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.     

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.     

Neuronal pathways in the guinea-pig lumbar sympathetic ganglia as revealed by immunohistochemistry

Tyrosine hydroxylase (TH)- and peptide-immunoreactivity of postganglionic neurons and of nerve fibres in guinea pig lumbar paravertebral sympathetic ganglia 2-4 after transection of the communicating rami and the visceral branches, respectively, were investigated by single- and double-labelling techniques. Six subpopulations of postganglionic neurons were discriminated immunohistochemically: two cell types, which were immunoreactive to only one of the applied antisera - TH, and vasoactive intestinal polypeptide (VIP); and four cell types in which immunoreactivity was colocalized - TH/neuropeptide Y (NPY), NPY/VIP, dynorphin/alpha-neoendorphin and dynorphin (alpha-neoendorphin)/NPY. Small intensely fluorescent (SIF) cells dependent on their location exhibited differential immunobehaviour to NPY-/dynorphin-(alpha-neoendorphin-) and TH-antisera. Immunoreactivity to substance P (SP), calcitonin gene-related peptide (CGRP), met-enkephalin-arg-phe (MEAP) and leu-enkephalin was present in nerve fibres but not in postganglionic neurons with frequent colocalization of SP/CGRP- and MEAP/leu-enkephalin- and, sometimes leu-enkephalin/SP- and dynorphin/SP-immunoreactivity. TH-immunoreactive intraganglionic nerve fibres were numerically more increased after cutting the visceral branches, than after transection of the communicating rami. Vice versa, NPY-, VIP-, dynorphin- and alpha-neoendorphin-immunoreactive nerve fibres were particularly increased in number after cutting the communicating rami. Many but not all of the nerve fibres exhibited colocalization of two of these peptides. SP-, CGRP-, and enkephalin-immunoreactive nerve fibres were not visibly affected by cutting the visceral branches but virtually disappeared after lesioning the communicating rami.     

Neuronal pathways in the guinea-pig lumbar sympathetic ganglia as revealed by immunohistochemistry

Summary Tyrosine hydroxylase (TH)- and peptide-immunoreactivity of postganglionic neurons and of nerve fibres in guinea pig lumbar paravertebral sympathetic ganglia 2–4 after transection of the communicating rami and the visceral branches, respectively, were investigated by single-and double-labelling techniques. Six subpopulations of postganglionic neurons were discriminated immunohistochemically: two cell types, which were immunoreactive to only one of the applied antisera — TH, and vasoactive intestinal polypeptide (VIP); and four cell types in which immunoreactivity was colocalized — TH/neuropeptide Y (NPY), NPY/VIP, dynorphin/α-neoendorphin and dynorphin (α-neoendorphin)/NPY. Small intensely fluorescent (SIF) cells dependent on their location exhibited differential immunobehaviour to NPY-/dynorphin-(α-neoendorphin-) and TH-antisera. Immunoreactivity to substance P (SP), calcitonin gene-related peptide (CGRP), met-enkephalin-arg-phe (MEAP) and leu-enkephalin was present in nerve fibres but not in postganglionic neurons with frequent colocalization of SP/CGRP- and MEAP/leu-enkephalin- and, sometimes leu-enkephalin/SP- and dynorphin/SP-immunoreactivity. TH-immunoreactive intraganglionic nerve fibres were numerically more increased after cutting the visceral branches, than after transection of the communicating rami. Vice versa, NPY-, VIP-, dynorphin- and α-neoendorphin-immunoreactive nerve fibres were particularly increased in number after cutting the communicating rami. Many but not all of the nerve fibres exhibited colocalization of two of these peptides. SP-, CGRP-, and enkephalin-immunoreactive nerve fibres were not visibly affected by cutting the visceral branches but virtually disappeared after lesioning the communicating rami.     

