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BACE1 initiates production of β-amyloid peptides (Aβ), which is associated with cognitive dysfunction in Alzheimer’s disease (AD) due to abnormal oligomerization and aggregation. While BACE1 inhibitors show strong reduction in Aβ deposition, they fail to improve cognitive function in patients, largely due to its role in synaptic function. We show that BACE1 is required for optimal release of synaptic vesicles. BACE1 deficiency or inhibition decreases synaptic vesicle docking in the synaptic active zones. Consistently, BACE1-null mice or mice treated with clinically tested BACE1 inhibitors Verubecestat and Lanabecestat exhibit severe reduction in hippocampal LTP and learning behaviors. To counterbalance this synaptic deficit, we discovered that BACE1-null mice treated with positive allosteric modulators (PAMs) of metabotropic glutamate receptor 1 (mGluR1), whose levels were reduced in BACE1-null mice and significantly improved long-term potentiation and cognitive behaviors. Similarly, mice treated with mGluR1 PAM showed significantly mitigated synaptic deficits caused by BACE1 inhibitors. Together, our data suggest that a therapy combining BACE1 inhibitors for reducing amyloid deposition and an mGluR1 PAM for counteracting BACE1-mediated synaptic deficits appears to be an effective approach for treating AD patients.

Syntaxin-binding protein 1 (STXBP1; also called MUNC18–1), encoded by STXBP1, is an essential component of the molecular machinery that controls synaptic vesicle docking and fusion. De novo pathogenic variants of STXBP1 cause a complex set of neurological disturbances, namely STXBP1 encephalopathy (STXBP1-E) that includes epilepsy, neurodevelopmental disorders and neurodegeneration. Several animal studies have suggested the contribution of GABAergic dysfunction in STXBP1-E pathogenesis. However, the pathophysiological changes in GABAergic neurons of these patients are still poorly understood. Here, we exclusively generated GABAergic neurons from STXBP1-E patient-derived induced pluripotent stem cells (iPSCs) by transient expression of the transcription factors ASCL1 and DLX2. We also generated CRISPR/Cas9-edited isogenic iPSC-derived GABAergic (iPSC GABA) neurons as controls. We demonstrated that the reduction in STXBP1 protein levels in patient-derived iPSC GABA neurons was slight (approximately 20%) compared to the control neurons, despite a 50% reduction in STXBP1 mRNA levels. Using a microelectrode array–based assay, we found that patient-derived iPSC GABA neurons exhibited dysfunctional maturation with reduced numbers of spontaneous spikes and bursts. These findings reinforce the idea that GABAergic dysfunction is a crucial contributor to STXBP1-E pathogenesis. Moreover, gene expression analysis revealed specific dysregulation of genes previously implicated in epilepsy, neurodevelopment and neurodegeneration in patient-derived iPSC GABA neurons, namely KCNH1, KCNH5, CNN3, RASGRF1, SEMA3A, SIAH3 and INPP5F. Thus, our study provides new insights for understanding the biological processes underlying the widespread neuropathological features of STXBP1-E.

Conspectusα-Synuclein (α-syn) is a key protein in the etiology of Parkinson's disease. In a disease state, α-syn accumulates as insoluble amyloid fibrils enriched in β-sheet structure. However, in its functional state, α-syn adopts an amphipathic helix upon membrane association and plays a role in synaptic vesicle docking, fusion, and clustering. In this Account, we describe our contributions made in the past decade toward developing a molecular understanding of α-syn membrane interactions, which are crucial for function and have pathological implications. Three topics are covered: α-syn membrane binding probed by neutron reflectometry (NR), the effects of membrane on α-syn amyloid formation, and interactions of α-syn with cellular membranes.NR offers a unique perspective by providing direct measurements of protein penetration depth. By the use of segmentally deuterated α-syn generated through native chemical ligation, the spatial resolution of specific membrane-bound polypeptide regions was determined by NR. Additionally, we used NR to characterize the membrane-bound complex of α-syn and glucocerebrosidase, a lysosomal hydrolase whose mutations are a common genetic risk factor for Parkinson's disease. Although phosphatidylcholine (PC) is the most abundant lipid species in mammalian cells, interactions of PC with α-syn have been largely ignored because they are substantially weaker compared with the electrostatically driven binding of negatively charged lipids. We discovered that α-syn tubulates zwitterionic PC membranes, which is likely related to its involvement in synaptic vesicle fusion by stabilization of membrane curvature. Interestingly, PC lipid tubules inhibit amyloid formation, in contrast to anionic phosphatidylglycerol lipid tubules, which stimulate protein aggregation. We also found that membrane fluidity influences the propensity of α-synuclein amyloid formation. Most recently, we obtained direct evidence of binding of α-syn to exocytic sites on intact cellular membranes using a method called cellular unroofing. This method provides direct access to the cytosolic plasma membrane. Importantly, measurements of fluorescence lifetime distributions revealed that α-syn is more conformationally dynamic at the membrane interface than previously appreciated. This exquisite responsiveness to specific lipid composition and membrane topology is important for both its physiological and pathological functions. Collectively, our work has provided insights into the effects of the chemical nature of phospholipid headgroups on the interplay among membrane remodeling, protein structure, and α-syn amyloid formation.

Efficient neurotransmitter release at the presynaptic terminal requires docking of synaptic vesicles to the active zone membrane and formation of fusion-competent synaptic vesicles near voltage-gated Ca2+channels. Rab3-interacting molecule (RIM) is a critical active zone organizer, as it recruits Ca2+channels and activates synaptic vesicle docking and priming via Munc13-1. However, our knowledge about Munc13-independent contributions of RIM to active zone functions is limited. To identify the functions that are solely mediated by RIM, we used genetic manipulations to control RIM and Munc13-1 activity in cultured hippocampal neurons from mice of either sex and compared synaptic ultrastructure and neurotransmission. We found that RIM modulates synaptic vesicle localization in the proximity of the active zone membrane independent of Munc13-1. In another step, both RIM and Munc13 mediate synaptic vesicle docking and priming. In addition, while the activity of both RIM and Munc13-1 is required for Ca2+-evoked release, RIM uniquely controls neurotransmitter release efficiency. However, activity-dependent augmentation of synaptic vesicle pool size relies exclusively on the action of Munc13s. Based on our results, we extend previous findings and propose a refined model in which RIM and Munc13-1 act in overlapping and independent stages of synaptic vesicle localization and release.SIGNIFICANCE STATEMENTThe presynaptic active zone is composed of scaffolding proteins that functionally interact to localize synaptic vesicles to release sites, ensuring neurotransmission. Our current knowledge of the presynaptic active zone function relies on structure-function analysis, which has provided detailed information on the network of interactions and the impact of active zone proteins. Yet, the hierarchical, redundant, or independent cooperation of each active zone protein to synapse functions is not fully understood. Rab3-interacting molecule and Munc13 are the two key functionally interacting active zone proteins. Here, we dissected the distinct actions of Rab3-interacting molecule and Munc13-1 from both ultrastructural and physiological aspects. Our findings provide a more detailed view of how these two presynaptic proteins orchestrate their functions to achieve synaptic transmission.

