localizes to the mouse NMJ, but its function there has been difficult to ascertain, perhaps due to redundancy with other EHD proteins. This study takes advantage of the fact that Past1 encodes the only Drosophila C-terminal EHD protein and define its role at the NMJ (Koles, 2015).

    Putting together the current observations at the NMJ and in S2 cells with previous results from other groups, a new working model is proposed for how Past1 functions in synaptic membrane elaboration. The first key observation is that Past1 is required for normal elaboration of the SSR and that this function depends on its ATP-binding and thus membrane-remodeling activity. Next, in wild-type SSR, Amph was found to localize to a domain proximal to the bouton, whereas Past1 and Synd localize to a more extended tubulovesicular domain. By contrast, in the absence of Past1, the SSR rearranges into highly organized subdomains, with a core of Synd surrounded by a shell of Amph (likely corresponding to membrane sheets. Amph was found to be required for the formation of the sheets (perhaps by regulating the tight curvature at the tips of these membrane structures) and for consolidation of Synd into nodules. Further, FRAP data indicate that the nodules result in significantly increased membrane flow within the SSR relative to wild-type SSR, suggesting reduced complexity. Finally, S2 cell data indicate that Past1 may activate the membrane-binding/remodeling activity of Synd (Koles, 2015).

    These results suggest a novel mechanism for SSR elaboration at the wild-type NMJ involving sequential steps of membrane remodeling. In this model, Amph localizes and generates membrane tubules proximal to the bouton, and Past1 and Synd work together to further elaborate the tubules distal to the bouton. Successive rounds of these events could lead to the growth and expansion of layers of reticulum. In past1 mutants, this process is severely compromised, resulting in nodules containing a core of inactive Synd packed by Amph-dependent membrane sheets (Koles, 2015).

    One issue that remains to be resolved is whether direct physical interactions among Past1, Amph, and Synd (within the SSR subdomains to which they colocalize) contribute to Past1-dependent membrane remodeling at the NMJ, as they do in other systems. S2 cell data suggest that Past1 and Synd functionally interact in vivo. However, no Past1-Amph or Past1-Synd complexes were detected using coprecipitation experiments in extracts from Drosophila larvae or S2 cells or with purified proteins, suggesting that either they do not directly interact or their interactions are not preserved in solution under the conditions tested. Genetic experiments at the NMJ using mutations that disrupt putative Past1-Synd and Past1-Amph interactions are unlikely to be informative because synd and amph single mutants exhibit no dramatic phenotype in SSR organization, perhaps due to redundancy with other membrane-remodeling proteins. In fact, in addition to Amph and Synd, it was found that the BAR proteins Cip4 and dRich are localized to nodules in past1 mutants, suggesting that multiple membrane-remodeling proteins are available to function in the Past1-dependent pathway. In the future, it will be important to build into a working model the additional roles of these and other SSR-localized membrane-remodeling proteins, as well as the timing of exocyst-dependent membrane addition (Koles, 2015 and references therein).

    The results demonstrate that postsynaptic Past1 plays critical roles in the structure and function of the Drosophila NMJ. Past1 mutant NMJs exhibit aberrant morphology and excess ghost boutons. These ghost boutons are unlikely to be due to defective clearance of excess neuronal membrane, since large amounts of neuronal debris were not observed. They are also unlikely to be related to excess ghost boutons seen in Wingless (Wg) signaling pathway mutants, since past1 mutants do not phenocopy many other aspects of reduced Wg signaling, including increased GluR levels, disrupted presynaptic function, and reduction in bouton number. The likeliest interpretation is that Past1 functions directly in SSR membrane elaboration, consistent with EM observations, and ghost boutons may arise when membrane nodules become too severe to allow SSR assembly around boutons that form toward the end of larval development (Koles, 2015).

    Another prominent synaptic phenotype that found in past1 mutants is a strong and specific reduction in localization of GluRIIA to postsynaptic specializations, resulting in decreased mEPSP amplitude. This decrease in GluRIIA could potentially arise by many mechanisms, including altered transcriptional or translational regulation or GluR traffic to or from the synapse. Indeed, expression of a dominant-negative EHD1 suppresses AMPA glutamate receptor recycling in hippocampal dendritic spines. Although there has been little evidence that Drosophila GluRs are regulated by membrane traffic, the data implicating the membrane-remodeling protein Past1 indicate that this may be the case. Finally, unlike the great majority of perturbations that reduce GluRIIA levels, past1 mutants surprisingly fail to compensate for this loss by homeostatic up-regulation of presynaptic release, suggesting that Past1 could be involved in relaying an as-yet-unidentified retrograde signal for synaptic homeostasis. Further work exploring mechanisms of GluRIIA regulation and retrograde signaling will be required to understand the role of Past1 in these events (Koles, 2015).

    The present data cannot distinguish whether the function of Past1 in GluR traffic or homeostasis is directly related to its role in SSR elaboration, and it is possible that membrane compartments independent of the SSR are required for these functions and are disrupted in the mutant. The finding that GluRIIA levels are still reduced in amph; past1 double mutants although SSR nodules are suppressed supports the conclusion that GluR localization defects are independent of aberrant SSR morphogenesis. Of note, many mutants with severely defective SSR and/or reduced GluR levels exhibit normal homeostasis (e.g., GluRIIA, which also has reduced SSR, Dlg, Gtaxin, and Pak1, suggesting that homeostasis is a specific function of Past1 rather than a general SSR- or GluR-related defect (Koles, 2015).

    Past1 represents the sole EHD homologue in Drosophila, whereas mammals express four EHD proteins with distinct functions. Of importance, many of the roles for EHD proteins at the NMJ and in muscle are likely to be conserved. Past1 localizes to the NMJ, the muscle cortex, and myotendinous junctions. However, unlike EHD1, Past1 does not significantly localize to t-tubules. The activities identified for Past1 at the Drosophila NMJ may inform mechanisms by which EHD2 participates in sarcolemmal repair at the muscle cortex, EHD3 functions in cardiac muscle physiology , and EHD1 and EHD4 act at the mouse NMJ (Mate, 2012). The current findings set the stage for uncovering how neuromuscular synapses are formed and elaborated and illustrate how cooperation between lipid-remodeling proteins can create highly complex membrane structures (Koles, 2015).

    Mucin-type core 1 glycans regulate the localization of neuromuscular junctions and establishment of muscle cell architecture in Drosophila

    T antigen (Galβ1-3GalNAcα1-Ser/Thr), a core 1 mucin-type O-glycan structure, is synthesized by Drosophila core 1 β1,3-galactosyltrasferase 1 (dC1GalT1) and is expressed in various tissues. dC1GalT1 synthesizes T antigen expressed in hemocytes, lymph glands, and the central nervous system (CNS) and dC1GalT1 mutant larvae display decreased numbers of circulating hemocytes and excessive differentiation of hematopoietic stem cells in lymph glands. dC1GalT1 mutant larvae have also been shown to have morphological defects in the CNS. However, the functions of T antigen in other tissues remain largely unknown. This study found that glycans contributed to the localization of neuromuscular junction (NMJ) boutons. In dC1GalT1 mutant larvae, NMJs were ectopically formed in the cleft between muscles 6 and 7 and connected with these two muscles. dC1GalT1 synthesized T antigen, which was expressed at NMJs. In addition, the function of mucin-type O-glycans in muscle cells was determined. In dC1GalT1 mutant muscles, myofibers and basement membranes were disorganized. Moreover, ultrastructural defects in NMJs and accumulation of large endosome-like structures within both NMJ boutons and muscle cells were observed in dC1GalT1 mutants. Taken together, these results demonstrated that mucin-type O-glycans synthesized by dC1GalT1 were involved in the localization of NMJ boutons, synaptogenesis of NMJs, establishment of muscle cell architecture, and endocytosis (Itoh, 2016).

    Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction

    Assembly, maintenance and function of synaptic junctions depend on extracellular matrix (ECM) proteins and their receptors. This study reports that Tenectin (Tnc), a Mucin-type protein with RGD motifs, is an ECM component required for the structural and functional integrity of synaptic specializations at the neuromuscular junction (NMJ) in Drosophila. Using genetics, biochemistry, electrophysiology, histology and electron microscopy, this study shows that Tnc is secreted from motor neurons and striated muscles and accumulates in the synaptic cleft. Tnc selectively recruits alphaPS2/betaPS integrin at synaptic terminals, but only the cis Tnc/integrin complexes appear to be biologically active. These complexes have distinct pre- and post-synaptic functions, mediated at least in part through the local engagement of the spectrin-based membrane skeleton: the presynaptic complexes control neurotransmitter release, while postsynaptic complexes ensure the size and architectural integrity of synaptic boutons. This study reveals an unprecedented role for integrin in the synaptic recruitment of spectrin-based membrane skeleton (Wang, 2018).

    The extracellular matrix (ECM) and its receptors impact every aspect of neuronal development, from axon guidance and migration to formation of dendritic spines and neuromuscular junction synaptic junctions and function. The heavily glycosylated ECM proteins provide anchorage and structural support for cells, regulate the availability of extracellular signals, and mediate intercellular communications. Transmembrane ECM receptors include integrins, syndecans and the dystrophin-associated glycoprotein complex. Integrins in particular are differentially expressed and have an extensive repertoire, controlling multiple processes during neural development. In adults, integrins regulate synaptic stability and plasticity. However, integrin roles in synapse development have been obscured by their essential functions throughout development. How integrins are selectively recruited at synaptic junctions and how they engage in specific functions during synapse development and homeostasis remain unclear (Wang, 2018).

    One way to confer specificity to ECM/integrin activities is to deploy specialized ECM ligands for the synaptic recruitment and stabilization of selective heterodimeric integrin complexes. For example, at the vertebrate NMJ, three laminins containing the β2 subunit (laminin 221, 421 and 521, that are heterotrimers of α2/4/5, β2 and γ1 subunits) are deposited into the synaptic cleft and basal lamina by skeletal muscle fibers and promote synaptic differentiation. However, only laminin 421 interacts directly with presynaptic integrins containing the α3 subunit and anchors a complex containing the presynaptic Cavα and cytoskeletal and active zone-associated proteins. Studies with peptides containing the RGD sequence, recognized by many integrin subtypes, have implicated integrin in the morphological changes and reassembly after induction of long-term potentiation (LTP). Several integrin subunits (α3, α5, α8, β1 and β2) with distinct roles in the consolidation of LTP have been identified, but the relevant ligands remain unknown (Wang, 2018).

    Drosophila neuromuscular junction (NMJ) is a powerful genetic system to examine the synaptic functions of ECM components and their receptors. In flies, a basal membrane surrounds the synaptic terminals only in late embryos; during development, the boutons ‘sink’ into the striated muscle, away from the basal membrane. The synaptic cleft relies on ECM to withstand the mechanical tensions produced by the muscle contractions. The ECM proteins, including laminins, tenascins/teneurins (Ten-a and -m) and Mind-the-gap (Mtg), interact with complexes of five integrin subunits (αPS1, αPS2, αPS3, βPS, and βν). The αPS1, αPS2 and βPS subunits localize to pre- and post-synaptic compartments and have been implicated in NMJ growth. The αPS3 and βν are primarily presynaptic and control activity-dependent plasticity. The only known integrin ligand at the fly NMJ is Laminin A, which is secreted from the muscle and signals through presynaptic αPS3/βν and Focal adhesion kinase 56 (Fak56) to negatively regulate the activity-dependent NMJ growth. Teneurins have RGD motifs, but their receptor specificities remain unknown. Mtg secreted from the motor neurons influences postsynaptic βPS accumulation, but that may be indirectly due to an essential role for Mtg in the organization of the synaptic cleft and the formation of the postsynaptic fields. The large size of these proteins and the complexity of ECM-integrin interactions made it difficult to recognize relevant ligand-receptor units and genetically dissect their roles in synapse development (Wang, 2018).