A dual role of SNAP‐25 as carrier and guardian of synaptic transmission

In this issue, Matteoli and colleagues show that SNAP-25 levels regulate the efficacy of presynaptic glutamate release and thereby alter short-term plasticity, with potential relevance for psychiatric diseases.EMBO reports(2013) 14 7, 645–651 doi:10.1038/embor.2013.75Control of exocytotic neurotransmitter release is essential for communication in the nervous system and for preventing synaptic abnormalities. The function of synaptosomal-associated protein of 25 kDa (SNAP-25) as a crucial component of the core machinery required for synaptic vesicle fusion is well established, but evidence is growing to suggest an additional modulatory role in neurotransmission. In this issue of EMBO reports, Antonucci et al show that the efficacy of evoked glutamate release is modulated by the expression levels of SNAP-25—a function that might relate to the ability of SNAP-25 to modulate voltage-gated calcium channels and presynaptic calcium ion concentration [1]. Altered synaptic transmission and short-term plasticity due to changes in SNAP-25 expression might have direct consequences for brain function and for the development of neuropsychiatric disorders.Communication between neurons is essential for brain function and occurs through chemical neurotransmission at specialized cell–cell contacts termed ‘synapses''. Within the nerve terminal of the presynaptic neuron electrical stimuli cause the opening of voltage-gated calcium channels (VGCCs), which results in the influx of calcium ions. This triggers the exocytic release of neurotransmitter by fusion of synaptic vesicles with the presynaptic membrane. Released neurotransmitter molecules are detected by specific receptors expressed by the postsynaptic neuron.Calcium-induced synaptic vesicle fusion requires complex assembly between the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) synaptobrevin 2, located on the synaptic vesicle, and the abundant plasma membrane SNAREs SNAP-25 and syntaxin 1, on the opposing presynaptic plasma membrane. SNARE complex assembly is tightly regulated by Sec1/Munc18-like proteins [2]. Further regulatory factors such as the synaptic vesicle calcium-sensing protein synaptotagmin 1 couple the SNARE machinery to presynaptic calcium influx. SNARE-mediated neurotransmitter release occurs preferentially at the active zone—a presynaptic membrane domain specialized for exocytosis within which VGCCs are positioned close to docked synaptic vesicles through a proteinaceous cytomatrix and associated cell adhesion molecules [3,4].Altered short-term plasticity due to changes in SNAP-25 expression might have direct consequences for brain function and for the development of neuropsychiatric disordersAn unresolved conundrum in synaptic transmission remains—the observation that SNARE proteins, such as SNAP-25, are among the most highly expressed, in copy number, presynaptic proteins, whilst only a handful of SNARE complexes are needed to drive the fusion of a single synaptic vesicle [5]. Why, then, are SNAREs such as SNAP-25 so abundant? One possible explanation might be that SNARE proteins, in addition to forming trans-SNARE complexes, assemble with other proteins, and such partitioning might regulate neurotransmission. For example, SNAP-25 has been shown to negatively regulate VGCCs in glutamatergic but not in GABAergic neurons [6]. A secondary regulatory function of SNAP-25 is also supported by its genetic association with synaptic abnormalities such as schizophrenia and attention deficit hyperactivity disorder (ADHD) in humans [7]. SNAP-25 expression is reduced twofold in the hippocampus and frontal lobe from schizophrenic patients [8] and in animal models for ADHD [9]. Thus, SNAP-25 expression levels might crucially regulate normal synaptic function.A new study in this issue of EMBO reports by Antonucci and colleagues investigates the consequences of reduced SNAP-25 expression on synaptic function in SNAP-25^+/− heterozygous (Het) mutant mice. By using patch clamp electrophysiology, Antonucci et al revealed a selective enhancement of glutamatergic but not GABAergic neurotransmission as a result of reduced SNAP-25 expression. Several other parameters including the amplitude and frequency of miniature excitatory and inhibitory currents were unaffected. These data indicate that reduced levels of SNAP-25, an essential component of the fusion machinery, selectively enhance evoked release of glutamate whilst synaptic connectivity and postsynaptic glutamate receptor sensitivity remain unaltered. Further electrophysiological experiments in hippocampal neurons