Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system

Following the fusion of synaptic vesicles with the presynaptic plasma membrane of nerve terminals by the process of exocytosis, synaptic-vesicle components are recycled to replenish the vesicle pool. Here we use a pH-sensitive green fluorescent protein to measure the residence time of VAMP, a vesicle-associated SNARE protein important for membrane fusion, on the surfaces of synaptic terminals of hippocampal neurons following exocytosis. The time course of VAMP retrieval depends linearly on the amount of VAMP that is added to the plasma membrane, with retrieval occurring between about 4 seconds and 90 seconds after exocytosis, and newly internalized vesicles are rapidly acidified. These data are well described by a model in which endocytosis appears to be saturable, but proceeds with an initial maximum velocity of about one vesicle per second. We also find that, following exocytosis, a portion of the newly inserted VAMP appears on the surface of the axon.     

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.     

A Highlights from MBoC Selection: Synaptobrevin-2 dependent regulation of single synaptic vesicle endocytosis

Evidence from multiple systems indicates that vesicle SNARE (soluble NSF attachment receptor) proteins are involved in synaptic vesicle endocytosis, although their exact action at the level of single vesicles is unknown. Here we interrogate the role of the main synaptic vesicle SNARE mediating fusion, synaptobrevin-2 (also called VAMP2), in modulation of single synaptic vesicle retrieval. We report that in the absence of synaptobrevin-2, fast and slow modes of single synaptic vesicle retrieval are impaired, indicating a role of the SNARE machinery in coupling exocytosis to endocytosis of single synaptic vesicles. Ultrafast endocytosis was impervious to changes in the levels of synaptobrevin-2, pointing to a separate molecular mechanism underlying this type of recycling. Taken together with earlier studies suggesting a role of synaptobrevin-2 in endocytosis, these results indicate that the machinery for fast synchronous release couples fusion to retrieval and regulates the kinetics of endocytosis in a Ca²⁺-dependent manner.     

Inhibition of exocytosis or endocytosis blocks activity-dependent redistribution of synapsin

The synaptic vesicle cycle encompasses the pre-synaptic events that drive neurotransmission. Influx of calcium leads to the fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter, closely followed by endocytosis. Vacated release sites are repopulated with vesicles which are then primed for release. When activity is intense, reserve vesicles may be mobilized to counteract an eventual decline in transmission. Recently, interplay between endocytosis and repopulation of the readily releasable pool of vesicles has been identified. In this study, we show that exo-endocytosis is necessary to enable detachment of synapsin from reserve pool vesicles during synaptic activity. We report that blockage of exocytosis in cultured mouse hippocampal neurons, either by tetanus toxin or by the deletion of munc13, inhibits the activity-dependent redistribution of synapsin from the pre-synaptic terminal into the axon. Likewise, perturbation of endocytosis with dynasore or by a dynamin dominant-negative mutant fully prevents synapsin redistribution. Such inhibition of synapsin redistribution occurred despite the efficient phosphorylation of synapsin at its protein kinase A/CaMKI site, indicating that disengagement of synapsin from the vesicles requires exocytosis and endocytosis in addition to phosphorylation. Our results therefore reveal hitherto unidentified feedback within the synaptic vesicle cycle involving the synapsin-managed reserve pool.     

Rabphilin regulates SNARE-dependent re-priming of synaptic vesicles for fusion

Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP-binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use-dependent depression, probably by a direct interaction with the SNARE protein SNAP-25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild-type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP-25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP-25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use-dependent depression, or has been artificially depleted by deletion of synaptobrevin.     

Polyunsaturated fatty acids influence synaptojanin localization to regulate synaptic vesicle recycling

