Antagonistic regulation of synaptic vesicle priming by Tomosyn and UNC-13

Priming of synaptic vesicles (SVs) is essential for synaptic transmission. UNC-13 proteins are required for priming. Current models propose that UNC-13 stabilizes the open conformation of Syntaxin, in which the SNARE helix is available for interactions with Synaptobrevin and SNAP-25. Here we show that Tomosyn inhibits SV priming. Tomosyn contains a SNARE motif, which forms an inhibitory SNARE complex with Syntaxin and SNAP-25. Mutants lacking Tomosyn have increased synaptic transmission, an increased pool of primed vesicles, and increased abundance of UNC-13 at synapses. Behavioral, imaging, and electrophysiological studies suggest that SV priming was reconstituted in unc-13 mutants by expressing a constitutively open mutant Syntaxin, or by mutations eliminating Tomosyn. Thus, priming is modulated by the balance between Tomosyn and UNC-13, perhaps by regulating the availability of open-Syntaxin. Even when priming was restored, synaptic transmission remained defective in unc-13 mutants, suggesting that UNC-13 is also required for other aspects of secretion.     

An Electrostatically Preferred Lateral Orientation of SNARE Complex Suggests Novel Mechanisms for Driving Membrane Fusion

Biological membrane fusion is a basic cellular process catalyzed by SNARE proteins and additional auxiliary factors. Yet, the critical mechanistic details of SNARE-catalyzed membrane fusion are poorly understood, especially during rapid synaptic transmission. Here, we systematically assessed the electrostatic forces between SNARE complex, auxiliary proteins and fusing membranes by the nonlinear Poisson-Boltzmann equation using explicit models of membranes and proteins. We found that a previously unrecognized, structurally preferred and energetically highly favorable lateral orientation exists for the SNARE complex between fusing membranes. This preferred orientation immediately suggests a novel and simple synaptotagmin-dependent mechanistic trigger of membrane fusion. Moreover, electrostatic interactions between membranes, SNARE complex, and auxiliary proteins appear to orchestrate a series of membrane curvature events that set the stage for rapid synaptic vesicle fusion. Together, our electrostatic analyses of SNAREs and their regulatory factors suggest unexpected and potentially novel mechanisms for eukaryotic membrane fusion proteins.     

Roles of SNARE proteins and synaptotagmin I in synaptic transmission: studies at the Drosophila neuromuscular synapse

The roles of SNARE proteins, i.e. neuronal Synaptobrevin (n-Syb), SNAP-25 and Syntaxin 1A (Syx 1A), and Synaptotagmin I (Syt I) in synaptic transmission have been studied in situ using mutant embryos or larvae that lack these molecules or have alterations in them. Because of the ease of genetic manipulation, the Drosophila neuromuscular synapse is widely used for these studies. The functional properties of synaptic transmission have been studied in mutant embryos using the patch-clamp technique, and in larvae by recording with microelectrodes. A major vesicular membrane protein, n-Syb, is indispensable for nerve-evoked synaptic transmission. Spontaneous synaptic currents (minis), however, are present even in embryos totally lacking n-Syb (N-SYB). Furthermore, Ca(2+)-independent enhancement of mini frequency induced by hypertonic sucrose solutions (hypertonicity response) is totally absent in N-SYB. Embryos that have defects in SNAP-25 (SNAP-25) have similar but milder phenotypes than N-SYB. The phenotype in synaptic transmission was most severe in the synapse lacking Syx 1A. Neither nerve-evoked synaptic currents nor minis occur in embryos lacking Syx 1A (SYX 1A). No hypertonicity response was observed in them. Syt I binds Ca(2+) in vitro and probably serves as a Ca(2+) sensor for nerve-evoked synaptic transmission, since nerve-evoked synaptic currents were greatly reduced in embryos lacking Syt I (SYT I). Also, Syt I has a role in vesicle recycling. Interestingly, the Ca(2+)-independent hypertonicity response is also greatly reduced in SYT I. Minis persist in mutant embryos lacking any of these proteins (n-Syb, SNAP-25 and Syt I), except Syx, suggesting that minis have a distinct fusion mechanism from that for fast and synchronized release. It appears that these SNARE proteins and Syt I are coordinated for fast vesicle fusion. Minis, on the other hand, do not require SNARE complex nor Syt I, but Syx is absolutely required for vesicle fusion. The SNARE complex and Syt I are indispensable for the hypertonicity response. None of these molecules seem to serve for selective docking of synaptic vesicles to the release site. For further studies on synaptic transmission, the Drosophila neuromuscular synapse will continue to be a useful model.     