Background: Gadolinium (Gd3+) is a chemical element belonging to the lanthanide group and commonly used in magnetic resonance imaging (MRI) as a contrast agent. However, recently, gadolinium has been reported deposition in the body after a patient receives multiple injections. Gadolinium is a potent block and competes with calcium diffusion into the presynaptic. There has not been a precise mechanism of gadolinium blocking calcium channel as a channel of calcium diffusion to presynaptic until now.Objective: This study aims to investigate the mechanism of calcium influx model and the effect of neurotransmitter release to the synaptic cleft influenced by the presence of Gd3+. Material and Methods: Monte Carlo Cell simulation was used to analyze simulation and also Blender was used to create and visualize the model for synapse. The synapse modeled by a form resembling the actual synapse base on a spherical shape. Results: The presence of gadolinium around the presynaptic has been disturbing diffusion of calcium influx presynaptic. The result shows that the presence of gadolinium around the presynaptic has caused a decrease in the amount of calcium influx presynaptic. These factors contribute to reducing the establishment of the active membrane, then the amount of synaptic vesicle docking and finally the amount of released neurotransmitter.Conclusion: Gadolinium and calcium compete with each other across of calcium channel. The presence of gadolinium has caused a chain effect for signal transmission at the chemical synapse, reducing the amount of active membrane, synaptic vesicle docking, and releasing neurotransmitter.

Neurotransmission is the basis of brain functions, and controllable neurotransmission tuning constitutes an attractive approach for interventions in a wide range of neurologic disorders and for synapse-based therapeutic treatments. Graphene-family nanomaterials (GFNs) offer promising advantages for biomedical applications, particularly in neurology. Our study suggests that reduced graphene oxide (rGO) serves as a neurotransmission modulator and reveals that the cellular oxidation of rGO plays a crucial role in this effect. We found that rGO could be oxidized via cellular reactive oxygen species (ROS), as evidenced by an increased number of oxygen-containing functional groups on the rGO surface. Cellular redox signaling, which involves NADPH oxidases and mitochondria, was initiated and subsequently intensified rGO oxidation. The study further shows that the blockage of synaptic vesicle docking and fusion induced through a disturbance of actin dynamics is the underlying mechanism through which oxidized rGO exerts depressant effects on neurotransmission. Importantly, this depressant effect could be modulated by restricting the cellular ROS levels and stabilizing the actin dynamics. Taken together, our results identify the complicated biological effects of rGO as a controlled neurotransmission modulator and can provide helpful information for the future design of graphene materials for neurobiological applications.

Active zones are molecular machines that control neurotransmitter release through synaptic vesicle docking and priming, and through coupling of these vesicles to Ca2+ entry. The complexity of active zone machinery has made it challenging to determine which mechanisms drive these roles in release. Here, we induce RIM+ELKS knockout to eliminate active zone scaffolding networks, and then reconstruct each active zone function. Re-expression of RIM1-Zn fingers positioned Munc13 on undocked vesicles and rendered them release-competent. Reconstitution of release-triggering required docking of these vesicles to Ca2+ channels. Fusing RIM1-Zn to CaVβ4-subunits sufficed to restore docking, priming and release-triggering without reinstating active zone scaffolds. Hence, exocytotic activities of the 80 kDa CaVβ4-Zn fusion protein bypassed the need for megadalton-sized secretory machines. These data define key mechanisms of active zone function, establish that fusion competence and docking are mechanistically separable, and reveal that active zone scaffolding networks are not required for release.

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.

Early life traumas lead to neuroprotection by preconditioning mechanisms. To determine which genes and pathways are most likely involved in specific adaptive effects, immature hippocampal cultures were exposed to a single high dose of glutamate (250 μM), NMDA (100 μM), or KA (300 μM) for 48 h (5–7 DIV) based on our prior “two hit” in vitro model of preconditioning. Transcriptome profiling and immunocytochemistry of gene candidates were performed 7 days later when cultured neurons mature (14 DIV). Many genes were up- and down- regulated involving distinct Ca2+-binding protein families, G-coupled proteins, various growth factors, synaptic vesicle docking factors, certain neurotransmitter receptors, heat shock, oxidative stress, and certain anti-apoptotic Bcl-2 gene members that influence neuronal survival. Immunohistochemistry showed a marked decrease in the number of Calb1 and Calm2 positive neurons following NMDA but not after glutamate exposure whereas ryanodine and Cav1.2 voltage gated channel expression was less affected. Survivors had marked increases in Calm2 immunostaining; however, high-density neural clusters observed in controls, were depleted after NMDA and partly diminished after glutamate. While NR1 mRNA expression was decreased in the microarray, specific antibodies revealed selective loss of the NR1C1 splice variant. Calm2 which can inactivate NMDA receptors by binding to C1 but not C2 regions of its NR1 subunit suggests that loss of the C1 splice variant will reduce co-regulation with Calm2 and alter NR1 trafficking, phosphorylation, and NMDA currents following early life NMDA exposure. A dramatic reduction in the density of GABAAα5 and GABAB receptor expressing neurons was observed after NMDA exposure but immunodensity measurements were unchanged as was the expression of the GABA synthesizing enzyme, GAD, suggesting that fast inhibitory neurotransmission and response to benzodiazepines and GABAB-mediated IPSPs may be preserved in matured survivors. Selective upregulation of Chat and CNRIP was detected after glutamate treatment suggesting this condition would decrease cholinergic and excitatory neurotransmission by decreasing Ach content and CB1 interacting protein function. This decrease likely contributes to memory and attention tasks deficits that follow a single early neurological insult. Diverse changes that follow overactivation of excitatory networks of immature neurons appear long-lasting or permanent and are expected to have profound effects on network function and adaptive responses to further insult.

Early exposure to anesthetics may interfere with synaptic development and lead to cognitive deficits. We previously demonstrated a decrease in vesicles docked at and within 100 nm from the presynaptic membrane in hippocampal nerve terminals of neonatal rats after anesthesia. Hence, we designed this study to assess the effects of neonatal anesthesia on Synapsin 1 (Syn 1) and Synaptotagmin 1 (Syt 1), two key regulators of vesicle docking and fusion. To test the link between changes in Syn 1 and Syt 1 and behavioral deficits observed after neonatal anesthesia, we also assessed retention memory and fear conditioning in adolescent rats after neonatal anesthesia. Pups received a combination of clinical anesthetics, then Syn 1 and Syt 1 mRNA and protein expression were determined at the peak (postnatal day 8, P8), part-way through (P12) and end of synaptogenesis (P24) in the CA1-subiculum by qPCR and Western Blotting. Anesthesia decreased Syn1 and Syt1 mRNA expression at P8 (p

International audience; Munc13-1 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggest that multiple copies of Munc13-1 form nanoassemblies in active zones of neurons. However, it is not known if such clustering of Munc13-1 is correlated with multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13-1 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13-1, and is indeed required for vesicle binding to bilayers containing Munc13-1. Purified Munc13-1 protein reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ~20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters containing a minimum of 6 copies of Munc13-1 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C 2 C domain of Munc13-1 is not required for Munc13-1 clustering, but is required for efficient vesicle capture. This capture is largely due to a combination of electrostatic and hydrophobic interactions between the C 2 C domain and the vesicle membrane.

Seizures are a common neonatal neurological disorder with an incidence of 1 to 5 in 1,000 live births. Genetic and metabolic epilepsies account for 10% to 12% of all neonatal seizures. Correct identification and diagnosis are important factors, as they carry treatment and management implications. Clinical history, neurological examination, seizure types, epilepsy syndromes, and electroencephalogram findings can be used to guide the diagnosis of epilepsy. Genetic and metabolic epilepsies in neonates can be categorized practically into two groups: amenably treatable disorders, and the most common genetic epilepsies. The treatable disorders primarily consist of inborn errors of metabolism that have a specific therapy. The most common genetic epilepsies include monogenic disorders, which usually result from channelopathies, synaptic vesicle docking/release defect, or dysfunction of cell signaling. A step-wise diagnostic approach to genetic and metabolic epilepsies is proposed in this article to aid clinicians in providing care for newborns with seizures. [ Pediatr Ann . 2020;50(6):e245–e253.]

