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Missense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson's disease. Here, we characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal-lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. We also establish multiple links between SVIP and VCP-dependent disease in our Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, we identify a human SVIP mutation and confirm the pathogenicity of this mutation in our Drosophila model. We propose a model for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles.

Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration. Here, we demonstrate that presynaptic homeostatic plasticity (PHP) is induced at degenerating neuromuscular junctions, mediated by an evolutionarily conserved activity of presynaptic ENaC channels in both Drosophila and mouse. To assess the consequence of eliminating PHP in a mouse model of ALS-like degeneration, we generated a motoneuron-specific deletion of Scnn1a, encoding the ENaC channel alpha subunit. We show that Scnn1a is essential for PHP without adversely affecting baseline neural function or lifespan. However, Scnn1a knockout in a degeneration-causing mutant background accelerated motoneuron loss and disease progression to twice the rate observed in littermate controls with intact PHP. We propose a model of neuroprotective homeostatic plasticity, extending organismal lifespan and health span.

We identify a set of common phenotypic modifiers that interact with five independent autism gene orthologs (RIMS1, CHD8, CHD2, WDFY3, ASH1L) causing a common failure of presynaptic homeostatic plasticity (PHP) in Drosophila. Heterozygous null mutations in each autism gene are demonstrated to have normal baseline neurotransmission and PHP. However, PHP is sensitized and rendered prone to failure. A subsequent electrophysiology-based genetic screen identifies the first known heterozygous mutations that commonly genetically interact with multiple ASD gene orthologs, causing PHP to fail. Two phenotypic modifiers identified in the screen, PDPK1 and PPP2R5D, are characterized. Finally, transcriptomic, ultrastructural and electrophysiological analyses define one mechanism by which PHP fails; an unexpected, maladaptive up-regulation of CREG, a conserved, neuronally expressed, stress response gene and a novel repressor of PHP. Thus, we define a novel genetic landscape by which diverse, unrelated autism risk genes may converge to commonly affect the robustness of synaptic transmission.

Epigenetic gene regulation shapes neuronal fate in the embryonic nervous system. Post-embryonically, epigenetic signaling within neurons has been associated with impaired learning, autism, ataxia, and schizophrenia. Epigenetic factors are also enriched in glial cells. However, little is known about epigenetic signaling in glia and nothing is known about the intersection of glial epigenetic signaling and presynaptic homeostatic plasticity. During a screen for genes involved in presynaptic homeostatic synaptic plasticity, we identified an essential role for the histone acetyltransferase and deubiquitinase SAGA complex in peripheral glia. We present evidence that the SAGA complex is necessary for homeostatic plasticity, demonstrating involvement of four new genes in homeostatic plasticity. This is also evidence that glia participate in presynaptic homeostatic plasticity, invoking previously unexplored intercellular, homeostatic signaling at a tripartite synapse. We show, mechanistically, SAGA signaling regulates the composition of and signaling from the extracellular matrix during homeostatic plasticity.

Presynaptic homeostatic plasticity (PHP) is an evolutionarily conserved form of adaptive neuromodulation and is observed at both central and peripheral synapses. In this work, we make several fundamental advances by interrogating the synapse specificity of PHP. We define how PHP remains robust to acute versus long-term neurotransmitter receptor perturbation. We describe a general PHP property that includes global induction and synapse-specific expression mechanisms. Finally, we detail a novel synapse-specific PHP expression mechanism that enables the conversion from short- to long-term PHP expression. If our 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.

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, we demonstrate 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 Krüppel-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. We propose that FRH includes mechanisms of proteostatic feedback that act in parallel with activity-driven feedback, with implications for the pathophysiology of human channelopathies.

