pgrp-sa.htm" Neuromuscular Junction

The Interactive Fly

Zygotically transcribed genes

Genes coding for Neuromuscular Junctional proteins

Presynaptic Active Zone

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

Postsynapse of the larval neuromuscular junction

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

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

Biology of the Presynapse
  • Transmission, Development, and Plasticity of Synapses - a review by Kathryn Harris & Troy Littleton
  • Genetic analysis in Drosophila reveals a role for the mitochondrial protein p32 in synaptic transmission
  • Pathogenic Huntington alters BMP signaling and synaptic growth through local disruptions of endosomal compartments
  • Myosin VI contributes to synaptic transmission and development at the Drosophila neuromuscular junction
  • Structural and molecular properties of insect type II motor axon terminals
  • A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity
  • Live observation of two parallel membrane degradation pathways at axon terminals
  • Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton
  • Acetylated alpha-tubulin K394 regulates microtubule stability to shape the growth of axon terminals
  • Drosophila neuronal injury follows a temporal sequence of cellular events leading to degeneration at the neuromuscular junction
  • Protein phosphatase 2A restrains DLK signaling to promote proper Drosophila synaptic development and mammalian cortical neuron survival
  • Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction
  • A pre-synaptic regulatory system acts trans-synaptically via Mon1 to regulate Glutamate receptor levels in Drosophila
  • Drosophila homolog of human KIF22 at the autism-linked 16p11.2 loci influences synaptic connectivity at larval neuromuscular junctions
  • Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction
  • Tao negatively regulates BMP signaling during neuromuscular junction development in Drosophila
  • AP2 Regulates Thickveins Trafficking to Attenuate NMJ Growth Signaling in Drosophila
  • The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila
  • Human APP gene expression alters active zone distribution and spontaneous neurotransmitter release at the Drosophila larval neuromuscular junction
  • In vivo single-molecule tracking at the Drosophila presynaptic motor nerve terminal
  • Characterization of developmental and molecular factors underlying release heterogeneity at Drosophila synapses
  • A neuropeptide signaling pathway regulates synaptic growth in Drosophila
  • Sphingolipids regulate neuromuscular synapse structure and function in Drosophila
  • Parvalbumin expression affects synaptic development and physiology at the Drosophila larval NMJ
  • GAL4 drivers specific for type Ib and type Is motor neurons in Drosophila
  • Oxidative stress induces overgrowth of the Drosophila neuromuscular junction
  • A circuit-dependent ROS feedback loop mediates glutamate excitotoxicity to sculpt the Drosophila motor system
  • Neuron-specific knockdown of Drosophila HADHB induces a shortened lifespan, deficient locomotive ability, abnormal motor neuron terminal morphology and learning disability
  • Non-enzymatic activity of the alpha-Tubulin acetyltransferase alphaTAT limits synaptic bouton growth in neurons
  • Drosophila motor neuron boutons remodel through membrane blebbing coupled with muscle contraction
  • Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila]
  • POU domain motif3 (Pdm3) induces wingless (wg) transcription and is essential for development of larval neuromuscular junctions in Drosophila
  • O-GlcNAcase contributes to cognitive function in Drosophila
  • Developmental arrest of Drosophila larvae elicits presynaptic depression and enables prolonged studies of neurodegeneration
  • The conserved alternative splicing factor Caper regulates neuromuscular phenotypes during development and aging
  • Fragile X Premutation rCGG Repeats Impair Synaptic Growth and Synaptic Transmission at Drosophila larval Neuromuscular Junction
  • Characterization of a novel stimulus-induced glial calcium wave in Drosophila larval peripheral segmental nerves and its role in PKG-modulated thermoprotection
  • cAMP signals in Drosophila motor neurons are confined to single synaptic boutons
  • Synaptic counts approximate synaptic contact area in Drosophila
  • Distinct molecular pathways govern presynaptic homeostatic plasticity
  • Rapid homeostatic modulation of transsynaptic nanocolumn rings
  • Vav independently regulates synaptic growth and plasticity through distinct actin-based processes
  • Loss of Activity-Induced Mitochondrial ATP Production Underlies the Synaptic Defects in a Drosophila Model of ALS
  • Influence of T-Bar on Calcium Concentration Impacting Release Probability
  • Determinants of synapse diversity revealed by super-resolution quantal transmission and active zone imaging

    Synaptic Proteins
  • The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization
  • Extended synaptotagmin localizes to presynaptic ER and promotes neurotransmission and synaptic growth in Drosophila
  • Presynaptic spinophilin tunes Neurexin signalling to control active zone architecture and function
  • Neurexin, neuroligin and wishful thinking coordinate synaptic cytoarchitecture and growth at neuromuscular junctions
  • Drosophila Cbp53E regulates axon growth at the meuromuscular junction
  • Misregulation of Drosophila Sidestep leads to uncontrolled wiring of the adult neuromuscular system and severe locomotion defects
  • Distinct regulation of transmitter release at the Drosophila NMJ by different isoforms of nemy
  • The guanine exchange factor Gartenzwerg and the small GTPase Arl1 function in the same pathway with Arfaptin during synapse growth
  • σ2-adaptin facilitates basal synaptic transmission and is required for regenerating endo-exo cycling pool under high frequency nerve stimulation in Drosophila
  • Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory
  • Hsc70-4 deforms membranes to promote synaptic protein turnover by endosomal microautophagy
  • Rab3-GEF controls active zone development at the Drosophila neuromuscular junction
  • The Strip-Hippo pathway regulates synaptic terminal formation by modulating actin organization at the Drosophila neuromuscular synapses
  • Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins
  • Presynaptic CamKII regulates activity-dependent axon terminal growth
  • Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function
  • Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability
  • Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca(2+) channels
  • The Ih channel gene promotes synaptic transmission and coordinated movement in Drosophila melanogaster
  • A Ca2+ channel differentially regulates Clathrin-mediated and activity-dependent bulk endocytosis
  • Neuroligin 4 regulates synaptic growth via the Bone morphogenetic protein (BMP) signaling pathway at the Drosophila neuromuscular junction
  • Inwardly rectifying potassium (Kir) channels represent a critical ion conductance pathway in the nervous systems of insects
  • The Neurexin-NSF interaction regulates short-term synaptic depression
  • Kinetochore proteins have a post-mitotic function in neurodevelopment
  • Secreted C-type lectin regulation of neuromuscular junction synaptic vesicle dynamics modulates coordinated movement
  • The ankyrin repeat domain controls presynaptic localization of Drosophila Ankyrin2 and is essential for synaptic stability
  • The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF
  • Regulation of excitation-contraction coupling at the Drosophila neuromuscular junction
  • The Putative Drosophila TMEM184B Ortholog Tmep Ensures Proper Locomotion by Restraining Ectopic Firing at the Neuromuscular Junction
  • Activity-induced synaptic structural modifications by Akt
  • Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila
  • Crucial Roles of Ubiquitin Carboxy-Terminal Hydrolase L1 in Motor Neuronal Health by Drosophila Model
  • Stochastic Properties of Spontaneous Synaptic Transmission at Individual Active Zones
  • Abnormal larval neuromuscular junction morphology and physiology in Drosophila prickle isoform mutants with known axonal transport defects and adult seizure behavior. J Neurogenet: 1-9
  • Loss of NF1 in Drosophila larvae causes tactile hypersensitivity and impaired synaptic transmission at the neuromuscular junction

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

    Homeostasis and plasticity at the presynapse
  • Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling
  • Phosphorylation of Complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity
  • The long 3'UTR mRNA of CaMKII is essential for translation-dependent plasticity of spontaneous release in Drosophila melanogaster
  • Reactive oxygen species regulate activity-dependent neuronal plasticity in Drosophila
  • The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity
  • The schizophrenia susceptibility gene dysbindin controls synaptic homeostasis
  • Structural and functional synaptic plasticity induced by convergent synapse loss in the Drosophila neuromuscular circuit
  • Molecular logic of synaptic diversity between Drosophila tonic and phasic motoneurons
  • 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
  • Separation of presynaptic CaV2 and CaV1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca2+ pump PMCA
  • Dysbindin links presynaptic proteasome function to homeostatic recruitment of low release probability vesicles
  • A Presynaptic ENaC Channel Drives Homeostatic Plasticity
  • Composition and control of a Deg/ENaC channel during presynaptic homeostatic plasticity
  • RIM-binding protein links synaptic homeostasis to the stabilization and replenishment of high release probability vesicles
  • RIM-binding protein couples synaptic vesicle recruitment to release site
  • Transsynaptic control of presynaptic Ca(2)(+) influx achieves homeostatic potentiation of neurotransmitter release
  • A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation
  • The auxiliary glutamate receptor subunit dSol-1 promotes presynaptic neurotransmitter release and homeostatic potentiation
  • Neto-alpha controls synapse organization and homeostasis at the Drosophila neuromuscular junction
  • The innate immune receptor PGRP-LC controls presynaptic homeostatic plasticity
  • Molecular interface of neuronal innate immunity, synaptic vesicle stabilization, and presynaptic homeostatic plasticity
  • An auxiliary subunit of the presynaptic calcium channel, α2δ-3, is required for rapid transsynaptic homeostatic signaling
  • Input-specific plasticity and homeostasis at the Drosophila larval neuromuscular junction
  • Endogenous tagging reveals differential regulation of Ca(2+) channels at single AZs during presynaptic homeostatic potentiation and depression
  • Target-wide induction and synapse type-specific robustness of presynaptic homeostasis
  • Activity-dependent global downscaling of evoked neurotransmitter release across glutamatergic inputs in Drosophila
  • FOXO Regulates Neuromuscular Junction Homeostasis During Drosophila Aging
  • Synaptotagmin 7 switches short-term synaptic plasticity from depression to facilitation by suppressing synaptic transmission
  • Active zone compaction correlates with presynaptic homeostatic potentiation
  • A comparison of three different methods of eliciting rapid activity-dependent synaptic plasticity at the Drosophila NMJ
  • Imidacloprid Impairs Glutamatergic Synaptic Plasticity and Desensitizes Mechanosensitive, Nociceptive, and Photogenic Response of Drosophila melanogaster by Mediating Oxidative Stress, Which Could Be Rescued by Osthole
  • Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation

    Distinguishing tonic and phasic synaptic function
  • Synaptic plasticity induced by differential manipulation of tonic and phasic motoneurons in Drosophila
  • The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity
  • 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 \

    Biology of the Periactive Zone
  • Synaptic development is controlled in the periactive zones of Drosophila synapses

    Biology of the Postsynapse
  • Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction
  • The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function
  • Mucin-type core 1 glycans regulate the localization of neuromuscular junctions and establishment of muscle cell architecture in Drosophila
  • Shank modulates postsynaptic wnt signaling to regulate synaptic development
  • The influence of postsynaptic structure on missing quanta at the Drosophila neuromuscular junction
  • Hebbian plasticity guides maturation of glutamate receptor fields in vivo
  • Dbo/Henji modulates synaptic dPAK to gate glutamate receptor abundance and postsynaptic response
  • Filamin, a synaptic organizer in Drosophila, determines glutamate receptor composition and membrane growth
  • TOR is required for the retrograde regulation of synaptic homeostasis at the Drosophila neuromuscular junction
  • The maintenance of synaptic homeostasis at the Drosophila neuromuscular junction is reversible and sensitive to high temperature
  • The Drosophila postsynaptic DEG/ENaC channel ppk29 contributes to excitatory neurotransmission
  • Synaptic excitation is regulated by the postsynaptic dSK channel at the Drosophila larval NMJ
  • Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila
  • Development of a tissue-specific ribosome profiling approach in Drosophila enables genome-wide evaluation of translational adaptations
  • Acute fasting regulates retrograde synaptic enhancement through a 4E-BP-dependent mechanism
  • Disparate postsynaptic induction mechanisms ultimately converge to drive the retrograde enhancement of presynaptic efficacy
  • A postsynaptic PI3K-cII dependent signaling controller for presynaptic homeostatic plasticity
  • Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction
  • Regulation of SH3PX1 by dNedd4-long at the Drosophila Neuromuscular Junction
  • Calcium-activated Calpain specifically cleaves Glutamate Receptor IIA but not IIB at the Drosophila neuromuscular junction
  • Ttm50 facilitates calpain activation by anchoring it to calcium stores and increasing its sensitivity to calcium
  • Cul3 and insomniac are required for rapid ubiquitination of postsynaptic targets and retrograde homeostatic signaling
  • Vps54 regulates Drosophila neuromuscular junction development and interacts genetically with Rab7 to control composition of the postsynaptic density
  • GTPase-activating protein TBC1D5 coordinates with retromer to constrain synaptic growth by inhibiting Bone Morphogenetic Protein signaling
  • Vesicle clustering in a living synapse depends on a synapsin region that mediates phase separation
  • Postsynaptic cAMP signalling regulates the antagonistic balance of Drosophila glutamate receptor subtypes
  • A Novel Neuron-Specific Regulator of the V-ATPase in Drosophila
  • AP2 Regulates Thickveins Trafficking to Attenuate NMJ Growth Signaling in Drosophila
  • The effects of doxapram (blocker of K2p channels) on resting membrane potential and synaptic transmission at the Drosophila neuromuscular junction
  • RNA-binding FMRP and Staufen sequentially regulate the coracle scaffold to control synaptic glutamate receptor and bouton development
  • Gamma-secretase promotes Drosophila postsynaptic development through the cleavage of a Wnt receptor

    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
  • The Drosophila beta-amyloid precursor protein homolog promotes synapse differentiation at the neuromuscular junction
  • 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
  • Excess glutamate release triggers subunit-specific homeostatic receptor scaling
  • Regulation of postsynaptic retrograde signaling by presynaptic exosome release
  • Macros to Quantify Exosome Release and Autophagy at the Neuromuscular Junction of Drosophila Melanogaster
  • A distinct perisynaptic glial cell type forms tripartite neuromuscular synapses in the Drosophila adult
  • The extracellular-regulated kinase effector Lk6 is required for Glutamate receptor localization at the Drosophila neuromuscular junction
  • Drosophila ortholog of intellectual disability-related ACSL4, inhibits synaptic growth by altered lipids
  • Activity-induced synaptic structural modifications by an activator of integrin signaling at the Drosophila neuromuscular junction
  • Two algorithms for high-throughput and multi-parametric quantification of Drosophila neuromuscular junction morphology
  • Myostatin-like proteins regulate synaptic function and neuronal morphology
  • Secreted tissue inhibitor of matrix metalloproteinase restricts trans-synaptic signaling to coordinate synaptogenesis
  • Notum coordinates synapse development via extracellular regulation of Wnt Wingless trans-synaptic signaling
  • Carrier of Wingless (Cow) regulation of Drosophila neuromuscular junction development
  • Retrograde semaphorin-plexin signalling drives homeostatic synaptic plasticity
  • Synapse-specific and compartmentalized expression of presynaptic homeostatic potentiation
  • Distinct homeostatic modulations stabilize reduced postsynaptic receptivity in response to presynaptic DLK signaling
  • A glutamate homeostat controls the presynaptic inhibition of neurotransmitter release
  • Neuronal glutamatergic synaptic clefts alkalinize rather than acidify during neurotransmission
  • Distinct Target-Specific Mechanisms Homeostatically Stabilize Transmission at Pre- and Post-synaptic Compartments
  • Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly
  • Dichotomous cis-regulatory motifs mediate the maturation of the neuromuscular junction by retrograde BMP signaling
  • Mayday sustains trans-synaptic BMP signaling required for synaptic maintenance with age>
  • The calcineurin regulator Sarah enables distinct forms of homeostatic plasticity at the Drosophila neuromuscular junction

    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
  • Inter-relationships among physical dimensions, distal-proximal rank orders, and basal GCaMP fluorescence levels in Ca(2+) imaging of functionally distinct synaptic boutons at Drosophila neuromuscular junctions
  • Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles
  • Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction
  • The HSPG glypican regulates experience-dependent synaptic and behavioral plasticity by modulating the non-canonical BMP pathway
  • The matricellular protein Drosophila CCN is required for synaptic transmission and female fertility

  • 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
  • Miniature neurotransmission is required to maintain Drosophila synaptic structures during ageing
  • Computational modeling predicts ephemeral acidic microdomains followed by prolonged alkalinization in the glutamatergic synaptic cleft
  • Bisphenol A affects neurodevelopmental gene expression, cognitive function, and neuromuscular synaptic morphology in Drosophila melanogaster
  • 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. This study demonstrated 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).

    Synapse formation is tightly associated with neuronal excitability. This study found striking synaptic overgrowth caused by Drosophila K(+)-channel mutations of the seizure and slowpoke genes, encoding Erg and Ca(2+)-activated large-conductance (BK) channels, respectively. These mutants display two distinct patterns of "satellite" budding from larval motor terminus synaptic boutons. Double-mutant analysis indicates that BK and Erg K(+) channels interact with separate sets of synaptic proteins to affect distinct growth steps. Post-synaptic L-type Ca(2+) channels, Dmca1D, and PSD-95-like scaffold protein, Discs large, are required for satellite budding induced by slowpoke and seizure mutations. Pre-synaptic cacophony Ca(2+) channels and the NCAM-like adhesion molecule, Fasciclin II, take part in a maturation step that is partially arrested by seizure mutations. Importantly, slowpoke and seizure satellites were both suppressed by rutabaga mutations that disrupt Ca(2+)/CaM-dependent adenylyl cyclase, demonstrating a convergence of K(+) channels of different functional categories in regulation of excitability-dependent Ca(2+) influx for triggering cAMP-mediated growth plasticity (Lee, 2014).

    Distinct satellite patterns induced by sloand sei mutations support the notion that the two K+ channels act on separate growth steps in concert with localized molecular partners. Double-mutant analysis leads to a minimal model involving functional interactions of Slo and Sei K+ channels with distinct assemblies of pre- and post-synaptic regulators in the sequential steps of synaptic growth and differentiation. Expression of slomutant phenotypes depends on scaffold protein, Dlg, and post-synaptic Dmca1D Ca2+ channels, both of which appear to be important for initial budding of satellites. Double-mutant analysis reveals a tight association between Sei, but not Slo, K+ channels and adhesion molecule, FasII, and pre-synaptic Cac Ca2+ and Para Na+ channels in initial satellite formation as well as the ensuing process. In the same vein, manipulations of pre-synaptic cAMP affect only sei-induced satellite formation, whereas slo satellites are more susceptible to modulations in post-synaptic cAMP signaling (Lee, 2014).

    Whereas these pre- and post-synaptic molecules can contribute to the initial growth of satellites in sloand sei mutants, they may also be important for further differentiation and stabilization of such intermediate structures. The stabilized satellites could accumulate over time and would facilitate their capture in fixed preparations. Immunohistochemical and electron-microscopic analyses has indicated that the majority of sloand sei satellites are well differentiated in molecular composition and ultrastructure. As live imaging studies have demonstrated, differentiation of early 'ghost boutons' occurs at a slow rate, taking hours to days. Consistently, preliminary live imaging indicates type B and M satellites abundant in mutants as stable structures with no active morphological changes over the observation period up to 1 h, during which new satellites were sighted budding from primary boutons after high K+ stimulation. Thus, the synaptic differentiation process involving Slo or Sei K+ channels and their interacting partners may occur at a slower time scale (Lee, 2014).

    The results demonstrate a more profound influence of post-synaptic molecules on initial induction of satellite formation and major pre-synaptic contribution in subsequent steps. This picture is in line with potential retrograde signaling during the sequential growth process. Recent studies at Drosophila larval NMJs have revealed significant contributions of retrograde factors, such as bone morphogenetic protein, to synaptic development and function. It will be important to examine whether and how these factors take part in particular steps of the proposed sequential growth process (Lee, 2014).

    There has been emerging evidence for colocalization of post-synaptic BK channels with L-type Ca2+ channels and with PSD-95 scaffold protein at vertebrate synapses. This genetic analysis thus demonstrates the functional significance of the homologous post-synaptic macromolecular association (Slo BK, Dmca1D/L-type Ca2+ channels, and Dlg/PSD-95) in synaptic growth at the Drosophila NMJ. Whether interactions among these players are also important for regulation of synaptic transmission awaits further investigations (Lee, 2014).

    It has been shown that seits1 mutants display increased spontaneous activities in the giant-fiber neuron and enhanced synaptic growth at larval NMJs when exposed to high temperature. However, this study observed synaptic overgrowth even at room temperature in seits2 and, to a lesser extent, seits2/seits1. DNA sequencing predicts truncated versus full-length polypeptides in the seits1 and seits2 alleles, respectively, which could explain the observed allele-dependent differences. Notably, altered pre-synaptic Ca2+ and cAMP regulation drastically suppressed sei phenotypes, but was ineffective on slo-induced overgrowth, suggesting significant interactions between Erg K+ channels and these pre-synaptic components, although pre-synaptic interaction of the Sei K+ channel with the Ca2+/cAMP pathway has not been well established in Drosophila. DNA sequence analysis suggests a putative cyclic nucleotide-binding domain in Sei K+ channels, similar to Eag that belongs to the same K+-channel family. Whether cAMP-dependent modification of sei phenotypes is related to the action of this putative domain should be further investigated in future studies (Lee, 2014).

    Multimeric assembly of K+ channels, including Sei Erg and Slo BK, has been implicated in regulating the channel properties. Indeed, seits2/+ and slo/+ larvae, presumably containing a mixture of mutated and WT subunits in their Erg and BK channels, display dominant mutational effects on satellite formation and associated synaptic growth. Importantly, pre-synaptic expression of a mutated sei transgene (UAS-seits2) in WT led to a similar, but less extreme, phenotype, confirming the pre-synaptic action of seits2 and its dominant effects in multimeric Sei channels (Lee, 2014).

    It is interesting to ask whether simply reducing the amount of Sei and Slo channel proteins may produce phenotypes similar to heterozygous seits2/+ and slo/+ animals. The RNA interference (RNAi) technique was used to test this possibility, using multiple combinations of GAL4 drivers and UAS-slo/sei-RNAi constructs, with Dicer-2 to facilitate RNA interference in some combinations. However, none of these combinations caused characteristic behavioral and physiological abnormalities of sei and slo. Only marginal and inconsistent synaptic growth phenotypes was observed among these combinations. For instance, the expression of sloand sei RNAi in motoneurons with the driver C164-GAL4 led to a slightly elevated satellite frequency, but the pan-neuronal driver C155-GAL4 produced even less overgrowth. Bouton formation was enhanced in these GAL4-UAS-RNAi combinations but not significantly above the elevated levels intrinsic to individual GAL4 and RNAi lines (Lee, 2014).

    The results suggest that dysfunctions induced by RNAi knockdown may not reproduce all aspects of mutant phenotypes. A match in protein levels or altered protein properties may be required to produce the phenotype of interest. At this time, the efficiency of these RNAi lines has not been documented. Since it was not possible to measure the levels of Slo and Sei proteins because of a lack of appropriate antibodies, it is not possible to determine the levels of each RNAi knockdown. The sloand sei mutations induced by a chemical mutagen, ethyl methanesulfonate, may affect the properties and/or the amount of the gene product. For example, seits2 mutants carry a point mutation near the pore domain of the channels, and thus may act as neomorphs that confer dominant effects in the heterozygote, a property difficult to be mimicked by RNAi knockdown (Lee, 2014).

    These results point out the critical role of cAMP signaling in the expression of both sloand sei mutant phenotypes and further highlight the profound functional consequences of altered excitability in neuronal plasticity. Activation of rut AC by activity-dependent accumulation of intracellular Ca2+ is pivotal in several forms of synaptic plasticity. For instance, in the Aplysia siphon-gill withdrawal reflex model, sensitizing stimuli increase cAMP levels and subsequently enhance transmission efficacy at sensorimotor synapses, and repeated conditioning induces sensory varicosity growth. Similarly, cAMP-dependent activation of protein kinase A in hippocampal slices is required for late-phase LTP that involves formation of new dendritic spines (Lee, 2014).

    At Drosophila larval NMJs, altered cAMP metabolism in rut and dnc mutants impairs synaptic transmission stability and post-tetanic potentiation. In addition, fewer docked vesicles and retarded reserve pool mobilization have been documented in these mutants, indicating vesicle targeting and cycling defects. Thus, it will be interesting to examine the possibility that suppression of sloand sei satellites by rut is associated with alterations in membrane recycling. Such studies can be facilitated by relevant mutations, such as shibire defective in Dynamin, which is responsible for vesicle pinch-off, or drp1 (Dynamin-related protein 1) defective in reserve pool mobilization (Lee, 2014).

    In summary, these observations reveal distinct patterns of satellite formation induced by sei and slomutations affecting two separate categories of K+ channels, which are apparently regulated by pre- and post-synaptic Ca2+/cAMP signaling, respectively. Together with previous studies, convergence on the Ca2+/CaM-activated cAMP synthesis by rut AC in the regulation of synaptic growth induced by a variety of K+ channel mutations further establishes a central role of rut AC in activity-dependent plasticity of synaptic function and growth (Lee, 2014).

    Separation of presynaptic CaV2 and CaV1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca2+ pump PMCA

    Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca2+ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)-triggered presynaptic Ca2+ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Cav2 homolog, Dmca1A or cacophony, and the Cav1 homolog, Dmca1D) but with spatial and functional separation. Cav2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Cav1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca2+ influx but has a small impact on release. Instead, L-type calcium currents through Cav1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca2+ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Cav1-mediated Ca2+ influx. The plasma membrane Ca2+ ATPase (PMCA) resides in between active zones and isolates Cav2-triggered release from Cav1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Cav2 may also contribute to. As L-type Cav1 channels also localize next to PQ-type Cav2 channels within axon terminals of some central mammalian synapses, it is proposed that Cav2, Cav1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals (Krick, 2021).

    Neuronal network function critically depends on the tight control of synaptic vesicle (SV) release probability at chemical synapses over wide ranges of activity regimes. At the same time, synaptic gain remains adjustable to render network function flexible. To maintain synapse function over time, SV recycling rates must be matched to vastly different activity patterns and synaptic gains. While SV release and recycling as well as their plasticity-related adjustments all include Ca2+-dependent steps, they operate in parallel but on different time scales. A tight spatial and temporal coordination of presynaptic Ca2+ signals and their effectors is thus needed for both the induction of changes in synaptic strength and the maintenance of robust synapse function. However, the mechanisms that effectively separate Ca2+ signals in time and space (e.g., through different voltage-gated calcium channels [VGCCs]) to allocate these to different presynaptic functions are not well understood (Krick, 2021).

    SV release probability depends on the sensitivity of the vesicular Ca2+ sensor and the positioning of VGCCs inside active zones (AZs). Various mechanisms that can tune release probability by modulating their precise localization or kinetic properties have been uncovered. Irrespective of such modulation, efficient Ca2+-triggered SV release through presynaptic VGCCs (mainly CaV2.1 and CaV2.2 in vertebrates) remains spatially restricted to a few hundred nanometers due to the limited abundance and brief opening of the channels and the presence of endogenous Ca2+ buffers. It is thus conceivable that Ca2+ signals originating within presynaptic terminals but outside AZs are engaged to tune SV recycling and plastic changes according to changes in activity (Krick, 2021).

    Apart from the need for fast activating and inactivating CaV2 channels (Drosophila Cacophony) for SV release, other types of VGCCs have been implicated in presynaptic plasticity. In GABAergic synapses, pharmacological blockade of CaV1 channels does not affect AP-induced SV release but converts posttetanic potentiation into synaptic depression. In hippocampal CA3 mossy fiber boutons or in synapses of the lateral amygdala, CaV2.3 and CaV1.2 channels are required for presynaptic long-term plasticity but are unable to trigger SV release (Krick, 2021).

