The Psychiatric Cell Map Initiative: A Convergent Systems Biological Approach to Illuminating Key Molecular Pathways in Neuropsychiatric Disorders.

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.

Retrograde semaphorin-plexin signalling drives homeostatic synaptic plasticity.

Homeostatic signalling systems ensure stable but flexible neural activity and animal behaviour. Presynaptic homeostatic plasticity is a conserved form of neuronal homeostatic signalling that is observed in organisms ranging from Drosophila to human. Defining the underlying molecular mechanisms of neuronal homeostatic signalling will be essential in order to establish clear connections to the causes and progression of neurological disease. During neural development, semaphorin-plexin signalling instructs axon guidance and neuronal morphogenesis. However, semaphorins and plexins are also expressed in the adult brain. 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.

Engineering a light-activated caspase-3 for precise ablation of neurons in vivo.

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.

Homeostatic control of presynaptic neurotransmitter release.

It is well established that the active properties of nerve and muscle cells are stabilized by homeostatic signaling systems. In organisms ranging from Drosophila to humans, neurons restore baseline function in the continued presence of destabilizing perturbations by rebalancing ion channel expression, modifying neurotransmitter receptor surface expression and trafficking, and modulating neurotransmitter release. This review focuses on the homeostatic modulation of presynaptic neurotransmitter release, termed presynaptic homeostasis. First, we highlight criteria that can be used to define a process as being under homeostatic control. Next, we review the remarkable conservation of presynaptic homeostasis at the Drosophila, mouse, and human neuromuscular junctions and emerging parallels at synaptic connections in the mammalian central nervous system. We then highlight recent progress identifying cellular and molecular mechanisms. We conclude by reviewing emerging parallels between the mechanisms of homeostatic signaling and genetic links to neurological disease.

Archaerhodopsin voltage imaging: synaptic calcium and BK channels stabilize action potential repolarization at the Drosophila neuromuscular junction.

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.

Snapin is critical for presynaptic homeostatic plasticity.

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.

RIM controls homeostatic plasticity through modulation of the readily-releasable vesicle pool.

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.

Stathmin is required for stability of the Drosophila neuromuscular junction.

Synaptic connections can be stably maintained for prolonged periods, yet can be rapidly disassembled during the developmental refinement of neural circuitry and following cytological insults that lead to neurodegeneration. To date, the molecular mechanisms that determine whether a synapse will persist versus being remodeled or eliminated remain poorly understood. Mutations in Drosophila stathmin were isolated in two independent genetic screens that sought mutations leading to impaired synapse stability at the Drosophila neuromuscular junction (NMJ). Here we demonstrate that Stathmin, a protein that associates with microtubules and can function as a point of signaling integration, is necessary to maintain the stability of the Drosophila NMJ. We show that Stathmin protein is widely distributed within motoneurons and that loss of Stathmin causes impaired NMJ growth and stability. In addition, we show that stathmin mutants display evidence of defective axonal transport, a common feature associated with neuronal degeneration and altered synapse stability. The disassembly of the NMJ in stathmin includes a predictable sequence of cytological events, suggesting that a common program of synapse disassembly is induced following the loss of Stathmin protein. These data define a required function for Stathmin during synapse maintenance in a model system in which there is only a single stathmin gene, enabling future genetic investigation of Stathmin function with potential relevance to the cause and progression of neuromuscular degenerative disease.

Negative regulation of active zone assembly by a newly identified SR protein kinase.

Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila) or ribbon (vertebrates) are believed to facilitate vesicle movement to the active zone (AZ) of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc) that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D). This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp) in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.

Activation and expansion of presynaptic signaling foci drives presynaptic homeostatic plasticity.

Presynaptic homeostatic plasticity (PHP) adaptively regulates synaptic transmission in health and disease. Despite identification of numerous genes that are essential for PHP, we lack a dynamic framework to explain how PHP is initiated, potentiated, and limited to achieve precise control of vesicle fusion. Here, utilizing both mice and Drosophila, we demonstrate that PHP progresses through the assembly and physical expansion of presynaptic signaling foci where activated integrins biochemically converge with trans-synaptic Semaphorin2b/PlexinB signaling. Each component of the identified signaling complexes, including alpha/beta-integrin, Semaphorin2b, PlexinB, talin, and focal adhesion kinase (FAK), and their biochemical interactions, are essential for PHP. Complex integrity requires the Sema2b ligand and complex expansion includes a ∼2.5-fold expansion of active-zone associated puncta composed of the actin-binding protein talin. Finally, complex pre-expansion is sufficient to accelerate the rate and extent of PHP. A working model is proposed incorporating signal convergence with dynamic molecular assemblies that instruct PHP.

NMDAR-dependent presynaptic homeostasis in adult hippocampus: Synapse growth and cross-modal inhibitory plasticity.