    This study reports the functional analysis of Tenectin (Tnc), an integrin ligand secreted from both motor neurons and muscles; Tnc accumulates at synaptic terminals and functions in cis to differentially engage presynaptic and postsynaptic integrin. tnc, which encodes a developmentally regulated RGD-containing integrin ligand, in a screen for ECM candidates that interact genetically with neto, a gene essential for NMJ assembly and function. This study found that Tnc selectively recruits the αPS2/βPS integrin at synaptic locations, without affecting integrin anchoring at muscle attachment sites. Dissection of Tnc functions revealed pre- and postsynaptic biologically active cis Tnc/integrin complexes that function to regulate neurotransmitter release and postsynaptic architecture. Finally, the remarkable features of this selective integrin ligand were explored to uncover a novel synaptic function for integrin, in engaging the spectrin-based membrane skeleton (Wang, 2018).

    The ECM proteins and their receptors have been implicated in NMJ development, but their specific roles have been difficult to assess because of their early development functions and the complexity of membrane interactions they engage. This study has shown that Tnc is a selective integrin ligand that enables distinct pre- and post-synaptic integrin activities mediated at least in part through the local engagement of the spectrin-based cortical skeleton. First, Tnc depletion altered NMJ development and function and correlated with selective disruption of αPS2/βPS integrin and spectrin accumulation at synaptic terminals. Second, manipulation of Tnc and integrin in neurons demonstrated that presynaptic Tnc/integrin modulate neurotransmitter release; spectrin mutations showed similar disruptions of the presynaptic neurotransmitter release. Third, postsynaptic Tnc influenced the development of postsynaptic structures (bouton size and SSR complexity), similar to integrin and spectrin. Fourth, presynaptic Tnc/integrin limited the accumulation and function of postsynaptic Tnc/integrin complexes. Fifth, secreted Tnc bound integrin complexes at cell membranes, but only the cis complexes were biologically active; trans Tnc/integrin complexes can form but cannot function at synaptic terminals and instead exhibited dominant-negative activities. These observations support the model that Tnc is a tightly regulated component of the synaptic ECM that functions in cis to recruit αPS2/βPS integrin and the spectrin-based membrane skeleton at synaptic terminals and together modulate the NMJ development and function (Wang, 2018).

    Tnc appears to fulfill unique, complementary functions with the other known synaptic ECM proteins at the Drosophila NMJ. Unlike Mtg, which organizes the active zone matrix and the postsynaptic domains, Tnc does not influence the recruitment of iGluRs and other PSD components. LanA ensures a proper adhesion between the motor neuron terminal and muscle and also acts retrogradely to suppress the crawling activity-dependent NMJ growth. The latter function requires the presynaptic βν integrin subunit and phosphorylation of Fak56 via a pathway that appears to be completely independent of Tnc. Several more classes of trans-synaptic adhesion molecules have been implicated in either the formation of normal size synapses, for example Neurexin/Neuroligin, or in bridging the pre- and post-synaptic microtubule-based cytoskeleton, such as Teneurins. However, genetic manipulation of Tnc did not perturb synapse assembly or microtubule organization, indicating that Tnc functions independently from these adhesion molecules. Instead, Tnc appears to promote expression and stabilization of αPS2/βPS complexes, which in turn engage the spectrin-based membrane skeleton (SBMS) at synaptic terminals. On the presynaptic side these complexes modulate neurotransmitter release. On the postsynaptic side, the Tnc-mediated integrin and spectrin recruitment modulates bouton morphology. A similar role for integrin and spectrin in maintaining tissue architecture has been reported during oogenesis; egg chambers with follicle cells mutant for either integrin or spectrin produce rounder eggs (Wang, 2018).

    The data are consistent with a local function for the Tnc/βPS-recruited SBMS at synaptic terminals; this is distinct from the role of spectrin in endomembrane trafficking and synapse organization. Embryos mutant for spectrins have reduced neurotransmitter release, a phenotype shared by larvae lacking presynaptic Tnc or βPS integrin. However, Tnc perturbations did not induce synapse retraction and axonal transport defects as seen in larvae with paneuronal α- or β- spectrin knockdown. Spectrins interact with ankyrins and form a lattice-like structure lining neuronal membranes in axonal and interbouton regions. This study found that Tnc manipulations did not affect the distribution of Ankyrin two isoforms (Ank2-L and Ank2-XL) in axons or at the NMJ; also loss of ankyrins generally induces boutons swelling, whereas Tnc perturbations shrink the boutons and erode bouton-interbouton boundaries. Like tnc, loss of spectrins in the striated muscle shows severe defects in SSR structure. Lack of spectrins also disrupts synapse assembly and the recruitment of glutamate receptors. In contrast, manipulations of tnc had no effect on PSD size and composition. Instead, tnc perturbations in the muscle led to boutons with altered size and individualization and resembled the morphological defects seen in spectrin tetramerization mutants, spectrinR22S. spectrinR22S mutants have more subtle defects than tnc, probably because spectrin is properly recruited at NMJs but fails to crosslink and form a cortical network. Spectrins are also recruited to synaptic locations by Teneurins, a pair of transmembrane molecule that form trans-synaptic bridges and influence NMJ organization and function. Drosophila Ten-m has an RGD motif; this study found that βPS levels were decreased by 35% at ten-mMB mutant NMJs. Thus, Ten-m may also contribute to the recruitment of integrin and SBMS at the NMJ, a function likely obscured by the predominant role both play in cytoskeleton organization (Wang, 2018).

    Previous work has shown that α-Spectrin is severely disrupted at NMJs with suboptimal levels of Neto, such as neto109- a hypomorph with 50% lethality. These mutants also had sparse SSR, reduced neurotransmitter release, as well as reduced levels of synaptic βPS. In this genetic background, lowering the dose of tnc should further decrease the capacity to accumulate integrin and spectrin at synaptic terminals and enhance the lethality. This may explain the increased synthetic lethality detected in the genetic screen (Wang, 2018).

    In flies or vertebrates, the ECM proteins that comprise the synaptic cleft at the NMJ are not fully present when motor neurons first arrive at target muscles. Shortly thereafter, the neurons, muscles and glia begin to synthesize, secrete and deposit ECM proteins. At the vertebrate NMJ, deposition of the ECM proteins forms a synaptic basal lamina that surrounds each skeletal myofiber and creates a ~ 50 nm synaptic cleft. In flies, basal membrane contacts the motor terminal in late embryos, but is some distance away from the synaptic boutons during larval stages. Nonetheless, the NMJ must withstand the mechanical tensions produced by muscle contractions. The current data suggest that Tnc is an ideal candidate to perform the space filling, pressure inducing functions required to engage integrin and establish a dynamic ECM-cell membrane network at synaptic terminals. First, Tnc is a large mucin with extended PTS domains that become highly O-glycosylated, bind water and form gel-like complexes that can extend and induce effects similar to hydrostatic pressure. In fact, Tnc fills the lumen of several epithelial tubes and forms a dense matrix that acts in a dose-dependent manner to drive diameter growth. Second, the RGD and RGD-like motifs of Tnc have been directly implicated in αPS2/βPS-dependent spreading of S2 cells (Fraichard, 2010). Third, secreted Tnc appears to act close to the source, presumably because of its size and multiple interactions. In addition to the RGD motifs, Tnc also contains five complete and one partial vWFC domains, that mediate protein interactions and oligomerization in several ECM proteins including mucins, collagens, and thrombospondins. The vWFC domains are also found in growth factor binding proteins and signaling modulators such as Crossveinless-2 and Kielin/Chordin suggesting that Tnc could also influence the availability of extracellular signals. Importantly, Tnc expression is hormonally regulated during development by ecdysone (Fraichard, 2010). Tnc does not influence integrin responsiveness to axon guidance cues during late embryogenesis; unlike integrins, the tnc mutant embryos have normal longitudinal axon tracks. Instead, Tnc synthesis and secretion coincide with the NMJ expansion and formation of new bouton structures during larval stages. Recent studies have reported several mucin-type O-glycosyltransferases that modulate integrin signaling and intercellular adhesion in neuronal and non-neuronal tissues, including the Drosophila NMJ. Tnc is likely a substrate for these enzymes that may further regulate Tnc activities (Wang, 2018).

    In flies as in vertebrates, integrins play essential roles in almost all aspects of synaptic development. Early in development, integrins have been implicated in axonal outgrowth, pathfinding and growth cone target selection. In adult flies, loss of αPS3 integrin activity is associated with the impairment of short-term olfactory memory. In vertebrates, integrin mediates structural changes involving actin polymerization and spine enlargement to accommodate new AMPAR during LTP, and ‘lock in’ these morphological changes conferring longevity for LTP. Thus far, integrin functions at synapses have been derived from compound phenotypes elicited by use of integrin mutants, RGD peptides, or enzymes that modify multiple ECM molecules. Such studies have been complicated by multiple targets for modifying enzymes and RGD peptides and by the essential functions of integrin in cell adhesion and tissue development (Wang, 2018).

    In contrast, manipulations of Tnc, which affects the selective recruitment of αPS2/βPS integrin at synaptic terminals, have uncovered novel functions for integrin and clarified previous proposals. This study demonstrated that βPS integrin is dispensable for the recruitment of iGluRs at synaptic sites and for PSD maintenance. An unprecedented role was reveaked for integrin in connecting the ECM of the synaptic cleft with spectrin, in particular to the spectrin-based membrane skeleton. These Tnc/integrin/spectrin complexes are crucial for the integrity and function of synaptic structures. These studies uncover the ECM component Tnc as a novel modulator for NMJ development and function; these studies also illustrate how manipulation of a selective integrin ligand could be utilized to reveal novel integrin functions and parse the many roles of integrins at synaptic junctions (Wang, 2018).

    The HSPG glypican regulates experience-dependent synaptic and behavioral plasticity by modulating the non-canonical BMP pathway

    Under food deprivation conditions, Drosophila larvae exhibit increases in locomotor speed and synaptic bouton numbers at neuromuscular junctions (NMJs). Octopamine, the invertebrate counterpart of noradrenaline, plays critical roles in this process; however, the underlying mechanisms remain unclear. This study shows that a glypican (Dlp) negatively regulates type I synaptic bouton formation, postsynaptic expression of GluRIIA, and larval locomotor speed. Starvation-induced octopaminergic signaling decreases Dlp expression, leading to increases in synapse formation and locomotion. Dlp is expressed by postsynaptic muscle cells and suppresses the non-canonical BMP pathway, which is composed of the presynaptic BMP receptor Wit and postsynaptic GluRIIA-containing ionotropic glutamate receptor. During starvation, decreases in Dlp increase non-canonical BMP signaling, leading to increases in GluRIIA expression, type I bouton number, and locomotor speed. These results demonstrate that octopamine controls starvation-induced neural plasticity by regulating Dlp and provides insights into how proteoglycans can influence behavioral and synaptic plasticity (Kamimura, 2019).

    Food deprivation induces behavioral and synaptic plasticity in Drosophila larvae through octopamine signaling. This study demonstrates that octopamine signaling dynamically regulates Dlp expression at the NMJ during starvation. Dlp was found to inhibits postsynaptic GluRIIA expression, and starvation and octopamine function were found to reduce postsynaptic Dlp expression. The inhibition of Dlp results in increased GluRIIA expression, which this study proposes is maintained for several hours during starvation by the activation of the non-canonical BMP pathway. Increased GluRIIA expression causes increased type I bouton growth at the NMJ and increased starvation-dependent larval locomotion. It has previously been shown that during the non-starvation or normal development of NMJs, postsynaptic GluRIIA expression and type I bouton growth are regulated by distinct biochemical pathways (Sulkowski, 2016). GluRIIA/GluRIIB postsynaptic composition is regulated by non-canonical BMP signaling, while type I bouton growth is regulated independently by canonical BMP signaling. However, this study finds that during starvation-dependent growth, GluRIIA expression and type I bouton growth become interrelated. Starvation increases GluRIIA expression in a Dlp-dependent manner, and type I bouton growth requires GluRIIA expression. It is not certain why this change in regulation occurs, but it is noted that independent regulatory mechanisms allow more flexibility for NMJ formation during normal development, while starvation-dependent growth may simply require a quick and transient increase in synaptic activity that may not need to be as flexible. This study found that Dlp regulates starvation-dependent changes at least in part through the regulation of non-canonical BMP signaling. However, non-canonical BMP signaling is likely only one component of how Dlp regulates NMJ plasticity and larval locomotion. This study finds that GluRIIA amounts increase upon the suppression of Dlp even in wit null mutants, indicating that BMP signaling functions to maintain increases in GluRIIA, but not necessarily to induce it. Thus, Dlp must inhibit GluRIIA expression through a pathway that is independent of non-canonical BMP signaling. Furthermore, although starvation-dependent increases in type I boutons depend on Wit and GluRIIA, there is also a component to this increase that is Wit and GluRIIA independent. This suggests that Dlp may also regulate type I boutons through a GluRIIA-independent mechanism (Kamimura, 2019).