    Differential functions of CaV2 and CaV1 channels in neurotransmitter release versus other Ca2+-dependent presynaptic processes can hardly be explained just by different coupling distances to SVs, since there are also situations where loose coupling is predominant. Moreover, compared with CaV2.1 and CaV2.2, CaV1 channels display higher conductances, suggesting that additional mechanisms are required to allocate CaV1-related Ca2+ signals to specific presynaptic functions while avoiding interference with SV release. SV recycling also includes regulation by presynaptic Ca2+ signals but operates mostly at different subsynaptic sites and at slower time scales than Ca2+-triggered SV release. It is hypothesized that activity-dependent regulation of SV recycling employs CaV1-dependent Ca2+ entry and that active mechanisms exist to regulate the relative contributions of CaV2 and CaV1 channels to SV release versus recycling. These hypotheses are addressed at the Drosophila larval neuromuscular junction (NMJ), an established model for glutamatergic synapse function (Krick, 2021).

    Neuronal network function critically depends on the tight control of synaptic vesicle (SV) release probability at chemical synapses over wide ranges of activity regimes. At the same time, synaptic gain remains adjustable to render network function flexible. To maintain synapse function over time, SV recycling rates must be matched to vastly different activity patterns and synaptic gains. While SV release and recycling as well as their plasticity-related adjustments all include Ca2+-dependent steps, they operate in parallel but on different time scales. A tight spatial and temporal coordination of presynaptic Ca2+ signals and their effectors is thus needed for both the induction of changes in synaptic strength and the maintenance of robust synapse function. However, the mechanisms that effectively separate Ca2+ signals in time and space (e.g., through different voltage-gated calcium channels [VGCCs]) to allocate these to different presynaptic functions are not well understood (Krick, 2021).

    SV release probability depends on the sensitivity of the vesicular Ca2+ sensor and the positioning of VGCCs inside active zones (AZs). Various mechanisms that can tune release probability by modulating their precise localization or kinetic properties have been uncovered. Irrespective of such modulation, efficient Ca2+-triggered SV release through presynaptic VGCCs (mainly CaV2.1 and CaV2.2 in vertebrates) remains spatially restricted to a few hundred nanometers due to the limited abundance and brief opening of the channels and the presence of endogenous Ca2+ buffers. It is thus conceivable that Ca2+ signals originating within presynaptic terminals but outside AZs are engaged to tune SV recycling and plastic changes according to changes in activity (Krick, 2021).

    Apart from the need for fast activating and inactivating CaV2 channels for SV release, other types of VGCCs have been implicated in presynaptic plasticity. In GABAergic synapses, pharmacological blockade of CaV1 channels does not affect AP-induced SV release but converts posttetanic potentiation into synaptic depression. In hippocampal CA3 mossy fiber boutons or in synapses of the lateral amygdala, CaV2.3 and CaV1.2 channels are required for presynaptic long-term plasticity but are unable to trigger SV release (Krick, 2021).

    Differential functions of CaV2 and CaV1 channels in neurotransmitter release versus other Ca2+-dependent presynaptic processes can hardly be explained just by different coupling distances to SVs, since there are also situations where loose coupling is predominant. Moreover, compared with CaV2.1 and CaV2.2, CaV1 channels display higher conductances, suggesting that additional mechanisms are required to allocate CaV1-related Ca2+ signals to specific presynaptic functions while avoiding interference with SV release. SV recycling also includes regulation by presynaptic Ca2+ signals but operates mostly at different subsynaptic sites and at slower time scales than Ca2+-triggered SV release. It is hypothesized that activity-dependent regulation of SV recycling employs CaV1-dependent Ca2+ entry and that active mechanisms exist to regulate the relative contributions of CaV2 and CaV1 channels to SV release versus recycling. These hypotheses are addressed at the Drosophila larval neuromuscular junction (NMJ), an established model for glutamatergic synapse function (Krick, 2021).

    The data show strict functional separation of AP-triggered neurotransmitter release by Cav2 and activity-dependent modulation of SV recycling and short-term plasticity by Cav1 VGCCs. Although task sharing and partial redundancy among Cav2 isoforms is known for mammalian synapses, and the dynamic regulation of their relative abundance within AZs can add to synaptic plasticity, insight into mechanisms that allow for the separate regulation of different aspects of presynaptic function by Cav2 and Cav1 channels is currently sparse (Krick, 2021).

    Ultrastructural support for the coexistence of Cav2 and Cav1 channels has been obtained in rat hippocampal neurons, where Cav2 localizes to AZs and Cav1 outside AZs, largely as is found for Drosophila. Moreover, pharmacological data in mammals indicate that Cav1 and Cav2 VGCCs separately control SV release and synaptic plasticity. In synapses of the amygdala, Cav1 is not required for SV release but for presynaptic forms of LTP; in GABAergic basket cells, Cav1 is not required for evoked release but for posttetanic potentiation; and at mouse neuromuscular synapses, anatomical and physiological data indicate the presence of both presynaptic Cav1 and Cav2 channels, but again with little contribution of Cav1 to evoked SV release. Therefore, studies of different synapse types in various species support the idea that multiple fundamental aspects of presynaptic function are executed in parallel on the basis of spatially separated VGCCs with different kinetics and conductances. This study provides a mechanism for functional separation in the small space of the axon terminal (Krick, 2021).

    The fast activation and inactivation kinetics of Cav2 channels in the AZ seem well suited for tight excitation-release coupling, and Cav2 activation mediates release mostly in an all or none fashion, though dynamic modulation of channel-SV coupling to adjust release probability is reported. By contrast, Cav1 channels typically have larger single-channel conductances and slower inactivation kinetics, suggesting that they are well suited to cope with the need for relatively high Ca2+ and the slow time course of endocytic vesicle retrieval (Krick, 2021).

    Endocytosis regulation by activity-dependent Ca2+ influx is discussed for mammalian and invertebrate synapses. At the Drosophila NMJ, separate Ca2+ entry routes for differential exo- and endocytosis regulation have been postulated, and the SV-associated calcium channel Flower has been suggested to contribute to this function. This study identified Cav1 channels within the periphery of AZs as a distinct entry route for Ca2+-dependent augmentation of SV endocytosis. Although the precise underlying mechanisms remain to be investigated, an attractive hypothesis is that Cav1 may serve as an activity-dependent switch to direct recycling into different SV pools. In basket cells, Cav1 mediated Ca2+ influx has been speculated to mobilize vesicles into the releasable pool to maintain synaptic transmission during high-frequency bursting. Similarly, at the mouse NMJ, pharmacological blockade of L-type Cav1 channels decreases FM2-10 loading and quantal release upon high-frequency stimulation. This is in line with the findings of increased synaptic depression, reduced SV reacidification, decreased FM1-43 uptake, and reduced PSC recovery after RRP depletion upon reduction of presynaptic Cav1 function. However, the effects of Cav1-kd manifest within a few seconds. Unless recycling and SV reformation are ultrafast, this seems too fast for SV reuse. In cultured hippocampal neurons, for example, SVs are not reused during the first 200 APs, irrespective of stimulation frequency between 5 and 40 Hz. However, given that endocytic proteins can also function in release site clearance, reduced endocytosis in Cav1-kd may increase synaptic depression and decrease recovery from RRP depletion indirectly as a result of reduced release site clearance. Additional effects of Cav1 channels on other steps in the SV cycle, such as SV priming, can also not be excluded (Krick, 2021).

    For the mouse NMJ, it has been inferred that Cav1 activity directs recycled SVs into a high-probability release pool. Ultrastructural analysis of Drosophila synapses has also revealed two different recycling modes, one that depends on external Ca2+ and directs recycled SVs to AZs and another one that does not depend on external Ca2+ and replenishes other SV pools. Taken together, peri-AZ localization of presynaptic Cav1 channels as found in hippocampus and at the Drosophila NMJ may provide a common control mechanism to direct SV recycling to different pools in an activity-dependent manner. Protection of AZs by the peri-AZ PMCA provides a mechanism to maintain mean quantal content, and thus coding reliability, in the face of Ca2+-mediated endocytosis regulation (Krick, 2021).

    As in many mammalian neurons, in Drosophila motoneurons, Cav1 channels localize also to dendrites to boost excitatory synaptic input. Therefore, cooperative functions of Cav1 channels in different subneuronal compartments coordinate firing and SV recycling rates. Moreover, as in spinal motoneurons, Drosophila Cav1 channel function is modulated by biogenic amines, thus providing means for integrative regulation of motoneuron excitability and SV recycling rates in the context of internal state and behavioral demands (Krick, 2021).

    This study shows that 1) axon terminal Cav1 segregates into the peri-AZ compartment to augment SV endocytosis, and 2) PMCA, rather than directly acting on Ca2+ entering through Cav2, actively controls Cav1-dependent Ca2+ changes, thereby enabling side-by-side Ca2+ domains with profiles that meet the different requirements for SV release and recycling. This is consistent with reports on spatially restricted expression and/or regulation of PMCA in small T lymphocytes as a means to steer Ca2+-dependent processes specifically within cellular microdomains. In consequence, this study proposes to expand the concept of controlling release probability by presynaptic Ca2+ buffering systems after nanodomain collapse, which has been scrutinized in many studies, with the idea of nanodomain protection from presynaptic Ca2+ signals originating outside the AZ (Krick, 2021).

    PMCAs have high Ca2+ affinity and can accelerate Ca2+ clearance on millisecond timescales. While isolating AZs from Ca2+ influx through Cav1, PMCA otherwise does not affect the spatiotemporal properties of AZ Ca2+ nanodomains, because transmission amplitudes are not altered by PMCA-kd in the absence of Cav1 channels. Instead, it ensures stable release probability in the face of presynaptic Ca2+ signals that augment SV recycling, shape APs, and control synaptic plasticity. In contrast to soluble Ca2+ buffers and fixed ones in the AZ, the membrane-bound peri-AZ PMCA can be regulated on short time scales (e.g., by downstream effectors of Ca2+ and phospholipids). In addition, release from autoinhibition by binding of Ca2+/calmodulin, which is conserved across phyla, provides a molecular memory due to the slow time course of calmodulin release, allowing PMCA to persist in a preactivated state and to respond instantaneously to the next Ca2+ signal. Therefore, PMCA-mediated control of SV release probability is likely adjusted by the local activity at the synaptic terminal. The data show that changes in PMCA-dependent AZ protection largely impact SV release probability by allowing or preventing functional coupling of Cav1 channels with readily releasable SVs. It is proposed that the distant localization of Cav1 channels and PMCA in between AZs enables effective and versatile regulation of synaptic strength on a short time scale. In fact, theoretical considerations and recent studies on Cav2.1 dynamic coupling in hippocampal synapses and on differential spacing of Cav2 channels in cerebellar synapses suggest that modulation of SV release probability favors loose coupling of VGCCs to SV. Thus, regulation of presynaptic PMCA activity emerges as an effective means to dynamically regulate plasticity and SV recycling rates downstream of Cav1 (Krick, 2021).

    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).

    Determinants of synapse diversity revealed by super-resolution quantal transmission and active zone imaging

    Neural circuit function depends on the pattern of synaptic connections between neurons and the strength of those connections. Synaptic strength is determined by both postsynaptic sensitivity to neurotransmitter and the presynaptic probability of action potential evoked transmitter release (P(r)). Whereas morphology and neurotransmitter receptor number indicate postsynaptic sensitivity, presynaptic indicators and the mechanism that sets P(r) remain to be defined. To address this, this study developed QuaSOR, a super-resolution method for determining P(r) from quantal synaptic transmission imaging at hundreds of glutamatergic synapses at a time. P(r) was mapped onto super-resolution 3D molecular reconstructions of the presynaptic active zones (AZs) of the same synapses at the Drosophila larval neuromuscular junction (NMJ). P(r) varies greatly between synapses made by a single axon, the contribution of key AZ proteins to P(r) diversity was quantified; one of these, Complexin, was found to suppress spontaneous and evoked transmission differentially, thereby generating a spatial and quantitative mismatch between release modes. Transmission is thus regulated by the balance and nanoscale distribution of release-enhancing and suppressing presynaptic proteins to generate high signal-to-noise evoked transmission (Newman, 2022).

    The operation of neural circuits depends on the synaptic connections between neurons. To understand how neural circuits process and store information, one needs to understand the molecular mechanisms that govern the synaptic transmission and distribute synaptic weights across large numbers of connections. While determinants of postsynaptic strength (e.g. dendritic spine size, postsynaptic scaffold size, number of postsynaptic receptors) are well characterized, the presynaptic determinants are not as clear. The relationship between synapse morphology and presynaptic action potential (AP)-evoked neurotransmitter release probability (Pr) is weak as is the dependence of Pr on specific elements of the transmitter release apparatus, the active zone (AZ) (Newman, 2022).

    To understand how presynaptic machinery governs quantal transmission, one needs to measure Pr at identified synapses whose molecular constituents and organization can be analyzed directly. Three approaches have been used to measure transmission at multiple identified synapses. Postsynaptic quantal (i.e. single synaptic vesicle resolution) imaging with Ca2+ indicators detects flux through ionotropic receptors as a proxy for the excitatory postsynaptic response, biosensors detect released neurotransmitters, and presynaptic synaptopHluorins detect vesicle fusion3. However, the diffraction-limited nature of these imaging paradigms makes it difficult to assign transmission events to particular synapses when AZs are densely arrayed (Newman, 2022).

    To overcome these limitations, a combination of super-resolution imaging modalities were developed to precisely relate quantal transmission to synaptic architecture at the glutamatergic model synapse of the Drosophila NMJ. The logic of stochastic single-molecule super-resolution localization microscopy was used to develop Quantal Synaptic Optical Reconstruction (“QuaSOR”), analogous to recent super-resolution imaging of transmission in neuronal culture with synaptopHluorin and iGluSnFR. QuaSOR resolved both action potential evoked and spontaneous quantal transmission events to individual synapses, even in regions where the synapses are crowded. QuaSOR allowed maping locations of quantal transmission, quantifing Pr using failure analysis, and measuring the frequency of spontaneous transmission (Fs) at hundreds of synapses simultaneously throughout the NMJ, under physiological conditions. QuaSOR analysis was followed by super-resolution molecular imaging of presynaptic AZ proteins, enabling spatial averaging of protein and transmission localizations that revealed new aspects of synaptic release mechanisms (Newman, 2022).

    Pr was found to have a high power dependence on the quantity of the presynaptic voltage-gated Ca2+ channel Cacophony (Cac), consistent with the power dependence of quantal content on Ca2+. Pr also had a strong dependence on the scaffolding protein Bruchpilot (Brp), which organizes the AZ and anchors synaptic vesicles near the site of release. However, Cac and Brp together accounted for only a minor fraction of the variance in Pr, indicating that other important factors control and diversify AP-evoked release. A clue about one additional contributor came from an observation that evoked and spontaneous transmission modes are mismatched spatially and quantitatively. This led to an investigation of Complexin (Cpx), whose Drosophila homolog is a powerful inhibitor of spontaneous transmitter release and which contains subdomains that both facilitate and inhibit evoked release. As the Cpx/Brp ratio increased, Pr declined. When Cpx was knocked down, the mismatch between spontaneous and evoked transmission disappeared. Additionally, Pr was higher compared to control synapses with the same Brp content. It is concluded that the interplay between release-promoting Cac and Brp and release-suppressing Cpx sets presynaptic transmission strength, generates synapse-to-synapse diversity, and enhances quantal signal-to-noise by suppressing spontaneous release at the site of maximal evoked release. The results demonstrate how super-resolution structure/function imaging can reveal the mechanisms of regulation of synaptic function (Newman, 2022).

    To understand the mechanisms that regulate synaptic strength and generate synapse diversity, this study set out to develop a new set of super-resolution imaging tools that together would allow relaying quantal transmission to presynaptic molecular composition in an intact model synapse. Imaging of Ca2+ influx through ionotropic glutamate receptors, with a postsynaptically targeted reporter, provided a quantal-resolution proxy for the excitatory postsynaptic current (EPSC), and QuaSOR analysis increased spatial resolution sufficiently to resolve synapses even in dense areas of the Drosophila NMJ. QuaSOR makes it possible to determine Pr directly by failure analysis under physiological Ca2+, i.e. at physiological Pr, avoiding reliance on estimation based on the ratio between evoked and spontaneous EPSC amplitudes (problematic in view of the finding that the sites of evoked and spontaneous transmission are segregated within the synapse), fits of amplitude distributions or analysis of variance. Post-hoc super-resolution presynaptic axon reconstructions enabled correlation of transmission to the molecular composition and nano-architecture of the presynaptic AZ for thousands of synapses (Newman, 2022).

    Earlier work suggested that, despite a common history of activity and postsynaptic target, transmission varies greatly between the synapses of a single Ib motor axon. QuaSOR assignment of transmission events to identified synapses showed this to be the case across thousands of synapses and revealed that the heterogeneity is even greater than previously thought, with Pr ranging over at least 100-fold, from <0.005 to 0.6. Half of the synapses are very weak (Pr < 0.02) and AP-evoked transmission is dominated by a small fraction of higher Pr synapses during low levels of activity. This large pool of low-Pr synapses could operate as a reserve that would be recruited to sustain transmission during long, high-frequency AP bursts, such as occur during locomotion (Newman, 2022).

    Previous studies at the NMJ demonstrated a positive relationship between Pr and both Cac and Brp. The ability to relate quantal transmission to multi-color 3D-STORM clarifies the nature of this relationship, by showing that Pr increases with the ~5th power of Cac, both in wildtype synapses and in synapses of a rab3 mutant whose AZs are enlarged, consistent with the power-dependence of release on Ca2+. Cac and Brp levels were also correlated with one another, consistent with Brp recruiting Cac to the AZ46. Although they are strong determinants, Cac and Brp only account for a fraction of the variance of Pr, indicating that other factors are at play. When AZs were expanded by the rab3 mutant to include more Brp and Cac, Pr increased to higher values, while maintaining the shallow Fs-Pr relation, the displacement of spontaneous transmission to locations outside the sites of evoked and the high power dependence of Pr on Cac. These observations are consistent with a mechanism that tunes Pr by regulating the size of the Brp scaffold and the number of Cac channels (Newman, 2022).

    In considering other potential regulators of presynaptic strength, it is necessary to take into account an almost complete lack of correspondence between evoked and spontaneous transmission in WT animals. Most startlingly was a complete suppression of spontaneous transmission at the site of maximal evoked transmission. This segregation is only possible to detect with these analysis tools and agrees with evidence from the use-dependent block that spontaneous and evoked release activate distinct populations of glutamate receptors in hippocampal neurons and the Drosophila NMJ. The observations reveal that this separation arises not only from synapse specialization, as proposed in earlier studies but from physical segregation of evoked and spontaneous transmission within the synapse. This spatial mismatch is remarkably consistent with recent iGluSnFR mapping of spontaneous and evoked transmission events in cultured hippocampal synapses, suggesting that segregation of transmission modes within the synapse may be a general phenomenon (Newman, 2022).

    It was considered that a factor that regulates both spontaneous and evoked release could be responsible for their spatial mismatch. Cpx has been shown to regulate both spontaneous and evoked release in complicated and contradictory ways. In vitro, Cpx interacts with the coiled-coiled domains of the SNARE complex to inhibit fusion and is displaced by Ca2+-bound synaptotagmin to trigger AP-evoked release. The mammalian isoforms of Cpx contain both fusogenic and inhibitory domains. Pan-neuronal removal of Cpx in Drosophila reduces postsynaptic response amplitude, suggesting that Cpx promotes evoked release. In contrast, expression of Drosophila Cpx in mammalian neurons suppresses evoked release. Cpx may also adjust the relationship between release and internal Ca2+ concentration through its role as an adapter that helps determine the composition of the release apparatus. Cpx is broadly distributed in the axon, enriched at the AZ and most densely concentrated in the Brp annular core. As the Cpx/Brp ratio within the AZ core rises, the Pr of Ib synapses decreases. This suggests that Cpx in the AZ core, which is positioned to interact with SNARE complexes, inhibits evoked release. Consistent with this relationship, Cpx knockdown increases the dependence of Pr on Brp so that at equivalent Brp levels Pr is higher when Cpx is knocked down and low Brp synapses with no detected transmission events become active (Newman, 2022).

    Knockdown of Cpx increased Fs by ~11-fold at Ib synapses and ~66-fold at Is synapses, indicating that Cpx suppresses spontaneous transmission more strongly than evoked transmission. In light of this and of the findings that: (a) Cpx density is highest in the Brp annular core, where Cac is also located, and where AP-evoked vesicle fusion is therefore expected to take place, (b) spontaneous transmission is suppressed at the site of maximal evoked transmission, (c) spontaneous and evoked transmission are poorly correlated, and (d) knockdown of Cpx eliminates the spatial and quantitative mismatch between spontaneous and evoked transmission. It is proposed that Cpx within the AZ core partly suppresses evoked release and completely suppresses spontaneous release. This differential suppression can preserve vesicles that are docked near Ca2+ channels in a state that is ready for release when the AP arrives, yielding a higher signal-to-noise for AP-evoked transmission over background spontaneous transmission (Newman, 2022).

    It is striking how knockdown of Cpx converts the relationship between Pr and Fs to near 1:1 and the spatial relationship of spontaneous and evoked transmission to coincident. This suggests that spontaneous and evoked release rates are, after all, governed by common factors. Brp levels were reduced in the CpxKD, possibly reflecting a compensatory mechanism that keeps the Pr of Ib synapses at near WT levels, as shown in recent focal extracellular recordings from Ib boutons. While Cpx in the Brp annular core suppresses Pr, this study found that higher bulk Cpx around the AZ is associated with higher Pr. This bulk Cpx likely reflects prenylated Cpx that is associated with endosomes and synaptic vesicles, which links vesicles to Brp69, and so may reflect higher vesicle content (Newman, 2022).

    Together, QuaSOR and super-resolution molecular imaging of AZs reveals that the balance between the quantity and nanoscale localizations of Cac, Brp, and Cpx contribute to a wide diversity in release dynamics for synapses that otherwise share common pre-post pairing and activity history. This heterogeneity could serve to maintain a deep pool of reserve synapses upon which the system can draw under diverse physiological demands (Newman, 2022).

    Kinetochore proteins have a post-mitotic function in neurodevelopment

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

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

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

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

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

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

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

    Synapse assembly and subsequent neurotransmission strength are dependent on secreted bidirectional signaling pathways, which communicate between presynaptic and postsynaptic partners to coordinate synaptic development, function and plasticity. Both synaptic cleft and the surrounding extracellular perisynaptic space (the 'synaptomatrix') are characterized by heavy glycosylation, which has been shown over the last half-century with extensive lectin imaging. Synaptic signals are both mediated by, and regulated via, secreted glycoproteins (GPs), proteoglycans (PGs) and endogenous glycan-binding lectins. Disruption of these synaptic glycan-binding and modifying mechanisms is linked to numerous heritable neurological diseases, which include autism spectrum disorder (ASD) and intellectual disability (ID) states such as fragile X syndrome (FXS), movement and degeneration disorders such as hereditary spastic paraplegias (HSPs), and most broadly, the rapidly expanding congenital disorders of glycosylation. Secreted lectins play important roles in synaptic processes, including the secreted Ca2+-binding (C-type) lectins first identified by Kurt Drickamer. For example, the Drosophila Mind-the-Gap (MTG) patterns synaptomatrix glycans, regulates glutamate receptor (GluR) clustering, and modulates trans-synaptic signaling to control neurotransmission strength (Bhimreddy, 2021).

    This study has identified a second C-type lectin, Lectin-galC1 (LGC1), in a whole-genome transgenic Drosophila RNAi screen for genes regulating synapse structure and function at the neuromuscular junction (NMJ). LGC1 is one of 33 C-type lectins encoded by the Drosophila genome, which are classified by their binding to extracellular β-galactoside glycans via a single characteristic Ca2+-dependent carbohydrate-binding domain (CBD). Similar to the many other lectins encoded by the Drosophila genome, LGC1 exists within a gene cluster with two other duplicated C-type lectin gene homologs. However, these two neighbors, with homologies of 59% and 24% respectively, show both differential expression and function. Previous LGC1 studies have revealed roles in development and cellular defense, showing enhanced expression in imaginal discs during pupation, binding to surface polysaccharide chains on gram-negative bacteria, and upregulation in response to injury due to an upstream nuclear factor κB-binding site, which is a common regulator of immune defense genes. Consistent with other glycosylation mechanisms, LGC1 has a pleiotropic role in neurotransmission (Scott and Panin, 2014). The current RNAi screen identification of LGC1 indicated critical roles in the neuromusculature, and specifically at NMJ synapses, with increased neurotransmission strength following LGC1 knockdown (Dani, 2012b). The goal of the current study was to pursue this lead by generating selective tools to test LGC1 requirements in coordinated movement and NMJ neurotransmission mechanisms (Bhimreddy, 2021).

    This study generated a LGC1 loss-of-function null allele using CRISPR/Cas9 genome editing by creating a frame-shift near the beginning of the coding sequence. To characterize neuromuscular roles, this null was combined with a characterized LGC1 deficiency. A LGC1::GFP fusion and specific LGC1 antibody were generated to reveal LGC1 protein at the NMJ, in presynaptic boutons and secreted into the extracellular synaptomatrix. Consistent with this NMJ localization, it was found that loss of LGC1 caused faster locomotory muscle peristalsis and much more rapid coordinated movement. Previously, LGC1 RNAi was used to show increased NMJ excitatory junction current (EJC) transmission, but no change in synaptic architecture (Dani, 2012b). The current study confirmed these preliminary findings in null mutants employing confocal imaging and two-electrode voltage-clamp (TEVC) electrophysiology. Although the loss of LGC1 did not alter NMJ architecture, it did increase synapse number. Consistently, LGC1 mutants showed higher spontaneous synaptic vesicle (SV) fusion rates as well as elevated motor nerve stimulation-evoked EJC amplitudes. To test mechanisms, FM1-43 dye imaging was used to reveal faster SV cycling dynamics with smaller SV pools in the mutants. With high-frequency stimulation (HFS), faster depression and slower recovery were found. Both confocal and electron microscopy revealed smaller membrane-associated SV pools at mutant active zones, consistent with faster activity-dependent SV cycling. Taken together, these results indicate that LGC1 plays a role in presynaptic SV release dynamics to cap NMJ neurotransmission strength and thus limit motor function (Bhimreddy, 2021).

    Anterograde and retrograde signals secreted into the NMJ synaptomatrix by both motor neuron and muscle cells orchestrate intercellular communication at the synapse. The signaling ligands, their receptors and other molecules in the synaptomatrix environment are all very highly glycosylated, and mutation of many genes affecting glycosylation has strong effects on both coordinated movement and NMJ synaptic function. Glycan-binding lectins interact with the NMJ synaptomatrix at many levels, to effectively modulate NMJ neurotransmission strength. In the extracellular space, lectins serve to regulate the distribution of glycans (as scaffolds, co-receptors and signaling platforms), thereby directing secreted and transmembrane protein. For example, the secreted C-type lectin Mind-the-Gap (MTG) was extensively characterized at the Drosophila NMJ. MTG regulates glycan distribution, secreted trans-synaptic signaling ligands, synaptic position-specific (PS) integrins and the postsynaptic glutamate receptor (GluR) field, as well as the downstream internal Drosophila Pix–Pak–Dock signaling cascade and the critical DLG synaptic scaffold. These secreted MTG C-type lectin roles controlling postsynaptic function suggest that similar C-type lectin roles may govern presynaptic function (Bhimreddy, 2021).