Homeostatic plasticity (HP) encompasses a suite of compensatory physiological processes that counteract neuronal perturbations, enabling brain resilience. Currently, we lack a complete description of the homeostatic processes that operate within the mammalian brain. Here, we demonstrate that acute, partial AMPAR-specific antagonism induces potentiation of presynaptic neurotransmitter release in adult hippocampus, a form of compensatory plasticity that is consistent with the expression of presynaptic homeostatic plasticity (PHP) documented at peripheral synapses. We show that this compensatory plasticity can be induced within minutes, requires postsynaptic NMDARs, and is expressed via correlated increases in dendritic spine volume, active zone area, and docked vesicle number. Further, simultaneous postsynaptic genetic reduction of GluA1, GluA2, and GluA3 in triple heterozygous knockouts induces potentiation of presynaptic release. Finally, induction of compensatory plasticity at excitatory synapses induces a parallel, NMDAR-dependent potentiation of inhibitory transmission, a cross-modal effect consistent with the anti-epileptic activity of AMPAR-specific antagonists used in humans.

The BMP ligand Gbb gates the expression of synaptic homeostasis independent of synaptic growth control.

Inhibition of postsynaptic glutamate receptors at the Drosophila NMJ initiates a compensatory increase in presynaptic release termed synaptic homeostasis. BMP signaling is necessary for normal synaptic growth and stability. It remains unknown whether BMPs have a specific role during synaptic homeostasis and, if so, whether BMP signaling functions as an instructive retrograde signal that directly modulates presynaptic transmitter release. Here, we demonstrate that the BMP receptor (Wit) and ligand (Gbb) are necessary for the rapid induction of synaptic homeostasis. We also provide evidence that both Wit and Gbb have functions during synaptic homeostasis that are separable from NMJ growth. However, further genetic experiments demonstrate that Gbb does not function as an instructive retrograde signal during synaptic homeostasis. Rather, our data indicate that Wit and Gbb function via the downstream transcription factor Mad and that Mad-mediated signaling is continuously required during development to confer competence of motoneurons to express synaptic homeostasis.

NF-kappaB, IkappaB, and IRAK control glutamate receptor density at the Drosophila NMJ.

NF-kappaB signaling has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. However, the cellular and molecular activity of NF-kappaB signaling within the nervous system remains to be clearly defined. Here, we show that the NF-kappaB and IkappaB homologs Dorsal and Cactus surround postsynaptic glutamate receptor (GluR) clusters at the Drosophila NMJ. We then show that mutations in dorsal, cactus, and IRAK/pelle kinase specifically impair GluR levels, assayed immunohistochemically and electrophysiologically, without affecting NMJ growth, the size of the postsynaptic density, or homeostatic plasticity. Additional genetic experiments support the conclusion that cactus functions in concert with, rather than in opposition to, dorsal and pelle in this process. Finally, we provide evidence that Dorsal and Cactus act posttranscriptionally, outside the nucleus, to control GluR density. Based upon our data we speculate that Dorsal, Cactus, and Pelle could function together, locally at the postsynaptic density, to specify GluR levels.

Synaptic homeostasis is consolidated by the cell fate gene gooseberry, a Drosophila pax3/7 homolog.

In a large-scale screening effort, we identified the gene gooseberry (gsb) as being necessary for synaptic homeostasis at the Drosophila neuromuscular junction. The gsb gene encodes a pair-rule transcription factor that participates in embryonic neuronal cell fate specification. Here, we define a new postembryonic role for gooseberry. We show that gsb becomes widely expressed in the postembryonic CNS, including within mature motoneurons. Loss of gsb does not alter neuromuscular growth, morphology, or the distribution of essential synaptic proteins. However, gsb function is required postembryonically for the sustained expression of synaptic homeostasis. In GluRIIA mutant animals, miniature EPSP (mEPSP) amplitudes are significantly decreased, and there is a compensatory homeostatic increase in presynaptic release that restores normal muscle excitation. Loss of gsb significantly impairs the homeostatic increase in presynaptic release in the GluRIIA mutant. Interestingly, gsb is not required for the rapid induction of synaptic homeostasis. Furthermore, gsb seems to be specifically involved in the mechanisms responsible for a homeostatic increase in presynaptic release, since it is not required for the homeostatic decrease in presynaptic release observed following an increase in mEPSP amplitude. Finally, Gsb has been shown to antagonize Wingless signaling during embryonic fate specification, and we present initial evidence that this activity is conserved during synaptic homeostasis. Thus, we have identified a gene (gsb) that distinguishes between rapid induction versus sustained expression of synaptic homeostasis and distinguishes between the mechanisms responsible for homeostatic increase versus decrease in synaptic vesicle release.

Clathrin dependence of synaptic-vesicle formation at the Drosophila neuromuscular junction.