    Octopamine is required to maintain the basal expression levels of postsynaptic Dlp, and thus it positively regulates Dlp expression under normal rearing conditions. However, after food deprivation, octopamine signaling turns into a negative regulator of Dlp expression during the early phase of starvation. In the late phase of starvation, octopamine signaling reverts back to being a positive regulator. Koon (2012) and Koon (2011) revealed that type II bouton formation was not only positively regulated by Octβ2R autoreceptors but also negatively regulated by Octβ1R autoreceptors. To explain the acute growth of type II bouton growth after starvation, they postulated that Octβ1R exhibited a higher affinity for octopamine than did Octβ2R. Under normal rearing conditions, high-affinity Octβ1R may be dominantly activated by low concentrations of octopamine. Under this condition, low-affinity Octβ2R may be weakly activated. Thus, basal levels of type II bouton growth are determined by the balanced activities of these antagonistic octopamine receptors. After food deprivation, high concentrations of octopamine released from type II endings may strongly activate positive Octβ2R receptors, leading to the acute growth of type II boutons. A similar mechanism may be involved in the regulation of Dlp expression. One possibility is that high-affinity octopamine receptors (HOctRs) and low-affinity octopamine receptors (LOctRs) at type I synaptic regions positively and negatively, respectively, regulate the postsynaptic localization of Dlp. If this is the case, then the basal postsynaptic levels of Dlp may be affected by the dominant activation of positive HOctRs. In the early phase of starvation, during which high levels of octopamine are present, postsynaptic Dlp levels may be decreased by the activation of negative LOctRs. In the late phase of starvation, octopamine levels may be decreased by the exhaustion of octopaminergic neurons, leading to the dominant activation of HOctRs again. This may result in increases in postsynaptic Dlp expression. Thus, the flexible roles of octopamine signaling may be explained by differences in the octopamine receptors mainly activated in each phase. Future studies are needed to identify the octopamine receptors that regulate Dlp expression (Kamimura, 2019).

    It remains unclear how octopamine signaling at type I synaptic regions regulates postsynaptic Dlp levels. Broadie and collaborators revealed that ECM metalloproteinases (Mmp1 and Mmp2) and the tissue inhibitor of metalloproteinases (Timp) play critical roles in synapse formation and activity-dependent plasticity in the Drosophila NMJ (Dear, 2016; Dear, 2017; Shilts, 2017). Mmp2 from the presynaptic and postsynaptic compartments has been shown to negatively regulate synaptic Dlp levels at the NMJ, possibly by the proteolytic cleavage of Dlp core proteins (Wang, 2014). However, Mmp1 is known to associate with Dlp via heparan sulfate (HS) chains and positively regulate Dlp expression levels at type I endings (Dear, 2016). Octopamine signaling after food deprivation may activate the proteolytic activity of Mmp2 at type I synaptic regions, leading to a decrease in postsynaptic Dlp levels. Mmp 1 may be activated in the later phase of starvation, leading to an increase in postsynaptic Dlp levels. A previous study revealed that Timp regulated the activities of these MMPs at the NMJ to restrict trans-synaptic Gbb signaling (Shilts, 2017). Thus, complex cooperation among Mmps, Timp, and Dlp may be involved in the experience-dependent plasticity of the NMJ. The local translation of Dlp may also be involved, particularly in the later phase (Kamimura, 2019).

    This study has revealed that Dlp suppressed the GluRIIA-mediated non-canonical BMP signaling pathway, which forms a positive feedback loop between presynaptic pMad accumulation and postsynaptic type A iGluR expression (Sulkowski, 2016). Thus, decreases in Dlp expression after food deprivation may acutely promote this signaling pathway, leading to rapid increases in type A iGluR and pMad accumulation at the NMJ. However, Dlp expression was increased during the late phase of starvation, which may have suppressed this BMP pathway. A positive feedback loop may lead to a runaway condition with no brakes. In this sense, Dlp appears to critically regulate the levels of this GluRIIA-mediated BMP signaling as a brake during starvation. How, then, does Dlp regulate non-canonical BMP signaling? Since Dlp exclusively localizes at postsynaptic regions, it may directly associate with GluRIIA and destabilize type A iGluRs at synapses. After food deprivation, the postsynaptic expression levels of Dlp decrease, and thus type A iGluRs may be stabilized, leading to the promotion of presynaptic local Wit signaling and pMad accumulation. It is also conceivable that Dlp inhibits the trans-synaptic association between presynaptic BMP receptor complexes and postsynaptic type A iGluRs proposed by Sulkowski (2016). Many of the functions of HSPGs are mediated by HS moieties, which bind with various signaling molecules in a sulfation pattern-dependent manner. The development and function of the NMJ are known to be regulated in a complex manner by HS co-polymerase (encoded by ttv), N-deacetylase/N-sulfotransferase (encoded by sfl), HS 6-O-sulfotransferase, and extracellular sulfatase (Sulf1) (Kamimura, 2019).

    These findings suggest that various signaling pathways at the NMJ are intricately regulated by structural changes in HS chains. The structure of HS chains on Dlp may be altered under specific conditions such as starvation, and Dlp may selectively regulate a particular signaling pathway among multiple target pathways in an HS sulfation pattern-dependent manner. LAR is known to associate with Dlp and Sdc through HS chains at the developing NMJ, in which Dlp showed higher affinity for LAR than Sdc, possibly because of differences in HS structures. Presynaptic LAR is activated by postsynaptic Sdc, leading to type I bouton growth, whereas Dlp competitively inhibits this signaling. Therefore, the decrease observed in Dlp expression after food deprivation may disinhibit LAR signaling and promote type I bouton growth. In addition, Dlp has been shown to regulate Wg signaling through the HS moieties during NMJ development. Wg is secreted from presynaptic terminal and glia and participates in synaptic plasticity. Therefore, it is also possible that Wg and Dlp regulate the starvation-induced synaptic and behavioral plasticities. Furthermore, a recent study revealed that neurexin synaptic organizing proteins are expressed as HSPGs. HS modifications in presynaptic neurexins are critical for high-affinity interactions with the postsynaptic ligands neuroligins and the leucine-rich repeat transmembrane neuronal proteins (LRRTMs), and are essential for synapse development at the mouse hippocampus. Drosophila Neurexin (Dnrx) is also an HSPG and positively regulates larval locomotion speed and type I bouton growth at NMJs in an HS-dependent manner. Furthermore, it has been suggested that Dnrx, Neuroligin (Dnlg1), and Wit form signaling complexes at the NMJ. Therefore, presynaptic Dnrx and postsynaptic Dlp may competitively regulate type I bouton formation and larval locomotor activity as antagonistic HSPGs, in which the HS chains of Dlp may inhibit the Dnrx-Dnlg1 interaction in a sulfation pattern-dependent manner. Further studies are needed to examine whether these pathways and HS modifications regulate starvation-induced synaptic and behavioral plasticities (Kamimura, 2019).

    Only three type II octopaminergic neurons bilaterally innervate most of the body wall muscles in each abdominal segment, suggesting that type II octopaminergic neurons globally regulate the properties of type I terminals, coordinating the activities of the body wall musculature. This is similar to the mammalian brain noradrenergic system. Noradrenaline-producing neurons mainly cluster in a small nucleus called the locus coeruleus, which is located in the pons with global projections throughout the brain. The locus coeruleus noradrenergic system is activated by many stressors. For example, noradrenaline is released in many brain regions during emotional arousal and enhances long-term memory, an extreme example of which is post-traumatic stress disorder (PTSD). It has been shown that that noradrenaline facilitated the synaptic delivery of GluR1-containing AMPA receptors in the hippocampus, which lowered the threshold for long-term potentiation (LTP), thereby enhancing learning and memory. This is similar to the starvation-induced synaptic localization of type A iGluRs in the Drosophila NMJ, which is regulated by Dlp in an octopamine-dependent manner. Thus, further studies are needed to investigate whether the emotional enhancement of memory is regulated by glypican in the hippocampus. A previous study reported that astrocyte-secreted glypican 4 induced the release of the AMPA receptor clustering factor neuronal pentraxin 1 from presynaptic terminals through the signaling of RPTPδ, a LAR family receptor protein tyrosine phosphatase. This induced the formation of active synapses by recruiting AMPA receptors to postsynaptic sites. In addition, noradrenaline has been shown to activate astrocyte networks in the cortex and enhance their responsiveness to local changes in neuronal activity. Therefore, noradrenaline may stimulate the secretion of glypican 4 from astrocytes in a neuronal activity-dependent manner, thereby enhancing active synapse formation. In this case, the direction of glypican 4 activities appears to be opposite that of Dlp. However, glypicans often display opposing activities in a context-dependent manner. Thus, the interplay among Dlp, type A iGluR, LAR, and octopamine at the Drosophila NMJ may become a simple model to study the molecular mechanisms underlying emotion-enhanced memory formation and thus PTSD (Kamimura, 2019).

    In the adult mammalian brain, the synapses of a subset of neurons are enwrapped with a specialized chondroitin sulfate proteoglycan-rich ECM called the perineuronal net (PNN). PNNs are mainly composed of tenascin-R, hyaluronan, and lectican family proteoglycans such as aggrecan, versican, and neurocan. The ECM enwrapping type I synaptic boutons at the Drosophila larval NMJ is an HSPG-based ECM containing Dlp, syndecan, perlecan, laminin, and the secreted lectin Mind-the-Gap. The orthologs of hyaluronan synthase and lectican family proteoglycans are not found in Drosophila, and therefore an HSPG-based ECM may be used at the synapses of Drosophila. In spite of their different compositions, both synaptic ECMs play critical roles in experience-induced neural plasticity, as shown by the present study and previous studies on mammalian ocular dominance plasticity. This study has revealed that Dlp regulated the localization of type A iGluRs during starvation. PNNs also contribute to the synaptic localization of AMPA receptors. Detailed comparative studies are needed to reveal functional and structural similarities between the PNNs and the ECM of the Drosophila NMJ (Kamimura, 2019).

    Abnormalities in the expression of glypicans have been linked to various neurological disorders, such as autism spectrum disorders (ASDs), schizophrenia, attention-deficit/hyperactivity disorder (ADHD), and neuroticism. Dysfunctions in the noradrenergic system have also been reported in ASDs, schizophrenia, and ADHD. Thus, studies on the octopamine-mediated control of Dlp expression may contribute to a deeper understanding of the molecular mechanisms responsible for the pathogeneses of these disorders (Kamimura, 2019).

    Shank modulates postsynaptic wnt signaling to regulate synaptic development

    Prosap/Shank scaffolding proteins regulate the formation, organization, and plasticity of excitatory synapses. Mutations in SHANK family genes are implicated in autism spectrum disorder and other neuropsychiatric conditions. However, the molecular mechanisms underlying Shank function are not fully understood, and no study to date has examined the consequences of complete loss of all Shank proteins in vivo. This study characterized the single Drosophila Prosap/Shank family homolog. Shank is enriched at the postsynaptic membrane of glutamatergic neuromuscular junctions and controls multiple parameters of synapse biology in a dose-dependent manner. Both loss and overexpression of Shank result in defects in synaptic bouton number and maturation. It was found that Shank regulates a noncanonical Wnt signaling pathway in the postsynaptic cell by modulating the internalization of the Wnt receptor Fz2. This study identifies Shank as a key component of synaptic Wnt signaling, defining a novel mechanism for how Shank contributes to synapse maturation during neuronal development (Harris, 2016).

