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).

  • Transmission, Development, and Plasticity of Synapses - a review by Kathryn Harris & Troy Littleton

    Biology of the Presynapse
  • The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity
  • Genetic analysis in Drosophila reveals a role for the mitochondrial protein p32 in synaptic transmission
  • 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
  • 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
  • Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila
  • Presynaptic biogenesis requires axonal transport of lysosome-related vesicles
  • The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization
  • Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling
  • Structural and molecular properties of insect type II motor axon terminals
  • 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
  • A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity
  • 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
  • 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 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
  • 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
  • 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
  • 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
  • Na+ /H+ -exchange via the Drosophila vesicular glutamate transporter (DVGLUT) mediates activity-induced acid efflux from presynaptic terminals
  • 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 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
  • The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila
  • Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function
  • High-probability neurotransmitter release sites represent an energy-efficient design
  • Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles
  • Phosphorylation of Complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity
  • Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability
  • Fife organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release
  • The long 3'UTR mRNA of CaMKII is essential for translation-dependent plasticity of spontaneous release in Drosophila melanogaster
  • 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
  • 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
  • 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
  • Characterization of developmental and molecular factors underlying release heterogeneity at Drosophila synapses
  • Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability
  • 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
  • Reactive oxygen species regulate activity-dependent neuronal plasticity in Drosophila
  • Neuron-specific knockdown of Drosophila HADHB induces a shortened lifespan, deficient locomotive ability, abnormal motor neuron terminal morphology and learning disability

    Homeostasis and the biology of the Presynapse
  • 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
  • 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
  • A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation
  • 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
  • Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling

    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

    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
  • 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

    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

  • 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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    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 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).

    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, 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, 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, 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, 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, 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, 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, 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, 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).

    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, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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; Bergquistet, 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, 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, 2016). Because there seem to be several similarities between the mouse NMJ and the Drosophila NMJ, it is possible that PHP at the insect NMJ can also be rapidly reversed (Yeates, 2017).

    It is instructive to consider mammalian synaptic preparations and study how homeostatic forms of synaptic plasticity are turned on and off. Groundbreaking work on cultured excitatory vertebrate synapses showed that in response to activity deprivation (or promotion), synapses employ scaling mechanisms by adding (or subtracting) AMPA-type glutamate receptors to counteract the perturbation. Bidirectional scaling suggested that reversible mechanisms likely dictate homeostatic scaling processes. Complementary studies testing scaling reversibility have borne out this prediction. Additionally, evidence for reversible forms of homeostatic scaling have been found in rodent sensory systems, such as auditory synapses after hearing deprivation (and restoration to reverse) and in the visual cortex after light deprivation. Collectively, the vertebrate and invertebrate studies support the notion that reversible fine-tuning is an efficient strategy used to stabilize activities in metazoan nervous systems. One advantage offered by the Drosophila system is a genetic toolkit to uncover possible reversibility factors. Partial or failed homeostatic signaling at high temperatures (Yeates, 2017).

    Are there environmental limitations for homeostatic potentiation at the Drosophila NMJ? Clearly there are. The data suggest that a combination of high temperature (30°C-32°C) plus impaired glutamate receptor function can severely limit the NMJ's ability to compensate for reduced neurotransmitter sensitivity. High temperature alone does not seem to be a severe enough restriction to impair PHP, because Gal4 driver controls at 30°C have somewhat reduced quantal size, but a fully (or nearly fully) offsetting increase in QC. Likewise, reduced glutamate receptor function alone does not appear to be a sufficient barrier to impair PHP. For example, quantal size is severely diminished when the dominant negative UAS-GluRIIAM/R transgene is expressed at 25°C, but PHP is nevertheless intact (Yeates, 2017).

    It is not clear what the molecular or anatomic basis of this limit on PHP is. It is known that it is not an issue of NMJ excitation at high temperatures, because evoked neurotransmission for WT (or driver control) NMJs remains remarkably robust over a range of temperatures, including 30°C. Nor does it seem to be an elimination of PHP in general, because PHP was still present in the case of GluRIIASP16 animals raised at 30°C, revealing a distinction between the null GluRIIA condition and the dominant-negative GluRIIA condition. The limitation seems to be on homeostatic signaling that supports PHP at high temperatures in the face of the dominant-negative transgene expression (Yeates, 2017).

    Temperature effects on neurophysiology are well documented. Recent work in crustaceans demonstrates that robust and reliable circuits such as the neurons driving the rhythmicity stomatogastric nervous system can fail under extreme temperature challenges. For the Drosophila NMJ, prior studies of larval development documented a significant enhancement of synaptic arborization when larvae were raised at higher temperatures. Additional studies have shown that NMJ growth plasticity can be additionally affected by mutations that affect neuronal excitability. Given the backdrop of these data, it is not unreasonable to hypothesize that the tolerable limits of synaptic activity challenge could be different at different temperatures (Yeates, 2017).

    For the current experiments, 29°C-32°C represents a potential failure zone for homeostatic potentiation at the Drosophila NMJ. It must be noted that the data suggest that the coping capacity of the NMJ is dependent on genotype. WT NMJs cope at all temperatures. By contrast, for dominant-negative GluRIIA-expressing NMJs, 29°C is a point at which PHP becomes partial, and 30°C is a point at which it fails. For GluRIIASP16 subunit deletion NMJs, there is robust, but partial PHP at 30°C, not unlike the compensation seen for the dominant-negatives at 29°C. GluRIIASP16 NMJs fail to execute PHP only when temperature is pushed to an extreme range, such as 32°C. Why do these differences persist? It is not clear. The answer could relate to the well-documented temperature-induced alterations in NMJ growth, or alternatively, a limited availability of synaptic factors that are needed to cope with a double challenge of high temperature and particular impairment of glutamate receptor function. Future molecular and physiologic work will be needed to unravel those possibilities in the contexts of different genetic backgrounds and culturing conditions (Yeates, 2017).

    The Drosophila postsynaptic DEG/ENaC channel ppk29 contributes to excitatory neurotransmission

    The protein family of Degenerin/Epithelial Sodium Channels (DEG/ENaC) is comprised of diverse animal-specific, non-voltage-gated ion channels that play important roles in regulating cationic gradients across epithelial barriers. However, the specific neurophysiological functions of most DEG/ENaC-encoding genes remain poorly understood. This study demonstrates that ppk29 contributes specifically to the postsynaptic modulation of excitatory synaptic transmission at the larval neuromuscular junction (NMJ). Electrophysiological data indicate that the function of ppk29 in muscle is necessary for normal postsynaptic responsivity to neurotransmitter release, and for normal coordinated larval movement. The ppk29 mutation does not affect gross synaptic morphology and ultrastructure, which indicates the observed phenotypes are likely due to defects in glutamate receptor function. Together, these data indicate that DEG/ENaC ion channels play a fundamental role in the postsynaptic regulation of excitatory neurotransmission (Hill, 2017).

    The identities and functions of genes that regulate neuronal synaptic functions in health and disease remains a major goal of neuroscience research. Although the principle molecular mechanisms that regulate synapse formation and function are relatively well understood, mechanisms for synaptic plasticity, especially at the physiological timescale, are still mostly unknown. This study describes a novel role for pickpocket (ppk) 29, a member of the degenerin/epithelial sodium channel (DEG/ENaC) family of non-voltage-gated sodium channels, in the postsynaptic modulation of baseline excitatory neurotransmission at the Drosophila larval neuromuscular junction (NMJ) (Hill, 2017).

    Members of the DEG/ENaC family are exclusively found in animal genomes. They function as trimeric cation channels, which are expressed in both neuronal and non-neuronal tissues. DEG/ENaC channels can be gated by diverse extracellular stimuli, including extracellular ligands and mechanical forces (Ben-Shahar, 2011; Eastwood, 2012; Hill, 2017 and references therein).

    Several studies in invertebrate and mammalian species suggest that some DEG/ENaC family members also directly contribute to synaptic functions (Younger, 2013; Urbano, 2014; Ievglevskyi, 2016; Miller-Fleming, 2016), which may explain their reported contributions to long-term potentiation, learning and memory (Wemmie, 2002), and addiction (Kreple, 2014). In addition, mutations in DEG/ENaC-encoding genes have been implicated in neuropathologies such as multiple sclerosis and epilepsy (Wemmie, 2013). However, whether the observed neuronal and behavioral phenotypes of mutations in DEG/ENaC-encoding genes are due to presynaptic or postsynaptic processes is not well understood (Hill, 2017).

    In contrast to mammalian genomes, which typically harbor eight to nine independent DEG/ENaC-encoding genes, the genome of the fruit fly Drosophila melanogaster encodes >30 independent family members, named ppk genes. Analyses of mutations in several ppk genes indicates that Drosophila DEG/ENaC channels contribute to diverse sensory functions such as salt taste, water sensing, and the detection of mating pheromone. In addition, some ppk genes have been implicated in the maintenance of synaptic homeostasis (Younger, 2013). Nevertheless, the specific molecular mechanisms by which these channels exert their synaptic functions remain elusive (Hill, 2017).

    This study reports that ppk29, which has been reported previously as a neuronally enriched Drosophila DEG/ENaC subunit implicated in pheromone-sensing functions (Thistle, 2012; Mast, 2014; Vijayan, 2014; Yuan, 2014), is also required for normal neurotransmission at the larval NMJ, a model glutamatergic synapse, via postsynaptic processes, possibly via modulation of postsynaptic glutamate receptors (Hill, 2017).

    Despite their emerging importance in neurological and cognitive pathologies, the precise neurophysiological functions of DEG/ENaC channels remain elusive. This study demonstrates that a Drosophila DEG/ENaC-encoding gene, ppk29, is required for normal synaptic functions. However, in contrast to the ppk11/ppk16 complex (Younger, 2013), ppk29 action is restricted to the postsynaptic site and is associated with baseline spontaneous neurotransmission but not PhTX-dependent synaptic homeostasis. Therefore, it is proposed that individual DEG/ENaC-like channels may play independent roles in regulating synaptic functions, which may explain some of the contradicting reports about their functions in the mammalian synapse (Hill, 2017).

    Previous studies have shown that some DEG/ENaC-encoding genes are expressed in human skeletal muscles, with their function remaining unknown. In Caenorhabditis elegans, the DEG/ENaC-encoding gene unc-105 is also expressed in muscles, where it is important for proper muscle organization, growth, and contraction. The current data indicate that, in addition to the contributions of DEG/ENaC proteins to muscle development and physiology, they also contribute to excitatory neurotransmission via postsynaptic processes in muscle. Nonetheless, because the fly NMJ is glutamatergic, the findings presented in this study could also provide important insights about postsynaptic functions of DEG/ENaC signaling in glutamatergic central synapses of vertebrates (Hill, 2017).

    The postsynaptic impact of ppk29 on excitatory signaling may be mediated directly by PPK29 or indirectly via interaction with other proteins. Since currently available tools did not allow localization of PPK29 to specific subcellular compartments, it is too early to conclude whether the observed phenotypes in the ppk29 mutants are the consequence of a direct synaptic function or possibly of an indirect function via its action in other subcellular domains. It is also noted the that PPK29 may play more than one role in muscles and, therefore, that the electrophysiological and behavioral data may not be mediated through the same mechanism (Hill, 2017).

    Nevertheless, one intriguing way that PPK29 might directly contribute to synaptic transmission is by acting as a direct postsynaptic receptor for glutamate or other molecules that are coreleased during spontaneous excitatory neurotransmission. For example, in mouse brain slices, extracellular protons increase with the stimulation of glutamatergic inputs, which can activate acid-sensitive ion channels, which are also members of the DEG/ENaC family (Du, 2014). Therefore, although it is not known whether PPK29 is an acid-activated channel, the corelease of protons with glutamate during spontaneous neurotransmission at the Drosophila NMJ may directly activate PPK29 channels (Hill, 2017).

    The ppk29 gene may also affect neurotransmission indirectly through the modulation of expression or function of other synaptic proteins. Postsynaptic glutamate receptors are promising candidates to mediate an indirect impact of ppk29 on synaptic physiology since these ionotropic receptors are the main mediators of excitatory neurotransmission at the larval NMJ. This study found that ppk29 mutant animals exhibit decreased spontaneous amplitude and current flow, suggesting altered function of the postsynaptic ionotropic GluRs, which may be mediated by direct interaction between PPK29 and the GluR complex, or by indirect interaction via other postsynaptic signaling mechanisms. In line with this hypothesis, direct physical interactions between other DEG/ENaC proteins and potassium channels and sodium/chloride cotransporters have been reported in mammalian systems. It is further hypothesized that the observed differences in GluR expression levels are due to compensatory transcriptional changes. It is not known whether the ppk29 mutation independently impacts both GluRIIA and GluRIIB. Yet, previous studies have shown that genetic manipulation of expression levels of either GluRIIA or GluRIIB affect expression levels of the other (Marrus, 2004); therefore, the ppk29 mutation may directly or indirectly impact one or both subunit types (Hill, 2017).

    To date, studies of spontaneous neurotransmitter release at the Drosophila larval NMJ have suggested that its main function is to regulate the development and maintenance of excitatory synaptic transmission by regulating presynaptic morphology and the postsynaptic clustering of glutamate receptors. However, spontaneous neurotransmitter release at central synapses has also been shown to impact local protein synthesis in dendrites as well as dendritic summation of EPSPs at much shorter timescales. This study has identified an important function for DEG/ENaC channels at the physiological timescale, which has an impact on both neurophysiological and behavioral phenotypes. Although it is not known how the molecular action of PPK29 might affect synapses and behavior, it is argued that these findings about the contribution of DEG/ENaC-encoding genes to spontaneous excitatory neurotransmission at the Drosophila larval NMJ may serve as an excellent model for understanding the function of spontaneous baseline excitatory neurotransmission in regulating synaptic organization. Better understanding of these processes at the physiological timescale is essential for understanding behavioral and neural plasticity in health and disease (Hill, 2017).

    Synaptic excitation is regulated by the postsynaptic dSK channel at the Drosophila larval NMJ

    In the mammalian CNS, the postsynaptic small-conductance Ca2+-dependent K+ (SK) channel has been shown to reduce postsynaptic depolarization and limit Ca2+ influx through NMDA receptors. To examine further the role of the postsynaptic SK channel in synaptic transmission, its action was studied at the Drosophila larval NMJ. Repetitive synaptic stimulation produced an increase in postsynaptic membrane conductance leading to depression of EPSP amplitude and hyperpolarization of the resting membrane potential (RMP). This reduction in synaptic excitation was due to the postsynaptic Drosophila SK (dSK) channel; synaptic depression, increased membrane conductance and RMP hyperpolarization were reduced in dSK mutants or after expressing a Ca2+ buffer in the muscle. Ca2+ entering at the postsynaptic membrane was sufficient to activate dSK channels based upon studies in which the muscle membrane was voltage clamped to prevent opening voltage-dependent Ca2+ channels. Increasing external Ca2+ produced an increase in resting membrane conductance and RMP that was not seen in dSK mutants or after adding the glutamate-receptor blocker philanthotoxin. Thus, it appeared that dSK channels were also activated by spontaneous transmitter release and played a role in setting membrane conductance and RMP. In mammals, dephosphorylation by protein phosphatase 2A (PP2A) increased the Ca2+ sensitivity of the SK channel; PP2A appeared to increase the sensitivity of the dSK channel since PP2A inhibitors reduced activation of the dSK channel by evoked synaptic activity or increased external Ca2+. It is proposed that spontaneous and evoked transmitter release activate the postsynaptic dSK channel to limit synaptic excitation and stabilize synapses (Gertner, 2014).

    Stimulation of the neuromuscular synapses at 20 Hz resulted in an increase in resting Gin (input conductance), and a decrease in synaptic excitation due to membrane hyperpolarization and a decrease in EPSP amplitude. Simulations predicted that the increase in resting Gin during stimulation accounted for about one-third of the depression of EPSP amplitude due to shunting of the synaptic current. Synaptic depression is generally held to be due to a reduction in transmitter release; thus the remaining decrease in EPSP amplitude may have resulted from a decline in transmitter release. Nonetheless, evidence is provided for a change in postsynaptic conductance contributing to synaptic depression. The increase in Gin and RMP was consistent with activation of a gKCa; this was further supported by the reduced increase in Gin and RMP seen after expressing th Ca2+ buffer parvalbumin in the muscle (Gertner, 2014).

    Larval muscle fibers contain two types of Ca2+-dependent K+ channels producing gCF and gCS. gCF was found to result from the slowpoke channel, a BK-type channel showing voltage- and Ca2+-dependent activation and voltage-dependent inactivation, and the dSK channel was proposed to produce gCS (Abou Tayoun, 2011). Western blots showed a single dSK isoform in larval brain and muscle; a Western blot of adult fly brains showed two closely spaced bands, which could have represented two dSK isoforms or a single isoform along with its posttranslationally altered form (Abou Tayoun, 2011). SK channels are not voltage-gated, and calmodulin acts as their Ca2+ sensor, resulting in a relatively high Ca2+ sensitivity. Repetitive synaptic stimulation activated the postsynaptic dSK channels, since the increase in Gin and RMP was eliminated in dSK or 24B/dSKDN larvae, but not in slo1 larva. As expected, there was reduced synaptic depression in dSK mutants and PV-expressing larvae; however, it was surprising that synaptic depression was completely eliminated. This suggests that there was also less depression of transmitter release in these larvae (Gertner, 2014).

    It is assumed that the dSK channel is activated during evoked transmitter release in vivo, since the 20-Hz stimulation applied in this study likely falls within the physiological range of activity. The Ib synapses on muscle fiber 6 fire on average 20–30 Hz, and the Is terminals fire less than 10 Hz in dissected larvae; however, these firing frequencies may be greater in vivo since the waves of contraction are slow in dissected larvae, probably due to lack of appropriate sensory input. Also, since HL3 with low Ca2+ was used for the stimulation procedure, the postsynaptic Ca2+ influx was less than would occur under physiological conditions (Gertner, 2014).

    Ca2+ entering at the postsynaptic membrane was sufficient to activate dSK channels, since evoked transmitter release produced an increase in Gin even when the muscle was voltage clamped at −60 mV. Under these conditions, it is expected that Ca2+ entry is limited to glutamate receptors, since voltage-dependent Ca2+ channels in larval muscle open at membrane potentials more positive than −30 mV. However, it cannot be ruled that there was a voltage drop across the subsynaptic reticulum (SSR), such that voltage-dependent Ca2+ channels near the glutamate receptors remained unclamped. In this case, both the glutamate receptors and nearby Ca2+ channels could have been the source of Ca2+ that activated the dSK channel. In fact, dSK channels in larval muscle can be activated by Ca2+ entering through voltage-dependent Ca2+ channels since depolarization of the muscle in the absence of synaptic activity produced an increase in gCS. Additional evidence that the glutamate receptor can act as the Ca2+ source comes from the activation of the dSK channel by spontaneous transmitter release; here, it seems unlikely that the SSR resistance would be great enough for quantal currents to open voltage-dependent Ca2+ channels. It appears that dSK channels exist at the postsynaptic membrane near the glutamate receptors, and this is consistent with findings in mammals, where the SK channel has been shown to be closely coupled to postsynaptic nicotinic acetylcholine receptors and NMDA receptors (Gertner, 2014).

    The dSK channels were activated at rest and were responsible for the previously reported dependence of the RMP on external Ca2+. This was based upon the finding that the increase in Gin and RMP typically seen when increasing external Ca2+ from 0 to 1.5 mM was not seen in dSK larvae. In fact, it appeared that activation of the dSK channel is responsible for much of the normal variability in the RMP, since resting Gin and the RMP were positively correlated in wild-type larvae, but not in dSK larvae. Spontaneous transmitter release apparently activates dSK channels to set the resting Gin and RMP, since blocking the glutamate receptors prevented the rise in Gin and RMP produced by increasing external Ca2+. This is a novel function for spontaneous transmitter release, but not entirely unexpected, since spontaneous transmitter release produced postsynaptic Ca2+ transients similar in amplitude to evoked release (Gertner, 2014).

    The spontaneous Ca2+ transients must have produced an increase in resting Gin that outlasted the Ca2+ signal, since the frequency of spontaneous events in this muscle is usually about 1 Hz, and the spontaneous Ca2+ transients had a decay time constant of ∼50 ms. The increase in Gin produced by repetitive synaptic stimulation also appeared to outlast the increase in [Ca2+]i, since it persisted for about 10 min after stimulation. Previous studies have found that after 5 s of 10-Hz stimulation, postsynaptic [Ca2+]i decayed with a time constant of about 100 ms, and this decay was largely due to the plasma membrane Ca2+ ATPase, which appeared to be highly enriched in the SSR. The SSR may limit Ca2+ diffusion to enhance the amplitude of postsynaptic Ca2+ transients; however, it seems likely that it also reduces their duration by providing efficient postsynaptic Ca2+ extrusion. Thus it seems improbable that postsynaptic [Ca2+]i remained elevated for 10 min after the stimulation train (Gertner, 2014).

    There was an increase in leakage conductance (gL) in dSK larvae. This is consistent with previous studies showing covariation of ion conductances. In particular, reduced activation of Ca2+-dependent K+ currents in Drosophila cultured neurons resulted in a compensatory upregulation of the transient K+ current, apparently due to increased expression of transient K+ current channels. Although the channels responsible for gL have not been characterized in larval muscle, they may include ORK1 which was identified as a K+ leak channel in Drosophila and appears to be expressed in adult muscle; it is a member of the two-pore domain K+ channels identified in mammals. The nonselective cation channel NALCN produces the Na+ leak conductance in mammalian neurons and its ortholog na is expressed in Drosophila neurons, raising the possibility that it could also be responsible for the Na+ leak conductance in larval muscle (Gertner, 2014).

    In mammals, CK2 and PP2A are constitutively bound to the SK channel and regulate the phosphorylation state of calmodulin, which determines the Ca2+ sensitivity and deactivation rate of the SK channel. Dephosphorylation of calmodulin by PP2A can reduce EC50 to 0.3 μM Ca2+ and phosphorylation of the SK channel by CK2 can increase EC50 to 2.0 μM. An increase in [Ca2+]i should shift the balance toward dephosphorylation and produce an increase in Ca2+ sensitivity, since CK2 cannot phosphorylate calmodulin when the channel is open. The current results showed that the dSK channel is also regulated by PP2A, since inhibiting PP2A reduced the increase in Gin and RMP produced by repetitive stimulation or increasing external Ca2+. During repetitive stimulation, the increase in postsynaptic [Ca2+]i may have increased the Ca2+ sensitivity of the dSK channel. Similarly, it may be that the Ca2+ transients produced by spontaneous transmitter release produced an increase in Ca2+ sensitivity so that the dSK channel is partially activated by resting [Ca2+]i. If the Ca2+-dependent increase in Ca2+ sensitivity persisted, this could explain the lasting increase in Gin seen after the end of 20-Hz stimulation and after spontaneous Ca2+ transients (Gertner, 2014).

    The current results demonstrate that Ca2+ entering at the postsynaptic membrane during transmitter release provides negative feedback on synaptic excitation. This could rapidly stabilize the synapse and dampen the effects of changes in impulse activity, transmitter release or postsynaptic sensitivity. The effect of gSK on synaptic excitation was modeled using a Gsyn of 500 nS. The resting gSK in 1 mM (HL3) and 1.5 mM external Ca2+ (HL3.1) would make the EPSP peak about 3 mV and 14 mV more negative, respectively, mainly due to hyperpolarization of the RMP. An increase in gSK from 0 to 1 μS (approximately the value seen after 20 Hz stimulation) made the EPSP peak 22 mV more negative due to both RMP hyperpolarization and a decrease in EPSP amplitude. Activation of the dSK channel by evoked transmitter release is consistent with this negative-feedback mechanism, but what is the function of dSK channel activation by spontaneous transmitter release? minEPSP frequency is elevated after repetitive synaptic activity, and this could produce a persistent reduction in synaptic excitation. Also, since greater minEPSP frequency and amplitude would predict a larger EPSP, spontaneous transmitter release could set gSK to oppose strong synaptic excitation (Gertner, 2014).

    This action of the postsynaptic dSK channel is similar to that found at mammalian central nervous system synapses, where the SK channel counteracts synaptic changes: experiments blocking SK channels with apamin, or overexpressing them, showed that activation of SK channels increased the threshold for the induction of LTP and impaired learning. At these synapses, it was proposed that SK channels act specifically to restrict Ca2+ entry through NMDA receptors limiting LTP and learning and memory. The current findings show that dSK channels play a more general role in regulating synaptic excitation (Gertner, 2014).

    The dSK channel could act in concert with forms of homeostatic synaptic plasticity; these have involved compensatory changes in transmitter release at the NMJ, and most operate over long time scales. A failure of the dSK channel to maintain appropriate synaptic excitation could result in compensatory changes in transmitter release; this is supported by the apparent decrease in transmitter release seen when dSK channel activity was reduced in dSK mutants or by expressing PV in the muscle. This is consistent with previous findings that expressing additional K+ channels in Drosophila larval muscle resulted in a homeostatic increase in transmitter release (Gertner, 2014).

    At mammalian central synapses, the SK channel reduced the amplitude of the EPSC; single EPSPs and EPSCs were larger after adding the SK-channel blocker apamin. Thus the SK channel was rapidly activated during synaptic transmission so that the resultant EPSC was a composite of the inward current through the postsynaptic receptor and outward current through the SK channel. This study did not directly examined whether the dSK channel influences the amplitude of the EPSC; this is made difficult since apamin does not block dSK channels. Evidence is available that dSK currents reduced the duration of the EPSC, but further experiments are required to characterize fully the contribution of the dSK current to the EPSC (Gertner, 2014).

    The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency

    The TGFβ pathway has essential roles in embryonic development, organ homeostasis, tissue repair and disease. These diverse effects are mediated through the intracellular effectors SMAD2 and SMAD3 (hereafter SMAD2/3), whose canonical function is to control the activity of target genes by interacting with transcriptional regulators. Therefore, a complete description of the factors that interact with SMAD2/3 in a given cell type would have broad implications for many areas of cell biology. This study describes the interactome of SMAD2/3 in human pluripotent stem cells. This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, a functional interaction was identified with the METTL3-METTL14-WTAP complex, which mediates the conversion of adenosine to N6-methyladenosine (m6A) on RNA4. SMAD2/3 promotes binding of the m6A methyltransferase complex to a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor gene NANOG, priming them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulation of the epitranscriptome. These aspects of TGFβ signalling could have far-reaching implications in many other cell types and in diseases such as cancer (Bertero, 2018)

    The Drosophila postsynaptic DEG/ENaC channel ppk29 contributes to excitatory neurotransmission

    Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila

    Mechanisms underlying synaptic differentiation, which involves neuronal membrane and cytoskeletal remodeling, are not completely understood. This study performed a targeted RNAi-mediated screen of Drosophila BAR-domain proteins and identified islet cell autoantigen 69 kDa (dICA69) as one of the key regulators of morphological differentiation of larval neuromuscular junction (NMJ). Drosophila ICA69 colocalizes with α-Spectrin at the NMJ. The conserved N-BAR domain of dICA69 deforms liposomes in vitro. Full length and ICAC but not the N-BAR domain of dICA69 which induces filopodia in cultured cells. Consistent with its cytoskeleton regulatory role, dICA69 mutants show reduced α-Spectrin immunoreactivity at the larval NMJ. Manipulating levels of dICA69 or its interactor dPICK1 alters synaptic level of ionotropic glutamate receptors (iGluRs). Moreover, reducing dPICK1 or dRab2 levels phenocopies dICA69 mutation. Interestingly, dRab2 regulates not only synaptic iGluR but also dICA69 levels. Thus, these data suggest that: a) dICA69 regulates NMJ organization through a pathway that involves dPICK1 and dRab2, and b) dRab2 genetically functions upstream of dICA69 and regulates NMJ organization and targeting/retention of iGluRs by regulating dICA69 levels (Mallik 2017).