Altered complexin expression in psychiatric and neurological disorders: cause or consequence?

Complexins play a critical role in the control of fast synchronous neurotransmitter release. They operate by binding to trimeric SNARE complexes consisting of the vesicle protein Synaptobrevin and the plasma membrane proteins Syntaxin and SNAP-25, which are key executors of membrane fusion reactions. SNARE complex binding by Complexins is thought to stabilize and clamp the SNARE complex in a highly fusogenic state, thereby providing a pool of readily releasable synaptic vesicles that can be released quickly and synchronously in response to an action potential and the concomitant increase in intra-synaptic Ca(2+) levels. Genetic elimination of Complexins from mammalian neurons causes a strong reduction in evoked neurotransmitter release, and altered Complexin expression levels with consequent deficits in synaptic transmission were suggested to contribute to the etiology or pathogenesis of schizophrenia, Huntington's disease, depression, bipolar disorder, Parkinson's disease, Alzheimer's disease, traumatic brain injury, Wernicke's encephalopathy, and fetal alcohol syndrome. In the present review I provide a summary of available data on the role of altered Complexin expression in brain diseases. On aggregate, the available information indicates that altered Complexin expression levels are unlikely to have a causal role in the etiology of the disorders that they have been implicated in, but that they may contribute to the corresponding symptoms.     

Syntaphilin: a syntaxin-1 clamp that controls SNARE assembly

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. We have cloned and characterized a protein called syntaphilin that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis.     

The Neurexin/N-Ethylmaleimide-sensitive Factor (NSF) Interaction Regulates Short Term Synaptic Depression

Although Neurexins, which are cell adhesion molecules localized predominantly to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their roles in the regulation of synaptic vesicle release during repetitive nerve stimulation are unknown. Here, we show that nrx mutant synapses exhibit rapid short term synaptic depression upon tetanic nerve stimulation. Moreover, we demonstrate that the intracellular region of NRX is essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, we find that the intracellular region of NRX interacts with N-ethylmaleimide-sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. We further map the binding sites of each molecule and demonstrate that the NRX/NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. Our results reveal a previously unknown role of NRX in the regulation of short term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of NRX in synaptic vesicle release.     

Modulation of the SNARE core complex by dopamine

Communication between nerve cells in the brain occurs primarily through specialized junctions called synapses. Recently, many details of synaptic transmission have emerged. The identities of specific proteins important for synaptic vesicle release have now been established. We have investigated three synaptic proteins, VAMP (vesicle associated membrane protein; also called synaptobrevin), syntaxin, and SNAP25 (synaptosomal associated protein of 25kDa) as possible targets in the dopamine-mediated modulation of synaptic function in rat striatal slices. These three proteins form a SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptors) core complex that is known to be essential for synaptic transmission. Although it is envisioned that the SNAREs undergo dynamic and cyclic interactions to elicit synaptic vesicle release, their precise functions in neurotransmission remains unknown. We have examined SNARE complexes in intact rat striatal slices. Cellular proteins were solubilized, separated electrophoretically by SDS-PAGE, and then identified immunologically. Application of dopamine to striatal slices results in SNAREs favoring the SNARE core complex, a complex which forms spontaneously in the absence of crosslinking agents, rather than the monomer form. In addition, rapid crosslinking of dopamine-treated striatal slices demonstrates that the SNARE complex is increased 4 fold in dopamine treated striatal slices compared with control slices. Haloperidol blocked the dopamine-induced change in the core complex. These results suggest that changes in the activities of SNAREs may be involved in the underlying cellular mechanisms(s) of dopamine-regulated synaptic plasticity of the striatum.     