    The postsynaptic density (PSD) of excitatory synapses contains a complex and dynamic arrangement of proteins, allowing the cell to respond to neurotransmitter and participate in bidirectional signaling to regulate synaptic function. Prosap/Shank family proteins are multidomain proteins that form an organizational scaffold at the PSD. Human genetic studies have implicated SHANK family genes as causative for autism spectrum disorder (ASD) (Uchino and Waga, 2013; Guilmatre, 2014), with haploinsufficiency of SHANK3 considered one of the most prevalent causes (Betancur and Buxbaum, 2013). Investigations of Shank in animal models have identified several functions for the protein at synapses, including regulation of glutamate receptor trafficking, the actin cytoskeleton, and synapse formation, transmission, and plasticity (Grabrucker, 2011; Jiang and Ehlers, 2013). However, phenotypes associated with loss of Shank are variable, and it has been challenging to fully remove Shank protein function in vivo as a result of redundancy between three Shank family genes and the existence of multiple isoforms of each Shank. There is a single homolog of Shank in Drosophila (Liebl and Featherstone, 2008), presenting the opportunity to characterize the function of Shank at synapses in vivo in null mutant animals (Harris, 2016).

    Wnt pathways play important roles in synaptic development, function, and plasticity. Like Shank and several other synaptic genes, deletions and duplications of canonical Wnt signaling components have been identified in individuals with ASD. A postsynaptic noncanonical Wnt pathway has been characterized at the Drosophila glutamatergic neuromuscular junction (NMJ), linking release of Wnt by the presynaptic neuron to plastic responses in the postsynaptic cell. In this Frizzled-2 (Fz2) nuclear import (FNI) pathway, Wnt1/Wg is secreted by the neuron and binds its receptor Fz2 in the postsynaptic membrane. Surface Fz2 is then internalized and cleaved, and a C-terminal fragment of Fz2 (Fz2-C) is imported into the nucleus in which it interacts with ribonucleoprotein particles containing synaptic transcripts. Mutations in this pathway result in defects of synaptic development at the NMJ (Harris, 2016 and references therein).

    In this study, a null allele of Drosophila Shank was created, allowing investigation of the consequences of removing all Shank protein in vivo. Loss of Shank is shown to impair synaptic bouton number and maturity and results in defects in the organization of the subsynaptic reticulum (SSR), a complex system of infoldings of the postsynaptic membrane at the NMJ. It was also demonstrated that overexpression of Shank has morphological consequences similar to loss of Shank and that Shank dosage is critical to synaptic development. Finally, the results indicate that Shank regulates the internalization of Fz2 to affect the FNI signaling pathway, revealing a novel connection between the scaffolding protein Shank and synaptic Wnt signaling (Harris, 2016).

    By generating Drosophila mutants completely lacking any Shank protein, this study identified a novel function of this synaptic scaffolding protein in synapse development. Aberrant expression of Shank results in defects affecting synapse number, maturity, and ultrastructure, and a subset of these defects is attributable to a downregulation of a noncanonical Wnt signaling pathway in the postsynaptic cell (Harris, 2016).

    The defects observed in Shank mutants are mostly consistent with defects described from in vivo and in vitro rodent models of Shank. Synaptic phenotypes reported from Shank mutants vary, likely reflecting incomplete knockdown of Shank splice variants, and heterogeneity in the requirement for Shank between the different brain regions and developmental stages analyzed (for review, see Jiang and Ehlers, 2013). Nevertheless, taken collectively, analyses of Shank1-Shank3 mutant mice indicate that Shank genes regulate multiple parameters of the structure and function of glutamatergic synapses, including the morphology of dendritic spines and the organization of proteins in the PSD (Harris, 2016 and references therein).

    By removing all Shank protein in Drosophila, this study identified essential functions for Shank at a model glutamatergic synapse. Shank mutants exhibit prominent abnormalities in synaptic structure, including a decrease in the total number of synaptic boutons, which results in an overall decrease in the number of AZs. In addition, a subset of synaptic boutons fails to assemble a postsynaptic apparatus. Finally, even in mature boutons, the SSR has fewer membranous folds and makes less frequent contact with the presynaptic membrane, indicating a defect in postsynaptic development. The SSR houses and concentrates important synaptic components near the synaptic cleft, including scaffolding proteins, adhesion molecules, and glutamate receptors. Thus, defects in SSR development can affect the assembly and regulation of synaptic signaling platforms. These findings indicate that Shank is a key regulator of synaptic growth and maturation (Harris, 2016).

    The findings also indicate that gene dosage of Shank is critical for normal synapse development at Drosophila glutamatergic NMJs. The morphological phenotypes that were observed scale with the level of Shank expression, with mild phenotypes seen with both 50% loss and moderate overexpression of Shank, and severe phenotypes seen with both full loss and strong overexpression of Shank. The observation of synapse loss in heterozygotes of the Shank null allele is significant, because haploinsufficiency of SHANK3 is well established as a monogenic cause of ASD (Harris, 2016).

    Consistent with the observation that excess Shank is detrimental, duplications of the SHANK3 genomic region (22q13) are known to cause a spectrum of neuropsychiatric disorders. Large duplications spanning SHANK3 and multiple neighboring genes have been reported in individuals with attention deficit-hyperactivity disorder (ADHD), schizophrenia, and ASD. Smaller duplications, spanning SHANK3 and only one or two adjacent genes, have been reported in individuals with ADHD, epilepsy, and bipolar disorder. Furthermore, duplication of the Shank3 locus in mice results in manic-like behavior, seizures, and defects in neuronal excitatory/inhibitory balance. Thus, the requirement for proper Shank dosage for normal synaptic function may be a conserved feature (Harris, 2016).

    One unexpected finding from this study was the identification of a previously unappreciated aspect of Shank as a regulator of Wnt signaling. Shank regulates the internalization of the transmembrane Fz2 receptor, thus affecting transduction of Wnt signaling from the plasma membrane to the nucleus. Downregulation of this pathway is implicated in impaired postsynaptic organization, including supernumerary GBs and SSR defects. The physical proximity of Shank and Fz2 at the postsynaptic membrane suggests that Shank directly or indirectly modulates the internalization of Fz2. Shank is a scaffolding protein with many binding partners that could contribute to such an interaction. One intriguing possibility is the PDZ-containing protein Grip. Shank2 and Shank3 have been reported to bind Grip1. Furthermore, Drosophila Grip transports Fz2 to the nucleus on microtubules to facilitate the FNI pathway (Ataman, 2006). Thus, an interaction between Shank, Fz2, and Grip to regulate synaptic signaling is an attractive model (Harris, 2016).

    Although loss of Shank is associated with impaired internalization of the Fz2 receptor, how excess Shank leads to FNI impairment remains an open question. One possibility is that an increase in the concentration of the Shank scaffold at the synapse physically impedes the transport of Fz2 or other components of the pathway or saturates binding partners that are essential for Fz2 trafficking. Both overexpression and loss of function of Shank ultimately lead to a failure to accumulate the cleaved Fz2 C terminus within the nucleus, in which it is required to interact with RNA binding proteins that facilitate transport of synaptic transcripts to postsynaptic compartments. Although Shank and Wnt both play important synaptic roles, this study is the first demonstration of a functional interaction between Shank and Wnt signaling at the synapse (Harris, 2016).

    Intriguingly, no obvious defects were found in glutamate receptor levels or distribution in the absence of Shank. This was surprising given the role for Shank in regulating the FNI pathway, and downregulation of FNI was shown previously to lead to increased GluR field size. Several studies have reported changes in the levels of AMPA or NMDA receptor subunits in Shank mutant mice, although others have also observed no changes. Levels of metabotropic glutamate receptors are also affected in some Shank mutant models. Moreover, transfected Shank3 can recruit functional glutamate receptors in cultured cerebellar neurons. It is possible that Drosophila Shank mutants have defects in GluRs that are too subtle to detect with current methodology. Another possibility is that Shank is involved in signaling mechanisms that are secondary to FNI and that lead to compensatory changes in GluRs at individual synapses. Indeed, the results are consistent with Shank having additional functions at the synapse in addition to its role in FNI, particularly affecting synaptic bouton number. In conclusion, this study has fpind that the sole Drosophila Shank homolog functions to regulate synaptic development in a dose-dependent manner, providing a new model system to further investigate how loss of this scaffolding protein may underlie neurodevelopmental disease (Harris, 2016).

    The influence of postsynaptic structure on missing quanta at the Drosophila neuromuscular junction

    Synaptic transmission requires both pre- and post-synaptic elements for neural communication. The postsynaptic structure contributes to the ability of synaptic currents to induce voltage changes in postsynaptic cells. At the Drosophila neuromuscular junction (NMJ), the postsynaptic structure, known as the subsynaptic reticulum (SSR), consists of elaborate membrane folds that link the synaptic contacts to the muscle, but its role in synaptic physiology is poorly understood. This study investigated the role of the SSR with simultaneous intra- and extra-cellular recordings that allow identification of the origin of spontaneously occurring synaptic events. Data from Type 1b and 1s synaptic boutons, which have naturally occurring variations of the SSR, were compared with genetic mutants that up or down-regulate SSR complexity. Some synaptic currents do not result in postsynaptic voltage changes, events that were called 'missing quanta'. The frequency of missing quanta is positively correlated with SSR complexity in both natural and genetically-induced variants. Rise-time and amplitude data suggest that passive membrane properties contribute to the observed differences in synaptic effectiveness. It is concluded that electrotonic decay within the postsynaptic structure contributes to the phenomenon of missing quanta. Further studies directed at understanding the role of the SSR in synaptic transmission and the potential for regulating 'missing quanta' will yield important information about synaptic transmission at the Drosophila NMJ (Nguyen, 2016).

    Hebbian plasticity guides maturation of glutamate receptor fields in vivo

    Synaptic plasticity shapes the development of functional neural circuits and provides a basis for cellular models of learning and memory. Hebbian plasticity describes an activity-dependent change in synaptic strength that is input-specific and depends on correlated pre- and postsynaptic activity. Although it is recognized that synaptic activity and synapse development are intimately linked, a mechanistic understanding of the coupling is far from complete. Using Channelrhodopsin-2 to evoke activity in vivo, this study investigated synaptic plasticity at the glutamatergic Drosophila neuromuscular junction. Remarkably, correlated pre- and postsynaptic stimulation increased postsynaptic sensitivity by promoting synapse-specific recruitment of GluR-IIA-type glutamate receptor subunits into postsynaptic receptor fields. Conversely, GluR-IIA was rapidly removed from synapses whose activity failed to evoke substantial postsynaptic depolarization. Uniting these results with developmental GluR-IIA dynamics provides a comprehensive physiological concept of how Hebbian plasticity guides synaptic maturation and sparse transmitter release controls the stabilization of the molecular composition of individual synapses (Ljaschenko, 2013).

    Repeated light-triggered neurotransmitter release from presynaptic active zones provoked synaptic depression via a decrease in quantal content. Interestingly, muscle depolarization itself also led to a drop in quantal content despite bypassing synapses (Post animals). The latter observation is highly reminiscent of homeostatic communication whereby a retrograde pathway of inverted polarity operates to increase quantal content in response to reduced muscle excitability. Future studies can now test whether molecular components involved in the homeostatic upregulation of quantal content also contribute to its downregulation following postsynaptic ChR2 stimulation. (Ljaschenko, 2013).