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

Presynaptic homeostatic plasticity (PHP) compensates for impaired postsynaptic neurotransmitter receptor function through a rapid, persistent adjustment of neurotransmitter release, an effect that can exceed 200%. An unexplained property of PHP is the preservation of short-term plasticity (STP), thereby stabilizing activity-dependent synaptic information transfer. We demonstrate that the dramatic potentiation of presynaptic release during PHP is achieved while simultaneously maintaining a constant ratio of primed to super-primed synaptic vesicles, thereby preserving STP. Mechanistically, genetic, biochemical and electrophysiological evidence argue that a constant ratio of primed to super-primed synaptic vesicles is achieved by the concerted action of three proteins: Unc18, Syntaxin1A and RIM. Our data support a model based on the regulated availability of Unc18 at the presynaptic active zone, a process that is restrained by Syntaxin1A and facilitated by RIM. As such, regulated vesicle priming/super-priming enables PHP to stabilize both synaptic gain and the activity-dependent transfer of information at a synapse.

Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward-an initiative aimed at mapping the physical and genetic interaction networks of these conditions and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.

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

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

The circuitry of the brain is characterized by cell heterogeneity, sprawling cellular anatomy, and astonishingly complex patterns of connectivity. Determining how complex neural circuits control behavior is a major challenge that is often approached using surgical, chemical, or transgenic approaches to ablate neurons. However, all these approaches suffer from a lack of precise spatial and temporal control. This drawback would be overcome if cellular ablation could be controlled with light. Cells are naturally and cleanly ablated through apoptosis due to the terminal activation of caspases. Here, we describe the engineering of a light-activated human caspase-3 (Caspase-LOV) by exploiting its natural spring-loaded activation mechanism through rational insertion of the light-sensitive LOV2 domain that expands upon illumination. We apply the light-activated caspase (Caspase-LOV) to study neurodegeneration in larval and adult Drosophila Using the tissue-specific expression system (UAS)-GAL4, we express Caspase-LOV specifically in three neuronal cell types: retinal, sensory, and motor neurons. Illumination of whole flies or specific tissues containing Caspase-LOV-induced cell death and allowed us to follow the time course and sequence of neurodegenerative events. For example, we find that global synchronous activation of caspase-3 drives degeneration with a different time-course and extent in sensory versus motor neurons. We believe the Caspase-LOV tool we engineered will have many other uses for neurobiologists and others for specific temporal and spatial ablation of cells in complex organisms.

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). Here, we demonstrate that the ppk1 gene encodes an essential subunit of this Deg/ENaC channel, functioning in motoneurons for the rapid induction and maintenance of PHP. We provide genetic and biochemical evidence that PPK1 functions together with PPK11 and PPK16 as a presynaptic, hetero-trimeric Deg/ENaC channel. Finally, we highlight tight control of Deg/ENaC channel expression and activity, showing increased PPK1 protein expression during PHP and evidence for signaling mechanisms that fine tune the level of Deg/ENaC activity during PHP.

Presynaptic homeostatic plasticity (PHP) controls synaptic transmission in organisms from Drosophila to human and is hypothesized to be relevant to the cause of human disease. However, the underlying molecular mechanisms of PHP are just emerging and direct disease associations remain obscure. In a forward genetic screen for mutations that block PHP we identified mctp (Multiple C2 Domain Proteins with Two Transmembrane Regions). Here we show that MCTP localizes to the membranes of the endoplasmic reticulum (ER) that elaborate throughout the soma, dendrites, axon and presynaptic terminal. Then, we demonstrate that MCTP functions downstream of presynaptic calcium influx with separable activities to stabilize baseline transmission, short-term release dynamics and PHP. Notably, PHP specifically requires the calcium coordinating residues in each of the three C2 domains of MCTP. Thus, we propose MCTP as a novel, ER-localized calcium sensor and a source of calcium-dependent feedback for the homeostatic stabilization of neurotransmission.