in culture showed that elevated glutamatergic transmission was probably due to increased release probability rather than changes in the number of fusion-prone, so-called ‘readily releasable synaptic vesicles''. This effect was occluded by pharmacologically induced calcium entry bypassing VGCCs, suggesting that altered calcium influx might underlie the differences in evoked glutamate release between wild-type and SNAP-25 Het neurons. As schizophrenia and ADHD are associated with changes in short-term plasticity, a paradigm reflecting presynaptic function, Antonucci et al analysed neurotransmission by paired-pulse stimulation—a protocol whereby two closely paired stimuli are applied within a 50 ms time interval. Wild-type neurons showed significant short-term facilitation, that is, a stronger response to the second stimulus as a result of increased calcium levels in the presynaptic compartment. By contrast, Het neurons had a reduced response to the second stimulus. Such paired-pulse depression is commonly viewed as a sign of increased release probability, which occurs when the first stimulus induces a partial depletion of release-ready synaptic vesicles during paired stimulation. As a consequence, the second stimulus evokes a comparably reduced response [3]. The switch from paired-pulse facilitation to depression was not fully reproduced in hippocampal slices from wild-type and Het mice, although facilitation seemed to be attenuated in SNAP-25 Het slices. One possible explanation for the apparent discrepancy between cultured neurons taken from newborn animals and acute slices from adult mice is the constant postnatal increase in SNAP-25 expression in SNAP-25 Het mice [10], which might partly counteract the defects caused by heterozygosity. Consistent with this explanation are data from rescue experiments by Antonucci et al, which showed that altered neurotransmission and defects in short-term plasticity in Het neurons can be gradually recovered in parallel with increased SNAP-25 expression. Moreover, cultured neurons show substantially higher levels of endogenous activity compared with acute slice preparations, leading to possible changes in the partitioning of SNAP-25 between SNARE complexes and association with VGCCs. Further experiments are clearly required to resolve these issues. Irrespective of these potential caveats, the combined data support the hypothesis that alterations in SNAP-25 expression underlie regulatory changes in neurotransmission, resulting in altered short-term plasticity and possibly disease.Many open questions remain. In particular, the precise mechanisms underlying elevated glutamatergic transmission and presynaptic plasticity under conditions of reduced SNAP-25 expression remain elusive. It has been shown before that free SNAP-25 inhibits Cav2.1-type VGCCs [6], an effect reversed by overexpression of synaptotagmin 1, which might associate with SNAP-25. Conversely, SNAP-25 occludes negative regulation of Cav2.2 VGCCs by free syntaxin 1 [3]. Hence, it is tempting to speculate that differential partitioning of SNAP-25 between free, SNARE-, synaptotagmin 1- and VGCC-complexed forms could regulate evoked neurotransmission (Fig 1). In this scenario, reduced SNAP-25 expression in Het animals and in schizophrenic and ADHD patients would be sufficient to sustain SNARE-mediated synaptic vesicle fusion but partially releases VGCCS from SNAP-25-mediated inhibition. This would result in elevated calcium influx and facilitated neurotransmission. Additional levels of regulation could be imposed by developmental switching between alternatively spliced ‘a'' and ‘b'' isoforms of SNAP-25 [11], age-dependent alterations in presynaptic protein turnover and post-translational modifications.Open in a separate windowFigure 1Effect of presynaptic SNAP-25 levels on calcium-induced glutamate release. Top: in wild-type (WT) neurons, SNARE-mediated calcium-triggered synaptic vesicle fusion is negatively regulated by complex formation between SNAP-25 and VGCCs. Bottom: reduced SNAP-25 expression in heterozygotes (Het;^+/−) partly releases VGCCs from SNAP-25-mediated clamping, resulting in elevated calcium influx through VGCCs and increased glutamate release through SNARE-mediated calcium-triggered synaptic vesicle fusion. Note that many key exocytotic proteins have been omitted for clarity. SNAP-25, synaptosomal-associated protein of 25 kDa; SNARE, soluble NSF attachment protein receptor; VGCC. voltage-gated calcium channel.Future studies need to address these possibilities, and their relationship to cognitive impairments and synaptic diseases, such as schizophrenia and ADHD.     

CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25

SNAP-25 is a component of the SNARE complex that is involved in membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between SNAP-25 and cytoplasmic Lek1 (cytLEK1), a protein previously demonstrated to associate with the microtubule network. The binding domains within each protein were defined by yeast two-hybrid, coimmunoprecipitation, and colocalization studies. Confocal analyses reveal a high degree of colocalization between the proteins. In addition, the endogenous proteins can be isolated as a complex by immunoprecipitation. Further analyses demonstrate that cytLEK1 and SNAP-25 colocalize and coprecipitate with Rab11a, myosin Vb, VAMP2, and syntaxin 4, components of the plasma membrane recycling pathway. Overexpression of the SNAP-25-binding domain of cytLEK1, and depletion of endogenous Lek1 alters transferrin trafficking, consistent with a function in vesicle recycling. Taken together, our studies indicate that cytLEK1 is a link between recycling vesicles and the microtubule network through its association with SNAP-25. This interaction may play a key role in the regulation of the recycling endosome pathway.     

Differential Phosphorylation of Syntaxin and Synaptosome-Associated Protein of 25 kDa (SNAP-25) Isoforms

Abstract : The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N -ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca²⁺ - and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca²⁺ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or α-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

rbSec1A and B colocalize with syntaxin 1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin

rbSec1 is a mammalian neuronal protein homologous to the yeast SEC1 gene product which is required for exocytosis. Mutations in Sec1 homologues in the nervous systems of C. elegans and D. melanogaster lead to defective neurotransmitter secretion. Biochemical studies have shown that recombinant rbSec1 binds syntaxin 1 but not SNAP-25 or synaptobrevin/VAMP, the two proteins which together with syntaxin 1 form the synaptic SNARE complex. In this study we have examined the subcellular localization of rbSec1 and the degree of interaction between rbSec1 and syntaxin 1 in situ. rbSec1, which we show here to be represented by two alternatively spliced isoforms, rbSec1A and B, has a widespread distribution in the axon and is not restricted to the nerve terminal. This distribution parallels the localization of syntaxin 1 and SNAP-25 along the entire axonal plasmalemma. rbSec1 is found in a soluble and a membrane-associated form. Although a pool of rbSec1 is present on the plasmalemma, the majority of membrane-bound rbSec1 is not associated with syntaxin 1. We also show that rbSec1 is not part of the synaptic SNARE complex or of the syntaxin 1/SNAP-25 complex we show to be present in non-synaptic regions of the axon. Thus, in spite of biochemical studies demonstrating the high affinity interaction of rbSec1 and syntaxin 1, our results indicate that rbSec1 and syntaxin 1 are not stably associated. They also suggest that the function of rbSec1, syntaxin 1, and SNAP-25 is not restricted to synaptic vesicle exocytosis at the synapse.     

CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity

A neuron forms thousands of presynaptic nerve terminals on its axons, far removed from the cell body. The protein CSPα resides in presynaptic terminals, where it forms a chaperone complex with Hsc70 and SGT. Deletion of CSPα results in massive neurodegeneration that impairs survival in mice and flies. In CSPα-knockout mice, levels of presynaptic SNARE complexes and the SNARE protein SNAP-25 are reduced, suggesting that CSPα may chaperone SNARE proteins, which catalyse synaptic vesicle fusion. Here, we show that the CSPα-Hsc70-SGT complex binds directly to monomeric SNAP-25 to prevent its aggregation, enabling SNARE-complex formation. Deletion of CSPα produces an abnormal SNAP-25 conformer that inhibits SNARE-complex formation, and is subject to ubiquitylation and proteasomal degradation. Even in wild-type mouse terminals, SNAP-25 degradation is regulated by synaptic activity; this degradation is decreased by CSPα overexpression, and enhanced by CSPα deletion. Thus, SNAP-25 function is maintained during rapid SNARE cycles by equilibrium between CSPα-dependent chaperoning and ubiquitin-dependent degradation, revealing unique protein quality-control machinery within the presynaptic compartment.     

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.     