    This study demonstrates that ICA69 regulates NMJ structural organization and synaptic levels of glutamate receptor clusters. The findings suggest a model in which Rab2 functions genetically upstream of ICA69 to regulate its synaptic level, which in turn regulates the Spectrin cytoskeleton and iGluRs at the NMJ (Mallik, 2017).

    The requirement of ICA69 for Drosophila NMJ organization is strongly supported by its enrichment in the postsynaptic Spectrin-rich scaffold. Consistent with this idea, ICA69 mutants or animals with downregulated ICA69 levels show reduced arborization and bouton numbers at the NMJ. Several studies have shown that cytoskeletal regulation is a key process for NMJ development. Multiple lines of evidence suggest that ICA69 promotes NMJ growth by regulating the cytoskeletal network surrounding the SSR. First, ICA69 is highly enriched at the NMJ in the same microdomain as Spectrin. Second, ICA69 induces filopodia in cultured cells and relocalizes positive regulators of actin polymerization at the filopodia. Third, mutation in ICA69 significantly reduces α-Spectrin levels. The Actin-Spectrin scaffold at the postsynapse has been implicated in regulation of NMJ organization in postembryonic development in Drosophila. This study reveals a crucial requirement of ICA69 in regulating synaptic α-Spectrin levels and indicates that ICA69 is required for the assembly of Actin-Spectrin scaffolds surrounding the SSR. Whether localization and/or stability of postsynaptic Spectrin-Actin scaffold depends on direct interaction between scaffold components and ICA69 or on some unknown signaling mechanism needs to be further investigated (Mallik, 2017).

    For the proper establishment of NMJ connections, neurons as well as muscles require trafficking of various synaptic proteins. Rab GTPases and their regulators are considered to be some of the most important signaling molecules for intracellular trafficking. Interestingly, nearly half of all the Drosophila Rab proteins function specifically in neurons and a few of them localize to the NMJs. ICA69 physically associates with Rab2 and has been suggested as one of its effectors in regulating dense core vesicle maturation in Caenorhabditis elegans. This study found that Rab2 endogenous regulatory sequence-driven Rab2EYFP is detectable in the larval muscles as punctate structures, suggesting its involvement in NMJ organization. This idea is supported by four compelling pieces of evidence. First, ubiquitous or muscle-specific knockdown of Rab2 phenocopies ICA69 mutants. Second, knockdown of Rab2 significantly reduces synaptic α-Spectrin levels. Third, Rab2 directly regulates synaptic ICA69 levels. Fourth, co-expressing an ICA69 transgene and Rab2 RNAi rescues the morphological defects of Rab2 RNAi. Based on these observations, it is suggested that Drosophila Rab2 functions genetically upstream of ICA69. Like Rab2, PICK1 depletion reduced synaptic ICA69 levels and phenocopied the NMJ morphological defects observed in ICA69 mutants or after Rab2 depletion. Moreover, simultaneous knockdown of ICA69 and PICK1 or of ICA69 and Rab2 did not show an additive effect on the NMJ structural defects. These observations support the notion that ICA69, PICK1 and Rab2 might function in the same genetic pathway to regulate NMJ structural organization (Mallik, 2017).

    In mammalian neurons, ICA69 is, surprisingly, not enriched at the synapses and negatively regulates AMPA receptor trafficking. Hence, it is expected that ICA69 mutants would have normal, if not more, iGluR clusters at the NMJ. Contrary to this expectation, reducing the ICA69 level resulted in reduced GluRIIA as well as GluRIIB glutamate receptor clusters. A recent study has shown that ICA69 and PICK1 stability is interdependent in Drosophila brain. Thus, it is likely that iGluR clusters at the NMJ are regulated by levels of ICA69 and PICK1 in muscles (Mallik, 2017).

    How does ICA69 reduce iGluR levels both in knockdown and overexpression scenarios? It is suggested that the endogenous stoichiometry of ICA69 and PICK1 is crucial for normal synaptic targeting of iGluRs at the Drosophila NMJ. Reducing ICA69 destabilizes the ICA69-PICK1 heteromeric complex thereby reducing PICK1 availability for synaptic targeting of iGluRs. Overexpression of ICA69 forms more of the ICA69-PICK1 inhibitory complexes, which reduces synaptic targeting of iGluRs. Hence, the idea that the endogenous level of ICA69 is crucial for maintaining normal glutamate receptor clusters at the synapses is supported (Mallik, 2017).

    The data suggest that ~40% simultaneous reduction of GluRIIA/IIB/III at Drosophila NMJ synapses has no major consequence on larval synaptic physiology. Three possibilities are suggested to explain this. First, the relative levels of GluRIIA and GluRIIB subunits are crucial for determining the efficacy of synaptic transmission at the Drosophila NMJ synapse . The decrease for each of the GluRIIA, -IIB and -III subunits in the ICA69 mutant is almost identical; ~40% compared with controls. This hints towards a homeostatic compensatory mechanism whereby ~60% of the receptor subunits are sufficient to form enough functional receptor complexes, which can maintain normal synaptic strength. Second, the amount of GluRIII is reflective of the sum of GluRIIA and -IIB complexes together, and GluRIII is essential for the localization of GluRIIA and IIB subunits. A 40% decrease in GluRIII staining correlates well with an identical decrease in GluRIIA and -IIB staining. It is plausible that there is essentially negligible change in functional glutamate receptor assembly at ICA69 mutant synapses. Third, ICA69 possibly plays a role in trafficking glutamate receptors to the postsynaptic density and is not rate limiting in the formation of functional glutamate receptor complexes. Thus, ICA69 mutants exhibit normal synaptic physiology without embracing other compensatory mechanisms such as reduced quantal size or increased quantal content (Mallik, 2017).

    How might the iGluR levels relate to the NMJ growth? A tight correlation exists between the amount of synaptic glutamate receptors and the NMJ morphology. Downregulation of iGluRs in muscles has been shown to reduce the number of boutons. Similarly, hypomorphic mutants of GluRIII or GluRIIA have reduced bouton numbers. Consistent with this, overexpression of GluRIIA induces arborization and bouton number. Moreover, mutants with altered synaptic iGluR levels also show altered bouton numbers. For instance, neto and filamin (cheerio) mutants show reduced iGluR levels and bouton numbers. One of the possible mechanisms by which glutamate receptors can alter the NMJ morphology is through regulation of synaptic phospho-MAD levels. As the iGluRs (for instance, GluRIID) have also been shown to localize in central neuropil, it remains a possibility that the endogenous pattern of central electrical activity could also play crucial roles in sculpting the NMJ during development (Mallik, 2017).

    Development of a tissue-specific ribosome profiling approach in Drosophila enables genome-wide evaluation of translational adaptations

    Recent advances in next-generation sequencing approaches have revolutionized understanding of transcriptional expression in diverse systems. However, measurements of transcription do not necessarily reflect gene translation, the process of ultimate importance in understanding cellular function. To circumvent this limitation, biochemical tagging of ribosome subunits to isolate ribosome-associated mRNA has been developed. However, this approach, called TRAP, lacks quantitative resolution compared to a superior technology, ribosome profiling. This study reports the development of an optimized ribosome profiling approach in Drosophila. First, successful ribosome profiling was demonstrate from a specific tissue, larval muscle, with enhanced resolution compared to conventional TRAP approaches. Next the ability of this technology to define genome-wide translational regulation was validated. This technology was leveraged to test the relative contributions of transcriptional and translational mechanisms in the postsynaptic muscle that orchestrate the retrograde control of presynaptic function at the neuromuscular junction. Surprisingly, no evidence was found that significant changes in the transcription or translation of specific genes are necessary to enable retrograde homeostatic signaling, implying that post-translational mechanisms ultimately gate instructive retrograde communication. Finally, it was shown that a global increase in translation induces adaptive responses in both transcription and translation of protein chaperones and degradation factors to promote cellular proteostasis. Together, this development and validation of tissue-specific ribosome profiling enables sensitive and specific analysis of translation in Drosophila (Chen, 2017).

    Acute fasting regulates retrograde synaptic enhancement through a 4E-BP-dependent mechanism

    While beneficial effects of fasting on organismal function and health are well appreciated, little is known about the molecular details of how fasting influences synaptic function and plasticity. Genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, these findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. These results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits (Kauwe, 2016).

    Many forms of dietary restriction can reduce cellular stress, improve organismal health, and in many instances extend lifespan in a number of model organisms. A major cellular function that is highly sensitive to nutrient intake from yeast to mammals is cap-dependent translation under the regulation of the target of rapamycin (TOR). TOR promotes cap-dependent translation primarily through phosphorylation of 4E-BPs (eukaryotic initiation factor 4E binding proteins) and p70 S6Ks (S6 ribosomal protein kinases). Phosphorylation of 4E-BP suppresses its ability to bind and inhibit the interaction between eIF4E (eukaryotic initiation factor 4E) and the initiation factor eIF4G, a critical step for translation initiation. In addition to the regulation by TOR, 4E-BP undergoes upregulation in response to dietary restriction and starvation. Together these two responses result in a strong inhibition of protein synthesis and act as a metabolic brake. Multiple lines of evidence suggest that fasting-induced increase in ketone bodies influences neuronal excitability and aspects of neurotransmitter release; however, little is known about how different forms of dietary restriction, by influencing protein translation, can exert an effect on the regulation of synaptic function and plasticity (Kauwe, 2016).

    At the Drosophila larval neuromuscular junction (NMJ), the genetic removal of GluRIIA, one of five glutamate receptor subunits, reduces the postsynaptic response to unitary release of neurotransmitter. As a result of this reduced response to neurotransmitter, a retrograde signal is triggered in the postsynaptic muscle that ultimately leads to a compensatory enhancement in presynaptic release from the motor neuron, a process that is conserved at the vertebrate NMJs. The maintenance of this homeostatic synaptic compensation or retrograde synaptic enhancement is highly sensitive to postsynaptic cap-dependent translation in Drosophila; mutations in either Target of Rapamycin (TOR) or eIF4E can dominantly suppress the synaptic compensation in GluRIIA mutants (Penney, 2012). Interestingly, postsynaptic overexpression of TOR or S6K, in an otherwise wild-type muscle, is also sufficient to trigger a retrograde enhancement in presynaptic neurotransmitter release, suggesting that normal synaptic strength may be affected by a postsynaptic signal from the muscle (Penney, 2012; Kauwe, 2016 and references therein).

    Previous findings have demonstrated that postsynaptic translation plays a critical role in the regulation of retrograde synaptic enhancement at the NMJ. Therefore, in light of the effect of dietary restriction on TOR-dependent translation, this study set out to investigate the consequence of nutrient restriction on retrograde synaptic compensation in GluRIIA mutants. Electrophysiological analysis indicates that acute fasting, but not amino acid restriction, blocks this retrograde synaptic compensation. This block is not merely due to reduced TOR activity, but rather a result of transcriptional upregulation of postsynaptic 4E-BP under the control of the transcription factor Foxo. These results indicate that the retrograde regulation of synaptic strength at the NMJ depends on the balance between 4E-BP and TOR (Kauwe, 2016).

    A few hours of fasting can have a strong impact on retrograde synaptic enhancement at the Drosophila larval NMJ. Removal of food source acutely activates 4E-BP transcription in postsynaptic muscles in a Foxo-dependent manner, thereby leading to the inhibition of retrograde synaptic enhancement at the NMJ. The results indicate that Foxo and 4E-BP act cell autonomously in postsynaptic muscles to exert a retrograde negative regulation on presynaptic neurotransmitter release. Future studies are needed to test whether fasting-induced alterations in insulin signaling underlie the transcriptional upregulation of 4E-BP via its effect on Foxo in postsynaptic muscles. While 4E-BP-mediated suppression of synaptic enhancement as a result of fasting could be considered undesirable during development, it can be beneficial under conditions of abnormally high synaptic activity. As such, 4E-BP-mediated inhibition of retrograde synaptic enhancement and the subsequent dampening of circuit activity might provide an explanation for the beneficial effects of fasting in reducing seizures in some cases. Similarly, in cases where dysregulation of TOR activity is thought to underlie abnormal circuit activity, such as in TSC models, intermittent fasting could potentially dampen the increase in synaptic release through a 4E-BP-dependent inhibition, thereby stabilizing neuronal circuits (Kauwe, 2016).

    In addition to its role as a molecular responder to stress, 4E-BP exerts a constitutive negative regulation on presynaptic neurotransmitter release at the NMJ. Electrophysiological analysis of loss-of-function mutant larvae indicates that 4E-BP functions in postsynaptic muscles to constitutively provide a retrograde negative influence on synaptic strength. In light of these findings, a two-pronged scheme is proposed for the retrograde regulation of synaptic strength at the NMJ. On the one hand, a positive input from TOR is mediated through S6K/eIF4A and eIF4E to enhance postsynaptic translation. Synaptic compensation in GluRIIA mutant larva appears to rely mostly on this axis as evidenced by strong sensitivity to S6K heterozygosity and no change in the proportion of phosphorylated 4E-BP versus non-phosphorylated 4E-BP levels. Opposing this positive input, 4E-BP inhibits translation by sequestering eIF4E and adjusting the degree of retrograde compensation. Indeed, loss of 4E-BP leads to a strong increase in quantal content that is highly sensitive to eIF4E heterozygosity but not sensitive to S6K heterozygosity. The balance between these two forces reveals itself also when 4E-BP loss-of-function mutants are rescued by a non-phosphorylatable 4E-BP transgene. In this combination TOR can no longer inhibit 4E-BP, and this study finds that the presynaptic neurotransmitter release is lower than wild-type, similarly to what is observed in TOR hypomorphic mutants (Penney, 2012). A working model is proposed in which the negative force of 4E-BP is under constant check via phosphorylation by TOR, and the positive input from TOR/S6K is constitutively countered by 4E-BP’s ability to sequester eIF4E, a dynamic duel that ensures a tight regulation of synaptic strength (Kauwe, 2016).

    Disparate postsynaptic induction mechanisms ultimately converge to drive the retrograde enhancement of presynaptic efficacy

    Retrograde signaling systems are fundamental modes of communication synapses utilize to dynamically and adaptively modulate activity. However, the inductive mechanisms that gate retrograde communication in the postsynaptic compartment remain enigmatic. This study investigated retrograde signaling at the Drosophila neuromuscular junction, where three seemingly disparate perturbations to the postsynaptic cell trigger a similar enhancement in presynaptic neurotransmitter release. This study shows that the same presynaptic genetic machinery and enhancements in active zone structure are utilized by each inductive pathway. However, all three induction mechanisms differ in temporal, translational, and CamKII activity requirements to initiate retrograde signaling in the postsynaptic cell. Intriguingly, pharmacological blockade of postsynaptic glutamate receptors, and not calcium influx through these receptors, is necessary and sufficient to induce rapid retrograde homeostatic signaling through CamKII. Thus, three distinct induction mechanisms converge on the same retrograde signaling system to drive the homeostatic strengthening of presynaptic neurotransmitter release (Goel, 2017).

    The Drosophila neuromuscular junction (NMJ) is an established system to study retrograde synaptic signaling. At this model glutamatergic synapse, genetic or pharmacological perturbations to postsynaptic receptor functionality trigger retrograde signaling that instructs the neuron to precisely increase presynaptic neurotransmitter release, maintaining stable levels of synaptic strength. This process is termed presynaptic homeostatic potentiation (PHP) and can be induced through two distinct disruptions to postsynaptic glutamate receptor functionality. First, acute pharmacological blockade of receptors by application of philanthotoxin-433 (PhTx) reduces miniature excitatory postsynaptic potential (mEPSP) amplitude, initiating rapid expression of PHP (increase in quantal content) within 10 min. Second, genetic loss of the postsynaptic glutamate receptor subunit GluRIIA leads to a similar reduction in mEPSP amplitudes over chronic timescales (days) and a similar expression of PHP. Although these perturbations each disrupt receptors and lead to adaptive increases in presynaptic neurotransmitter release, PhTx- and GluRIIA-mediated PHP signaling exhibit important differences. First, some genes have been identified that are only necessary for GluRIIA-dependent PHP expression, whereas PHP is robustly expressed following acute PhTx application in larvae with mutations in these genes (Frank, 2009, Kauwe, 2016, Penney, 2016, Spring, 2016, Tsurudome, 2010). In addition, PhTx-induced PHP expression is translation-independent (Frank, 2006), whereas GluRIIA-induced PHP is blocked by inhibitions to postsynaptic translation through loss of the translational regulator target of rapamycin (Tor) (Kauwe, 2016, Penney, 2012). Although several genes and mechanisms necessary for the expression of PHP in the presynaptic neuron have been identified, far less is known about the mechanistic differences in postsynaptic transduction between PhTx- and GluRIIA-induced PHP signaling (Goel, 2017).

    Recently, a novel manipulation to the postsynaptic muscle that does not affect glutamate receptors was demonstrated to induce retrograde PHP signaling at the Drosophila NMJ. This was accomplished by postsynaptic overexpression of the non-specific translational regulator Tor (Tor-OE) (Penney, 2012), which leads to a chronic and global increase in muscle protein synthesis (Chen, 2017). Although Tor-OE does not functionally affect glutamate receptors, somehow the increased muscle protein synthesis is converted into an instructive retrograde signal that appears to induce an enhancement in presynaptic glutamate release of a magnitude comparable with that observed in PhTx- and GluRIIA-mediated PHP (Penney, 2012). Although PhTx application, loss of GluRIIA, and Tor-OE each induce a similar enhancement in presynaptic release, to what extent they utilize separate or shared postsynaptic induction pathways, retrograde signaling systems, and modulations to presynaptic function is not known (Goel, 2017).

    This study has characterized PHP signaling and expression when induced through PhTx application, loss of GluRIIA, and Tor-OE. This analysis has revealed that a common retrograde signaling system drives similar homeostatic adaptations in the presynaptic terminal but that separate inductive pathways differentially respond to glutamate receptor perturbation, Ca2+/calmodulin-dependent protein kinase II (CamKII) activity, and protein synthesis (Goel, 2017).

    There appears to be a core set of genes necessary for both acute and chronic PHP expression, including ones involved in the homeostatic modulation of synaptic vesicle trafficking, presynaptic excitability, calcium channel activity, and active zone remodeling. However, other genes appear to be dispensable for this core program and may rather be involved in secondary functions, such as maintaining PHP expression over chronic timescales or supporting other aspects of homeostatic adaptation. Interestingly, the existence of multiple retrograde signaling pathways may be one reason for the failure of forward genetic approaches to identify any individual genes required in the muscle for the core process of PHP induction, suggesting some level of redundancy. This convergence of diverse induction mechanisms in the postsynaptic cell enables multiple pathways to detect and respond to homeostatic challenges by feeding into a unitary retrograde signaling system that potentiates presynaptic neurotransmitter release to stabilize synaptic strength (Goel, 2017).

    CamKII activity plays a crucial role in gating diverse forms of synaptic plasticity. At the Drosophila NMJ, transgenic manipulations that affect postsynaptic CamKII activity have been reported to modulate the expression of PHP in GluRIIA mutants (Haghighi, 2003, Newman, 2017). The current results indicate that pCamKII levels are reduced to similar levels in GluRIIA mutants or following acute pharmacological receptor blockade, consistent with CamKII activity being capable of modulation in seconds at postsynaptic compartments. An attractive model would be that a reduction in calcium influx, either over 10 min or during chronic timescales, triggers diminished pCamKII levels and activates PHP signaling. However, this study found that pharmacological blockade of receptors is necessary and sufficient to reduce pCamKII levels at postsynaptic densities, independent of extracellular calcium, and that incubation in calcium-free saline alone is not sufficient to acutely induce PHP expression. Although there are several indications that reduced calcium influx in the postsynaptic muscle over chronic timescales likely contributes to PHP signaling, perhaps necessitating translation-dependent pathways, a calcium-independent system drives the acute expression of PHP following PhTx application, implying a distinct mechanism (Goel, 2017).

    Two possibilities are considered to explain how PhTx application to glutamate receptors is transduced into PHP retrograde signaling without requiring calcium signaling through extracellular sources. First, PhTx binding to receptors may induce a conformational perturbation, distinct from ion influx through the receptor, to initiate PHP signaling. Such a mechanism could operate through a metabotropic mechanism, which would be unanticipated but not unprecedented. For example, at mammalian central synapses, the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long term depression (LTD) does not require calcium influx through NMDA, but, rather, pharmacological perturbation to the receptor is sufficient. A metabotropic pathway has been proposed. Further, mammalian kainate receptors, to which the Drosophila glutamate receptors are homologous, are also capable of signaling through metabotropic mechanisms. Thus, pharmacological perturbation to GluRIIA-containing receptors could, in principle, initiate PHP signaling through an undefined metabotropic mechanism. However, at present, there is no evidence for such a mechanism in Drosophila (Goel, 2017).

    Alternatively, pharmacological disruption of glutamate receptors may lead to local signaling at the NMJ through interactions with scaffolds such as Discs large (DLG)/PSD-95 and Dalcium/calmodulin dependent serine protein kinase (CASK). These scaffolds are known to be in complexes with CamKII and capable of modulating CamKII activity and phosphorylation at the subsynaptic reticulum (SSR). Intriguingly, defects in the elaboration of the SSR have recently been reported to disrupt retrograde homeostatic plasticity (Koles, 2015). CamKII signaling during PHP appears to be restricted to postsynaptic densities of type 1b boutons (Newman, 2017), suggesting that compartmentalized signaling at the SSR orchestrates local PHP signal transduction. In contrast, Tor-OE is capable of initiating PHP signaling independent of pCamKII reduction, where it promotes translation throughout the cell. This implies that protein synthesis modulates retrograde signaling downstream of or in parallel to CamKII signal transduction but ultimately feeds back into local post-translational signaling pathways. Future experiments probing the interactions between glutamate receptors, postsynaptic scaffolds, translation, and CamKII activity will clarify the signaling at this compartmentalized synapse (Goel, 2017).

    The finding that PHP can be acutely induced by pharmacological perturbation of glutamate receptors and not through reductions in calcium influx over rapid timescales may help to explain perplexing observations regarding the phenomenology of PhTx-mediated PHP. For example, it was noted that PHP can be induced and expressed by a 10-min incubation of PhTx with only mEPSP events occurring. Although a reduction in calcium during these mEPSP events was discussed as a possible induction mechanism, estimates are that, at most, six mEPSP events occur per active zone during this induction time, a very low level and frequency of activity to reliably and robustly produce PHP expression. Indeed, a recent study demonstrated that mEPSP events account for a very small fraction (<1%) of the total postsynaptic calcium signal at individual NMJs (Newman, 2017), making a reduction in calcium even more implausible to explain acute PHP induction. Hence, pharmacological perturbation of postsynaptic glutamate receptors, rather than a reduction in calcium through these receptors, is an attractive mechanism to explain the characteristics of the acute induction of PHP by PhTx and raises interesting questions for future studies about how pharmacological receptor perturbation is transduced into PHP induction (Goel, 2017).

    Why might a single retrograde signaling system exist to homeostatically stabilize synaptic strength at the Drosophila NMJ? In central neurons, diverse forms of synaptic plasticity, including Hebbian and homeostatic, dynamically operate over multiple timescales to bi-directionally adjust synaptic strength. Further, translation-dependent and independent processes also contribute to retrograde homeostatic signaling in the hippocampus following AMPA receptor blockade. In contrast, the NMJ is built for stable excitation and is acutely sensitive to reductions in receptor function. However, when neurotransmitter sensitivity in muscle is enhanced by increased receptor expression, no retrograde signaling system exists to homeostatically downregulate presynaptic efficacy. Thus, the muscle is endowed with multiple signaling systems to respond to perturbations but appears limited to signal retrograde increases in neurotransmitter release. Hence, a single retrograde signaling system might provide an efficient means to ensure non-additive potentiation in synaptic strength and prevent hyper-excitation when conflicting signals and multiple inductive mechanisms are simultaneously activated (Goel, 2017).

    A postsynaptic PI3K-cII dependent signaling controller for presynaptic homeostatic plasticity

    Presynaptic homeostatic plasticity stabilizes information transfer at synaptic connections in organisms ranging from insect to human. By analogy with principles of engineering and control theory, the molecular implementation of PHP is thought to require postsynaptic signaling modules that encode homeostatic sensors, a set point, and a controller that regulates transsynaptic negative feedback. The molecular basis for these postsynaptic, homeostatic signaling elements remains unknown. In this study, an electrophysiology-based screen of the Drosophila kinome and phosphatome defines a postsynaptic signaling platform that includes a required function for PI3K-cII, PI3K-cIII and the small GTPase Rab11 during the rapid and sustained expression of PHP. Evidence is presented that PI3K-cII localizes to Golgi-derived, clathrin-positive vesicles and is necessary to generate an endosomal pool of PI(3)P that recruits Rab11 to recycling endosomal membranes. A morphologically distinct subdivision of this platform concentrates postsynaptically where it is proposed to functions as a homeostatic controller for retrograde, trans-synaptic signaling (Hauswirth, 2018).

    Homeostatic signaling systems stabilize the functional properties of individual neurons and neural circuits through life. Despite widespread documentation of neuronal homeostatic signaling, many fundamental questions remain unanswered. For example, given the potent action of homeostatic signaling systems, how can neural circuitry be modified during neural development, learning, and memory? Although seemingly contradictory, the homeostatic signaling systems that stabilize neural function throughout life may actually enable learning-related plasticity by creating a stable, predictable background upon which learning-related plasticity is layered. Therefore, defining the underlying molecular mechanisms of homeostatic plasticity may not only be informative about the mechanisms of neurological disease, these advances may be informative regarding how complex neural circuitry is able to accomplish an incredible diversity of behaviorally relevant tasks and, yet, retain the capacity for life-long, learning-related plasticity (Hauswirth, 2018).

    Neuronal homeostatic plasticity encompasses a range of compensatory signaling that can be sub-categorized based upon the cellular processes that are controlled, including ion channel gene expression, neuronal firing rate, postsynaptic neurotransmitter receptor abundance and presynaptic vesicle release. Presynaptic homeostatic potentiation (PHP) is an evolutionarily conserved form of neuronal homeostatic control that is expressed at the insect, rodent and human neuromuscular junctions (NMJ) and has been documented at mammalian central synapses. PHP is initiated by the pharmacological inhibition of postsynaptic neurotransmitter receptors. The homeostatic enhancement of presynaptic vesicle release can be detected in a time frame of seconds to minutes, at both the insect and mouse NMJ. This implies the existence of postsynaptic signaling systems that can rapidly detect the disruption of neurotransmitter receptor function and convert this into retrograde, trans-synaptic signals that accurately adjust presynaptic neurotransmitter release. Notably, the rapid induction of PHP is transcription and translation independent, and does not include a change in nerve terminal growth or active zone number (Hauswirth, 2018).

    There has been considerable progress identifying presynaptic effector molecules responsible for the expression of PHP. There has also been progress identifying postsynaptic signaling molecules that control synaptic growth at the Drosophila NMJ as well as the long-term, translation-dependent maintenance of PHP. However, to date, nothing is known about the postsynaptic signaling systems that initiate and control the rapid induction and expression of PHP (Hauswirth, 2018).

    This paper reports the completion of an unbiased, forward genetic screen of the Drosophila kinome and phosphatome, and the identification of a postsynaptic signaling system for the rapid expression of PHP that is based on the activity of postsynaptic Phosphoinoside-3-Kinase (PI3K) signaling. There are three classes of PI3-Kinases, all of which phosphorylate the 3 position of phosphatidylinositol (PtdsIns). Class I PI3K catalyzes the conversion of PI(4,5)P2 to PI(3,4,5)P3 (PIP3) at the plasma membrane, enabling Akt-dependent control of cell growth and proliferation, and participating in the mechanisms of long-term potentiation. Class II and III PI3Ks (PI3K-cII and PI3K-cIII, respectively) both catalyze the conversion of PI to PI(3)P, which is a major constituent of endosomal membranes. PI(3)P itself may be a signaling molecule with switch like properties, functioning in the endosomal system as a signaling integrator. The majority of PI(3)P is synthesized by PI3K-cIII, which is involved in diverse cellular processes. By contrast, the cellular functions of PI3K-cII remain less well defined. PI3K-cII has been linked to the release of catecholamines, immune mediators, insulin, surface expression and recycling of integrins, and GLUT4 translocation to the plasma membrane, a mediator of metabolic homeostasis in muscle cells. This study demonstrates that Class II and Class III PI3K-dependent signaling are necessary for the rapid expression of PHP, controlling signaling from Rab11-dependent, recycling endosomes. By doing so, this study defines a postsynaptic signaling platform for the rapid expression of PHP and defines a novel action of PI3K-cII during neuronal homeostatic plasticity. This is the first established postsynaptic function for PI3K-cII at a synapse in any organism (Hauswirth, 2018).