Synchronous release at neuronal synapses is accomplished by a machinery that senses calcium influx and fuses the synaptic vesicle and plasma membranes to release neurotransmitters. Previous studies suggested the calcium sensor Synaptotagmin (Syt) is a facilitator of vesicle docking and both a facilitator and inhibitor of fusion. On phospholipid monolayers, the Syt C2AB domain spontaneously oligomerized into rings that are disassembled by Ca2+, suggesting Syt rings may clamp fusion as membrane-separating “washers” until Ca2+-mediated disassembly triggers fusion and release (Wang et al., 2014). Here we combined mathematical modeling with experiment to measure mechanical properties of Syt rings and to test this mechanism. Consistent with experiment, the model quantitatively recapitulates observed Syt ring-induced dome and volcano shapes on phospholipid monolayers, and predicts rings are stabilized by anionic phospholipid bilayers or bulk solution with ATP. The selected ring conformation is highly sensitive to membrane composition and bulk ATP levels, a property that may regulate vesicle docking and fusion in ATP-rich synaptic terminals. We find the Syt molecules hosted by a synaptic vesicle oligomerize into a halo, unbound from the vesicle, but in proximity to sufficiently PIP2-rich plasma membrane (PM) domains the PM-bound trans Syt ring conformation is preferred. Thus, the Syt halo serves as landing gear for spatially directed docking at PIP2-rich sites that define the active zones of exocytotic release, positioning the Syt ring to clamp fusion and await calcium. Our results suggest the Syt ring is both a Ca2+-sensitive fusion clamp and a high-fidelity sensor for directed docking.SignificanceSynchronous neurotransmitter release relies on directed docking of synaptic vesicles at active zones in axon terminals, where calcium influx activates membrane fusion and release. In vitro, the calcium sensor Synaptotagmin oligomerizes into rings disassembled by calcium. Here, experiment and modeling suggest the Synaptotagmin molecules hosted by an undocked vesicle oligomerize into a tethered, unbound halo in ATP-rich synaptic terminals. The halo directs vesicle docking to PIP2-rich plasma membrane domains in active zones, where the trans-bound ring conformation is favored, interposed between the membranes to clamp fusion until calcium triggers ring disassembly and neurotransmitter release. The mechanism exploits the extreme sensitivity of Synaptotagmin ring binding preferences to solution and membrane composition, with ~15 -fold-enhanced sensitivity for rings of ~15 molecules.

Synaptic vesicle (SV) docking is a dynamic multi-stage process that is required for efficient neurotransmitter release in response to nerve impulses. Although the steady-state SV docking likely involves the cooperation of Synaptotagmin-1 (Syt1) and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), where and how the docking process initiates remains unknown. Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) can interact with Syt1 and SNAREs to contribute to vesicle exocytosis. In the present study, using the CRISPRi-mediated multiplex gene knockdown and 3D electron tomography approaches, we show that in mouse hippocampal synapses, SV docking initiates at similar to 12 nm to the active zone (AZ) by Syt1. Furthermore, we demonstrate that PI(4,5)P2 is the membrane partner of Syt1 to initiate SV docking, and disrupting their interaction could abolish the docking initiation. In contrast, the SNARE complex contributes only to the tight SV docking within 0-2 nm. Therefore, Syt1 interacts with PI(4,5)P2 to loosely dock SVs within 2-12 nm to the AZ in hippocampal neurons.

Munc13 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggested that multiple copies of Munc13 form nanoassemblies in active zones of neurons. However, it is not known if such clustering is an inherent self-assembly property of Munc13 or whether Munc13 clusters indirectly by multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13, and is indeed required for vesicle binding to bilayers containing Munc13. Pure Munc13 reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ∼20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters a minimum of 6 copies of Munc13 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C2C domain of Munc13 is not required for Munc13 clustering, but is required for efficient vesicle capture.

Phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) is critical for synaptic vesicle docking and fusion and generation of the second messengers, diacylglycerol and inositol-1,4,5-trisphosphate. PI-4,5-P2 can be generated by two families of kinases: type 1 phosphatidylinositol-4-phosphate 5-kinases, encoded by PIP5K1A, PIP5K1B and PIP5K1C, and type 2 phosphatidylinositol-5-phosphate 4-kinases, encoded by PIP4K2A, PIP4K2B, and PIP4K2C. While the roles of the type 1 enzymes in brain function have been extensively studied, the roles of the type 2 enzymes are poorly understood. Using selective antibodies validated by genetic deletion of pip4k2a or pip4k2b in mouse brain, we characterized the location of the enzymes, PI5P4Kα and PI5P4Kß, encoded by these genes. In mice, we demonstrate that PI5P4Kα is expressed in adulthood, whereas PI5P4Kß is expressed early in development. PI5P4Kα localizes to white matter tracts, especially the corpus callosum, and at a low level in neurons, while PI5P4Kß is expressed in neuronal populations, especially hippocampus and cortex. Dual labeling studies demonstrate that PI5P4Kα co-localizes with the oligodendrocyte marker, Olig2, whereas PI5P4Kß co-localizes with the neuronal marker, NeuN. Immunohistochemical subcellular distribution studies demonstrate that PI5P4Kα and PI5P4Kß are expressed in the early endosome system. Ultrastructural analysis demonstrates that both kinases are contained in axon terminals and dendritic spines adjacent to the synaptic membrane, which support a potential role in synaptic transmission. Immunohistochemical analysis of macaque and human brain tissue demonstrate a conserved pattern for PI5P4Kα and PI5P4Kß. These results highlight the diverse cell-autonomous expression of PI5P4Kα and PI5P4Kß and support further exploration into their role in synaptic function in the brain.

Peterson and Heil [Hear. Res., In Press] have argued that the statistics of spontaneous spiking in auditory nerve fibers (ANFs) can be best explained by a model with a limited number of synaptic vesicle docking (release) sites ( ∼ 4 ) and a relatively-long average redocking time ( ∼ 16–17 ms) for each of the sites. In this paper we demonstrate how their model can be: i) generalized to also describe sound-driven ANF responses and ii) incorporated into a well-established and widely-used model of the entire auditory periphery [Zilany et al., J. Acoust. Soc. Am. 135, 283–286, 2014]. The responses of the new model exhibit substantial improvement in several measures of ANF spiking statistics, and predicted physiological forward-masking and rate-level functions from the new model structure are shown to also better match published physiological data.

The synaptosomal-associated protein SNAP25 is a key player in synaptic vesicle docking and fusion and has been associated with multiple psychiatric conditions, including schizophrenia, bipolar disorder, and attention-deficit/hyperactivity disorder. We recently identified a promoter variant inSNAP25,rs6039769, that is associated with early-onset bipolar disorder and a higher gene expression level in human prefrontal cortex. In the current study, we showed that this variant was associated both in males and females with schizophrenia in two independent cohorts. We then combinedin vitroandin vivoapproaches in humans to understand the functional impact of the at-risk allele. Thus, we showedin vitrothat thers6039769C allele was sufficient to increase theSNAP25transcription level. In a postmortem expression analysis of 33 individuals affected with schizophrenia and 30 unaffected control subjects, we showed that theSNAP25b/SNAP25aratio was increased in schizophrenic patients carrying thers6039769at-risk allele. Last, using genetics imaging in a cohort of 71 subjects, we showed that male risk carriers had an increased amygdala–ventromedial prefrontal cortex functional connectivity and a larger amygdala than non-risk carriers. The latter association has been replicated in an independent cohort of 121 independent subjects. Altogether, results from these multilevel functional studies are bringing strong evidence for the functional consequences of this allelic variation ofSNAP25on modulating the development and plasticity of the prefrontal–limbic network, which therefore may increase the vulnerability to both early-onset bipolar disorder and schizophrenia.SIGNIFICANCE STATEMENTFunctional characterization of disease-associated variants is a key challenge in understanding neuropsychiatric disorders and will open an avenue in the development of personalized treatments. Recent studies have accumulated evidence that the SNARE complex, and more specifically the SNAP25 protein, may be involved in psychiatric disorders. Here, our multilevel functional studies are bringing strong evidence for the functional consequences of an allelic variation ofSNAP25on modulating the development and plasticity of the prefrontal–limbic network. These results demonstrate a common genetically driven functional alteration of a synaptic mechanism both in schizophrenia and early-onset bipolar disorder and confirm the shared genetic vulnerability between these two disorders.

