How synapsin I may cluster synaptic vesicles

Synapsin I is the most abundant brain phosphoprotein present in conventional synapses of the CNS. Knockout and rescue experiments have demonstrated that synapsin is essential for clustering of synaptic vesicles (SVs) at active zones and the organization of the reserve pool of SVs. However, in spite of intense efforts it remains largely unknown how exactly synapsin I performs this function. It has been proposed that synapsin I in its dephosphorylated state may tether SVs to actin filaments within the cluster from where SVs are released in response to activity-induced synapsin phosphorylation. Recent studies, however, have failed to detect actin filaments inside the vesicle cluster at resting central synapses. Instead, proteins with established functional roles in SV recycling have been found within this presynaptic compartment. Here we discuss potential alternative mechanisms of synapsin I-dependent SV clustering in the reserve pool.     

Munc13-1 is a Ca2+-phospholipid-dependent vesicle priming hub that shapes synaptic short-term plasticity and enables sustained neurotransmission

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Preserved synaptic vesicle recycling in hippocampal neurons in a mouse Alzheimer's disease model

A recently described triple-transgenic mouse model (3xTg, PS1(M146V), APP(Swe), and tau(P301L)) develops a neuropathology similar to the brains of Alzheimer's disease patients including progressive deposits of plaques and tangles [Neuron 39 (2003) 409]. These mice also show age-related deficits in hippocampal synaptic plasticity that occurs before the development of plaques and tangles. Here we report unchanged synaptic vesicle recycling, as measured by FM1-43 release, in the hippocampal neurons of the 3xTg mice. Expression levels of presynaptic protein synaptophysin and of proteins involved in synaptic vesicle recycling including AP180, dynamin I, and synaptotagmin I also remain unaffected. These data suggest that the synaptic deficits observed in the 3xTg neurons may not arise from the preserved synaptic vesicle recycling.     

Synaptotagmin-7 controls the size of the reserve and resting pools of synaptic vesicles in hippocampal neurons

Continuous neurotransmitter release is subjected to synaptic vesicle availability, which in turn depends on vesicle recycling and the traffic of vesicles between pools. We studied the role of Synaptotagmin-7 (Syt-7) in synaptic vesicle accessibility for release in hippocampal neurons in culture. Synaptic boutons from Syt-7 knockout (KO) mice displayed normal basal secretion with no alteration in the RRP size or the probability of release. However, stronger stimuli revealed an increase in the size of the reserve and resting vesicle pools in Syt-7 KO boutons compared with WT. These data suggest that Syt-7 plays a significant role in the vesicle pool homeostasis and, consequently, in the availability of vesicles for synaptic transmission during strong stimulation, probably, by facilitating advancing synaptic vesicles to the readily releasable pool.     

Differences in the osmotic fragility of recycling and reserve synaptic vesicles from the cholinergic electromotor nerve terminals of Torpedo and their possible significance for vesicle recycling

In this study we demonstrate differences in the osmotic fragility of two metabolically and physically heterogeneous synaptic vesicle populations from stimulated electromotor nerve terminals. When synaptic vesicles isolated on sucrose density gradients are submitted to solutions of decreasing osmolarity 50% of VP₂-type vesicles lysed at (mean + S.E. (number of experiments)) 332 ± 14 (4) mosM and 50% of VP₁-type vesicles lysed at 573 ± 8 (3) mosM. These results indicate that recycling vesicles are more resistant to hypo-osmotic lysis and they are consistent with our earlier conclusion that changes in water content on recycling are secondary to changes in the content of the osmotically active small-molecular-mass constituents acetylcholine and ATP.     

Immunoisolation and subfractionation of synaptic vesicle proteins

Within recent years, the advances in proteomics techniques have resulted in considerable novel insights into the protein expression patterns of specific tissues, cells, and organelles. The information acquired from large-scale proteomics approaches indicated, however, that the proteomic analysis of whole cells or tissues is often not suited to fully unravel the proteomes of individual organellar constituents or to identify proteins that are present at low copy numbers. In addition, the identification of hydrophobic proteins is still a challenge. Therefore, the development of techniques applicable for the enrichment of low-abundance membrane proteins is essential for a comprehensive proteomic analysis. In addition to the enrichment of particular subcellular structures by subcellular fractionation, the spectrum of techniques applicable for proteomics research can be extended toward the separation of integral and peripheral membrane proteins using organic solvents, detergents, and detergent-based aqueous two-phase systems with water-soluble polymers. Here, we discuss the efficacy of a number of experimental protocols. We demonstrate that the appropriate selection of physicochemical conditions results in the isolation of synaptic vesicles of high purity whose proteome can be subfractionated into integral membrane proteins and soluble proteins by several phase separation techniques.     

神经元突触囊泡循环的分子机理

神经末梢突触囊泡循环包括锚靠、出胞、入胞及囊泡再生等步骤,由囊泡、轴浆及突触前膜的多种蛋白质的级联反应介导,其关键步骤的分子模型的确立,为进一步了解神经系统高级活动奠定了基础。     

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.     