The lipid polyunsaturated fatty acids are highly enriched in synaptic membranes, including synaptic vesicles, but their precise function there is unknown. Caenorhabditis elegans fat-3 mutants lack long-chain polyunsaturated fatty acids (LC-PUFAs); they release abnormally low levels of serotonin and acetylcholine and are depleted of synaptic vesicles, but the mechanistic basis of these defects is unclear. Here we demonstrate that synaptic vesicle endocytosis is impaired in the mutants: the synaptic vesicle protein synaptobrevin is not efficiently retrieved after synaptic vesicles fuse with the presynaptic membrane, and the presynaptic terminals contain abnormally large endosomal-like compartments and synaptic vesicles. Moreover, the mutants have abnormally low levels of the phosphoinositide phosphatase synaptojanin at release sites and accumulate the main synaptojanin substrate phosphatidylinositol 4,5-bisphosphate at these sites. Both synaptobrevin and synaptojanin mislocalization can be rescued by providing exogenous arachidonic acid, an LC-PUFA, suggesting that the endocytosis defect is caused by LC-PUFA depletion. By showing that the genes fat-3 and synaptojanin act in the same endocytic pathway at synapses, our findings suggest that LC-PUFAs are required for efficient synaptic vesicle recycling, probably by modulating synaptojanin localization at synapses.     

Peculiarities of synaptic vesicle recycling in frog and mouse motor nerve terminals

Using electrophysiology and fluorescence microscopy with dye FM 1-43, a comparative study of peculiarities of neurotransmitter secretion, synaptic vesicle exo-endocytosis and recycling has been carried out in nerve terminals (NT) of the skin-sternal muscle of the frog Rana ridibunda and of the white mouse diaphragm muscle during a long-term high-frequency stimulation (20 imp/s). The obtained data have allowed identifying three synaptic vesicle pools and two recycling ways in the motor NT. In the frog NT, the long-term high-frequency stimulation induced consecutive expenditure of the pool ready to release, the mobilizational, and reserve vesicle pools. The exocytosis rate exceeded markedly the endocytosis rate; the slow synaptic vesicle recycling with replenishment of the reserve pool was predominant. In the mouse NT, only the vesicles of the ready to release and the mobilizational pools, which are replenished predominantly by fast recycling, were exocytosed. The exo- and endocytosis occurred practically in parallel, while vesicles of the reserve pool did not participate in the neurotransmitter secretion. It is suggested that evolution of the motor NT from the poikilothermal to homoiothermal animals went by the way of a decrease of the vesicle pool size, the more economic expenditure and the more effective reuse of synaptic vesicles owing to the high rates of endocytosis and recycling. These peculiarities can provide in NT of homoiothermal animals a long maintenance of neurotransmitter secretion at the steady and sufficiently high level to preserve reliability of synaptic transmission in the process of the high-frequency activity.     

Sweeping model of dynamin activity. Visualization of coupling between exocytosis and endocytosis under an evanescent wave microscope with green fluorescent proteins

Vesicle recycling through exocytosis and endocytosis is mediated by a coordinated cascade of protein-protein interactions. Previously, exocytosis and endocytosis were studied separately so that the coupling between them was understood only indirectly. We focused on the coupling of these processes by observing the secretory vesicle marker synaptobrevin and the endocytotic vesicle marker dynamin I tagged with green and red fluorescent proteins under an evanescent wave microscope in pheochromocytoma cells. In control cells, many synaptobrevin-expressing vesicles were found as fluorescent spots near the plasma membrane. Upon electrical stimulation, many of these vesicles showed an exocytotic response as a transient increase in fluorescence intensity followed by their disappearance. In contrast, fluorescent dynamin appeared as clusters increasing slowly in number upon stimulation. The clusters of fluorescent dynamin moved around beneath the plasma membrane for a significant distance. Simultaneous observations of green fluorescent dynamin and red fluorescent synaptobrevin indicated that more than 70% of the exocytotic responses of synaptobrevin had no immediate dynamin counterpart at the same site. From these findings it was concluded that dynamin-mediated recycling is not directly coupled to exocytosis but rather completed by a scanning movement of dynamin for the sites of invaginating membrane destined to endocytosis.     

Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses.

We have investigated mechanisms in postendocytic processing of synaptic vesicles at hippocampal synapses, using synaptobrevin/vesicle-associated membrane protein (VAMP) tagged with variants of the green fluorescent protein. Following exocytosis, VAMP is retrieved at synaptic and adjoining axonal regions. Retrieved VAMP-containing vesicles return to synaptic vesicle clusters at a rate slower than endocytosis. Vesicles containing a different protein, synaptophysin, recluster at a similar rate, suggesting common vesicular intermediates for the two proteins. Activity prolongs the time taken by endocytosed vesicles to return to synapses. Exogenous calcium buffers slow endocytosis but have no additional effect on the time course of reclustering. In contrast, the protein kinase inhibitor staurosporine does not affect endocytosis but slows reclustering. Finally, since VAMP can move freely on surface membranes, sustained synaptic activity leads to mixing of this vesicular component between adjacent synapses.     

The effects of hydrogen sulfide on the processes of exo- and endocytosis of synaptic vesicles in the mouse motor nerve endings

The effects of sodium hydrosulfide (NaHS), the donor of hydrogen sulfide (H2S), on the exo/endocytosis cycle of synaptic vesicles in the motor nerve ending of the mouse diaphragm were studied using intracellular microelectrode technique and fluorescent microscopy. NaHS increased the frequency of miniature end-plate potentials (MEPPs), without changing their amplitude-time parameters. NaHS also increased the amplitude of the evoked postsynaptic responses during single stimulation (0.3 Hz), which was the evidence of the enhanced synaptic vesicle exocytosis. During high-frequency stimulation (50 Hz), NaHS induced more significant decline of neurotransmitter release, probably due to the lower rate of synaptic vesicle mobilization from recycling pool to exocytic sites. NaHS also decreased the uptake of the fluorescent endocytic dye FM 1–43, which indicated the reduced endocytosis of synaptic vesicles. Thus, the H₂S donor increases exocytosis and decreases the processes of synaptic vesicle endocytosis and mobilization in the mouse motor nerve ending.     

Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.     

A slowed classical pathway rather than kiss-and-run mediates endocytosis at synapses lacking synaptojanin and endophilin

The extent to which a "kiss-and-run" mode of endocytosis contributes to synaptic-vesicle recycling remains controversial. The only genetic evidence for kiss-and-run at the synapse comes from mutations in the genes encoding synaptojanin and endophilin, proteins that together function to uncoat vesicles in classical clathrin-mediated endocytosis. Here we have characterized the endocytosis that persists in null alleles of Drosophila synaptojanin and endophilin. In response to high-frequency stimulation, the synaptic-vesicle pool can be reversibly depleted in these mutants. Recovery from this depletion is slow and indicates the persistence of an impaired form of classical endocytosis. Steady-state exocytosis rates reveal that endocytosis saturates in mutant neuromuscular terminals at approximately 80 vesicles/s, 10%-20% of the wild-type rate. Analyses of quantal size, FM1-43 loading, and dynamin function further demonstrate that, even in the absence of synaptojanin or endophilin, vesicles undergo full fusion and re-formation. Therefore, no genetic evidence remains to indicate that synaptic vesicles undergo kiss-and-run.     

Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release

During constitutive endocytosis, internalized membrane traffics through endosomal compartments. At synapses, endocytosis of vesicular membrane is temporally coupled to action potential-induced exocytosis of synaptic vesicles. Endocytosed membrane may immediately be reused for a new round of neurotransmitter release without trafficking through an endosomal compartment. Using GFP-tagged endosomal markers, we monitored an endosomal compartment in Drosophila neuromuscular synapses. We showed that in conditions in which the synaptic vesicles pool is depleted, the endosome is also drastically reduced and only recovers from membrane derived by dynamin-mediated endocytosis. This suggests that membrane exchange takes place between the vesicle pool and the synaptic endosome. We demonstrate that the small GTPase Rab5 is required for endosome integrity in the presynaptic terminal. Impaired Rab5 function affects endo- and exocytosis rates and decreases the evoked neurotransmitter release probability. Conversely, Rab5 overexpression increases the release efficacy. Therefore, the Rab5-dependent trafficking pathway plays an important role for synaptic performance.     

Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes.

Existing data support the hypothesis that insulin triggers the exocytosis of small vesicles containing the GluT4 isoform of the glucose transporter. The data also suggest that these vesicles reform through endocytosis of GluT4. These processes resemble those described for synaptic vesicles after depolarization of nerve cells. To determine whether GluT4 vesicles are related to synaptic vesicles, rat adipocyte low density microsomes (LDM), which are rich in GluT4 vesicles, were screened for the synaptic vesicle proteins synaptotagmin, synaptophysin, SV2, p29, rab3, and VAMP (synaptobrevin) by immunoblotting. Two polypeptides that reacted with antibodies against the VAMPs were identified, one with the same apparent size as the two isoforms of VAMP in the brain (18 kDa) and one that was slightly smaller (17 kDa). These members of the VAMP family were highly enriched in GluT4 vesicles isolated by immunoadsorption and translocated from the LDM to the plasma membrane in response to insulin. With the exception of rab3, which was observed in the LDM but was not localized in the GluT4 vesicles, the other synaptic vesicle proteins were not detected. The presence of the VAMPs in both GluT4 and synaptic vesicles suggests that the genesis and/or exocytosis of these two types of vesicles involve shared processes.     

