Synaptic Vesicle Exocytosis

Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the “active zone”) that includes Munc13 and RIM proteins as central components.Synaptic vesicles are uniform organelles of ∼40 nm diameter that constitute the central organelle for neurotransmitter release. Each presynaptic nerve terminal contains hundreds of synaptic vesicles that are filled with neurotransmitters. When an action potential depolarizes the presynaptic plasma membrane, Ca²⁺-channels open, and Ca²⁺ flows into the nerve terminal to trigger the exocytosis of synaptic vesicles, thereby releasing their neurotransmitters into the synaptic cleft (Fig. 1). Ca²⁺ triggers exocytosis by binding to synaptotagmin; after exocytosis, vesicles are re-endocytosed, recycled, and refilled with neurotransmitters. Recycling can occur by multiple parallel pathways, either by fast recycling via local reuse of vesicles (“kiss-and-run” and “kiss-and-stay”), or by slower recycling via an endosomal intermediate (Fig. 1).Open in a separate windowFigure 1.The synaptic vesicle cycle. A presynaptic nerve terminal is depicted schematically as it contacts a postsynaptic neuron. The synaptic vesicle cycle consists of exocytosis (red arrows) followed by endocytosis and recycling (yellow arrows). Synaptic vesicles (green circles) are filled with neurotransmitters (NT; red dots) by active transport (neurotransmitter uptake) fueled by an electrochemical gradient established by a proton pump that acidifies the vesicle interior (vesicle acidification; green background). In preparation to synaptic exocytosis, synaptic vesicles are docked at the active zone, and primed by an ATP-dependent process that renders the vesicles competent to respond to a Ca²⁺-signal. When an action potential depolarizes the presynaptic membrane, Ca²⁺-channels open, causing a local increase in intracellular Ca²⁺ at the active zone that triggers completion of the fusion reaction. Released neurotransmitters then bind to receptors associated with the postsynaptic density (PSD). After fusion pore opening, synaptic vesicles probably recycle via three alternative pathways: local refilling with neurotransmitters without undocking (“kiss-and-stay”), local recycling with undocking (“kiss-and-run”), and full recycling of vesicles with passage through an endosomal intermediate. (Adapted from Südhof 2004.)Due to their small size, synaptic vesicles contain a limited complement of proteins that have been described in detail (Südhof 2004; Takamori et al. 2006). Although the functions of several vesicle components remain to be identified, most vesicle components participate in one of three processes: neurotransmitter uptake and storage, vesicle exocytosis, and vesicle endocytosis and recycling. In addition, it is likely that at least some vesicle proteins are involved in the biogenesis of synaptic vesicles and the maintenance of their exquisite uniformity and stability, but little is known about how vesicles are made, and what determines their size.     

Organization of Brain Synaptic Vesicle Proteins

Abstract: The topographical arrangement of proteins and glycoproteins of mouse brain synaptic vesicles was studied with trypsin and galactose oxidase, reagents known to be impermeable with respect to other membranes. Incubation of vesicles with trypsin at a concentration of 1 μg/ml extensively degraded seven polypeptides of molecular weights (M.W.) (×10^-3) 125, 107, 95, 83, 70, 60, and 36; higher concentrations degraded two additional species of 75,000 and 46,000 M.W., while leaving unaffected polypeptides of M.W. 66,000, 55,000, 33,000, 26,000, 22,000, 19,000, and 16,000. All of the trypsin-sensitive species of greater than 70,000 M.W. stained positively with the periodic acid-Schiff reagent; several other glycoproteins, all of M.W. less than 70,000, were identified, and all of these were insensitive to trypsin. Galactose oxidase-NaB³H₄ treatment of synaptic vesicles heavily and exclusively labeled material of greater than 70,000 M.W. All of the polypeptides studied were sensitive to each reagent when the synaptic vesicles were first treated with detergents. Extraction of vesicles with 0.05 M-NaOH partially or completely removed a wide variety of polypeptides, including most of those in the M.W. range 46,000–83,000; none of the glycoproteins was solubilized. Essentially the opposite results were obtained when the vesicles were extracted with 0.5% Triton X-100. Most of the vesicle's species were insensitive to several bisimidate cross-linking reagents. These results suggest that: (a) The polypeptides of M.W. 125K, 107K, 95K, 83K, 75K, 70K, 60K, 46K, and 36K are externally oriented in the vesicle, whereas those of 66K, 55K, 33K, 26K, 22K, 19K, and 16K are internally oriented; (b) the vesicles contain two classes of glycoproteins, one consisting of high-molecular-weight, externally oriented species that are rich in galactose, and the other consisting of low-molecular-weight, internally oriented species of relatively low galactose content; (c) the vesicles contain a large class of nonglycosylated species that are relatively loosely attached to the membrane; and (d) most of the vesicles' polypeptides are probably freely mobile in the membrane. The organization of synaptic vesicle proteins is compared with that of the proteins of synaptosomal plasma membrane, with which the vesicle is believed to fuse.     

Vesicular Glutamate Transporter 1 Orchestrates Recruitment of Other Synaptic Vesicle Cargo Proteins during Synaptic Vesicle Recycling

A long standing question in synaptic physiology is how neurotransmitter-filled vesicles are rebuilt after exocytosis. Among the first steps in this process is the endocytic retrieval of the transmembrane proteins that are enriched in synaptic vesicles (SVs). At least six types of transmembrane proteins must be recovered, but the rules for how this multiple cargo selection is accomplished are poorly understood. Among these SV cargos is the vesicular glutamate transporter (vGlut). We show here that vGlut1 has a strong influence on the kinetics of retrieval of half of the known SV cargos and that specifically impairing the endocytosis of vGlut1 in turn slows down other SV cargos, demonstrating that cargo retrieval is a collective cargo-driven process. Finally, we demonstrate that different cargos can be retrieved in the same synapse with different kinetics, suggesting that additional post-endocytic sorting steps likely occur in the nerve terminal.     

Synapse-Assembly Proteins Maintain Synaptic Vesicle Cluster Stability and Regulate Synaptic Vesicle Transport in Caenorhabditis elegans

The functional integrity of neurons requires the bidirectional active transport of synaptic vesicles (SVs) in axons. The kinesin motor KIF1A transports SVs from somas to stable SV clusters at synapses, while dynein moves them in the opposite direction. However, it is unclear how SV transport is regulated and how SVs at clusters interact with motor proteins. We addressed these questions by isolating a rare temperature-sensitive allele of Caenorhabditis elegans unc-104 (KIF1A) that allowed us to manipulate SV levels in axons and dendrites. Growth at 20° and 14° resulted in locomotion rates that were ∼3 and 50% of wild type, respectively, with similar effects on axonal SV levels. Corresponding with the loss of SVs from axons, mutants grown at 14° and 20° showed a 10- and 24-fold dynein-dependent accumulation of SVs in their dendrites. Mutants grown at 14° and switched to 25° showed an abrupt irreversible 50% decrease in locomotion and a 50% loss of SVs from the synaptic region 12-hr post-shift, with no further decreases at later time points, suggesting that the remaining clustered SVs are stable and resistant to retrograde removal by dynein. The data further showed that the synapse-assembly proteins SYD-1, SYD-2, and SAD-1 protected SV clusters from degradation by motor proteins. In syd-1, syd-2, and sad-1 mutants, SVs accumulate in an UNC-104-dependent manner in the distal axon region that normally lacks SVs. In addition to their roles in SV cluster stability, all three proteins also regulate SV transport.     

Synaptic Vesicle Dynamics: A Simple Model of Phasic Release

We present a simple model of phasic neurotransmitter release whichreproduces the salient features of chemical neurotransmission. The synapticvesicle cycle has been modelled as a set of biochemical reactionsrepresented by a system of coupled differential equations. These equationshave been solved analytically to obtain the time dependent behaviour of thesystem on perturbation from the steady state. The scheme of the synapticvesicle network has been emphasized and its role in determining some of themajor experimentally observed properties of synaptic transmission has beendiscussed, which includes the biphasic decay of the rate neurotransmitterrelease even under sustained stimulation. Another interesting outcome ofthis theoretical exercise is the saturation of total release with thecalcium dependent rate constant. The theoretically calculated values oftotal release fit very well into a sigmoidal saturating function with afourth order cooperativity exponent similar to the empiricalDodge–Rahamimoff equation. It appears that the synaptic vesiclenetwork itself is responsible for some of the major properties associatedwith chemical neurotransmission.     

Synapsin Isoforms and Synaptic Vesicle Trafficking

Synapsins were the first presynaptic proteins identified and have served as the flagship of the presynaptic protein field. Here we review recent studies demonstrating that different members of the synapsin family play different roles at presynaptic terminals employing different types of synaptic vesicles. The structural underpinnings for these functions are just beginning to be understood and should provide a focus for future efforts.     

The Vesicle Protein SAM-4 Regulates the Processivity of Synaptic Vesicle Transport

Axonal transport of synaptic vesicles (SVs) is a KIF1A/UNC-104 mediated process critical for synapse development and maintenance yet little is known of how SV transport is regulated. Using C. elegans as an in vivo model, we identified SAM-4 as a novel conserved vesicular component regulating SV transport. Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104. Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport. Genetic analyses suggest that SAM-4, SYD-2/liprin-α and the KIF1A/UNC-104 motor function in the same pathway to regulate SV transport. Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.     

Signaling for Vesicle Mobilization and Synaptic Plasticity

The hypothesis that release of classical neurotransmitters and neuropeptides is facilitated by increasing the mobility of small synaptic vesicles (SSVs) and dense core vesicles (DCVs) could not be tested until the advent of methods for visualizing these secretory vesicles in living nerve terminals. In fact, fluorescence imaging studies have only since 2005 established that activity increases secretory vesicle mobility in motoneuron terminals and chromaffin cells. Mobilization of DCVs and SSVs appears to be due to liberation of hindered vesicles to promote quicker diffusion. However, F-actin and synapsin, which have been featured in mobilization models, are not required for activity-dependent increases in the mobility of DCVs or SSVs. Most recently, the signaling required for sustained mobilization has been identified for Drosophila motoneuron DCVs and shown to increase synaptic transmission. Specifically, presynaptic endoplasmic reticulum ryanodine receptor-mediated Ca2+ release activates Ca2+/calmodulin-dependent kinase II to mobilize DCVs and induce post-tetanic potentiation (PTP) of neuropeptide release in the Drosophila neuromuscular junction. The shared signaling for increasing vesicle mobility and PTP links vesicle mobilization and synaptic plasticity.     

Fusion of Endosomes Involved in Synaptic Vesicle Recycling

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca²⁺ chelator EGTA. Endosome fusion was restored by the addition of Ca²⁺ with an optimum at a free Ca²⁺ concentration of 0.3 × 10⁻⁶ M. Other divalent cations did not substitute for Ca²⁺. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca²⁺. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca²⁺ from the endosomal interior.     

Tomosyn Inhibits Synaptic Vesicle Priming in Caenorhabditis elegans

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.     

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye ¹; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K⁺). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1A_i-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1A_iv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining). Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy ^2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.Open in a separate windowClick here to view.^{(49M, flv)}     

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.     

Synaptic Vesicle Proteins and Neuronal Plasticity in Adrenergic Neurons

The neurons in the superior cervical ganglion are active in plasticity and re-modelling in order to adapt to requirements. However, so far, only a few studies dealing with synaptic vesicle related proteins during adaptive processes have been published. In the present paper, changes in content and expression of the synaptic vesicle related proteins in the neurons after decentralization (cutting the cervical sympathetic trunk) or axotomy (cutting the internal and external carotid nerves) were studied. Immunofluorescence studies were carried out using antibodies and antisera against integral membrane proteins, vesicle associated proteins, NPY, and the enzymes TH and PNMT. For colocalization studies, the sections were simultaneously double labelled. Confocal laser scanning microscopy was used for colocalization studies as well as for semi-quantification analysis, using the computer software. Westen blot analysis, in situ 3'-end DNA labelling, and in situ hybridization were also employed. After decentralization of the ganglia several of the synaptic vesicle proteins (synaptotagmin I, synaptophysin, SNAP-25, CLC and GAP-43) were increased in the iris nerve terminal network, but with different time patterns, while TH-immunoreactivity had clearly decreased. In the ganglia, these proteins had decreased at 1 day after decentralization, probably due to degeneration of the pre-ganglionic nerve fibres and terminals. At later intervals, these proteins, except SNAP-25, had increased in the nerve fibre bundles and re-appeared in nerve fibres outlining the principal neurons.     

突触泡蛋白2研究进展

突触泡蛋白2(SV2)是一类跨膜糖蛋白,定位于脊椎动物神经元及内分泌细胞,与神经递质的释放、内分泌泡胞吐作用、突触泡稳态的维持、神经肌肉接头的形成及肾上腺素能受体α2C的定位密切相关。最近还发现SV2是肉毒神经毒素BoNT/A的受体,介导BoNT/A进入神经元。SV2可作为突触泡标记蛋白,广泛应用于生物学研究及肿瘤诊断。此外,SV2还是抗癫痫药物的作用靶标。     

Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking

Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed.     

Tubulin: An Integral Protein of Mammalian Synaptic Vesicle Membranes

Abstract: The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis, by two-dimensional gel electrophoresis and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the α and β subunits. In the presence of ethyleneglycol bis (aminoethyl ether)- N, N' -tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the α-tubulin whereas the β subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of α-tubulin than of the β subunit. Non-ionic detergents such as Triton X-100 or Nonidet P-40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that α-tubulin is an integral vesicle membrane protein, whereas most of the β sub-unit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.     

Enhanced Synaptic Vesicle Traffic in Hippocampus of Phenytoin-Resistant Kindled Rats

Aim: Intractable epilepsy is characterized of seizure resistance to the anti-epileptic drugs. The underlying mechanisms are still elusive. Alterations of synaptic vesicle traffic may be one of the candidate mechanisms. Methods: Phenytoin-resistant and phenytoin-non resistant epileptic rats were selected in the amygdala kindled adult male Wistar rats. Synaptotagmin-I and clathrin were determined by cDNA microarry analysis and Western blotting in the hippocampus of phenytoin-resistant and phenytoin-nonresistant kindled rats, which were associated with the exocytosis and endocytosis of the synaptic vesicle traffic. Results: Microarry analysis showed both synaptotagmin-I and clathrin mRNA were up-regulated at least 3.06 fold accompanied with their correspondent proteins increased by 52.3 ± 6.4 % and 76.7 ± 12.4 % respectively in the hippocampus of phenytoin-resistant rats as compared with those in phenytoin-nonresistant rats. There were no significant differences in plasma phenytoin concentrations between the two groups. Conclusions: The increased expressions of synaptotagmin-I and clathrin in the hippocampus of phenytoin-resistant kindled rats play a role in the development of intractable epilepsy.     

Hydrodynamic Molecular Weight of Solubilized Cholinergic Synaptic Vesicle Glycoprotein ATPase

The Torpedo californica electric organ synaptic vesicle glycoprotein ATPase was solubilized with octaethyleneglycoldodecyl ether and stabilized with phosphatidylserine. The complex was analyzed by size exclusion chromatography and band sedimentation velocity ultracentrifugation in water/glycerol and deuterium oxide/glycerol density gradients. The complex was found to have a Stokes' radius of 79 +/- 0.7 A, a sedimentation velocity coefficient at 20 degrees C in water of 6.8 +/- 0.2S, a partial specific volume of 0.81 +/- 0.01 cm3/g, and a frictional coefficient of 1.6. The molecular weight of the solubilized complex was calculated to be 320,000 +/- 7,000 and that of the protein 210,000 +/- 9,000. The relationship of this latter value to the major transport ATPase types is discussed.