    In a previous systematic transgenic RNAi screen for NMJ modulator genes, a new secreted C-type lectin was uncovered that strongly controls neurotransmission strength (Dani, 2012b). Lectin-galC1 (LGC1) is an ideal candidate for modulation of synaptic function due to its characterized Ca2+-binding (C-type) and glycan-binding properties as a secreted protein. To create a LGC1-specific null mutation, CRISPR/Cas9 homology-directed DNA repair was used to create a targeted frame-shift mutation. This LGC1 null mutant was paired with a deletion removing the C-type lectin gene cluster containing LGC1 to characterize a new specific LGC1 antibody and to show that LGC1 is locally secreted at the NMJ. Then the effects were studied of LGC1 loss on coordinated movement, which intimately depends on both NMJ synaptic architecture and function. Consistent with the initial discovery of increased NMJ neurotransmission following LGC1 RNAi knockdown (Dani, 2012b), and earlier studies demonstrating LGC1 Ca2+-dependent binding functions, this study reports that LGC1-null mutants exhibit accelerated movement. LGC1 loss increases both the rate of locomotion and the speed to complete complex coordinated behaviors when challenged. These findings suggest LGC1 acting locally at the NMJ is important for modulating locomotion and, particularly, complex coordinated motor function (Bhimreddy, 2021).

    LGC1 is secreted at the NMJ, as demonstrated using non-permeabilized labeling, and concentrated within the extracellular synaptomatrix surrounding synaptic boutons; a space characterized by striking glycan accumulation. LGC1-null mutants have absolutely no alterations in NMJ structure. This is surprising, since NMJ overelaboration is a common feature of glycosylation mutations. For example, mutations in the key phosphomannomutase type 2 (PMM2) glycosylation enzyme strongly alter synaptomatrix pauci-mannose glycans to cause significant NMJ architectural expansion, including more synaptic branching and supernumerary boutons. Similarly, loss of UDP-sugar precursors, which are required for synaptic glycosylation also results in obvious NMJ structural overelaboration, with both increased branching and synaptic bouton number (Jumbo-Lucioni, 2016). However, synaptic structure and function are independently regulated, with separable glycan roles. Consistently, LGC1-null mutants exhibit an elevated number of synapses within otherwise normal NMJ boutons, thereby exhibiting greater synaptic density. LGC1 nulls have more presynaptic BRP-positive active zones (AZs) and more apposed postsynaptic GluR fields, suggesting that a higher level of synaptic connectivity contributes to elevated neurotransmission strength (Bhimreddy, 2021).

    LGC1 limits NMJ function by reducing SV fusion and evoked transmission. An elevated LGC1 EJC amplitude reflects an increase in number of released vesicles (quantal content) in a more mobile readily releasable pool (RRP). Since spontaneous mEJC amplitude does not change in LGC1 nulls, an increase in postsynaptic GluR number or function is ruled out as a contributing factor. In addition to increased synapse density, it was hypothesized that there would be higher SV fusion probability from altered SV cycling in LGC1 mutants. FM1-43 dye imaging shows that LGC1 loss results in a smaller cycling SV pool with higher cycling rate. A smaller SV pool could negate the effects of increased synapse number and SV fusion probability. However, most SVs do not participate detectably in transmission cycling, and smaller SV pools can show high transmission due to increased synaptic density and SV cycling rates. Moreover, altered SV endocytosis–exocytosis cycling efficacy can cause altered synaptic depression/recovery with high-frequency stimulation (HFS). Stimulation frequency can change SV cycling dynamics, with higher frequencies triggering differential SV release and recovery mechanisms. Consequently, NMJ function (depression and recovery) was assayed during varying levels of frequency stimulation, to stress neurotransmission machinery, and thus gain insight into the LGC1 mutant SV cycling mechanisms (Bhimreddy, 2021).

    In LGC1 nulls, HFS causes more rapid fatigue and slower recovery. Consistent with this, smaller SV cycling pools with higher turnover rates can increase basal transmission strength while impairing transmission maintenance during HFS demand. Thus, the smaller cycling SV pool in LGC1 mutants can maintain elevated basal transmission while exhibiting impaired replenishment of SV pools and maintenance of transmission during HFS. Other glutamatergic synapse classes maintain a small cycling RRP for high probability SV release during basal stimulation conditions, yet are unable to maintain transmission under conditions of greater demand. Likewise, LGC1 nulls exhibit reduced membrane-associated SV pools at both the light microscope and electron microscope levels, including lower SV density at AZs. Other Drosophila mutants with increased NMJ neurotransmission can similarly show presynaptic SV depletion in TEM ultrastructural studies. At the AZ, SVs near the presynaptic membrane make up the RRP and linked cycling pools, but internal SV pools can be held in 'reserve' or serve other functions apart from neurotransmitter release. Overall, these findings are consistent with LGC1 mutants exhibiting a smaller and faster SV cycling pool, with altered SV cycle dynamics during and following HFS stress (Bhimreddy, 2021).

    Similar to what is seen with LGC1 mutants, the loss of a number of C-type lectin domain (CTLD) proteins causes synaptic defects. For example, Mincle-deficient mice suppress TNF induction, which lowers glutamate release from central synapses, resulting in a direct modulation of neurotransmission strength. Although LGC1 appears to have the opposite effect, the Drosophila TNF homolog (Eiger) downregulates excitatory amino acid transporters 1 and 2 (EAAT1/2), and the EAAT1 mutation impairs locomotory peristaltic movement. This show'that LGC1 limits locomotory peristaltic movement, suggesting a possible relationship between LGC1, Eiger and EAAT1 may be a useful avenue to explore in future studies. Another CTLD protein is Caenorhabditis elegans CLEC-38, which has been shown to regulate presynaptic organization and mediate changes in the integral SV protein Synaptobrevin. As the vesicle SNARE (V-SNARE), Synaptobrevin drives SV fusion with the presynaptic membrane AZ to directly mediate neurotransmitter release. Since LGC1 mutants display neurotransmission defects comparable to CLEC-38 mutants, such as similar increased SV fusion and impaired SV cycling, the exploration of downstream LGC1 interactions and Synaptobrevin regulation in the presynaptic terminal could offer more insight into the mechanisms by which LGC1 produces neurotransmission changes (Bhimreddy, 2021).

    In conclusion, this study provides insight into the roles of a novel C-type lectin at a glutamatergic neuromuscular synapse. Beyond the previously characterized roles in development and immunity, this study found LGC1 secreted at the NMJ synapse modulates presynaptic function. Along with acting to limit synapse number and neurotransmission strength, LGC1 also regulates SV cycling to increase SV availability during basal levels of use, but decrease SV availability during high levels of use. In resting basal conditions, LGC1 caps NMJ neurotransmission strength to limit coordinated motor function. With intense stimulation, LGC1 loss causes more fatigue and decreases the recovery rate, indicating impaired activity-dependent SV cycling. This study revealed functional defects with electrophysiology, and visualized SVs with both FM1-43 dye and TEM electron microscopy imaging. In future studies, it is planned to dissect LGC1 mechanisms by focusing on SV pools and trafficking, as well as testing the possible involvement in signaling via Eiger, EAAT1 and downstream SV Synaptobrevin changes. This work indicates that LGC1-dependent signaling fine-tunes the multiple presynaptic pools that exchange SVs differentially based on activity levels and release demand to control SV release probability. Beyond this LGC1 mechanism, this new model will allow further exploration of SV cycling-dependent NMJ function in neuromuscular disease states (Bhimreddy, 2021).

    The ankyrin repeat domain controls presynaptic localization of Drosophila Ankyrin2 and is essential for synaptic stability

    The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. This study addresses the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. A P[acman] based rescue approach was used to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. Specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, these data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance (Weber, 2019).

    Giant ankyrins serve as central organizing adaptor molecules in both invertebrate and vertebrate neurons. This study provides first insights into the control of synaptic localization and function of giant Ankyrins by the N-terminal ankyrin repeat domain in vivo. By selectively deleting subdomains of the ARD of ank2 this study unravel critical requirements of specific regions of the ARD for the synaptic localization of the neuronal specific giant isoforms Ank2-L and Ank2-XL. The data demonstrate that the N-terminal domain controls not only synaptic targeting of individual isoforms but also contributes to synaptic localization of the alternative isoform. The functional requirements of Ank2-L and Ank2-XL for synaptic stability and microtubule organization clearly correlate with ARD-dependent regulation of protein abundance at the presynaptic terminal, with individual subdomains providing unique functional features (Weber, 2019).

    The N-terminal ARD mediates membrane-association of Ankyrins and is essential for the subcellular localization and organization of transmembrane binding partners. Prior work largely focused on the organizational roles of giant Ankyrins at the AIS and nodes of Ranvier in vertebrate neurons. In vivo analysis of ARD deletions now revealed a critical importance of this domain for the localization of Ank2-L at the presynaptic terminal of motoneurons. The synaptic localization of Ank2-L depends on AnkD2-4 (repeats 7-24) while the first domain (repeats 1-6) only had a minor impact on protein abundance. Interestingly, axonal targeting of Ank2-L was only partially affected by these deletions. These results indicate that separate and distinctive mechanisms exist in vivo that enable selective localization of giant Ankyrins in axons and within the presynaptic motoneuron terminal. As similar localization defects were observed already at earlier stages of development it argues that AnkD2-4 are essential for initial synaptic localization. Previous work demonstrated that a fusion protein comprising the specific C-terminal tail domain of Ank2-L efficiently localizes to both axonal and synaptic areas. The demonstration that synaptic localization of full length Ank2-L requires an intact ARD indicates that subcellular distribution is precisely regulated and not achieved by passive distribution within the neuron. Of particular interest is the observation that the first six ankyrin repeats are dispensable for synaptic localization of Ank2-L in the ank2ΔL mutant background. The recent characterization of the structure of the human ARD demonstrated distinct and independent binding sites for voltage-gated sodium channels (Nav1.2) and for L1-family CAMs (Neurofascin) in vitro and in cultured neurons. Interestingly, in cultured hippocampal neurons the L1-family CAM binding sites within AnkD2 (ankyrin repeats 8-9 and 11-13) are essential for clustering of the 270 kDa AnkG isoform at the AIS. In contrast, AnkG lacking the Nav1.2 binding site within AnkD1 (ankyrin repeats 2-6) efficiently localized to the AIS but failed to cluster Nav channels at the AIS. Thus, similar to the situation at the vertebrate AIS, the synaptic localization of Drosophila Ank2-L largely depends on interactions with L1-family CAMs or alternative transmembrane proteins that occupy binding sites within the central and C-terminal part of the ARD. This localized L1-CAM-Ankyrin complex can then serve as an assembly platform for additional molecules like voltage-gated sodium channels that acquired Ankyrin-binding capacities during chordate evolution to determine the physiological properties of specific subcellular neuronal compartments. While the AnkD1 of Drosophila ank2 had the least impact on synaptic stability phenotypes it will be interesting to identify putative binding proteins that may provide specific functional properties as absence of this domain resulted in decreased survival and locomotion of adult flies (Weber, 2019).

    In contrast to Ank2-L, the deletion of individual parts of the ARD had smaller effects on synaptic targeting of Ank2-XL in the ank2ΔXL mutant background. In this study, only the deletion of AnkD3 (repeats 13-18) resulted in a significant reduction of axonal and synaptic localization. However, the analysis revealed a striking dependence of Ank2-XL on the presence of the intact ARD of Ank2-L. Impairments of Ank2-L localization, including the minor effects caused by deletion of AnkD1, resulted in a severe reduction of synaptic Ank2-XL levels. This shows that Ank2-XL localizes via Ank2-L to the presynaptic terminal consistent with prior observations. Interestingly, deletions of parts of the ARD of Ank2-XL also significantly reduced synaptic Ank2-L levels indicating a critical co-dependence of Ank2-L and Ank2-XL at the NMJ. This interaction may depend on direct interactions between the two Ankyrin isoforms, potentially mediated within the ARD. However, it is equally possible that incorporation of mutated Ankyrins with altered binding properties resulted in a disruption of the larger Ankyrin-Spectrin scaffold that in turn leads to reduced targeting of the other isoform. Indeed, in vertebrate axons it has been demonstrated that different affinities of specific giant Ankyrins control the isoform-specific incorporation into selective axonal compartments like the nodes of Ranvier (Weber, 2019).

    At a functional level, a strong correlation was observed between synaptic Ank2-L level and the control of synaptic stability. At all muscle groups analyzed it was observed that deletion of AnkD1 domain had the mildest impact on synaptic stability consistent with the least impairments of synaptic Ank2-L localization. Importantly, the analysis of synaptic stability at muscle 4 also revealed that AnkD3 is most critical for synaptic maintenance. Despite an almost identical reduction in Ank2-L level compared to AnkD2 and 4 the rescue construct lacking the third domain was unable to restore any synaptic stability of ank2ΔL mutant animals. Interestingly, this assay also highlighted the specificity of the individual ankyrin repeats within the ARD. The exchange of second domain sequences with analogous sequences of the first domain failed to restore both Ank2-L localization and the synaptic stability phenotype of ank2ΔL mutants. The failure to support synaptic stability is at least in part due to a failure to organize and stabilize synaptic cell adhesion molecules. This analysis demonstrated that AnkD2 and 3 are critical for normal clustering of the NCAM homolog Fas II and of the L1 CAM homolog Nrg. This is consistent with prior observations that Ank2-L supports synaptic organization of both Fas II and Nrg and the co-dependence of Ank2-L and these CAMs at the synapse. This function is evolutionary conserved as mutations in the second and third binding site of vertebrate AnkG failed to restore clustering of the L1 CAM family members Neurofascin or Nr-CAM at the AIS of hippocampal neurons (Weber, 2019).

    In contrast to the clear requirements of the ARD for presynaptic targeting of Ank2-L the same mutations only partially affected localization of Ank2-XL. Consistently, only mild disruptions were observed of the presynaptic microtubule cytoskeleton and of the synaptic bouton organization compared to the ank2ΔXL mutation. The effects were most severe when deleting AnkD2 despite the fact that Ank2-XL protein levels were more compromised in AnkD3 mutants. As the large C-terminal repeat region of Ank2-XL is essential for the interaction with microtubules these results indicate that inappropriate ARD-dependent complex assembly may influence the functional properties of Ank2-XL at the NMJ (Weber, 2019).

    Together, this in vivo analysis of the ARD of Drosophila giant Ankyrins uncovered a structural basis for presynaptic localization and provided a genetic basis for the identification of regulatory mechanisms controlling structural synaptic plasticity via the selective Ankyrin complex assembly (Weber, 2019).

    The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF

    Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, this study used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. Sixteen candidates were identified that met the established criteria: a significant change of at least two-fold increase on ubiquitination, with at least two unique peptides identified. Amongst those candidates, Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release, was identified. Using a pulldown approach that relies on the overexpression and stringent isolation of a GFP-fused construct, Comt/NSF was validated to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. The possible functional relevance of this modification was tested using Ari-1 loss of function mutants that displayed a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared to controls in a Ca(2+) dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous versus evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. These results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination (Ramirez, 2021).

    Neurotransmitter release is mediated by a set of protein-protein interactions that include the N-ethylmaleimide sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). These proteins assemble into a tripartite complex in order to elicit synaptic vesicle fusion, which is formed by one synaptic vesicle membrane SNARE protein (v-SNARE), Synaptobrevin, and two plasma membrane SNARE proteins (t-SNAREs), Syntaxin and the 25-kDa synaptosome-associated protein. Following vesicle fusion, the tripartite SNARE complex disassembles by the activities of NSF and SNAPs. Free t-SNAREs from the plasma membrane can then participate in new priming reactions, while the v-SNAREs can be incorporated into recycled synaptic vesicles. These interactions, also routinely used for intracellular vesicle trafficking in all cell types, are conserved across species, including Drosophila (Ramirez, 2021).

    Deviations on the rate of neurotransmitter release are at the origin of multiple neural diseases, including Parkinson's disease. Under physiological conditions, the Leucine-rich repeat Serine/Threonine-protein kinase 2 (LRRK2) phosphorylates NSF to enhance its ATPase activity, which facilitates the disassembly of the SNARE complex. However, the most common Parkinson's disease mutation in LRRK2 causes an excess of kinase activity that interferes with the vesicle recycling. Similarly, α-Synuclein, another Parkinson's disease protein, alters neurotransmitter release by preventing the v-SNARE vesicle-associated membrane protein (VAMP)-2, also known as Synaptobrevin-2, from joining the SNARE complex cycle. Correct neural functioning, therefore, requires delicate regulation in vesicle trafficking. This regulation can be achieved by posttranslational modifications, such as ubiquitination. In fact, ubiquitination of certain proteins can affect their activity or life span. At the presynaptic side, for example, increased neurotransmitter release correlates with decreased protein ubiquitination. Similarly, acute pharmacological proteasomal inhibition causes rapid strengthening of neurotransmission (Ramirez, 2021).

    Ariadne 1 is an E3 ubiquitin-ligase, first identified in Drosophila, from a conserved gene family defined by two C3HC4 Ring fingers separated by a C6HC in-Between-Rings domain (the RBR motif). Ari-1 had been described to be essential for neuronal development, and its mutants reported to exhibit reduced eye rhabdomere surface and endoplasmic reticulum, as well as aberrant axonal pathfinding. However, despite its importance, no neuronal substrates have been reported so far. Only three Ari-1 substrates have been postulated, either in cultured cells or in vitro, while three Parkin substrates were reported to interact with Ari-1 in COS-1 cells. For this reason, with the aim to identify neuronal Ari-1 substrates, advantage was taken of two methodologies. The first one, the bioUb strategy, allows the identification of hundreds of ubiquitinated proteins from neuronal tissues. The system relies on the overexpression of a tagged ubiquitin that bears a 16 amino acid long biotinylatable peptide, which can be biotinylated by the Escherichia coli biotin holoenzyme synthetase enzyme (BirA) in neurons in vivo. Remarkably, this approach can be efficiently applied to identify neuronal E3 ligase substrates. In contrast, the second methodology favors the isolation of GFP-tagged proteins under denaturing conditions to further characterize their ubiquitination pattern under the presence or absence of an E3 ligase (Ramirez, 2021).

    This study has combined the bioUb strategy with the overexpression of Ari-1 and identified 16 putative neuronal substrates of Ari-1. Among those, focus was placed on Comatose (Comt), the fly NSF orthologue, due to its relevance in normal and pathological function at the synapse. By the isolation of GFP-tagged Comt from Drosophila photoreceptor neurons overexpressing Ari-1, this study confirmed Comt/NSF as an Ari-1 ubiquitin substrate and showed that it is mostly monoubiquitinated. Furthermore, Ari-1 loss-of-function mutants displayed lower rate of spontaneous neurotransmitter release, but enhanced evoked release, due to failures at the presynaptic side. These defects in the mutants are compatible with a deregulation of Com/NSF activity. Altogether, these data show that Ari-1 regulates neurotransmitter release by controlling Comt/NSF activity through ubiquitination (Ramirez, 2021).

    This study identified sixteen novel putative substrates of Ari-1 in Drosophila photoreceptor neurons in vivo by means of an unbiased proteomic approach. Remarkably, despite Ari-1 being recently shown to regulate the positioning of the cell nucleus in muscles via a direct interaction with Parkin (Tan, 2018), as well as to interact with some Parkin substrates, there is no overlap between the substrates identified for Ari-1 and those previously identified for Parkin in flies. Taken together, the available data suggest that the 16 targets identified in this study are specifically regulated by Ari-1 in Drosophila photoreceptor neurons and that this E3 ligase has a wide functional repertoire (Ramirez, 2021).

    This study focused on Comt, an ATPase required for the maintenance of the neurotransmitter release. Ubiquitination of proteins involved in vesicle trafficking and neurotransmitter release had been previously reported. Similarly, the importance of the ubiquitination machinery for the proper neuronal function has also been demonstrated. The alterations produced on synaptic transmission by ubiquitination are typically attributed to an acute control of synaptic protein turnover. However, many of these presynaptic proteins have been reported to be mainly mono- or di-ubiquitinated, a type of ubiquitin modification that is not usually associated with protein degradation. In line with this, the results showed that Comt/NSF is preferentially monoubiquitinated by Ari-1/ARIH1, suggesting that Ari-1/ARIH1 could be regulating Comt/NSF activity, rather than its life span or expression levels (Ramirez, 2021).

    Ari-1 mutations result in abnormal synaptic function at the larval stage, a result consistent with a regulatory function of NSF. All mutant alleles examined exhibit a reduced frequency of spontaneous synaptic release. In addition, ari-12 mutants exhibit a large calcium-dependent evoked release. Analysis of the mechanism for enhanced evoked release in ari-12 suggests that the primary defect consists in an increased probability of vesicle fusion in response to calcium entry in the presynaptic side. First, by comparing the amplitude and time course of spontaneously occurring postsynaptic events in mutant and control animals, the possibility of a postsynaptic modification was ruled out. Since no significant differences were found, it is concluded that the receptor field size and kinetic properties of postsynaptic receptors are normal in the mutant (Ramirez, 2021).

    The ari-1 functional defects could result from alterations of synaptic transmission during development; therefore, the number of synaptic contacts was quantified, assuming that most release sites occur within varicosities. No significant difference was observed between mutant and control. Although this study does not include electron microscopy quantification of synaptic vesicles, th confocal microscopy and electrophysiology data point toward an upregulation of release from single synapses (Ramirez, 2021).

    The failure analysis from single varicosities represents direct evidence that, at relatively low calcium concentrations, mutant terminals release more quanta than controls in response to an action potential. Whether increased quantal release could be explained on the basis of more release sites being concentrated on mutant terminals was examined. The focal recordings using saturating calcium concentrations argue against this possibility. When mutant terminals are exposed to high calcium, in order to increase the likelihood that all active zones within the bouton will release a quantum, EJP amplitudes in the mutant are indistinguishable from that of controls. These data suggest that the number of release sites in mutant and control terminals is similar and favor the hypothesis that, at physiological calcium concentrations, the probability of vesicle fusion upon calcium entry is increased in the mutant (Ramirez, 2021).

    It was found that ari-1 mutants have opposite effects on spontaneous and evoked release. Classically, the two modes of vesicular release have been considered to represent a single exocytotic process that functions at different rates depending on the Ca2+ concentration. However, recent work challenges this idea and supports the alternative model where spontaneous and evoked response might come from different vesicles pools. Several experimental evidences indicate that both forms of release may represent separate fusion pathways. Employing a state-of-the-art optical imaging in larval NMJ, it has been shown that evoked and spontaneous release can be segregated across active zones. Thus, three types of active zones could be defined: those that only release vesicles in response to a rise of intracellular calcium (evoked release), a second population that only participates in spontaneous release, and a third small proportion (around 4%) that participates in both evoked and spontaneous release. This result advocates for a different molecular and spatial segregation of both modes of release (Ramirez, 2021).

    Differential content or activity of regulatory SNARE binding proteins could discriminate between spontaneous and evoked release. It has been shown that the presence of the Vamp-7 isoform could participate in this differential release. Vamp-7 preferentially labels vesicles unresponsive to stimulation, and it colocalizes only partially with the endogenous synaptic vesicle glycoprotein Sv2 and the vesicular glutamate transporter Vglut1, suggesting that this vesicle pool does not support evoked transmitter release. Recently, it was shown that the double knockout mouse for Synaptobrevin genes, syb1 and syb2, results in a total block of evoked release, while spontaneous release was increased in both frequency and quantal size without changes in the number of docked vesicles at the active zone, confirming the idea that evoked and spontaneous releases are differentially regulated. Interestingly, Vamp-7 was found by MS to be less ubiquitinated when Ari-1 is overexpressed, suggesting that Ari-1 mutants could be favoring evoked release through NSF and reducing spontaneous release through Vamp7. Thus, Ari-1 could be acting as a repressor and activator of evoked and spontaneous release, respectively. All together, these results evidence a new layer of complexity over the actual fine-tuning of synaptic transmission. A physiological regulatory mechanism for both types of release has been recently demonstrated for inhibitory synapses at the trapezoid body, an important brain area in auditory integration. In this nucleus, activation of metabotropic glutamate receptor mGluR1 differentially modulates both spontaneous and evoked release in both GABAergic and Glycynergic synapses (Ramirez, 2021).

    Early functional studies of NSF employing the fly thermo-sensitive mutant allele comtts17, have reported a reversible reduction of synaptic transmission. Consistent with a role of NSF on SNARE dissociation, this inhibition parallels an increase in the number of synaptic vesicles at the presynaptic terminal. At this point, it can only be speculated how specifically Ari-1/ARIH1 regulates Comt/NSF activity within the presynaptic terminal. Opposite to the role of NSF mutant comtts17, which impairs SNARE complex disassembly, a change that enhances NSF functionality due to the lack of its ubiquitination would favor the dissociation of the so-called trans-SNARE complex. Further, this would build up the number of SNARE complexes assembled per vesicle, thus increasing the efficiency of fusion machinery in a Ca2+-dependent manner. Consistent with this interpretation, it has been shown that fast release of a synaptic vesicle requires at least three SNARE complexes, whereas slower release may occur with fewer complexes (Ramirez, 2021).

    Interestingly, some of the additional putative substrates identified are also related to synapse physiology and neurotransmitter release. PPO1 is an enzyme with L-DOPA monooxygenase activity, hence, may be involved in the metabolism of dopamine neurotransmitter. Similarly, GstO3 is involved in glutathione metabolism, another type of neurotransmitter. Vha44 and Vha68-1 are components of the vacuolar proton-pump ATPase, whose mutations have been reported to impair neurotransmitter release. Vha44 has also been described as an enhancer of Tau-induced neurotoxicity, and CG15117, orthologue of human GUSB, has been associated with neuropathological abnormalities. The long recovery time from paralysis observed in comt6 ari2/comt6 females could result from the role of Ari-1 in the ubiquitination of these additional substrates, in addition to the role of Comt in tissues other than the nervous system (Ramirez, 2021).

    The data reported in this study may be relevant in the context of Parkinson's disease. It should be noted that most Parkinson's-related genes encode proteins involved in vesicle recycling and neurotransmitter release at the synapse. Thus, the kinase LRRK2 phosphorylates NSF to enhance its ATPase activity upon the SNARE complex and facilitate its disassembly. Pathological mutations in this protein, such as G2019S, cause an excess of kinase activity that interferes with vesicle recycling. Deregulated synaptic aggregates of α-Synuclein may target VAMP-2 hampering the formation of the SNARE complex. Parkin is a structural relative of Ari-1, based on their common Cysteine rich C3HC4 motif, which is also at the origin of some forms of Parkinson's disease. All these genes and their corresponding mechanisms of activity sustain the scenario in which several types of Parkinson's disease seem to result from a defective activity of the synapse. In this context, the role of Ari-1/ARIH1 emerges as a mechanism to regulate a key component of the SNARE complex, Comt/NSF. Conceivably, Ari-1/ARIH1 may become a suitable target for either diagnosis or pharmacological treatment of Parkinson's and related diseases (Ramirez, 2021). Regulation of excitation-contraction coupling at the Drosophila neuromuscular junction

    Larval muscle contraction force increases with stimulation frequency and duration, revealing substantial plasticity between 5 and 40 Hz. Fictive contraction recordings demonstrate endogenous motoneuron burst frequencies consistent with the neuromuscular system operating within the range of greatest plasticity. Genetic and pharmacological manipulation of critical components of pre- and post-synaptic Ca(2+) regulation significantly impact the strength and time-course of muscle contractions. A screen for modulators of the excitation-contraction machinery identified a FMRFa peptide, TPAEDFMRFa, and its associated signaling pathway that dramatically increases muscle performance. Drosophila serves as an excellent model for dissecting components of the excitation-contraction coupling machinery. This study developed and used a force transducer system to characterize excitation-contraction coupling at Drosophila larval neuromuscular junctions (NMJs), examining how specific neuronal and muscle manipulations disrupt muscle contractility. Muscle contraction force increased with motoneuron stimulation frequency and duration, showing considerable plasticity between 5-40 Hz and saturating above 50 Hz. Endogenous recordings of fictive contractions revealed average motoneuron burst frequencies of 20-30 Hz, consistent with the system operating within this plastic range of contractility. Temperature was also a key factor in muscle contractility, as force was enhanced at lower temperatures and dramatically reduced with increasing temperatures. Pharmacological and genetic manipulations of critical components of Ca(2+) regulation in both pre- and post-synaptic compartments impacted the strength and time-course of muscle contractions. A screen for modulators of muscle contractility led to identification and characterization of the molecular and cellular pathway by which the FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to correlate synaptic dysfunction, regulation, and modulation, to alterations in excitation-contraction coupling (Ormerod, 2021).