    Recently, it has become clear that the endosomal system has a profound influence on intracellular signaling and neural development. There is evidence that early and recycling endosomes can serve as sites of signaling intersection and may serve as signaling integrators and processors. Furthermore, protein sorting within recycling endosomes, and novel routes of protein delivery to the plasma membrane, may specify the concentration of key signaling molecules at the cell surface. The essential role of endosomal protein trafficking is underscored by links to synapse development and neurodegeneration. Yet, connections to homeostatic plasticity remain to be established. Based upon the data presented in this study and building upon prior work on endosomal signaling in other systems, it is speculated that postsynatpic PI3K-cII and Rab11-dependent recycling endosomes serve as as a postsynaptic 'homeostatic controller' that is essential for the specificity of retrograde, transsynaptic signaling (Hauswirth, 2018).

    The Drosophila kinome and phosphatome were screened for genes that control the rapid expression of PHP. This screen identified three components of a conserved, postsynaptic lipid signaling pathway that is essential for the robust expression of PHP including: (1) class II PI3K, (2) class III PI3K (Vps34) and a gene encoding the Drosophila orthologue of PI4K (not examined in detail in this study). Pi3K68D was shown to be is essential, postsynaptically for PHP. Pi3K68D resides on a Clathrin-positive membrane compartment that is positioned directly adjacent to Golgi membranes, throughout muscle and concentrated at the postsynaptic side of the synapse. Pi3K68D is necessary for the maintenance of postsynaptic PI(3)P levels and the recruitment of Rab11 to intracellular membranes, likely PI(3)P-positive recycling endosomes. Postsynaptic Rab11 and Vps34 knockdown block PHP in an unusual, calcium-dependent manner that phenocopies Pi3K68D. Thus, this study has identified a postsynaptic signaling platform, centered upon the formation of PI(3)P and Rab11-positive recycling endosomes, that is essential for PHP (Hauswirth, 2018).

    First it is considered whether postsynaptic Pi3K68D, Vps34 and Rab11 might alter PHP through modulation of postsynaptic glutamate receptor abundance. There is no consistent change in mEPSP amplitude in Pi3K68D mutants or following muscle-specific knockdown of Rab11 or Vps34 that could account for altered PHP. Therefore, functionally, there is no evidence for a change in glutamate receptor abundance at the postsynaptic membrane that could drive the phenotypic effects that were observe. Anatomically, data is presented examining GluR staining levels. In the Pi3K68D mutants, no change was found in GluRIIA levels. GluRIIA subunit containing receptors are the primary mediator of PhTx-dependent PHP. This study also reports a very modest (16%), though statistically significant, increase in GluRIIB levels. Based on these combined data, it seems unlikely that a change in GluR trafficking is a causal event leading to altered expression of PHP. It is noted that previous work showed limited GluRIIA receptor mobility within the PSD at the Drosophila NMJ. Thus, it is speculated that the function of Pi3K68D, Vps34 and Rab11 during PHP is not directly linked to postsynaptic GluR trafficking (Hauswirth, 2018).

    Any model to explain the role of PI3K, Vps34 and Rab11-dependent endosomal signaling during homeostatic plasticity must account for the phenotypic observation that PHP is only blocked at low extracellular concentrations. More specifically, in animals deficient for Pi3K68D, Rab11 or Vps34, PHP is fully expressed at elevated calcium, following PhTX application or in the GluRIIA mutant. However, PHP completely fails when extracellular calcium is acutely decreased (following induction) below 0.7 mM [Ca2+]e. Clearly, the PHP induction mechanisms remain fully intact. Instead, the presynaptic expression of PHP has been rendered calcium-dependent. It is important to note that PHP can be fully induced in the absence of extracellular calcium, so the concentration of calcium itself is not the defect. In addition, this study documents trans-heterozygous interactions of Pi3K68D with presynaptic rim and dmp, arguing for the loss of trans-synaptic signaling and a specific function of Pi3K68D in the mechanisms of PHP. In very general terms, it is concluded that PI3K and Rab11-dependent endosomal signaling platform is necessary to enable the normal expression of PHP. Ultimately, some form of retrograde signaling must be defective due to either: 1) the absence of a retrograde signal that should have normally participated in PHP or 2) the presence of an aberrant or inappropriate signal that dominantly obstructs normal PHP expression. Both of these ideas in greater depth (Hauswirth, 2018).

    First, the possibility is considered that the absence of postsynaptic PI3K and Rab11 signaling could alter the molecular composition or development of the presynaptic terminal due to the persistent absence of a retrograde signal that controls generalized synapse development or growth. Several observations demonstrate that impaired PHP is not a secondary consequence of a general defect in synapse development. Three independent postsynaptic manipulations are reported (postsynaptic expression of kinase dead Pi3K68D, postsynaptic knockdown of Rab11, and postsynaptic knockdown of Vps34) that have no effect on presynaptic release at any [Ca2+]e, yet block PHP at low [Ca2+]e. In addition, no obvious defect was found in anatomical synapse development (Hauswirth, 2018).

    Next, the possibility is considered that postsynaptic PI3K and Rab11 signaling eliminate a retrograde signal that is specific for PHP. It was recently demonstrated that Semaphorin2b (Sema2b) and PlexinB (PlexB) define a retrograde signal at the Drosophila NMJ that is necessary for PHP. However, both Sema2b and PlexB are essential for the rapid induction of PHP, inclusive of experiments at low and elevated extracellular calcium. Further, acute application of recombinant Sema2b is sufficient to fully induce PHP. Since the induction of PHP remains fully intact in the Pi3K68D mutant, and since PHP is rendered calcium sensitive, it suggests that altered Sema2b secretion is not the cause of impaired PHP in the Pi3K68D mutant. Nevertheless, this possibility will be directly tested in the future (Hauswirth, 2018).

    Next, the possibility is considered that the loss of PI3K and Rab11 signaling causes aberrant or inappropriate retrograde signaling, thereby impairing the expression of PHP. This is a plausible scenario because the induction of presynaptic homeostatic plasticity suffers from a common problem inherent to many intra-cellular signaling systems: two incompatible outcomes (1. presynaptic homeostatic potentiation and 2. presynaptic homeostatic depression -- PHD) are produced from a common input, and it remains unclear how signaling specificity is achieved. The topic of signaling specificity has been studied in several systems. One system, budding yeast, is a good example. Different pheromone concentrations can induce several distinct behaviors in budding yeast despite having a common input (pheromone concentration) and underlying signaling systems. Signaling specificity degrades in the background of mutations that affect Map Kinase scaffolding proteins. In a similar fashion, presynaptic homeostatic plasticity is induced by a change in mEPSP amplitude. A decrease in mEPSP amplitude causes the induction of PHP, whereas an increase in mEPSP amplitude causes the induction of presynaptic homeostatic depression (PHD). If a common sensor is employed to detect deviations in average mEPSP amplitude, how is this converted into the specific induction of either PHP or PHD? It has been shown that PHD and PHP can be sequentially induced. But, it remains unknown what would happen if the mechanisms of PHP and PHD were simultaneously induced. Under normal conditions this would never occur because mEPSP amplitudes cannot be simultaneously increased and decreased. But, if signaling specificity were degraded in animals lacking postsynaptic PI3K or Rab11, then the expression of PHP and PHD might coincide and create a mechanistic clash within the presynaptic terminal (Hauswirth, 2018).

    Signaling and recycling endosomes are, in many respects, ideally suited to achieve signaling specificity during homeostatic plasticity. Signaling specificity can be achieved by mechanisms including sub-cellular compartmentalization of pathways, physically separating signaling elements with protein scaffolds, or through mechanisms of cross-pathway inhibition. Well-established mechanisms of protein sorting within recycling endosomes could physically compartmentalize signaling underlying PHP versus PHD. Alternatively, recycling endosomes can serve as a focal point for signal digitization, integration, and, perhaps, cross-pathway inhibition. Thus, it is proposed that the loss of postsynaptic PI3K and Rab11 compromises the function of the postsynaptic endosomal platform that this study has identified, thereby degrading homeostatic signaling specificity. As such, this platform could be considered a 'homeostatic controller' that converts homeostatic error signaling into specific, homeostatic, retrograde signaling for either PHP or PHD (Hauswirth, 2018).

    Other models are considered, but are not favored. It remains formally possible that the calcium-sensitivity of PHP expression could be explained by a partially functioning PHP signaling system. This seems unlikely given that the same phenotype is observed in four independent genetic manipulations including a null mutation in Pi3K68D, postsynaptic expression of kinase dead Pi3K68D, postsynaptic knockdown of Rab11, and postsynaptic knockdown of Vps34. Furthermore, prior experiments examining hypomorphic and trans-heterozygous genetic interactions among essential PHP genes suggest that PHP is either diminished across the entire calcium spectrum or fully functional. So, there is no evidence that partial disruption of PHP could account for calcium-sensitive expression of PHP. Finally, the experiments argue against the possibility that compensatory changes in Vps34 expression partially rescue the Pi3K68D mutant phenotype (Hauswirth, 2018).

    Another common signaling module that emerged from the genetic screen is considered. Both CamKII and CamKK were identified as potential hits. The identification of CamKII is supported by prior work showing the expression of dominant negative CamKII transgenes disrupt the long-term maintenance of PHP in the GluRIIA mutant background (Haghighi, 2003). It has been assumed that postsynaptic calcium is used to detect the PhTX or GluRIIA-dependent perturbation. But, the logic remains unclear. PHP is induced by diminished GluR function and, therefore, diminished postsynaptic calcium influx. This should diminish activation of CamKII and yet, loss of CamKII blocks PHP. An interesting alternative model is that calcium and calmodulin-dependent kinase activity facilitate the function of the postsynaptic endosomal membrane system. Both calcium and calmodulin are necessary for endosomal membrane fusion. In this manner, the action of CamKK and CamKII would be entirely consistent with the identification of Class II/III PI3K and Rab11 as homeostatic plasticity genes (Hauswirth, 2018).

    This study has uncovered novel postsynaptic mechanisms that drive homeostatic plasticity. Eventually, continued progress in this direction may make it possible to not only reveal how stable neural function is achieved throughout life, but to uncover new rules that are essential for the processing of information throughout the nervous system. In particular, PHP has a very large dynamic range, whether one considers data from Drosophila or human NMJ or mammalian central synapses. The homeostatic control of presynaptic release can achieve a 7-fold change in synaptic gain, and yet retains the ability to offset even small changes in postsynaptic neurotransmitter receptor function. Thus, it is expected that the regulatory systems that achieve PHP will be complex and have a profound impact on brain function. This study has defined a postsynaptic signaling system responsible for the rapid expression of PHP and a novel, albeit speculative, model is proposed for the postsynaptic control of PHP, taking into account the need for signaling specificity. The identification of this pathway paves the way for future advances in understanding how homeostatic signaling is designed and implemented at a cellular and molecular level (Hauswirth, 2018).

    Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction

    The Akt family of serine-threonine kinases integrates a myriad of signals governing cell proliferation, apoptosis, glucose metabolism, and cytoskeletal organization. Akt affects neuronal morphology and function, influencing dendrite growth and the expression of ion channels. Akt is also an integral element of PI3Kinase-target of rapamycin (TOR)-Rheb signaling, a pathway that affects synapse assembly in both vertebrates and Drosophila. Recent findings demonstrated that disruption of this pathway in Drosophila is responsible for a number of neurodevelopmental deficits that may also affect phenotypes associated with tuberous sclerosis complex, a disorder resulting from mutations compromising the TSC1/TSC2 complex, an inhibitor of TOR. Therefore, this study examined the role of Akt in the assembly and physiological function of the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse that displays developmental and activity-dependent plasticity. The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR). Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin (Syntaxin18), a Drosophila t-SNARE protein in a manner independent of the mislocalization of GluRIIA. These findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboratio (Lee, 2013).

    This study explored Akt function in synapse development and function using a well-characterized model system, the Drosophila neuromuscular junction. There is a single Akt homolog in Drosophila, Akt1, facilitating the genetic and cellular studies of Akt function in synapse assembly (see Model for Akt1's regulatory role at the NMJ). Akt1 was specifically required for the correct assembly of A-type glutamate receptors. Reductions of Akt1 function either by mutation or RNA interference resulted in a loss of GluRIIA at the synapse paired with accumulation into intracellular structures. Reduction of Akt1 influenced the levels and localization of proteins shown to affect GluRIIA, Dorsal, and Cactus. Therefore, Akt1 could affect GluRIIA at least in part via control of these proteins. Akt1 was also required for the normal expansion of a specialized postsynaptic membrane compartment, the SSR. Evidence is provided that Akt1 mediates its effects on SSR via control of the t-SNARE Gtaxin. RNA interference of Gtaxin did not affect GluRIIA localization, showing that the control of SSR expansion and glutamate receptor composition mediated by Akt1 occurs via different molecular mechanisms (Lee, 2013).

    The analysis of Akt1 reported in this study examined physiological, morphological, and cellular phenotypes, using both traditional Akt1 mutant alleles and cell-type directed knockdown achieved with either of two different UAS-Akt1RNAi lines. The results from these different genetic tools were consistent and showed that Akt1 function is critical for both GluRIIA localization and SSR expansion. In particular, combinations of Akt1 alleles resulted in the redistribution of GluRIIA into intracellular bands, a phenotype found to be even more pronounced in muscle-directed RNAi of Akt1. This remarkable phenotype was also observed in larvae expressing both Akt1RNAi and a UAS-transgene-derived GluRIIA-RFP in the muscle, the latter detected by either endogenous fluorescence or anti-RFP antibody. It was of note that fluorescent signal from the GluRIIA-RFP was reduced at the synapse but receptor mislocalization to intracellular compartments was detected only with anti-RFP antibody. Akt1-dependent events were clearly required for the proper formation of the folded RFP domain of the recombinant GluRIIA protein while the polypeptide, detected with the anti-RFP antibody was present and redirected to an alternative cellular location, as was observed for the endogenous GluRIIA. These data implicate Akt1 in processes of folding, stabilization, or assembly of GluRIIA (Lee, 2013).

    A number of experiments were conducted to evaluate if Akt1 was required for the localization of specific postsynaptic proteins, or rather served a more generalized role in directing a variety of proteins to this membrane specialization. The correct localization of GluRIIB, GluRIIC, Basigin, Discs large, andSyndapin in animals with Akt1 knockdown in the muscle demonstrated that Akt1 has specific targeting functions for GluRIIA and is not a general factor for delivery of all postsynaptic proteins. Levels of these postsynaptic proteins were reduced in Akt1RNAi bearing animals, not surprisingly given the substantial size reduction in the SSR (Lee, 2013).

    At the Drosophila NMJ, two types of glutamate receptors have been defined by their distinct compositions and physiological properties. The shifting between A- and B-type receptors provides a mechanism for modulating postsynaptic responses to variable presynaptic inputs during development. There is considerable evidence that modulation of GluRIIA and B representation at the NMJ is governed by different signaling systems. Coracle, a homolog of protein 4.1 in Drosophila, has been shown to specifically influence the targeting of GluRIIA but not IIB. A physical interaction between Coracle and GluRIIA was essential for actin-dependent trafficking of GluRIIA-containing vesicles to the plasma membrane. Conversely, DLG has been shown to be required for GluRIIB but not GluRIIA localization at the NMJ. The current finding supports the conclusion that A and B receptor subunits are differentially regulated and show that Akt1 serves a role in A but not B subunit control (Lee, 2013).

    There is evidence that the assembly and localization of GluRIIA into the postsynaptic density at the NMJ is accomplished following delivery to the plasma membrane. This conclusion is based upon the observation that fluorescence photobleaching of the entire muscle delays accumulation of new GluRIIA to synaptic sites more so than local bleaching at the NMJ (Rasse, 2005). The effects of Akt1 on GluRIIA localization could therefore be mediated by either regulated delivery of GluRIIA-containing vesicles to the plasma membrane, or by affecting the localization to the postsynaptic density following insertion into the plasma membrane. The accumulation of GluRIIA into an intracellular membrane compartments argues for a trafficking-based mechanism. This model is further supported by the results from the developmental timing experiments, where Akt1 function was removed during different stages in synapse assembly. Loss of Akt1 in a 2 day window early in development produced the phenotypes observed with continuous loss of Akt1, whereas a 2 day loss in third instar did not. If Akt1 simply served to retain GluRIIA at the synapse, there should have been time for new synthesis to repopulate the NMJ. Therefore, a model is favored where Akt1 affects developmental processes required for the selective delivery of GluRIIA from the endoplasmic reticulum into functional receptor units that arrive at the plasma membrane. It is notable that in mammalian systems, Akt is critical for the insulin-stimulated exocytosis of glucose transporter containing vesicles to the plasma membrane. Perhaps Akt1 governs similar exocytic processes at synapses. Akt1 signaling has also shown to be essential for AMPA receptor trafficking in hippocampal neurons, further supporting a role for Akt1 in trafficking of synaptic proteins (Lee, 2013).

    A striking phenotype of animals with reduced Akt1 function in muscles was a severe reduction in the SSR and disruption of intracellular membrane organization. These phenotypes were similar to those found in a Gtaxin mutant and suggested the possibility that Akt1 and Gtaxin are involved in the same cellular process. A number of observations reported in this study indicate Akt1 activity is mediated at least in part by control of Gtaxin. First, Gtaxin levels at the SSR are greatly reduced in animals with reduced Akt1 function in the muscle cells. Second, muscle-directed overexpression of a constitutively active form of Akt1 (Akt1CA) produced ectopic membranous structures; a phenotype also observed with Gtaxin overexpression and elevated levels of Gtaxin. Third, inhibition of Gtaxin blocks the effects of the constitutively active Akt1 in the muscle cell. Gtaxin does contain a consensus site for Akt1 phosphorylation and could therefore be a direct target of Akt1 kinase activity in regulating SNARE complex assembly (Lee, 2013).

    The regulatory roles of Akt1 in glutamate receptor composition and postsynaptic membrane expansion could be accomplished through separate or identical downstream effectors. The fact that Gtaxin mutants did not disrupt GluRIIA distribution suggests different downstream effectors regulated by Akt1. The regulation of GluRIIA localization by Akt1 does not involve Gtaxin but could be mediated via Dorsal and Cactus. Dorsal and Cactus influence glutamate receptor delivery and are known effectors of Akt activity in mammalian cells. The levels of both Dorsal and Cactus were reduced in animals with knockdown of Akt1 in the muscle. Notably, in some animals expressing Akt1RNAi in the muscle, Dorsal showed an altered intracellular distribution that overlapped with the mislocalized GluRIIA. However, because Dorsal and Cactus mutants are not reported to mislocalize GluRIIA into intracellular bands, Akt1 is likely to have additional downstream targets that influence GluRIIA localization and delivery to the postsynaptic specialization (Lee, 2013).

    Physiological measures of synaptic transmission showed that Akt1 function is required for normal synapse function. Akt1 transheterozygous mutants (Akt11/Akt104226) showed reduced EJP amplitudes and altered decay kinetics of the EJP. These same phenotypes were observed in animals with muscle-specific inhibition of Akt1 function, with the severity correlating to the degree of Akt1 inhibition. These changes in EJP kinetics were not accompanied by alterations of nonvoltage-dependent membrane capacitance or resistance, suggesting that voltage-gated channels contributing to EJP rise and decay times may be affected by Akt1. These findings contrast published work with Akt1 mutant animals describing changes in long-term depression but not in EJP properties. However, it is noted that the physiological studies reported in this paper were conducted at a higher Ca2+ concentration, which could account for these different measures of EJP properties in Akt1 mutants. It is important to point out that the physiological changes documented in this study observed in both Akt1 mutant larvae as well as animals with RNA interference of Akt1 in the muscle cell. The physiological changes observed in Akt1 compromised animals are logical consequences of observed changes in NMJ composition. Loss of GluRIIA-containing receptors and an overall decrease in functional GluRs at the synapse could decrease the EJP amplitude. The altered EJP decay pattern in animals with reduced Akt1 is consistent with the involvement of Gtaxin, as has been documented in this study. Gtaxin mutants showed similar changes in EJP decay, indicating that this feature of Akt1 mediated physiological change is associated with the consequences of compromising the function of this t-SNARE (Lee, 2013).

    There is a precedent for Akt-mediated regulation of neurotransmitter receptor localization to the cell surface. The NMDA receptor subunit NR2C is developmentally regulated in cerebellar granule cells and Akt-mediated phosphorylation is critical for cell surface expression of NR2C-containing receptors. Akt has also proven to be important in the elaboration of dendritic complexity in Drosophila sensory neurons, suggesting that this kinase is of general importance in the control of nervous system receptive fields. Selective control of Akt or its downstream targets could provide a powerful method of influencing synaptic transmission and the receptive properties of neurons (Lee, 2013).

    Regulation of SH3PX1 by dNedd4-long at the Drosophila Neuromuscular Junction

    Drosophila Nedd4 (dNedd4) is a HECT E3 ubiquitin ligase present in two major isoforms: short (dNedd4S) and long (dNedd4Lo), with the latter containing two unique regions (N-terminus and Middle). While dNedd4S promotes neuromuscular synaptogenesis (NMS), dNedd4Lo inhibits it and impairs larval locomotion. To explain how dNedd4Lo inhibits NMS, mass spectrometry was performed to find its binding partners and identified SH3PX1, which binds dNedd4Lo unique Middle region. SH3PX1 contains SH3, PX and BAR domains and is present at neuromuscular junctions, where it regulates active zone ultrastructure and presynaptic neurotransmitter release. This study demonstrates direct binding of SH3PX1 to the dNedd4Lo Middle region (which contains a Pro rich sequence) in vitro and in cells, via the SH3PX1-SH3 domain. In Drosophila S2 cells, dNedd4Lo overexpression reduces SH3PX1 levels at the cell periphery. In vivo overexpression of dNedd4Lo post-synaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo post-synaptically and are heterozygous for a null mutation in SH3PX1 display increased neurotransmission compared to dNedd4Lo or SH3PX1 mutant larvae alone, suggesting a compensatory effect from the remaining SH3PX1 allele. These results suggest a postsynaptic - specific regulation of SH3PX1 by dNedd4Lo (Wasserman, 2018).

    Calcium-activated Calpain specifically cleaves Glutamate Receptor IIA but not IIB at the Drosophila neuromuscular junction

    Calpains are calcium-dependent, cytosolic proteinases active at neutral pH. They do not degrade but cleave substrates at limited sites. Calpains are implicated in various pathologies, such as ischemia, injuries, muscular dystrophy, and neurodegeneration. Despite so, the physiological function of calpains remains to be clearly defined. Using the neuromuscular junction of Drosophila of both sexes as a model, RNAi screening was performed, and calpains were found to negatively regulated protein levels of the glutamate receptor GluRIIA but not GluRIIB. Calpains enrich at the postsynaptic area, and the calcium-dependent activation of calpains induced cleavage of GluRIIA at Q788 of its C terminus. Further genetic and biochemical experiments revealed that different calpains genetically and physically interact to form a protein complex. The protein complex was required for the proteinase activation to downregulate GluRIIA. These data provide a novel insight into the mechanisms by which different calpains act together as a complex to specifically control GluRIIA levels and consequently synaptic function (Metwally, 2019).

    Calpains are a family of calcium-activated cytoplasmic cysteine proteases, which are ubiquitously expressed in various mammalian tissues and are functionally active at neutral pH. Members of the calpain family act in pathological processes associated with calcium overload, such as ischemia and Alzheimer's disease. For example, calpains cleave multiple synaptic proteins, including both inotropic and metabotropic glutamate receptors in excitotoxic conditions, as well as in synaptic plasticity (Chan, 1999; Xu, 2007; Doshi, 2009; Baudry, 2016). However, little is known of the precise physiological function of calpain-dependent cleavage of target proteins at synapses during normal development (Metwally, 2019).

    Calpains exist in organisms ranging from bacteria to humans. To date, 15 different calpains have been identified in mammals and four in Drosophila. Unlike most proteases, calpains do not destroy but cleave their substrates at limited sites to modulate their function. Calpain 1 (μ-calpain) and calpain 2 (m-calpain) are the most ubiquitous and well-studied calpain family members. They are activated in vitro by micromolar and millimolar concentrations of calcium, respectively. Conventional calpains 1 and 2 are heterodimers composed of a large catalytic subunit with four domains (dI-dIV) and a small regulatory subunit with two domains (dV and dVI). Heterodimerization of the large and small subunits occurs through a unique interaction between their C-terminal domains. There are two calcium-binding sites within the crystal structure of the enzymatically active domain II of μ-calpain. These sites, together with domains IV and VI, interact with calcium and are required for full enzymatic activity. Calpastatin is an endogenous calpain inhibitor that binds and inhibits calpains via its calpain-inhibitor domains when the proteases are activated by calcium (Hanna, 2008). Unlike mammalian calpains, Drosophila calpains comprise a large subunit only and calpastatin has not been identified in Drosophila (Metwally, 2019).

    The efficiency of neurotransmission is determined by the level of neurotransmitter receptors at the postsynaptic densities (PSDs). However, the molecular mechanism by which the level of neurotransmitter receptors at synapses is regulated is not well understood. At Drosophila neuromuscular junction (NMJ) synapses, there are two subtypes of glutamate receptors (GluR), GluRIIA and GluRIIB, which are developmentally, biophysically, and pharmacologically distinct. For example, the postsynaptic sensitivity to glutamate is much higher at synapses enriched for GluRIIA than at synapses enriched for GluRIIB. Previous studies show that GluRIIA and GluRIIB receptors are differentially regulated via independent pathways, although the molecular mechanisms that control different subtypes of GluRs remain elusive. To uncover new pathways that control GluR expression at NMJ synapses, a genetic screen was carried out identified a critical role was identified for calpains in specifically regulating the abundance of GluRIIA, but not GluRIIB, at the Drosophila NMJs. Valpains enrich at the postsynaptic area and cleave GluRIIA, but not GluRIIB, at its C-terminal in a calcium-dependent manner. As calcium is a highly versatile intracellular signal that regulates many different cellular functions, the finding of calpain proteinases activated by calcium under physiological conditions at NMJ synapses offers novel insights into the myriad of calcium-mediated processes at other cellular contexts (Metwally, 2019).

    Neurotransmission efficiency is determined by the level of neurotransmitter receptors at the PSDs. Therefore, the regulation of glutamate receptors at synapses has been under intensive studies. However, the molecular mechanism responsible for regulating the expression of neurotransmitter receptors at synapses is still poorly understood. Using genetic screening, together with optogenetic manipulation and calcium treatment, this study has revealed that GluRIIA protein levels were specifically and negatively regulated by calcium-dependent calpains during development (Metwally, 2019).