Amytrophic lateral sclerosis (ALS) is a neurodegenerative disorder that has been associated with multiple genetic lesions, including mutations in the gene FUS (Fused in Sarcoma), an RNA/DNA-binding protein. Expression of the ALS-associated human FUS in C. elegans results in mislocalization and aggregation of FUS outside the nucleus, and leads to impaired neuromuscular behaviors. However, the mechanisms by which mutant FUS disrupts neuronal health and function remain partially understood. Here we investigated the impact of ALS-associated FUS on motor neuron health using correlative light and electron microscopy, electron tomography, and electrophysiology. Expression of ALS-associated FUS impairs synaptic vesicle docking at neuromuscular junctions, and leads to the emergence of a population of large and electron-dense filament-filled endosomes. Electrophysiological recording of neuromuscular transmission revealed reduced transmission from motor neurons to muscles. Together, these results suggest a potential direct or indirect role of human FUS in the organization of synaptic vesicles, and reduced transmission from motor neurons to muscles.Summary statementAn ALS-associated mutation in a trafficking protein disrupts the organization of the C. elegans neuromuscular junction.

Munc13-1 plays a crucial role in neurotransmitter release. We recently proposed that the C-terminal region encompassing the C1, C2B, MUN and C2C domains of Munc13-1 (C1C2BMUNC2C) bridges the synaptic vesicle and plasma membranes through interactions involving the C2C domain and the C1-C2B region. However, the physiological relevance of this model has not been demonstrated. Here we show that C1C2BMUNC2C bridges membranes through opposite ends of its elongated structure. Mutations in putative membrane-binding sites of the C2C domain disrupt the ability of C1C2BMUNC2C to bridge liposomes and to mediate liposome fusion in vitro. These mutations lead to corresponding disruptive effects on synaptic vesicle docking, priming, and Ca2+-triggered neurotransmitter release in mouse neurons. Remarkably, these effects include an almost complete abrogation of release by a single residue substitution in this 200 kDa protein. These results show that bridging the synaptic vesicle and plasma membranes is a central function of Munc13-1.

Neurotransmitter release requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes by SNARE proteins syntaxin-1 (Stx1), synaptosomal-associated protein 25 (SNAP-25), and synaptobrevin-2 (Syb2). In mammalian systems, loss of SNAP-25 or Syb2 severely impairs neurotransmitter release; however, complete loss of function studies for Stx1 have been elusive due to the functional redundancy between Stx1 isoforms Stx1A and Stx1B and the embryonic lethality of Stx1A/1B double knock-out (DKO) mice. Here, we studied the roles of Stx1 in neuronal maintenance and neurotransmitter release in mice with constitutive or conditional deletion of Stx1B on an Stx1A-null background. Both constitutive and postnatal loss of Stx1 severely compromised neuronal viability in vivo and in vitro, indicating an obligatory role of Stx1 for maintenance of developing and mature neurons. Loss of Munc18-1, a high-affinity binding partner of Stx1, also showed severely impaired neuronal viability, but with a slower time course compared with Stx1A/1B DKO neurons, and exogenous Stx1A or Stx1B expression significantly delayed Munc18-1-dependent lethality. In addition, loss of Stx1 completely abolished fusion-competent vesicles and severely impaired vesicle docking, demonstrating its essential roles in neurotransmission. Putative partial SNARE complex assembly with the SNARE motif mutant Stx1A(AV) (A240V, V244A) was not sufficient to rescue neurotransmission despite full recovery of vesicle docking and neuronal survival. Together, these data suggest that Stx1 has independent functions in neuronal maintenance and neurotransmitter release and complete SNARE complex formation is required for vesicle fusion and priming, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. SIGNIFICANCE STATEMENT Syntaxin-1 (Stx1) is a component of the synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and is essential for neurotransmission. We present the first detailed loss-of-function characterization of the two Stx1 isoforms in central mammalian neurons. We show that Stx1 is fundamental for maintenance of developing and mature neurons and also for vesicle docking and neurotransmission. We also demonstrate that neuronal maintenance and neurotransmitter release are regulated by Stx1 through independent functions. Furthermore, we show that SNARE complex formation is required for vesicle fusion, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. Therefore, our work provides insights into differential functions of Stx1 in neuronal maintenance and neurotransmission, with the latter explored further into its functions in vesicle docking and fusion.

Synaptic vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new vesicles must refill vacated release sites. How many vesicles can fuse at a single active zone, where they fuse within the active zone, and how quickly they are replaced with new vesicles is not well-established. To capture synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple vesicles can fuse at a single active zone; this multivesicular release is augmented by increasing the extracellular calcium concentration. Synchronous fusions are distributed throughout the active zone, whereas asynchronous fusions are biased toward the center of the active zone. Immediately after stimulation a large fraction of vesicles become undocked. Between 8 and 14 ms, new vesicles are recruited to the plasma membrane and fully replenish the docked pool, but docking of these vesicles is transient and they either undock or fuse within 100 ms. These results demonstrate that recruitment of synaptic vesicles to release sites is rapid and reversible.

The glomerular visceral epithelial cell (podocyte) is characterized as a specialized structure of the interdigitating foot processes, covering the outer side of the glomerular basement membrane (GBM). The neighboring foot processes are connected by a slit diaphragm, which is a key structure regulating the barrier function of the glomerular capillary wall to prevent proteinuria. We have previously reported that synaptic vesicle protein 2 B (SV2B) is expressed in the podocyte and that the expression is clearly decreased in nephrotic models. However, the precise function of SV2B in the podocyte is unclear. To investigate the role of SV2B in maintaining the podocyte function and to better understand the function of the neuron-like vesicle expressing SV2B in the podocyte, we analyzed them with SV2B knockout (KO) mice. An increase in the amount of proteinuria, effacement of the foot process of the podocyte, and alterations of the GBM were detected in SV2B KO mice. It was also found that the expression of CD2AP, nephrin, and NEPH1, the functional molecules of the slit diaphragm, and laminin, a critical component of the GBM, is clearly altered in SV2B KO mice. Synaptotagmin and neurexin, which have a role in the synaptic vesicle docking in neurons, are downregulated in the kidney cortex of SV2B KO mice. We have previously reported that neurexin interacts with CD2AP, and the present study shows that SV2B interacts with CD2AP. These findings suggest that the SV2B-neurexin complex is involved in the formation and maintenance of the slit diaphragm. In addition, SV2B is densely expressed close to the cell surface in the presumptive podocyte in the early stage of glomerulogenesis. These results suggest that SV2B has an essential role in the formation and maintenance of the glomerular capillary wall.