    The Putative Drosophila TMEM184B Ortholog Tmep Ensures Proper Locomotion by Restraining Ectopic Firing at the Neuromuscular Junction

    TMEM184B is a putative seven-pass membrane protein that promotes axon degeneration after injury. TMEM184B mutation causes aberrant neuromuscular architecture and sensory and motor behavioral defects in mice. The mechanism through which TMEM184B causes neuromuscular defects is unknown. This study employed Drosophila melanogaster to investigate the function of the closely related gene, Tmep (CG12004), at the neuromuscular junction. Tmep was shown to be required for full adult viability and efficient larval locomotion. Tmep mutant larvae have a reduced body contraction rate compared to controls, with stronger deficits in females. In recordings from body wall muscles, Tmep mutants show substantial hyperexcitability, with many postsynaptic potentials fired in response to a single stimulation, consistent with a role for Tmep in restraining synaptic excitability. Additional branches and satellite boutons at Tmep mutant neuromuscular junctions are consistent with an activity-dependent synaptic overgrowth. Tmep is expressed in endosomes and synaptic vesicles within motor neurons, suggesting a possible role in synaptic membrane trafficking. Using RNAi knockdown, this study show that Tmep is required in motor neurons for proper larval locomotion and excitability, and that its reduction increases levels of presynaptic calcium. Locomotor defects can be rescued by presynaptic knockdown of endoplasmic reticulum calcium channels or by reducing evoked release probability, further suggesting that excess synaptic activity drives behavioral deficiencies. This work establishes a critical function for Tmep in the regulation of synaptic transmission and locomotor behavior (Cho, 2022).

    Activity-induced synaptic structural modifications by Akt

    The activity-dependent regulation of synaptic structures plays a key role in synaptic development and plasticity; however, the signaling mechanisms involved remain largely unknown. The serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K), plays a pivotal role in a wide range of physiological functions. This study focused on the importance of Akt in rapid synaptic structural changes after stimulation at the Drosophila neuromuscular junction, a well-studied model synapse. Compared with wild-type larvae, akt mutants showed significantly reduced muscle size and an increased number of boutons per area, suggesting that Akt is required for proper pre- and postsynaptic growth. In addition, the level of cysteine string protein (CSP) was significantly increased, and its distribution was different in akt mutants. After high K(+) single stimulation, the CSP level of akt mutant NMJs increased dramatically compared with that of wild-type NMJs. Interestingly, ghost boutons without postsynaptic specialization were found in akt mutant NMJs, and the number of these boutons was significantly increased by patterned stimulation. In contrast, the postsynaptic change in the subsynaptic reticulum (SSR) in the akt mutant occurred independent of stimulation. These results suggest that Akt functions in both pre- and postsynaptic growth and differentiation, and in particular, presynaptic action occurs in an activity-dependent manner (Kim, 2022).

    Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila

    O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. This study manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrated an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG (Fenckova, 2022).

    Crucial Roles of Ubiquitin Carboxy-Terminal Hydrolase L1 in Motor Neuronal Health by Drosophila Model

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) plays an important role in the ubiquitin-proteasome system and is distributed mostly in the brain. Previous studies have shown that mutated forms or reduction of UCH-L1 are related to neurodegenerative disorders, but the mechanisms of pathogenesis are still not well understood. To study its roles in motor neuronal health, the Drosophila model in which dUCH, a homolog of human UCH-L1, was specifically knocked down in motor neurons. RThe reduction of Drosophila ubiquitin carboxyl-terminal hydrolase (dUCH) in motor neurons induced excessive reactive oxygen species production and multiple aging-like phenotypes, including locomotive defects, muscle degeneration, enhanced apoptosis, and shortened longevity. In addition, there is a decrease in the density of the synaptic active zone and glutamate receptor area at the neuromuscular junction. Interestingly, all these defects were rescued by vitamin C treatment, suggesting a close association with oxidative stress. Strikingly, the knockdown of dUCH at motor neurons exhibited aberrant morphology and function of mitochondria, such as mitochondrial DNA (mtDNA) depletion, an increase in mitochondrial size, and overexpression of antioxidant enzymes. This research indicates a new, possible pathogenesis of dUCH deficiency in the ventral nerve cord and peripheral nervous systems, which starts with abnormal mitochondria, leading to oxidative stress and accumulation aging-like defects in general. Taken together, by using the Drosophila model, these findings strongly emphasize how the UCH-L1 shortage affects motor neurons and further demonstrate the crucial roles of UCH-L1 in neuronal health (Huynh, 2022).

    Stochastic Properties of Spontaneous Synaptic Transmission at Individual Active Zones

    Using postsynaptically tethered calcium sensor GCaMP, this study investigated spontaneous synaptic transmission at individual active zones (AZs) at the Drosophila (both sexes) neuromuscular junction. Optical monitoring of GCaMP events coupled with focal electrical recordings of synaptic currents revealed "hot spots" of spontaneous transmission, which corresponded to transient states of elevated activity at selected AZs. The elevated spontaneous activity had two temporal components, one at a timescale of minutes and the other at a subsecond timescale. To investigate the mechanisms of elevated activity, focused was first placed on the protein Complexin, which binds the SNARE protein complex and serves to clamp spontaneous fusion. Overexpression of Drosophila complexin largely abolished the high-activity states of AZs, while complexin deletion drastically promoted it. How presynaptic Ca(2+) transients affect the states of elevated activity was investiged at individual AZs. Ca(2+) influx was blocked or promoted pharmacologically, and also Ca(2+) release was promoted from internal stores. These experiments coupled with computations revealed that Ca(2+) transients can trigger bursts of spontaneous events from individual AZs or AZ clusters at a subsecond timescale. Together, these results demonstrated that spontaneous transmission is highly heterogeneous, with transient hot spots being regulated by the SNARE machinery and Ca(2+) (Astacio, 2022).

    Abnormal larval neuromuscular junction morphology and physiology in Drosophila prickle isoform mutants with known axonal transport defects and adult seizure behavior. J Neurogenet: 1-9

    Previous studies have demonstrated the striking mutational effects of the Drosophila planar cell polarity gene prickle (pk) on larval motor axon microtubule-mediated vesicular transport and on adult epileptic behavior associated with neuronal circuit hyperexcitability. Mutant alleles of the prickle-prickle (pk(pk)) and prickle-spiny-legs (pk(sple)) isoforms (hereafter referred to as pk and sple alleles, respectively) exhibit differential phenotypes. While both pk and sple affect larval motor axon transport, only sple confers motor circuit and behavior hyperexcitability. However, mutations in the two isoforms apparently counteract to ameliorate adult motor circuit and behavioral hyperexcitability in heteroallelic pk/pk/pk(sple) flies. The consequences of altered axonal transport was further investigated in the development and function of the larval neuromuscular junction (NMJ). Robust dominant phenotypes were uncovered in both pk and sple alleles, including synaptic terminal overgrowth (as revealed by anti-HRP and -Dlg immunostaining) and poor vesicle release synchronicity (as indicated by synaptic bouton focal recording). However, recessive alteration of synaptic transmission was observed only in pk/pk larvae, i.e. increased excitatory junctional potential (EJP) amplitude in pk/pk but not in pk/+ or sple/sple. Interestingly, for motor terminal excitability sustained by presynaptic Ca(2+) channels, both pk and sple exerted strong effects to produce prolonged depolarization. Notably, only sple acted dominantly whereas pk/+ appeared normal, but was able to suppress the sple phenotypes, i.e. pk/sple appeared normal. Thesd observations contrast the differential roles of the pk and sple isoforms and highlight their distinct, variable phenotypic expression in the various structural and functional aspects of the larval NMJ (Ueda, 2022).

    Loss of NF1 in Drosophila larvae causes tactile hypersensitivity and impaired synaptic transmission at the neuromuscular junction

    Autism Spectrum Disorder (ASD) is a neurodevelopmental condition in which the mechanisms underlying its core symptomatology are largely unknown. Studying animal models of monogenic syndromes associated with ASD, such as neurofibromatosis type 1 (NF1), can offer insights into its aetiology. This study shows that loss of function of the Drosophila NF1 ortholog results in tactile hypersensitivity following brief mechanical stimulation in the larva (mixed sexes), paralleling the sensory abnormalities observed in individuals with ASD. Mutant larvae also exhibit synaptic transmission deficits at the glutamatergic neuromuscular junction (NMJ), with increased spontaneous but reduced evoked release. While the latter is homeostatically compensated for by a postsynaptic increase in input resistance, the former is consistent with neuronal hyperexcitability. Indeed, diminished expression of NF1 specifically within central cholinergic neurons induces both excessive neuronal firing and tactile hypersensitivity, suggesting the two may be linked. Furthermore, both impaired synaptic transmission and behavioural deficits are fully rescued via knockdown of Ras proteins. These findings validate NF1(-/-) Drosophila as a tractable model of ASD with the potential to elucidate important pathophysiological mechanisms (Dyson, 2022).

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

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

    FOXO Regulates Neuromuscular Junction Homeostasis During Drosophila Aging

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

    Active zone compaction correlates with presynaptic homeostatic potentiation

    Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. This study use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. This study found compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier (Mretiani, 2021).

    A comparison of three different methods of eliciting rapid activity-dependent synaptic plasticity at the Drosophila NMJ

    The Drosophila NMJ is a system of choice for investigating the mechanisms underlying the structural and functional modifications evoked during activity-dependent synaptic plasticity. Because fly genetics allows considerable versatility, many strategies can be employed to elicit this activity. This study compared three different stimulation methods for eliciting activity-dependent changes in structure and function at the Drosophila NMJ. It was found that the method using patterned stimulations driven by a K+-rich solution creates robust structural modifications but reduces muscle viability, as assessed by resting potential and membrane resistance. It is argued that, using this method, electrophysiological studies that consider the frequency of events, rather than their amplitude, are the only reliable studies. These results were contrasted with the expression of CsChrimson channels and red-light stimulation at the NMJ, as well as with the expression of TRPA channels and temperature stimulation. With both these methods reliable modifications were observed of synaptic structures, along with consistent changes in electrophysiological properties. Indeed, a rapid appearance was observed of immature boutons that lack postsynaptic differentiation, and a potentiation of spontaneous neurotransmission frequency. Surprisingly, a patterned application of temperature changes alone is sufficient to provoke both structural and functional plasticity. In this context, temperature-dependent TRPA channel activation induces additional structural plasticity but no further increase in the frequency of spontaneous neurotransmission, suggesting an uncoupling of these mechanisms (Maldonado-Diaz, 2021).

    Imidacloprid Impairs Glutamatergic Synaptic Plasticity and Desensitizes Mechanosensitive, Nociceptive, and Photogenic Response of Drosophila melanogaster by Mediating Oxidative Stress, Which Could Be Rescued by Osthole

    Imidacloprid (IMD) is a widely used neonicotinoid-targeting insect nicotine acetylcholine receptors (nAChRs). However, off-target effects raise environmental concerns, including the IMD's impairment of the memory of honeybees and rodents. Although the down-regulation of inotropic glutamate receptor (iGluR) was proposed as the cause, whether IMD directly manipulates the activation or inhibition of iGluR is unknown. Using electrophysiological recording on fruit fly neuromuscular junction (NMJ), this study found that IMD of 0.125 and 12.5 mg/L did not activate glutamate receptors nor inhibit the glutamate-triggered depolarization of the glutamatergic synapse. However, chronic IMD treatment attenuated short-term facilitation (STF) of NMJ by more than 20%. Moreover, by behavioral assays, it was found that IMD desensitized the fruit flies' response to mechanosensitive, nociceptive, and photogenic stimuli. Finally, the treatment of the antioxidant osthole rescued the chronic IMD-induced phenotypes. It was clarified that IMD is neither agonist nor antagonist of glutamate receptors, but chronic treatment with environmental-relevant concentrations impairs glutamatergic plasticity of the NMJ of fruit flies and interferes with the sensory response by mediating oxidative stress (Liu, 2022).

    Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation

    Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. This study employed minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13 (GFSTF) 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13(GFSTF), Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13(GFSTF) subclusters that move toward the AZ center during PHP with unaltered Unc-13(GFSTF) protein levels (Dannhauser, 2023).

    Synaptic development is controlled in the periactive zones of Drosophila synapses

    A cell-adhesion molecule Fasciclin 2 (FAS2), which is required for synaptic growth and Still life (SIF), an activator of RAC, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. BetaPS integrin and discs large (DLG), both involved in synaptic development, also decorated the zone. However, Shibire (SHI), the Drosophila dynamin that regulates endocytosis, was found in the distinct region. Mutant analyses showed that sif genetically interacted with Fas2 in synaptic growth and that the proper localization of SIF required FAS2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. It is proposed that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively (Sone, 2000).

    This study characterized the periactive zone in three respects. Initially, the periactive zone was defined on the basis of the distribution patterns of SIF and FAS2, both clearly surrounding the electron-dense region marked with anti-DPAK antibody. The concentric staining patterns that represent pairs of the periactive zone and electron-dense region were often separated from the adjacent pairs and therefore suggested that these two regions constitute a structural unit in the NMJ. Secondly, the biochemical properties of the molecules found in the periactive zone characterize this zone. Cell adhesion molecules, FAS2, and integrin, were found on the plasma membrane, and DLG and SIF were found inside the membrane of the zone, although βPS integrin and DLG were more widely distributed, at least on the postsynaptic side. These findings suggest that the periactive zone may link the information from cell adhesion molecules to the intracellular signaling pathways. Finally, the role for the periactive zone was suggested by the genetic evidence for the molecules localized in the zone. The mutant analyses in this and the previous studies have shown that all these molecules are involved in synaptic development. In Fas2 null mutants, neuromuscular synapses fail to grow and eventually retract. In the mutants affecting βPS integrin, the growth of larval NMJ is increased or decreased dependent on the alleles. The mutation in dlg caused the reduction in size of the subsynaptic reticulum in postsynapses and the increase in the number of active zones in presynapses. Also, the sif mutation exhibited a modulatory effect on synaptic growth when combined with the Fas2 mutation as shown in this report. Taken together, all gene products localized in the peri-active zone participate in the synaptic development. These findings provide genetic evidence suggesting that the periactive zone serves as a membrane domain for the structural control of synaptic terminals (Sone, 2000).

    In addition, the data indicate that the periactive zone is distinct from the zone for endocytosis recently reported. Staining for SHI, which is in a donut-like pattern, does not overlap with most of the FAS2-positive region, and is nearly within the hole of the FAS2 rings. This observation suggests that endocytosis actively occurs near or within the electron-dense region, which is consistent with the previous finding that one type of endocytosis occurs near the active zone in the Drosophila optic lobe synapses. Although it is possible that endocytosis occurs in the periactive zone at some frequency, these data suggest that the major role for the zone is directed to events other than shi-dependent endocytosis (Sone, 2000).

    Genetic data suggested that FAS2 and SIF are components in the related signaling pathways that locally function in the periactive zone. The altered localization of SIF in the Fas2 mutants suggests that a certain level of FAS2 expression is required to maintain the configuration of the intracellular molecules including SIF in the periactive zone. Further evidence that shows the functional interaction between SIF and FAS2 was also obtained by mutant analyses. While each of the sif and Fas2 mutations causes the reduction in bouton number, the sif and Fas2 double mutations exhibited a suppressive genetic interaction in synaptic growth. This suppression suggests that the two pathways for FAS2 and SIF signals are both involved in synaptic development and the balance between the pathways is important for the regulation of synaptic growth. These notions were supported by the exacerbated reduction of the bouton number when SIF was overexpressed in the Fas2e76 background (Sone, 2000).

    It has been shown that FAS2 mediates synaptic stabilization, the varying extent of which seems to cause the increase or decrease in synaptic growth. The current data may be therefore consistent with the idea that SIF acts to regulate synaptic stabilization that is mediated by FAS2 or other adhesion molecules. Moreover, SIF may modulate synaptic growth in an inhibitory manner when the FAS2-mediated synapse stabilization is reduced. It may be further noteworthy that the Fas2e76 sifES11 double mutants exhibit low viability and are difficult to maintain as a stock even as heterozygotes. This observation may be also interpreted as indicating that eliminating the doses of both sif and Fas2 impairs a regulatory cascade that is established by the balance of the two signaling pathways (Sone, 2000).

    In summary, the cell adhesion molecule FAS2 and intracellular signaling molecule SIF interact with each other, both controlling synaptic development in the periactive zone. This finding enforces the idea that the periactive zone is the functional membrane domain where various types of proteins constitute signaling networks or protein complexes that control synapse formation. This notion is further supported by the fact that DLG regulates the localization of FAS2 in the periactive zone. In addition, FAS2, in turn, may function to organize the arrangement of the molecules including SIF in the periactive zones (Sone, 2000).

    An in vitro assay shows that SIF catalyzes the guanine- nucleotide exchange reaction for mammalian RAC1. This is consistent with the previous observation that SIF induces ruffling membranes in human KB cells, as does the constitutive active form of RAC1. In addition, Drosophila RHO family G proteins are more than 85% identical in amino acid sequences to the corresponding mammalian G proteins. These lines of evidence suggest that SIF activates Drosophila RAC in the periactive zones. As SIF contains multiple domains that potentially mediate interaction with other molecules, RAC and several other molecules may be recruited to make a protein complex in the zone. In mice, TIAM1 and STEF have been identified as GEFs that specifically activate RAC1, and both are highly related to SIF in domain organization and amino acid sequence in several domains, indicating that these two proteins are likely to be the mouse orthologs of SIF. Interestingly, both Tiam1 and Stef are expressed in the brain, and Tiam1 expression is observed in the adult hippocampus. It would be important therefore to examine whether TIAM1 and STEF are localized in the synaptic terminals (Sone, 2000).

    RAC is well known as a regulator of the actin-based cytoskeleton and cell adhesion in various cells. RAC is also implicated in the structural changes of nerve terminals including growth cones and dendrites. Therefore, in the periactive zones, activated RAC may locally regulate the processes of the structural change in the synaptic terminals, which include reorganization of the actin-based cytoskeleton and cell adhesion (Sone, 2000).

    Previous studies have shown that RAC acts in the neurite outgrowth of neuroblastoma cells that depends on the signal from integrin on the cell surface. The mammalian SIF homolog TIAM1, which functions as a RAC GEF, recruits integrin to specific adhesive contacts at the cell periphery. Moreover, expression of TIAM1 increases cadherin-mediated cell adhesion in epithelial MDCK cells. Therefore, there appear to be signaling links between the RAC and cell-adhesion molecules. This study shows that SIF activates RAC, sif genetically interacts with Fas2 in synaptic growth and the SIF localization was perturbed in the Fas2 mutants. Taken together, these data suggest that the SIF-RAC pathway is linked to the cell-adhesion molecule FAS2 in close vicinity in the periactive zone (Sone, 2000).

    This study has indicated the periactive zone as a region for the control of synaptic development. The periactive zone surrounds the active zone, which is the site for vesicle exocytosis or neurotransmission. This concentric organization suggests that the two zones specialized for the different cellular functions constitute an elemental unit for the presynaptic structure. Investigation of how these zones are incorporated into the synaptic bouton during development will be interesting but remains to be carried out (Sone, 2000).

    The segregated distribution of the two zones suggests that the mechanisms controlling synaptic development and neurotransmission may be separable. This view was supported by the mutant analyses for FAS2 and SIF; both mutations affect structural properties of synapses without changing basic electrophysiological functions. In the NMJs of Fas2 mutants, the bouton number is decreased or increased depending on the alleles but the total synaptic strength is maintained at the normal level (Sone, 2000).

    Functional strength of the synapse is regulated only through the activity of a transcription factor, cAMP-response-element- binding protein (CREB), which functions independently of FAS2. Also in sif mutants, basic electrophysiological properties of NMJs are normal. These observations clearly contrast with the mutant phenotypes for the proteins controlling vesicle exocytosis: synaptotagmin, cysteine string protein, n-synaptobrevin and syntaxin 1A. All these mutants show the impaired EJPs. Taken together, these results indicate that synaptic development and neurotransmission are genetically separable phenomena and are regulated by independent pathways. It is proposed that these genetically separable phenomena are spatially segregated into the two zones on the presynaptic plasma membrane, although the possibility that the two zones interact with each other cannot be excluded (Sone, 2000).

    Thia study identified new members to be added to the periactive zone proteins. In these studies, Drosophila highwire protein and its C. elegans homolog, RPM-1, were demonstrated to function in the growth or structural development of synapses, and highwire was found to localize in the periactive zone. Discovery of these proteins further enforces the current view and highlights the importance of periactive zones for synaptic growth and stability (Sone, 2000).

    In the nervous system, various cell-adhesion molecules and signaling molecules may provide characteristic attributes for each synapse. In addition to the molecules described in this article, cadherins and catenins are likely to play such a role in synapses. Notably, N-cadherin and SIF are localized laterally to the active zones in the neuron-neuronal synapses of the mammalian and Drosophila brain, respectively. This suggests that the presence of the specialized area surrounding the active zone is a general feature among synapses. Further examination of signaling pathways that function locally in the periactive zone would provide insights as to how synapses grow or retract during development and plasticity (Sone, 2000).

    Sphingolipids regulate neuromuscular synapse structure and function in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Oxidative stress induces overgrowth of the Drosophila neuromuscular junction

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

    Reactive oxygen species regulate activity-dependent neuronal plasticity in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Drosophila motor neuron boutons remodel through membrane blebbing coupled with muscle contraction

    Wired neurons form new presynaptic boutons in response to increased synaptic activity, however the mechanism(s) by which this occurs remains uncertain. Drosophila motor neurons (MNs) have clearly discernible boutons that display robust structural plasticity, being therefore an ideal system in which to study activity-dependent bouton genesis. This study showed that in response to depolarization and in resting conditions, MNs form new boutons by membrane blebbing, a pressure-driven mechanism that occurs in 3-D cell migration, but not previously described to occur in neurons. Accordingly, F-actin is decreased in boutons during outgrowth, and non-muscle myosin-II is dynamically recruited to newly formed boutons. Furthermore, muscle contraction plays a mechanical role, which is hypothesized to promotes bouton addition by increasing MN confinement. Overall, this study identified a mechanism by which established circuits form new boutons allowing their structural expansion and plasticity, using trans-synaptic physical forces as the main driving force (Fernandes, 2023).

    Dissection of the mechanisms that regulate activity-dependent bouton formation is critical to understand remodeling strategies required for neuronal growth and wiring. Using live imaging of the Drosophila larval NMJ, a system in which 3-D intercellular and biophysical interactions are preserved, this study showed that bouton formation occurred by membrane blebbing, a mechanism widely used in 3-D cellular migration but, to our knowledge, never reported to be used by neurons to remodel. This study showed that new boutons induced by elevated activity, also called ghost boutons (GB), are bona fide blebs, whose growth does not rely on actin polymerization but is rather pressure driven. Additionally, it was observed that activity-dependent bouton formation was frequently associated with muscle activity and that blocking muscle contraction significantly decreased GB frequency after stimulation. Moreover, the manipulation of synaptic activity and/or muscle contraction resulted in predictable changes in bouton formation in response to stimulation, with a progressive increase in new bouton numbers with higher levels of synaptic activity and/or muscle contraction (Fernandes, 2023).

    The results suggest that muscle contraction plays a mechanical role in activity-dependent bouton formation. At the NMJ, the MN is deeply imbedded in the muscle, which by contracting can directly alter neuronal confinement. It is hypothesized that with elevated activity, and in response to a still unclear pre and/or postsynaptic signal to initiate bouton outgrowth, MNs add new boutons by membrane blebbing, and muscle contraction is required to increase neuronal confinement. This confinement results in increased pressure onto the MN membrane, powering bouton outgrowth at places putatively primed for bouton formation. In accordance, it is known that blebs are favored in conditions of high confinement, namely in tissues where cells encounter increased mechanical resistance. Furthermore, cells in compressive 3-D environments naturally go through stiffness gradients, which can polarize the recruitment of molecules required to or that facilitate blebbing in spots of high membrane tension. Likewise, at the NMJ it is possible that regions of higher activity produce more signals for inducing synaptic growth, which is supported by previous studies that showed that synaptic plasticity at the NMJ, including GB formation, requires activity-dependent secretion of postsynaptic signals such as BMP ligands (Maverick and Gbb) and Ca2+-sensitive vesicle regulator Syt-IV, presynaptic Wg and Syt-I mediated NT release and postsynaptic glutamate receptor function. It will be interesting to investigate whether activation of pathways downstream of these factors can converge to determine sites primed for bouton initiation with synaptic activity, by recruiting initiation factors and/or changing cytoskeletal dynamics at these regions (Fernandes, 2023).

    Cells migrating in confined environments typically display rounder morphologies and use hydrostatic blebs for movement. Interestingly, it has been shown that neurons from mouse central nervous system (CNS) growing in 3-D matrices, rather than 2-D surfaces, displayed GCs that had an amoeboid-like morphology, characterized by very low adhesive interactions and extensive rounded deformations of the body-wall, challenging the paradigm of the lamellipodia based GC. In the brain, even though there are no muscle compressive forces, synaptic boutons are equally confined and surrounded by other neuronal, astrocytic, or microglial processes. Interestingly, a recent study showed that in the mammalian brain, enlargement of dendritic spines produces mechanical pressure onto boutons thereby enhancing NT release, thus contributing to synaptic strength (a force reported to be comparable to that of muscle contraction). This exciting discovery is in line with the hypothesis that mechanical force directly regulates synaptic function, or as in the case of this study, formation of new boutons. It will be interesting to study how neurons can sense and respond to mechanical signals provided by the cellular environment, and if pathways used for mechanosensing and transduction, likely regulating adhesion and contacts with extracellular partners, are coupled with synaptic transmission machinery during plasticity (Fernandes, 2023).

    While the molecular identity of the signal that leads to bouton initiation remains to be discovered, it is well established that blebs nucleate when a small patch of membrane is detached from the actin cortex. This can happen either as a direct consequence of buildup in hydrostatic pressure (that detaches membrane to cortex binding proteins) or by formation of local gaps, resulting from rupture of the cortex in regions of high membrane energy (where accumulation of MyoII helps to weaken the cortex). Our data suggests that, although at the NMJ the two mechanisms may coexist, with elevated activity boutons tend to form mainly because of compressive forces exerted by the muscle. Supporting this, it was found that activity-induced bouton formation was usually fast and correlated with visible muscle contraction, while events with low or no visible muscle contraction were slower and occasional. Additionally, post-stimulation, even though MyoII puncta were observed preceding bouton formation in ~35% of events, boutons were found forming with low MyoII levels and decreasing or inactivation of MyoII did not prevent bouton formation, which suggests that MyoII was not required to propel bouton growth induced by NMJ stimulation (although this result can not directly be extended to a null scenario). Interestingly, MyoII was recruited to ghost boutons in stimulated and unstimulated NMJs, which may explain cases where boutons formed with lessened muscle contractions. The fact that boutons always formed without filamin, which links actin to membrane, further supported that actin polymerization is not required for bouton initiation. Altogether a model is favored in which boutons form in regions of high pressure by detachment of membrane from the actin cortex and that local MyoII activation can facilitate the weaking of the cortex at these sites. Importantly, regardless of the exact signal used to initiate activity-dependent bouton formation, this study showed that growth and stabilization of these boutons requires both actin and MyoII local rearrangements analogous to what is reported for cellular blebs: weakened actomyosin cortex in early-stage boutons and enrichment after expansion (Fernandes, 2023).