    Pairing pre- and postsynaptic depolarizations repetitively (pre and post) triggered a synapse-specific increase in postsynaptic GluR-IIA-type GluRs. Hence, correlated activity initiated a Hebbian form of synaptic plasticity at the Drosophila NMJ. A comparison of the cluster size distributions following brief and long pulses suggests two phases of plasticity. The first phase of activity-induced plasticity (15 ms pulses) promotes an evenly distributed increase in the number of clusters. Therefore, despite an increase in total number, the average size of GluR-IIA clusters is not significantly altered. The next phase (2 s pulses) then leads to an increase mainly in the number of large clusters, and hence the average GluR-IIA cluster size increases. Neurotransmitter pr varies across active zones at the NMJ. Because the size of GluR clusters is largest opposite high-pr active zones, it is to be expected that functional recordings of synaptic currents preferentially sample large receptor fields. For this reason, GluR-IIA incorporation likely remained below the detection threshold in electrophysiological recordings following brief light pulses (Ljaschenko, 2013).

    In view of the unchanged total number of receptor fields (anti-GluR-IID staining) and active zones, paired stimulation did not appear to give rise to the formation of new synapses. Instead, GluR-IIA was likely incorporated into receptor fields with previously undetectable IIA levels (Ljaschenko, 2013).

    In vivo imaging suggests that positive feedback initially promotes GluR-IIA incorporation during synapse growth and that GluR-IIA entry is specifically restrained with further maturation, whereas the rate of GluR-IIB recruitment remains constant. The physiological signals that guide these synapse-specific molecular dynamics are unknown. It is argued that the Hebbian mechanism identified in the present study represents the signal that promotes GluR-IIA entry during synapse development. This is consistent with the observed increase in small clusters following short pulses. Furthermore, in this framework, paired pre- and postsynaptic stimulation would be able to override the inhibition of GluR-IIA incorporation at relatively mature receptor fields and thereby restore the 'juvenile behavior' of the PSDs (Ljaschenko, 2013).

    At the developing Drosophila NMJ, receptor field growth is accompanied by BRP-dependent, active zone maturation. Correspondingly, large receptor fields are found opposite high-pr active zones that are rich in BRP. Therefore, small, growing receptor fields opposite immature, low-pr active zones will tend to be exposed to glutamate only when pr is elevated, e.g., during trains of action potentials. Because a large number of other synapses will also be active at these time points, transmitter release will coincide with strong postsynaptic depolarization, leading to Hebbian GluR-IIA incorporation (Ljaschenko, 2013).

    A comprehensive model conversely demands a signal to remove GluR-IIA from mature receptor fields in order to describe their diminished rate of IIA incorporation in vivo and to limit receptor-field growth. It was reasoned that such a physiological cue could be provided by sparse (i.e., unsynchronized) transmitter release that preferentially occurs at high-pr, mature synapses and does not trigger substantial muscle depolarization. This hypothesis is experimentally supported by GluR-IIA removal from synapses when muscle depolarization is prevented during neurotransmission (Ljaschenko, 2013).

    This study introduces a physiological model in which GluR-IIA is increased at simultaneously active synapses via Hebbian plasticity and is decreased at solitarily active synapses. Such solitary activity may be provided by spontaneous transmitter release (i.e., minis). The physiological function of minis has been controversially discussed. The results suggest that they contribute to 'taming the beast'; in other words, restraining the extent of Hebbian plasticity. The model can account for developmental, synapse-specific receptor subunit dynamics, and explains why GluR-IIA levels are higher opposite low-pr Ib motorneurons than opposite high-pr Is motorneurons. This conceptual framework can account for an increase in GluR-IIA following chronic activity elevation and is consistent with low synaptic IIA levels in the presence of ambient extracellular glutamate, although, intriguingly, sustained glutamate exposure also affects GluR-IIB (Ljaschenko, 2013).

    Trains of action potentials are likely the physiological equivalent of paired pre- and postsynaptic depolarization, which simply triggers the Hebbian change more efficiently than solely presynaptic ChR2 stimulation. Notably, rapid GluR-IIA exit can be acutely provoked. This observation is compatible with fast GluR dynamics in mammals, which can operate on a timescale of minutes and well below. Hence, rapid receptor trafficking also occurs in Drosophila, though this probably remains concealed when receptor exit is not explicitly provoked during time-lapse imaging of synapse development in vivo (Ljaschenko, 2013).

    Perhaps most conspicuously, activity-dependent bidirectional GluR-IIA mobility is reminiscent of subunit-specific AMPA receptor trafficking at mammalian central synapses, which mediates manifold forms of synaptic plasticity. Local activity has been shown to drive synapse-specific accumulation of GluR1 AMPA receptors. Whereas high-frequency stimulation triggers LTP and synaptic GluR1 incorporation, low-frequency stimulation triggers LTD and GluR1 removal. Collectively, these considerations support the notion that fundamental mechanisms of synaptic plasticity have been strongly conserved during evolution (Ljaschenko, 2013).

    Dbo/Henji modulates synaptic dPAK to gate glutamate receptor abundance and postsynaptic response

    In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. This study analyzed mutant synapses of henji, also known as diablo (dbo), at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. It is proposed that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK (Wang, X., 2016).

    Coordinated action and communication between pre- and postsynapses are essential in maintaining synaptic strength and plasticity. Presynaptic strength or release probability of synaptic vesicles involves layers of regulation including vesicle docking, fusion, and recycling, as well as endocytosis and exocytosis. Also, how postsynapses interpret the signal strength from presynapses depends largely on the abundance of neurotransmitter receptors at the synaptic membrane. During long-term potentiation, lateral diffusion of extrasynaptic AMPA receptor to synaptic sites is accelerated and the exocytosis of AMPAR is enhanced near the postsynaptic density (PSD), causing an accumulation of synaptic receptors. In contrast, under the long-term depression condition, synaptic AMPAR is reduced by hastened endocytosis. While molecular mechanisms are proposed to play roles in regulating and fine-tuning postsynaptic glutamate receptor (GluR) abundance in plasticity models, the developmental regulation of GluR abundance at the synaptic surface still needs to be elucidated. Synapses at the Drosophila neuromuscular junction (NMJ) use glutamate as the neurotransmitter, and have properties reminiscent of mammalian central excitatory synapses. Homologous to vertebrate AMPAR and kainate receptors, Drosophila GluR subunits assemble as tetramers to gate ion influx. Each functional receptor contains essential subunits (GluRIIC, GluRIID and GluRIIE) and either GluRIIA or GluRIIB; therefore, synaptic GluRs can be classified according to their subunit compositions as either A- or B-type receptors. These two types of receptors exhibit distinct developmental and functional properties. Newly-formed PSDs tend to accumulate more GluRIIA channels, while the IIA/IIB ratio becomes more balanced when PSDs mature. In addition, GluRIIB channels have much faster desensitization kinetics, which results in smaller quantal size than GluRIIA channels. Therefore, the synaptic composition of these two types of GluRs greatly influences the postsynaptic interpretation of neuronal activities. The Drosophila homolog of p21-activated kinase (dPAK) regulates GluRIIA abundance at the PSD; GluRIIA receptor clusters at the postsynaptic membrane are strongly reduced in dpak mutants. However, overexpression of dPAK in postsynapses is not sufficient to increase GluRIIA cluster size, suggesting that dPAK activity in regulating GluRIIA abundance is tightly controlled (Wang, X., 2016).

    Ubiquitination and deubiquitination play critical roles in regulating synaptic functions. In loss-of-function mutants for highwire, a gene encoding a conserved E3 ubiquitin ligase, NMJs overgrow, producing supernumerary synaptic boutons. This phenotype is duplicated by overexpression of the deubiquitinating enzyme Fat facets (Faf) in presynapses. These studies underline the importance of balanced ubiquitination in synapse formation and function. Cullin-RING ubiquitin ligases (CRLs) are large protein complexes that confer substrate ubiquitination. Importantly, CRLs promote ubiquitination through substrate receptors that provide specific recognition of substrates for ubiquitination. The BTB-Kelch proteins are suggested to be the substrate receptors for Cul3-scaffolded CRLs. This study identified a BTB-Kelch-containing protein, Henji, also known as Dbo, which regulates NMJ growth and synaptic activity by restricting the clustering of GluRIIA. Synaptic size of henji mutants was significantly expanded, as viewed under transmission electron microscopy (TEM). Immunostaining for dPAK and GluRIIA also suggests larger areas of PSDs in the absence of Henji, and the intensity of each fluorescent punctum becomes stronger, indicating abnormal accumulation of these PSD proteins. By genetically reducing one gene dosage of dpak in henji mutants, GluRIIA accumulation and abnormal bouton morphology was suppressed. In contrast, reducing the gluriia gene dosage in henji mutants restored bouton morphology but failed to suppress dPAK accumulation. Thus, Henji regulates bouton morphology and GluRIIA clustering levels likely through a control of dPAK. Interestingly, while overexpression of dPAK, either constitutively active or dominantly negative, had no effects on GluRIIA clustering, overexpression of these dPAK forms in henji mutants modulated GluRIIA levels, indicating that Henji limits the action of dPAK to regulate GluRIIA synaptic abundance. Henji localized to the subsynaptic reticulum (SSR) surrounding synaptic sites, consistent with the idea that Henji functions as a gatekeeper for synaptic GluRIIA abundance (Wang, X., 2016).

    This study shows that Henji functions at the postsynapse to regulate synaptic development and function at the NMJ. The PSD area is expanded and GluRIIA clusters abnormally accumulate at the PSD. Genetic evidences are provided to support that the elevation of GluRIIA synaptic abundance is at least partially caused by a corresponding accumulation of dPAK in henji mutants. Henji is sufficient to downregulate dPAK and GluRIIA levels and the Kelch repeats of Henji play the most critical role in this process. Henji tightly gates dPAK in regulating GluRIIA abundance, as dPAK enhances GluRIIA cluster abundance only when Henji is absent. Therefore, this study has identified a specific negative regulation of dPAK at the postsynaptic sites that contributes to the PSD formation and GluR cluster formation at the NMJ (Wang, X., 2016).

    PAK proteins transduce various signaling activities to impinge on cytoskeleton dynamics. Through kinase activity-dependent and -independent mechanisms, PAK regulates not only actin- and microtubule-based cytoskeletal rearrangement but also the activity of motors acting on these cytoskeletal tracks. In mammalian systems, PAKs participate in many synaptic events including dendrite morphogenesis, neurotransmitter receptor trafficking, synaptic strength modulation, and activity-dependent plasticity. Pathologically, PAK dysregulation also contributes to serious neurodegenerative diseases, Huntington's disease and X-linked mental retardation (Wang, X., 2016).

    At Drosophila NMJs, dPAK has divergent functions; loss of dpak causes a dramatic reduction in both Dlg and GluRIIA synaptic abundance, but the underlying molecular mechanisms have not been revealed. The current data show that Henji functions to restrict GluRIIA clustering but has no effect on Dlg levels, suggesting that Henji regulates one aspect of dPAK activities, probably via the SH2/SH3 adaptor protein Dock. Alternatively, Henji may function to limit dPAK protein levels locally near the postsynaptic region, rendering its influence on GluRIIA clustering, while dPAK that regulates Dlg may localize outside of the Henji-enriched region. Supporting this idea, Henji is specifically enriched around the SSR region instead of dispersed throughout the muscle cytosol. Moreover, ectopic Myc-dPAK localized at the postsynapse only when henji was mutated, indicating that Henji regulates dPAK postsynaptic localization (Wang, X., 2016).

    The interaction with Rac, Cdc42, or both triggers autophosphorylation and subsequent conformational changes of PAK, resulting in kinase activation. The myristoylated dPAK that has been shown to be active in growth cones failed to enhance GluRIIA abundance at the NMJ. This result shows that dPAK is necessary to regulate GluRIIA synaptic abundance, but is itself tightly regulated at the synaptic protein level or the kinase activity. Indeed, evidence is provided to show specific negative regulation of dPAK by Henji; overexpression of dPAK CA that could not enhance GluRIIA abundance in WT larvae further increased the already enhanced GluRIIA levels in the henji mutant. Similar to the CA form, the DN form also showed no effect on GluRIIA when simply overexpressed in the WT background, but exhibited strong suppression of GluRIIA in the henji mutant background. Thus, regardless of the possible conformational differences between the CA and DN forms, Henji appears to confer a constitutive negative regulation of dPAK at postsynapses, suggesting a tight control that could be at subcellular localization. In contrast to CA and DN forms, activation of dPAK requires binding to Rac1 and Cdc42, and subsequent protein phosphorylation. This additional layer of regulation may serve as a limiting factor rendering dPAK WT from recruiting GluRIIA to PSDs regardless in WT or henji mutant background (Wang, X., 2016).