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

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

Lysosomes are classically viewed as vesicular structures to which cargos are delivered for degradation. Here, we identify a network of dynamic, tubular lysosomes that extends throughout Drosophila muscle, in vivo. Live imaging reveals that autophagosomes merge with tubular lysosomes and that lysosomal membranes undergo extension, retraction, fusion and fission. The dynamics and integrity of this tubular lysosomal network requires VCP, an AAA-ATPase that, when mutated, causes degenerative diseases of muscle, bone and neurons. We show that human VCP rescues the defects caused by loss of Drosophila VCP and overexpression of disease relevant VCP transgenes dismantles tubular lysosomes, linking tubular lysosome dysfunction to human VCP-related diseases. Finally, disruption of tubular lysosomes correlates with impaired autophagosome-lysosome fusion, increased cytoplasmic poly-ubiquitin aggregates, lipofuscin material, damaged mitochondria and impaired muscle function. We propose that VCP sustains sarcoplasmic proteostasis, in part, by controlling the integrity of a dynamic tubular lysosomal network.

Homeostatic signaling stabilizes synaptic transmission at the neuromuscular junction (NMJ) of Drosophila, mice, and human. It is believed that homeostatic signaling at the NMJ is bi-directional and considerable progress has been made identifying mechanisms underlying the homeostatic potentiation of neurotransmitter release. However, very little is understood mechanistically about the opposing process, homeostatic depression, and how bi-directional plasticity is achieved. Here, we show that homeostatic potentiation and depression can be simultaneously induced, demonstrating true bi-directional plasticity. Next, we show that mutations that block homeostatic potentiation do not alter homeostatic depression, demonstrating that these are genetically separable processes. Finally, we show that homeostatic depression is achieved by decreased presynaptic calcium channel abundance and calcium influx, changes that are independent of the presynaptic action potential waveform. Thus, we identify a novel mechanism of homeostatic synaptic plasticity and propose a model that can account for the observed bi-directional, homeostatic control of presynaptic neurotransmitter release.

Here we define activities of RIM-binding protein (RBP) that are essential for baseline neurotransmission and presynaptic homeostatic plasticity. At baseline, rbp mutants have a ∼10-fold decrease in the apparent Ca(2+) sensitivity of release that we attribute 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 ∼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. We propose 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.

The strength and dynamics of synaptic transmission are determined, in part, by the presynaptic action potential (AP) waveform at the nerve terminal. The ion channels that shape the synaptic AP waveform remain essentially unknown for all but a few large synapses amenable to electrophysiological interrogation. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying synaptic biology, but it is not amenable to presynaptic electrophysiology. Here, we demonstrate that Archaerhodopsin can be used to quantitatively image AP waveforms at the Drosophila NMJ without disrupting baseline synaptic transmission or neuromuscular development. It is established that Shaker mutations cause a dramatic increase in neurotransmitter release, suggesting that Shaker is predominantly responsible for AP repolarization. Here we demonstrate that this effect is caused by a concomitant loss of both Shaker and slowpoke (slo) channel activity because of the low extracellular calcium concentrations (0.2-0.5 mM) used typically to assess synaptic transmission in Shaker. In contrast, at physiological extracellular calcium (1.5 mM), the role of Shaker during AP repolarization is limited. We then provide evidence that calcium influx through synaptic CaV2.1 channels and subsequent recruitment of Slo channel activity is important, in concert with Shaker, to ensure proper AP repolarization. Finally, we show that Slo assumes a dominant repolarizing role during repetitive nerve stimulation. During repetitive stimulation, Slo effectively compensates for Shaker channel inactivation, stabilizing AP repolarization and limiting neurotransmitter release. Thus, we have defined an essential role for Slo channels during synaptic AP repolarization and have revised our understanding of Shaker channels at this model synapse.

At synapses in organisms ranging from fly to human, a decrease in postsynaptic neurotransmitter receptor function elicits a homeostatic increase in presynaptic release that restores baseline synaptic efficacy. This process, termed presynaptic homeostasis, requires a retrograde, trans-synaptic signal of unknown identity. In a forward genetic screen for homeostatic plasticity genes, we identified multiplexin. Multiplexin is the Drosophila homolog of Collagen XV/XVIII, a matrix protein that can be proteolytically cleaved to release Endostatin, an antiangiogenesis signaling factor. Here we demonstrate that Multiplexin is required for normal calcium channel abundance, presynaptic calcium influx, and neurotransmitter release. Remarkably, Endostatin has a specific activity, independent of baseline synapse development, that is required for the homeostatic modulation of presynaptic calcium influx and neurotransmitter release. Our data support a model in which proteolytic release of Endostatin signals trans-synaptically, acting in concert with the presynaptic CaV2.1 calcium channel, to promote presynaptic homeostasis.