    This model explains the formation of new synaptic boutons by a physical process-membrane blebbing, which in addition to the more studied transcriptional or biochemical factors, allows fast, on demand expansion of the NMJ. Whether axons are always competent for bleb-dependent bouton expansion or require priming by cytoskeletal regulation remains to be elucidated. Moreover, considering the conservation of presynaptic cytoskeletal components and bouton ultrastructure throughout evolution, it is postulated that this mechanism of synapse remodeling can be present in other organisms, including vertebrates. Overall, the finding that bouton addition at the NMJ occurs as result of a MN-muscle physical interplay highlights the importance of intercellular cooperation during plastic changes. Circuit remodeling as a response to experience requires rapid modulations of bouton number. We speculate that the regulation of confinement by non-neuronal cells (muscle or glia) can be a mechanism widely used by the nervous system to coordinate local activity-dependent structural changes in neurons with its surrounding 3-D cellular microenvironment. Future studies will elucidate whether the understanding of the biochemical and mechanical relations between neurons and their neighboring cells during structural plasticity can help design new strategies to remodel neuronal circuits that have been impaired by neurodevelopmental or neurodegenerative diseases (Fernandes, 2023).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    O-GlcNAcase contributes to cognitive function in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Molecular logic of synaptic diversity between Drosophila tonic and phasic motoneurons
    Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features are poorly understood. To identify molecular pathways that contribute to synaptic diversity, single-neuron Patch-seq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated that synaptic active zones in phasic motoneurons are more compact and display enhanced Ca(2+) influx compared with their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications, and intracellular Ca(2+) buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes (Jetti, 2023).

    Although pathways driving neuronal development and function are well characterized, how diversity in neuronal subtypes across the brain is established and maintained is poorly understood. With the advent of single-cell transcriptomics, mRNA expression profiles that contribute to neuronal morphology, connectivity, and function are being identified. One feature of diversity is the unique synaptic properties observed across neuronal classes. Synapses represent fundamental building blocks of information processing, exhibiting distinct synaptic vesicle (SV) release probability (Pr), response kinetics, and short-term plasticity. Notable examples of synaptic diversity include Caenorhabditis elegans (C. elegans) AWC and ASH olfactory neurons, invertebrate tonic and phasic motoneurons (MNs), zebrafish ON and OFF bipolar cells, and mammalian hippocampal, cerebellar, and auditory neurons. Functional heterogeneity in synaptic transmission contributes to temporal coding, processing of multisensory information, and circuit computations. Despite these roles, the differentially expressed genes (DEGs) that collectively specify differences in synaptic structure and output are largely unknown (Jetti, 2023).

    Neurons displaying tonic or phasic synaptic output are conserved from invertebrates to mammals and often co-innervate postsynaptic targets. Tonic and phasic synapses increase the robustness of information processing by acting as high- and low-pass filters, respectively. Drosophila larvae contain MNs with tonic or phasic output that co-innervate muscles and form glutamatergic neuromuscular junctions (NMJs). Tonic MNs have bigger 'type Ib' boutons and typically innervate single muscles to act as primary drivers of contraction. Phasic MNs form smaller 'type Is' boutons that innervate and coordinate contraction of muscle subgroups. Phasic MN active zones (AZs) display higher Pr and show synaptic depression during stimulation. In contrast, tonic MNs have weaker synaptic output and undergo facilitation. Tonic and phasic MNs also show differences in morphology, inputs, membrane excitability, and synaptic plasticity. The molecular logic that governs these differences is largely unknown, providing a system to characterize DEGs that establish and maintain these distinct properties. This study combined single-neuron Patch-seq RNA profiling with quantal imaging, stimulated emission depletion (STED) nanoscopy, transmission electron microscopy (TEM), optogenetics, electrophysiology, and genetic manipulations to identify and dissect molecular components contributing to the structural and functional diversity of Drosophila tonic and phasic MNs. RNA sequencing (RNA-seq) identified distinct transcriptional profiles for each MN subtype, while genetic analyses indicate multiple DEGs contribute to functional and morphological differences in AZ organization, Pr, and Ca 2+ buffering (Jetti, 2023).

    This study describes the nanoscopic, transcriptomic, and post-translational signatures that contribute to distinct features encoding neuropeptide receptors, synaptic cleft proteins, axonal pathfinding and cell adhesion molecules, membrane excitability and ionic balance regulators, and the Arl8 and Unc104 axonal transport factors, were also differentially expressed. Together, these data provide a framework for characterizing molecular mechanisms contributing to unique features of tonic and phasic synaptic diversity. This analysis is consistent with a model in which AZ structural differences and altered Ca 2+ entry and buffering contribute to release differences between Ib and Is MNs. Given that phasic Is synapses have enhanced synaptic strength and higher Pr, their unique stellate AZ architecture and smaller cytomatrix, together with shorter T-bars and larger SVs, could contribute to distinct AZ Ca2+ dynamics and alterations in coupling Ca 2+ influx to SV fusion. Previous studies demonstrated that changes in the coupling distance between SVs and Ca 2+ channels can alter synaptic strength at a number of synapse types. Increased expression of the Cbp53E Ca 2+ buffer in Is MNs lowers resting [Ca 2+ ] and restricts facilitation, which, together with enhanced Ca 2+ influx, provides a synergistic mechanism with the more compact Is AZ nanostructure that could enhance release. Prior studies identified additional differences in SV availability and fusogenicity that also contribute to differences in Ib and Is release properties. Reduced expression of Brp, the decoy SNARE Tomosyn, and the fusion clamp Complexin at Is terminals alters the size of SV pools and their availability for fusion, indicating that Ib synapses have fewer fusogenic SVs available to respond to Ca 2+ entry, in part due to higher expression of these proteins. No changes in Brp, Tomosyn, or Complexin mRNA levels were detected in Isoform-Patch-seq, suggesting that post-translational degradation or altered trafficking underlies differences in their abundance at Is synapses (Jetti, 2023).

    Multiple studies indicate that PTMs, such as ubiquitylation, glycosylation, phosphorylation, acetylation, and sumoylation, impact synapse organization and function by modifying protein stability and localization. Isoform-Patch-seq indicated that sialylation and ubiquitination are differentially regulated in Ib and Is MNs. Disruptions of other E3 ubiquitin ligases like Highwire alter synaptic growth at Drosophila NMJs, though they impact both Is and Ib terminals. Identifying targets of the Skp2-CG9003 E3 ligase complex should clarify mechanisms by which cell-type-specific proteolysis contributes to synaptic morphology and function. Sialylation is critical for organization of the cell-surface proteome, regulation of cell adhesion, and control of neuronal excitability. Lack of sialylation impairs surface expression of multiple ion channels and cell adhesion proteins, and further studies should identify the specific targets regulating Ib synaptic organization (Jetti, 2023).

    In summary, these data highlight multiple pathways that contribute to synaptic diversity between tonic and phasic MNs, while providing a resource to characterize additional DEGs that specify other structural and functional features of these neuronal subtypes. Neurons with tonic and phasic output represent an evolutionarily conserved design principle, and it will be interesting to determine whether similar DEGs are involved in other species. Recent RNA profiling indicates that Drosophila visual neurons differentially express sialyltransferases and proteolytic regulators, while mammalian cortical GABAergic interneuron subtypes differentially express sialyltransferases that alter the cell-surface proteome. As such, cell-type-specific PTMs may represent a widely used mechanism to generate neuronal diversity beyond their role in Drosophila tonic and phasic MNs (Jetti, 2023).

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

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

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

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

    Characterization of a novel stimulus-induced glial calcium wave in Drosophila larval peripheral segmental nerves and its role in PKG-modulated thermoprotection

    Insects, as poikilotherms, have adaptations to deal with wide ranges in temperature fluctuation. Allelic variations in the foraging gene that encodes a cGMP dependent protein kinase, were discovered to have effects on behavior in Drosophila by Dr. Marla Sokolowski in 1980. This single gene has many pleiotropic effects and influences feeding behavior, metabolic storage, learning and memory and has been shown to affect stress tolerance. PKG regulation affects motoneuronal thermotolerance in Drosophila larvae as well as adults. While the focus of thermotolerance studies has been on the modulation of neuronal function, other cell types have been overlooked. Because glia are vital to neuronal function and survival, this study determine if glia play a role in thermotolerance as well. In this investigation, a novel calcium wave was discovered at the larval NMJ and set out to characterize the wave's dynamics and the potential mechanism underlying the wave prior to determining what effect, if any, PKG modulation has on the thermotolerance of glia cells. Using pharmacology, it was determined that calcium buffering mechanisms of the mitochondria and endoplasmic reticulum play a role in the propagation of the novel glial calcium wave. By coupling pharmacology with genetic manipulation using RNA interference (RNAi), it was found that PKG modulation in glia alters thermoprotection of function as well as glial calcium wave dynamics (Krill, 2021).

    cAMP signals in Drosophila motor neurons are confined to single synaptic boutons

    The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. This study suggests a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, this study demonstrates the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, evidence is provided that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons (Maiellaro, 2016).

    The cyclic AMP (cAMP) pathway plays fundamental roles in the nervous system, where it is prominently involved in synaptic plasticity and memory formation. Previous studies in vertebrate and invertebrate models have shown that cAMP can propagate from dendrites to the cell body of neurons, in line with the properties of a small diffusible molecule. However, a local mode of action for cAMP has also been proposed, whereby cAMP signals are localized to the periphery of neurons, namely, dendrites, creating a cAMP microdomain. While the existence of cAMP microdomains in neuronal dendrites is disputed based on the experimental and theoretical data, very little is known about possible cAMP compartmentation in axons and how this may exert local effects at the presynaptic site. In particular, it is unclear how biochemical signals may spread from presynaptic boutons through the axon (Maiellaro, 2016).

    To investigate this question in vivo, the neuromuscular junction (NMJ) of Drosophila melanogaster was used, that displays different forms of synaptic plasticity, many of which are dependent on cAMP signaling. Both structural and functional properties of larval neuromuscular synapses are heterogeneous, varying between boutons belonging to the same motor neuron. How such site-specific synaptic differentiation may be achieved at high spatial resolution is currently unknown, though it is tempting to speculate that local cAMP signals play a role. Thus, the developing Drosophila NMJ is a powerful model to investigate the role of cAMP in synaptic plasticity under physiological conditions (Maiellaro, 2016).

    This study focused on glutamatergic type Ib motor neurons, which are structurally regulated via G-protein-coupled receptors (GPCRs) for octopamine. Stimulation of these receptors has been shown to induce synaptic bouton outgrowth via a cAMP- and CREB-dependent pathway. Therefore, this study set out to measure spatiotemporal patterns of octopamine-induced cAMP signals in these neurons. To this end, a FRET (Forster resonance energy transfer)-based sensor for cAMP (Epac1-camps) was expressed that has previously been used to image cAMP levels in central Drosophila neurons. The results reveal that cAMP signals are confined to their initiation site, the individual synaptic bouton, and suggest a highly efficient local mechanism for controlling site-specific synaptic plasticity (Maiellaro, 2016).

    This study genetically expressed the cAMP sensor, Epac1-camps, at the Drosophila NMJ to monitor and quantify spatiotemporal cAMP dynamics induced by octopamine stimulation. The results reveal an unexpectedly high degree of cAMP compartmentalization in motor neurons, which may serve as a basis for local synaptic plasticity, with cAMP FRET signals being ultimately limited to single synaptic boutons. Three cAMP signaling compartments were identified within the motor neuron: boutons, axon, and cell body. For each cellular compartment, the particular mechanism responsible for the segregation of cAMP increases is described. Specifically, this study found that boutons constitute the most reactive compartment of the motor neuron in terms of cAMP accumulation. The results demonstrate that the production of cAMP is heterogeneous among boutons, with stronger responses to octopamine in large boutons rather than in smaller ones, possibly related to the increased synaptic strength measured at large boutons. Moreover, the activity and the specific localization of PDEs within the synaptic bouton prevent the propagation of cAMP to the cell body and its diffusion from one bouton to the next (Maiellaro, 2016).

    In contrast to the boutons, octopamine receptors seem to be absent or inaccessible in the axon, and PDEs were not detected. Accordingly, cAMP FRET signals recorded in the axon differ from those in boutons in terms of amplitude and kinetics. Hence, the axon emerges as the second independent, but not isolated, cAMP signaling compartment. Interestingly, and in contrast to dendrites, cAMP did not propagate along the axon. It remains to be clarified whether this may relate to functional differences between axons and dendrites, between species, or between specific neuron types. Finally, the cell body was determined as the third cAMP signaling compartment within the motor neuron. The cell body has a very low sensitivity toward octopamine, and it was demonstrated that cAMP FRET signals generated in the cell body are not affected by the cell body's physical isolation from the axon. High PDE activity in the cell body contributes to this local suppression of cAMP signaling and may prevent spillover activation of cAMP effectors in the cell body and in the nucleus (Maiellaro, 2016).

    Evidence of cAMP microdomains restricted to single boutons provides a biophysical basis for the local control of synaptic plasticity. Spatially constrained cAMP changes help to establish differences in morphology and synaptic content of boutons, suggesting that local cAMP, bouton structure, and synapse formation are intimately linked. The observed confinement of cAMP supports the notion that individual synaptic boutons may represent largely autonomous signaling units, which can receive and integrate signals independently of the other (Maiellaro, 2016).

    The concept that cAMP could act as a local messenger was postulated almost 40 years ago. The existence of cAMP microdomains has been demonstrated in other cell types (e.g., cardiomyocytes). However, in neurons, there are contradicting experimental lines of evidence and simulations concerning the existence of cAMP microdomains. The current data clearly show that cAMP can act as a local messenger upon physiological stimulation of a neuron. The detailed spatiotemporal analysis of the dynamics of cAMP reveals that this messenger can be restricted at the micrometer level to induce highly localized physiological responses (Maiellaro, 2016).

    Synaptic counts approximate synaptic contact area in Drosophila

    The pattern of synaptic connections among neurons defines the circuit structure, which constrains the computations that a circuit can perform. The strength of synaptic connections is costly to measure yet important for accurate circuit modeling. Synaptic surface area has been shown to correlate with synaptic strength, yet in the emerging field of connectomics, most studies rely instead on the counts of synaptic contacts between two neurons. This study quantified the relationship between synaptic count and synaptic area as measured from volume electron microscopy of the larval Drosophila central nervous system. The total synaptic surface area, summed across all synaptic contacts from one presynaptic neuron to a postsynaptic one, can be accurately predicted solely from the number of synaptic contacts, for a variety of neurotransmitters. These findings support the use of synaptic counts for approximating synaptic strength when modeling neural circuits (Barnes, 2021).

    Distinct molecular pathways govern presynaptic homeostatic plasticity

    Presynaptic homeostatic plasticity (PHP) stabilizes synaptic transmission by counteracting impaired neurotransmitter receptor function through neurotransmitter release potentiation. PHP is thought to be triggered by impaired receptor function and to involve a stereotypic signaling pathway. However, this study demonstrates that different receptor perturbations that similarly reduce synaptic transmission result in different responses at the Drosophila neuromuscular junction. While receptor inhibition by the glutamate receptor (GluR) antagonist γ-D-glutamylglycine (γDGG) is not compensated by PHP, the GluR inhibitors Philanthotoxin-433 (PhTx) and Gyki-53655 (Gyki) induce compensatory PHP. Intriguingly, PHP triggered by PhTx and Gyki involve separable signaling pathways, including inhibition of distinct GluR subtypes, differential modulation of the active-zone scaffold Bruchpilot, and short-term plasticity. Moreover, while PHP upon Gyki treatment does not require genes promoting PhTx-induced PHP, it involves presynaptic protein kinase D. Thus, synapses not only respond differentially to similar activity impairments, but achieve homeostatic compensation via distinct mechanisms, highlighting the diversity of homeostatic signaling (Nair, 2021).

    This study demonstrates that neurotransmitter receptor impairment by different GluR antagonists (PhTx and Gyki) induces PHP via distinct mechanisms, including differential inhibition of GluR subtypes, differential modulation of an active zone scaffold, and short-term plasticity during PHP. Importantly, genetic evidence is provided that separable molecular mechanisms promote PHP in response to Gyki and PhTx treatment. On the other hand, the GluR antagonist γDGG did not produce PHP, despite a robust inhibition of postsynaptic receptors. Together, the data suggest that distinct molecular mechanisms mediate PHP in response to GluR inhibition by different antagonists and that GluR inhibition per se is not sufficient for PHP expression (Nair, 2021).

    The results contrast with prevalent models of homeostatic synaptic plasticity, which rest on the assumption that synaptic activity changes induce homeostatic signaling. In the case of PHP, the magnitude of the homeostatic increase in presynaptic release scales with the decrease in the amplitude of postsynaptic miniature events. Based on these data, it was proposed that reduced ion flux through the receptors triggers a homeostatic signaling cascade in the postsynaptic cell, which is relayed to the presynaptic compartment where it adjusts release. A prediction that directly follows is that any receptor perturbation with similar effects on the amplitude of synaptic miniature events produces a homeostatic response via similar underlying mechanisms. This study observed that two GluR antagonists, PhTx and Gyki, induced PHP, while one antagonist, γDGG, did not. All three perturbations similarly decreased quantal size (q), indicating a similar reduction of ion flux through the receptors. Although it cannot be excluded that γDGG may have directly blocked PHP expression, the results suggest that ion flux is unlikely the sole signal responsible for PHP induction at the Drosophila NMJ. This agrees with recent observations at the Drosophila and mouse NMJ indicating that Ca2+ flux through the receptor is likely dispensable for PHP induction (Goel, 2017; Wang, 2018). How could pharmacological receptor perturbation induce PHP independent of ion flux through the receptor? One intriguing possibility is that perturbation-specific conformational changes of the receptor may be involved in PHP signaling. Conformational changes of kainate-, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)-, and N-Methyl-d-aspartate (NMDA)-type GluRs at mammalian synapses are known to signal independent of ion flux in the context of synaptic plasticity, possibly through metabotropic signaling. It is also known that different antagonists stabilize different conformational states of AMPA receptors. Thus, it is conceivable that distinct antagonists may trigger different PHP signaling pathways, depending on the different signaling partners that could be recruited by different conformational states. This hypothesis requires further investigation in relation to molecular signaling underlying PHP induction (Nair, 2021).

    Although PhTx and Gyki robustly induced PHP through an increase in RRP size, several differences were observed between PhTx- and Gyki-induced PHP. First, PhTx and Gyki differentially affected GluR subtypes. Whereas PhTx mainly inhibits GluRIIA-containing receptors, Gyki reduced mEPSP amplitude at either GluRIIA or GluRIIB mutant NMJs. Since mEPSPs at the Drosophila NMJs are mediated by GluRIIA- and GluRIIB-containing receptors, these results indicate that Gyki may act on both types of receptor complexes. An intriguing possibility to be tested in the future is that PhTx-dependent receptor inhibition may trigger PHP by affecting a signaling module associated with GluRIIA-containing receptor complexes, whereas Gyki-induced PHP may be triggered by mechanisms beyond GluRIIA-dependent signaling. Second, while Gyki similarly accelerated the decay of miniature excitatory postsynaptic currents (mEPSCs) and EPSCs, the EPSC decay was slower than that of mEPSCs upon PhTx application. The mismatch between the mEPSC and EPSC decay kinetics after PhTx treatment may either result from presynaptic changes, such as the recruitment of synaptic vesicles with a low pr during the EPSC decay phase, or different effects on GluR desensitization, saturation, or diffusion during evoked release. Regardless, as this mismatch was not observed after Gyki treatment, it either reflects a difference in antagonist action or in Gyki- and PhTx-induced PHP. Third, Gyki, but not PhTx, resulted in altered short-term plasticity, indicative of a decrease in pr or increased GluR desensitization and/or saturation. Fourth, while PhTx increased Brp-fluorescence intensity, indicating elevated Brp abundance, Gyki did not. Although it cannot be excluded that Brp modulation at a different time point with regard to Gyki application, this observation suggests differential regulation of this core active zone protein during PhTx- and Gyki-induced PHP. Fifth, this study revealed that Gyki application induced PHP in six mutants that were previously shown to block PHP upon PhTx treatment. Sixth, this study identified a gene (PKD) that is required for Gyki-induced PHP, but not PhTx-induced, PHP. Thus, although the possibility cannot be excluded of partially overlapping molecular mechanisms, these data demonstrate separable molecular pathways between Gyki- and PhTx-induced PHP (Nair, 2021).

    Diverging homeostatic signaling has been observed in the context of acute versus chronic receptor perturbations. At the Drosophila NMJ, mutants with intact acute PHP in response to PhTx application displayed impaired chronic PHP upon GluRIICRNAi expression. However, it is difficult to separate whether these differences emerge because of differences in the nature or the timescale of receptor perturbation. Since the curremt experiments focus on acute perturbations, the results support the idea that distinct homeostatic signaling pathways can be triggered depending on the specific way receptors have been perturbed. PhTx, γDGG, and Gyki inhibit AMPA receptors via different modes of antagonism. While PhTx is an activity-dependent pore blocker, γDGG acts as a low-affinity competitive antagonist and Gyki is an allosteric inhibitor. PhTx and γDGG inhibit Drosophila GluRs with characteristics similar to AMPA receptor inhibition. Similarly, the conserved Gyki-interacting residues between rat and Drosophila GluRs highlight the possibility that Gyki could also act similarly on Drosophila and mammalian GluRs. Thus, PhTx, γDGG, and Gyki may inhibit Drosophila GluRs via different modes of antagonism, which may be involved in triggering distinct homeostatic signaling pathways. Differential homeostatic signaling has also been observed in the context of firing rate homeostasis, where molecular responses depend on whether the protein or the conductance of a sodium channel protein is eliminated. Thus, distinct molecular signaling mechanisms that are specific to the nature, rather than the functional effects of a perturbation, may be a general theme in the context of neuronal homeostasis (Nair, 2021).

    This study also implicated presynaptic PKD signaling in synaptic homeostasis. Although PKD has been linked to various intracellular processes, such as vesicle sorting, endocytosis, and the regulation of the actin cytoskeleton, its synaptic function is less explored. Recently, PKD was associated with synaptic plasticity (Oueslati Morales, 2020). The current data clearly demonstrate that presynaptic PKD is required for Gyki-induced PHP, but not for PhTx-induced PHP and establish that separable molecular pathways govern Gyki- and PhTx-induced PHP. However, PKD was required for PHP only at low extracellular Ca2+ concentration, similar to a number of genes supporting PhTx-induced PHP. This indicates that PKD signaling may contribute to the robustness of PHP under conditions of low pr. It is worth highlighting that the PHP defect observed upon presynaptic PKD knockdown and in PKD hypomorphs may not be fully penetrant, likely because of the genetic nature of the perturbation, and/or genetic compensation by other members of the Protein Kinase C/Calmodulin-Dependent Kinase family. This genetic compensation is particularly pronounced in PKD null mutants, which precluded the analysis after complete loss of PKD function (Nair, 2021).

    Unlike PhTx, Gyki induced PHP in the fully dissected NMJ preparation, which is more amenable to electrophysiology and imaging approaches, and thus allowed probing the dynamics of PHP induction. It is currently unclear why PhTx-induced PHP cannot be observed in the full preparation. One hypothesis is that the muscles/synapses may be stretched in the full preparation, which could lead to a disruption of signaling domains relevant for PhTx-induced PHP. A second possibility could be the potential disruption of neuro-glial signaling that is important for PhTx-induced PHP in the full preparation (Wang, 2020). The fact that Gyki results in PHP after full dissection demonstrates that PHP induction is not limited to the 'semi-intact' preparation per se. It will be interesting to investigate whether different molecular pathways promoting PHP in response to Gyki and PhTx treatment may be differentially affected by the type of the preparation. In this regard, Gkyi induced PHP in Multiplexin (dmp) mutants, which were shown to disrupt PhTx-dependent PHP and neuro-glia signaling (Wang, 2020; Nair, 2021 and regerences therein).

    Gyki application to the fully dissected preparation revealed very rapid, low-latency PHP induction kinetics, which maintained EPSP amplitudes constant despite Gyki-induced mEPSP amplitude reduction. However, due to the sampling interval of 35 s, shorter latencies cannot be ruled out. In addition, the exact latency of PHP could not be estimated because of the relatively slow kinetics of mEPSP reduction in these experiments. The reversible GluR block by Gyki also revealed rapid, low-latency PHP reversal after Gyki washout, consistent with previous observations at mouse cerebellar mossy fiber boutons or at mouse NMJs. The results imply continuous, bidirectional PHP signaling that compensates for receptor impairment within ∼35 s, similar to observations at the mouse NMJ. It will be interesting to explore the molecular mechanisms underlying the induction of distinct PHP pathways and whether distinct molecular pathways also rapidly stabilize synaptic efficacy at other synapses (Nair, 2021).

    Although the results indicate that PHP signaling depends on specific receptor perturbations, the mechanisms of antagonism of the used antagonists have not been studied for Drosophila GluRs. Moreover, the lack of PHP upon γDGG treatment may result from off-target inhibition of PHP signaling cannot be excluded, nor that Gyki acts independent of GluRs to induce PHP. However, given Gyki's known mechanism of allosteric antagonism of mammalian AMPARs, and its potent reduction of mEPSP amplitudes at the Drosophila NMJ, this is considered unlikely. Furthermore, given the evolutionary conservation of Gyki-interacting residues in Drosophila GluR subunits, it is likely that Gyki acts on all Drosophila GluR subunits. However, the effect of Gyki on individual receptor subunits cannot be tested, because GluRIIC-E subunits are essential for receptor formation. Homozygous Drosophila lines harboring mutations in genes required for PhTx-induced PHP were used to test whether PhTx and Gyki activate different downstream signaling pathways. This analysis did not systematically investigate the effects of potential off-site mutations that may have accumulated over time in these fly lines. However, most strains were kept over a balancer chromosome, and Gyki-induced PHP proceeded normally in six mutants that were previously shown to disrupt PhTx-induced PHP, suggesting that a major contribution of off-target mutations is unlikely (Nair, 2021).

    Rapid homeostatic modulation of transsynaptic nanocolumn rings

    Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization and how synaptic nano-architecture is transsynaptically regulated to enable stable synaptic transmission remain enigmatic. Using time-gated stimulated emission-depletion microscopy at the Drosophila neuromuscular junction, it was found that presynaptic nanorings formed by the active-zone scaffold Bruchpilot (Brp) align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor approximately four transsynaptically aligned Brp-GluR nanocolumns. Similar nanocolumn rings are formed by the presynaptic protein Unc13A and GluRs. Intriguingly, acute GluR impairment triggers transsynaptic nanocolumn formation on the minute timescale during homeostatic plasticity. Distinct phases of structural transsynaptic homeostatic plasticity were revealed, with postsynaptic GluR reorganization preceding presynaptic Brp modulation. Finally, homeostatic control of transsynaptic nano-architecture and neurotransmitter release requires the auxiliary GluR subunit Neto. Thus, transsynaptic nanocolumn rings provide a substrate for rapid homeostatic stabilization of synaptic efficacy (Muttathukunnel, 2022).

    This study has identified a stereotypic arrangement of transsynaptically aligned molecular nanocolumns that is regulated in a modular and sequential fashion during homeostatic plasticity at the Drosophila NMJ. Moreover, a GluR subtype-specific nano-organization was revealed and it was discovered that the auxiliary GluR subunit Neto is required for rapid homeostatic modulation of transsynaptic nanocolumn number and neurotransmitter release (Muttathukunnel, 2022).