    The structural feature suggests that Henji could function as a conventional substrate receptor of the Cul3-based E3 ligase complex. At Drosophila wing discs, Dbo functions as a Cul3-based E3 ligase to promote Dishevelled (Dsh) downregulation. Similar to the henji alleles, it was confirmed that the dbo [Δ25.1] allele and dbo RNAi were competent to induce dPAK and GluRIIA accumulation at the postsynapse. An immunoprecipitation experiment detected Henji and dPAK in the same complex, and dPAK also forms a complex with the C-terminal substrate-binding Kelch-repeats region. However, no notable or consistent increase was detected in Henji-dependent dPAK poly-ubiquitination in both S2 cells and larval extracts. Also, the Cul3-binding BTB domain of Henji seems dispensable in the suppression of dPAK levels in henji mutants. Importantly, Cul3 knockdown in muscle cells failed to cause any accumulation of GluRIIA and dPAK at the NMJ. Sensitive genetic interaction between henji and Cul3 failed to induced dPAK and GluRIIA accumulation. Dbo functions together with another BTB-Kelch protein Kelch (Kel) to downregulate Dsh. However, Kel negatively regulates GluRIIA levels without affecting dPAK localization at the postsynaptic site. This data argues that Kel functions in a distinct pathway to Henji in postsynaptic regulation of GluRIIA. Taken together, no direct evidence was found to support that dPAK is downregulated by Henji through ubiquitination-dependent degradation. Alternately, Henji could bind dPAK near the postsynaptic region and this interaction may block the recruitment or localization of dPAK onto postsynaptic sites. Under this model, dPAK is less restricted and has a higher propensity to localize at postsynaptic sites in the absence of Henji, resulting in synaptic accumulation of dPAK and GluRIIA expansions (Wang, X., 2016).

    As many synaptic events require rapid responses, local regulation of protein levels becomes crucial in synapses. To achieve accurate modulation, certain synaptic proteins should be selectively controlled under different developmental or environmental contexts. Indeed, emerging evidence shows that various aspects of synapse formation and function are under the control of the ubiquitin proteasome system (UPS), including synapse formation, morphogenesis, synaptic pruning and elimination, neurotransmission, and activity-dependent plasticity. In particular, the membrane abundance of postsynaptic GluR that modulates synaptic function can be regulated by components of the UPS. When Apc2, the gene encoding Drosophila APC/C E3 ligase, is mutated, GluRIIA shows excess accumulation but the molecular mechanism was not elucidated. Similarly, loss of the substrate adaptor BTB-Kelch protein KEL-8 in C. elegans also results in the stabilization of GLR-1-ubiquitin conjugates. However, no evidence shows direct ubiquitination and degradation of GLR-1 by KEL-8. Also, absence of the LIN-23-APC/C complex in C. elegans affects GLR-1 abundance at postsynaptic sites without altering the level of ubiquitinated GLR-1. Therefore, GLR-1 receptor endocytosis and recycling or ubiquitination and degradation of GLR-1-associated scaffold proteins are proposed to be the underlying mechanism for E3 ligase regulation. In mammals, endocytosis of AMPAR can be influenced by poly-ubiquitination and degradation of the prominent postsynaptic scaffold protein PSD-95 (Wang, X., 2016).

    This study describes a novel regulation by the BTB-Kelch protein Henji on synaptic GluRIIA levels. By limiting GluRIIA synaptic levels, Henji modulates the postsynaptic output in response to presynaptic glutamate release. In the absence of Henji, quantal size is elevated, coinciding with an increase in the postsynaptic GluRIIA/GluRIIB ratio. In a previous study, increases in the GluRIIA/GluRIIB ratio by overexpressing a GluRIIA transgene in the muscle or by reducing the gene copy of gluriib promote NMJ growth, but co-expression of both GluRIIA and GluRIIB did not alter the bouton number. Combined with the current findings, those data provide a link between an increased GluRIIA-mediated postsynaptic response and bouton addition at NMJs. However, satellite boutons were not detected following GluRIIA overexpression. One possibility is that satellite boutons are considered as immature boutons and their appearance may indicate the tendency for NMJ expansion, as in the case of excess BMP signaling. Failure to become mature boutons may be caused by the lack of cooperation with other factors such as components of the presynaptic endocytic pathway, actin cytoskeleton rearrangement or neuronal activity. No significant alterations in endocytosis and the BMP pathway in the henji mutant. Nevertheless, it cannot be ruled out that Henji may modulate other presynaptic events that are defective in henji mutants to interfere with bouton maturation (Wang, X., 2016).

    Filamin, a synaptic organizer in Drosophila, determines glutamate receptor composition and membrane growth

    Filamin is a scaffolding protein that functions in many cells as an actin-crosslinker. FLN90, an isoform of the Drosophila ortholog Filamin/cheerio that lacks the actin-binding domain, is shown in this study to govern the growth of postsynaptic membrane folds and the composition of glutamate receptor clusters at the larval neuromuscular junction. Genetic and biochemical analyses reveal that FLN90 is present surrounding synaptic boutons. FLN90 is required in the muscle for localization of the kinase dPak and, downstream of dPak, for localization of the GTPase Ral and the exocyst complex to this region. Consequently, Filamin is needed for growth of the subsynaptic reticulum. In addition, in the absence of filamin, type-A glutamate receptor subunits are lacking at the postsynapse, while type-B subunits cluster correctly. Receptor composition is dependent on dPak, but independent of the Ral pathway. Thus two major aspects of synapse formation, morphological plasticity and subtype-specific receptor clustering, require postsynaptic Filamin (Lee, 2016).

    Proper postsynaptic function depends on appropriate localization of receptors and signaling molecules. Scaffolds such as PSD-95/SAP90 and members of the Shank family are critical to achieving this organization. While usually without intrinsic enzymatic activity, scaffolds recruit, assemble, and stabilize receptors and protein networks through multiple protein-protein interactions: they can bind to receptors, postsynaptic signaling complexes, and the cytoskeleton at the postsynaptic density. Mutations in these proteins are associated with neuropsychiatric disorders. While understanding synapse assembly has begun, much remains to be investigated (Lee, 2016).

    The Drosophila larval neuromuscular junction (NMJ) is a well-studied and genetically accessible glutamatergic synapse. Transmission is mediated by AMPA-type receptors, and several postsynaptic proteins important for its development and function have related proteins at mammalian synapses, including the PDZ-containing protein Discs-Large (DLG) and the kinase Pak. In addition, the postsynaptic membrane forms deep invaginations and folds called the subsynaptic reticulum (SSR), which are hypothesized to create subsynaptic compartments comparable to dendritic spines. Recently, we found that the SSR is a plastic structure whose growth is regulated by synaptic activity. This phenomenon may be akin to the use-dependent morphological changes, such as growth of dendritic spines, that occur postsynaptically in mammalian brain. The addition of membrane and growth of the SSR requires the exocyst complex to be recruited to the synapse by the small GTPase Ral; the SSR fails to form in ral mutant larvae. Moreover, the localization of Ral to a region surrounding synaptic boutons is likely to direct the selective addition of membrane to this domain. Ral thus provided a tractable entry point for better understanding postsynaptic assembly. The mechanism for localizing the Ral pathway, however, was unknown. The present study determined that Ral localization is dependent on cheerio, a gene encoding filamin, which is critical for proper development of the postsynapse (Lee, 2016).

    Filamin is a family of highly conserved protein scaffolds with a long rod-like structure of Ig-like repeats. With over 90 identified binding partners, some of which are present also at the synapse, mammalian filamin A (FLNA) is the most abundant and commonly studied filamin. Filamin can bind actin and has received the most attention in the context of actin cytoskeletal organization. Drosophila filamin, encoded by the gene cheerio (cher), shares its domain organization and 46% identity in amino acid sequence with human FLNA. Drosophila filamin has a well-studied role in ring canal formation during oocyte development, where it recruits and organizes actin filaments. This study now shows that filamin has an essential postsynaptic role at the fly NMJ. An isoform of this scaffold protein that lacks the actin-binding domain acts via dPak to localize GluRIIA receptors and Ral; filamin thereby orchestrates both receptor composition and membrane growth at the synapse (Lee, 2016).

    Filamin is a highly conserved protein whose loss of function is associated with neurodevelopmental disorders. In humans, mutations in the X-linked FLNA cause periventricular heterotopia, a disorder of cortical malformation with a wide range of clinical manifestations such as epilepsy and neuropsychiatric disturbances. Studies in rodent models have shown that abnormal filamin expression causes dendritic arborization defects in a TSC mouse and that filamin influences neuronal proliferation. Filamin is present in acetylcholine receptor clusters at the mammalian NMJ, but its function there is unknown. In lysates of the mammalian brain, filamin associates with known synaptic proteins such as Shank3, Neuroligin 3, and Kv4.2. A recent report indicated that filamin degradation promotes a transition from immature filopodia to mature dendritic spines, a phenomenon that is likely to be related to the actin-bundling properties of the long isoform of filamin. Data in the present study have uncovered a novel pathway that does not require the actin-binding domain of filamin. In this pathway, postsynaptically localized filamin, via Pak, directs two distinct effector modules governing synapse development and plasticity: (1) the Ral-exocyst pathway for activity-dependent membrane addition and (2) the composition of glutamate receptor clusters. These pathways determine key structural and physiological properties of the postsynapse (Lee, 2016).

    Although loss of filamin had diverse effects on synapse assembly, they were selective. Muscle-specific knockdown or the cherQ1415sd allele disrupted type-A but not type-B GluR localization at the postsynaptic density. Likewise, the phenotypes for muscle filamin were confined to the postsynaptic side: the presynaptic active zone protein Brp and overall architecture of the nerve endings were not altered by muscle-specific knockdown. The specificity of its effect on particular synaptic proteins, and the absence of the actin-binding region in FLN90, suggests that filamin's major mode of action here is not overall cytoskeletal organization, but rather to serve as a scaffold for particular protein-protein interactions (Lee, 2016).

    Analysis of the distribution of the SSR marker Syndapin and direct examination of the subsynaptic membrane by electron microscopy revealed that formation of the SSR required filamin. Genetic analysis uncovered a sequential pathway for SSR formation from filamin to the Pak/Pix/Rac signaling complex, to Ral, to the exocyst complex and consequent membrane addition. The SSR is formed during the second half of larval life and may be an adaptation for the low input resistance of third-instar muscles. Like dendritic spines, the infoldings of the SSR create biochemically isolated compartments in the vicinity of postsynaptic receptors and may shape physiological responses, although first-order properties of the synapse, such as mini- or EPSP amplitude are little altered in mutants that lack an SSR. The formation of the SSR requires transcriptional changes driven by Wnt signaling and nuclear import, proteins that induce membrane curvature (such as Syndapin, Amphiphysin, and Past1), and Ral-driven, exocyst-dependent membrane addition. The activation of Ral by Ca2+ influx during synaptic transmission allows the SSR to grow in an activity-dependent fashion. The localization of Ral to the region surrounding the bouton appears crucial to determining the site of membrane addition because Ral localization precedes SSR formation and exocyst recruitment and because exocyst recruitment occurred selectively surrounding boutons even when Ca2+-influx occurred globally in response to calcimycin. This study has now shown that Ral localization, and consequently exocyst recruitment, membrane growth, and the presence of the SSR marker Syndapin, are all dependent on a local action of filamin at the synapse. FLN90, the filamin short isoform, localized to sites of synaptic contact and indeed surrounded the boutons just as does Ral. When this postsynaptic filamin was removed by muscle-specific filamin knockdown or the cherQ1415sd allele, the downstream elements of the pathway, Pak, Rac, Ral, the exocyst, and Syndapin, were no longer synaptically targeted. The mislocalization is not a secondary effect of loss of the SSR but likely a direct consequence of filamin loss, as Pak and Ral can localize subsynaptically even in the absence of the SSR. Unlike the likely mode of action of nuclear signaling by Wnt, the delocalization of Ral was not a consequence of altered protein production; its expression levels did not change. Thus filamin may be viewed as orchestrating the formation of the SSR and directing it to the region surrounding synaptic boutons (Lee, 2016).