Ion channel gene expression can vary substantially among neurons of a given type, even though neuron-type-specific firing properties remain stable and reproducible. The mechanisms that modulate ion channel gene expression and stabilize neural firing properties are unknown. In Drosophila, we demonstrate that loss of the Shal potassium channel induces the compensatory rebalancing of ion channel expression including, but not limited to, the enhanced expression and function of Shaker and slowpoke. Using genomic and network modeling approaches combined with genetic and electrophysiological assays, we demonstrate that the transcription factor Krüppel is necessary for the homeostatic modulation of Shaker and slowpoke expression. Remarkably, Krüppel induction is specific to the loss of Shal, not being observed in five other potassium channel mutants that cause enhanced neuronal excitability. Thus, homeostatic signaling systems responsible for rebalancing ion channel expression can be selectively induced after the loss or impairment of a specific ion channel.

The brain is astonishing in its complexity and capacity for change. This has fascinated scientists for more than a century, filling the pages of this journal for the past 25 years. But a paradigm shift is underway. It seems likely that the plasticity that drives our ability to learn and remember can only be meaningful in the context of otherwise stable, reproducible, and predictable baseline neural function. Without the existence of potent mechanisms that stabilize neural function, our capacity to learn and remember would be lost in the chaos of daily experiential change. This underscores two great mysteries in neuroscience. How are the functional properties of individual neurons and neural circuits stably maintained throughout life? And, in the face of potent stabilizing mechanisms, how can neural circuitry be modified during neural development, learning, and memory? Answers are emerging in the rapidly developing field of 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). We demonstrate that ppk11 and ppk16 are necessary in presynaptic motoneurons for both the acute induction and long-term maintenance of synaptic homeostasis. We show that 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.

Rab3 interacting molecules (RIMs) are evolutionarily conserved scaffolding proteins that are located at presynaptic active zones. In the mammalian nervous system, RIMs have two major activities that contribute to the fidelity of baseline synaptic transmission: they concentrate calcium channels at the active zone and facilitate synaptic vesicle docking/priming. Here we confirm that RIM has an evolutionarily conserved function at the Drosophila neuromuscular junction and then define a novel role for RIM during homeostatic synaptic plasticity. We show that loss of RIM disrupts baseline vesicle release, diminishes presynaptic calcium influx, and diminishes the size of the readily-releasable pool (RRP) of synaptic vesicles, consistent with known activities of RIM. However, loss of RIM also completely blocks the homeostatic enhancement of presynaptic neurotransmitter release that normally occurs after inhibition of postsynaptic glutamate receptors, a process termed synaptic homeostasis. It is established that synaptic homeostasis requires enhanced presynaptic calcium influx as a mechanism to potentiate vesicle release. However, despite a defect in baseline calcium influx in rim mutants, the homeostatic modulation of calcium influx proceeds normally. Synaptic homeostasis is also correlated with an increase in the size of the RRP of synaptic vesicles, although the mechanism remains unknown. Here we demonstrate that the homeostatic modulation of the RRP is blocked in the rim mutant background. Therefore, RIM-dependent modulation of the RRP is a required step during homeostatic plasticity. By extension, homeostatic plasticity appears to require two genetically separable processes, the enhancement of presynaptic calcium influx and a RIM-dependent modulation of the RRP.