The presynaptic active zone protein Munc13 is essential for neurotransmitter release, playing key roles in vesicle docking and priming. Mechanistically, it is thought that the C2A domain of Munc13 inhibits the priming function by homodimerization, and that RIM disrupts the autoinhibitory homodimerization forming monomeric priming-competent Munc13. However, it is unclear whether the C2A domain mediates other Munc13 functions in addition to this inactivation–activation switch. Here, we utilize mutations that modulate the homodimerization and heterodimerization states to define additional roles of the Munc13 C2A domain. Using electron microscopy and electrophysiology in hippocampal cultures, we show that the C2A domain is critical for additional steps of vesicular release, including vesicle docking. Optimal vesicle docking and priming is only possible when Munc13 heterodimerizes with RIM via its C2A domain. Beyond being a switching module, our data suggest that the Munc13-RIM heterodimer is an active component of the vesicle docking, priming and release complex., The interaction between RIM and the C2A domain of Munc13 is known to be required for synaptic vesicle priming. Here the authors show new implications of the C2A domain of Munc13, through its dynamic interaction with RIM, in orchestrating a wide range of modulatory operations that shape vesicle docking, priming and neurotransmitter release.

Background The synaptosomal associated protein SNAP25 is crucial for synaptic vesicle docking and fusion and has been associated with multiple psychiatric conditions. We recently identified a promoter variant in SNAP25, rs6039769, associated with bipolar disorder and gene expression in prefrontal cortex. Methods Here, we performed a genetic association study using this variation on two independent cohorts of 288 and 173 subjects with schizophrenia and 315 unaffected control individuals. We replicated our results using data from the schizophrenia group of the Psychiatric Genomics Consortium (PGC). Functional consequences combined both in vitro and post-mortem gene expression analysis on 30 patients with schizophrenia and 33 unaffected controls. Structural and functional magnetic resonance imaging in regards to SNAP25 genotypes was performed on 71 subjects and replicated on 121 individuals. Results We showed that rs6039769 was associated with schizophrenia in the two cohorts of patients as well as in the Caucasian population of the PGC cohort and demonstrated in vitro this variant was sufficient to increase the SNAP25 transcription level. In a postmortem expression analysis, we showed that the SNAP25b:SNAP25a ratio was increased in patients carrying the at-risk allele. Imaging genetics studies revealed that risk carriers had an increased amygdala-ventromedial prefrontal cortex functional connectivity and a larger amygdala. Discussion This multi-level functional study brings strong evidence for the functional consequences of this SNAP25 variation on the prefrontal-limbic network and the increased risk of developing associated psychiatric disorders.

Next-generation sequencing has accelerated the identification of disease genes in many rare genetic disorders including early-onset epileptic encephalopathies (EOEEs). While many of these disorders are caused by neuronal channelopathies, the role of synaptic and related neuronal proteins are increasingly being described. Here, we report a 6-year-old girl with unexplained EOEE characterized by multifocal seizures and profound global developmental delay. Recessive inheritance was considered due to parental consanguinity and Irish Traveller descent. Exome sequencing was performed. Variant prioritization identified a homozygous nonsense variant in the N-ethylmaleimide-sensitive factor attachment protein, beta (NAPB) gene resulting in a premature stop codon and 46% loss of the protein. NAPB plays a role in soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-complex dissociation and recycling (synaptic vesicle docking). Knockout mouse models of the murine ortholog Napb have been previously reported. These mice develop recurrent post-natal epileptic seizures in the absence of structural brain changes. The identification of a disease-causing variant in NAPB further recognizes the importance of the SNARE complex in the development of epilepsy and suggests that this gene should be considered in patients with unexplained EOEE.

Death-associated protein kinase (DAPK) undergoes activation in response to various death stimuli, and they have been associated with an increase in DAPK catalytic activity. One of the most prominent features of DAPK-induced cell death is the effect on the cytoskeleton, including loss of matrix attachment, and membrane blebbing. One known cytoskeletal-associated substrate of DAPK is the myosin-II light chain, phosphorylated by DAPK on Ser(19), thus stabilizing actin stress fibres. Moreover, paxillin, a component of focal adhesions, was found to be localized in close proximity to the tips of the DAPK-positive filaments, indicating that stress fibres containing DAPK extend to focal contacts. Forced expression of DAPK in multiple cell types results in morphological changes such as cell rounding, membrane blebbing, shrinking and detachment. During directed migration, DAPK functions as a potent inhibitor of cell polarization, as evidenced by its perturbation of the formation of static protrusion at the leading edge. Furthermore, DAPK inhibits random migration by suppressing directional persistence. One of the studies considered DAPK as an anoikis inducer. Others showed that DAP-kinase inhibits the activities of cell surface integrins by converting them into an inactive conformation. Biochemical experiments have established the DAPK binding to Syntaxin1 and its subsequent phosphorylation at Ser(188) in a Ca(2+) dependent manner. This phosphorylation event has been shown to decrease the binding of Syntaxin to MUNC18-1, a protein critically involved in synaptic vesicle docking. Here, we have investigated the structural interactions that modulate DAPK phosphorylation with Syntaxin and its functional role in binding to the MUNC18-1 to regulate vesicle docking. This review will summarize our current knowledge of the role of DAPK on cytoskeleton reorganization and report the mechanisms that regulate these changes.

Structure and function of presynaptic terminals are critical for the transmission and processing of neuronal signals. Trans-synaptic signaling systems instruct the differentiation and function of presynaptic release sites but their downstream mediators are only beginning to be understood. Here, we identify the intracellular mSYD1A (mouse Synapse-Defective-1A) as a novel regulator of presynaptic function in mice. mSYD1A forms a complex with presynaptic receptor tyrosine phosphatases and controls tethering of synaptic vesicles at synapses. mSYD1A function relies on an intrinsically disordered domain that interacts with multiple structurally-unrelated binding partners, including the active zone protein liprin-α2 and nsec1/munc18-1. In mSYD1A knock-out mice, synapses assemble in normal numbers but there is a significant reduction in synaptic vesicle docking at the active zone and an impairment of synaptic transmission. Thus, mSYD1A is a novel regulator of presynaptic release sites at central synapses.

In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near-complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane.

Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca2+-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a ‘primed’ state that does not fuse but is ready for fast fusion upon Ca2+ influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion. DOI: http://dx.doi.org/10.7554/eLife.13696.001, eLife digest In the brain, neurons communicate with each other using small molecules called neurotransmitters. Electrical signals in one neuron trigger the release of the neurotransmitters, which then bind to receptor proteins on another neuron nearby. Neurotransmitters are packaged into small compartments called synaptic vesicles and are released from the neuron when these vesicles fuse with the membrane that surrounds the cell. Many proteins are involved in regulating this process to ensure that neurotransmitters are released at the right place and time. A large protein called Munc13 plays an important role in the release of neurotransmitters. It contains many different regions, including a long domain called MUN and three additional domains called C1, C2B and C2C among others. However, it is not clear how all these domains work together to control neurotransmitter release. Here Liu, Seven et al. address this question using purified proteins inserted into membranes as well as experiments in neurons from mice. The experiments show that the C1, C2B and C2C domains all play key roles in neurotransmitter release. Together with the MUN domain, these three domains help to form bridges between synaptic vesicles and the membrane surrounding the neuron. These bridges could help other proteins involved in neurotransmitter release to form a group that induces vesicle fusion. Liu, Seven et al.’s findings also suggest that Munc13 proteins cooperate with other proteins to form a 'primed' state in which a synaptic vesicle is ready to rapidly fuse with a neuron’s membrane when triggered to do so by an electrical signal. A future challenge is to find out how the proteins that form this primed state promote vesicle fusion. DOI: http://dx.doi.org/10.7554/eLife.13696.002