Distinct requirements for the AP-3 adaptor complex in pigment granule and synaptic vesicle biogenesis in Drosophila melanogaster

The AP-3 adaptor protein complex has been implicated in the biogenesis of lysosome-related organelles, such as pigment granules/melanosomes, and synaptic vesicles. Here we compare the relative importance of AP-3 in the biogenesis of these organelles in Drosophila melanogaster. We report that the Drosophila pigmentation mutants orange and ruby carry genetic lesions in the σ3 and β3-adaptin subunits of the AP-3 complex, respectively. Electron microscopy reveals dramatic reductions in the numbers of electron-dense pigment granules in the eyes of these AP-3 mutants. Mutant flies also display greatly reduced levels of pigments housed in these granules. In contrast, electron microscopy of retinula cells reveals numerous synaptic vesicles in both AP-3 mutant and wild-type flies, while behavioral assays show apparently normal locomotor ability of AP-3 mutant larvae. Together, these results demonstrate that Drosophila AP-3 is critical for the biogenesis of pigment granules, but is apparently not essential for formation of a major population of synaptic vesicles in vivo. Received: 1 February 2000 / Accepted: 10 April 2000     

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.     

The synaptic ribbons of the guinea-pig pineal gland under normal and experimental conditions

Summary Synaptic ribbons (SR), functionally enigmatic structures of mammalian pinealocytes, were studied electron microscopically with regard to number, intracellular localization and topographical relationships, both under normal and experimental conditions. Pineal glands of guinea-pigs serving as controls contained 1.75 ribbon fields/unit area in the males and 2.58 in the females. In animals subjected to continuous illumination for 64 days the number of ribbon fields increased 20-fold in the males and 9-fold in the females. Continuous darkness (26 to 70 days) had varying effects; in some animals SR increased either strongly or moderately, in others they appeared unchanged. Under continuous illumination a higher percentage of ribbon fields bordered the cell membrane than in the controls. Moreover, paired ribbon fields occurred. The topographical analysis revealed that 98 % of the ribbon fields bordering the cell membrane lay opposite another pinealocyte and the remainder opposite nerve fibres, blood vessels and collagenous fibres. It is suggested that SR of mammalian pinealocytes do not represent non-functioning phylogenetic relics but true organelles possibly involved in coupling adjacent pinealocytes functionally.This study was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn.     

Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis

The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.     

Drosophila homologue of Eps15 is essential for synaptic vesicle recycling

The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. We have identified a hypomorphic Eps15 mutant in Drosophila which shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the alpha-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of alpha-Adaptin.     

Identification of a Synaptic Vesicle Antigen (Mr 86,000) Conserved Between Torpedo and Rat

Antisera were raised in guinea pigs to synaptic vesicles purified from the electric organ of Torpedo marmorata. In cholinergic nerve terminals from Torpedo the major antigens identified had Mr 300,000-150,000, 86,000, and 18,000. The Mr 86,000 antigen was conserved between Torpedo and rat, where it is neuron-specific and concentrated in nerve terminals. When rat brain synaptosomes are subfractionated the antigen is associated with synaptic vesicles. The antigen is not found in the cytoskeleton and in the vesicle-free cytosol. Immunohistochemical localization of the antigen in rat shows it to be associated with synapses in diaphragm, cerebellum, hippocampus, and cerebral cortex. The staining pattern of the antigen indicates that the antigen is not cholinergic-specific. The function of the Mr 86,000 antigen remains to be identified.     

Characterisation of phosphatidylcholine/phosphatidylinositol sonicated vesicles. Effects of phospholipid composition on vesicle size

Phosphatidylinositol (PI), dipalmitoylphosphatidylcholine (DPPC) and mixed lipid (DPPC plus PI) sonicated vesicles have been prepared covering a range of composition. The vesicles were characterised by gel filtration, electron microscopy and photon correlation spectroscopy. The dimensions of the vesicles as measured by electron microscopy were in good accord with those obtained from photon correlation spectroscopy measurements. The number average diameters of the vesicles increase on increasing the PI content and range from approx. 30–80 nm as the weight % of PI is increased from 0 to 100. Gel filtration on Sepharose 4B columns gave anomalous results indicating that PI-containing vesicles were retarded on the gel possibly due to an interaction between the inositol headgroup and the gel matrix. Electrophoretic measurements on multilamellar vesicles show that the surface charge density increases with the PI content of the vesicles upto 50 weight % PI and remains constant thereafter. The radii of sonicated vesicles also increase with PI content which reflects a decreasing liposome curvature with increasing surface charge density.     

Identification and characterization of SV31, a novel synaptic vesicle membrane protein and potential transporter

Synaptic vesicle proteins govern all relevant functions of the synaptic vesicle life cycle, including vesicle biogenesis, vesicle transport, uptake and storage of neurotransmitters, and regulated endocytosis and exocytosis. In spite of impressive progress made in the past years, not all known vesicular functions can be assigned to defined protein components, suggesting that the repertoire of synaptic vesicle proteins is still incomplete. We have identified and characterized a novel synaptic vesicle membrane protein of 31 kDa with six putative transmembrane helices that, according to its membrane topology and phylogenetic relation, may function as a vesicular transporter. The vesicular allocation is demonstrated by subcellular fractionation, heterologous expression, immunocytochemical analysis of brain sections and immunoelectron microscopy. The protein is expressed in select brain regions and contained in subpopulations of nerve terminals that immunostain for the vesicular glutamate transporter 1 and the vesicular GABA transporter VGaT (vesicular amino acid transporter) and may attribute specific and as yet undiscovered functions to subsets of glutamatergic and GABAergic synapses.     

Key role of clathrin-mediated endocytosis in synaptic vesicle recycling

Using novel fluorescent markers, virus-induced modulation of amphiphysin 1 expression, and electron microscopy, we demonstrated that clathrin-mediated endocytosis is the main mechanism of synaptic vesicle retrieval; a hypothesis on the role of a fast “kiss-and-run” mechanism has not been supported. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 388–389, July–October, 2007.