    Extensive studies have demonstrated robust homeostatic regulation at the Drosophila NMJ. Genetic or pharmacological manipulations that impair the protein level or activity of postsynaptic GluRs cause a compensatory increase in presynaptic action potential-evoked neurotransmitter release; the increase in evoked release precisely compensates for the decrease in GluR sensitivity and, thereby, maintains a normal level of transmission. This study has shown that calpains negatively regulated mEJP amplitude and frequency at NMJs, consistent with previous studies demonstrating that quantal size and mEJP frequency are increased when GluRIIA is overexpressed. The increase in mEJP amplitude and frequency in calpain mutants can be explained by the elevated levels of GluRIIA and Brp protein at synapses. GluRIIA mutant exhibits a large decrease in mEJP amplitude with no change in EJP amplitude, probably due to homeostatic regulation, whereas overexpression of GluRIIA leads to an increase in mEJP amplitude with an increase of EJP amplitude, indicating that there is no compensatory downregulation of quantal content. In the latter case, different effects on EJP amplitude were observed in a more quantitative manner by using a different gene dosage of GluRIIA. Specifically, increased EJP amplitude is observed at synapse overexpressing a low level of GluRIIA, whereas overexpression of GluRIIA at a high level leads to normal EJP amplitude. This latter case is consistent with the finding that a high threefold increase of GluRIIA level in calpains mutant did not lead to upregulation of EJP amplitude. It is possible that the increase in mEJP amplitude is compensated by a downregulation of quantal content to maintain normal EJP amplitude in calpain mutants. The finding of normal EJP amplitudes regardless of upregulation or downregulation of GluRIIA abundance upon calpain manipulations further support homeostatic regulation at Drosophila NMJ (Metwally, 2019).

    Previous studies showed that GluRIIA and GluRIIB exhibit competing effects and interdependence as a result of the selective absence of GluRIIA or GluRIIB type at NMJ. If the primary effect of calpains was specifically on GluRIIA, we would expect to see an opposite change in GluRIIB level. However, this is not what we observed. Instead, we observed that GluRIIB was normally localized and its levels were unchanged when GluRIIA was upregulated, resulting from calpain mutation or knockdown, suggesting a distinct regulation of subunit composition by calpains at the NMJ (Metwally, 2019).

    At the Drosophila NMJ, the postsynaptic calcium has been speculated to regulate the sensitivity and synaptic localization of GluRIIA. However, how calcium regulates GluRIIA at NMJ is yet to be investigated. The findings in the present study showed a critical role of calcium in regulating postsynaptic GluRIIA levels via calpain. In Drosophila muscles and S2 cells, calcium-dependent activation of calpain resulted in the C terminus cleavage of GluRIIA but not GluRIIB. It is noted that the percentage of cleaved GluRIIA by calpains is low by Western analysis. This low cleavage efficiency is consistent with previous findings. However, immunostaining showed apparent changes in GluRIIA level at NMJ upon manipulating calpain expressions. Multiple possibilities could explain the discrepancy between low cleavage efficiency by Western analysis and apparent changes in GluRIIA level by immunostaining. First, synaptic GluRIIA is part of the total GluRIIA and calpain may preferentially cleave a subset of synaptic GluRIIA, which is physically accessible and sensitive to calpain cleavage due to specific post-translational modifications. The enzymatic activity of calpains could also be differentially regulated at different cellular locations. Alternatively, calpain may indirectly regulate GluRIIA level by other mechanism, such as translation regulation (Metwally, 2019).

    There are a few reports that calpain is involved in the regulation of protein synthesis. For example, ischemia-induced calpain activation targets translation initiation factor 4G1 for cleavage, leading to reduced translation. On the other hand, calpain stimulates protein synthesis by truncating B56a, the regulatory subunit of PP2A which inhibits the Akt/mTOR/S6 signaling during mGluR-dependent LTD. Although multiple independent lines of evidence support that Drosophila calpains specifically target GluRIIA for cleavage, there is still a possibility that calpain negatively regulates GluRIIA level through other unknown mechanisms, including translational regulation (Metwally, 2019).

    Where does GluRIIA get cleaved by calpains within the cell? As calpains are enriched in the postsynaptic area where calcium transients are produced by spontaneous and evoked transmitter release, it is possible that calpain cleaves GluRIIA at the postsynaptic area. Alternatively, calpain might cleave GluRIIA at the endoplasmic reticulum when it is synthesized. As an obvious decrease in GluRIIA intensity was observed upon calcium treatment for a short time of 30 min, the first scenario is favored, in which calpain cleaves GluRIIA mostly at the postsynaptic site (Metwally, 2019).

    Calpain interacts with and cleaves dozens of target proteins. The present studyfound that calpains specifically target GluRIIA but not other synaptic proteins at the Drosophila NMJ terminals. A physical interaction between calpain and GluRIIA in a calcium-dependent manner. This is the first demonstration of calcium-facilitated interaction of calpain with its target protein in cultured cells. It will be of interest to test whether other substrates of calpains identified so far also interact with calpains in a calcium-dependent manner (Metwally, 2019).

    Calpain activation is triggered by abnormally high calcium levels associated with pathologies, such as ischemic insults and neurodegeneration, but the in vivo role of calpain under physiological conditions is largely unknown. This genetic and optogenetic results indicate that calcium-induced activation of calpains is likely to occur under certain physiological conditions at NMJ synapses during development. As calcium signaling is involved in a variety of molecular and cellular processes in development and disease, the current findings offer a novel insight into the role of calcium signaling pathways. How and where calpains are activated by calcium within a cell remain to be elucidated (Metwally, 2019).

    Multiple independent lines of evidence showed that different calpains, at least calpains A, B, and D, form a complex in which each is essential for the normal proteinase activity to control GluRIIA level. Drosophila calpain B was originally demonstrated to work as monomers based on in vitro studies of Escherichia coli-produced recombinant protein (Park, 2008), rather than as heterodimers, such as mammalian calpains 1 and 2. However, the current results showed that different calpains acted together in vivo to downregulate GluRIIA. Specifically, this study showed that different calpains colocalized and interacted physically in vivo and in cultured cells. Drosophila calpains do not form homodimers, at least for calpain A (this study) and calpain B (Park, 2008), but instead formed multimers consisting of different calpains, although the domain mediating calpain-calpain interaction remains to be defined. The finding of a calpain complex is supported by a previous study demonstrating that different calpains in Drosophila function synergistically downstream of calcium transients to trigger dendritic pruning of sensory neurons during metamorphosis (Kanamori, 2013). Similarly, mammalian calpains 8 and 9, which are specifically expressed in the gastrointestinal tract, interact physically to form a protein complex called 'gastric calpain' involving in gastric mucosal defense. Thus, in addition to the conventional calpain heterodimers, such as calpains 1 and 2, consisting of a large and a small subunit, heterodimers or heteromultimers of different calpains without a small subunit are emerging as a new mode of calpain activity regulation (Metwally, 2019).

    Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth

    The molecular pathways involved in retrograde signal transduction at synapses and the function of retrograde communication are poorly understood. This study demonstrates that postsynaptic calcium 2+ ion (Ca2+) influx through glutamate receptors and subsequent postsynaptic vesicle fusion trigger a robust induction of presynaptic miniature release after high-frequency stimulation at Drosophila neuromuscular junctions. An isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), serves as a postsynaptic Ca2+ sensor to release retrograde signals that stimulate enhanced presynaptic function through activation of the cyclic adenosine monophosphate (cAMP)-cAMP-dependent protein kinase pathway. Postsynaptic Ca2+ influx also stimulates local synaptic differentiation and growth through Syt 4-mediated retrograde signals in a synapse-specific manner (Yoshihara, 2005).

    Neuronal development requires coordinated signaling to orchestrate pre- and postsynaptic maturation of synaptic connections. Synapse-specific enhancement of synaptic strength as occurs during long-term potentiation, as well as compensatory homeostatic synaptic changes, have been suggested to require retrograde signals for their induction. Although retrograde signaling has been implicated widely in synaptic plasticity, the molecular mechanisms that transduce postsynaptic Ca2+ signals during enhanced synaptic activity to alterations in presynaptic function are poorly characterized. Because postsynaptic Ca2+ is essential for synapse-specific potentiation, it is important to characterize how Ca2+ can regulate retrograde communication at synapses (Yoshihara, 2005).

    To dissect the mechanisms underlying activity-dependent synaptic plasticity, test were performed to see whether newly formed Drosophila glutamatergic neuromuscular junctions (NMJs), which have ∼30 active zones, show physiological changes after 100-Hz stimulation (5-1552+ chelator EGTA from the patch pipette caused a modest suppression of HFMR, whereas the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) induced strong suppression by 2.5 min of perfusion. Longer perfusion with BAPTA for 5 min before stimulation abolished HFMR, indicating HFMR is induced after postsynaptic Ca2+ influx (Yoshihara, 2005).

    Ca2+-induced vesicle fusion in presynaptic terminals provides a temporally controlled and spatially restricted signal essential for synaptic communication. Postsynaptic vesicles within dendrites have been visualized by transmission electron microscopy, and dendritic release of several neuromodulators has been reported. To test whether postsynaptic vesicle fusion might underlie the Ca2+-dependent release of retrograde signals, postsynaptic vesicle recycling was blocked by using the dominant negative shibirets1 mutation, which disrupts endocytosis at elevated temperatures. shibirets1 was expressed specifically in postsynaptic muscles by driving a UAS-shibirets1 transgene with muscle-specific myosin heavy chain (Mhc)-Gal4, keeping presynaptic activity intact. At the permissive temperature (23°C), high-frequency stimulation induced normal HFMR. However, raising the temperature to 31°C suppressed HFMR in the presence of postsynaptic shibirets1, whereas wild-type animals displayed normal HFMR at 31°C. Basic synaptic properties in Mhc-Gal4, UAS-shibirets1 animals were not affected at either the permissive or the restrictive temperature. The suppression of HFMR is not due to irreversible damage induced by postsynaptic UAS-shibirets1 expression, because a second high-frequency stimulation after recovery to the permissive temperature induced normal HFMR (Yoshihara, 2005).

    The synaptic vesicle protein synaptotagmin 1 (Syt 1) is the major Ca2+ sensor for vesicle fusion at presynaptic terminals but is not localized postsynaptically. It has recently been shown that another isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), is present in the postsynaptic compartment (Adolfsen, 2004), suggesting Syt 4 might function as a postsynaptic Ca2+ sensor. Syt 4 immunoreactivity is observed in a punctate pattern surrounding presynaptic terminals, suggesting Syt 4 is present on postsynaptic vesicles. Postsynaptic vesicle recycling was blocked by using the UAS-shibirets1 transgene driven with Mhc-Gal4. Without a temperature shift, Syt 4-containing vesicles showed their normal postsynaptic distribution surrounding presynaptic terminals. When the temperature was shifted to 37oC for 10 min in the presence of high-K+ saline containing 1.5 mM Ca2+ to drive synaptic activity, Syt 4-containing vesicles translocated to the plasma membrane. After recovery at 18oC for 20 min, postsynaptic vesicles returned to their normal position. Removing extracellular Ca2+ during the high-K+ stimulation resulted in vesicles that did not translocate to the postsynaptic membrane (Yoshihara, 2005).

    To further test whether the Syt 4 vesicle population undergoes fusion with the postsynaptic membrane as opposed to mediating fusion between intracellular compartments, transgenic animals were constructed expressing a pH-sensitive green fluorescent protein (GFP) variant (ecliptic pHluorin) fused at the intravesicular N terminus of Syt 4. Ecliptic pHluorin increases its fluorescence 20-fold when exposed to the extracellular space from the acidic lumen of intracellular vesicles during fusion. Expression of Syt 4-pHluorin in postsynaptic muscles resulted in intense fluorescence at specific subdomains in the postsynaptic membrane, defining regions where Syt 4 vesicles undergo exocytosis. The fluorescence was not diffusely present over the postsynaptic membrane but directed to restricted compartments. Mhc-Gal4, UAS-Syt 4-pHluorin larvae were costained with antibodies against the postsynaptic density protein, DPAK, and nc82, a monoclonal antibody against a presynaptic active zone protein. Syt 4-pHluorin colocalized with DPAK and localized adjacent to nc82, demonstrating that Syt 4-pHluorin translocates from postsynaptic vesicles to the plasma membrane at postsynaptic densities opposite presynaptic active zones (Yoshihara, 2005).

    To examine the function of Syt 4-dependent postsynaptic vesicle fusion, the phenotypes of a syt 4 null mutant (syt 4BA1) and a syt 4 deficiency (rn16) were tested. Mutants lacking Syt 4 hatch from the egg case 21 hours after egg laying at 25oC, similar to wild type, and grow to fully mature larvae that pupate and eclose with a normal time course. To determine whether postsynaptic vesicle fusion triggered by Ca2+ influx is required for HFMR, the effects of high-frequency stimulation in syt 4 mutants were analyzed. In contrast to controls, the increase of miniature release was eliminated in syt 4 mutants. Postsynaptic expression of a UAS-syt 4 transgene completely restored HFMR in the null mutant, demonstrating that postsynaptic Syt 4 is required for triggering enhanced presynaptic function. Presynaptic expression of a UAS-syt 4 transgene did not restore HFMR. In addition, postsynaptic expression of a mutant Syt 4 with neutralized Ca2+-binding sites in both C2A and C2B domains did not rescue HFMR, indicating that retrograde signaling by Syt 4 requires Ca2+ binding (Yoshihara, 2005).

    The large increase in miniature frequency observed during HFMR is similar to the enhancement of presynaptic release after activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) described in Aplysia and Drosophila. Bath application of forskolin, an activator of adenylyl cyclase, results in a robust enhancement of miniature frequency at Drosophila NMJs similar to that observed during HFMR, suggesting retrograde signals may function to increase presynaptic cAMP. To test the role of the cAMP-PKA pathway in HFMR, DC0 mutants were assayed for the presence of HFMR. DC0 encodes the major catalytic subunit of PKA in Drosophila and has been implicated in olfactory learning. Similar to the lack of forskolin-induced miniature induction, DC0 null mutants lacked HFMR. Bath application of forskolin in syt 4 mutants resulted in enhanced miniature frequency, suggesting activation of the cAMP pathway can bypass the requirement for Syt 4 in synaptic potentiation (Yoshihara, 2005).

    To further explore the role of retrograde signaling at Drosophila synapses, the role of activity was tested in synapse differentiation and growth. During Drosophila embryonic development, presynaptic terminals undergo a stereotypical structural change from a flat path-finding growth cone into varicose synaptic terminals through dynamic reconstruction. Such developmental changes in synaptic structure may share common molecular mechanisms with morphological changes induced during activity-dependent plasticity. Synaptic transmission was eliminated by using a deletion mutation that removes the postsynaptic glutamate receptors, DGluRIIA and DGluRIIB (referred to as GluRs). Postsynaptic currents normally induced by nerve stimulation were completely absent in the mutants (gluR). Miniatures were also eliminated, even at elevated extracellular Ca2+ concentrations of 4 mM. In the absence of GluRs, the presynaptic morphology of motor terminals is abnormal, even though GluRs are only expressed in postsynaptic muscles. GluR-deficient terminals maintain a flattened growth cone-like structure and fail to constrict into normal synaptic varicosities. Synaptic development was also assayed in a null mutant of the presynaptic plasma membrane t-SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) receptor], syntaxin (syx), which eliminates neurotransmitter release, providing an inactive synapse similar to that in the gluR mutant. syx null mutants also have abnormal growth cone-like presynaptic terminals with less varicose structure (Yoshihara, 2005).

    Because activity is required for synapse development, whether Syt 4-dependent vesicle fusion may be required, similar to its role in acute retrograde signaling during HFMR, was tested. Physiological analysis revealed that the amplitude of evoked currents in mutants lacking Syt 4 was moderately reduced compared with wild type, suggesting weaker synaptic function or development. Similar to the morphological phenotype of the gluR mutant, syt 4 null mutant embryos showed defective presynaptic differentiation. Nerve terminals lacking Syt 4 displayed reduced varicose structure, whereas wild-type terminals have already formed individual varicosities at this stage of development. Postsynaptic expression with a UAS-syt 4 transgene rescued the physiological and morphological phenotypes. Syt 4 Ca2+-binding deficient mutant transgenes did not rescue either the morphological immaturity or the reduced amplitude of evoked currents, even though Syt 4 immunoreactivity at the postsynaptic compartment was restored by muscle-specific expression of the mutant syt 4 transgene, similar to the wild-type syt 4 transgene and endogenous Syt 4 immunoreactivity (Yoshihara, 2005).

    Mammalian syt 4 was originally identified as an immediate-early gene that is transcriptionally up-regulated by nerve activity in certain brain regions. Therefore, this study analyzed gain-of-function phenotypes caused by postsynaptic Syt 4 overexpression specifically in muscle cells to increase the probability of postsynaptic vesicle fusion. Syt 4 overexpression induced overgrowth of presynaptic terminals in mature third instar larvae, in contrast to overexpression of Syt 1, which does not traffic to Syt 4-containing postsynaptic vesicles. In addition to synaptic overgrowth, Syt 4 overexpression occasionally induced the formation of abnormally large varicosities. Postsynaptic overexpression of the Syt 4 Ca2+-binding mutant did not induce synaptic overgrowth, indicating that retrograde signaling by Syt 4 also requires Ca2+ binding to promote synaptic growth (Yoshihara, 2005).

    To determine whether the cAMP-PKA pathway is important in activity-dependent synaptic growth, the effects of PKA on synaptic morphology were assayed. Expression of constitutively active PKA presynaptically using a motor neuron-specific Gal4 driver induced not only synaptic overgrowth but also larger individual varicosities in mature third instar larvae, similar to those induced by postsynaptic overexpression of Syt 4. These observations are consistent with the presynaptic overgrowth observed in the learning mutant, dunce, which disrupts the enzyme that degrades cAMP, and with studies in Aplysia implicating PKA in synaptic varicosity formation. The loss-of-function phenotype of PKA mutants (DC0B3) were characterized at the embryonic NMJ to compare with gluR and syt 4 mutants. Presynaptic terminals in the DC0 mutant were morphologically aberrant, with abnormal growth cone-like features and less varicose structure. Postsynaptic expression of a constitutively active PKA transgene in the DC0 or syt 4 mutant backgrounds rescued the immature morphology, suggesting activation of PKA is downstream of Syt 4-dependent release of retrograde signals (Yoshihara, 2005).

    Similar to the role of Syt 1-dependent synaptic vesicle fusion in triggering synaptic transmission at individual synapses, Syt 4-dependent vesicle fusion might trigger synapse-specific plasticity and growth. To test synapse specificity, advantage was taken of the specific properties of the Drosophila NMJ at muscle fibers 6 and 7, where two motorneurons innervate both muscle fibers 6 and 7 during development. Syt 4 was expressed specifically in embryonic muscle fiber 6 but not muscle fiber 7 by using the H94-Gal4 driver. If Syt 4-dependent retrograde signals induce general growth of the motorneuron, one would expect to see a proliferation of synapses on both muscle fibers. Alternatively, if Syt 4 promoted local synaptic growth, one would expect specific activation of synapse proliferation only on target muscle 6, releasing the Syt 4-dependent signal. UAS-syt 4 driven by H94-Gal4 increased innervation on muscle fiber 6 compared with that on muscle fiber 7 in third instar larvae. Control experiments with Syt 4 Ca2+-binding deficient mutant transgenes, or a transgene encoding Syt 1, did not result in proliferation. Thus, synaptic growth can be preferentially directed to specific postsynaptic targets where Syt 4-dependent retrograde signals predominate, allowing differential strengthening of active synapses via local rewiring (Yoshihara, 2005).

    On the basis of the results described in this study, a local feedback model is proposed for activity-dependent synaptic plasticity and growth at Drosophila NMJs. Synapse-specific Ca2+ influx triggers postsynaptic vesicle fusion through Syt 4. Fusion of Syt 4-containing vesicles with the postsynaptic membrane releases locally acting retrograde signals that activate the presynaptic terminal, likely through the cAMP pathway. Active PKA then triggers cytoskeletal changes by unknown effectors to induce presynaptic growth and differentiation. Moreover, PKA is well known to facilitate neurotransmitter release directly, triggering a local synaptic enhancement of presynaptic release as shown in HFMR. Therefore, postsynaptic vesicular fusion might initiate a positive feedback loop, providing a localized activated synaptic state that can be maintained beyond the initial trigger (Yoshihara, 2005).

    As a general mechanism for memory storage, Hebb postulated that potentiated synapses maintain an activated state until structural changes occur to consolidate alterations in synaptic strength. The current results demonstrate that acute plasticity and synapse-specific growth require Syt 4-dependent retrograde signaling at Drosophila NMJs. The feedback mechanism described in this study could be a molecular basis for both input-specific postsynaptic tagging and an output-specific presynaptic mark or tag for long-lasting potentiation. The regenerative nature of a positive feedback signal allows individual synapses to be tagged in a discrete all-or-none manner until synaptic rewiring is completed. The synaptic tag is maintained as a large increase in miniature frequency at Drosophila NMJs, suggesting a previously unknown role for miniature release in neuronal function. The spatial resolution for input and output specificity would result from the accuracy insured by Ca2+-dependent vesicle fusion and subsequent diffusion, similar to the precision of presynaptic neurotransmitter release (Yoshihara, 2005).

    Postsynaptic Syntaxin 4 negatively regulates the efficiency of neurotransmitter release

    Signaling from the postsynaptic compartment regulates multiple aspects of synaptic development and function. Syntaxin 4 (Syx4) is a plasma membrane t-SNARE that promotes the growth and plasticity of Drosophila neuromuscular junctions (NMJs) by regulating the localization of key synaptic proteins in the postsynaptic compartment. This study describes electrophysiological analyses and reports that loss of Syx4 leads to enhanced neurotransmitter release, despite a decrease in the number of active zones. A requirement is described for postsynaptic Syx4 in regulating several presynaptic parameters, including Ca(2+) cooperativity and the abundance of the presynaptic calcium channel Cacophony (Cac) at active zones. These findings indicate Syx4 negatively regulates presynaptic neurotransmitter release through a retrograde signaling mechanism from the postsynaptic compartment (Harris, 2018).

    Syntaxin 4 regulates multiple aspects of synaptic biology. This study reports that loss of Syx4 leads to synaptic enhancement, a surprising finding give that Syx4 mutant synapses have significantly fewer active zones than the control animals. An increase in evoked release and a reduction in paired-pulse facilitation were observed in Syx4 mutants. Two mechanisms were identified that are likely to contribute to the increase in neurotransmission: an increase in the levels of the presynaptic Ca2+ channel Cac at individual active zones, and a decrease in Ca2+ cooperativity. These two potentiation mechanisms could be linked – for example, an increase in Cac channels, leading to changes in Ca2+ influx and the spatial arrangement of the channels, could contribute to changes in the sensitivity of the exocytotic machinery to Ca2+. However, the possibility cannot be ruled out that they are distinct phenomena. As all of these phenotypes are rescued by postsynaptic, but not presynaptic, expression of Syx4, the data indicate a retrograde signaling mechanism by which Syx4 regulates active zones (Harris, 2018).

    Cac clustering at active zones is regulated by components of the active zone cytomatrix, including Brp. Of these, Brp has the largest effect, with an approximate 50% reduction in Cac levels at active zones of brp null mutants. Although Syx4 mutants have no obvious defects in the size or intensity of Brp clusters, the distribution other cytomatrix proteins has not yet been examined. One mechanism for Cac regulation downstream of Syx4 signaling could be through changes in the levels of other active zone cytomatrix components (Harris, 2018).

    One possible explanation for the potentiation observed in Syx4 mutants is that it is the result of homeostatic compensation. Many studies have described homeostatic mechanisms of potentiation and depression at the fly NMJ. In presynaptic homeostatic potentiation (PHP), perturbations that inhibit the function of postsynaptic glutamate receptors by acute pharmacological blockade or genetic loss, though most studies have not reported Cac levels. Presynaptic homeostatic depression (PHD) is a distinct phenomenon in which overexpression of the vesicular glutamate transporter, resulting in more glutamate packaged per synaptic vesicle, is offset by compensatory decreases in neurotransmitter release. PHD has been shown to involve a decrease in presynaptic Ca2+ influx and a decrease in Cac levels at active zones. Thus, the synapse employs multiple mechanisms during homeostatic plasticity, including regulation of Cac channels and Ca2+ influx (Harris, 2018).

    An important distinction is that during homeostatic compensation the compensatory changes typically restore muscle depolarization precisely, whereas in Syx4 mutants a significant enhancement of neurotransmission is seen, well beyond control levels. Nevertheless, it may be interesting to investigate whether any of the known homeostatic pathways are required for potentiation in Syx4 mutants. It is also possible that Syx4 itself is engaged in homeostatic mechanisms. For example, if Syx4 is involved in downregulating Cac channels, it could potentially participate in a previously described PHD mechanism, which would therefore be impaired in Syx4 mutant animals. Syx4 could also interact with other retrograde pathways that affect presynaptic release probability, such as signaling through the importin Imp13, which functions postsynaptically to regulate release probability and presynaptic intracellular Ca2+ (Harris, 2018).

    One intriguing observation from the current study is that paired-pulse facilitation is enhanced, compared to controls, when Syx4 is expressed postsynaptically in Syx4 mutants. This is surprising since simple overexpression of Syx4 in a wildtype animal does not affect facilitation. While it is not yet understood how this enhanced facilitation arises, one possibility could be differential expression of Syx4 isoforms. All of the overexpression and rescue experiments described in this study were conducted using a full-length Syx4 cDNA (Syx4A). However, there is a second isoform of Syx4 (Syx4B), encoding a shorter N-terminus, which is redundant with the A isoform with respect to all other phenotypes characterized for Syx4 to date. However no evidence was found that Syt4 or Nlg participate with Syx4 to regulate the Syx4 presynaptic enhancement phenotypes. Thus, it is likely that Syx4, as a postsynaptic t-SNARE, mediates the release of additional retrograde signals, and that multiple overlapping Syx4-dependent pathways are involved in establishing normal synaptic morphology, plasticity, and function (Harris, 2018).

    This study has described electrophysiological analysis of animals lacking the postsynaptic t-SNARE Syx4. Syx4 mutants exhibit synaptic enhancement accompanied by presynaptic changes at active zones, including an increase in presynaptic Ca2+ channels at active zones and a decrease in Ca2+ cooperativity. All of these features are rescued by restoring postsynaptic Syx4. It is concluded that retrograde pathways regulated by Syx4 inhibit active zone potentiation, and that Syx4 modulates multiple postsynaptic signaling pathways with overlapping function (Harris, 2018).

    Molecular interface of neuronal innate immunity, synaptic vesicle stabilization, and presynaptic homeostatic plasticity

    This study define a homeostatic function for innate IMD immune signaling within neurons. A genetic analysis of the innate immune signaling genes IMD, IKKbeta, Tak1, and Relish demonstrates that each is essential for presynaptic homeostatic plasticity (PHP). Subsequent analyses define how the rapid induction of PHP (occurring in seconds) can be coordinated with the life-long maintenance of PHP, a time course that is conserved from invertebrates to mammals. This study defines a novel bifurcation of presynaptic innate immune signaling. Tak1 (Map3K) acts locally and is selective for rapid PHP induction. IMD, IKKbeta, and Relish are essential for long-term PHP maintenance. This study then define how Tak1 controls vesicle release. Tak1 stabilizes the docked vesicle state, which is essential for the homeostatic expansion of the readily releasable vesicle pool. This represents a mechanism for the control of vesicle release, and an interface of innate immune signaling with the vesicle fusion apparatus and homeostatic plasticity (Harris, 2018).

    This study has tested the core signaling components of an innate immune signaling pathway (IMD signaling) for a role during presynaptic homeostatic plasticity. The data support a model in which IMD signaling bifurcates downstream of the presynaptic innate immune receptor PGRP-LC to achieve immediate, local modulation of the presynaptic release apparatus via Tak1, and prolonged maintenance of the homeostatic response via the transcription factor Relish. This model allows the innate immune signaling system to rapidly alter presynaptic release (seconds to minutes) and simultaneously initiate a Rel-dependent consolidation of PHP. It is noteworthy that the consolidation of PHP can persist for months in insects and decades in humans. It has become clear that the molecular mechanisms responsible for the rapid induction and sustained expression are genetically separable. These findings provide an explanation for how the rapid induction and sustained expression of PHP are mechanistically coordinated (Harris, 2018).