    Previous work demonstrated that a cluster of voltage-gated Ca2+ channels localizes to the Brp ring center at the Drosophila NMJ. Furthermore, Unc13A, a molecule suggested as a molecular correlate of presynaptic release sites, forms ring-like arrays in close proximity to Brp C termini and GluRs. In light of these findings, the results are consistent with a model in which Ca2+ influx at the Brp/AZ center induces neurotransmitter release in the nanocolumn rings. Given that the neurotransmitter content released by a single synaptic vesicle does not activate all GluRs of a given PSD at the Drosophila NMJ and that Drosophila GluRs have a low glutamate affinity, neurotransmitter release may predominantly activate GluRs that are aligned to presynaptic release sites. Some evidence suggests that synaptic transmission predominantly occurs within transsynaptic nanocolumns. Hence, the transsynaptic nanocolumn rings discovered in this study may reflect subsynaptic transmission modules that are activated by a common Ca2+-channel cluster. Future work is needed to relate the molecular nanocolumn topography to synaptic physiology, for example, by assessing how many GluRs are activated by neurotransmitter release from a single synaptic vesicle. In this regard, the slight offset between Unc13A and GluR rings may indicate that a given release site may not only activate a single aligned GluR cluster but also neighboring GluR clusters, consistent with physiology data (Muttathukunnel, 2022).

    GluR subunit composition and GluR location with regard to release sites are important factors determining synaptic efficacy. At the Drosophila NMJ, the ratio of slowly and rapidly desensitizing GluRIIA- and GluRIIB-containing receptors is a key regulator of quantal size. This study revealed that transsynaptic nanocolumns harbor a mix of GluRIIA- and GluRIIB-containing receptors, and that ambient receptors, which represent almost half of the GluRs within a PSD, mainly incorporate the GluRIIB subunit. The persistence of transsynaptic nanocolumn rings in GluRIIA and GluRIIB mutants implies that neither of these subunits alone is sufficient for ring formation or transsynaptic alignment. Previous work revealed no defects in spontaneous or AP-evoked synaptic transmission upon GluRIIA overexpression or after GluRIIB loss . Thus, two genetic manipulations that mainly decrease ambient receptor abundance, but not receptors inside the nanocolumn ring, do not induce a corresponding decrease in synaptic transmission. This indicates that synaptic transmission is largely confined to transsynaptic nanocolumn rings and/or that synaptic transmission outside the rings is dominated by rapidly desensitizing GluRIIB-containing receptors. Moreover, the observation of increased mEPSP amplitudes in GluRIIBSP5 mutants suggests that GluRIIB-containing receptors surrounding the nanocolumns have the potential to negatively regulate synaptic transmission by replacing GluRIIA-containing receptors within the nanocolumns (Muttathukunnel, 2022).

    A variety of auxiliary subunits control GluR assembly, trafficking, and function. The auxiliary GluR subunit Neto has been implicated in GluR clustering at the Drosophila NMJ (Kim, 2021). This uncovered modular ring arrays of Neto-β that transsynaptically align with Brp C termini, suggesting that this auxiliary GluR subunit is a postsynaptic element of transsynaptic nanocolumn rings. The persistence of transsynaptic nanocolumn rings in hypomorphic neto109 mutants suggests that neto is not crucial for ring formation or transsynaptic alignment, or that the remaining Neto was sufficient for transsynaptic nanocolumn ring formation. In contrast to neto109 mutants, in which both Neto-α and Neto-β levels are reduced, loss of Neto-α does not decrease GluR levels or mEPSP amplitude, suggesting that this Neto isoform either does not stabilize GluRs at the Drosophila NMJ or that there is a compensation by Neto-β. While reduced levels of ambient receptors do not impair synaptic transmission in case of GluRIIA overexpression or in GluRIIBSP5 mutants, the decreased GluR abundance within the rings of neto109 mutants correlates with a decrease in spontaneous and AP-evoked synaptic transmission, again implying that synaptic transmission predominantly occurs within the rings (Muttathukunnel, 2022).

    GluR impairment at the Drosophila NMJ induces a homeostatic increase in release, and there is evidence for the modulation of presynaptic nano-architecture during this form of homeostatic plasticity. A previous study reported increased GluR levels upon sustained pharmacological GluR inhibition for several days. This study demonstrates GluR modulation within 5 min after pharmacological GluR impairment that precedes the modulation of Brp, as well as Neto-β. Although it cannot be excluded that other molecules are modulated prior to GluRs, or that small changes in Brp or Neto-&beta could not be resolved; after PhTX treatment for 5 min, the data imply that GluR modulation precedes Neto-β and presynaptic regulation during homeostatic plasticity. Furthermore, GluR and Brp fluorescence intensity changes detected with confocal microscopy preceded the increase in GluR and Brp cluster numbers at STED resolution. This could either indicate that small nanostructural changes could not be detected with STED microscopy or that the modulation of transsynaptic nano-architecture lags behind the regulation of GluR and Brp levels or distribution. Similar to the data obtained with confocal microscopy, the increase in GluR cluster number preceded Brp cluster regulation upon GluR perturbation, again indicative of a temporal sequence of transsynaptic changes during PHP. Interestingly, while GluR, but not Brp cluster number increased 15 min after PhTX treatment, a larger fraction of transsynaptically aligned Brp clusters was noted. This suggests that transsynaptic nanocolumn formation likely precedes Brp cluster formation. The temporal sequence of GluR and Brp regulation may also explain the existence of GluR clusters within the ring that are not opposed by Brp. Together, these findings are consistent with a model of coordinated, transsynaptic, and modular structural plasticity during PHP that results in the addition of transsynaptic nanocolumns to the ring (Muttathukunnel, 2022).

    Apparent changes in GluR fluorescence intensity, GluR cluster number, or homeostatic potentiation of release upon pharmacological GluR perturbation in hypomorphic neto109 mutants were not observed. This shows that wild-type Neto levels are required for homeostatic control of GluRs and presynaptic release. GluR inhibition also led to a slight but significant increase in Brp fluorescence intensity in neto109 mutants, which was less pronounced than in wild type. The defect in PHP seen in neto109 mutants could thus arise from impaired GluR and/or Brp regulation. Although the genetic data establish a causal relationship between the homeostatic regulation of transsynaptic nanocolumns and presynaptic release, future work is required to scrutinize the relationship between transsynaptic nano-architecture and synaptic transmission, and to dissect the molecular mechanisms controlling transsynaptic nano-architecture and its homeostatic regulation. In this regard, it will be exciting to explore which molecules are involved in transsynaptic alignment and ring formation. Synaptic cell-adhesion molecules, such as neurexins and neuroligins, represent obvious candidates (Muttathukunnel, 2022).

    Vav independently regulates synaptic growth and plasticity through distinct actin-based processes

    Modulation of presynaptic actin dynamics is fundamental to synaptic growth and functional plasticity; yet the underlying molecular and cellular mechanisms remain largely unknown. At Drosophila NMJs, the presynaptic Rac1-SCAR pathway mediates BMP-induced receptor macropinocytosis to inhibit BMP growth signaling. This study shows that the Rho-type GEF Vav acts upstream of Rac1 to inhibit synaptic growth through macropinocytosis. Evidence is presented that Vav-Rac1-SCAR signaling has additional roles in tetanus-induced synaptic plasticity. Presynaptic inactivation of Vav signaling pathway components, but not regulators of macropinocytosis, impairs post-tetanic potentiation (PTP) and enhances synaptic depression depending on external Ca2+ concentration. Interfering with the Vav-Rac1-SCAR pathway also impairs mobilization of reserve pool (RP) vesicles required for tetanus-induced synaptic plasticity. Finally, treatment with an F-actin-stabilizing drug completely restores RP mobilization and plasticity defects in Vav mutants. It is proposed that actin-regulatory Vav-Rac1-SCAR signaling independently regulates structural and functional presynaptic plasticity by driving macropinocytosis and RP mobilization, respectively (Park, 2022).

    Loss of Activity-Induced Mitochondrial ATP Production Underlies the Synaptic Defects in a Drosophila Model of ALS

    Mutations in the gene encoding vesicle-associated membrane protein B (VAPB) cause a familial form of amyotrophic lateral sclerosis (ALS). Expression of an ALS-related variant of vapb (vapbP58S) in Drosophila motor neurons results in morphologic changes at the larval neuromuscular junction (NMJ) characterized by the appearance of fewer, but larger, presynaptic boutons. Although diminished microtubule stability is known to underlie these morphologic changes, a mechanism for the loss of presynaptic microtubules has been lacking. By studying flies of both sexes, this study demonstrate the suppression of vapbP58S) -induced changes in NMJ morphology by either a loss of endoplasmic reticulum (ER) Ca(2+) release channels or the inhibition Ca(2+)/calmodulin (CaM)-activated kinase II (CaMKII). These data suggest that decreased stability of presynaptic microtubules at vapbP58S NMJs results from hyperactivation of CaMKII because of elevated cytosolic [Ca(2+)]. The Ca(2+) dyshomeostasis is attributed to delayed extrusion of cytosolic Ca(2+) Suggesting that this defect in Ca(2+) extrusion arose from an insufficient response to the bioenergetic demand of neural activity, depolarization-induced mitochondrial ATP production was diminished in vapbP58S neurons. These findings point to bioenergetic dysfunction as a potential cause for the synaptic defects in vapbP58S -expressing motor neurons (Karagas, 2022).

    Influence of T-Bar on Calcium Concentration Impacting Release Probability

    The relation of form and function, namely the impact of the synaptic anatomy on calcium dynamics in the presynaptic bouton, is a major challenge of present (computational) neuroscience at a cellular level. The Drosophila larval neuromuscular junction (NMJ) is a simple model system, which allows studying basic effects in a rather simple way. This synapse harbors several special structures. In particular, in opposite to standard vertebrate synapses, the presynaptic boutons are rather large, and they have several presynaptic zones. In these zones, different types of anatomical structures are present. Some of the zones bear a so-called T-bar, a particular anatomical structure. The geometric form of the T-bar resembles the shape of the letter 'T' or a table with one leg. When an action potential arises, calcium influx is triggered. The probability of vesicle docking and neurotransmitter release is superlinearly proportional to the concentration of calcium close to the vesicular release site. It is tempting to assume that the T-bar causes some sort of calcium accumulation and hence triggers a higher release probability and thus enhances neurotransmitter exocytosis. In order to study this influence in a quantitative manner, a typical T-bar geometry was constructed and the calcium concentration close to active zones (AZs) with and without T-bars was compared. Indeed, a substantial influence was found of the T-bar structure on the presynaptic calcium concentrations close to the AZs, indicating that this anatomical structure increases vesicle release probability. Therefore, this study reveals how the T-bar zone implies a strong relation between form and function. This study answers the question of experimental studies concerning the sense of the anatomical structure of the T-bar (Knodel, 2022).

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

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

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

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

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

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

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

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

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

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

    The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity

    Synaptic vesicle release probability (P(r)) is a key presynaptic determinant of synaptic strength established by cell intrinsic properties and further refined by plasticity. To characterize mechanisms that generate P(r) heterogeneity between distinct neuronal populations, this study examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in P(r) and synaptic plasticity. The decoy SNARE Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing P(r) to generate decreased levels of synaptic vesicle fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic P(r) at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation (PHP) (Sauvola, 2021).

    Ca2+-dependent fusion of synaptic vesicles (SVs) is the primary mechanism for neurotransmission and is mediated by the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family. Following an action potential, SNARE proteins located on the SV and plasma membrane zipper into an energetically favorable coiled-coil bundle to induce SV fusion. Neurotransmitter release results in a postsynaptic response that varies in size depending on the strength of the synapse, which can be regulated from both pre-and post-synaptic compartments. The postsynaptic cell controls sensitivity to neurotransmitters by governing receptor field composition, while the presynaptic neuron establishes the probability (Pr) of SV fusion. Highly stereotyped differences in Pr exist across neurons, with many neuronal populations broadly classified as tonic or phasic depending on their spiking patterns, Pr and short-term plasticity characteristics. How cell-intrinsic properties establish differences in presynaptic Pr between neuronal classes, and how release strength is further refined via plasticity, remain incompletely understood (Sauvola, 2021).

    The Drosophila melanogaster larval neuromuscular junction (NMJ) provides a robust genetic system for characterizing mechanisms mediating synaptic communication and tonic versus phasic release properties. Larval body wall muscles are co-innervated by two glutamatergic motoneuron populations that drive locomotion, including the tonic-like Ib and phasic-like Is subtypes. Tonic Ib terminals display lower initial Pr and sustained release during stimulation, whereas phasic Is terminals show higher intrinsic Pr and rapid depression (Lu, 2016; Newman, 2017). The Drosophila NMJ also undergoes robust presynaptic homeostatic potentiation (PHP) that rapidly increases Pr to compensate for disruptions to postsynaptic glutamate receptor (GluR) function. In addition to intrinsic release differences, the Ib and Is subtypes display distinct capacity for PHP. How tonic and phasic neurons differentially regulate Pr during normal synaptic communication and plasticity is largely unknown (Sauvola, 2021).

    The highly conserved SNARE regulatory protein Tomosyn negatively controls SV release and has been proposed to participate in synaptic plasticity. Tomosyn has an N-terminal WD40 repeat domain and a C-terminal SNARE motif with homology to the SV v-SNARE Synaptobrevin 2 (Syb2). Tomosyn inhibits presynaptic release by binding the t-SNAREs Syntaxin1 (Syx1) and SNAP-25 to prevent Syb2 incorporation into the SNARE complex fusion machinery (Sauvola, 2021).

    To further examine the role of Tomosyn in synaptic transmission and plasticity, CRISPR was used to generate mutations in the sole Drosophila tomosyn gene. Structure-function analysis revealed the SNARE domain is critical for release inhibition, while the scaffold region promotes enrichment of Tomosyn to SV-rich sites. Despite enhanced evoked release, tomosyn mutants fail to maintain high levels of SV output during sustained stimulation due to rapid depletion of the immediately releasable SV pool. Tomosyn is highly enriched at Ib synapses and generates tonic neurotransmission properties characterized by low Pr and sustained release in this population of motoneurons. Indeed, optogenetic stimulation and optical quantal analysis demonstrate an exclusive role for Tomosyn in regulating intrinsic release strength in tonic motoneurons. PHP expression primarily occurs at tonic synapses and is abolished in tomosyn mutants, suggesting Tomosyn is also essential for acute PHP expression. Together, these data indicate Tomosyn mediates the tonic properties of Ib motoneurons by suppressing Pr to slow the rate of SV usage, while decreasing Tomosyn suppression enables Pr enhancement during PHP. Conversely, the absence of Tomosyn in Is motoneurons facilitates phasic release properties by enabling an intrinsically high Pr that quickly depletes the releasable SV pool, resulting in rapid synaptic depression and reduced capacity for PHP (Sauvola, 2021).

    The findings reported in this study indicate the conserved presynaptic release suppressor Tomosyn functions in setting presynaptic output and plasticity differences for a tonic/phasic pair of motoneurons that co-innervate Drosophila larval muscles. CRISPR-generated mutations in Drosophila tomosyn revealed synchronous, asynchronous and spontaneous SV release are all elevated in the absence of the protein. While single evoked responses were enhanced, rapid depression of release was observed during train stimulation, suggesting loss of Tomosyn biases synapses toward a more phasic pattern of SV release. To directly test whether Tomosyn plays a unique role in tonic synapses, Ib and Is motoneurons were separately stimulated using optogenetics to measure their isolated contributions. These experiments revealed a 4-fold increase in output from Ib neurons with no change to Is release. Optical quantal analysis confirmed the Ib specific effect of Tomosyn and demonstrated enhanced evoked responses in tomosyn is due to higher intrinsic Pr across the entire AZ population. Endogenously-tagged Tomosyn was more abundant at Ib synapses than Is, consistent with Tomosyn's role in regulating Ib release. Together, these data indicate the intrinsically high Pr and rapid depression normally found in Is motoneurons is due in part to a lack of Tomosyn inhibition of SV usage at phasic synapses. High-frequency stimulation experiments demonstrate Tomosyn does not regulate the size of the immediately releasable SV pool (IRP) but rather regulates IRP usage to ensure sustained availability of SVs during prolonged stimulation, as the IRP is strongly biased towards early release in tomosyn mutants. A model is proposed where Drosophila synapses are more phasic in release character by default, with tonic release requiring higher levels of Tomosyn to generate a fusion bottleneck that enables extended periods of stable release by slowing the rate of SV usage (Sauvola, 2021).

    How Tomosyn normally suppresses SV release has been unclear. The most widely hypothesized mechanism is that Tomosyn competes with Syb2 for binding t-SNAREs. By forming fusion-incompetent SNARE complexes that must be disassembled by NSF, a pool of t-SNAREs is kept in reserve and can be mobilized by alleviating Tomosyn inhibition. Indeed, enhanced SNARE complex formation was found in Drosophila tomosyn mutants, consistent with the model that Tomosyn's SNARE domain acts as a decoy SNARE to inhibit productive SNARE complex assembly. Expression of the Tomosyn scaffold alone failed to rescue the null phenotype, while overexpression of the scaffold had no effect on evoked release. As such, these data indicate that while the scaffold is required for full Tomosyn function, it does not directly inhibit fusion. These observations are consistent with the mechanism proposed in C. elegans, but differ from studies in cultured mammalian cells suggesting the scaffold acts as an independent release suppressor by inhibiting Syt1. Characterization of Drosophila tomosyn/syt1 double mutants demonstrated Tomosyn suppresses release independent of Syt1, arguing the scaffold must serve a function that enhances the inhibitory activity of the SNARE domain independent of Syt1. Indeed, this study found the Tomosyn SNARE motif was mislocalized without the WD40 scaffold, arguing this region indirectly supports Tomosyn's inhibitory activity by ensuring proper localization so the SNARE domain can compete for t-SNARE binding. Similar to studies in C. elegans and mammals, this study found Drosophila Tomosyn co-localized with other SV proteins. Human Tomosyn transgenes also rescued elevated evoked and spontaneous release in tomosyn mutants, indicating functional conservation of its inhibitory properties. Overexpression of either Drosophila or human Tomosyn in a wildtype background also decreased release, demonstrating presynaptic output can be bi-directionally controlled by varying Tomosyn expression levels (Sauvola, 2021).

    In addition to intrinsic release differences between tonic and phasic motoneurons, this study found Tomosyn also controls presynaptic homeostatic potentiation (PHP). This form of synaptic plasticity occurs when presynaptic motoneurons upregulate Pr and quantal content to compensate for decreased GluR function and smaller quantal size. Inducing PHP with the allosteric GluR inhibitor Gyki revealed Tomosyn is required for expression of this form of acute PHP at Ib terminals. Removing Tomosyn inhibition at Ib synapses generates a ~ 4 fold enhancement in evoked release, more than sufficient to compensate for a twofold reduction in evoked response size from two equally contributing motoneurons. Indeed, AZ Pr mapping revealed Ib synapses potentiate in the presence of Gyki while Is terminals showed no change, indicating enhanced release from Ib is sufficient to homeostatically compensate for Gyki-induced decreases in quantal size. Although future studies will be required to determine the molecular cascade through which Tomosyn mediates PHP expression, prior work indicates PKA phosphorylation of Tomosyn reduces its SNARE binding properties and decreases its inhibition of SV release. Given Gyki-induced PHP expression requires presynaptic PKD (Nair, 2020), an attractive hypothesis is that PKD phosphorylates Tomosyn and reduces its ability to inhibit SNARE complex formation. Similar to tomosyn mutants, this could promote SV availability by generating a larger pool of free t-SNAREs to support enhanced docking of SVs at AZs. Increased docking would elevate single AZ Pr by increasing the number of fusion-ready SVs upon Ca2+ influx, similar to the effect observed with quantal imaging (Sauvola, 2021).

    Despite the importance of Tomosyn in regulating release character between tonic and phasic motoneurons, tomosyn null mutants are viable into adulthood. As such, the entire range of Tomosyn expression can be used by distinct neuronal populations in vivo to set presynaptic output. Tonic Ib terminals shift towards phasic release with no effect on Is output in tomosyn null mutants, resulting in a collapse of presynaptic release diversity between these two neuronal subgroups. Like tomosyn, null mutants in syt7 are viable and show dramatically enhanced evoked release. Tomosyn/syt7 double mutants show even greater increases in release output, arguing multiple non-essential presynaptic proteins can independently fine tune synaptic strength within the presynaptic terminal. Together, these experiments demonstrate Tomosyn is a highly conserved release inhibitor that varies in expression between distinct neuronal subtypes to regulate intrinsic Pr and plasticity, providing a robust mechanism to generate presynaptic diversity across the nervous system (Sauvola, 2021).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Dual separable feedback systems govern firing rate homeostasis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Presynaptic biogenesis requires axonal transport of lysosome-related vesicles

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

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

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

    Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    A Presynaptic ENaC Channel Drives Homeostatic Plasticity

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Structural and molecular properties of insect type II motor axon terminals

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

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

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

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

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

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

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

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

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

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

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

    GTPase-activating protein TBC1D5 coordinates with retromer to constrain synaptic growth by inhibiting Bone Morphogenetic Protein signaling

    Formation and plasticity of neural circuits rely on precise regulation of synaptic growth. At Drosophila neuromuscular junction (NMJ) Bone Morphogenetic Protein (BMP) signaling is critical for many aspects of synapse formation and function. The evolutionarily-conserved retromer complex and its associated GTPase-activating protein TBC1D5 are critical regulators of membrane trafficking and cellular signaling. However, their functions in regulating the formation of NMJ are less understood. This study reports that TBC1D5 is required for inhibition of synaptic growth, and loss of TBC1D5 leads to abnormal presynaptic terminal development, including excessive satellite boutons and branch formation. Ultrastructure analysis reveals that the size of synaptic vesicles and the density of subsynaptic reticulum are increased in TBC1D5 mutant boutons. Disruption of interactions of TBC1D5 with Rab7 and retromer phenocopies the loss of TBC1D5. Unexpectedly, this study found that TBC1D5 is functionally linked to Rab6, in addition to Rab7, to regulate synaptic growth. Mechanistically, this study showed that loss of TBC1D5 leads to upregulated BMP signaling by increasing the protein level of BMP type II receptor Wit at NMJ. Overall, these data establish that TBC1D5 in coordination with retromer constrains synaptic growth by regulating Rab7 activity, which negatively regulates BMP signaling through inhibiting Wit level (Zhou, 2022).

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

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

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

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

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

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

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

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

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

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

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

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

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

    Postsynaptic cAMP signalling regulates the antagonistic balance of Drosophila glutamate receptor subtypes

    The balance among different subtypes of glutamate receptors (GluRs) is crucial for synaptic function and plasticity at excitatory synapses. This study shows that the two subtypes of GluRs (A and B) expressed at Drosophila neuromuscular junction synapses mutually antagonize each other in terms of their relative synaptic levels and affect subsynaptic localization of each other. Upon temperature shift-induced neuromuscular junction plasticity, GluR subtype A increased but subtypeB decreased with a timecourse of hours. Inhibition of the activity of GluR subtype A led to imbalance of GluR subtypes towards more GluRIIA. To gain a better understanding of the signalling pathways underlying the balance of GluR subtypes, an RNA interference screen of candidate genes was performed and postsynaptic-specific knockdown of dunce, which encodes cAMP phosphodiesterase, was found to increase levels of GluR subtype A but decreased subtype B. Furthermore, bidirectional alterations of postsynaptic cAMP signalling resulted in the same antagonistic regulation of the two GluR subtypes. These findings thus identify a direct role of postsynaptic cAMP signalling in control of the plasticity-related balance of GluRs (Zhao, 2020).

    A negative correlation between subtype A and B receptors has been reported previously at Drosophila NMJs. However, the mechanism by which the antagonistic balance of different subtypes of GluRs is regulated remains unclear. The present study revealed that bidirectional alterations of cAMP levels in the postsynaptic muscle cells alter the balance of GluR subtypes in a cell-autonomous manner. This study thus provides new insights into the mechanism underlying synaptic plasticity by altering the balance of GluR subtypes (Zhao, 2020).

    Most previous conventional microscopy studies have reported substantial colocalization or differential localization of GluRIIA and GluRIIB. This study reports an apparently non-overlapping localization of GluRIIA and GluRIIB at the postsynaptic densities (PSDs) of NMJ synapses (see The subtype B forms a doughnut-shaped ring, with a smaller subtype A ring in the centre). Although clear interpretations are lacking for the distinct localization of GluRIIA and GluRIIB at PSDs, there could be two possibilities: either different classes of receptors might be associated with specific interacting proteins that could mediate, directly or indirectly, the concentric localization of GluR subtypes at PSDs or concentric rings of GluR subtypes A and B in wild-type larvae might associate with their specific biophysical properties. Desensitization is the process by which receptors are inactivated in the prolonged presence of an agonist; it occurs faster in response to a lower concentration of agonist. On the postsynaptic side, GluR subtype A exhibits slower desensitization kinetics than GluR subtype B. It is therefore speculated that the slower desensitization of subtype A receptors might be caused, in part, by a higher concentration of glutamate released on the presynaptic side, because subtype A rings are more closely juxtaposed to presynaptic Cacophony calcium channels than subtype B rings (Zhao, 2020).

    This study showed that subtype A rings become enlarged (both the inner and outer ring diameters are increased) when the synaptic levels of GluRIIA are increased, whereas subtype B rings are enlarged in a specific manner, i.e., the inner diameter decreases, but the outer diameter of the ring increases when the level of GluRIIB is increased. A simple explanation for the enlarged GluR rings might therefore be increased synaptic levels of GluRIIA or GluRIIB. Given that GluR-enriched PSDs are confined to specific spatial domains by cell adhesion molecules and the spectrin-actin network, it is possible that an increase in the level of one subtype of GluR might take up the space left by a reduced level of the other (Zhao, 2020).

    Synaptic plasticity is the ability of synapses to strengthen or weaken over time, in response to increases or decreases in their activity. It is well established that GluRs are involved in synaptic plasticity at excitatory synapses. However, it is not entirely known how different types or subtypes of GluRs are involved in synaptic plasticity. Drosophila glutamatergic NMJs with two subtypes of GluRs, rather than mammalian NMJs with multiple subtypes of GluRs, are an effective model for studying synaptic plasticity. Hyperexcitable double mutants of eag sh show persistent strengthening of larval NMJs, which represents long-term plasticity. This study found that increased presynaptic release by warm-activated TrpA1 led to increased GluRIIA but normal GluRIIB, which was consistent with increased GluRIIA in eag sh double mutants. In addition, increased GluRIIA but decreased GluRIIB were observed in high temperature-induced synaptic plasticity of long-term strengthening of neurotransmission. Thus, it is speculated that increased presynaptic release might result in long-term plasticity by enhancing postsynaptic responses through increased GluRIIA (Zhao, 2020).

    Increased GluRIIA is consistently observed in different models of synaptic plasticity. However, this study observed normal and reduced GluRIIB in TrpA1- and high temperature-induced synaptic plasticity, respectively. The discrepant changes in GluRIIB levels in different models of synaptic plasticity could be caused by different timescales, such as a limited time (8 h) for elevating presynaptic release through activating TRPA1 versus 4 days for raising larvae at 27°C, or the change in the level of GluRIIB might be too low to be detected by overexpressing TRPA1, or both (Zhao, 2020).

    Whether the antagonistic balance of GluRs is actively (as a functional requirement) or passively (as a physical competition) regulated depends on specific conditions. It appeared that GluRIIA and GluRIIB competed with each other for the essential subunits when the expression levels of either GluRIIA or GluRIIB were changed, consistent with previous reports. These results support a passive competition between GluRIIA and GluRIIB. However, an actively regulated antagonistic balance of GluRs also occurs. When the essential subunit GluRIIC, GluRIID or GluRIIE was limited, both GluRIIA and GluRIIB decreased. If only passive regulation of GluRIIA and GluRIIB occurs, GluR subtype A and B decreased at similar levels would be expected. However, the ratio of GluRIIA to GluRIIB increased, indicating that the GluRIIA subtype is maintained preferentially when the total GluRs are limited and supporting an active regulation of the balance between GluRIIA and GluRIIB. Given that GluRIIA is mainly responsible for the postsynaptic responses, the relative increase in GluRIIA when an essential subunit of GluRs was knocked down might be a functional compensation for the decrease of synaptic strength (Zhao, 2020).

