Munc18-1 binding to the neuronal SNARE complex controls synaptic vesicle priming

Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. Munc18-1 binds to the SNARE syntaxin-1 folded into a closed conformation and to SNARE complexes containing open syntaxin-1. Understanding which steps in fusion depend on the latter interaction and whether Munc18-1 competes with other factors such as complexins for SNARE complex binding is critical to elucidate the mechanisms involved. In this study, we show that lentiviral expression of Munc18-1 rescues abrogation of release in Munc18-1 knockout mice. We describe point mutations in Munc18-1 that preserve tight binding to closed syntaxin-1 but markedly disrupt Munc18-1 binding to SNARE complexes containing open syntaxin-1. Lentiviral rescue experiments reveal that such disruption selectively impairs synaptic vesicle priming but not Ca²⁺-triggered fusion of primed vesicles. We also find that Munc18-1 and complexin-1 bind simultaneously to SNARE complexes. These results suggest that Munc18-1 binding to SNARE complexes mediates synaptic vesicle priming and that the resulting primed state involves a Munc18-1–SNARE–complexin macromolecular assembly that is poised for Ca²⁺ triggering of fusion.     

The synaptic vesicle protein CSP alpha prevents presynaptic degeneration

Cysteine string protein alpha (CSPalpha)--an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40 chaperones--is thought to regulate Ca2+ channels and/or synaptic vesicle exocytosis. We now show that, in young mice, deletion of CSPalpha does not impair survival and causes no significant changes in presynaptic Ca2+ currents or synaptic vesicle exocytosis as measured in the Calyx of Held synapse. At 2-4 weeks of age, however, CSPalpha-deficient mice develop a progressive, fatal sensorimotor disorder. The neuromuscular junctions and Calyx synapses of CSPalpha-deficient mice exhibit increasing neurodegenerative changes, synaptic transmission becomes severely impaired, and the mutant mice die at approximately 2 months of age. Our data suggest that CSPalpha is not essential for the normal operation of Ca2+ channels or exocytosis but acts as a presynaptic chaperone that maintains continued synaptic function, raising the possibility that enhanced CSPalpha function could attenuate neurodegenerative diseases.     

The vesicular SNARE Synaptobrevin is required for Semaphorin 3A axonal repulsion

Attractive and repulsive molecules such as Semaphorins (Sema) trigger rapid responses that control the navigation of axonal growth cones. The role of vesicular traffic in axonal guidance is still largely unknown. The exocytic vesicular soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) Synaptobrevin 2 (Syb2) is known for mediating neurotransmitter release in mature neurons, but its potential role in axonal guidance remains elusive. Here we show that Syb2 is required for Sema3A-dependent repulsion but not Sema3C-dependent attraction in cultured neurons and in the mouse brain. Syb2 associated with Neuropilin 1 and Plexin A1, two essential components of the Sema3A receptor, via its juxtatransmembrane domain. Sema3A receptor and Syb2 colocalize in endosomal membranes. Moreover, upon Sema3A treatment, Syb2-deficient neurons failed to collapse and transport Plexin A1 to cell bodies. Reconstitution of Sema3A receptor in nonneuronal cells revealed that Sema3A further inhibited the exocytosis of Syb2. Therefore, Sema3A-mediated signaling and axonal repulsion require Syb2-dependent vesicular traffic.     

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.     

All three components of the neuronal SNARE complex contribute to secretory vesicle docking

Before exocytosis, vesicles must first become docked to the plasma membrane. The SNARE complex was originally hypothesized to mediate both the docking and fusion steps in the secretory pathway, but previous electron microscopy (EM) studies indicated that the vesicular SNARE protein synaptobrevin (syb) was dispensable for docking. In this paper, we studied the function of syb in the docking of large dense-core vesicles (LDCVs) in live PC12 cells using total internal reflection fluorescence microscopy. Cleavage of syb by a clostridial neurotoxin resulted in significant defects in vesicle docking in unfixed cells; these results were confirmed via EM using cells that were prepared using high-pressure freezing. The membrane-distal portion of its SNARE motif was critical for docking, whereas deletion of a membrane-proximal segment had little effect on docking but diminished fusion. Because docking was also inhibited by toxin-mediated cleavage of the target membrane SNAREs syntaxin and SNAP-25, syb might attach LDCVs to the plasma membrane through N-terminal assembly of trans-SNARE pairs.     