We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx. DOI: http://dx.doi.org/10.7554/eLife.17262.001, eLife digest Reliable communication between neurons is essential for the brain to work properly. This is accomplished by tightly controlling how chemical messengers, called neurotransmitters, move between neurons. Neurotransmitters are typically packaged into bubble-like structures called synaptic vesicles and are released only when the neuron receives an input electrical signal. A set of proteins orchestrates the release of the neurotransmitters from the neuron, which happens after the synaptic vesicles fuse with the cell membrane. Synaptotagmin, a protein found on the surface of the synaptic vesicle, plays many roles in neurotransmitter release. It helps to attach the synaptic vesicle to the cell membrane and also prevents the vesicles from fusing to the membrane in the absence of an appropriate input signal. Most importantly, it detects when the electrical signal arrives at the neuron by binding to calcium ions that flood the cell following the input signal. This triggers the rapid fusion of the vesicles to the cell membrane. It is not clear how Synaptotagmin is able to carry out its different roles and in particular, control how neurotransmitters are released as calcium ions enter the cell. Zanetti et al. have now used a technique called negative stain electron microscopy to investigate how Synaptotagmin molecules taken from mammals arrange themselves on the surface of a membrane. In this technique, individual Synaptotagmin proteins on the surface of a synthetic membrane are chemically marked and their structure is imaged using an electron beam. Using this approach under conditions resembling those in cells, Zanetti et al. found that 15–20 copies of Synaptotagmin came together and formed ring-like structures on the membrane surface. These ring structures were rapidly broken apart when calcium ions were added to them. Further investigations suggest that the ring structures form when synaptic vesicles first attach to a membrane. Overall, it appears that the Synaptotagmin rings act as washers or spacers to prevent the vesicle from fusing to the cell membrane until the rings are disrupted by the arrival of calcium ions. Future studies are now needed to investigate whether the ring structures form inside cells and whether they act together with other proteins involved in neurotransmitter release. DOI: http://dx.doi.org/10.7554/eLife.17262.002

Dual-specificity tyrosine(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) is a protein kinase that might be responsible for mental retardation and early onset of Alzheimer's disease in Down's syndrome patients. Dyrk1A plays a role in many cellular pathways through phosphorylation of diverse substrate proteins; however, its role in synaptic vesicle exocytosis is poorly understood. Munc18-1, a central regulator of neurotransmitter release, interacts with Syntaxin 1 and X11α. Syntaxin 1 is a key soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein involved in synaptic vesicle docking/fusion events, and X11α modulates amyloid precursor protein processing and β amyloid generation. In this study, we demonstrate that Dyrk1A interacts with and phosphorylates Munc18-1 at the Thr(479) residue. The phosphorylation of Munc18-1 at Thr(479) by Dyrk1A stimulated binding of Munc18-1 to Syntaxin 1 and X11α. Furthermore, the levels of phospho-Thr(479) -Munc18-1 were enhanced in the brains of transgenic mice over-expressing Dyrk1A protein, providing in vivo evidence of Munc18-1 phosphorylation by Dyrk1A. These results reveal a link between Munc18-1 and Dyrk1A in synaptic vesicle trafficking and amyloid precursor protein processing, suggesting that up-regulated Dyrk1A in Down's syndrome and Alzheimer's disease brains may contribute to some pathological features, including synaptic dysfunction and cognitive defect through abnormal phosphorylation of Munc18-1.

Although genetic studies have demonstrated that β-amyloid protein (Aβ) plays a pivotal role in Alzheimer's disease (AD) pathogenesis, how aging contributes to AD onset remains unclear. Moreover, growing evidence suggests that Aβ-independent mechanisms, such as altered intracellular signaling cascades and impaired neurotransmitter release, also are likely involved in this process. Cytoplasmic dynein, a microtubule-based motor protein, mediates minus end–directed vesicle transport via interactions with dynactin, another microtubule-associated protein. We previously showed that normal aging attenuates the interaction between dynein-dynactin complexes in monkey brain and that dynein dysfunction reproduces age-dependent endocytic disturbances, resulting in intracellular Aβ accumulation. In this study, we report that dynein dysfunction disrupts not only retrograde transport of neurotrophic receptors but also anterograde transport of synaptic vesicles, which occurs concomitantly with an increase in Rab3 GTPase levels. Additionally, synaptic vesicle docking was perturbed via enhanced endocytosis. Dynein dysfunction also induced neuritic swelling, which is accompanied by a significant accumulation of neurofilaments. Moreover, we also confirmed that the dynein dysfunction–related disturbances are associated with aging in monkey brains and that age-dependent endocytic disturbances precede Aβ abnormality. These findings suggest that dynein dysfunction can alter neuronal activity via endocytic disturbances and may underlie age-dependent impairment of cognitive function. Moreover, in the presence of other risk factors, such as intracellular Aβ accumulation, dynein dysfunction may contribute to the development of AD.

The neuronal acceptor SNARE complex that functions as the receptor for synaptic vesicle docking and fusion at the presynaptic membrane is composed of the single-span transmembrane protein syntaxin-1A and the palmitoylated soluble protein SNAP-25. Previously, we explored interactions that promote the formation of syntaxin-1A clusters in membranes. Cholesterol activates clustering in native and model membranes, and its depletion in neuroendocrine cells results in a homogeneous distribution of the protein. However, as little as 1 mol % phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)) or 20 mol % phosphatidylserine was found to disperse syntaxin-1A clusters [Murray, D. H., and Tamm, L. K. (2009) Biochemistry 48, 4617-4625]. Strong evidence suggests that syntaxin-1A and its synaptic vesicle cognate synaptobrevin both interact directly with PI-4,5-P(2) and that this interaction activates fusion. However, the molecular details of this interaction and its relationship to the partial dispersion of syntaxin-1A clusters remain largely unexplored. Hence, we mutated the polybasic juxtamembrane motif of syntaxin-1A and found several residues that partially or fully abrogate the electrostatic interaction with PI-4,5-P(2). We further show that even in the presence of physiological concentrations of phosphatidylserine, the PI-4,5-P(2)-syntaxin interaction is sufficiently strong to disrupt syntaxin-1A clustering. The stereochemistry of PI-4,5-P(2) is not critical for this interaction as other polyphosphoinositides have similar effects. Forming an acceptor SNARE complex between syntaxin-1A and SNAP-25 weakens but does not abrogate cholesterol/PI-4,5-P(2)-controlled cluster formation. Potential consequences of these interactions with respect to synaptic vesicle fusion are discussed.

Summary At the presynaptic active zone, Ca 2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca 2+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses—mostly lacking the ribbon—showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca 2+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca 2+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca 2+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca 2+ channels and vesicles, and (2) promote vesicle replenishment.

Synapsins are a family of neuron-specific phosphoproteins involved in synaptic vesicle docking, synaptogenesis, and synaptic plasticity. Previous studies have reported an increase in synapsin II protein by dopaminergic agents in the striatum, medial prefrontal cortex, and nucleus accumbens. This study investigated the mechanistic pathway involved in synapsin II regulation by dopaminergic drugs using primary midbrain neurons to determine which of several transcription factors regulates synapsin II expression. Protein kinase A (PKA) participation in the signaling pathway was examined using selective PKA inhibitors, which reduced synapsin II expression in cell cultures while dopaminergic agents were unable to increase synapsin II in the presence of the PKA inhibitor. Transcription factor involvement was further investigated using separate cultures treated with antisense deoxyoligonucleotides (ADONs) against the following transcription factors: activating protein 2 alpha (AP-2alpha), early growth response factor 1 (EGR-1), or polyoma enhancer activator-3 (PEA-3). Selective knockdown of AP-2alpha by ADONs reduced synapsin II levels, whereas treatment with EGR-1 and PEA-3 ADONs did not affect synapsin II expression. Furthermore, dopaminergic agents were no longer able to influence synapsin II concentrations following AP-2alpha knockdown. Collectively, these results indicate that a cyclic adenosine-3',5'-monophosphate/PKA-dependent mechanism involving the AP-2alpha transcription factor is likely responsible for the increase in neuronal synapsin II following dopamine D1 receptor stimulation or dopamine D2 receptor inhibition.