BACKGROUND: Among the most prominent molecular constituents of a recycling synaptic vesicle is the clathrin triskelion, composed of clathrin light chain (Clc) and clathrin heavy chain (Chc). Remarkably, it remains unknown whether clathrin is strictly necessary for the stimulus-dependent re-formation of a synaptic vesicle and, conversely, whether clathrin-independent vesicle endocytosis exists at the neuronal synapse. RESULTS: We employ FlAsH-FALI-mediated protein photoinactivation to rapidly (3 min) and specifically disrupt Clc function at the Drosophila neuromuscular junction. We first demonstrate that Clc photoinactivation does not impair synaptic-vesicle fusion. We then provide electrophysiological and ultrastructural evidence that synaptic vesicles, once fused with the plasma membrane, cannot be re-formed after Clc photoinactivation. Finally, we demonstrate that stimulus-dependent membrane internalization occurs after Clc photoinactivation. However, newly internalized membrane fails to resolve into synaptic vesicles. Rather, newly internalized membrane forms large and extensive internal-membrane compartments that are never observed at a wild-type synapse. CONCLUSIONS: We make three major conclusions. (1) FlAsH-FALI-mediated protein photoinactivation rapidly and specifically disrupts Clc function with no effect on synaptic-vesicle fusion. (2) Synaptic-vesicle re-formation does not occur after Clc photoinactivation. By extension, clathrin-independent "kiss-and-run" endocytosis does not sustain synaptic transmission during a stimulus train at this synapse. (3) Stimulus-dependent, clathrin-independent membrane internalization exists at this synapse, but it is unable to generate fusion-competent, small-diameter synaptic vesicles.

A postsynaptic spectrin scaffold defines active zone size, spacing, and efficacy at the Drosophila neuromuscular junction.

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.

SVIP is a molecular determinant of lysosomal dynamic stability, neurodegeneration and lifespan.

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.

Discrete residues in the c(2)b domain of synaptotagmin I independently specify endocytic rate and synaptic vesicle size.

It has been demonstrated that synapses lacking functional synaptotagmin I (Syt I) have a decreased rate of synaptic vesicle endocytosis. Beyond this, the function of Syt I during endocytosis remains undefined. Here, we demonstrate that a decreased rate of endocytosis in syt(null) mutants correlates with a stimulus-dependent perturbation of membrane internalization, assayed ultrastructurally. We then separate the mechanisms that control endocytic rate and vesicle size by mapping these processes to discrete residues in the Syt I C(2)B domain. Mutation of a poly-lysine motif alters vesicle size but not endocytic rate, whereas the mutation of calcium-coordinating aspartate residues (syt-D3,4N) alters endocytic rate but not vesicle size. Finally, slowed endocytic rate in the syt-D3,4N animals, but not syt(null) animals, can be rescued by elevating extracellular calcium concentration, supporting the conclusion that calcium coordination within the C(2)B domain contributes to the control of endocytic rate.

Mechanisms underlying the rapid induction and sustained expression of synaptic homeostasis.

Homeostatic signaling systems are thought to interface with the mechanisms of neural plasticity to achieve stable yet flexible neural circuitry. However, the time course, molecular design, and implementation of homeostatic signaling remain poorly defined. Here we demonstrate that a homeostatic increase in presynaptic neurotransmitter release can be induced within minutes following postsynaptic glutamate receptor blockade. The rapid induction of synaptic homeostasis is independent of new protein synthesis and does not require evoked neurotransmission, indicating that a change in the efficacy of spontaneous quantal release events is sufficient to trigger the induction of synaptic homeostasis. Finally, both the rapid induction and the sustained expression of synaptic homeostasis are blocked by mutations that disrupt the pore-forming subunit of the presynaptic Ca(V)2.1 calcium channel encoded by cacophony. These data confirm the presynaptic expression of synaptic homeostasis and implicate presynaptic Ca(V)2.1 in a homeostatic retrograde signaling system.

Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo.

Neurotransmission requires a balance of synaptic vesicle exocytosis and endocytosis. Synaptotagmin I (Syt I) is widely regarded as the primary calcium sensor for synaptic vesicle exocytosis. Previous biochemical data suggest that Syt I may also function during synaptic vesicle endocytosis; however, ultrastructural analyses at synapses with impaired Syt I function have provided an indirect and conflicting view of the role of Syt I during synaptic vesicle endocytosis. Until now it has not been possible experimentally to separate the exocytic and endocytic functions of Syt I in vivo. Here, we test directly the role of Syt I during endocytosis in vivo. We use quantitative live imaging of a pH-sensitive green fluorescent protein fused to a synaptic vesicle protein (synapto-pHluorin) to measure the kinetics of endocytosis in sytI-null Drosophila. We then combine live imaging of the synapto-pHluorins with photoinactivation of Syt I, through fluorescein-assisted light inactivation, after normal Syt I-mediated vesicle exocytosis. By inactivating Syt I only during endocytosis, we demonstrate that Syt I is necessary for the endocytosis of synaptic vesicles that have undergone exocytosis using a functional Syt I protein.