    The canonical function of innate immunity is to recognize invading pathogens or non-normal molecular patterns and induce a rapid, inflammatory reaction that is sustained for as long as the invasion persists. During an innate immune response, Tak1-dependent signaling turns on more rapidly, and turns off more rapidly, than Rel-mediated transcription. As such, Tak1 signaling can be considered as a feedforward activator of the cellular immune response. Based on the data, it is proposed that innate immune signaling in the nervous system is transfigured to detect non-normal neurophysiology, although the molecular event that reports altered neural (synaptic) function and is subsequently detected by pre-synaptic PGRP-LC remains a mystery. In the current study, Tak1 is placed at the presynaptic release site where homeostatic plasticity is rapidly induced. Thus, just as in canonical innate immunity, Tak1 is ideally situated to act as a feedforward potentiometer that controls vesicle fusion, thereby achieving a rapid compensatory change in presynaptic release following postsynaptic glutamate receptor inhibition. Subsequent activation of Rel- mediated transcription provides a sustained response that can be maintained for the duration of the perturbation (Harris, 2018).

    Evidence is provided that Tak1 has a potent, constitutive function to stabilize the docked vesicle pool, defined both electrophysiologically and at the EM level. This can explain the dramatic defect in spontaneous miniature release frequency, which occurs without a change in the total number of presynaptic release sites. Fewer docked vesicles can explain the defect in EPSC amplitude despite normal action-potential-induced calcium influx. It was also demonstrated that vesicles can be mobilized to the release site during a stimulus train, temporarily achieving WT release rates before declining back to a reduced baseline. This effect reflects a stimulus-dependent re-population of the docked vesicle pool, as demonstrated by EM analysis directly following a train of APs. The stimulus, and presumably calcium-dependent, re-population of the docked vesicle pool explains why there is pronounced short-term synaptic facilitation in the Tak1 mutants compared to short-term depression in WT. Essentially, the number of docked vesicles is increased in a stimulus-dependent manner, something that does not occur in WT, based on EM analysis. Finally, analysis of recovery from vesicle depletion pinpoints the time at which Tak1 activity becomes essential. The recovery of synaptic transmission is completely normal in Tak1 for the first 500 ms following a stimulus train. It is only after this time point that recovery becomes defective (Harris, 2018).

    As a working model, it is proposed that Tak1 functions to inhibit the rate of vesicle undocking and, thereby, persistently stabilizes the docked/primed vesicle pool. Immediately following a stimulus train, calcium- dependent mechanisms actively mobilize vesicles to the active zone and drive calcium-dependent vesicle docking and priming. Thus, calcium-dependent potentiation of the forward rate of docking/priming transiently overcomes the enhanced un-docking rate caused by loss of Tak1 and the number of docked vesicles is restored to WT levels. As intra-terminal calcium levels drop following the stimulus train, the rates of docking and un-docking re-equilibrate in the Tak1 mutant, reaching a steady state with many fewer docked vesicles compared to WT (Harris, 2018).

    This model can also explain the failure of PHP in a Tak1 mutant. The rapid induction of PHP occurs following inhibition of postsynaptic glutamate receptors and is expressed in response to a single AP. This requires an expansion of the resting pool of docked/primed vesicles and this cannot be achieved in the absence of Tak1. Accordingly, enhanced Tak1 activity, following activation of the innate immune receptor PGRP, would inhibit the rate of vesicle un-docking and potentiate the pool of docked/primed vesicles, necessary for the rapid induction of PHP. Thus, Tak1 functions as a molecular potentiometer controlling the docked/primed vesicle pool during PHP. A question for future experimentation concerns how baseline Tak1 activity is established to achieve highly reproducible, baseline synaptic transmission in WT (Harris, 2018).

    Most of the knowledge regarding the organization of innate immune signaling pathways is based on assays that detect the nuclear translocation of Rel/NF-κB, or quantify Rel/NF-κB-mediated transcription. Thus, the logic and spatial organization of innate immune signaling in neurons has yet to be clearly defined. The data argue for several novel features of signaling organization in a neuron, beyond the local synaptic action of Tak1. In canonical innate immune signaling, receptor activation catalyzes the assembly of an intracellular complex that includes Imd, Tak1, and IKKβ. Based on the evidence in Drosophila motoneurons, and consistent with published models of IMD signaling, it is proposed that activation of the PGRP receptor drives assembly of the IMD signaling complex that includes a poly-ubiquitin chain as well as the three proteins IMD, Tak1, and IKKβ. Then, it is suggested that IKKβ is necessary for this signaling complex to either assemble correctly or signal efficiently. Thus, IKKβ is necessary for both the short- and long-term forms of PHP, just like the PGRP receptor. Finally, once this complex is assembled and activated, two independent signal transduction cascades are initiated. One requires Tak1 and acts locally within the presynaptic terminal to achieve the rapid induction of PHP. The second pathway requires the function of IMD and signals via Rel-mediated transcription, necessary for the long-term maintenance of PHP. It is noted, however, that other models for IKKβ activation should also be considered. Finally, the data raise an important question regarding how signaling is conveyed from synaptic IKKβ to nuclear Rel, a topic of future studies (Harris, 2018).

    Postsynaptic glutamate receptors regulate local BMP signaling at the Drosophila neuromuscular junction

    Effective communication between pre- and post-synaptic compartments is required for proper synapse development and function. At the Drosophila neuromuscular junction (NMJ), a retrograde BMP signal functions to promote synapse growth, stability and homeostasis and coordinates the growth of synaptic structures. Retrograde BMP signaling triggers accumulation of the pathway effector pMad in motoneuron nuclei and at synaptic termini. Nuclear pMad, in conjunction with transcription factors, modulates the expression of target genes and instructs synaptic growth; a role for synaptic pMad remains to be determined. This study reports that pMad signals are selectively lost at NMJ synapses with reduced postsynaptic sensitivities. Despite this loss of synaptic pMad, nuclear pMad persisted in motoneuron nuclei, and expression of BMP target genes was unaffected, indicating a specific impairment in pMad production/maintenance at synaptic termini. During development, synaptic pMad accumulation followed the arrival and clustering of ionotropic glutamate receptors (iGluRs) at NMJ synapses. Synaptic pMad was lost at NMJ synapses developing at suboptimal levels of iGluRs and Neto, an auxiliary subunit required for functional iGluRs. Genetic manipulations of non-essential iGluR subunits revealed that synaptic pMad signals specifically correlate with the postsynaptic type-A glutamate receptors. Altering type-A receptor activities via protein kinase A (PKA) revealed that synaptic pMad depends on the activity and not the net levels of postsynaptic type-A receptors. Thus, synaptic pMad functions as a local sensor for NMJ synapse activity and has the potential to coordinate synaptic activity with a BMP retrograde signal required for synapse growth and homeostasis (Sulkowski, 2013).

    Previous work has described Neto as the first nonchannel subunit required for the clustering of iGluRs and formation of functional synapses at the Drosophila NMJ. Neto and iGluR complexes associate in the striated muscle and depend on each other for targeting and clustering at postsynaptic specializations. This study shows that Neto/iGluR synaptic complexes induce accumulation of pMad at synaptic termini in an activity-dependent manner. The effect of Neto/iGluR clusters on BMP signaling is selective, and limited to synaptic pMad; nuclear accumulation of pMad appears largely independent of postsynaptic glutamate receptors. This study demonstrates that synaptic pMad mirrors the activity of postsynaptic type-A receptors. As such, synaptic pMad may function as an acute sensor for postsynaptic sensitivity. Local fluctuations in synaptic pMad may provide a versatile means to relay changes in synapse activity to presynaptic neurons and coordinate synapse activity status with synapse growth and homeostasis (Sulkowski, 2013).

    Drosophila NMJs maintain their evoked potentials remarkably constant during development, from late embryo to the third instar larval stages. This coordination between motoneuron and muscle properties requires active trans-synaptic signaling, including a retrograde BMP signal, which promotes synaptic growth and confers synaptic homeostasis. Nuclear pMad accumulates in motoneurons during late embryogenesis. However, embryos mutant for BMP pathway components hatch into the larval stages, indicating that BMP signaling is not required for the initial assembly of NMJ synapses and instead modulates NMJ growth and development. This study demonstrates that synaptic accumulation of pMad follows GluRIIA arrival at nascent NMJs and depends on optimal levels of synaptic Neto and iGluRs. As type-A receptors have been associated with nascent synapses, and type-B receptors mark mature NMJs, accumulation of synaptic pMad appears to correlate with a growing phase at NMJ synapses. Furthermore, synaptic pMad correlates with the activity and not the net levels of postsynaptic type-A receptors. In fact, expression of a GluRIIA variant with a mutation in the putative ion conduction pore triggered reduction of synaptic pMad levels. Thus, synaptic pMad functions as a molecular sensor for synapse activity and may constitute an important element in synapse plasticity (Sulkowski, 2013).

    The synaptic pMad pool has been localized primarily to the presynaptic compartment. However, a contribution for postsynaptic pMad to the pool of synaptic pMad is also possible. Postsynaptic pMad accumulates in response to glia-secreted Mav, which regulates gbb expression and indirectly modulates the Gbb-mediated retrograde signaling (Fuentes-Medel, 2012). RNAi experiments revealed that knockdown of mad in muscle induces a decrease in synaptic pMad, albeit much reduced in amplitude compared with knockdown of mad in motoneurons (Fuentes-Medel, 2012). Also, knockdown of wit in motoneurons, but not in muscle, and knockdown of put in muscle, but not in motoneurons, triggers reduction of synaptic pMad (Fuentes-Medel, 2012). Intriguingly, the synaptic pMad is practically abolished in GluRIIA and neto109 mutants and cannot be further reduced by additional decrease in Mad levels. Whereas loss of postsynaptic pMad could be due to a Mav-dependent feedback mechanism that controls Gbb secretion from the muscle, the absence of presynaptic pMad demonstrates a role for GluRIIA and Neto in modulation of BMP retrograde signaling (Sulkowski, 2013).

    As BMP signals are generally short lived, synaptic pMad probably reflects accumulation of active BMP/receptor complexes at synaptic termini. Recent evidence suggests that BMP receptors traffic along the motoneuron axons, with Gbb/receptors complexes moving preferentially in a retrograde direction. By contrast, Mad does not appear to traffic. Thus, Mad is likely to be phosphorylated and maintained locally by a pool of active Gbb/BMP receptor complexes that remain at synaptic termini for the time postsynaptic type-A receptors are active (Sulkowski, 2013).

    The activity of type-A glutamate receptors may control synaptic pMad accumulation (1) indirectly via activity-dependent changes that are relayed to both pre- and postsynaptic cells, or (2) directly by influencing the production and signaling of varied Gbb ligand forms or by localizing Gbb activities. For example, inhibition of postsynaptic receptor activity induces trans-synaptic modulation of presynaptic Ca2+ influx. Such Ca2+ influx changes may trigger events that induce a local change in synaptic pMad accumulation. One possibility is that changes in Ca2+ influx may recruit Importin-β11 at presynaptic termini, which in turn mediate synaptic pMad accumulation (Sulkowski, 2013).

    At the Drosophila NMJ, Gbb is secreted in the synaptic cleft from both pre- and postsynaptic compartments. The secretion of Gbb is regulated at multiple levels, transcriptionally and post-translationally. Furthermore, the Gbb prodomain could be processed at several cleavage sites to generate Gbb ligands with varying activities. The longer, more active Gbb ligand retains a portion of the prodomain that could influence the formation of Gbb/BMP receptor complexes. Synaptic pMad may result from signaling by selective forms of Gbb. Or type-A receptors could modulate secretion and processing of Gbb in an activity-dependent manner. Understanding the function of different pools and active forms of Gbb within the synaptic cleft will help explain the multiple roles for Gbb at Drosophila NMJs (Sulkowski, 2013).

    Alternatively, active postsynaptic type-A receptor complexes may directly engage and stabilize presynaptic Gbb/BMP receptor signaling complexes via trans-synaptic interactions. CUB domains can directly bind BMPs; thus Neto may utilize its extracellular CUB domains to engage Gbb and/or presynaptic BMP receptors. As synaptic pMad mirrors active type-A receptors, such trans-synaptic complexes will depend on Neto in complexes with active type-A receptors. No capture has yet been shown of a direct interaction between Gbb and Neto CUB domains in co-immunoprecipitation experiments. Nonetheless, a trans-synaptic complex that depends on the activity of type-A receptors could offer a versatile means for relaying synapse activity status to the presynaptic neuron via fast assembly and disassembly (Sulkowski, 2013).

    Irrespective of the strategy that correlates synaptic pMad pool with the active type-A receptor/Neto complexes, further mechanisms must act to maintain the Gbb/BMP receptor complexes at synapses and protect them from endocytosis and retrograde transport. Such mechanisms must be specific, as general modulators of BMP receptors endocytosis impact both synaptic and nuclear pMad. A candidate for differential control of BMP/receptor complexes is Importin-β11. Loss of synaptic pMad in importin-β11 is rescued by neuronal expression of activated BMP receptors, by blocking retrograde transport, but not by neuronal expression of Mad. As Mad does not appear to traffic, presynaptic Importin-β11 must act upstream of the BMP receptors, perhaps to stabilize active Gbb/BMP receptor complexes at the neuron membrane. By contrast, local pMad cannot be restored at Neto-deprived NMJs by overactivation of presynaptic BMP receptors or by blocking retrograde transport. As neto and gbb interact genetically, it is tempting to speculate that postsynaptic Neto/type-A complexes localize Gbb activities and stabilize Gbb/BMP receptor complexes from the extracellular side. Additional extracellular factors, for example heparan proteoglycans, or intracellular modulators, such as Nemo kinase, may control the distribution of sticky Gbb molecules within the synaptic cleft and their binding to BMP receptors, or may stabilize Gbb/BMP receptor complexes at synaptic termini (Sulkowski, 2013).

    Synaptic pMad may act locally and/or in coordination with the transcriptional control of BMP target genes to ensure proper growth and development of the synaptic structures. A presynaptic pool of pMad maintained by Importin-β11 neuronal activities ensures normal NMJ structure and function. Like importin-β11, GluRIIA and Neto-deprived synapses show a significantly reduced number of boutons. Intriguingly, the absence of GluRIIA induces up to 20% reduction in bouton numbers, whereas knockdown of GluRIIB does not appear to affect NMJ growth. Although the amplitude of the growth phenotypes observed in normal culturing conditions (25°C) was modest, this phenomenon may explain the requirement for GluRIIA reported for activity-dependent NMJ development (at 29°C). Furthermore, knockdown of Neto or any iGluR essential subunit affect synaptic pMad and NMJ growth in a dose-dependent manner. Not significant changes were found in nuclear pMad or expression of BMP target genes in GluRIIA or Neto-deprived animals, but the restoration of synaptic pMad by presynaptic constitutively active BMP receptors rescues the morphology and physiology of importin-β11 mutant NMJs. The smaller NMJs observed in the absence of local pMad may reflect a direct contribution of synaptic pMad to retrograde BMP signaling, a pathway that provides an instructive signal for NMJ growth. Thus, BMP signaling may integrate synapse activity status with the control of synapse growth (Sulkowski, 2013).

    Synaptic pMad may also contribute to synapse stability. Mutants in BMP signaling pathway have an increased number of 'synaptic footprints': regions of the NMJ where the terminal nerve once resided and has retracted. It has been proposed that Gbb binding to its receptors activates the Williams Syndrome-associated Kinase LIMK1 to stabilize the NMJ. Synaptic pMad may further contribute to the stabilization of synapse contacts by engaging in interactions that anchor the Gbb/BMP receptor complexes at synaptic termini. During neural tube closure, local pSmad1/5/8 mediates stabilization of BMP signaling complexes at tight junction via binding to apical polarity complexes. Flies may utilize a similar anchor mechanism that relies on pMad-mediated interactions for stabilizing BMP signaling complexes and other components at synaptic junctions. Local active BMP signaling complexes are thought to function in this manner in the maintenance of stemness and in epithelial-to-mesenchymal transition (Sulkowski, 2013).

    Separate from its role in synapse growth and stability, BMP signaling is required presynaptically to maintain the competence of motoneurons to express homeostatic plasticity. The requirements for BMP signaling components for the rapid induction of presynaptic response may include a role for synaptic pMad in relaying acute perturbations of postsynaptic receptor function to the presynaptic compartment. At the very least, attenuation of local pMad signals, when postsynaptic type-A receptors are lost or inactive, may release local Gbb/BMP receptor complexes and allow them to traffic to neuron soma and increase the BMP transcriptional response, promoting expression of presynaptic components and neurotransmitter release. In addition, synaptic pMad-dependent complexes may influence the composition and/or activity of postsynaptic glutamate receptors. Although future experiments will be needed to address the nature and function of local pMad-containing complexes, the current findings clearly demonstrate that synaptic pMad constitutes an exquisite monitor of synapse activity status, which has the potential to relay information about synapse activity to both pre- and postsynaptic compartments and contribute to synaptic plasticity. As BMP signaling plays a crucial role in synaptic growth and homeostasis at the Drosophila NMJ, the use of synaptic pMad as a sensor for synapse activity may enable the BMP signaling pathway to monitor synapse activity then function to adjust synaptic growth and stability during development and homeostasis (Sulkowski, 2013).

    MAN1 restricts BMP signaling during synaptic growth in Drosophila

    Bone morphogenic protein (BMP) signaling is crucial for coordinated synaptic growth and plasticity. This study shows that the nuclear LEM-domain protein MAN1 is a negative regulator of synaptic growth at Drosophila larval and adult neuromuscular junctions (NMJs). Loss of MAN1 is associated with synaptic structural defects, including floating T-bars, membrane attachment defects, and accumulation of vesicles between perisynaptic membranes and membranes of the subsynaptic reticulum. In addition, MAN1 mutants accumulate more heterogeneously sized vesicles and multivesicular bodies in larval and adult synapses, the latter indicating that MAN1 may function in synaptic vesicle recycling and endosome-to-lysosome trafficking. Synaptic overgrowth in MAN1 is sensitive to BMP signaling levels, and loss of key BMP components attenuate BMP-induced synaptic overgrowth. Based on these observations, it is proposed that MAN1 negatively regulates accumulation and distribution of BMP signaling components to ensure proper synaptic growth and integrity at larval and adult NMJs (Laugks, 2016).

    Jelly belly trans-synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

    In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ), and a conserved intercellular signaling function was been postulated. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans-synaptic signaling. This study shows that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb-Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild-type postsynaptic Alk expression in Alk partial loss-of-function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb-Alk signaling triggers the Ras-MAP kinase cascade in both pre- and postsynaptic compartments. These novel roles for Jeb-Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis (Rohrbough, 2013).

    The results support an anterograde signaling model in which presynaptically secreted Jeb activates postsynaptic Alk. The data to support this hypothesis derives from multiple tests. First, immunolabeling shows Jeb is concentrated within presynaptic boutons, while Alk is present in the surrounding postsynaptic subsynaptic reticulum (SSR) (Rohrbough, 2011). Second, targeted postsynaptic Alk expression in Alk LOF mutants is sufficient to rescue synaptic transmission defects, a strong demonstration that Alk is required in the postsynaptic muscle to regulate neurotransmission. Third, post-synaptic inhibtion of Alk by tissue specific RNAi results in 2- fold increased accumulation of perisynaptic Jeb. Fourth, the MARCM clonal approach demonstrates Jeb may be required within presynaptic motor neurons to regulate postsynaptic molecular assembly. Fifth, elevated presynaptic Jeb expression activates postsynaptic Ras/MAPK/ERK activation, while inhibition of postsynaptic Alk reduces Ras/MAPK/ERK activitation (Rohrbough, 2013).

    In structurally normal NMJs, strong effects on neurotransmission were found as a consequence of perturbations in Jeb-Alk signaling. The clearest, most consistent results derive from techniques that activate or inhibit Jeb-Alk signaling postsynaptically. Postsynaptic hyperactivation of Alk weakens NMJ synaptic transmission. This functional phenotype parallels the negative regulation of synaptic growth by postsynaptic Alk activation. Consistent with the inhibitory effect of Alk activation on neurotransmission, enhanced neurotransmission was observed as a consequence of muscle specific reductions in Alk levels by transgenic RNAi. Additional confirmation for Alk-dependent inhibition of neurotransmission is provided by analysis of a hypomorphic temperature sensitive allele of Alk. Partial loss of Alk function results in strongly increased NMJ neurotransmission. The implication is that Alk activity limits or negatively regulates synaptic strength. It was also shown that muscle-specific Alk expression in the strongest alkts/alkf01491 partial loss of function genotype rescues reduced neurotransmission to near wild-type levels, a conclusive demonstration that postsynaptic Alk function negatively regulates the strength of NMJ neurotransmission. This function is novel: Jeb-Alk transynaptic signaling is the only known negative regulator of synaptic transmission (Rohrbough, 2013).

    Presynaptic manipulation of Jeb yields less strong though still consistent results. Transmission is uneffected by increased pan-neuronal Jeb expression, though this activates Ras/MAPK/ERK both centrally and presynaptically at the NMJ and, to a lesser degree, within the postsynaptic muscle. Motor neuron electrical activity activates neuronal Ras/MAPK/ERK signaling, and this presynaptic Ras/MAPK/ERK activation is positively linked to both structural and functional NMJ synaptic remodeling. Motor neuron specific over expression of Jeb does produce a modest but statistically significant reduction in neuromuscular transmission. Ectopic expression of Jeb in muscle results in substantial inhibiton of neuromuscular transmission. One hypothesis that may account for the diffence between panneuronal and motor neuron or muscle specific manipulation of Jeb-Alk signalling is that the effects of manipulating pan-neuronal Jeb represent a composite of central and peripheral effects on the motor neuron. In first instar larvae it was found that both jeb and alk mutants display impaired central output to motor neurons most consistent with a central synaptic defect (Rohrbough, 2011). The integrated physiologic function subserved by Jeb-Alk signaling in the NMJ, which has yet to be determined, will provide the essential context for interpretting these results (Rohrbough, 2013).

    The novel inhibitory role of Jeb-Alk signaling in NMJ transmission implies that it is part of a transynaptic regulatory network that integrates neuronal activity and responses with other homeostatic mechanisms. This study provides indirect evidence that Jeb secretion is regulated. The physiologic regulation of Jeb secretion is a critical missing component of understanding how Jeb-Alk signaling fits into the regulation of synaptic plasticity. Jeb-Alk signaling regulates postembryonic NMJ synaptic growth and patterning Jeb-Alk signaling is not required for embryonic NMJ synaptogenesis or differentiation, although jeb and alk null mutants display impaired locomotion and reduced NMJ transmission (Rohrbough and Broadie 2011). At later developmental stages, removing Jeb in motor neurons strongly disrupts late larval NMJ synaptic terminal architecture and bouton morphology. Postsynaptic Dlg scaffolding and GluR clustering are strongly perturbed in association with jeb mutant terminals. The mosaic analysis supports a cell-autonomous, anterograde signaling function for Jeb. One mechanistic hypothesis is that Jeb-Alk nerve-to-muscle signaling regulates NMJ morphogenesis by recruiting or regulating cell adhesion molecules (CAMs). In the developing adult visual system, anterograde Jeb-Alk signaling induces the expression of postsynaptic adhesion molecules Dumbfounded/Kirre, Roughest/IrreC and Flamingo to shape the optic neuropil target environment. At the larval NMJ, adhesion molecules such as fasciclins and integrins regulate activity-dependent synaptic growth and structural remodeling. The current results imply that Jeb-Alk signaling either directly regulates Dlg localization or indirectly drives Dlg-dependent postsynaptic differentiation. Dlg has demonstrated roles in NMJ morphogenesis and GluR expression and field regulation, and directly binds and regulates fasciclin II and βPS integrin. Future work will test the hypothesis that Jeb-Alk signaling organizes or regulates adhesion receptors and postsynaptic scaffolding to control bouton differentiation and shape functional synaptic architecture (Rohrbough, 2013).

    In other systems, Jeb-Alk signaling has been studied primarily at the level of behavior. In C. elegans, the Jeb homolog Hen-1 was identified in a forward genetic behavioral screen for impaired ability to integrate conflicting sensory input (Ishihara, 2002). The Hen-1 phenotype is non-developmental and can be rescued only by adult Hen-1 expression. There is no uniquely identified mammalian Jeb/Hen-1 homolog, but ALK is expressed in the mammalian nervous system during development and at maturity. Alk is expressed in the mouse hippocampus and Alk loss of function enhances behavioral performance in tests dependent on hippocampal function. Similarly, Drosophila learning has shown a dependence on the Ras/MAPK/ERK cascade, which is activated by Jeb-Alk signaling and is probably inhibited by Drosophila neurofibromin (dNf1). Genetic or pharmacologic inhibtion of Jeb-Alk signaling enhances associative learning while increased Jeb-Alk signaling or loss of dNf1 impairs learning. Inhibition of Alk rescues dNf1 mutant learning deficits. These studies suggest that the Jeb-Alk trans-synaptic pathway acts in concert with other, negative regulators of Ras/MAPK/ERK signaling to balance developmental and learning-related synaptic structural and functional changes. Strikingly, a whole-genome association study recently identified human ALK as one of a small number of genes associated with sporadic amyotrophic lateral sclerosis (ALS), a devistating neurodegerative disease of central motor units. If Alk has a conserved inhibitory role in synaptic physiological regulation, hypofunctional human Alk variants may result in augmented motor unit activity and contribute to excitotoxicity and progressive motor unit degeneration in ALS. Pharmacologic activation of Alk has already been hypothesized to have therapeutic benefit in treating ALS. Further insight from future studies should be gained into the mechanism by which the Jeb-Alk signaling pathway regulates synaptic adaptivity in both normal and pathological states (Rohrbough, 2013).

    Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling

    Spinal muscular atrophy (SMA), a devastating neurodegenerative disorder characterized by motor neuron loss and muscle atrophy, has been linked to mutations in the Survival Motor Neuron (SMN) gene. Based on an SMA model developed in Drosophila, which displays features that are analogous to the human pathology and vertebrate SMA models, the fibroblast growth factor (FGF) signaling pathway was functionally linked to the Drosophila homologue of SMN, Smn through interaction of the FGF receptor breathless with Smn. This study functionally characterize this relationship and demonstrates that Smn activity regulates the expression of FGF signaling components and thus FGF signaling. Furthermore, it was shown that alterations in FGF signaling activity are able to modify the neuromuscular junction defects caused by loss of Smn function and that muscle-specific activation of FGF is sufficient to rescue Smn-associated abnormalities (Sen, 2011).

    Given the variability of the SMA phenotype and the proven relationship between the severity of the disease and small changes in wild-type SMN activity, there is a significant possibility that any modifiers of SMN activity, either direct or indirect, will have therapeutic value. To systematically explore the genome for genes that are capable of modulating SMN function in vivo, advantage was taken of the existence of an SMA model offered by Drosophila to search for Smn genetic interactors. The model that was developed is based on the lethality and an associated neuromuscular junction phenotype linked to loss of Smn function, a phenotype remarkably similar to the NMJ phenotype reported for human patients. Though the role of SMN in biogenesis of snRNPs has been well documented, its regulators and downstream effectors have not been systematically delineated, nor has the link between mutations in SMN and the specific loss of motor neurons seen in SMA patients been uncovered. It may be the case that the specificity of this phenotype is reflective of either specialized SMN functions at the NMJ or a particular sensitivity of motor neurons to the loss of SMN activity. Among the genes the genetic strategy revealed as Smn loss of function modifiers was breathless, encoding an FGF receptor, thus establishing a link between Smn and the FGF pathway (Sen, 2011).

    Importantly, in addition to this link, it was also found that FGF signaling is independently involved in NMJ morphogenesis, a function demonstrated in vertebrates but not previously attributed to this pathway in Drosophila despite extensive characterization of its essential role in branching morphogenesis of the tracheal system, migration of multiple cell types, as well as the proper patterning of the mesoderm. The morphological effects that were observed, caused by the modulation of several pathway elements, plainly reveal an involvement of FGF signaling at the NMJ, a role confirmed by the electrophysiological analyses. The down-regulation of FGF signals in muscle results in a reduction of bouton numbers and is associated with increased mEJP amplitudes. The opposite effect is observed when FGF signaling is increased in muscles, suggesting that FGF signaling inversely regulates quantal size. Thus, FGF perturbation in muscle alters both presynaptic growth and specific aspects of synaptic transmission. These observations imply the existence of functional trans-synaptic homeostatic mechanisms, which have been previously shown to compensate for similar changes by increasing presynaptic bouton numbers and transmitter release. However, in this specific instance, only synaptic growth (bouton number) but not transmitter release (quantal content) is affected, the precise mechanisms for which remain unclear. Moreover, the fact that mEJP amplitudes are affected suggests that postsynaptic receptivity to glutamate release from the presynapse is altered. Similar quantal size phenotypes have been observed in several instances previously. For instance, postsynaptic PKA and NF-kappaB are known to regulate quantal size through changes in DGluRs. Directly altering the expression of various GluR subunits also predictably influences quantal size. The genetic interaction this study has demonstrated between FGF and Smn can be described as an epistatic relationship in which the FGF pathway functions downstream of Smn and is consistent with the observation that neuromuscular defects associated with loss of Smn function in muscle can be rescued by muscle-specific activation of FGF signaling. Intriguingly, the relationship described in this study between Smn and FGF is valid beyond the NMJ, as loss of Smn function genetic mosaics in the wing disc clearly result in the down-regulation of FGF signaling. Although the precise molecular mechanism underlying this relationship is still elusive, Smn activity affects transcript and protein levels of the FGF receptor, as well as the expression of additional elements of the FGF pathway. Whether this defines a cascade of interrelated events or whether each of these changes reflects an independent Smn-related regulatory event remains to be determined. Given the fact that Smn mutants in Drosophila display altered postsynaptic currents and severely compromised postsynaptic receptor clustering in muscles, it is conceivable that FGF signaling represents a link between Smn activity and postsynaptic glutamate receptor levels (Sen, 2011).

    It should be noted that a link between SMN and the FGF pathway has been suggested by a series of studies in vertebrates where a molecular interaction between an FGF-2 isoform and the SMN protein has been described.These studies raise the possibility that FGF-2 may negatively interfere with SMN complex function through SMN itself. Such observations would, on first appearance, suggest that the epistatic relationship between SMN and FGF signaling in vertebrate cells may be the reverse of what was observed in Drosophila. In point of fact however, the differences in the experimental parameters and approaches between these studies do not allow meaningful comparisons (Sen, 2011).

    An important question raised by the above phenotypic analyses is whether the abnormalities associated with FGF and/or Smn perturbations reflect developmental or maintenance issues. It may be the case that the larval system in Drosophila is not ideally suited to differentiate between these alternatives as larval tissue is destined to undergo programmed cell death (histolysis) during metamorphosis. One advantage that flies do offer, however, is the ability to dissociate the development of the adult neuromuscular system from its maintenance as the entirety of its development occurs during the pupal stage, before emergence of the adult. Thus, the Drosophila pupa/adult may provide a platform to address these issues, as Drosophila displays Smn-dependent adult phenotypes. In light of the relationship that was established between Smn and FGF signaling and the known involvement of FGF signaling in the development of both the larval and adult musculature, it will be particularly interesting to examine the effects of modulating FGF activity on the aforementioned processes. Such studies may be of particular relevance to SMA where it is quite difficult to discern the developmental consequences of SMN loss in humans, as neurodegenerative symptoms displayed by patients may obscure basic problems resulting from altered developmental programs such as neuronal pathfinding, initial NMJ formation, etc (Sen, 2011).

    In vertebrates, synaptic development and maintenance use at least three distinct signaling mechanisms: the TGF-β, wingless, and FGF pathways. In Drosophila, it is noteworthy that the first two have been demonstrated to function in a similar fashion at the NMJ. Remarkably, the genetic screens involving Smn have identified elements of all three of these pathways as modifiers of Smn-related phenotypes. These connections are considered particularly significant as they raise the possibility that Smn may serve as a node, integrating signaling events crucial for NMJ function, potentially leaving this structure particularly vulnerable to the loss of Smn. Though further correspondence between the Drosophila model and the human condition remains to be determined, the Smn-FGF relationship observed in Drosophila raises the possibility that pharmacological manipulation of FGF signals might mitigate SMN motor neuron-related abnormalities (Sen, 2011).

    Drosophila Neuroligin3 regulates neuromuscular junction development and synaptic differentiation

    Neuroligins (Nlgs) are a family of cell adhesion molecules thought to be important for synapse maturation and function. Studies in mammals have shown that different Nlgs have different roles in synaptic maturation and function. The functions of Drosophila Neuroligin1 (DNlg1), DNlg2, and DNlg4 have also been examined. This study reports the role of DNlg3 in synaptic development and function by using Drosophila neuromuscular junctions (NMJs) as a model system. DNlg3 was found to be expressed in both CNS and NMJs where it was largely restricted to the postsynaptic site. By generating and examining dnlg3 mutants, the mutants mutants were found to exhibit an increased bouton number and reduced bouton size compared to the wild-type. Consistent with alterations in bouton properties, pre- and postsynaptic differentiations were also affected including abnormal synaptic vesicle endocytosis, increased PSD length and reduced GluRIIA recruitment. Additionally, synaptic transmission was reduced. Altogether, this study shows that DNlg3 is required for NMJ development, synaptic differentiation and function (Xing, 2014).

    Anterograde Activin signaling regulates postsynaptic membrane potential and GluRIIA/B abundance at the Drosophila neuromuscular junction

    Members of the TGF-beta superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. This study analyzed whether the Activin branch of the TGF-beta superfamily also contributes to NMJ development and function. Elimination of the Activin/TGF-beta type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. These results demonstrate that anterograde Activin/TGF-beta signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling via Glass bottom boat and Wishful thinking, these findings indicate that the two branches of the TGF-beta superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function (Kim, 2014).

    Numerous reports have now implicated the Activin/TGF-β and BMP branches of the TGF-β superfamily in regulating neuronal development, synaptic plasticity and cognitive behavior. Accordingly, members from both subfamilies are widely expressed in the nervous system and are co-expressed in multiple regions of vertebrate and invertebrate brains. It is therefore quite likely that ligands of both subfamilies co-exist within the extracellular space and in some cases, act on the same neurons. Lending support to this idea, pyramidal neurons in the CA3 region of the rat hippocampus are known to accumulate both phosphorylated Smad2 and Smad1/5/8, transcriptional transducers of the canonical Activin/TGF-β and BMP-type signaling, respectively. The activation of these two closely-related signaling pathways in common sets of neurons, or different cells of a common neuronal circuit raises the intriguing question of whether the two pathways play different or redundant roles during neuronal development and function (Kim, 2014).

    This study utilized the Drosophila neuromuscular junction to address this issue since ligands of both Activin/TGF-β and BMP families are expressed in both muscle and motor neurons. The data, together with previous studies on the role of BMP signaling at the NMJ, clearly demonstrate that the two pathways influence NMJ synaptogenesis in different ways. The Activin/TGF-β pathway is necessary for achieving the proper densities of GluRIIA, GluRIIB and Dlg in postsynaptic muscle membrane, while the BMP pathway has a smaller effect on the distribution of these postsynaptic proteins. In addition, the Activin/TGF-β pathway was dispensable for maintaining overall synaptic growth and homeostasis, both of which are strongly affected by mutations in the BMP pathway. In addition, tissue-specific rescue experiments indicate that the postsynaptic reception of Activin/TGF-β signaling is important in regulating synaptic GluR abundance, whereas BMP signal reception is known to act in the presynaptic motor neurons to promote synaptic growth. These observations suggest that each pathway influences NMJ synapse development and function by acting mainly in either the pre- or postsynaptic cell (Kim, 2014).

    Interestingly, the BMP and Activin/TGF-β pathways have also been recently found to control different aspects of the Drosophila innate immune response (Clark, 2011). In this case BMP signaling suppresses the expression of multiple antimicrobial peptide genes following wounding, whereas the Activin/TGF-β pathway limits melanization after bacterial infection in adult flies. Therefore, it appears that the division of labor between these subpathways is not limited to just the nervous system, rather it may be the norm when these related signaling pathways act in concert to regulate a common biological process (Kim, 2014).

    The fact that the pathways actually differ in how they affect a complex biological process is not surprising given that the different R-Smads are likely to have different selectivity in gene activation. Within motor neurons, BMP signaling promotes microtubule formation in axons and directly regulates expression of trio, a Rac GEF, that acts as a major regulator of actin cytoskeleton in many types of cells. Thus, it is likely that BMP signaling modulates synaptic growth, in part, by changing the structure and dynamics of the actin and microtubule cytoskeleton within motor neurons. BMP signaling also regulates the transcription of twit, a gene encoding a L-6 neurotoxin-like molecule that controls the frequency of presynaptic spontaneous vesicle release (Kim, 2012; Kim, 2014 and references therein).

    Targets of Drosophila Activin/TGF-β signaling in any tissue are less well characterized. Within the central brain, glial-derived Myo signals through Smox to control expression of the Ecdysone B1 receptors in remodeling mushroom body neurons. However, it is not clear if EcR-B1 is a direct or indirect target of smox transcriptional regulation. It is also unclear if Ecdysone signaling plays a role in regulating synaptogenesis at the NMJ, although it may play a role during metamorphic remodeling of the NMJ as it does for the mushroom body neurons. The only other known targets of Smox are InR, Pi3K and Akt, all of which are Insulin signaling components and are reduced in the Drosophila prothoracic gland in the absence of Activin/TGF-β signaling. Once again the effect may be indirect, but this finding is interesting since Insulin signaling components have been shown to control synaptic clustering of GluRs (Kim, 2014).

    The clustering of GluRs and Dlg at the NMJ have been shown to be regulated by both transcriptional and post-transcriptional mechanisms. For example, a recent genetic screen identified longitudinals lacking (lola), a BTN-Zn finger transcription factor, as an essential regulator of GluR and dPak expression in muscles. In contrast, the current studies on Activin/TGF-β signaling suggest, at least for GluRIIA, that this pathway functions at the post-transcriptional level since this study found that overexpression of glurIIA-gfp using an exogenous promotor and transcriptional activator does not lead to an enrichment of GluRIIAGFP at synaptic sites of Activin/TGF-β pathway mutants. This phenotype is reminiscent of that found for certain mutants in the NF-κB signaling system. Loss of Dorsal (an NF-κB homolog), Cactus (an IκB related factor), or Pelle (an IRAK kinase) leads to a substantial reduction of GluRIIA and a slight reduction of Dlg postsynaptic localization at the NMJ and a concomitant reduction in mEJP size. In addition, as was found for loss of Activin/TGF-β signaling, exogenously-expressed GluRIIA-myc did not reach the synaptic surface in NF-κB signaling mutants consistent with a possible role of Activin/TGF-β signaling in regulating NF-κB signaling. However, even if future studies show that the relationship is true, the Activin/TGF-β pathway likely regulates additional factors since its loss also affects GluRIIB levels and muscle resting potential, neither of which is altered in NF-κB pathway mutants. Interestingly, the regulation of GluRIIB levels by Activin/TGF-β signaling does appear to be at the level of transcription, indicating that this signaling pathway likely affects GluR clustering at the NMJ via both transcriptional and post-transcriptional mechanisms (Kim, 2014).

    Analysis of Activin/TGF-β signaling at the NMJ, coupled with previous studies on BMP signaling and the novel ligand Maverick, indicates that TGF-β ligands are produced in, and act upon, all three cell types that contribute to NMJ function, specifically the motor neuron, wrapping glia, and muscle (see Model of controlling NMJ development and function by Activin/TGF-β and BMP pathways). This leads to the important issue of how directionality of TGF-β signaling at the NMJ is regulated. One possibility is that ligands are sequestered, either inside the secreting cells or on their surfaces, so that they have limited access to receptors on the opposing pre or postsynaptic membrane. For example, Gbb is produced both in muscle and motor neurons, leading to the issue of how directional signaling from muscle to motor neurons is achieved. On the postsynaptic muscle, Gbb release is potentiated by dRich (Rho GTPase activating protein at 92B), a Cdc42 selective Gap while in the presynaptic neuron Crimpy, a Drosophila homolog of the vertebrate Crim1 gene, has been shown to bind to a precursor form of Gbb. The Gbb/Crimpy complex is thought to either interfere with secretion or activation of motor neuron-derived Gbb thus ensuring that only muscle-derived Gbb activates the retrograde BMP signal at the NMJ. Since there are a large number of characterized TGF-β superfamily binding proteins, Drosophila homologs of some of these factors such as the BMP binding proteins Cv-2, Sog, Tsg and Dally, or the Activin-binding protein Follistatin, may sequester and regulate levels of active ligands within the NMJ. Sequestering mechanisms may also provide direction control by facilitating autocrine as opposed to juxtacrine signaling. If ligand-binding proteins are associated with the membrane surface of the ligand-producing cell, they may facilitate delivery of the ligand to receptors on the producing cell, thus enhancing autocrine signaling. It is interesting in this regard that in the developing Drosophila retina, Actβ appears to signal in an autocrine fashion to control photoreceptor connectivity in the brain (Kim, 2014).

    Activin-type ligands are secreted from glia, motor neuron and muscle. The Activin-type ligands induce Babo-mediated phosphorylation of Smox that facilitates association with Med. In the muscle, the phospho-Smox/Med complexes activate the transcription of glurIIB and an unknown factor controlling post-transcriptional process or stability of glurIIA mRNA. In the motor neuron, the phospo-Smox/Med complex controls spontaneous release of synaptic vesicles via unknown mechanism(s). On the other hand, glia-secreted Mav stimulates Mad phosphorylation in the muscle resulting in increased gbb transcription. Gbb protein is released from the muscle and binds Tkv/Sax and Wit complex on the motor neuron leading to an accumulation of phospho-Mad in the nuclei by an unknown mechanism. The resultant phospho-Mad/Med complex activates the transcription of trio whose product promotes synaptic bouton formation (Kim, 2014).

    Another important mechanism to control signal direction is likely to be tissue-specific receptor expression. For example, Wit is highly enriched in motor neurons compared to muscle, and this may help ensure that Gbb released from the postsynaptic muscle signals to the presynaptic motor neuron. Type I receptor diversity may be even more important in controlling directionality since at least 2 isoforms of Tkv and three isoforms of Babo have been identified. In the case of Babo, Activin-like ligands have a clear preference for signaling through different receptor isoforms, and these isoforms show differential tissue expression (Kim, 2014).

    An additional factor to be considered in understanding TGF-β superfamily signal integration within different NMJ cell types is the possibility of canonical versus non-canonical and/or cross-pathway signaling. For example, in mushroom body neurons Babo can signal in a non-Smad dependent manner through Rho1, Rac and LIM kinase1 (LIMK1) to regulate axon growth and target recognition. Whether this mechanism, or another non-canonical pathway is operative at the NMJ is unclear. Cross-pathway signaling has also recently been identified in Drosophila. In this example, loss of Smox protein in the wing disc has been shown to up-regulate Mad activity in a Babo-dependent manner. Double mutants of babo and smox suppress the cross-pathway signal. As is described in this study, smox protein null mutations lead to significantly more severe GluR and mEJP defects than strong babo mutations alone, and this phenotype is suppressed in double mutants. Thus, as in wing discs, loss of Smox protein likely leads to ectopic Mad activity in muscles that further decrease GluR expression and/or localization at the NMJ. Consistent with this view, this study found that loss of Mad actually increases GluRIIB localization, suggesting that Mad acts negatively to regulate GluRIIB in muscle. One possible model to explain the Smox/Mad data is that normally the Babo/Smox signal inhibits Mad signaling which is itself a repressive signal for GluR accumulation. Thus, in babo mutants, total GluR levels decrease due to the loss of smox and therefore an increase in the repressive Mad signal. In the smox protein null mutant even more repressive Mad signal is generated by Babo further hyperactivating Mad activity leading to even lower levels of GluR accumulation. In medea mutants the activity of both pathways is reduced thereby returning the level of GluR levels close to normal. Additional experiments employing various single and double mutants, together with tissue-specific expression of various ligands, receptor isoforms and ligand-binding proteins will be needed to fully elucidate how vectorial TGF-β signaling is accomplished at the NMJ. Likewise, the identifcation of directly responding target genes and how they are influenced by both Smox and Mad signals is needed to fully appreciate how these two TGF-β signaling branches regulates NMJ functional activity (Kim, 2014).

    Regulation of postsynaptic retrograde signaling by presynaptic exosome release

    Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. This study shows that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling (Korkut, 2013).

    This study shows that Syt4 protein functions in postsynaptic muscles to mediate activity-dependent presynaptic growth and potentiation of quantal release. However, to mediate this function Syt4 needs to be transferred from presynaptic terminals to postsynaptic muscle sites. Evidence is presented that, most likely, the entire pool of postsynaptic Syt4 is derived from presynaptic cells. Like the Wnt binding protein, Evi, Syt4 is packaged in exosomes, which provides a mechanism for the unusual transfer of transmembrane proteins across cells. Taken together, these studies support a novel mechanism for the presynaptic control of a retrograde signal, through the presynaptic release of exosomes containing Syt4 (Korkut, 2013).

    Larval NMJs continuously generate new synaptic boutons and their corresponding postsynaptic specializations, ensuring constant synaptic efficacy despite the continuous growth of muscle cells. This precise matching of pre- and postsynaptic compartments is regulated by electrical activity, which induces a retrograde signal in muscle to stimulate new presynaptic growth. This process is likely to fine-tune the magnitude of the retrograde signal in specific nerve terminal-muscle cell pairs, each with a characteristic size. Given that most larval muscle cells are innervated by multiple motorneurons, this mechanism may also enable spatial coincidence to ensure the synaptic specificity of plasticity, making certain that only those activated synapses within a cell become structurally regulated (Korkut, 2013).

    A distinct perisynaptic glial cell type forms tripartite neuromuscular synapses in the Drosophila adult

    Studies of Drosophila flight muscle neuromuscular synapses have revealed their tripartite architecture and established an attractive experimental model for genetic analysis of glial function in synaptic transmission. This study defined a new Drosophila glial cell type, designated peripheral perisynaptic glia (PPG), which resides in the periphery and interacts specifically with fine motor axon branches forming neuromuscular synapses. Identification and specific labeling of PPG was achieved through cell type-specific RNAi-mediated knockdown (KD) of a glial marker, Glutamine Synthetase 2 (GS2). In addition, comparison among different Drosophila neuromuscular synapse models from adult and larval developmental stages indicated the presence of tripartite synapses on several different muscle types in the adult. In contrast, PPG appear to be absent from larval body wall neuromuscular synapses, which do not exhibit a tripartite architecture but rather are imbedded in the muscle plasma membrane. Evolutionary conservation of tripartite synapse architecture and peripheral perisynaptic glia in vertebrates and Drosophila suggests ancient and conserved roles for glia-synapse interactions in synaptic transmission (Strauss, 2015).

    The extracellular-regulated kinase effector Lk6 is required for Glutamate receptor localization at the Drosophila neuromuscular junction

    The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). This study used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. The data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. It was also found that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. (Hussein, 2016).

    This study is the first to provide information on the properties and regulation of the Drosophila protein kinase LK6. Its catalytic domain is strikingly similar to those of mammalian Mnks; similar to them, in mammalian cells LK6 can bind to ERK, can be activated by ERK signalling and can phosphorylate eIF4E. This occurs at the physiological site, Ser209. The MAPK-binding motif of LK6 is of the type previously shown to bind ERK but not p38 MAPK. Consistent with this, when expressed in mammalian cells, LK6 is not activated by stimuli that turn on p38 MAPK (Hussein, 2016).

    It is more challenging to perform similar experiments in Drosophila cells owing to the difficulty in transfecting, e.g. S2 cells with high efficiency. However, importantly, this study shows that LK6 also interacts with the ERK homologue Rolled, but not with the Drosophila p38 homologue. The results, furthermore, show that LK6 is activated by Phorbol myristate acetate (PMA), but not by arsenite, which activates p38 MAPK. The regulatory properties of LK6 thus appear to be similar in mammalian and Drosophila cells, indicating that the specificity of the MAPK-interaction motifs is probably similar in both mammals and Diptera. Similar to Mnk1 and Mnk2a, LK6 is primarily, if not exclusively, cytoplasmic. It does contain a basic region of the type that, in Mnk1 and Mnk2, can bind to the nuclear shuttling protein importin-α. It therefore seems probable that either (1) it contains an NES, which ensures its efficient re-export from the nucleus, or (2) the basic region is not accessible to importin-α. The lack of effect of LMB on the localization of LK6 rules out the operation of a CRM1-type NES of the kind found in Mnk1, although the very long C-terminal extension of LK6 might contain an LMB-insensitive NES (Hussein, 2016).

    By analogy with the Mnks, it is probable that the N-terminal polybasic region of LK6 mediates its binding to eIF4G and could also interact with importin-α. Given that full-length LK6 shows less efficient binding to eIF4G when compared with Mnk1, it also seems possible that it binds importin-α less efficiently, which may contribute to the finding that LK6 is cytoplasmic. It has been shown previously that even the much shorter C-terminus of Mnk2a impedes access to the N-terminal basic region in that protein, so it is entirely possible that the much larger C-terminal part of LK6 has a similar effect. This could explain why the fragment of LK6 that lacks the C-terminus bound better to eIF4G than did the full-length protein. It may also be that the low degree of binding reflects the fact that the association of LK6 with the heterologous human protein was being studied, rather than with Drosophila eIF4G. Repeated attempts have been made to use the available antisera to examine the association of LK6 with eIF4G in S2 cells, but without success. Comparison of the polybasic region of LK6 with those of Mnk1 and Mnk2a (which do bind eIF4G and importin-α), and recent results for mutants with alterations in these features, do not reveal any difference that might obviously explain the decreased ability of LK6 to bind mammalian eIF4G. As argued above, the C-terminus of LK6 may also impair its activation by ERK, based on the observation that the catalytic domain is more effectively activated than a mutant of the full-length protein that also lacks the ERK-binding motif (Hussein, 2016).

    The results support the idea that LK6 is a Drosophila eIF4E kinase. LK6 can phosphorylate eIF4E in vitro and its overexpression in cells leads to increased phosphorylation of endogenous eIF4E. Furthermore, the activation of LK6 by ERK signalling but not by p38 MAPK signalling correlates well with the observed behaviour of the phosphorylation of eIF4E in PMA- or arsenite-treated Drosophila cells, and the fact that LK6 is activated by stimuli that stimulate ERK but is not activated by stimuli that activate p38 MAPK, in HEK-293 cells. The ability of LK6 to bind eIF4G also supports the contention that it can act as an eIF4E kinase in vivo (Hussein, 2016).

    The observation that phosphorylation of the endogenous eIF4E in S2 cells is increased by PMA but not by arsenite is consistent with the regulatory properties of LK6 and with the notion that LK6 may phosphorylate eIF4E in these cells. The fact that it is the only close homologue of the Mnks in the fruitfly genome is also consistent with this notion. Phosphorylation of eIF4E has previously been shown to play an important role in growth in this organism and in its normal development. The current data show that LK6 can phosphorylate Drosophila eIF4E in vitro, consistent with the idea that LK6 acts as an eIF4E kinase in this organism. The dsRNAi data that was obtained, which show that two different interfering dsRNAs directed against LK6 each markedly decrease eIF4E phosphorylation in S2 cells, offer strong support to the conclusion that LK6 acts as an eIF4E kinase in Drosophila. Unfortunately, the poor quality of the available anti-LK6 antisera prevented assessing whether the incomplete nature of the loss of phosphorylation of eIF4E reflects incomplete elimination of LK6 expression (Hussein, 2016).

    Previous genetic studies have linked LK6 to Ras signalling in Drosophila. This agrees very well with the finding that LK6 is activated by ERK signalling, since ERK lies downstream of Ras. LK6 was first identified as interacting with microtubules and centrosomes. Overexpression of LK6 led to defects in microtubule organization, indicative of their increased stability. The connections between the phosphorylations of eIF4E and microtubules are not immediately obvious. However, it is entirely possible that LK6 has additional substrates that interact with microtubules or are components of centrosomes and their phosphorylation may be important in the regulation of, for example, mitosis. Numerous microtubule-associated proteins are indeed phosphorylated. Microtubules undergo massive reorganization during mitosis and this involves an array of phosphorylation events and protein kinases. It may therefore be relevant that LK6 is activated by mitogenic signalling (i.e. through ERK and thus Ras) (Hussein, 2016).

    Drosophila ortholog of intellectual disability-related ACSL4, inhibits synaptic growth by altered lipids

    Nervous system development and function are tightly regulated by metabolic processes, including the metabolism of lipids such as fatty acids (FAs). Mutations in long-chain acyl-CoA synthetase 4 (ACSL4) are associated with non-syndromic intellectual disabilities. A previous study reported that Acsl, the Drosophila ortholog of mammalian ACSL3 and ACSL4, inhibits neuromuscular synapse growth by suppressing transforming growth factor-beta/bone morphogenetic protein (BMP) signaling. This study reports that Acsl regulates the composition of FAs and membrane lipid, which in turn affect neuromuscular junction (NMJ) synapse development. Acsl mutant brains had decreased abundance of C16:1 fatty acyls; restoration of Acsl expression abrogated NMJ overgrowth and the increase in BMP signaling. A lipidomic analysis revealed that Acsl suppressed the levels of three lipid raft components in the brain, including mannosyl glucosylceramide (MacCer), phosphoethanolamine ceramide, and ergosterol. MacCer level was elevated in Acsl mutant NMJs and along with sterol promoted NMJ overgrowth but was not associated with the increase in BMP signaling in the mutants. These findings suggest that Acsl inhibits NMJ growth by stimulating C16:1 and concomitantly suppressing raft-associated lipid levels (Huang, 2016).

    Lipids are essential membrane components that have crucial roles in neural development and function. Dysregulation of lipid metabolism underlies a wide range of human neurological diseases including neurodegeneration and intellectual disability. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is the first gene in fatty acid metabolism associated with non-syndromic intellectual disability. ACSL4 protein has two variants: a ubiquitously expressed short form, and a brain-specific long form that is highly expressed in the hippocampus, a crucial region for memory. Indeed, ACSL4 has been shown to play an important role in synaptic spine formation. However, it is unclear how mutations in ACSL4 lead to intellectual disability (Huang, 2016).

    There are 26 genes encoding acyl-CoA synthetases (ACSs) in humans. Each of the enzymes has distinct substrate preferences for fatty acid with various lengths of aliphatic carbon chains. In contrast, there are 13 ACS genes in the Drosophila genome. ACSs convert free fatty acids into acyl-CoAs for lipid synthesis, fatty acid degradation or membrane lipid remodeling. For example, ACSL4 converts long-chain fatty acids (LCFAs; aliphatic tails longer than 12 carbons), preferentially arachidonic acid (C20:4), into LCFA-CoAs that are incorporated into glycerol-phospholipids (GPLs) and neutral lipids in non-neuronal cells. The mechanism of how fatty acids and fatty-acid-modifying enzymes affect lipid composition, and thereby modulate development processes, is beginning to be understood in lower model organisms (Huang, 2016 and references therein).

    Synaptic growth is required for normal brain function such as learning and memory. Many neurological disorders including intellectual disability are associated with synaptic defects. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying the mechanisms that regulate synaptic growth. Drosophila Acsl, also known as dAcsl, is the ortholog of mammalian ACSL3 and ACSL4 (Zhang, 2009). It has been reported that Acsl affects axonal transport of synaptic vesicles and inhibits NMJ growth by inhibiting bone morphogenetic protein (BMP) signaling However, how Acsl affects lipid metabolism and the role of Acsl-regulated lipid metabolism in synapse development are largely unknown (Huang, 2016).

    This study demonstrates that Acsl positively regulates the abundance of the LCFA palmitoleic acid (C16:1) in the brain. Reduced levels of C16:1 in Acsl mutants led to NMJ overgrowth and enhanced BMP signaling. A lipidomic analysis revealed that mannosyl glucosylceramide (MacCer), phosphoethanolamine ceramide (CerPE, the Drosophila analog of sphingomyelin) and ergosterol levels were increased in Acsl mutant brains. Genetic and pharmacological analyses further showed that the increased level of MacCer and sterol underlie the NMJ overgrowth in Acsl mutants in a pathway parallel to BMP signaling. These results indicate that Acsl regulates fatty acid and sphingolipid levels to modulate growth signals and NMJ growth, providing insight into the pathogenesis of ACSL4-related intellectual disability (Huang, 2016).

    Like ACSL4, Acsl primarily associates with the ER and facilitates fatty acid incorporation into lipids ; impairment of Acsl activity reduces the abundance of its substrate LCFAs in lipids. Although this study did not directly determine the substrate preference of Acsl, fatty acid analysis suggests that both Acsl and ACSL4 positively regulate C16:1 abundance in Drosophila brain. The two proteins also have conserved functions in other processes, such as lipid storage, axonal transport and synaptic development (Huang, 2016 and references therein).

    The similar NMJ overgrowth in desat1 and Acsl mutants suggests that the normal fatty acid composition is essential for proper development of synapses. The rescue effect of C16:1, together with the genetic interaction between Acsl and desat1, indicates that reduced C16:1 contributes to NMJ overgrowth in Acsl mutants. It has been previously reported that the synaptic overgrowth in Acsl mutants is due in part to an elevation of BMP signaling resulting from defects in endocytic recycling and BMP receptor inactivation. Endosomes are membrane compartments that are regulated by various membrane lipids, particularly the conversion between different PtdIns species, such as PI(3)P, PI(4,5)P2, PI(3,4,5)P3 and so on. The current findings suggest that increased BMP signaling in Acsl mutants is associated with an imbalance in fatty acid composition, specifically, a decrease in C16:1. It is thus possible that proper fatty acid composition is necessary for the normal conversion and localization of endosomal lipids (e.g., PtdIns species), affecting endosomal recycling and BMP receptor inactivation. Future studies will examine the regulation of specific fatty acids such as C16:1 in endosomal recycling and BMP signaling (Huang, 2016).

    Acsl primarily associates with the ER in multiple cell types including motor neurons and participates in lipid synthesis. In Drosophila, most of acyl chains in GPLs are C16 and C18 LCFAs without VLCFAs. In contrast, sphingolipids contain higher levels of VLCFAs than LCFAs as acyl chains. Thus, most LCFA-CoAs are channeled into GPLs whereas VLCFA-CoAs are mainly incorporated into sphingolipids in Drosophila. This study found that Acsl positively regulates the production of C16:1-containing GPLs, as well as the level of PtdEth, the most abundant GPL in the brain. Presumably, fatty acids that are less preferred by Acsl could be channeled into lipids by other ACSs, and might show increased abundance because of a compensatory mechanism in Acsl mutants, which could contribute to the elevation in VLCFA-containing sphingolipids (Huang, 2016).

    In addition to ER localization, ACSL4 and Acsl also localize to peroxisomes in a few non-neuronal cells, suggesting a role for these proteins in the activation of VLCFAs for peroxisomal degradation. Indeed, in animal models and patients with impaired peroxisomal function, accumulation of VLCFAs or increased levels of lipid species with longer fatty acid chains is observed. Thus, a defect in peroxisomal VLCFA degradation might underlie the elevation in sphingolipid species with VLCFA chains in Acsl mutant brains (Huang, 2016).

    Alternatively, Acsl might affect lipid composition through a pathway that is independent of fatty acid incorporation. For example, as degradation of sphingolipids occurs primarily within lysosomes, a defect in lysosomal degradation might lead to an accumulation of sphingolipids and sterols in Acsl mutants. In addition, fatty acids and acyl-CoAs are ligands of many transcription factors, and ACSL3 activates the transcription of lipogenic genes in rat hepatocytes. Thus, Acsl might transcriptionally regulate genes encoding enzymes involved in the metabolism of lipids such as MacCer, CerPE and PtdEth. However, the detailed mechanism of how Acsl affects lipid class composition, especially the downregulation of raft-related MacCer and CerPE by Acsl in the nervous system, remains to be elucidated (Huang, 2016).

    The data showed that elevation of the raft-related lipids MacCer and sterol facilitate NMJ overgrowth in Acsl mutants. Moreover, MacCer promotes bouton formation in a pathway parallel to BMP signaling, at least in part. It is unclear how these raft-related lipids regulate synaptic growth. It is likely that MacCer and sterol might interact with raft-associated growth signaling pathways. Larval NMJ development is mediated by multiple growth factors and downstream signaling cascades. For instance, Wingless (Wg) (Wnt1 in mammals) is a raft-associated protein that activates signaling pathways essential for NMJ growth and synaptic differentiation. It is therefore possible that the level or activity of some raft-associated growth factors is increased in Acsl mutants, thereby promoting NMJ overgrowth. Further investigation is needed to dissect the regulatory mechanisms of raft-related lipids, particularly MacCer, in promoting synaptic growth and bouton formation. It also would be of interest to address how Acsl-regulated lipids regulate neurotransmission in conjunction with synapse development (Huang, 2016).

    Activity-induced synaptic structural modifications by an activator of integrin signaling at the Drosophila neuromuscular junction

    Activity-induced synaptic structural modification is crucial for neural development and synaptic plasticity, but the molecular players involved in this process are not well defined. This study reports that a protein named Shriveled, Shv, regulates synaptic growth and activity-dependent synaptic remodeling at the Drosophila neuromuscular junction. Depletion of Shv causes synaptic overgrowth and an accumulation of immature boutons. Shv physically and genetically interacts with βPS integrin. Furthermore, Shv is secreted during intense, but not mild, neuronal activity to acutely activate integrin signaling, induce synaptic bouton enlargement, and increase postsynaptic glutamate receptor abundance. Consequently, loss of Shv prevents activity-induced synapse maturation and abolishes post-tetanic potentiation, a form of synaptic plasticity. These data identify Shv as a novel trans-synaptic signal secreted upon intense neuronal activity to promote synapse remodeling through integrin receptor signaling (Yeun Lee, 2017)

    Two algorithms for high-throughput and multi-parametric quantification of Drosophila neuromuscular junction morphology

    The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal (Castells-Nobau, 2017).

    Myostatin-like proteins regulate synaptic function and neuronal morphology

    Growth factors of the TGF-beta superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of both Myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. This study shows that Myoglianin (MYO), the Drosophila homolog of Myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both Myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. This study also shows that Myoglianin acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth (Augustin, 2017).

    Growth factors regulate many aspects of tissue development, growth and metabolism. Myostatin and GDF11 are highly homologous members of the TGF-β superfamily of growth factors. While GDF11 plays a role in a variety of systems, the role of Myostatin appears to be confined to skeletal and cardiac muscles (Augustin, 2017).

    Despite the previously described roles of MYO in neural remodelling and synapse refinement (Awasaki, 2011; Yu, 2013) very little is known about the impact of Myoglianin on synaptic physiology. This study first established muscle-derived MYO as a negative regulator of both spontaneous and evoked response at the NMJ, demonstrating its role as a broad regulator of synaptic transmission. The highly coordinated apposition of active zones and glutamate receptors underlies their ability to regulate synaptic strength and plasticity of the larval NMJ. This study has shown that muscle expression of myo inversely affects the NMJ quantity of Brp and GluRIIA, critical pre-and post-synaptic proteins, and determinants of evoked neurotransmitter release and quantal size (i.e., postsynaptic sensitivity to presynaptically released transmitter), respectively. While it is possible that MYO exerts its influence on synaptic strength through other mediators, GluRIIA and Brp are their likely downstream effectors. The electrophysiological results, obtained using the GAL4-UAS system for targeted manipulation of myo, differ from the ones obtained recently using a genetic null myo mutant showing slightly reduced miniature amplitudes (Kim, 2014). The likely explanation is that compensatory effects happen in other tissues in the tissue-specific knockdown animals that cannot occur in genetic nulls, especially for systemic type factors. The other possible explanation is differential cross regulation between different (MYO-like) ligands in genetic null vs tissue knockdown animals. These results thus indicate the relevance of tissue specificity of MYO action, and of myo expression levels, in regulating synaptic function, and emphasize the need for caution when interpreting results from different types of gene manipulations (Augustin, 2017).

    myo expression was detected in the glial cells of the larval neuromuscular junction. While Drosophila NMJ contains at least 2 subtypes of glia, myo expression appears confined to the 'repo-positive' subtype both in the central (Awasaki, 2011) and peripheral nervous system. The dual muscle and glial presence makes MYO ideally positioned for regulating NMJ function. Due to the small size of the compartment, however, glia-derived MYO likely has a modulatory role at the neuromuscular junction (Augustin, 2017).

    This study also found that muscle suppression of Myoglianin, a Drosophila homolog of Myostatin and GDF11, promotes increased larval weight and body-wall muscle size in developing larvae, resembling the effect of Myostatin knockdown in mammals. Interestingly, pan-glial expression of myo negatively affected larval wet weight, but not the size of somatic myofibers, suggesting previously unsuspected systemic roles for glial cells (Augustin, 2017).

    Smad2 is a mediator of MYO action on both evoked response and postsynaptic sensitivity, with MAD having a minor effect on the latter. While MAD primarily functions as a cytoplasmic transducer of BMP signalling, it has been demonstrated that, under certain conditions, MAD can be phosphorylated in response to Activin pathway activation (Peterson, 2012; Augustin, 2017 and references therein).

    This study detected elevated levels of phosphorylated Akt and GSK-3/Shaggy in larval somatic muscles of animals with reduced myo expression in this tissue. In flies and mammals, the Akt- mTOR axis promotes skeletal muscle growth, and phosphorylation-induced inhibition of GSK-3/Shaggy induces hypertrophy in skeletal myotube. The effects of attenuated myo expression on larval tissue size, however, do not appear to be mediated by Smad2 (or MAD) activation as their overexpression does not reverse the weight phenotype in 'low myo' background. Indeed, 'non-Smad' signalling pathways have been demonstrated for various TGF-β ligands in vertebrates and Drosophila. In addition to its role as an inhibitor of the NMJ growth and active zone formation in developing Drosophila larvae, GSK-3β is also a critical promoter of synaptic plasticity, possibly through regulation of glutamate receptor function or trafficking. This work has revealed Shaggy as a mediator of reduced MYO action, and as a negative regulator of synaptic strength at the larval NMJ. While MYO likely affects both sides of the synapse directly, an unlikely but possible scenario is that presynaptic motoneuron responds to a retrograde signal released from muscle/glial cells at the NMJ in response to an induction by MYO. An attractive hypothesis is that MYO negatively regulates presynaptic release directly, in conjunction with muscle-secreted Gbb, a positive regulator of neuromuscular synapse development and growth. The effects of MYO could also be mediated through the transmembrane protein Plum, previously proposed to regulate connectivity at the larval NMJ by sequestrating Myoglianin (Yu, 2013; Augustin, 2017 and references therein).

    Myostatin negatively regulates synaptic function and neuronal morphology This study found that injections of Myostatin into rapidly growing larvae abolish the positive effect of myo down-regulation on NMJ strength and composition, and reverse the elevated muscle p-Akt levels. Furthermore, both Myostatin and GDF11 surpressed the growth of neuronal processes and perturbed the formation of synapses in cultured brain neurons, suggesting a direct action on neurons and regulation of synaptogenesis beyond neuromuscular junctions. Recently, Myostatin transcript and protein were detected in the mouse hippocampus and olfactory system neurons, respectively, and Myostatin type I (Alk4/5) and type II (ActIIB) receptors were found to be expressed in the mammalian nervous system. The current results therefore expand on these findings, suggesting functional relevance for Myostatin in both peripheral and central nervous system, and beyond its action as a canonical regulator of skeletal muscle growth. These novel roles remain to be further explored (Augustin, 2017).

    This study expanded analysis of the functional relevance of MYO in the nervous system by demonstrating its importance in a non-NMJ synapse. Specifically, Myoglianin plays a role in the development of a mixed electro-chemical synapse in the Drosophila escape response pathway, likely by regulating the density of shakB-encoded gap junctions at the GF-TTMn synapse (Blagburn, 1999). These findings implicate MYO as a broad negative regulator of neuronal function across the nervous system and developmental stages (Augustin, 2017).

    This work thus reveals broad and novel roles for anti-myogenic TGF-β superfamily of proteins in the nervous system and suggests new targets for interventions into synaptic function across species (Augustin, 2017).

    Secreted tissue inhibitor of matrix metalloproteinase restricts trans-synaptic signaling to coordinate synaptogenesis

    Synaptogenesis is coordinated by trans-synaptic signals that traverse the specialized synaptomatrix between pre- and postsynaptic cells. Matrix metalloproteinase (Mmp) activity sculpts this environment, balanced by secreted Tissue inhibitors of Mmp (Timp). This study used the reductionist Drosophila matrix metalloproteome to test consequences of eliminating all Timp regulatory control of Mmp activity at the neuromuscular junction (NMJ). Using in situ zymography, Timp was found to limit Mmp activity at the NMJ terminal and shape extracellular proteolytic dynamics surrounding individual synaptic boutons. In newly-generated timp null mutants, NMJs exhibit architectural overelaboration with supernumerary synaptic boutons. With cell-targeted RNAi and rescue studies, postsynaptic Timp was found to limit presynaptic architecture. Functionally, timp nulls exhibit compromised synaptic vesicle cycling, with reduced, lower fidelity activity. NMJ defects manifest in impaired locomotor function. Mechanistically, Timp was found to limit BMP trans-synaptic signaling and the downstream synapse-to-nucleus signal transduction. Pharmacologically restoring Mmp inhibition in timp nulls corrects BMP signaling and synaptic properties. Genetically restoring BMP signaling in timp nulls corrects NMJ structure and motor function. Thus, Timp inhibition of Mmp proteolytic activity restricts BMP trans-synaptic signaling to coordinate synaptogenesis (Shilts, 2017).

    The synaptic cleft is populated with a complex extracellular network of secreted and transmembrane proteins, yet little is known about the extracellular mechanisms that act to shape this critical cellular interface. This synaptomatrix contains integrin, heparan sulfate proteoglycan and cognate receptors for a host of known secreted and transmembrane ligands. Extracellular proteins in the cleft are extensively remodeled in parallel with intercellular changes that accompany synaptic maturation and activity-dependent plasticity. Extracellular matrix metalloproteinase (Mmp) enzymes catalyze synaptic remodeling by proteolytically cleaving the secreted and transmembrane substrates regulating synapse structural integrity, and modulating intercellular signaling between presynaptic and postsynaptic partners. Given the powerful organizing effects of these proteases, their activity must be tightly regulated. One key mechanism is secretion of tissue inhibitors of Mmps (Timps), which restrict Mmp activity to proper spatial and temporal windows. Whenever Mmp regulation is disrupted, developmental abnormalities and disease often result. Improper Mmp expression and activity is implicated in a range of neurological disorders, including schizophrenia, addiction, epilepsy and autism spectrum disorder (ASD). As the most common heritable ASD and intellectual disability, Fragile X syndrome (FXS) underscores the importance of preserving Mmp balance to control proper synaptic structure and function. Importantly, the Mmp inhibitor minocycline alleviates synaptic and behavioral phenotypes in FXS disease models, and has shown promise in clinical trials for human patients, showing that elevated Mmp activity is causally linked to FXS neuropathology (Shilts, 2017).

    Since Mmp dysregulation produces pronounced synaptic defects in disease states, it was hypothesized that loss of the endogenous Mmp control mediated by Timp would disrupt synapse architecture and function. This study took advantage of the simplified Drosophila melanogaster matrix metalloproteome to test consequences of genetically ablating all Timp regulatory control over Mmp activity. In contrast to mammals, which have at least 24 Mmps and four partially redundant Timps, Drosophila has a single secreted Mmp (Mmp1), a single membrane-anchored Mmp (Mmp2) and a single Timp, all of which are highly conserved and can interact with their respective human homologs. In the Drosophila nervous system, Mmps direct both axonal targeting and dendritic remodeling. Recently, Mmp1 and Mmp2 were found to regulate trans-synaptic signaling at the neuromuscular junction (NMJ) to modulate synaptic structure and function. Moreover, trans-synaptic signaling dysregulation has been causally linked to synaptic defects in the Drosophila FXS model. One trans-synaptic pathway important for both synaptic structure and function involves bone morphogenetic protein (BMP) signaling via the Glass Bottom Boat (Gbb) ligand. Gbb secreted from the muscle regulates NMJ structure, whereas Gbb secreted from the motor neuron regulates neurotransmission. Gbb ligand in the extracellular space surrounding synaptic boutons activates downstream phosphorylated Mothers Against Decapentaplegic (pMAD) signal transduction locally at the synapse and distantly within motor neuron nuclei of the central nervous system. Synaptic pMAD is associated with assembly of functional neurotransmission machinery at the NMJ, whereas the accumulation of nuclear pMAD promotes NMJ growth. It is hypothesized that the balance of Mmp proteolytic activity controlled by Timp guides trans-synaptic signaling pathways to modulate both NMJ synaptic structure and function (Shilts, 2017).

    To test this hypothesis, the first ever timp-specific loss-of-function null alleles were generated using CRISPR/Cas9 genome editing. Generation of specific mutations was previously intractable due to a conserved nested relationship that places the timp gene within an intron of the important synaptic synapsin gene. Previously work has shown that Timp localizes in the NMJ perisynaptic space (Dear, 2016), where it shows a co-dependent relationship with both secreted Mmp1 and membrane-anchored Mmp2. The current study employed in situ zymography in living NMJs to show that Timp inhibits synaptic Mmp function and regulates the dynamics of Mmp proteolytic activity in the extracellular space surrounding synaptic boutons. Loss of Timp regulation removes a restraint on synaptic architecture, resulting in the overelaboration of boutons. Using transgenic RNA interference (RNAi) and rescue, Timp secretion from the postsynaptic muscle was shown to be required to regulate the presynaptic motor neuron architecture. In parallel, Timp was also found to control synaptic function. By employing FM1-43 dye imaging, this study found Timp modulates the speed and fidelity of the synaptic vesicle (SV) cycle driving synaptic neurotransmission, and impairs the coordinated muscle peristalsis output of neuromuscular activity. In testing trans-synaptic signaling pathways, Timp function was found to act to restrict BMP signaling, with timp null mutants showing elevated Gbb ligand levels in the extracellular space surrounding synaptic boutons and increased downstream pMAD signal transduction at both the synapse and within motor neuron nuclei. Inhibiting proteolytic activity with minocycline treatment in timp null mutants restores normal BMP signaling and significantly corrects NMJ properties and output motor function. Genetically restoring normal BMP signaling in timp null mutants corrects NMJ architecture and functional motor output, indicating that aberrant trans-synaptic signaling is the causal mechanism. Taken together, these results show that neuromuscular synapses require a responsive balance of Mmp activity controlled by Timp inhibition to restrict the BMP trans-synaptic signaling that modulates NMJ structure and function (Shilts, 2017).

    Remodeling of the synaptic extracellular environment is a highly dynamic process, demanding precise spatiotemporal control in response to specific developmental and activity-dependent signals. Mmp proteolytic activity is an ideal node of regulation for the necessary responsive kinetics and specificity, with Timps controlling the timing, duration and spatial specificity of enzyme function. Taking advantage of the simplified matrix metalloproteome of Drosophila, with only a single functionally conserved Timp, it was possible to eliminate all Timp function with one mutation. Using site-directed CRISPR/Cas9, the first timp null allele was generated, with targeted mutation of the timp gene, without disrupting the synapsin gene in which it is nested. Although nested genetic placement does not imply a functional relationship, the highly conserved nesting of timp in synapsin occurs across vertebrates and invertebrates, which are separated by hundreds of millions of years of evolution. The evolutionary conservation of timp nesting in synapsin is interesting, since synapsin encodes a key synaptic regulator and there is evidence of co-regulation of genes nested with Timps. In addition to timp loss-of-function mutants, no viable Mmp gain-of-function has yet been reported in Drosophila. Thus, the new CRISPR-induced timp null is a tool to characterize total Timp function as well as generally elevated Mmp activity as seen in the nervous system. Currently, there are relatively few reports concerning Timp loss in the nervous system. In mice, TIMP-2 knockout causes motor deficits and expanded NMJ branching, and TIMP-1 overexpression reduces outgrowth in cortical cells, supporting findings of this tudy. In Drosophila, Timp overexpression inhibits NMJ growth, which again complements the current findings (Shilts, 2017).

    This study uncovered key roles for Timp in controlling synaptic Mmp activity, thereby regulating NMJ structure, function and output. Muscle-secreted Timp limits synaptic Mmp proteolytic activity and shapes the distribution of Mmp activity within the synaptomatrix. This local regulation of Mmp functional dynamics has not been reported in neuronal synapses, but is consistent with known roles of Timp in non-neuronal contexts. This study found that postsynaptic Timp limits presynaptic NMJ architecture and bouton formation. This is surprising given that individual Mmp knockdown similarly limits synaptic structure in flies and mice, but may suggest that both loss and gain of Mmp function converge phenotypically or that, collectively, Timp repression of multiple Mmp activities acts as a brake on synaptic growth. Timp also was found to regulate synaptic function, by facilitating SV endocytosis and maintaining SV cycle fidelity. In comparison, mmp mutants elevate transmission strength, also by altered SV cycling dynamics, consistent with Timp repression of Mmp function. Timp enables faster and higher fidelity muscle contraction peristalsis, driving coordinated locomotion. Motor defects have consistently been found across a range of Mmp manipulations, although molecular mechanisms had not been identified. Taken together, these results complete a characterization of the entire Drosophila matrix metalloproteome in controlling neuromuscular synapse structure and function. The timp null synaptic phenotypes prompt a re-assessment: Mmps are not simply negative regulators of synaptic differentiation, but can promote structural development within a context-dependent mechanism. This work shows Timp and Mmps interact to sculpt synapse form and function (Shilts, 2017).

    Timp limits BMP trans-synaptic signals mediating communication between the muscle and motor neuron, with Timp loss elevating Gbb ligand levels. BMP ligands are well known to be sequestered by extracellular molecules, and proteolytic cleavage of these extracellular antagonists controls the distributions of signaling activity in multiple cellular contexts. In Drosophila neurons, Mmp2 regulates motor axon pathfinding and fasciculation via Mmp2-mediated proteolytic cleavage of the ECM Fibrillin/Fibulin-related Faulty Attraction (Frac) protein to enable BMP signaling. Similarly, this study found elevated BMP trans-synaptic signaling in timp mutants with Mmp proteolytic hyperactivity. Gbb secretion from the postsynaptic muscle regulates NMJ architecture, whereas Gbb released from the presynaptic motor neuron regulates neurotransmission function. These roles are consistent with the misregulation of synaptic structure and SV cycle function, respectively, seen in timp mutants with elevated Gbb signaling. The accumulation of Gbb in the perisynaptic synaptomatrix of timp null mutants drives downstream activation of pMAD signal transduction in both motor neuron synaptic terminals and motor neuron nuclei. This is consistent with pMAD activation of transcriptional programs for coordinating synapse structural and functional differentiation. Gbb secreted from the postsynaptic muscle is regulated by Timp that is also secreted from the muscle, which provides control for motor neuron terminals to expand in response to muscle growth and activity-dependent plasticity. In contrast, Mmps come from both presynaptic and postsynaptic cells. Thus, directional Timp control acts as a specific muscle-derived mechanism to regulate Gbb trans-synaptic signaling (Shilts, 2017).

    Elevated BMP Gbb signaling in a Drosophila model of Troyer syndrome, a hereditary spastic paraplegia (HSP) disease, causes strikingly similar NMJ synaptic structural and functional defects to those seen upon loss of Timp. Like the timp null mutants, spartin mutants that are causatively associated with Troyer syndrome exhibit expanded synaptic arbors and decreased FM1-43 dye SV endocytic loading with impaired motor function. Importantly, Fragile X Mental Retardation Protein (FMRP) is a downstream effector of Spartin function, limiting BMP Gbb signaling. Loss of FMRP causes Fragile X syndrome (FXS), and reducing non-canonical BMP signaling alleviates the synaptic defects in Drosophila and mouse FXS disease models. Likewise, targeted mutation of the FXS-related S6 kinase (S6K) similarly results in both expanded synaptic architecture and decreased SV endocytosis at the Drosophila NMJ, once again resembling timp null phenotypes. As in timp mutants, there are also clear precedents for mutations of other key regulatory proteins increasing NMJ functional variability to compromise motor output function. These findings with timp demonstrate the utility of variability as a metric to uncover regulatory nodes that preserve the functional resiliency of the nervous system (Shilts, 2017).

    By pharmacologically correcting timp null phenotypes with the characterized Mmp inhibitor minocycline, this study has shown that mutant defects are causally linked to Mmp hyperactivity. Alleviation of timp null phenotypes is robust, albeit partial, which may reflect experimental limitations of the drug administration, or possibly reveal other Mmp-independent Timp functions. In particular, behavioral assays of motor function show conspicuous, albeit partial, rescue, which may be evidence of an Mmp-independent contribution to motor function or, more likely, that the precise spatiotemporal dynamics of Timp at the NMJ are necessary for proper motor function. In rats, transient proteolytic activity in the synaptomatrix accompanies long-term potentiation and dendrite maturation , which corroborates the current model that Timp dynamically restricts synaptic modulation through localized ECM proteolysis. Crucially, pharmacologically balancing Mmp activity in timp null mutants with minocycline treatment restores BMP trans-synaptic signaling, and genetically correcting BMP signaling prevent synaptic and movement defects. These findings support the model that Mmp activity in the synaptomatrix, under regulation by Timp, limits BMP trans-synaptic signals, thereby controlling NMJ synaptogenesis and functional motor output (Shilts, 2017).

    These studies provide an avenue for possible therapeutic treatments in a range of neurological disease states with elevated Mmp activity. In particular, Mmp hyperactivity has been causally implicated in FXS and related ASD conditions. The synaptic cytoarchitectural phenotypes of timp mutants phenocopy the Drosophila FXS model, and trans-synaptic signaling defects are causative in synaptic structural and functional defects in this disease model, including BMP signaling. By re-creating the elevated Mmp activity characterizing neurological disease conditions such as FXS, the timp genetic tools developed in this study provide insights into fundamental synaptic mechanisms with direct clinical relevance. In future studies, timp manipulations will be combined with established Drosophila disease models in order to more fully dissect contributions of Mmp-dependent trans-synaptic signaling impairments in different neurological disease states (Shilts, 2017).

    Notum coordinates synapse development via extracellular regulation of Wnt Wingless trans-synaptic signaling

    Synaptogenesis requires orchestrated communication between pre- and postsynaptic cells via coordinated trans-synaptic signaling across the extracellular synaptomatrix. The first discovered Wnt signaling ligand Drosophila Wingless (Wg; Wnt-1 in mammals) plays critical roles in synaptic development, regulating synapse architecture as well as functional differentiation. This study investigated synaptogenic functions of the secreted extracellular deacylase Notum, which restricts Wg signaling by cleaving an essential palmitoleate moiety. At the glutamatergic neuromuscular junction (NMJ) synapse, Notum secreted from the postsynaptic muscle was found to act to strongly modulate synapse growth, structural architecture, ultrastructural development and functional differentiation. In notum nulls, upregulated extracellular Wg ligand and nuclear trans-synaptic signal transduction was found, as well as downstream misregulation of both pre- and postsynaptic molecular assembly. Structural, functional and molecular synaptogenic defects are all phenocopied by Wg over-expression, suggesting Notum acts solely through inhibiting Wg trans-synaptic signaling. Moreover, these synaptic development phenotypes are suppressed by genetically correcting Wg levels in notum null mutants, indicating that Notum normally functions to coordinate synaptic structural and functional differentiation via negative regulation of Wg trans-synaptic signaling in the extracellular synaptomatrix (Kopke, 2017).