    The second major feature of the filamin phenotype was the large reduction in the levels of the GluRIIA receptor subunit from the postsynaptic membranes. GluRIIA and GluRIIB differ in their electrophysiological properties and subsynaptic distribution. Because type B GluRs, which contain the IIB subunit, desensitize more rapidly than type A, the relative abundance of type A and type B GluRs is a key determinant of postsynaptic responses and changes with synapse maturation. The selective decrease in GluRIIA at filamin-null NMJs is likely a consequence of dPak mislocalization: filamin-null NMJs lack synaptic dPak, and dPak null NMJs lack synaptic GluRIIA. Moreover, the first-order electrophysiological properties at NMJs lacking filamin resembled those reported at NMJs missing dPak. In the current study, though, only the change in mEPSP frequency was statistically significant. At filamin-null NMJs, the decrease in GluRIIA is accompanied by an increase in GluRIIB, suggestive of a partial compensation that could account for the relatively normal synaptic transmission. Because the IIA and IIB subunits differ in desensitization kinetics and regulation by second messengers, functional consequences of filamin loss may become more apparent with more extensive physiological characterizations at longer time scales (Lee, 2016).

    While both SSR growth and receptor composition required the kinase activity of dPak, receptor composition was independent of Ral and thus represents a distinct branch of the pathway downstream of dPak. As with Ral, the loss of GluRIIA from the synapse was due to delocalization and not a change in expression of the protein, consistent with unaltered GluRIIA transcripts in dPak null animals. Thus filamin, via dPak, alters proteins with functional significance for the synapse as well as its structural maturation (Lee, 2016).

    Mammalian filamin, via its many Ig-like repeats, has known scaffold functions in submembrane cellular compartments and filamin is therefore likely also to serve as a scaffold at the fly NMJ. These epistasis data indicate that filamin recruits Ral through recruitment of a signaling complex already known to function at the fly NMJ: dPak and its partners dPix and Rac. Mammalian filamin is reported to directly interact with Ral during filopodia formation, however the details of their interaction at the fly NMJ are less clear. Because Ral localization requires filamin to recruit dPak and dPix and specifically requires the kinase activity of dPak, it is possible that either Ral or filamin need to be phosphorylated by dPak to bind one another. Mammalian filamin interacts with some components of the Pak signaling complex and is a substrate of Pak. This study has now shown that Drosophila filamin and PAK interact when coexpressed in HEK cells, and thus a direct scaffolding role for FLN90 in the recruitment of Pak and the organization of the postsynapse is likely (Lee, 2016).

    The overlapping but different distributions of filamin and its downstream targets indicate that its scaffolding functions must undergo regulation by additional factors. The proteins discussed in this study take on either of two patterns at the synapse. Some, like Ral, Syndapin, and filamin itself, are what can be termed subsynaptic and, like the SSR, envelope the entire synaptic bouton. Others, like dPak and its partners and the GluRIIA proteins, are concentrated in much smaller regions, immediately opposite presynaptic active zones, where the postsynaptic density (PSD) is located. It is hypothesized that filamin interacts with additional proteins, including potentially transsynaptic adhesion proteins, that localize filamin to the subsynaptic region and also govern to which of the downstream effectors it will bind. Indeed, it appears paradoxical that dPak, though predominantly at the postsynaptic density is nonetheless required for Ral localization throughout the subsynaptic region. If dPak is needed to phosphorylate either filamin or Ral to permit Ral localization, the phosphorylations outside the PSD may be due to low levels of the dPak complex in that region; synaptic dPak was previously shown to be a relatively mobile component of the PSD (Lee, 2016).

    Filamin was the first nonmuscle actin-crosslinking protein to be discovered. With an actin-binding domain at the N terminus, the long isoform of filamin and its capacity to integrate cellular signals with cytoskeletal dynamics have subsequently been the focus of the majority of the filamin literature. At the NMJ, however, this was not the case. Several lines of evidence indicated that the short FLN90 isoform of filamin, which lacks the actin-binding domain, plays an essential role in postsynaptic assembly. First, the short FLN90 isoform was the predominant and perhaps the only isoform of filamin found expressed in the muscles. Second, both endogenous and overexpressed FLN90 localized subsynaptically. Third, loss of the short isoform disrupted localization of postsynaptic components while lack of just the long isoform had little or no effect. Lastly, exogenous expression of just the short isoform in filamin null background sufficiently rescued the defect in SSR growth. The modest postsynaptic phenotypes of the cher1allele, which predominantly disrupts the long isoform, may be due to small effects of the allele on expression of the short isoform or may be an indirect consequence of the presence of the long isoform in the nerve terminals (Lee, 2016).

    The existence of the short isoform has been reported in both flies and mammals and may be produced either by transcriptional regulation or calpain-mediated cleavage. The short isoform can be a transcriptional co-activator, but its functional significance and mechanisms of action have been largely elusive. The short isoform has little or no affinity for actin, but most of the known sites for other protein-protein interactions are shared by both isoforms. Thus the structure of FLN90, with nine predicted Ig repeats and likely protein-protein interactions, is consistent with a scaffolding function to localize key synaptic molecules independent of interactions with the actin cytoskeleton (Lee, 2016).

    This study has introduced filamin as a major contributor to synapse development and organization. The severity of the phenotypes indicates filamin has a crucial role that is not redundant with other scaffolding proteins. The effects of filamin encompass several much-studied aspects of the Drosophila NMJ: the clustering and subunit subtype of glutamate receptors and the plastic assembly of specialized postsynaptic membrane structures. The pathways that govern these two phenomena diverge downstream of Pak kinase activity and are dependent on filamin for the proper localization of key signaling modules in the pathways. By likely acting as a scaffold protein, the short isoform of filamin may function as a link between cell surface proteins, as yet unidentified, and postsynaptic proteins with essential localizations to and functions at the synapse. Because many of the components of these pathways at the fly NMJ are also present at mammalian synapses and can interact with mammalian filamin, a parallel set of functions in CNS dendrites merits investigation (Lee, 2016).

    TOR is required for the retrograde regulation of synaptic homeostasis at the Drosophila neuromuscular junction

    Homeostatic mechanisms operate to stabilize synaptic function; however, little is known about how they are regulated. Exploiting Drosophila genetics, a critical role was uncovered for the target of rapamycin (TOR) in the regulation of synaptic homeostasis at the Drosophila larval neuromuscular junction. Loss of postsynaptic TOR disrupts a retrograde compensatory enhancement in neurotransmitter release that is normally triggered by a reduction in postsynaptic glutamate receptor activity. Moreover, postsynaptic overexpression of TOR or a phosphomimetic form of S6 ribosomal protein kinase, a common target of TOR, can trigger a strong retrograde increase in neurotransmitter release. Interestingly, heterozygosity for eIF4E, a critical component of the cap-binding protein complex, blocks the retrograde signal in all these cases. These findings suggest that cap-dependent translation under the control of TOR plays a critical role in establishing the activity dependent homeostatic response at the NMJ (Penney, 2012).

    A growing consensus by neurobiologists suggests that a balance exists between forces that promote and those that hinder synaptic growth and function, ensuring proper synaptic connectivity and functional stability in the nervous system. A robust retrograde signaling mechanism at the Drosophila NMJ carries out the task of adjusting synaptic strength in response to a reduction in postsynaptic receptor function in GluRIIA mutants. Genetic analysis suggests that postsynaptic activity of TOR plays a key role in the ability of this retrograde signaling to carry out its function. The current findings are consistent with a model in which TOR, through activation of S6K and inhibition of 4E-BP, ensures the efficiency of cap-dependent translation in muscles and allows for the retrograde compensation to take place (Penney, 2012).

    Interestingly, a moderate to strong reduction in TOR activity in the TorE161K/TorΔP mutant combination does not influence normal synaptic growth and has only a mild effect on baseline synaptic transmission. However, the findings indicate that once synaptic activity is compromised, i.e., in GluRIIA mutants, TOR becomes critical for the retrograde induction of homeostatic signaling. Furthermore, the findings suggest that TOR activity is required throughout larval development, as its inhibition by rapamycin for 12 hr during late stages of larval development is sufficient to block the retrograde signal. In addition, it was found that TOR can induce a retrograde increase in neurotransmitter release in wild-type animals, indicating that TOR can also act as an instructive force to regulate synaptic strength. These results together lead one to envision that under metabolic stress, during dietary restriction or as a result of aging perhaps, TOR could function as a modulator of neuronal function. As such, the identification of TOR as a key player in establishing retrograde signaling across synapses offers new insights into how defects in this aspect of translational regulation may underlie the destabilization of synaptic activity in neural circuits leading to abnormal neural function and behavior associated with diseases such as tuberous sclerosis complex (TSC), autism, mental retardation, and schizophrenia, where regulation of TOR activity may be altered. In fact, in animal models for TSC, hyperactivity in circuits and the susceptibility to epileptic activity can be diminished in response to rapamycin treatment. Based on the current results, it is conceivable that in TSC animals, when TOR is upregulated, synaptic activity in circuits is enhanced due to the retrograde action of TOR on neurotransmitter release, in a manner independent of growth related phenotype associated with TOR gain of function. Therefore, the results reveal a role for TOR in the retrograde regulation of neurotransmitter release in neurons, an avenue to explore aimed at potential therapeutic approaches (Penney, 2012).

    Based on genetic interaction experiments and biochemical assessment, it is concluded that TOR normally acts downstream of synaptic activity. It was observed that postsynaptic phosphorylation of S6K, a bona fide TOR target, is increased in GluRIIA mutants, suggesting that TOR signaling may be upregulated in these mutants. Consistently, the genetic experiments show that removal of one gene copy of either Tor or S6k is sufficient to block the homeostatic response in GluRIIA mutants. Furthermore, when TOR is overexpressed in GluRIIA mutants no additional increase in quantal content is observed. This lack of an additive effect suggests that a common molecular pathway may be utilized by GluRIIA mutants and larvae overexpressing TOR. This is further supported by the observations that the enhancement in neurotransmission in response to TOR (or S6K) overexpression and that triggered in GluRIIA loss of function are both highly dependent on wild-type availability of eIF4E. These results together support the idea that TOR functions downstream of synaptic activity at the NMJ. Further experiments are needed to understand how changes in synaptic activity may regulate the activity of TOR (Penney, 2012).

    The findings are consistent with a growing body of evidence that implicates the involvement of TOR/S6K in the regulation of synaptic plasticity in mammals. The results indicate that TOR/S6K may be exerting their function through a retrograde mechanism to enhance neurotransmission. As such, the findings reveal a novel mode of action for TOR, through which it can modulate circuit activity in higher organisms. Further experiments are required to verify if this mode of action is conserved in higher organisms (Penney, 2012).

    One potential way in which general translational mechanisms can lead to specific changes in synaptic function is through localized translation. In both vertebrates and invertebrates, local postsynaptic translation is required for normal synaptic plasticity and is itself modulated by synaptic function. This is perhaps best demonstrated in cultured hippocampal neurons, where local protein synthesis at postsynaptic sites is regulated by postsynaptic activity. It appears that this modulation is, at least in part, mediated by the action of synaptic activity on the function of elongation factor, eEF2. Interestingly, blocking NMDA mediated miniatures leads to phosphorylation and inhibition of eEF2 (Sutton, 2007). The current findings are consistent with a role for miniature synaptic activity in the regulation of postsynaptic translation: in GluRIIA mutants, a reduction in miniature amplitude (and perhaps in postsynaptic calcium influx) leads to an upregulation of TOR activity as evident in the increase in S6K phosphorylation. However, at this point it cannot be conclusively show that the effect of postsynaptic TOR is localized. Further experiments are needed to verify whether these changes occur at specific postsynaptic loci at the NMJ (Penney, 2012).