High- and Low-Mobility Stages in the Synaptic Vesicle Cycle

Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhibit little mobility. Despite the differences in mobility, both recently endocytosed and mature vesicles are exchanged between synapses. Electrical stimulation does not seem to affect the mobility of the two types of vesicles. After exocytosis, the vesicle material is mobile in the plasma membrane, although the movement appears to be somewhat limited. Increasing the proportion of fused vesicles (by stimulating exocytosis while simultaneously blocking endocytosis) leads to substantially higher mobility. We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement.     

Stimulus-dependent dynamic homo- and heteromultimerization of synaptobrevin/VAMP and synaptophysin

Synaptophysin and synaptobrevin/VAMP are abundant synaptic vesicle proteins that form homo- and heterooligomers. We now use chemical cross-linking in synaptosomes, pinched-off nerve terminals that are capable of stimulus-dependent neurotransmitter release, to investigate whether these complexes are regulated. We show that in synaptosomes treated with three stimuli that induce exocytosis (a depolarizing K(+) solution, the excitatory neurotoxin alpha-latrotoxin, or the Ca(2+)-ionophore ionomycin), the homo- and heteromultimerization of synaptophysin and synaptobrevin is increased up to 6-fold. Whereas at rest less than 10% of the total synaptobrevin and synaptophysin could be chemically cross-linked into homo- and heteromeric complexes, after stimulation up to 25% of synaptobrevin and synaptophysin are present in homo- and heteromultimers, suggesting that a large fraction of these synaptic vesicle proteins physiologically participate in such complexes. The increase in multimerization of synaptophysin and synaptobrevin was only observed in intact but not in lysed synaptosomes and could not be inhibited by general kinase or phosphatase inhibitors. The stimulus dependence of synaptophysin and synaptobrevin multimers indicates that the complexes are not composed of a fixed multisubunit structure, for example, as an ion channel, but represent distinct functional states of synaptobrevin and synaptophysin that are modulated in parallel with synaptic vesicle exo- and endocytosis.     

Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes

The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.     

Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23

The SNARE complex, consisting of synaptobrevin, syntaxin, and SNAP-25, is essential for calcium-triggered exocytosis in neurosecretory cells. Little is known, however, about how developmentally regulated isoforms and other cognate SNARE components regulate vesicular fusion. To address this question, we examined neuroexocytosis from chromaffin cells of Snap25 null mice rescued by the two splice variants SNAP-25a and SNAP-25b and the ubiquitously expressed homolog SNAP-23. In the absence of SNAP-25, vesicle docking persisted, but primed vesicle pools were empty and fast calcium-triggered release abolished. Single vesicular fusion events showed normal characteristics, except for a shorter duration of the fusion pore. Overexpression of SNAP-25a, SNAP-25b, and SNAP-23 resulted in three distinct phenotypes; SNAP-25b induced larger primed vesicle pools than SNAP-25a, whereas SNAP-23 did not support a standing pool of primed vesicles. We conclude that three alternative SNARE components support exocytosis, but they differ in their ability to stabilize vesicles in the primed state.     

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

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

神经末梢突触囊泡释放神经递质过程的调控蛋白

神经末梢突触囊泡释放神经递质是一个复杂且受到精细调控的过程,涉及多种蛋白质间的相互作用。位于突触囊泡膜上的突触囊泡蛋白／突触囊泡相关膜蛋白（synaptobrevin/VAMP),与位于突触前膜上的syntaxin和突触小体相关蛋白SNAP－25,三者聚合形成的可溶性N－甲基马来酰胺敏感因子（NSF）附着蛋白受体（SNARE）核心复合物是突触囊泡胞吐过程中的核心成分。本文主要围绕参与空触囊泡胞吐过程,以及调节SNARE核心复合物的形成,解离及其功能的蛋白质,并对突触囊泡胞吐过程的分子模型作一概述。