The C2 domains of synaptotagmin--partners in exocytosis

Rapid signaling between neurons relies on the Ca(2+)-triggered exocytosis of neurotransmitters. Release is mediated by 'kiss-and-run' or complete fusion of secretory organelles with the plasma membrane. Current models indicate that exocytosis is regulated by synaptotagmin I (syt) and mediated by SNARE (soluble NSF-attachment protein receptor) proteins. Syt senses Ca(2+) via two conserved motifs, C2A and C2B. C2B engages a wider array of effector molecules than C2A and appears to play a more crucial role in synaptic transmission. However, it has recently become clear that the tandem C2 domains of syt influence each another in unexpected ways. Here, we focus on recent structure-function studies that are beginning to provide insights into the mechanism through which the C2 domains of syt trigger exocytosis.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

The SNAREs vti1a and vti1b have distinct localization and SNARE complex partners

Two mammalian proteins, vtila and vtilb, are homologous to the yeast Q-SNARE Vtilp which is part of several SNARE complexes in different transport steps. In vitro experiments suggest distinct functions for vtila and vtilb. Here we compared the subcellular localization of endogenous vtila and vtilb by immunofluorescence and immuno-electron microscopy. Both proteins had a distinct but overlapping localization. vtila was found predominantly on the Golgi and the TGN, vtilb mostly on tubules and vesicles in the TGN area and on endosomes. vti1a coimmunoprecipitated with VAMP-4, syntaxin 6, and syntaxin 16. These four SNAREs could assemble into a SNARE complex of conserved structure because one SNARE motif of each subgroup is present. vtila-beta, VAMP-4, syntaxin 6, and syntaxin 16 are coenriched with small synaptic vesicles and with clathrin-coated vesicles isolated from rat brain synaptosomes. Therefore, this SNARE complex may have a role in synaptic vesicle biogenesis or recycling.     

SNAP-29-mediated modulation of synaptic transmission in cultured hippocampal neurons

Identifying the molecules that regulate both the recycling of synaptic vesicles and the SNARE components required for fusion is critical for elucidating the molecular mechanisms underlying synaptic plasticity. SNAP-29 was initially isolated as a syntaxin-binding and ubiquitously expressed protein. Previous studies have suggested that SNAP-29 inhibits SNARE complex disassembly, thereby reducing synaptic transmission in cultured superior cervical ganglion neurons in an activity-dependent manner. However, the role of SNAP-29 in regulating synaptic vesicle recycling and short-term plasticity in the central nervous system remains unclear. In the present study, we examined the effect of SNAP-29 on synaptic transmission in cultured hippocampal neurons by dual patch clamp whole-cell recording, FM dye imaging, and immunocytochemistry. Our results demonstrated that exogenous expression of SNAP-29 in presynaptic neurons significantly decreased the efficiency of synaptic transmission after repetitive firing within a few minutes under low and moderate frequency stimulations (0.1 and 1 Hz). In contrast, SNAP-29 did not affect the density of synapses and basal synaptic transmission. Whereas neurotransmitter release was unaffected during intensive stimulation, recovery after synaptic depression was impaired by SNAP-29. Furthermore, knockdown of SNAP-29 expression in neurons by small interfering RNA increased the efficiency of synaptic transmission during repetitive firing. These findings suggest that SNAP-29 acts as a negative modulator for neurotransmitter release, probably by slowing recycling of the SNARE-based fusion machinery and synaptic vesicle turnover.     

Modeling of the potential coiled-coil structure of snapin protein and its interaction with SNARE complex

Autism is a developmental disability causing learning and memory disorder. The heart of the search for a cure for this syndrome is the need to understand dendrite branch patterning, a process crucial for proper synaptic transmission. Due to the association of snapin with the SNARE complex and its role in synaptic transmission it is reported as a potential drug target for autism therapies. We wish to impart the noesis of the 3D structure of the snapin protein, and in this chase we predict the native structure from its sequence of amino acid residues using the classical Comparative protein structure modeling methods. The predicted protein model can be of great assistance in understanding the structural insights, which is necessary to understand the protein function. Understanding the interactions between snapin and SNARE complex is crucial in studying its role in the neurotransmitter release process. We also presented a computational model that shows the interaction between the snapin and SNAP-25 protein, a part of the larger SNARE complex.     