    GluRIIA mutants is critically dependent on the efficiency of the cap-binding protein complex but is less sensitive to the availability of the ternary complex, which mediates the binding of Met-tRNAiMet to the 40S ribosomal subunit (Sonenberg, 2009). Similarly, while very strong inhibition of translation at the level of elongation using cycloheximide can block the retrograde compensation, revealing that retrograde compensation relies on de novo protein synthesis, moderate genetic interference with translation elongation does not interfere with retrograde signaling. These results together highlight the critical role of the cap-dependent protein complex in the retrograde regulation of synaptic strength (Penney, 2012).

    The major task of the cap-binding complex is binding to the 5′UTR of the mRNA and unwinding it, so that the ribosome can interact with the mRNA and initiate translation (Ma, 2009). The results suggest that this stage of translation is the most critical for the induction of retrograde compensation. As the results suggest, once the 5′UTR is unwound, changes in the availability of the ternary complex and translation elongation are less critical for the induction of retrograde signaling. On the other hand, TOR would have a two-fold function in this scenario: one through its inhibitory action on 4E-BP, promoting eIF4Es ability to bind the 5′ cap structure, and another through its activation of S6K, which would ultimately increase the helicase ability of eIF4A to unwind mRNA 5′UTR secondary structure. This notion was supported by the results of an in vivo reporter assay showing a significant increase in translation of a reporter that bore a complex 5′UTR in response to TOR overexpression. Based on the current findings, it is speculated that perhaps genes with highly structured 5′UTRs are among the mRNAs triggered when postsynaptic activity is reduced in GluRIIA mutants or when TOR is overexpressed in muscles. The next challenge is to identify and characterize these genes, a discovery that will likely lead to a better understanding of how homeostatic mechanisms are regulated at the synapse (Penney, 2012).

    The maintenance of synaptic homeostasis at the Drosophila neuromuscular junction is reversible and sensitive to high temperature

    Homeostasis is a vital mode of biological self-regulation. The hallmarks of homeostasis for any biological system are a baseline set point of physiological activity, detection of unacceptable deviations from the set point, and effective corrective measures to counteract deviations. Homeostatic synaptic plasticity (HSP) is a form of neuroplasticity in which neurons and circuits resist environmental perturbations and stabilize levels of activity. One assumption is that if a perturbation triggers homeostatic corrective changes in neuronal properties, those corrective measures should be reversed upon removal of the perturbation. This study tests the reversibility and limits of HSP at the well-studied Drosophila melanogaster neuromuscular junction (NMJ). At the Drosophila NMJ, impairment of glutamate receptors causes a decrease in quantal size, which is offset by a corrective, homeostatic increase in the number of vesicles released per evoked presynaptic stimulus, or quantal content. This process has been termed presynaptic homeostatic potentiation (PHP). Taking advantage of the GAL4/GAL80TS/UAS expression system, PHP was triggered by expressing a dominant-negative glutamate receptor subunit at the NMJ. PHP was then reversed by halting expression of the dominant-negative receptor. The data show that PHP is fully reversible over a time course of 48-72 h after the dominant-negative glutamate receptor stops being genetically expressed. As an extension of these experiments, it was found that when glutamate receptors are impaired, neither PHP nor NMJ growth is reliably sustained at high culturing temperatures (30-32°C). These data suggest that a limitation of homeostatic signaling at high temperatures could stem from the synapse facing a combination of challenges simultaneously (Yeates, 2017).

    Homeostasis is a strong form of biological regulation. It permits individual cells or entire systems of cells to maintain core physiologic properties that are compatible with life. In the nervous system, decades of study have shown that while synapses and circuits are inherently plastic, they also possess robust homeostatic regulatory systems to maintain physiologic stability. Homeostatic plasticity in the nervous system is a non-Hebbian strategy to counteract challenges to neuronal function that may threaten to disrupt essential neuronal and circuit activities (Turrigiano, 2017). Depending on the synaptic preparation examined and the environmental challenge presented to the synapse, homeostatic responses may be executed via compensatory adjustments to presynaptic neurotransmitter release (Cull-Candy, 1980; Petersen, 1997; Murthy, 2001; Thiagarajan, 2005; Frank, 2006; Davis, 2015), postsynaptic neurotransmitter receptor composition (O'Brien, 1998; Turrigiano, 1998; Rongo , 1999; Turrigiano, 2008), neuronal excitability (Marder, 2002; Marder, 2006; Marder and Bucher, 2007; Bergquist, 2010; Parrish, 2014), or even developmentally, via changes in synaptic contact formation and maintenance (Davis and Goodman, 1998; Burrone, 2002; Wefelmeyer, 2016; Yeates, 2017 and references therein).

    Bidirectionality has been documented in several homeostatic systems, perhaps most prominently in the case of synaptic scaling of neurotransmitter receptors. For vertebrate neuronal culture preparations -- such as cortical neurons or spinal neurons—global silencing of network firing can induce increases in excitatory properties, such as increased AMPA-type glutamate receptor accumulation; by contrast, global enhancement of activity can induce the opposite type of response (O'Brien, 1998; Turrigiano, 1998; Wierenga, 2005; Turrigiano, 2008). Bidirectionality is also a key feature underlying homeostatic alterations of neurotransmitter release at peripheral synapses such as the neuromuscular junction (NMJ). At the NMJs of Drosophila melanogaster and mammals, impairing neurotransmitter receptor function postsynaptically results in diminished sensitivity to single vesicles of transmitter. Electrophysiologically, this manifests as decreased quantal size. NMJs respond to this challenge by enhancing neurotransmitter vesicle release (Cull-Candy, 1980; Plomp, 1992, 1995; Petersen, 1997; Davis, 1998; Frank, 2009). By contrast, perturbations that enhance quantal size (for example, overexpression of a vesicular neurotransmitter transporter in Drosophila) can result in decreased quantal content (Daniels, 2004; Gavino, 2015; Yeates, 2017 and references therein).

    Synapses and circuits possess myriad solutions to assume appropriate functional outputs in the face of perturbations (Marder, 2002; Marder, 2006). Therefore, a corollary to bidirectional regulation is that homeostatic forms of regulation should also be reversible. There are experimental difficulties of presenting and removing a synaptic challenge in the context of a living synapse, so homeostatic reversibility has not been rigorously studied in an in vivo system or over extended periods of developmental time. Understanding how homeostatic regulatory systems are reversibly turned on and off could have profound implications for elucidating fundamental properties of circuit regulation (Yeates, 2017).

    This study exploits the Drosophila NMJ as a living synapse to test homeostatic reversibility. At the Drosophila NMJ, a canonical way to challenge synapse function is through glutamate receptor impairment (Frank, 2014), either genetically (Petersen, 1997) or pharmacologically (Frank, 2006). Impairments of muscle glutamate receptor function decrease quantal size. Decreased quantal size spurs muscle-to-nerve signaling that ultimately results in a homeostatic increase in presynaptic vesicle release, a process that has been termed presynaptic homeostatic potentiation (PHP). The most widely used experimental homeostatic challenges to Drosophila NMJ function are not easily reversed. These challenges include genetic deletion of the glutamate receptor subunit GluRIIA (Petersen, 1997) and the mostly irreversible pharmacological inhibition of glutamate receptors with Philanthotoxin-433 (PhTox; Frank, 2006; Yeates, 2017 and references therein).

    For this study, a way was engineered to challenge NMJ function in vivo for significant periods of time, verify the effectiveness of the challenge at a defined developmental time point, remove the challenge, and then assess the homeostatic capacity of the NMJ at a later developmental time point. By using the temporal and regional gene expression targeting (TARGET) GAL4/GAL80TS/UAS expression system (McGuire, 2003) to temporally control the expression of a dominant-negative GluRIIA receptor subunit (DiAntonio, 1999), this study found that homeostatic potentiation of neurotransmitter release is fully reversible. In the course of conducting these studies, a high temperature limitation of homeostatic potentiation at the NMJ was uncovered (Yeates, 2017).

    Thus study presents evidence that PHP at the Drosophila neuromuscular synapse is a reversible process. In doing so, prior findings were confirmed showing that there is a tight inverse relationship between quantal amplitude and QC at the NMJ. Those findings were complemented with the results of temperature shift experiments. PHP is measurable at an early stage of larval development and can be erased over a matter of days. Interestingly, at high temperatures, PHP induced by impairing glutamate receptor function either fails or falls short of full compensation. This failure appears to correlate with impaired NMJ growth in the same animals. Why is reversibility slow after dominant-negative GluRIIAM/R removal (Yeates, 2017)?

    There was a robust expression of PHP for NMJs of MHC-Gal4 >> UAS-GluRIIAM/R larvae with the TubP-Gal80TS transgene raised at 29°C for 48 h after egg-laying. Once the expression of the dominant-negative UAS-GluRIIAM/R transgene was halted, this expression of PHP was erased over a slow 48- to 72-h period (Yeates, 2017).

    If PHP is a readily reversible homeostatic process, why is there a days-long time lag to reverse it? The data likely reflect a constraint of the dominant-negative GluRIIAM/R experimental perturbation, rather than the NMJ's capacity to respond quickly to the changed environment. In a prior study, researchers expressed functional, tagged GluRIIA transgenic subunits at the NMJ and performed fluorescence recovery after photobleaching (FRAP) experiments. Those experiments demonstrated that receptor turnover rates at the Drosophila NMJ are extremely slow: it appears that once postsynaptic densities (PSDs) reach a critical size, GluRIIA subunits are stably incorporated. For the current study, this likely means that the temperature downshifts in the reversibility experiments represented an opportunity for the NMJ to incorporate endogenous WT GluRIIA into a significant number of new PSDs while it continued to grow. Given sufficient growth, the endogenously expressed GluRIIA would gradually overcome the previously incorporated dominant-negative GluRIIAM/R subunits. This would restore electrophysiological parameters to normal levels, which is consistent with the data. Reversibility of rapid and sustained forms of homeostatic plasticity (Yeates, 2017).

    The majority of recent studies about synaptic homeostasis at the Drosophila NMJ have emphasized that presynaptic adjustments to neurotransmitter release properties must occur within minutes of drug-induced (PhTox) postsynaptic receptor inhibition to induce a rapid and offsetting response to PhTox challenge. Some important parameters uncovered through these studies include regulation of presynaptic Ca2+ influx; regulation of the size of the readily releasable pool (RRP) of presynaptic vesicles; control of presynaptic SNARE-mediated fusion events; control of neuronal excitability; and recently, implication of endoplasmic reticulum calcium-sensing activities; presynaptic glutamate receptor activity ; and finally, identification of a retrograde, trans-synaptic signaling system governed by the Semaphorin 2b ligand and the Plexin B receptor (Yeates, 2017).

    For almost all of the cases in which a mutation or an experimental condition blocks the short-term induction of homeostatic signaling, the same perturbation has also been shown to block its long-term maintenance. Interestingly, however, the converse is not true. Additional studies have shown that the long-term consolidation (or expression) of homeostatic signaling at the NMJ can be genetically uncoupled from its induction. Select molecules seem to be dedicated to a long-term maintenance program that involves protein translation and signaling processes in both the neuron and the muscle. Recent data suggest that such long-term processes may take 6 h or more to take full effect (Yeates, 2017).

    As more molecular details about HSP are elucidated, it will be interesting to test whether the rapid induction and sustained consolidation of PHP can be reversed by similar or separate mechanisms, and what the time courses of those reversal mechanisms are. At the mouse NMJ, reversibility was recently demonstrated pharmacologically. d-Tubocurarine was applied at a subblocking concentration to impair postsynaptic acetylcholine receptors. Within seconds of drug application, QC increased—and then within seconds of drug washout, it decreased again (Wang, X., 2016). Follow-up experiments suggested that those rapid, dynamic changes in PHP dynamics at the mouse NMJ were mediated by a calcium-dependent increase in the size of the RRP of presynaptic vesicles (Wang, X., 2016). Because there seem to be several similarities between the mouse NMJ and the Drosophila NMJ, it is possible that PH