At the mouse neuromuscular junction, activation of adenosine A1 and P2Y receptors inhibits acetylcholine release by an effect on voltage dependent calcium channels related to spontaneous and evoked secretion. However, an effect of purines upon the neurotransmitter-releasing machinery downstream of Ca2+ influx cannot be ruled out. An excellent tool to study neurotransmitter exocytosis in a Ca2+-independent step is the hypertonic response. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of the specific adenosine A1 receptor agonist 2-chloro-N6-cyclopentyl-adenosine (CCPA) and the P2Y12-13 agonist 2-methylthio-adenosine 5′-diphosphate (2-MeSADP) on the hypertonic response. Both purines significantly decreased such response (peak and area under the curve), and their effect was prevented by specific antagonists of A1 and P2Y12-13 receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(methylthioethyl)]-2-[3,3,3-trifluoropropyl]thio-5′-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid, tetrasodium salt (AR-C69931MX), respectively. Moreover, incubation of preparations only with the antagonists induced a higher response compared with controls, suggesting that endogenous ATP/ADP and adenosine are able to modulate the hypertonic response by activating their specific receptors. To search for the intracellular pathways involved in this effect, we studied the action of CCPA and 2-MeSADP in hypertonicity in the presence of inhibitors of several pathways. We found that the effect of CPPA was prevented by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) while that of 2-MeSADP was occluded by the protein kinase C antagonist chelerythrine and W-7. On the other hand, the inhibitors of protein kinase A (N-(2[pbromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide, H-89) and phosphoinositide-3 kinase (PI3K) (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride, LY-294002) did not modify the modulatory action in hypertonicity of both purines. Our results provide evidence that activation of A1 and P2Y12-13 receptors by CCPA and 2-MeSADP inhibits ACh release from mammalian motor nerve terminals through an effect on a Ca2+-independent step in the cascade of the exocytotic process. Since presynaptic calcium channels are intimately associated with components of the synaptic vesicle docking and fusion processes, further experiments could clarify if the actions of purines on calcium channels and on secretory machinery are related. Fil: Veggetti, Mariela Iris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Muchnik, Salomon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Losavio, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina

Docking to the plasma membrane prepares vesicles for rapid release. Here, we describe a mechanism for dense core vesicle docking in neurons. In Caenorhabditis elegans motor neurons, dense core vesicles dock at the plasma membrane but are excluded from active zones at synapses. We have found that the calcium-activated protein for secretion (CAPS) protein is required for dense core vesicle docking but not synaptic vesicle docking. In contrast, we see that UNC-13, a docking factor for synaptic vesicles, is not essential for dense core vesicle docking. Both the CAPS and UNC-13 docking pathways converge on syntaxin, a component of the SNARE (soluble N-ethyl-maleimide–sensitive fusion protein attachment receptor) complex. Overexpression of open syntaxin can bypass the requirement for CAPS in dense core vesicle docking. Thus, CAPS likely promotes the open state of syntaxin, which then docks dense core vesicles. CAPS function in dense core vesicle docking parallels UNC-13 in synaptic vesicle docking, which suggests that these related proteins act similarly to promote docking of independent vesicle populations.

Background Onabotulinumtoxin type A (BoNT-A) has been found to reduce pain in chronic migraine. The aim of the present study was to ask if BoNT-A can interact directly on sensory mechanisms in the trigeminal ganglion (TG) using an organ culture method. Methods To induce inflammation, rat TGs were incubated for 24 hrs with either the mitogen MEK1/2 inhibitor U0126, BoNT-A or NaCl. After this the TGs were prepared for immunohistochemistry. Sections of the TG were then incubated with primary antibodies against CGRP (neuronal transmitter), iNOS (inflammatory marker), IL-1β (Interleukin 1β), SNAP-25 (synaptic vesicle docking protein) or SV2-A (Botulinum toxin receptor element). Results We report that CGRP, iNOS, IL-1β, SNAP-25 and SV2-A were observed in fresh TG with a differential distribution. Interestingly, NaCl organ culture of the TG resulted in enhanced expression of CGRP and SNAP-25 in neurons and iNOS in SGCs. Co-incubation with U0126 or BoNT-A retained the increased expression of SNAP-25, while it decreased the IL-1β immunoreactivity in neurons. The iNOS expression in SGCs returned to levels observed in fresh specimens. Moreover, we observed no alteration SV2-A expression in SGCs. Thus, the overall picture is that both U0126 and BoNT-A have the ability to modify the expression of certain molecules in the TG. Conclusion We hypothesize that chronic migraine might be associated with some degree of inflammation in the TG that could involve both neurons and SGCs. It is clinically well recognized that treatment with corticosteroids will reduce the symptoms of chronic migraine; however this remedy is associated with long-term side effects. Understanding the mechanisms involved in the expressional alterations may suggest novel ways to modify the changes and indicate novel therapeutics. The results of the present work illustrate one way by which BoNT-A may modify these expressional alterations.

Transmitter release at synapses between nerve cells is spatially restricted to active zones, where synaptic vesicle docking, priming, and Ca2+-dependent fusion take place in a temporally highly coordinated manner. Munc13s are essential for priming synaptic vesicles to a fusion competent state, and their specific active zone localization contributes to the active zone restriction of transmitter release and the speed of excitation-secretion coupling. However, the molecular mechanism of the active zone recruitment of Munc13s is not known. We show here that the active zone recruitment of Munc13 isoforms Munc13-1 and ubMunc13-2 is regulated by their binding to the Rab3A-interacting molecule RIM1alpha, a key determinant of long term potentiation of synaptic transmission at mossy fiber synapses in the hippocampus. We identify a single point mutation in Munc13-1 and ubMunc13-2 (I121N) that, depending on the type of assay used, strongly perturbs or abolishes RIM1alpha binding in vitro and in cultured fibroblasts, and we demonstrate that RIM1alpha binding-deficient ubMunc13-2(I121) is not efficiently recruited to synapses. Moreover, the levels of Munc13-1 and ubMunc13-2 levels are decreased in RIM1alpha-deficient brain, and Munc13-1 is not properly enriched at active zones of mossy fiber terminals of the mouse hippocampus if RIM1alpha is absent. We conclude that one function of the Munc13/RIM1alpha interaction is the active zone recruitment of Munc13-1 and ubMunc13-2.

Apolipoprotein E4 (APOE4) allele is a major risk factor for late-onset familial and sporadic Alzheimer disease (AD). The mechanism of action of APOE in the etiology of AD remains unclear. Using gene expression (microarray) analysis of human hippocampus from APOE3/3 AD and APOE4/4 AD cases, we found different gene transcription patterns between APOE4/4 and APOE3/3 AD cases. The expression of APOE4/4 alleles, in comparison to APOE3/3, is associated with upregulation of multiple gene transcripts encoding cell growth suppresser or arrest, signal transduction, myelinogenesis, cell adhesion and migration, heavy metal metabolism and detoxification. Whereas the APOE4 gene expression is associated with downregulation of gene transcripts involved in mitochondrial oxidative phosphorylation and energy metabolism, synaptic vesicle docking and fusing, and synaptic plasticity compared to APOE3. These mechanisms may contribute increased risk for AD and for cognitive dysfunction in AD patients who carry the APOE4 allele(s).