    In addition to the antagonism of GluRIIA and GluRIIB reported in this study, there are a few reports on the regulation of synaptic levels of single GluR subunits. For example, GluRIIA but not GluRIIB receptors are anchored at the PSD by the actin-associated Coracle (Chen, 2005) and are regulated by a signalling pathway involving the Rho-type GEF (Pix) and its effector, Pak kinase (Albin, 2004). Recent studies also showed specific upregulation of GluRIIA but not GluRIIB when the calcium-dependent proteinase calpains were mutated (Zhao, 2020).

    A previous study showed that the numbers of terminal varicosities and branches were increased in dnc but not rut mutants. Given that elevated cAMP levels induced an antagonistic balance of GluRs at the postsynaptic side, it was important to test whether the antagonistic balance of GluRs was associated with NMJ overgrowth. The results showed that the number of varicosities remained normal when dnc or rut was knocked down by RNAi in the postsynaptic muscles, suggesting that an alteration in the cAMP pathway at the postsynaptic side did not affect NMJ development (Zhao, 2020).

    The importance of the GluR subtype balance in synaptic plasticity has been documented in mammals. The major forms of AMPA receptors in the hippocampus include GluA1/2 and GluA2/3 heteromers, in addition to GluA1 homomers. The relative abundance of GluA1- and GluA2-containing receptors is a well-established determinant of synaptic plasticity in diverse brain circuits; GluA1-containing receptors are recruited to synapses after long-term potentiation, whereas GluA2-containing receptors are required for long-term depression. Together with the mammalian findings, the results support the notion that the GluR subtype balance contributes to synaptic plasticity at excitatory synapses (Zhao, 2020).

    It is widely known that cAMP signalling plays an important role in regulating synaptic plasticity by increasing presynaptic neurotransmitter release. However, it is not known whether the cAMP pathway acts postsynaptically in regulating the ratio of GluRs, which plays a crucial role in synaptic plasticity. The present study showed, for the first time, that the cAMP pathway regulates the balance of different GluR subtypes on the postsynaptic side; either increased or reduced cAMP leads to an altered ratio of GluR subtypes at Drosophila NMJ synapses. Thus, an optimal level of cAMP in postsynaptic muscles might be required for the normal ratio of synaptic GluR subtypes (Zhao, 2020).

    When cAMP levels are elevated, cAMP binds to the regulatory subunits of PKA and liberates catalytic subunits that then become active. Active PKA in muscles decreases the activity of GluRIIA in Drosophila (Davis, 1998). Thus, an increase in the level of synaptic GluRIIA might compensate for the reduced activity of GluRIIA caused by overexpression of wild-type or constitutively active PKA. Conversely, inhibition of PKA activity in muscles causes a significant increase in the average amplitude of miniature excitatory junctional currents, consistent with the notion that PKA negatively regulates the activity of GluRIIA. Surprisingly, an increase was observed in synaptic GluRIIA when the cAMP level was downregulated in rut mutants or when PKA was knocked down by RNAi. It appears that the negative regulation of GluRIIA activity by PKA is not sufficient to account for the increase of GluRIIA at NMJ synapses (Zhao, 2020).

    Analysis of western blots showed that the protein level of GluRIIA increased significantly, regardless of whether postsynaptic cAMP pathway was up- (dnc RNAi and PKAOE) or downregulated (rut RNAi and PKA RNAi), suggesting that the similar antagonistic balance of GluR subtypes induced by both up- and downregulation of cAMP might be caused by an elevated protein level of GluRIIA the cAMP pathway regulates the antagonism between GluRIIA and GluRIIB at two distinct steps, GluRIIA activity and protein level. Exactly how bidirectional changes of cAMP lead to a similar alteration of GluR subtypes remains to be investigated (Zhao, 2020).

    Although Dnc and Rut regulate cAMP levels in opposite directions, physiological studies in Drosophila have shown that activity-dependent short-term plasticity is altered in a similar manner at larval NMJs in both dnc and rut mutants. Specifically, synaptic facilitation and post-tetanic potentiation are both weakened, indicating that the bidirectional change of cAMP signalling might result in similar abnormalities in synapse plasticityn. The mechanisms underlying synaptic facilitation and post-tetanic potentiation are exclusively presynaptic. Synaptic facilitation and post-tetanic potentiation both result from increased presynaptic calcium concentrations, leading to an enhanced release of neurotransmitters. A bell-shaped model was proposed to explain this mode of regulation, i.e. mutations in dnc and rut, which regulate cAMP levels in opposite directions, result in a similar plasticity phenotype. It is proposed that the bell-shaped model might also explain a similar increase in GluRIIA at NMJ synapses caused by bidirectional changes in cAMP levels in postsynaptic muscles (Zhao, 2020).

    The antagonistic balance of GluRIIA and GluRIIB is induced by the postsynaptic cAMP/PKA pathway. However, whether the antagonism between GluRIIA and GluRIIB requires the cAMP/PKA pathway is unclear. GluRIIA or GluRIIB nulls were recombined with postsynaptic RNAi knockdown of PKA (i.e. inhibition of the cAMP pathway). It is noted that PKA null mutants are lethal at the first larval stage and thus cannot be used for the genetic interaction assay. Compared with simple null mutants of GluRIIA (or GluRIIB), PKA RNAi in the mutant background of GluRIIA (or GluRIIB) did not change the synaptic levels of GluRIIB (or GluRIIA), suggesting that the antagonistic balance of GluRIIA and GluRIIB does not require the cAMP pathway at the postsynaptic side. Thus, an altered cAMP pathway leads to the antagonistic balance of GluRIIA and GluRIIB, but the antagonistic balance of GluRIIA and GluRIIB appears not to be dependent on the cAMP pathway, at least for the antagonism induced by null mutations of GluRIIA or GluRIIB, or the remaining PKA upon RNAi knockdown is sufficient to support the antagonistic balance of GluRIIA and GluRIIB (Zhao, 2020).

    It will be of great interest to determine how the cAMP-PKA-GluR signalling pathway acts on the postsynaptic side to contribute to synaptic plasticity and whether this pathway is also effective and conserved in mammalian central synapses (Zhao, 2020).

    A Novel Neuron-Specific Regulator of the V-ATPase in Drosophila

    The V-ATPase is a highly conserved enzymatic complex that ensures appropriate levels of organelle acidification in virtually all eukaryotic cells. While the general mechanisms of this proton pump have been well studied, little is known about the specific regulations of neuronal V-ATPase. This study characterized CG31030, a previously uncharacterized Drosophila protein predicted from its sequence homology to be part of the V-ATPase family. In contrast to its ortholog ATP6AP1/VhaAC45 which is ubiquitous, it was observed that CG31030 expression is apparently restricted to all neurons, and using CRISPR/Cas9-mediated gene tagging, that it is mainly addressed to synaptic terminals. CG31030 is essential for fly survival and this protein co-immunoprecipitates with identified V-ATPase subunits, and in particular ATP6AP2. Using a genetically-encoded pH probe (VMAT-pHluorin) and electrophysiological recordings at the larval neuromuscular junction, it showed that CG31030 knock-down induces a major defect in synaptic vesicle acidification and a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single synaptic vesicle. These defects were associated with severe locomotor impairments. Overall, these data indicate that CG31030, which was renamed VhaAC45-related protein (VhaAC45RP), is a specific regulator of neuronal V-ATPase in Drosophila that is required for proper synaptic vesicle acidification and neurotransmitter release (Dulac, 2021).

    AP2 Regulates Thickveins Trafficking to Attenuate NMJ Growth Signaling in Drosophila

    Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. This study shows that α2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila. Ultrastructural analysis of α2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. Mutations in α2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the α2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of α2-adaptin mutations. A model is proposed in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth (Choudhury, 2022).

    The effects of doxapram (blocker of K2p channels) on resting membrane potential and synaptic transmission at the Drosophila neuromuscular junction

    The resting membrane potential of most cells is maintained by potassium K2p channels. The pharmacological profile and distribution of various K2p channel subtypes in organisms are still being investigated. The Drosophila genome contains 11 subtypes; however, their function and expression profiles have not yet been determined. Doxapram is clinically used to enhance respiration in humans and blocks the acid-sensitive K2p TASK subtype in mammals. The resting membrane potential of larval Drosophila muscle and synaptic transmission at the neuromuscular junction are pH sensitive. The present study investigated the effects of doxapram on membrane potential and synaptic transmission using intracellular recordings of larval Drosophila muscles. Doxapram (1 mM and 10 mM) depolarizes the muscle and appears to depolarize motor neurons, causing an increase in the frequency of spontaneous quantal events and evoked excitatory junction potentials. Verapamil (1 and 10 mM) paralleled the action of doxapram. These changes were matched by an extracellular increase in KCl (50 mM) and blocked by Cd(2+). It is assumed that the motor nerve depolarizes to open voltage-gated Ca(2+) channels in presynaptic nerve terminals because of exposure to doxapram. These findings are significant for building models to better understand the function of pharmacological agents that affect K2p channels and how K2p channels contribute to the physiology of tissues. Drosophila offers a genetically amenable model that can alter the tissue-specific expression of K2p channel subtypes to simulate known human diseases related to this family of channels (Vacassenno, 2023).

    RNA-binding FMRP and Staufen sequentially regulate the coracle scaffold to control synaptic glutamate receptor and bouton development

    Both mRNA-binding Fragile X Mental Retardation Protein (FMRP) and mRNA-binding Staufen regulate synaptic bouton formation and glutamate receptor (GluR) levels at the Drosophila neuromuscular junction (NMJ) glutamatergic synapse. This study tested whether these RNA-binding proteins (RBPs) act jointly in a common mechanism. Both dfmr1 and staufen mutants, and trans-heterozygous double mutants, were shown to display increased synaptic bouton formation and GluRIIA accumulation. With cell-targeted RNAi, a downstream Staufen role within postsynaptic muscle. With immunoprecipitation, this study showed that FMRP binds staufen mRNA to stabilize postsynaptic transcripts. Staufen is known to target actin-binding, GluRIIA anchor Coracle, and this study confirmed that Staufen binds to coracle mRNA. FMRP and Staufen were shown to act sequentially to co-regulate postsynaptic Coracle expression, and show Coracle, in turn, controls GluRIIA levels and synaptic bouton development. Consistently, this study found dfmr1, staufen and coracle mutants elevate neurotransmission strength. FMRP, Staufen and Coracle all suppress pMad activation, providing a trans-synaptic signaling linkage between postsynaptic GluRIIA levels and presynaptic bouton development. This work supports an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulating structural and functional synapse development (Song, 2022).

    This study reveals the mechanism of the established FMRP negative regulation of postsynaptic GluRIIA receptors and presynaptic bouton formation in the Drosophila FXS disease model. Specifically, the mRNA-binding FMRP-positive translational regulator binds to staufen mRNA as predicted, within the postsynaptic cell. Consequently, both dfmr1 and staufen mutants share the elevated GluRIIA level and bouton number phenotypes based on a common postsynaptic pathway function, and genetically interact as trans-heterozygotes to reproduce these phenotypes. Staufen acts as a dsRBP to bind coracle mRNA as predicted; both dfmr1 and staufen mutants exhibit elevated postsynaptic Coracle levels, and genetically interact as trans-heterozygotes to reproduce this phenotype. Coracle acts as a GluRIIA-binding anchoring scaffold within the postsynaptic domain to regulate local receptor accumulation (Chen, 2005). Consequently, dfmr1, staufen and coracle mutants all increase NMJ synaptic functional differentiation to elevate neurotransmission strength. Finally, the elevated postsynaptic GluRIIA levels mediate retrograde BMP receptor trans-synaptic signaling that induces pMad to drive new presynaptic bouton development. dfmr1, staufen and coracle mutants all exhibit elevated presynaptic pMad levels, thereby linking the postsynaptic GluRIIA accumulation and presynaptic supernumerary bouton formation defects shared by all of these mutants (Song, 2022).

    The staufen mutant increased synaptic Coracle levels, GluRIIA levels and bouton number are all internally consistent. In a previous study, opposite phenotypes were measured in staufenHL/Df(2R)Pcl7B, which reduces another 14 genes in heterozygous deficiency, including loci involved in neuronal development (e.g. grh, nopo). Importantly, this study similarly found reduced synaptic protein levels and bouton number in staufenHL/Df(2R)Pcl7B, suggesting that heterozygosity of one or more of the neighboring genes impairs synaptic development. However, this study showed that a staufen RNAi that reduces transcript levels by ~90% replicates the staufen mutant NMJ phenotypes of increased GluRIIA levels and synaptic bouton numbers. This was also replicated with a second, independent staufen RNAi line. Moreover, this study showed that the effect is entirely restricted to postsynaptic muscle RNAi, with no effect from presynaptic neuron RNAi, consistent with restricted postsynaptic Staufen function. In addition, postsynaptic staufen rescue of the staufen mutant restored normal synaptic bouton formation, with OE reducing GluRIIA levels in staufen mutants and rescuing GluRIIA levels in dfmr1 mutants. Both staufen mutants and postsynaptic staufen RNAi also share the arrested supernumerary satellite bouton development characterizing dfmr1 null mutants. These many independent lines of evidence confirm the results, and are consistent with the known parallel FMRP role in restricting GluRIIA levels and synaptic bouton formation (Song, 2022).

    To regulate Staufen, FMRP binds staufen mRNA and protects targeted staufen transcripts from degradation. FMRP contains at least three distinct RNA-binding domains (RBDs), and Staufen has five RBDs. Staufen reportedly binds a specific RNA hairpin structure formed by long 3' UTRs, but RIP shows that Staufen also binds mRNAs that are not predicted to generate this secondary structure. Although the decreased staufen mRNA levels in both dfmr1 mutants and muscle-targeted dfmr1 RNAi are predicted to be due to the lack of FMRP binding, it is also possible that other unregulated interactors cause the downregulated staufen mRNA expression (Shah et al., 2020). Localized labeling with an anti-Staufen antibody has been reported in the postsynaptic NMJ, which can be confirmed, but it was not possible to reduce labeling in staufen hypomorphic mutants. Therefore Staufen labeling was not shown in the current study. Moreover, western blots have been reported with the same anti-Staufen antibody; however, attempts were unsuccessful. Therefore qPCR was used to measure staufen mRNA levels. Staufen binds to coracle mRNA, but does so in a non-selective manner. This result is consistent with Staufen acting as a very broad spectrum dsRBP, and suggests that Staufen likely acts with a translational regulator partner to generate specificity. FMRP is very well established to partner with other RBPs to mediate the translational regulation of its target transcripts (Song, 2022). ------

    The postsynaptic Coracle scaffold acts in a GluRIIA local anchoring mechanism, presumably to link the receptors to the underlying actin cytoskeleton (Chen, 2005). The jointly elevated Coracle and GluRIIA levels in both dfmr1 and staufen mutants are consistent with this scaffold function. Because the dfmr1/+; staufen/+ trans-heterozygotes share this correlated Coracle and GluRIIA upregulation in the postsynaptic domain, a single common signaling pathway is indicated. Coracle also restricts terminal branching development in peripheral sensory neurons. Both coracle mutants and sensory neuron-targeted coracle RNAi also display increased dendritic branch and termini numbers. These phenotypes are similar to the expanded NMJ terminals and increased synaptic bouton development reported in this study. Importantly, both coracle loss of function (mutants and muscle-targeted RNAi) and gain of function (muscle-targeted OE) increase postsynaptic GluRIIA levels and generate supernumerary boutons. Likewise, the knockdown and OE of many other similar scaffolds are known to cause phenocopying defects. Some examples include the muscle chaperone UNC-45, the tight junction scaffold zonula occludens-1 (ZO-1) and synaptic UNC-13. Indeed, both coracle loss and OE similarly cause increased dendritic crossing in Drosophila sensory neurons, similar to the phenocopy of developmental defects reported in this study. Combining the roles of postsynaptic FMRP-Staufen-Coracle in GluRIIA clustering, it was reasoned that this pathway must be a regulatory determinant of synaptic functional development (Song, 2022).

    Removing FMRP, Staufen and Coracle strongly enhances functional synaptic differentiation and NMJ neurotransmission strength. This is consistent with expectations from the postsynaptic GluRIIA accumulation in all of these mutants. Elevated GluRIIA levels are well known to be associated with increased evoked functional responses and prolonged channel open times. A GluRIIA pore sequence (MQQ) critically required for the Drosophila channel Ca2+ permeability is conserved in mammalian receptors. This selectivity allows Ca2+-dependent participation in spontaneous (mEJC) and evoked (EJC) neurotransmission. Although enhanced evoked EJC amplitudes are typically accompanied by mEJC alterations, this study found that mEJC amplitude and frequency are unchanged in both the staufen and coracle mutants, and show only minimal changes in the dfmr1 mutants. Classically, both evoked and spontaneous neurotransmission were thought to be mediated by the same vesicles; however, more recent evidence has indicated that spontaneous and evoked neurotransmission have distinct machinery and vesicle pools. Postsynaptic receptors can be segregated into different compartments that are activated by either spontaneous or evoked release. This work supports this growing body of evidence for differential regulation. Importantly, GluRIIA has unique functions, modulating both presynaptic glutamate release and presynaptic bouton development (Song, 2022).

    The dfmr1, staufen and coracle mutants all showed upregulated presynaptic pMad correlated with postsynaptic activated GluRIIA accumulation. GluRIIA activation triggers presynaptic pMad signaling via BMP receptors surrounding active zones, which, in turn, stabilizes GluRIIA receptors in the postsynaptic domains. This trans-synaptic signaling mechanism induces new presynaptic bouton development. The targeted postsynaptic RNAi for all three genes confirms this intercellular link. Synaptic BMP signaling involves both the type I serine/threonine kinase receptors and the type II receptor Wit. Although BMP ligand Glass bottom boat (Gbb) signaling via Wit presynaptic receptors is well established at the NMJ to modulate synaptogenesis, the mechanism of presynaptic bouton formation induced by activated GluRIIA signaling does not involve canonical BMP signaling via Gbb. In the dfmr1 mutants, it is suggested that postsynaptic GluRIIA accumulation induces presynaptic bouton development via non-canonical GluRIIA-Wit trans-synaptic retrograde signaling. Similarly, the muscle postsynaptic glypican Dally-like protein (Dlp) negatively regulates NMJ synaptic development by inhibiting this same non-canonical BMP pathway through decreased activated GluRIIA expression. Postsynaptic GluRIIA clustering can thus trigger presynaptic bouton formation, although supernumerary boutons do not always induce reciprocal GluRIIA changes. It is concluded that an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulates intertwined structural and functional glutamatergic synapse development (Song, 2022).

    Gamma-secretase promotes Drosophila postsynaptic development through the cleavage of a Wnt receptor

    Developing synapses mature through the recruitment of specific proteins that stabilize presynaptic and postsynaptic structure and function. Wnt ligands signaling via Frizzled (Fz) receptors play many crucial roles in neuronal and synaptic development, but whether and how Wnt and Fz influence synaptic maturation is incompletely understood. This study showed that Fz2 receptor cleavage via the γ-secretase complex is required for postsynaptic development and maturation. In the absence of γ-secretase, Drosophila neuromuscular synapses fail to recruit postsynaptic scaffolding and cytoskeletal proteins, leading to behavioral deficits. Introducing presenilin mutations linked to familial early-onset Alzheimer's disease into flies leads to synaptic maturation phenotypes that are identical to those seen in null alleles. This conserved role for γ-secretase in synaptic maturation and postsynaptic development highlights the importance of Fz2 cleavage and suggests that receptor processing by proteins linked to neurodegeneration may be a shared mechanism with aspects of synaptic development (Restrepo, 2022).

    Postsynaptic development and maturation enable the essential transition from nascent, unreliable synapse to robust connection capable of high-fidelity neurotransmission. In Drosophila, the Wg ligand promotes neurodevelopment by activating postsynaptic Fz2 receptors, but the precise downstream events utilized by Fz2 to influence events like synaptic maturation remained controversial and unclear. This study found an unexpected developmental role for postsynaptic γ-secretase in enabling Fz2-mediated postsynaptic development via receptor cleavage. This role is also perturbed by AD patient-derived PSEN1 alleles in Drosophila, suggesting a link between AD and Wnts. These data first solve longstanding mysteries as to the significance of, and factors required for, Fz2 cleavage in postsynaptic maturation. Second, it suggests that proteolytic receptor cleavage may be a shared mechanism between developmental and degenerative processes, raising the possibility of a prior unappreciated neurodevelopmental component to AD (Restrepo, 2022).

    In the last 20 years, work at the Drosophila NMJ revealed the importance of Wnts in regulating synaptic function and development through Fz2 and multiple downstream pathways. The roles of each downstream pathway, however, remained unclear. In postsynaptic muscles, Fz2 signals via a Frizzled nuclear import (FNI) pathway resulting in nuclear import of cleaved Fz2 receptor. Three major questions about FNI remained unanswered: (1) what is its physiological significance, (2) how does the Fz2 cleavage site lend to its physiological role, and (3) what protease is necessary for Fz2 cleavage to promotes this physiological role? This work addresses each question, highlighting a role for FNI in postsynaptic development, the function of the cleavage site in synaptic maturation, and identifying γ-secretase as being required for Fz2 cleavage (Restrepo, 2022).

    First, it was shown that postsynaptic Fz2 cleavage (and not canonical Wnt signaling) specifically promotes postsynaptic maturation. This does not preclude a postsynaptic Fz2 function in presynaptic growth and active zone establishment. Instead, postsynaptic Fz2 may promote growth via canonical pathways; this will be an important area for future study. Second, identification of γ-secretase in promoting Fz2 cleavage is critical to understand downstream Fz2 pathways and begins to answer longstanding questions in synaptic neurodevelopment and postsynaptic maturation. As the developmental defects in γ-secretase mutants are suppressed by activating the postsynaptic FNI pathway, this indicates that the relevant cleavage target in maturation is Fz2. As maturation defects underlie neurodevelopmental disorders, further understanding the relevant machinery that promotes maturation can inform a grasp of neurodevelopment disease progression (Restrepo, 2022).

    Maturation of Drosophila central and NMJ synapses and mammalian dendritic spines all require γ-secretase. γ-secretase promotes axon guidance and early synaptic development, but its role in synaptic maturation is largely unknown. THe data suggest γ-secretase-dependent cleavage and translocation of signaling proteins may be a fundamental feature of postsynaptic development. Indeed, blocking Presenilin::Syt I interaction modestly reduces spine density and mature spines (Zoltowska, 2017). This is consistent with the current data, although the more general blockade of γ-secretase function has a larger effect on spine maturation. The mechanism remains unclear as γ-secretase modulates multiple downstream pathways, including Wnts. More work will be needed to determine if Wnt signaling is conserved in downstream signaling for synaptic maturation across evolution. In vertebrates, Fz5 and Fz8 are homologous to Drosophila Fz2 and contain the same consensus cleavage site. Fz8 is expressed in the nervous system , but its neuronal function and cleavage status remain unknown. Intriguingly, recent work showed that Fz5 can be cleaved in motor neuron-like NSC-34 cells. As Fz5 promotes activity-dependent synaptogenesis and neuronal survival, these data raise a tantalizing prospect that the γ-secretase / Wnt / Fz pathway is conserved in mammalian synaptic development (Restrepo, 2022).

    Identifying a role for γ-secretase and its mechanistic basis in neurodevelopment offers insight into the potential function of γ-secretase in disease. PSEN1 mutations are the most widely known genetic cause of Early-Onset Alzheimer Disease (EOAD), but thorough understanding of this genetic link is lacking. The data indicate that neurodevelopmental and neurodegenerative mechanisms may be united by a shared requirement for γ-secretase-dependent receptor processing. In AD, this adds a layer of connection between neurodevelopment and neurodegeneration via synapse-to-nucleus communication. In a model, γ-secretase cleavage enables Fz2-C generation and transition from synapse to nucleus. In AD, the synapse-to-nucleus signals AIDA-1, ATF4, and CRTC1 (that are protein partners of cleaved receptor products) are also altered and blocking the synapse-to-nucleus translocation of Jacob, a protein that couples activity to CREB signaling, suppresses pathogenic Aβ-induced impairments. Neurodevelopmental defects were observed in EOAD mutations indistinguishable from loss-of-function mutants that could be suppressed by restoring nuclear Fz2-C . It will be important for future study to determine the intersection of proteolytic processing, synapse-to-nucleus signaling, and Wnts on AD. The data raise a tantalizing possibility of connecting AD to a previously unappreciated neurodevelopmental process, which can potentially provide earlier hallmarks to assess disease progression and inform therapeutic strategies to ameliorate disease states (Restrepo, 2022).

    A prevailing mystery surrounding neurodegenerative diseases involves its onset. Although patients carry gene mutations all their lives, why do these disorders manifest later in life? A further challenge is to identify the earliest changes resulting from neurodegenerative disease gene mutations to better understand if symptoms are causative or correlative. The presence of synaptic maturation defects presents a hypothesis: if neurodegenerative disease risk genes are involved in synaptic maturation, the first reflection of mutations in those genes may be immature synapses that still function but lack the longevity of mature synapses. In advanced age, synapses constructed incorrectly during development may be the first to fail, leading to degeneration. AD models show reductions in postsynaptic proteins that precede neurodegeneration. Further, growing evidence suggests that neurodegenerative diseases have synaptopathic origins and early developmental defects. It is further intriguing that molecules which influence synaptogenesis, like Ephs, are modified by γ-secretase, potentially underlying further connection. Postsynaptic development and maturation may provide a unique angle to consider neurodegeneration, leading to earlier detection. With γ-secretase, maturation defects are suppressed by activating the downstream pathway; the data suggests analogous approaches may have clinical relevance. Whether synaptic NMJ phenotypes indicate a preclinical phenotype in disease models is an open question but examining postsynaptic maturation provides an opportunity to study shared mechanisms of neurodegenerative diseases, inform strategies to combat disease, and better understand neurodevelopment (Restrepo, 2022).

    The data indicates that γ-secretase activity is required for Fz2 cleavage. First, γ-secretase and Fz2 localize close enough for interaction. Second, Fz2 and γ-secretase colocalize in the same subcellular compartments and traffic similarly. Third, a structural model predicts that the cleavage site embeds in the membrane, keeping it within the 'lid' distance to be recognized as a cleavage site. Moving the cleavage site so it no longer abuts the membrane prevents the cleavage-dependent functions of Fz2. The most parsimonious interpretation supports Fz2 cleavage by γ-secretase. However, it is important to note that the data do not conclusively imply a direct cleavage event. Fz2 is not a canonical γ-secretase substrate for several reasons. First, γ-secretase typically cleaves single pass transmembrane proteins; Fz2 is a 7-pass transmembrane domain atypical G-protein coupled receptor (GPCR). This is not a requisite, however, as multipass transmembrane proteins like neuregulin-1 can be cleaved by γ-secretase. Second, γ-secretase typically cleaves in a processive fashion: the substrate ectodomain is first shed via an ADAM a disintegrin and metallprotease) enzyme or β-secretase, after which γ-secretase cleavage releases the substrate intracellular domain. There is no evidence that Fz2 undergoes ectodomain shedding and the receptor lacks consensus N-terminal cleavage sites. Third, γ-secretase typically cleaves in the plane of the membrane. The Fz2 cleavage site is cytosolic, although predicted to juxtapose the membrane. Recent years, however, identified noncanonical substrates that serve as exceptions to this rule (Restrepo, 2022).