The molecular mechanisms underlying the homeostatic modulation of presynaptic neurotransmitter release are largely unknown. We have previously used an electrophysiology-based forward genetic screen to assess the function of >400 neuronally expressed genes for a role in the homeostatic control of synaptic transmission at the neuromuscular junction of Drosophila melanogaster. This screen identified a critical function for dysbindin, a gene linked to schizophrenia in humans (Dickman and Davis, 2009). Biochemical studies in other systems have shown that Snapin interacts with Dysbindin, prompting us to test whether Snapin might be involved in the mechanisms of synaptic homeostasis. Here, we demonstrate that loss of snapin blocks the homeostatic modulation of presynaptic vesicle release following inhibition of postsynaptic glutamate receptors. This is true for both the rapid induction of synaptic homeostasis induced by pharmacological inhibition of postsynaptic glutamate receptors, and the long-term expression of synaptic homeostasis induced by the genetic deletion of the muscle-specific GluRIIA glutamate receptor subunit. Loss of snapin does not alter baseline synaptic transmission, synapse morphology, synapse growth, or the number or density of active zones, indicating that the block of synaptic homeostasis is not a secondary consequence of impaired synapse development. Additional genetic evidence suggests that snapin functions in concert with dysbindin to modulate vesicle release and possibly homeostatic plasticity. Finally, we provide genetic evidence that the interaction of Snapin with SNAP25, a component of the SNARE complex, is also involved in synaptic homeostasis.

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. Here we address 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, we reveal a retrograde, transsynaptic modulation of presynaptic Ca(2+) influx that is sufficient to account for the rapid induction and sustained expression of the homeostatic change in vesicle release. We show that 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, we establish that retrograde, transsynaptic modulation of presynaptic Ca(2+) influx through CaV2.1 channels is a key factor underlying the homeostatic regulation of neurotransmitter release.

The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. In this paper, we demonstrate that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. We then provide evidence that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. We further demonstrate that PDK1 is also a presynaptic protein, though it is distributed more broadly. We present a model in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release.

Loss of spectrin or ankyrin in the presynaptic motoneuron disrupts the synaptic microtubule cytoskeleton and leads to disassembly of the neuromuscular junction (NMJ). Here, we demonstrate that NMJ disassembly after loss of alpha-spectrin can be suppressed by expression of a Wld(S) transgene, providing evidence for a Wallerian-type degenerative mechanism. We then identify a second signaling system. Enhanced MAPK-JNK-Fos signaling suppresses NMJ disassembly despite loss of presynaptic alpha-spectrin or ankyrin2-L. This signaling system is activated after an acute cytoskeletal disruption, suggesting an endogenous role during neurological stress. This signaling system also includes delayed, negative feedback via the JNK phosphatase puckered, which inhibits JNK-Fos to allow NMJ disassembly in the presence of persistent cytoskeletal stress. Finally, the MAPK-JNK pathway is not required for baseline NMJ stabilization during normal NMJ growth. We present a model in which signaling via JNK-Fos functions as a stress response system that is transiently activated after cytoskeletal disruption to enhance NMJ stability, and is then shut off allowing NMJ disassembly during persistent cytoskeletal disruption.

Synaptic connections are established with characteristic, cell type-specific size and spacing. In this study, we document a role for the postsynaptic Spectrin skeleton in this process. We use transgenic double-stranded RNA to selectively eliminate alpha-Spectrin, beta-Spectrin, or Ankyrin. In the absence of postsynaptic alpha- or beta-Spectrin, active zone size is increased and spacing is perturbed. In addition, subsynaptic muscle membranes are significantly altered. However, despite these changes, the subdivision of the synapse into active zone and periactive zone domains remains intact, both pre- and postsynaptically. Functionally, altered active zone dimensions correlate with an increase in quantal size without a change in presynaptic vesicle size. Mechanistically, beta-Spectrin is required for the localization of alpha-Spectrin and Ankyrin to the postsynaptic membrane. Although Ankyrin is not required for the localization of the Spectrin skeleton to the neuromuscular junction, it contributes to Spectrin-mediated synapse development. We propose a model in which a postsynaptic Spectrin-actin lattice acts as an organizing scaffold upon which pre- and postsynaptic development are arranged.