The synaptic vesicle proteome

Synaptic vesicles are key organelles in neurotransmission. Vesicle integral or membrane-associated proteins mediate the various functions the organelle fulfills during its life cycle. These include organelle transport, interaction with the nerve terminal cytoskeleton, uptake and storage of low molecular weight constituents, and the regulated interaction with the pre-synaptic plasma membrane during exo- and endocytosis. Within the past two decades, converging work from several laboratories resulted in the molecular and functional characterization of the proteinaceous inventory of the synaptic vesicle compartment. However, up until recently and due to technical difficulties, it was impossible to screen the entire organelle thoroughly. Recent advances in membrane protein identification and mass spectrometry (MS) have dramatically promoted this field. A comparison of different techniques for elucidating the proteinaceous composition of synaptic vesicles revealed numerous overlaps but also remarkable differences in the protein constituents of the synaptic vesicle compartment, indicating that several protein separation techniques in combination with differing MS approaches are required to identify and characterize the synaptic vesicle proteome. This review highlights the power of various gel separation techniques and MS analyses for the characterization of the proteome of highly purified synaptic vesicles. Furthermore, the newly detected protein assignments to synaptic vesicles, especially those proteins which are new to the inventory of the synaptic vesicle proteome, are critically discussed.     

Docosahexaenoic acid promotes hippocampal neuronal development and synaptic function

Docosahexaenoic acid (DHA, 22:6 n -3), the major polyunsaturated fatty acid accumulated in the brain during development, has been implicated in learning and memory, but underlying cellular mechanisms are not clearly understood. Here, we demonstrate that DHA significantly affects hippocampal neuronal development and synaptic function in developing hippocampi. In embryonic neuronal cultures, DHA supplementation uniquely promoted neurite growth, synapsin puncta formation and synaptic protein expression, particularly synapsins and glutamate receptors. In DHA-supplemented neurons, spontaneous synaptic activity was significantly increased, mostly because of enhanced glutamatergic synaptic activity. Conversely, hippocampal neurons from DHA-depleted fetuses showed inhibited neurite growth and synaptogenesis. Furthermore, n -3 fatty acid deprivation during development resulted in marked decreases of synapsins and glutamate receptor subunits in the hippocampi of 18-day-old pups with concomitant impairment of long-term potentiation, a cellular mechanism underlying learning and memory. While levels of synapsins and NMDA receptor subunit NR2A were decreased in most hippocampal regions, NR2A expression was particularly reduced in CA3, suggesting possible role of DHA in CA3-NMDA receptor-dependent learning and memory processes. The DHA-induced neurite growth, synaptogenesis, synapsin, and glutamate receptor expression, and glutamatergic synaptic function may represent important cellular aspects supporting the hippocampus-related cognitive function improved by DHA.     

The impact of short term synaptic depression and stochastic vesicle dynamics on neuronal variability

Neuronal variability plays a central role in neural coding and impacts the dynamics of neuronal networks. Unreliability of synaptic transmission is a major source of neural variability: synaptic neurotransmitter vesicles are released probabilistically in response to presynaptic action potentials and are recovered stochastically in time. The dynamics of this process of vesicle release and recovery interacts with variability in the arrival times of presynaptic spikes to shape the variability of the postsynaptic response. We use continuous time Markov chain methods to analyze a model of short term synaptic depression with stochastic vesicle dynamics coupled with three different models of presynaptic spiking: one model in which the timing of presynaptic action potentials are modeled as a Poisson process, one in which action potentials occur more regularly than a Poisson process (sub-Poisson) and one in which action potentials occur more irregularly (super-Poisson). We use this analysis to investigate how variability in a presynaptic spike train is transformed by short term depression and stochastic vesicle dynamics to determine the variability of the postsynaptic response. We find that sub-Poisson presynaptic spiking increases the average rate at which vesicles are released, that the number of vesicles released over a time window is more variable for smaller time windows than larger time windows and that fast presynaptic spiking gives rise to Poisson-like variability of the postsynaptic response even when presynaptic spike times are non-Poisson. Our results complement and extend previously reported theoretical results and provide possible explanations for some trends observed in recorded data.     

Stable SNARE complex prior to evoked synaptic vesicle fusion revealed by fluorescence resonance energy transfer

Although it is clear that soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex plays an essential role in synaptic vesicle fusion, the dynamics of SNARE assembly during vesicle fusion remain to be determined. In this report, we employ fluorescence resonance energy transfer technique to study the formation of SNARE complexes. Donor/acceptor pair variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) are fused with the N termini of SNAP-25 and synaptobrevin, respectively. In vitro assembly of SNARE core complex in the presence of syntaxin shows strong fluorescence resonance energy transfer (FRET) between the CFP-SNAP-25 and YFP-synaptobrevin. Under the same conditions, CFP fused to the C terminus of SNAP-25, and YFP- synaptobrevin have no FRET. Adenovirus-mediated gene transfer is used to express the fusion proteins in PC12 cells and cultured rat cerebellar granule cells. Strong FRET is associated with neurite membranes and vesicular structures in PC12 cells co-expressing CFP-SNAP-25 and YFP-synaptobrevin. In cultured rat cerebellar granule cells, FRET between CFP-SNAP-25 and YFP-synaptobrevin is mostly associated with sites presumed to be synaptic junctions. Neurosecretion in PC12 cells initiated by KCl depolarization leads to an increase in the extent of FRET. These results demonstrate that significant amounts of stable SNARE complex exist prior to evoked synaptic vesicle fusion and that the assembly of SNARE complex occurs during vesicle docking/priming stage. Moreover, it demonstrates that FRET can be used as an effective tool for investigating dynamic SNARE interactions during synaptic vesicle fusion.     

Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation

Protein palmitoylation is a post-translational modification that affects a great number of proteins. In most cases, the enzymes responsible for this modification have not been identified. Some proteins use palmitoylation to attach themselves to membranes; however, palmitoylation also occurs in transmembrane proteins, and the function of this palmitoylation is less clear. Here we identify Swf1, a member of the DHHC-CDR family of palmitoyltransferases, as the protein responsible for modifying the yeast SNAREs Snc1, Syn8 and Tlg1, at cysteine residues close to the cytoplasmic end of their single transmembrane domains (TMDs). In an swf1Delta mutant, Tlg1 is mis-sorted to the vacuole. This occurs because unpalmitoylated Tlg1 is recognised by the ubiquitin ligase Tul1, resulting in its targeting to the multivesicular body pathway. Our results suggest that one role of palmitoylation is to protect TMDs from the cellular quality control machinery, and that Swf1 may be the enzyme responsible for most, if not all, TMD-associated palmitoylation in yeast.     

Proper synaptic vesicle formation and neuronal network activity critically rely on syndapin I

Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs.     

Profilin2 contributes to synaptic vesicle exocytosis, neuronal excitability, and novelty-seeking behavior

Profilins are actin binding proteins essential for regulating cytoskeletal dynamics, however, their function in the mammalian nervous system is unknown. Here, we provide evidence that in mouse brain profilin1 and profilin2 have distinct roles in regulating synaptic actin polymerization with profilin2 preferring a WAVE-complex-mediated pathway. Mice lacking profilin2 show a block in synaptic actin polymerization in response to depolarization, which is accompanied by increased synaptic excitability of glutamatergic neurons due to higher vesicle exocytosis. These alterations in neurotransmitter release correlate with a hyperactivation of the striatum and enhanced novelty-seeking behavior in profilin2 mutant mice. Our results highlight a novel, profilin2-dependent pathway, regulating synaptic physiology, neuronal excitability, and complex behavior.     

Intestinal inflammation modulates expression of the synaptic vesicle protein neuronal calcium sensor-1

The calcium-binding protein neuronal calcium sensor 1 (NCS-1) is involved in modulation of neurotransmitter release in the peripheral and central nervous systems. Since intestinal inflammation impairs neurotransmitter release, we evaluated the expression of NCS-1 in the normal rat colon and in dinitrobenzene sulfonic acid (DNBS)-induced colitis. Immunocytochemistry and Western blots showed high levels of NCS-1 in the myenteric plexus and in axons in the smooth muscle layers; 23 +/- 2% of myenteric neurons were NCS-1 positive, with staining restricted to the largest neurons. NCS-1-positive axons decreased to 13.3 +/- 0.4% of total axons by day 2 and dropped further to 7.0 +/- 0.1% by day 4, returning to control levels by day 16. Dual-label Western blot analysis showed that the expression of NCS-1 relative to PGP 9.5 decreased by 50% on day 4 but returned to control by day 16. The selective loss of NCS-1 during colitis may underlie the altered neural function seen in the inflamed intestine.     

The kinetics of synaptic vesicle pool depletion at CNS synaptic terminals

During sustained action potential (AP) firing at nerve terminals, the rates of endocytosis compared to exocytosis determine how quickly the available synaptic vesicle pool is depleted, in turn influencing presynaptic efficacy. Mechanisms, including rapid kiss-and-run endocytosis as well as local, preferential recycling of docked vesicles, have been proposed as a means to allow endocytosis and recycling to keep up with stimulation. We show here that, for CNS nerve terminals at physiological temperatures, endocytosis is sufficiently fast to avoid vesicle pool depletion during continuous AP firing at 10 Hz. This endocytosis-exocytosis balance persists for turnover of the entire releasable pool of vesicles and allows for efficient escape of FM 4-64, indicating that it is a non-kiss-and-run endocytic event. Thus, under physiological conditions, the sustained speed of vesicle membrane retrieval for the entire releasable pool appears to be sufficiently fast to compensate for exocytosis, avoiding significant vesicle pool depletion during robust synaptic activity.     

Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.

A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP-25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS-resistant SNARE core was compromised. This SDS-resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP-dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin-containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.     

The synaptic vesicle: cycle of exocytosis and endocytosis

Synaptic vesicles are clustered at the presynaptic terminal where they fuse and recycle in response to stimulation. Vesicles appear to be sorted into pools, but we do not yet understand how physiologically defined pools relate to morphological pools. The advent of dynamic imaging approaches has led to an appreciation of the regulation of vesicle mobility. Newly endocytosed vesicles are highly mobile but appear to become transiently trapped as they re-enter the recycling pool. Recent experiments indicate that endocytosis might have a constant rate, but limited capacity. How endocytosis is linked to exocytosis remains unclear, although calcium emerges as an important player.