SNAP-25 modulation of calcium dynamics underlies differences in GABAergic and glutamatergic responsiveness to depolarization

SNAP-25 is a component of the SNARE complex implicated in synaptic vesicle exocytosis. In this study, we demonstrate that hippocampal GABAergic synapses, both in culture and in brain, lack SNAP-25 and are resistant to the action of botulinum toxins type A and E, which cleave this SNARE protein. Relative to glutamatergic neurons, which express SNAP-25, GABAergic cells were characterized by a higher calcium responsiveness to depolarization. Exogenous expression of SNAP-25 in GABAergic interneurons lowered calcium responsiveness, and SNAP-25 silencing in glutamatergic neurons increased calcium elevations evoked by depolarization. Expression of SNAP-25(1-197) but not of SNAP-25(1-180) inhibited calcium responsiveness, pointing to the involvement of the 180-197 residues in the observed function. These data indicate that SNAP-25 is crucial for the regulation of intracellular calcium dynamics and, possibly, of network excitability. SNAP-25 is therefore a multifunctional protein that participates in exocytotic function both at the mechanistic and at the regulatory level.     

Chronic decentralization of the heart differentially remodels canine intrinsic cardiac neuron muscarinic receptors

The objective of the study was to determine if chronic interruption of all extrinsic nerve inputs to the heart alters cholinergic-mediated responses within the intrinsic cardiac nervous system (ICN). Extracardiac nerve inputs to the ICN were surgically interrupted (ICN decentralized). Three weeks later, the intrinsic cardiac right atrial ganglionated plexus (RAGP) was removed and intrinsic cardiac neuronal responses were evaluated electrophysiologically. Cholinergic receptor abundance was evaluated using autoradiography. In sham controls and chronic decentralized ICN ganglia, neuronal postsynaptic responses were mediated by acetylcholine, acting at nicotinic and muscarinic receptors. Muscarine- but not nicotine-mediated synaptic responses that were enhanced after chronic ICN decentralization. After chronic decentralization, muscarine facilitation of orthodromic neuronal activation increased. Receptor autoradiography demonstrated that nicotinic and muscarinic receptor density associated with the RAGP was unaffected by decentralization and that muscarinic receptors were tenfold more abundant than nicotinic receptors in the right atrial ganglia in each group. After chronic decentralization of the ICN, intrinsic cardiac neurons remain viable and responsive to cholinergic synaptic inputs. Enhanced muscarinic responsiveness of intrinsic cardiac neurons occurs without changes in receptor abundance.     

Negative regulation of neurotransmitter release by calpain: a possible involvement of specific SNAP-25 cleavage

Synaptic transmission is conducted by neurotransmitters released from presynaptic nerve terminals by means of Ca2+-dependent exocytosis of synaptic vesicles. Formation of a complex of soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE) proteins, including vesicle-associated membrane protein-2 (VAMP-2) in the synaptic vesicle membrane, and syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) in the plasma membrane, is essential for exocytosis. Ionomycin treatment of cultured rat cerebellar granule cells led to cleavage of SNAP-25, but not syntaxin 1 and VAMP-2, that was dependent on extracellular Ca2+. Cleavage was also induced by N-methyl-D-aspartate (NMDA) treatment, but not by depolarization. The use of various site-specific antibodies to SNAP-25, suggested that the cleavage site was in the N-terminal domain of SNAP-25. Calpain inhibitors abolished the Ca2+-dependent cleavage of SNAP-25 and markedly facilitated Ca2+-dependent glutamate (Glu) release from cerebellar granule cells. These results suggest that calpain may play an important role in the long-lasting regulation of synaptic transmission by suppressing neurotransmitter release, possibly through the proteolytic cleavage of SNAP-25.     