    The data suggest that GPCRs may be atypical γ-secretase cleavage targets. This broadens the potential repertoire of γ-secretase, underscoring its importance in diverse processes. However, alternate interpretations cannot be ruled out, where Fz2 cleavage requires ectodomain shedding or occurs via successive events. In the latter, Fz2 would still be a direct target of γ-secretase that follows a preceding event. Also, γ-secretase may cleave a secondary target that allows Fz2 to be cleaved. In all cases, the activity of γ-secretase is still required for Fz2 cleavage. Future study in a cell-free system will be required for formal proof. Regardless, the complete suppression of the γ-secretase developmental phenotypes by nuclear Fz2-C expression indicates the most relevant cellular substrate in promoting postsynaptic development and maturation via γ-secretase is cleaved Fz2-C (Restrepo, 2022).

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

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

    RIM-binding protein couples synaptic vesicle recruitment to release site

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    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 in this study suggest that dnrx and dnlg1 genetically interact with wit as well as the downstream effector of BMP signaling, Mad to allow both the organization and growth of NMJ synapses. The surprising finding that loss of dnrx and dnlg1 leads to decreased wit stability and that dnrx and dnlg1 are required for proper wit localization raises the possibility that these proteins function to coordinate trans-synaptic adhesion and synaptic growth. This is further strengthened from the observations that, in the absence of Wit, the synaptogenic activity from overexpression of dnrx did not manifest into an increased synaptic growth as is seen in the presence of wit. Loss of dnrx and dnlg1 also led to a reduction in the levels of other components of the BMP pathway, namely the Tkv and pMad. Together these findings are the first to demonstrate a functional coordination between trans-synaptic adhesion proteins dnrx and dnlg1 with wit receptor and BMP pathway members to allow precise synaptic organization and growth at NMJ. It would be of immense interest to investigate whether similar mechanisms might be operating in vertebrate systems (Banerjee, 2016).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Live observation of two parallel membrane degradation pathways at axon terminals

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    To characterize the location and function of SYT7 in Drosophila the CRISPR-Cas9 system was used to endogenously label the protein and generate null mutations in the Syt7 locus. The findings indicate SYT7 acts as a negative regulator of SV release, active zone probability of release (AZ Pr), the readily releasable pool (RRP) size, and RRP refilling. The elevated Pr across the AZ population in Syt7 mutants provides a robust explanation for why defects in asynchronous release and facilitation are observed in the absence of the protein, as SYT7 levels set the baseline for the amount of evoked release. SYT7's presence on an internal tubular membrane network within the peri-AZ positions the protein to interface with the SV cycle at multiple points to regulate membrane trafficking. In addition, increased SV release in animals lacking both SYT1 and SYT7 indicate the full complement of Ca2+ sensors that mediate the distinct phases of SV release remain unknown (Guan, 2020).

    Using a combination of synaptic physiology and imaging approaches, the findings indicate SYT7 acts to reduce SV recruitment and release. Minor defects in asynchronous release and facilitation were identified in Drosophila Syt7 mutants, as observed in mouse and zebrafish models. However, these defects are attrobited to reduced SV availability as a result of increased Pr in Syt7 mutants. Indeed, a key feature of facilitation is its critical dependence on initial Pr. Low Pr synapses increase SV fusogenicity as Ca2+ levels rise during paired-pulses or stimulation trains, resulting in short-term increases in Pr for SVs not recruited during the initial stimuli. In contrast, depression occurs at high Pr synapses due to the rapid depletion of fusion-capable SVs during the initial response. Prior quantal imaging at Drosophila NMJs demonstrated facilitation and depression can occur across different AZs within the same neuron, with high Pr AZs depressing and low Pr AZs facilitating. Given the elevated Pr in Syt7 mutants, the facilitation defects are likely related to differences in initial Pr and depletion of fusion-competent SVs available for release during the 2nd stimuli (Guan, 2020).

    A similar loss of SVs due to elevated Pr in Syt7 mutants would reduce fusogenic SVs that are available during the delayed phase of the asynchronous response. Syt1; Syt7 double mutants continue to show asynchronous fusion and facilitation, demonstrating there must be other Ca2+ sensors that mediate these components of SV release. The predominant localization of endogenous SYT7 to an internal tubular membrane compartment at the peri-AZ also places the majority of the protein away from release sites where it would need to reside to directly activate SV fusion. As such, the data indicate SYT7 regulates SV release through a distinct mechanism from SYT1 (Guan, 2020).

    It is also concluded that the remaining components of asynchronous fusion and facilitation must be mediated by an entirely different family of Ca2+-binding proteins than Synaptotagmins (or through Ca2+-lipid interactions). Of the seven Syt genes in the Drosophila genome, only 3 SYT proteins are expressed at the motor neuron synapses assayed in this study - SYT1, SYT4 and SYT7. For the remaining SYTs in the genome, SYT-α and SYT-β are expressed in neurosecretory neurons and function in DCV fusion. SYT12 and SYT14 lack Ca2+ binding residues in their C2 domains and are not expressed in motor neurons. In addition, SYT4 is found on exosomes and transferred to postsynaptic cells, where it participates in retrograde signaling. Syt1; Syt4 double mutants display the same SV fusion defects found in Syt1 mutants alone, indicating SYT4 cannot compensate for SYT1 function in SV release. As such, SYT1 and SYT7 are the only remaining SYT isoforms that could contribute to SV trafficking within Drosophila motor neuron terminals (Guan, 2020).

    A prior study using a Syt7 exon-intron hairpin RNAi did not result in an increase in evoked release. Although a reduction in ectopic expression of SYT7 in muscles could be seen with Mhc-GAL4 driving the UAS-Syt7 RNAi, the anti-SYT7 antisera does not recognize the endogenous protein in neurons using immunocytochemistry, preventing a determination of presynaptic SYT7 levels following neuronal RNAi. To further examine this issue, western blot analysis was performed with this RNAi and compared those used in the current study. The results confirmed that the RNAi line failed to reduce endogenous GFP-tagged SYT7, although the two commercial RNAi lines used in the current study were highly effective. Based on these data, it is concluded that the previous Syt7 UAS-RNAi line was ineffective in knocking down endogenous SYT7. Given the Syt7M1 and Syt7M2 alleles result in early stop codons and lack SYT7 expression by western blot analysis and display elevated levels of fusion, the data indicate SYT7 normally acts to suppress SV release as demonstrated by electrophysiology and optical Pr imaging. SYT7 overexpression reduces SV release even more, further confirming that the levels of SYT7 set the baseline amount of SV fusion at Drosophila NMJ synapses (Guan, 2020).

    Although the data indicate SYT7 is not the primary asynchronous or facilitation Ca2+ sensor in Drosophila, this study found it inhibits SV release in a dosage-sensitive manner. The reduction in SV release is not due to changes in the Ca2+ cooperativity of fusion or enhanced presynaptic Ca2+ entry, ruling out the possibility that SYT7 normally acts as a local Ca2+ buffer or an inhibitor of presynaptic voltage-gated Ca2+ channels. The reduction in release is also not due to altered endocytosis, as Syt7 mutants have a normal steady-state rate of SV cycling following depletion of the RRP. Instead, SYT7 regulates SV fusogenicity at a stage between SV endocytosis and fusion. Given the rapid enhanced refilling of the RRP observed in Syt7 mutants, and the suppression of RRP refilling following SYT7 overexpression, the data indicate SYT7 regulates releasable SVs in part through changes in SV mobilization to the RRP. Ca2+ is well known to control the replenishment rate of releasable SVs, with Calmodulin-UNC13 identified as one of several molecular pathways that accelerate RRP refilling in a Ca2+-dependent manner. The findings indicate SYT7 acts in an opposite fashion and slows RRP refilling, providing a Ca2+-dependent counter-balance for SV recruitment into the RRP. Although such an effect has not been described for mammalian SYT7, defects in RRP replenishment have been observed when both SYT1 and SYT7 are deleted or following high frequency stimulation trains (Guan, 2020).

    SYT7's role in restricting SV fusion and RRP size also affects spontaneous release. Although Syt7 mutants alone do not show elevated mini frequency, DoubleNull mutants have a 2-fold increase in spontaneous release. Similar increases in spontaneous release were observed at mammalian synapses lacking both SYT7 and SYT1 (or SYT2), with the effect being attributed to a dual role in clamping fusion in the absence of Ca2+ (Luo, 2017; Turecek, 2019). The current results indicate the elevation in spontaneous release at Drosophila synapses is a result of an increase in releasable SVs rather than a clamping function for SYT7. Following overexpression of SYT7, there is a reduction in the number of fusogenic SVs available for both evoked and spontaneous release. The dosage-sensitivity of the various phenotypes indicate SYT7 abundance is a critical node in controlling SV release rate. Indeed, mammalian SYT7 levels are post-transcriptionally modulated by γ-secretase proteolytic activity and APP, linking it to SV trafficking defects in Alzheimer's disease (Guan, 2020).

    How does SYT7 negatively regulate recruitment and fusion of SVs? The precise mechanism by which SYT7 reduces release and slows refilling of the RRP is uncertain given it is not enriched at sites of SV fusion. Although the possibility cannot be ruled out that a small fraction of the protein is found at AZs, SYT7 is predominantly localized to an internal membrane compartment at the peri-AZ where SV endocytosis and endosomal sorting occurs. SYT7 membrane tubules are in close proximity and could potentially interact with peri-AZs proteins, endosomes, lysosomes and the plasma membrane. Given its primary biochemical activity is to bind membranes in a Ca2+-dependent manner, SYT7 could mediate cargo or lipid movement across multiple compartments within peri-AZs. In addition, it is possible SYT7 tubules could form part of the poorly defined SV recycling endosome compartment. However, no change was observed in SV density or SV localization around AZs, making it unlikely SYT7 would be essential for endosomal trafficking of SVs. The best characterized regulator of the SV endosome compartment in Drosophila is the RAB35 GAP Skywalker (SKY). Although Sky mutations display some similarities to Syt7, including increased neurotransmitter release and larger RRP size, Syt7 lacks most of the well-described Sky phenotypes such as behavioral paralysis, FM1-43 uptake into discrete punctated compartments, cisternal accumulation within terminals and reduced SV density. In addition, no co-localization was found between SKY-GFP and SYT7RFP within presynaptic terminals (Guan, 2020).

    By blocking SV refilling with bafilomycin, the findings indicate the fast recovery of the RRP can occur via enhanced recruitment from the reserve pool and does not require changes in endocytosis rate. The phosphoprotein Synapsin has been found to maintain the reserve SV pool by tethering vesicles to actin filaments at rest. Synapsin interacts with the peri-AZ protein Dap160/Intersectin to form a protein network within the peri-AZ that regulates clustering and release of SVs. Synapsin-mediated phase separation is also implicated in clustering SVs near release sites. SYT7 could similarly maintain a subset of SVs in a non-releasable pool and provide a dual mechanism for modulating SV mobilization. Phosphorylation of Synapsin and Ca2+ activation of SYT7 would allow multiple activity-dependent signals to regulate SV entry into the RRP. As such, SYT7 could play a key role in organizing membrane trafficking and protein interactions within the peri-AZ network by adding a Ca2+-dependent regulator of SV recruitment and fusogenicity (Guan, 2020).

    Additional support for a role for SYT7 in regulating SV availability through differential SV sorting comes from recent studies on the SNARE complex binding protein CPX. Analysis of Drosophila Cpx mutants, which have a dramatic increase in minis, revealed a segregation of recycling pathways for SVs undergoing spontaneous versus evoked fusion. Under conditions where intracellular Ca2+ is low and SYT7 is not activated, spontaneously-released SVs do not transit to the reserve pool and rapidly return to the AZ for re-release. In contrast, SVs released during high frequency evoked stimulation when Ca2+ is elevated and SYT7 is engaged, re-enter the RRP at a much slower rate. This mechanism slows re-entry of SVs back into the releasable pool when stimulation rates are high and large numbers of SV proteins are deposited onto the plasma membrane at the same time, allowing time for endosomal sorting that might be required in these conditions. In contrast, SVs released during spontaneous fusion or at low stimulation rates would likely have less need for endosomal re-sorting. Given SYT7 restricts SV transit into the RRP, it provides an activity-regulated Ca2+-triggered switch for redirecting and retaining SVs in a non-fusogenic pool that could facilitate sorting mechanisms (Guan, 2020).

    Beyond SV fusion, presynaptic membrane trafficking is required for multiple signaling pathways important for developmental maturation of NMJs. In addition, alterations in neuronal activity or SV endocytosis can result in synaptic undergrowth or overgrowth. No defect was found in synaptic bouton or AZ number, indicating SYT7 does not participate in membrane trafficking pathways that regulate synaptic growth and maturation. However, a decrease in T-bar area and presynaptic Ca2+ influx in Syt7 mutants was found. Although it is unclear how these phenotype arise, they may represent a form of homeostatic plasticity downstream of elevated synaptic transmission. There is also ample evidence that SV distance to Ca2+ channels plays a key role in defining the kinetics of SV release and the size of the RRP, suggesting a change in such coupling in Syt7 mutants might contribute to elevations in Pr and RRP refilling. Further studies will be required to precisely define how SYT7 controls the baseline level of SV release at synapses (Guan, 2020).

    Synaptotagmin 7 switches short-term synaptic plasticity from depression to facilitation by suppressing synaptic transmission

    Short-term synaptic plasticity is a fast and robust modification in neuronal presynaptic output that can enhance release strength to drive facilitation or diminish it to promote depression. The mechanisms that determine whether neurons display short-term facilitation or depression are still unclear. This study shows that the Ca(2+)-binding protein Synaptotagmin 7 (Syt7) determines the sign of short-term synaptic plasticity by controlling the initial probability of synaptic vesicle (SV) fusion. Electrophysiological analysis of Syt7 null mutants at Drosophila embryonic neuromuscular junctions demonstrate loss of the protein converts the normally observed synaptic facilitation response during repetitive stimulation into synaptic depression. In contrast, overexpression of Syt7 dramatically enhanced the magnitude of short-term facilitation. These changes in short-term plasticity were mirrored by corresponding alterations in the initial evoked response, with SV release probability enhanced in Syt7 mutants and suppressed following Syt7 overexpression. Indeed, Syt7 mutants were able to display facilitation in lower [Ca(2+)] where release was reduced. These data suggest Syt7 does not act by directly sensing residual Ca(2+) and argues for the existence of a distinct Ca(2+) sensor beyond Syt7 that mediates facilitation. Instead, Syt7 normally suppresses synaptic transmission to maintain an output range where facilitation is available to the neuron (Fujii, 2021).

    Synaptotagmins (Syts) are a large family of Ca2+ binding proteins, with the Syt1 isoform functioning as the major Ca2+ sensor for synchronous synaptic vesicle (SV) fusion1. Ca2+ also controls presynaptic forms of short-term plasticity, with other Syt isoforms representing promising candidates to mediate these processes. Indeed, Synaptotagmin 7 (Syt7) has been reported to function in facilitation, a form of short-term plasticity that enhances synaptic transmission following consecutive action potentials. Facilitation is believed to be mediated by residual Ca2+ acting to enhance the number of SVs that are released during repetitive action potentials occurring within a short temporal window. Since Syt7 binds Ca2+ with high affinity and slow kinetics, which match requirements for facilitation, the protein has been hypothesized to act as the Ca2+ sensor for this form of presynaptic plasticity. However, the role of Syt7 in facilitation is still unclear (Fujii, 2021).

    Drosophila neuromuscular junctions (NMJs) provide an excellent system for testing the role of Syt7 in short-term synaptic plasticity. In particular, embryonic NMJs are highly plastic and allow stable recordings in high Ca2+ concentrations using the myosin heavy chain (Mhc) mutant background to prevent muscle contraction. Together with the lack of compensation that might occur at older synapses, these advantages provide highly reliable measurements of synaptic transmission. Indeed, analysis of Syt1 mutants at embryonic NMJs established this Syt isoform functions as the synchronous Ca2+ sensor for synaptic transmission. Although NMJs in many other species show depression, Drosophila embryonic NMJs are facilitative at physiological Ca2+ concentrations as shown here, similar to many mammalian central synapses such as those in the hippocampus. Using stable recordings from this facilitative and plastic synapse in Drosophila embryos, the absolute value of synaptic currents was quantified in Syt7 mutants and in animals overexpressing Syt7. While loss of Syt7 enhanced presynaptic output, overexpression of Syt7 suppressed release. These changes in the magnitude of presynaptic output were mirrored by changes in short-term presynaptic plasticity. High levels of Syt7 enabled robust facilitative responses while loss of Syt7 switched the normally facilitating synapse into one that displayed short-term depression. This work reveals that Syt7 normally reduces synaptic transmission to scale it to an appropriate range where facilitation is allowed, providing a bi-directional switch for short-term synaptic plasticity (Fujii, 2021).

    The current study indicates Syt7 is indispensable for facilitation across the physiological range of Ca2+ concentrations at Drosophila embryonic NMJs as previously shown for mammalian preparations (Jackman, 2016). In the absence of Syt7, the normally facilitating embryonic NMJ now displays depression. Following Syt7 overexpression, facilitation is greatly enhanced. These data indicate the major reason for defective facilitation in Syt7-/- mutants is due to loss of Syt7's ability to suppress release, which likely causes rapid SV depletion that is non-compatible with short-term synaptic facilitation. Likewise, overexpression of Syt7 reduces SV release and allows for enhanced facilitation. This role for Syt7 contrasts with current models proposed in mammals where Syt7 is hypothesized to bind residual Ca2+ to directly act as a facilitation Ca2+ sensor. Although the current data indicate Syt7 is not the primary Ca2+ sensor for facilitation, the possibility cannot be ruled out that Syt7 has dual roles in both suppressing and facilitating SV fusion as observed for Syt. If Syt7 has a dual role with C2A functioning for clamping and C2B for facilitation, the null mutant would lack both properties. It is possible the lack of clamping is the dominant phenotype, with any facilitative function being masked by SV depletion at higher Ca2+ concentrations. Thus, a Syt7-dependent component of facilitation cannot be ruled out. However, the presence of facilitation in Syt7-/- mutants at lower [Ca2+] indicates there is a facilitation sensor besides Syt7 that monitors residual Ca2+ to directly activate this form of short-term plasticity. Although a role for residual maternally supplied Syt7 at the embryonic stage in Syt7-/- mutants cannot be ruled out, there is no evidence from RNA profiling studies that indicate Syt7 is present at earlier stages of embryonic development prior to nervous system formation. Thus, it is unlikely residual Syt7 could sustain normal levels of facilitation as observed in low [Ca2+], consistent with the enhanced synaptic transmission observed across a broad [Ca2+] range in Syt7-/- mutants. Given facilitation is also present in low [Ca2+] in Syt7-/- mutants at the 3rd instar stage when any maternal contribution would be depleted, it is concluded that facilitation can occur in the complete absence of Syt7 under conditions where the initial response is reduced (Fujii, 2021).

    A key advantage of the Drosophila embryonic NMJ preparation is the ability to unambiguously monitor the absolute baseline values of synaptic strength even in high [Ca2+] using the non-contracting Mhc mutant. In this regard, it is clear that synaptic transmission at Drosophila embryonic NMJs is much stronger in Syt7-/- mutants at all Ca2+ concentrations tested. Moreover, the stronger transmission is due to higher release probability rather than an increased number of releasable SVs. Thus, the data predict that higher release probability leads to a lower facilitation ratio secondary to vesicle depletion. The precise mechanisms by which Syt7 suppresses SV release to enable facilitation will require further study. Beyond a potential clamping function for Syt7, the protein could alter local Ca2+ buffering or cause increased Ca2+ influx that could contribute to elevated SV release. Syt7 does not localize to SVs and may instead act from the plasma membrane or internal membrane compartments, allowing for several potential mechanisms for Syt7 to suppress release. Ca2+ binding to the C2A and C2B domains of Syt1 have been shown to have distinct functions in SV release, with C2B playing a dominant role in triggering SV fusion and C2A acting to clamp release. It is unclear if the C2A and C2B domains of Syt7 act similarly in Drosophila or have independent functions compared to Syt1. One possibility is that the C2A domain of Syt7 suppresses SV fusion and the C2B domain facilitates release, similar to Syt1. Structure function studies of Syt7 should help elucidate this biology in Drosophila, similar to prior studies of Syt1 function (Fujii, 2021).

    As suppression of SV release by Syt7 is dose-dependent (Guan, 2020), increasing levels of Syt7 would elevate the ratio of facilitation. These results suggest the degree of facilitation across distinct neuronal populations may be set by Syt7 levels similar to a potentiometer. The analysis of Syt7-/- nulls, heterozygotes and overexpression lines support such a model that changes release and short-term plasticity in a graded fashion. Depending on whether a synapse is facilitative or depressive, Syt7 expression could be modulated to gate plasticity to the level that most benefits the local circuit, similar to how Syt1 and Syt2 levels variably control release synchronicity across neuronal populations. Indeed, the squid giant synapse is facilitative only when Ca2+ is lowered from normal saline (artificial sea water), similar to Syt7-/- mutants, suggesting synapses that normally depress may have reduced levels of Syt7. Indeed, recent evidence suggests that species-specific differences in presynaptic plasticity in rodents is linked to the levels of Syt7. In shrews, the levels of Syt7 are lower in hippocampal CA3 synapses and they show reduced presynaptic plasticity. In contrast, Syt7 levels are much higher in mice, with their CA3 output synapses displaying far greater forms of presynaptic plasticity. Drosophila adults and 3rd instar larvae also have less facilitative NMJs than embryonic NMJs. This difference may contribute to the distinct effects of Syt7 on clamping spontaneous SV release that is observed between embryonic and 3rd instar NMJs. Mammalian studies identified redundant functions for Syt1 and Syt7 in clamping spontaneous fusion at inhibitory synapses. While reductions in Syt7 levels alone did not increase spontaneous SV release, removal of both Syt1 and Syt7 enhanced mini frequency to a far greater level that loss of Syt1 alone. In addition, a Syt7 transgene was able to rescue the elevated miniature frequency in Syt1 mutants. These differences in clamping properties were attributed to an insufficient level of Syt7 expression compared to Syt1. Differences in the Syt1/Syt7 ratio between Drosophila 3rd instar and embryonic NMJs may also contribute to distinct effects on spontaneous SV clamping observed in Syt1 and Syt7 mutants at these distinct developmental stages. In conclusion, controlling expression level of Syt7 provides an attractive mechanism for activity-dependent presynaptic scaling of release probability as a homeostat for both presynaptic output and short-term facilitation, similar to postsynaptic scaling mechanisms previously described for chronic forms of synaptic plasticity (Fujii, 2021).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Simulation of a sudden drop-off in distal dense core vesicle concentration in Drosophila type II motoneuron terminals

    Recent experimental observations have shown evidence of an unexpected sudden drop-off in the dense core vesicles (DCVs) content at the ends of certain types of axon endings. A mathematical model was developed that is based on the conservation of captured and transiting DCVs in boutons. The model consists of 77 ordinary differential equations and is solved using a standard Matlab solver. It was hypothesized that the drop in DCV content in distal boutons is due to an insufficient supply of anterogradely moving DCVs coming from the soma. This hypothesis was tested by modifying the flux of DCVs entering the terminal, and it was found that the number of most distal boutons left unfilled increases if the DCV flux entering the terminal is decreased. The number of anterogradely moving DCVs in the axon can be increased either by the release of a portion of captured DCVs into the anterograde component or by an increase of the anterograde DCV flux into the terminal. This increase could lead to having enough anterogradely moving DCVs such that they could reach the most distal bouton and then turn around by changing molecular motors that propel them. The model suggests that this could result in an increased concentration of resident DCVs in distal boutons beginning with bouton 2 (the most distal is bouton 1). This is because in distal boutons, DCVs have a larger chance to be captured from the transiting state as they pass the boutons moving anterogradely and then again as they pass the same boutons moving retrogradely (Kuznetsov, 2021).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    The Neurexin-NSF interaction regulates short-term synaptic depression

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    A neuropeptide signaling pathway regulates synaptic growth in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Acetylated alpha-tubulin K394 regulates microtubule stability to shape the growth of axon terminals

    Microtubules are essential to neuron shape and function. Acetylation of tubulin has the potential to directly tune the behavior and function of microtubules in cells. Although proteomic studies have identified several acetylation sites in α-tubulin, the effects of acetylation at these sites remains largely unknown. This includes the highly conserved residue lysine 394 (K394), which is located at the α-tubulin dimer interface. Using a fly model, this study showed that α-tubulin K394 is acetylated in the nervous system and is an essential residue. Acetylation-blocking mutation in endogenous α-tubulin, K394R, was found to perturb the synaptic morphogenesis of motoneurons and reduces microtubule stability. Intriguingly, the K394R mutation has opposite effects on the growth of two functionally and morphologically distinct motoneurons, revealing neuron-type-specific responses when microtubule stability is altered. Eliminating the deacetylase HDAC6 increases K394 acetylation, and the over-expression of HDAC6 reduces microtubule stability similar to the K394R mutant. Thus, these findings implicate α-tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis (Saunders, 2022).

    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).

    Misregulation of Drosophila Sidestep leads to uncontrolled wiring of the adult neuromuscular system and severe locomotion defects

    Holometabolic organisms undergo extensive remodelling of their neuromuscular system during metamorphosis. Relatively, little is known whether or not the embryonic guidance of molecules and axonal growth mechanisms are re-activated for the innervation of a very different set of adult muscles. This study shows that the axonal attractant Sidestep (Side) is re-expressed during Drosophila metamorphosis and is indispensable for neuromuscular wiring. Mutations in side cause severe innervation defects in all legs. Neuromuscular junctions (NMJs) show a reduced density or are completely absent at multi-fibre muscles. Misinnervation strongly impedes, but does not completely abolish motor behaviours, including walking, flying, or grooming. Overexpression of Side in developing muscles induces similar innervation defects; for example, at indirect flight muscles, it causes flightlessness. Since muscle-specific overexpression of Side is unlikely to affect the central circuits, the resulting phenotypes seem to correlate with faulty muscle wiring. It was further shown that mutations in beaten path Ia (beat), a receptor for Side, results in similar weaker adult innervation and locomotion phenotypes, indicating that embryonic guidance pathways seem to be reactivated during metamorphosis (Kinold, 2021).

    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).

    Protein phosphatase 2A restrains DLK signaling to promote proper Drosophila synaptic development and mammalian cortical neuron survival

    Protein phosphatase 2A (PP2A) is a major cellular phosphatase with many protein substrates. In post-mitotic neurons, the microtubule associated protein Tau is a particularly well-studied PP2A substrate as hyperphosphorylation of Tau is a hallmark of Alzheimer's disease. This study finds that activation of a single pathway can explain important aspects of the PP2A loss-of-function phenotype in neurons. PP2A inhibits activation of the neuronal stress kinase DLK and its Drosophila ortholog Wallenda. In the fly, PP2A inhibition activates a DLK/Wallenda-regulated transcriptional program that induces synaptic terminal overgrowth at the neuromuscular junction. In cultured mammalian neurons, PP2A inhibition activates a DLK-dependent apoptotic program that induces cell death. Contrary to expectations, in the absence of Tau PP2A inhibition still activates DLK and induces neuronal cell death, demonstrating that hyperphosphorylated Tau is not required for cell death in this model. Moreover, hyperphosphorylation of Tau following PP2A inhibition does not require DLK. Hence, loss of PP2A function in cortical neurons triggers two independent neuropathologies: 1) Tau hyperphosphorylation and 2) DLK activation and subsequent neuronal cell death. These findings demonstrate that inhibition of the DLK pathway is an essential function of PP2A required for normal Drosophila synaptic terminal development and mammalian cortical neuron survival (Hayne, 2022).

    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