Tag team action at the synapse

Communication between neurons relies on chemical synapses and the release of neurotransmitters into the synaptic cleft. Neurotransmitter release is an exquisitely regulated membrane fusion event that requires the linking of an electrical nerve stimulus to Ca(2+) influx, which leads to the fusion of neurotransmitter-filled vesicles with the cell membrane. The timing of neurotransmitter release is controlled through the regulation of the soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) proteins-the core of the membrane fusion machinery. Assembly of the fusion-competent SNARE complex is regulated by several neuronal proteins, including complexin and the Ca(2+)-sensor synaptotagmin. Both complexin and synaptotagmin bind directly to SNAREs, but their mechanism of action has so far remained unclear. Recent studies revealed that synaptotagmin-Ca(2+) and complexin collaborate to regulate membrane fusion. These compelling new results provide a molecular mechanistic insight into the functions of both proteins: complexin 'clamps' the SNARE complex in a pre-fusion intermediate, which is then released by the action of Ca(2+)-bound synaptotagmin to trigger rapid fusion.     

Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.

Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.     

神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.     

SNARE complex oligomerization by synaphin/complexin is essential for synaptic vesicle exocytosis

Synaphin/complexin is a cytosolic protein that preferentially binds to syntaxin within the SNARE complex. We find that synaphin promotes SNAREs to form precomplexes that oligomerize into higher order structures. A peptide from the central, syntaxin binding domain of synaphin competitively inhibits these two proteins from interacting and prevents SNARE complexes from oligomerizing. Injection of this peptide into squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion step of synaptic vesicle exocytosis. We propose that oligomerization of SNARE complexes into a higher order structure creates a SNARE scaffold for efficient, regulated fusion of synaptic vesicles.     

Delivering the goods: Activity-induced exocytosis and AMPA receptor insertion within a post-synaptic membrane compartment depends on syntaxin 4

The activity-dependent strengthening of neural transmission at individual synapses has long been postulated to underlie learning and memory in the brain, and current wisdom strongly suggests that molecular modifications within both the pre- and post-synaptic nerve terminals contribute to this strengthening process (i.e. long-term potentiation or LTP). At excitatory, glutamatergic synapses, the dynamic insertion and retrieval of ionotropic glutamate receptors into and from the post-synaptic plasma membrane have been implicated in synaptic plasticity, however, the site(s) for these trafficking events and the molecules involved have not be clearly elucidated. Biochemical studies have identified SNARE proteins as critical mediators of membrane fusion events in many cell types, including neurons, and several bacterial toxins are known to interfere with neurotransmission by disrupting the function of membrane-bound SNARE proteins, such as VAMP and syntaxin. Using high resolution imaging techniques, the authors have characterized the molecular processes underlying activity-driven membrane fusion events within dendritic spines, tiny membrane protrusions only 1-2 microns in size. Their data demonstrate that syntaxin 4 functions as a key SNARE protein for the exocytic insertion of glutamate receptors and the membrane trafficking events contributing to synaptic plasticity.     

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28''s SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.     

Membrane‐directed molecular assembly of the neuronal SNARE complex

Since the discovery and implication of N‐ethylmaleimide‐sensitive factor (NSF)‐attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full‐length recombinant t‐SNAREs and v‐SNARE present in opposing liposomes, leading to the assembly of a t‐/v‐SNARE ring complex. Using high‐resolution electron microscopy, the electron density maps and 3D topography of the membrane‐directed SNARE ring complex was determined at nanometre resolution. Similar to the t‐/v‐SNARE ring complex formed when 50 nm v‐SNARE liposomes meet a t‐SNARE‐reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t‐SNARE‐reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane‐directed t‐/v‐SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome‐reconstituted recombinant t‐/v‐SNARE proteins, and native v‐SNARE present in isolated SV membrane, the membrane‐directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.