Seven serologically related, but antigenically different, botulinum toxins (BoNTs) have been identified including types A, B, C, D, E, F, and G. The bacterium Clostridium botulinum along with some strains of Clostridium baratti and Clostridium butyricum are known to produce botulinum toxins responsible for 4 forms of botulism poisoning including food-borne botulism, inhalation botulism, wound botulism, and infant botulism. Botulism toxins consist of a heavy chain (100 kDa), responsible for binding to target cells, and a light chain (50 kDa) responsible for catalytic protein cleaving activity. Light chain has been identified as a zinc endopeptidase that cleaves proteins forming the synaptic vesicle docking and fusion complex (Simpson 1996; Lacy and Stevens 1997). The standard for detection of BoNT toxins is the mouse bioassay, which is able to detect as little as 0.02 ng of toxin. Strengths of the mouse bioassay include conceptual simplicity and sensitivity. While the non-selectivity of the mouse bioassay enables it to detect any BoNT serotype, additional neutralization assays are necessary to determine serotype. Other limitations of the mouse bioassay include expense, expertise related to maintaining mouse-rearing facilities, and time, because as much as 4 d may be required to obtain results (Hallis and others 1996; Witcome and others 1999). Several attempts to replace the mouse bioassay have been made. Methods that have been developed and hold promise for future replacement of the mouse bioassay include mass spectroscopy, immunoassays, polymerase chain reaction (PCR) assays, and assays based upon protease activities of BoNTs. Currently, no single assay appears to be capable of replacing the broadly applicable mouse bioassay.

Autonomic neurotransmission is thought to occur via a loose association between nerve varicosities and smooth muscle cells. In the gastrointestinal tract ultrastructural studies have demonstrated close apposition between enteric nerves and intramuscular interstitial cells of Cajal (ICC-IM) in the stomach and colon and ICC in the deep muscular plexus (ICC-DMP) of the small intestine. In the absence of ICC-IM, postjunctional neural responses are compromised. Although membrane specializations between nerves and ICC-IM have been reported, the molecular identity of these specializations has not been studied. Here we have characterized the expression and distribution of synapse-associated proteins between nerve terminals and ICC-IM in the murine stomach. Transcripts for the presynaptic proteins synaptotagmin, syntaxin, and SNAP-25 were detected. Synaptotagmin and SNAP-25-immunopositive nerve varicosities were concentrated in varicose regions of motor nerves and were closely apposed to ICC-IM but not smooth muscle. W/W(V) mice were used to examine the expression and distribution of synaptic proteins in the absence of ICC-IM. Transcripts encoding synaptotagmin, syntaxin, and SNAP-25 were detected in W/W(V) tissues. In the absence of ICC-IM, synaptotagmin and SNAP-25 were localized to nerve varicosities. Reverse transcriptase polymer chain reaction (RT-PCR) and immunohistochemistry demonstrated the expression of postsynaptic density proteins PSD-93 and PSD-95 in the stomach and expression levels of PSD-93 and PSD-95 were reduced in W/W(V) mutants. These data support the existence of synaptic specializations between enteric nerves and ICC-IM in gastric tissues. In the absence of ICC-IM, components of the synaptic vesicle docking and fusion machinery is trafficked and concentrated in enteric nerve terminals.

Opiate abuse has been shown to cause adaptive changes in presynaptic release and protein phosphorylation-mediated synaptic plasticity, but the underlying mechanisms remain unclear. Neuronal SNARE proteins serve as important regulatory molecules underlying neural plasticity in view of their major role in the process of neurotransmitter release. In the present study, the expression of SNAP-25, a t-SNARE protein essential for vesicle release, was found to be dramatically regulated in hippocampus after chronic morphine treatment, which was visualized with two-dimensional gel electrophoresis. The spots of SNAP-25 in the gel were shifted along the dimension of isoelectric point, indicating a likely change of the post-transcriptional modification. Immunoblotting analysis with specific antibody to Ser187, a protein kinase C (PKC) phosphorylation site of SNAP-25, revealed that the specific phosphorylation was correspondingly decreased, which was correlated with morphine-induced inhibition of PKC activity. Moreover, the level of ternary complex of SNARE proteins in either synaptosomes or PC12 cells was significantly reduced after chronic morphine treatment. This suggests a causal relationship between the inhibition of PKC-dependent SNAP-25 phosphorylation and the down-regulation of SNARE complex formation after chronic morphine treatment. Further analysis of SNARE complex formed by transfection of the wild-type or Ser187 mutants of SNAP-25 showed that only wild-type-formed complex was inhibited by morphine treatment. Thus, these results indicate that chronic morphine treatment inhibits phosphorylation of SNAP-25 at Ser187 and leads to a down-regulation of SNARE complex formation, which presents a potential molecular mechanism for the alteration of exocytotic process and neural plasticity during opiate abuse.

Synaptobrevin is a vesicle-associated membrane protein (VAMP) that is believed to play a critical role with presynaptic membrane proteins (SNAP-25 and syntaxin) during regulated synaptic vesicle docking and exocytosis of neurotransmitter at the central nervous system. Synaptic contacts between taste cells and nerve processes have been found to exist, but little is known about synaptic vesicle docking and neurotransmitter release at taste cell synapses. Previously we demonstrated that immunoreactivity to SNAP-25 is present in taste cells with synapses. Our present results show that synaptobrevin-2-like immunoreactivity (-LIR) is present in approximately 35% of the taste cells in rat circumvallate taste buds. Synaptobrevin-2-LIR colocalizes with SNAP-25-, serotonin-, and protein gene product 9.5-LIR. Synaptobrevin-2-LIR also colocalizes with immunoreactivity for type III inositol 1,4,5-triphosphate receptor (IP3R3), a taste-signaling molecule in taste cells. All IP3R3-LIR taste cells express synaptobrevin-2-LIR. However, approximately 27% of the synaptobrevin-2-LIR taste cells do not display IP3R3-LIR. We believe, based on ultrastructural and biochemical features, that both type II and type III taste cells display synaptobrevin-2-LIR. All of the synapses that we observed from taste cells onto nerve processes express synaptobrevin-2-LIR, as well as some taste cells without synapses. By using colloidal gold immunoelectron microscopy, we found that synaptobrevin-2-LIR is associated with synaptic vesicles at rat taste cell synapses. The results of this study suggest that soluble NSF attachment receptor (SNARE) machinery may control synaptic vesicle fusion and exocytosis at taste cell synapses.

Sec1-related proteins function in most, if not all, membrane trafficking pathways in eukaryotic cells. The Sec1-related protein required in neurons for synaptic vesicle exocytosis is UNC-18. Several models for UNC-18 function during vesicle exocytosis are under consideration. We have tested these models by characterizing unc-18 mutants of the nematode Caenorhabditis elegans. In the absence of UNC-18, the size of the readily releasable pool is severely reduced. Our results show that the near absence of fusion-competent vesicles is not caused by a reduction in syntaxin levels, by a mislocalization of syntaxin, by a defect in fusion or by a failure to open syntaxin during priming. Rather, we found a reduction of docked vesicles at the active zone in unc-18 mutants, suggesting that UNC-18 functions, directly or indirectly, as a facilitator of vesicle docking.