    In the developing nervous system, Wnt signaling ligands act as potent regulators of multiple stages of neuronal connectivity maturation, stabilization and synaptogenesis, including sculpting structural architecture and determining neurotransmission strength. Drosophila Wingless is secreted from presynaptic neurons and glia at the developing glutamatergic neuromuscular junction (NMJ), to bind Frizzled-2 (Fz2) receptors in both anterograde and autocrine signaling. In the postsynaptic muscle, Wg binding to Fz2 activates the Frizzled Nuclear Import (FNI) signaling pathway, which involves Fz2 endocytosis followed by Fz2 cleavage and Fz2 C-terminus nuclear import (Mathew, 2005). Fz2-C trafficked in nuclear ribonucleoprotein (RNP) granules regulates translation of synaptic mRNAs, thereby driving expression changes that modulate synapse structural and functional differentiation (Speese, 2012). In the presynaptic neuron, Wg binding to Fz2 activates a divergent canonical pathway inhibiting the Glycogen Synthase Kinase 3β (GSK3β) homolog Shaggy (Sgg) to regulate microtubule cytoskeleton dynamics via Microtubule-Associated Protein 1B (MAP1B) homolog Futsch. Futsch binding to microtubules regulates architectural changes in synaptic branching and bouton formation. Such multifaceted Wg functions require tight management throughout synaptic development (Kopke, 2017).

    A highly conserved extracellular Wg regulator is the secreted deacylase Notum. The notum gene was discovered in a Drosophila gain-of-function (GOF) mutant screen targeting wing development. Under scalloped-Gal4 control, notum GOF causes loss of the wing and duplication of the dorsal thorax. In the developing wing disc, Notum acts as a secreted, extracellular feedback inhibitor of Wg signaling. Notum function was recently re-defined as a carboxylesterase that cleaves an essential Wg lipid moiety (palmitoleic acid attached to conserved serine), leaving it unable to bind to Fz2 and activate downstream signaling (Kakugawa, 2015). This Wnt palmitoleate moiety is similarly cleaved by human Notum acting as a highly conserved secreted feedback antagonist in the extracellular space to inactivate Wnt signaling (Langton, 2016; Kakugawa, 2015). At the Drosophila NMJ, extracellular regulation of Wg trans-synaptic signaling has been found to play key roles in synaptogenesis (Dani, 2012b; Parkinson et al., 2013). For example, extracellular matrix metalloproteinase (MMP) enzymes cleave heparan sulfate proteoglycan (HSPG) co-receptors to regulate Wg trans-synaptic signaling that controls structural and functional synaptic development. Impairment of this mechanism is causative for Fragile X syndrome (FXS) synaptogenic defects. Similarly, misregulated extracellular mechanisms impair Wg trans-synaptic signaling in both Congenital Disorder of Glycosylation (CDG) and Galactosemia disease states, causing NMJ synaptogenic defects underlying coordinated movement disorders. Given these insights, this study investigated the putative roles for Notum as a new secreted Wg antagonist regulating synaptogenesis (Kopke, 2017).

    This study utilized the well-characterized Drosophila NMJ glutamate synapse model to study Notum requirements in synaptic development. Notum, secreted from muscle and glia, is resident in the extracellular space surrounding developing synaptic boutons, where it negatively regulates Wg trans-synaptic signaling. In notum mutants, extracellular Wg ligand levels and downstream Wg signaling are elevated. Null mutants display both increased synapse number and strength, altered synaptic vesicle cycling, and synaptic ultrastructural defects including a decrease in SSR/bouton ratio, decreased synaptic vesicle density and an increase in the size of vesicular organelles. Cell-targeted RNAi studies reveal both postsynaptic and perisynaptic requirements, with muscle and glial notum knockdown resulting in overelaborated NMJ architecture, but neuronal-driven notum knockdown causing no detectable effects on synaptogenesis. Null notum defects are all phenocopied by neuronal Wg overexpression, suggesting that synaptogenic phenotypes arise from lack of Wg inhibition. Consistently, genetically correcting Wg levels at the synapse in notum nulls alleviates synaptogenic phenotypes, demonstrating that Notum functions solely as a negative regulator of Wg signaling. Taken together, these results identify Notum as a secreted Wnt inhibitor resident in the extracellular synaptomatrix with critical functions regulating trans-synaptic Wnt signaling to coordinate structural and functional synaptogenesis (Kopke, 2017).

    Tightly coordinated trans-synaptic signals are required for proper development of the pre- and postsynaptic apparatus to ensure efficient communication at the synapse. This signaling is both coordinated and controlled in the extracellular space through the actions of secreted and transmembrane glycans, heparan sulfate proteoglycan (HSPG) co-receptors and secreted enzymes, such as matrix metalloproteinase (Mmp) classes. Wg (Wnt-1) mediates a critical trans-synaptic signaling pathway regulated by these extracellular synaptic mechanisms, with key roles in both structural and functional synaptogenesis. This study proposes that Notum is a novel extracellular regulator limiting Wg trans-synaptic signaling to control NMJ synaptogenesis. Wg is post-translationally modified by addition of palmitoleate on a conserved serine (S239) by membrane-bound O-acyltransferase (MBOAT) Porcupine. This lipidation event is required for Fz2 receptor binding and essential for signaling. At the synaptic interface, the GPI-anchored glypican Dally-like Protein (Dlp) regulates Wg trans-synaptic signaling, and Notum was initially described as cleaving such GPI-anchored glypicans from the cell surface, affecting their ability to interact with the Wg ligand. However, Notum was recently redefined as a secreted carboxylesterase, not a phospholipase (Kakugawa, 2015), with structural studies showing a hydrophobic pocket that binds and then cleaves palmitoleate (Kopke, 2017).

    Notum is consistently reported to act primarily as an extracellular Wg feedback inhibitor. The current studies support this function within the synaptomatrix during synaptogenesis. At the Drosophila NMJ, Wg is secreted from both presynaptic neurons and associated peripheral glia (Kerr, 2014), with the glial function specifically regulating synaptic transmission strength and postsynaptic glutamate receptor clustering. This analyses suggest that Notum is secreted from both postsynaptic muscle and peripheral glia, establishing a dynamic, Wg-like expression pattern surrounding synaptic boutons. In notum null mutants, Wg signaling is increased at the developing NMJ, revealed by both decreased Fz2 receptor in the synaptic membrane (Wg-driven endocytosis) and an increase in nuclear Fz2-C punctae (FNI pathway). These findings are consistent with Notum function limiting Wg signaling, as established in other developmental contexts. Notum appears to provide a fascinating directional regulation of Wg trans- synaptic signaling, affecting the anterograde FNI signaling pathway in muscles, but not the autocrine divergent canonical pathway in neurons. Despite the strong elevation in synaptic Wg ligand levels in notum null mutants, no evidence is seen of altered presynaptic MAP1B homolog Futsch or changes in the microtubule cytoskeleton. However, Notum strongly limits Fz2 C-terminus nuclear import into the postsynaptic nuclei, which is known to drive ribonucleoprotein (RNP) translational regulation of synaptic mRNAs to control synapse structural and functional differentiation (Kopke, 2017).

    Synaptic morphogenesis and architectural development is strongly perturbed in notum null mutants, including increased NMJ area, branching and bouton formation, consistent with Notum function inhibiting Wg trans-synaptic signaling. Elevating presynaptic Wg closely phenocopies notum synaptic defects, including expanded innervation area, more branching and supernumerary synaptic boutons. The results show that Notum secreted from muscle and peripheral glia controls Wg in the extracellular space, with targeted notum RNAi resulting in a similar NMJ expansion to notum nulls, whereas neuronal notum knockdown produces no effects. Interestingly, the glial-targeted RNAi increases boutons with no change in branching, whereas muscle knockdown has a stronger impact also affecting branching. Presynaptic Futsch/Map1B microtubule loops have been proposed to mediate Wg-dependent branching and bouton formation. However, neuronal Wg overexpression has no discernable effect on Futsch-positive microtubule loops. Consistently, Notum LOF also does not impact this pathway, with notum mutants displaying no change in Futsch-labeled looped, bundled, punctate or splayed microtubules. Wg binding to the presynaptic Fz2 receptor may activate another divergent Wnt pathway that does not involve Futsch. Alternatively, Wg signaling via muscle Fz2 may produce a retrograde signal back to the neuron to alter presynaptic development. To test these two possibilities, future studies will employ cell-targeted Fz2 knockdown in notum nulls to assay for suppression of the synaptic overgrowth phenotypes (Kopke, 2017).

    Measures of functional synaptic differentiation reveal elevated neurotransmission and faster motor output function with both notum knockout and Wg over-expression. These results are consistent with Notum function inhibiting Wg trans-synaptic signaling, and consistent with previously characterized roles of Wg in NMJ functional development. Notum LOF increases presynaptic function selectively with an elevated mEJC frequency, greater EJC quantal content and heightened synaptic vesicle release during maintained high- frequency stimulation. Some of these effects may map to the increased synaptic bouton numbers. Both Notum LOF and Wg GOF also cause NMJ boutons to spatially clump together, with ultrastructural studies showing multiple boutons sharing one SSR profile. These are not satellite boutons, but rather aberrantly developing boutons that may result in functional defects. Notum knockdown in glia does not cause detectable mEJC/EJC changes, although Wg from glia regulates NMJ functional properties. Interestingly, loss of Notum appears to improve motor performance, and repo-targeted notum RNAi shows that glial Notum contributes to this function. This is an unusual outcome in a mutant condition, and it is assumed that there must be a counter-balancing cost for increasing neuromuscular function. Live FM dye imaging reveals that notum mutants load less dye into synaptic boutons upon nerve stimulation, indicating a role in synaptic vesicle endocytosis and/or the developmental regulation of synaptic vesicle pool size. These results show Notum function limits Wg trans-synaptic signaling to control presynaptic differentiation critical for synapse function and motor output. As with Wg, the source of Notum (muscle vs. glia) appears to be important for distinct synaptogenic functions. Notum from peripheral glia regulates only bouton formation, whereas Notum from muscle regulates both NMJ growth and function (Kopke, 2017).

    Electron microscopy reveals a very strong decrease in synaptic vesicle density in notum null boutons, providing an explanation for the live FM1-43 dye imaging defects. One of the most striking ultrastructural phenotypes is numerous, enlarged synaptic vesicular bodies. These organelles are highly reminiscent of bulk endosomes, in which a large area of presynaptic membrane is internalized, and will subsequently bud off synaptic vesicles. This pathway is usually driven by intense stimulation during activity-dependent bulk endocytosis (ADBE), as first observed at the frog neuromuscular junction. This pathway is induced by high frequency trains of stimulation, and several proteins have been identified that affect the formation of bulk endosomes, including Syndapin and Rolling Blackout (RBO). At the Drosophila NMJ, conditional rbots mutants block ADBE, reducing the number and size of bulk endosomes (Vijayakrishnan, 2009). It will be interesting to test Wg GOF for enlarged endosomal structures, and study their involvement in Wg-dependent synaptic maturation. On the postsynaptic side, Notum also drives proper differentiation. Notum LOF reduces the postsynaptic DLG scaffold and postsynaptic SSR layering. The reduced SSR area in notum mutants is surprising, given that a reduction in postsynaptic Wg signaling also results in fewer SSR layers. However, SSR architecture has not been studied following Wg over-expression. Postsynaptic SSR formation may be sensitive to bidirectional Wg changes, and may be reduced if Wg is tipped in either direction (Kopke, 2017).

    Mechanistically, Notum controls both pre- and postsynaptic molecular assembly, with LOF defects phenocopied by Wg over-expression. The results are consistent with Notum function inhibiting Wg trans-synaptic signaling, and consistent with previously characterized roles for Wg in synaptic molecular development. This study analyzed both the presynaptic active zone protein Bruchpilot and the two postsynaptic GluR classes. Both presynaptic Brp and postsynaptic GluRs are misregulated in notum nulls, with an increase in synapse number but not density. Importantly, both Notum LOF and Wg GOF elevates synapse number. Consistently, Wnt7a over-expression in mouse cerebellar cells also increases the number of synaptic sites and causes accumulation of presynaptic proteins required for synaptic vesicle function. The increased synapse density per NMJ may compensate for reduced neurotransmission per bouton, leading to a net stronger overall NMJ function. In notum mutants, this could reconcile the elevated synaptic strength measured by electrophysiology compared to compromised single bouton function measured by FM dye imaging and impaired TEM ultrastructure. In any case, synaptic assembly during development is regulated by Notum function limiting Wg trans-synaptic signaling (Kopke, 2017).

    Genetically reducing Wg by combining a heterozygous wg null mutation into the homozygous notum null background reduces extracellular synaptic Wg back to control levels. Wg reduction suppresses synaptogenic defects, restoring increased NMJ area, branching and bouton numbers completely back to normal. Both notumKO and Wg GOF causes hyperactive movement, with roll-over speeds supporting synaptogenic defects of larger, stronger NMJs in both mutant conditions. However, notumKO motor function is only partially restored by correcting Wg levels. One explanation for incomplete rescue is that multiple Wnts may contribute to motor behavior. Serine lipidation is conserved for all Wnts, and at least two other Wnts have been suggested to act at the Drosophila NMJ (Wnt2, Wnt5). Wnts are the only secreted ligands suggested to be O-palmitoleated on a serine to function as Notum substrates (Kopke, 2017).

    Retrograde semaphorin-plexin signalling drives homeostatic synaptic plasticity

    Homeostatic signalling systems ensure stable but flexible neural activity and animal behaviour. Presynaptic homeostatic plasticity is a conserved form of neuronal homeostatic signalling that is observed in organisms ranging from Drosophila to human. Defining the underlying molecular mechanisms of neuronal homeostatic signalling will be essential in order to establish clear connections to the causes and progression of neurological disease. During neural development, semaphorin-plexin signalling instructs axon guidance and neuronal morphogenesis. However, semaphorins and plexins are also expressed in the adult brain. This study shows that semaphorin 2b (Sema2b) is a target-derived signal that acts upon presynaptic plexin B (PlexB) receptors to mediate the retrograde, homeostatic control of presynaptic neurotransmitter release at the neuromuscular junction in Drosophila. Further, Sema2b-PlexB signalling regulates presynaptic homeostatic plasticity through the cytoplasmic protein Mical and the oxoreductase-dependent control of presynaptic actin. It is proposed that semaphorin-plexin signalling is an essential platform for the stabilization of synaptic transmission throughout the developing and mature nervous system. These findings may be relevant to the aetiology and treatment of diverse neurological and psychiatric diseases that are characterized by altered or inappropriate neural function and behaviour (Orr, 2017).

    Semaphorins are a large family of secreted or membrane-associated signalling proteins and plexins serve as signal-transducing semaphorin receptors. Semaphorin-plexin signalling was initially described as mediating growth cone collapse. But, semaphorin-plexin signalling is far more diverse. Notably, semaphorins and plexins continue to be expressed in the mature brain, where their function remains mostly unknown. Semaphorins have been shown to be synaptic signalling proteins, but the activity of semaphorins has been limited to the control of neuroanatomical synapse formation and elimination. This study demonstrates that semaphorin-plexin signalling achieves retrograde, trans-synaptic control of presynaptic neurotransmitter release and homeostatic plasticity (Orr, 2017).

    A well-documented assay was used to induce presynaptic homeostatic plasticity (PHP), applying a sub-blocking concentration of the glutamate-receptor antagonist philanthotoxin-433 (PhTx; 15 μM) to significantly decrease the amplitude of average miniature excitatory postsynaptic potentials (mEPSPs; 0.3 μM [Ca2+]e) or miniature excitatory postsynaptic currents (mEPSCs; 1.5 μM [Ca2+]e). This postsynaptic perturbation induces a significant increase in presynaptic neurotransmitter release (the quantal content) that offsets the postsynaptic perturbation and restores normal muscle excitation. This offsetting increase in presynaptic neurotransmitter release is characteristic of PHP1. When this assay was used in larvae containing a null mutation in either the sema2b gene (sema2bC4) or the PlexB gene (PlexBKG0088), PHP was blocked. Consistent with this being a loss-of-function phenotype, heterozygous mutations (either sema2b/+ or PlexB/+) have normal PHP. Remarkably, a double-heterozygous mutant combination of sema2b/+ and PlexB/+ blocks PHP, consistent with both genes acting in concert to drive the expression of PHP (Orr, 2017).

    The long-term maintenance of PHP was investigated and the involvement of other semaphorin or Plexin gene family members. Deletion of a non-essential glutamate-receptor subunit (GluRIIA) induces a long-lasting form of PHP1. Long-term PHP is blocked in a sema2b;GluRIIA double mutant as well as in GluRIIA larvae expressing transgenic RNA interference (RNAi) to knockdown PlexB selectively in motor neurons. Next, the effect of mutations was separately tested in all of the remaining semaphorin and Plexin genes encoded in the Drosophila genomet. The sema2b and PlexB mutants are the only mutants that show disruption of PHP (Orr, 2017).

    Tissue-specific RNAi and transgenic rescue experiments were performed. Expression of UAS-Sema2b-RNAi in motor neurons (OK371-Gal4) had no effect on PHP, whereas expression in muscle (BG57-Gal4) blocked PHP. In addition, expression of UAS-sema2b in muscle rescues PHP in the sema2b-mutant background. Consistent with these data, sema2b was found to be expressed in muscle and Sema2b protein, expressed under endogenous promoter sequences, concentrates at postsynaptic membranes. Next, it was shown that motor neuron-specific expression of UAS-PlexB-RNAi blocks PHP, whereas muscle-specific expression does not. Motor neuron-specific expression of a previously characterized UAS-PlexBDN dominant-negative transgene, lacking the intracellular signalling domain, blocks PHP. RNA-sequencing analysis of purified motor neurons demonstrates PlexB expression in motor neurons. Finally, motor neuron-specific expression of a PlexB-myc transgene shows that PlexB traffics to the presynaptic nerve terminal. Taken together, these data indicate that Sema2b is a ligand originating in the muscle that acts via presynaptic PlexB to drive expression of PHP (Orr, 2017).

    If Sema2b is a retrograde signal that acts upon the presynaptic PlexB receptor, then it should be possible to reconstitute this retrograde signalling by acute application of Sema2b protein. Purified Sema2b protein was acutely applied to the neuromuscular junction (NMJ) of sema2b mutants following PhTx treatment to induce PHP. Sema2b protein (100 nM) was found to completely restores PHP in the sema2b mutant, but fails to restore PHP in the PlexB mutant. In addition, application of Sema2b protein is sufficient to potentiate baseline release, and this effect is also dependent upon PlexB. Finally, a membrane-tethered UAS-sema2b transgene, expressed in muscle, fails to rescue PHP, even though it is concentrated on the postsynaptic membranes. Together, these results indicate that Sema2b is a secreted, postsynaptic ligand that acts upon presynaptic PlexB to enable the expression of PHP. The possibility is acknowledged that PlexB could require a presynaptic co-receptor of, as yet, unknown identity (Orr, 2017).

    Given that acute application of Sema2b protein rescues PHP in the sema2b mutant, the failure of PHP in sema2b-mutant larvae cannot be a secondary consequence of altered NMJ development. Nonetheless, Sema2b-PlexB signalling is required for normal NMJ growth. Axon-targeting errors are rare at muscles 6/7, analysed at the third instar larval stage. This study demonstrated that the NMJs in sema2b and PlexB mutants are composed of fewer, larger synaptic boutons with no change in total NMJ area. The abundance of the active-zone-associated protein Bruchpilot (Brp) is unaltered in the sema2b mutant and the sema2b/+;;PlexB/+ double-heterozygous larvae, both of which block PHP. There is a significant decrease in total Brp staining in the PlexB mutant, an effect of unknown consequence. Qualitatively, the ring-like organization of Brp staining was similar across all genotypes, indicative of normal active-zone organization. Finally, there is no consistent difference in synapse ultrastructure across genotypes. Therefore, the Sema2b-PlexB-dependent control of bouton size may be a separate function of Sema2b-PlexB signalling, analogous to anatomical regulation by semaphorins in mammalian systems (Orr, 2017).

    PHP occurs through the potentiation of the readily releasable pool (RRP) of synaptic vesicles. Application of PhTx induces a doubling of the apparent RRP in wild-type larvae, an effect that is disrupted in both sema2b and PlexB mutants. Failure to potentiate the RRP is also shown as a failure to maintain the cumulative EPSC amplitude after PhTx application. A strong genetic interaction was subsequently shown with a mutation in the presynaptic scaffolding gene rab3-interacting molecule (rim), a PHP gene. Heterozygous mutations in rim, or in sema2b or PlexB have no effect on PHP. However, double-heterozygous combinations of rim/+ with either sema2b/+ or PlexB/+ strongly impaired the expression of PHP (sema2b/+,rim/+) or abolished PHP (rim/+;;PlexB/+). These data do not, however, reflect direct signalling between PlexB and Rim (Orr, 2017).

    To define how PlexB could modulate the RRP, known downstream signalling elements were tested. Mical is necessary for PHP. In Drosophila a single mical gene encodes a highly conserved multi-domain cytoplasmic protein that mediates actin depolymerization, achieved through redox modification of a specific methionine residue (Met44) in actin. Notably, prior genetic evidence has placed Mical downstream of both PlexA and PlexB signalling during axon guidance (Orr, 2017).

    An analysis of multiple mical mutations in larvae as well as transgenic rescue animals demonstrates that mical is necessary presynaptically for PHP. Mical protein is present presynaptically and presynaptic expression of a Mical-resistant UAS-Actin5C transgene, which interferes with Mical-mediated actin depolymerization, blocks PHP. This transgenic protein also concentrates within presynaptic boutons. Additional experiments reveal that the homeostatic expansion of RRP is blocked in mical mutants and when Mical-resistant UAS-Act5 is expressed presynaptically. Strong genetic interactions were found between mical and both the PlexB and rim mutants. Finally, anatomical experiments demonstrate that active zones are normal in the mical mutant, including in both light and electron microscopy experiments. It is proposed that Mical enables PlexB-mediated control of the RRP through the regulation of presynaptic actin (Orr, 2017).

    For half a century, evidence has underscored the importance of target-derived, retrograde signalling that controls presynaptic neurotransmitter release1. Gene discovery, based on forward genetics, indicates that PHP is controlled by the coordinated action of at least three parallel signalling systems. These data regarding Sema2b, PlexB and Mical can be generalized, then semaphorin-plexin signalling could represent a platform for retrograde, trans-synaptic, homeostatic control of presynaptic release, thereby stabilizing synaptic transmission and information transfer throughout the nervous systems of organisms ranging from Drosophila to humans (Orr, 2017).

    Synapse-specific and compartmentalized expression of presynaptic homeostatic potentiation

    Postsynaptic compartments can be specifically modulated during various forms of synaptic plasticity, but it is unclear whether this precision is shared at presynaptic terminals. Presynaptic Homeostatic Plasticity (PHP) stabilizes neurotransmission at the Drosophila neuromuscular junction, where a retrograde enhancement of presynaptic neurotransmitter release compensates for diminished postsynaptic receptor functionality. To test the specificity of PHP induction and expression, this study has developed a genetic manipulation to reduce postsynaptic receptor expression at one of the two muscles innervated by a single motor neuron. PHP can be induced and expressed at a subset of synapses, over both acute and chronic time scales, without influencing transmission at adjacent release sites. Further, homeostatic modulations to CaMKII, vesicle pools, and functional release sites are compartmentalized and do not spread to neighboring pre- or post-synaptic structures. Thus, both PHP induction and expression mechanisms are locally transmitted and restricted to specific synaptic compartments (Li, 2018a).

    Although the genes and mechanisms that mediate retrograde homeostatic potentiation have been intensively investigated, whether this process can be expressed and restricted to a subset of synapses within a single neuron has not been determined. This study has developed a manipulation that enables the loss of GluRs on only one of the two postsynaptic targets innervated by a Type Ib motor neuron at the Drosophila NMJ. The analysis of synaptic structure and function in this condition has revealed the spectacular degree of compartmentalization in postsynaptic signaling and presynaptic expression that ultimately orchestrate the synapse- specific modulation of presynaptic efficacy (Li, 2018a).

    Compartmentalization of postsynaptic PHP signaling GluRs are dynamically trafficked in postsynaptic compartments where they mediate the synapse-specific expression of Hebbian plasticity such as LTP and homeostatic plasticity, including receptor scaling. In contrast, homeostatic plasticity at the human, mouse, and fly NMJ is expressed through a presynaptic enhancement in neurotransmitter release, but is induced through a diminishment of postsynaptic neurotransmitter receptor functionality. Using biased expression of Gal4 to reduce GluR levels on only one of the two muscle targets innervated by a single motor neuron, this study demonstrates that the inductive signaling underlying PHP is compartmentalized at the postsynaptic density, and does not influence activity at synapses innervating the adjacent muscle (Li, 2018a).

    Postsynaptic changes in CaMKII function and activity have been associated with PHP retrograde signaling. Consistent with this compartmentalized inductive signaling, this study observed pCaMKII levels to be specifically reduced at postsynaptic densities of Ib boutons in which GluR expression is perturbed, while pCaMKII was unchanged at postsynaptic compartments opposite to Is boutons and at NMJs in the adjacent muscle with normal GluR expression. Further, postsynaptic overexpression of the constitutively active CaMKII occludes the expression of PHP. Similar synapse-specific control of postsynaptic CaMKII phosphorylation, modulated by activity, has been previously observed. As noted in other studies, this localized reduction in pCaMKII provides a plausible mechanism for the inductive PHP signaling restricted to and compartmentalized at Ib synapses (Li, 2018a).

    How does a perturbation to GluR function lead to a reduction in CaMKII activity that is restricted to postsynaptic densities opposing Type Ib boutons? Recent evidence suggests that distinct mechanisms regulate pCaMKII levels during retrograde PHP signaling depending on pharmacologic or genetic perturbation to glutamate receptors and the role of protein synthesis. Scaffolds at postsynaptic densities are associated in complexes with GluRs and CaMKII. Intriguingly, the scaffold dCASK is capable of modulating CaMKII activity at specific densities in an activity-dependent fashion. Further, CaMKII activity can regulate plasticity with specificity at subsets of synapses in Drosophila and other systems. Although intra-cellular 'cross talk' between Is and Ib boutons cannot be ruled out, as GluRIIA is reduced at postsynaptic sites of both neuronal subtypes, it is striking that reductions in pCaMKII are restricted to Ib postsynaptic compartments. An attractive model, therefore, is that the postsynaptic density isolates calcium signaling over chronic time scales to compartmentalize PHP induction. The membranous complexity and geometry of the SSR at the Drosophila NMJ may be the key to restricting calcium signaling at these sites, as this structure can have major impacts on ionic signaling during synaptic transmission. These properties, in turn, may lead to local modulation of CaMKII function. Interestingly, Drosophila mutants with defective SSR elaboration and complexity have been associated with defects in PHP expression. In the mammalian central nervous system, it is well established that dendritic spines function as biochemical compartments that isolate calcium signaling while enabling propagation of voltage changes, and it is tempting to speculate that the SSR may subserve similar functions at the Drosophila NMJ to enable synapse-specific retrograde signaling (Li, 2018a).

    The homeostatic modulation of presynaptic neurotransmitter release is compartmentalized at the terminals of Type Ib motor neurons. It was previously known that PHP can be acutely induced and expressed without any information from the cell body of motor neurons. The current data suggests that the signaling necessary for PHP expression is even further restricted to specific postsynaptic densities and presynaptic boutons, demonstrated through several lines of evidence. First, quantal content is specifically enhanced at boutons innervating muscle 6 in M6>GluRIIARNAi without measurably impacting transmission on the neighboring boutons innervating muscle 7. In addition, PHP can be acutely induced at synapses innervating muscle 7 despite PHP having been chronically expressed at muscle 6. Finally, the homeostatic modulation of the RRP and enhancement of the functional number of release sites is fully expressed regardless of whether PHP is induced at all Type Ib boutons or only a subset. Thus, PHP signaling is orchestrated at specific boutons according to the state of GluR functionality of their synaptic partners and does not influence neighboring boutons within the same motor neuron. Although the compartmentalized expression of PHP was not unexpected, there was precedent to suspect inter-bouton cros