Identification of SNAP-47, a novel Qbc-SNARE with ubiquitous expression

The SNARE proteins are essential components of the intracellular fusion machinery. It is thought that they form a tight four-helix complex between membranes, in effect initiating fusion. Most SNAREs contain a single coiled-coil region, referred to as the SNARE motif, directly adjacent to a single transmembrane domain. The neuronal SNARE SNAP-25 defines a subfamily of SNARE proteins with two SNARE helices connected by a longer linker, comprising also the proteins SNAP-23 and SNAP-29. We now report the initial characterization of a novel vertebrate homologue termed SNAP-47. Northern blot and immunoblot analysis revealed ubiquitous tissue distribution, with particularly high levels in nervous tissue. In neurons, SNAP-47 shows a widespread distribution on intracellular membranes and is also enriched in synaptic vesicle fractions. In vitro, SNAP-47 substituted for SNAP-25 in SNARE complex formation with the neuronal SNAREs syntaxin 1a and synaptobrevin 2, and it also substituted for SNAP-25 in proteoliposome fusion. However, neither complex assembly nor fusion was as efficient as with SNAP-25.     

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.     

Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.

The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Söllner et al. (1993b) Cell, 75, 409-418]. Here we demonstrate that the majority of synaptobrevin is bound to the vesicle protein synaptophysin in detergent extracts. No syntaxin I was found in this complex when synaptophysin-specific antibodies were used for immunoprecipitation. Conversely, no synaptophysin was associated with the synaptobrevin-syntaxin I complex when syntaxin-specific antibodies were used for immunoprecipitation. Thus, the synaptobrevin pool bound to synaptophysin is not available for binding to syntaxin I and SNAP-25, and vice versa. Synaptobrevin-synaptophysin binding was also demonstrated by chemical cross-linking in isolated nerve terminals. Furthermore, recombinant synaptobrevin II efficiently bound synaptophysin and its isoform synaptoporin, but not the more distantly related synaptic vesicle protein p29. Recombinant synaptobrevin I bound with similar efficiency, whereas the non-neuronal isoform cellubrevin displayed a lower affinity towards synaptophysin. Treatment with high NaCl concentrations resulted in a dissociation of the synaptobrevin-synaptophysin complex. In addition, the interaction of synaptobrevin with synaptophysin was irreversibly abolished by low amounts of SDS, while the interaction with syntaxin I was enhanced. We conclude that synaptophysin selectively interacts with synaptobrevin in a complex which excludes the t-SNAP receptors syntaxin I and SNAP-25, suggesting a role for synaptophysin in the control of exocytosis.     

Brain-Derived Neurotrophic Factor Increases the Stimulation-Evoked Release of Glutamate and the Levels of Exocytosis-Associated Proteins in Cultured Cortical Neurons from Embryonic Rats

Abstract: Differentiation and survival of neurons induced by neurotrophins have been widely investigated, but little has been reported about the long-term effect of brain-derived neurotrophic factor (BDNF) on synaptic transmission. Among many steps of neurotransmission, one important step is regulated release of transmitters. Therefore, the release of glutamate and GABA from cortical neurons cultured for several days with or without BDNF was measured by an HPLC-fluorescence method. Although BDNF had little effect on the basal release of glutamate, high K⁺-evoked release was greatly increased by BDNF. BDNF also tended to increase evoked release of GABA. Recently, several proteins involved in the step of "regulated release" have been identified. Thus, the effect of BDNF on the levels of these proteins was then investigated. Neurons were cultivated with or without BDNF, collected, and electrophoresed for western blotting. BDNF increased levels of synaptotagmin, synaptobrevin, synaptophysin, and rab3A, which were known as vesicle protein. Levels of syntaxin, SNAP-25, and β-SNAP were also increased by BDNF. In addition, the numbers of cored and clear vesicles in nerve terminals or varicosities were also increased by BDNF. These results raise the possibility that BDNF increases regulated release of neurotransmitters through the up-regulation of secretory mechanisms.     

Interactions between Presynaptic Calcium Channels and Proteins Implicated in Synaptic Vesicle Trafficking and Exocytosis

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.     

Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+

BACKGROUND: In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing core-constituted SNAREs as reaction partners, in fusion assays. RESULTS: By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca(2+) does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin. CONCLUSIONS: Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.     

Syndapin I, a Synaptic Dynamin-binding Protein that Associates with the Neural Wiskott-Aldrich Syndrome Protein

The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein–protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal.