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PubMed1993

Interactions of synapsin I with phospholipids: possible role in synaptic vesicle clustering and in the maintenance of bilayer structures.

Synapsin I is a synaptic vesicle-specific phosphoprotein composed of a globular and hydrophobic head and of a proline-rich, elongated and basic tail. Synapsin I binds with high affinity to phospholipid and protein components of synaptic vesicles. The head region of the protein has a very high surface activity, strongly interacts with acidic phospholipids and penetrates the hydrophobic core of the vesicle membrane. In the present paper, we have investigated the possible functional effects of the interaction between synapsin I and vesicle phospholipids. Synapsin I enhances both the rate and the extent of Ca(2+)-dependent membrane fusion, although it has no detectable fusogenic activity per se. This effect, which appears to be independent of synapsin I phosphorylation and localized to the head region of the protein, is attributable to aggregation of adjacent vesicles. The facilitation of Ca(2+)-induced liposome fusion is maximal at 50-80% of vesicle saturation and then decreases steeply, whereas vesicle aggregation does not show this biphasic behavior. Association of synapsin I with phospholipid bilayers does not induce membrane destabilization. Rather, 31P-nuclear magnetic resonance spectroscopy demonstrated that synapsin I inhibits the transition of membrane phospholipids from the bilayer (L alpha) to the inverted hexagonal (HII) phase induced either by increases in temperature or by Ca2+. These properties might contribute to the remarkable selectivity of the fusion of synaptic vesicles with the presynaptic plasma membrane during exocytosis.

Animals↗

PubMed2000

Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles.

Here, to study lipid-protein interactions that contribute to the biogenesis of regulated secretory vesicles, we have developed new approaches by which to label proteins in vivo, using photoactivatable cholesterol and glycerophospholipids. We identify synaptophysin as a major specifically cholesterol-binding protein in PC12 cells and brain synaptic vesicles. Limited cholesterol depletion, which has little effect on total endocytic activity, blocks the biogenesis of synaptic-like microvesicles (SLMVs) from the plasma membrane. We propose that specific interactions between cholesterol and SLMV membrane proteins, such as synaptophysin, contribute to both the segregation of SLMV membrane constituents from plasma-membrane constituents, and the induction of synaptic-vesicle curvature.

Animals↗

PubMed1991

Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans.

unc-104 encodes a novel kinesin paralog that may act as a microtubule-based motor in the nervous system. Neuronal cell lineages and axonogenesis are normal in unc-104 null mutants, but axons have few synaptic vesicles and make only a few small synapses. By contrast, neuron cell bodies have surfeits of similar vesicles tethered together within the cytoplasm. Based on behavioral and cellular phenotypes, we suggest that UNC-104 is a neuron-specific motor used for anterograde translocation of synaptic vesicles along axonal microtubules. Other membrane-bounded organelles are transported normally.

Adenosine Triphosphatases↗

PubMed2000

Assembly of SNARE core complexes prior to neurotransmitter release sets the readily releasable pool of synaptic vesicles.

Precise regulation of neurotransmitter release is essential for the normal function of neural networks, but the mechanisms involved are largely unclear. Using superfused synaptosomes, we have studied the readily releasable pool of synaptic vesicles, measured as the amount of release triggered by hypertonic sucrose. We show that activation of presynaptic metabotropic glutamate receptors by dihydroxyphenylglycine and stimulation of protein kinase C by phorbol esters enhance the readily releasable pool of glutamate. Although the molecular nature of the readily releasable pool is unknown, one possibility is that during its generation, SNARE proteins form full core complexes, and that core complex formation occurs prior to neurotransmitter release. To test this possibility, we employed N-ethylmaleimide (NEM), an inhibitor of the ATPase N-ethylmaleimide-sensitive factor that dissociates core complexes, to study the relation of the readily releasable pool to core complex assembly in synaptosomes. NEM induced a dose-dependent increase in the readily releasable pool of neurotransmitters but by itself did not trigger release. Direct measurements of core complexes confirmed that NEM caused an increase in the levels of SNARE core complexes under these conditions. Our data suggest that in the readily releasable pool of synaptic vesicles, SNARE proteins are fully assembled into core complexes, and that SNARE complex assembly is a target of presynaptic regulation.

Animals↗

PubMed1979

The lipid and protein content of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata purified to constant composition: implications for vesicle structure.

The lipid, protein, acetylcholine and ATP content of cholinergic synaptic vesicles isolated from the richly innervated electric organ of Torpedo marmorata and purified to constant composition has been determined. The number of vesicles present in the preparations has been estimated by quantitative electron microscopy and the mean composition of the vesicle deduced. The acetylcholine content of the purest preparations was considerably greater than that previously attained and reached a mean of 6.10 mmole/g of protein and 2.6 X 10(5) molecules/vesicle; the mean values, for all determinations, were 4.1 +/- S.E.M. 0.6 and 2.6 X 10(5) +/- S.E.M. 0.6 X 10(5) respectively. The lipid and protein content of the vesicle (about 140 and 80 ag/vesicle respectively) is relatively low, indicating a thin, lipid-rich membrane and a highly hydrated core of which not more than 1-2% can be occupied by protein. These findings are consistent with conclusions drawn from recent density determinations made at different osmotic pressures using penetrating and non-penetrating gradients.

Acetylcholine↗

PubMed1984

Demonstration of electron-dense material in clear synaptic vesicles using cationic ferrocenyl compounds.

Electron-dense material in clear synaptic vesicles in rat cerebral cortex and neuromuscular junctions of frog cutaneous pectoris muscle was demonstrated by using ferrocenyl cationics. Electron-dense spots were usually attached to the inner surface of the vesicular membrane. Control experiments (treatment with Triton X-100 or cetylpyridinium chloride; enzyme digestion with trypsin, hyaluronidase, neuraminidase, sulfatase and beta-glucuronidase) suggested that the electron-dense material is a glycoprotein.

Animals↗

PubMed1993

Ral and Rab3a are major GTP-binding proteins of axonal rapid transport and synaptic vesicles and do not redistribute following depolarization stimulated synaptosomal exocytosis.

We have employed high-resolution SDS polyacrylamide gels to demonstrate that there are two major low-molecular-weight GTP-binding proteins associated with axonal membranes including synaptic vesicles, rapid transported membranes and clathrin-coated vesicles. We demonstrate that one of the major proteins is Ral and that the other is Rab3A. Following the depolarization of synaptosomes resulting in increased neurotransmitter release, we see no significant dissociation of either Ral or Rab3a from synaptic vesicle derived membranes in contrast to results reported previously.

Amino Acid Sequence↗

PubMed1979

Adenosine triphosphate-dependent calcium uptake of synaptic vesicle fraction is largely due to contaminating microsomes.

Ca2+ uptake by synaptic vesicle fractions isolated from bovine caudatolenticular nuclei and from rat brain was studied. The purified vesicle fractions from both materials took up very little Ca2+ even in the presence of ATP and Mg2+, but the crude fractions took up Ca2+ actively, showing the maximum uptake around pH 7.0. Since the crude fractions were contaminated by microsomes, which are known to accumulate Ca2+ actively (Yoshida, H., Kadota, K., & Fujisawa, H. (1966) Nature 212, 291--292; Otsuka, M., Ohtsuki, I., & Ebashi, S. (1965) J. Biochem. 58, 188-190), the active uptake of Ca2+ appeared to be largely, if not wholly, due to microsomal contamination.

Adenosine Triphosphate↗

PubMed2005

Distribution of proteins associated with synaptic vesicle endocytosis in the mouse and goldfish retina.

Current models of synaptic transmission require retrieval of membrane from the presynaptic terminal following neurotransmitter exocytosis. Dynamin, a GTPase, is thought to be critical for this retrieval process. At ribbon synapses of retinal bipolar neurons, however, compensatory endocytosis does not require GTP hydrolysis, suggesting that endocytosis mechanisms may differ among synapses. To understand better the synaptic vesicle recycling at conventional and ribbon synapses, the distributions of dynamin and two associated proteins, amphiphysin and clathrin, were examined in the retinas of goldfish and mouse by using immunocytochemical methods. Labeling for dynamin, clathrin, and amphiphysin was distributed differentially among conventional and ribbon synapses in retinas of both species. Ribbon synapses of photoreceptors and most bipolar cells labeled only weakly for dynamin relative to conventional synapses. Amphyiphysin labeling was strong at many ribbon synapses, and labeling in rod terminals was stronger than in cone terminals in the mouse retina. Clathrin labeling was heterogeneous among ribbon synapses. Similarly to the case with amphiphysin, mouse rod terminals showed stronger clathrin labeling than cone terminals. Among conventional synapses, there was heterogeneous labeling for all three endocytic proteins. Some labeling for each protein might have been associated with postsynaptic terminals. The differential distribution of labeling for these proteins among identified synapses in the retina suggests considerable heterogeneity in the molecular mechanisms underlying synaptic membrane retrieval, even among synapses with similar active zone ultrastructure. Thus, as with exocytosis, mechanisms of synaptic membrane retrieval may be tuned by the precise complement of proteins expressed within the synaptic terminal.

Animals↗

PubMed2002

The synaptic vesicle protein synaptophysin: purification and characterization of its channel activity.

The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCl solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 +/- 13 pS at +60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K(+) over Cl(-) (P(K(+))/P(Cl(-)) > 8) and preferred K(+) over Li(+), Na(+), Rb(+), Cs(+), or choline(+). The synaptophysin channel is impermeable to Ca(2+), which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission.

Animals↗

PubMed1991

Calcium-dependent release of accumulated glutamate from synaptic vesicles within permeabilized nerve terminals.

We have studied glutamate release from synaptic vesicles in permeabilized synaptosomes, which were preloaded with [3H]glutamate in an ATP-dependent manner. The release was found to be calcium-dependent and to require a heat-labile cytosolic macromolecule factor for maximum activity. Maximal release occurred at 5 microM free Ca2+ and within 5 min. Of the other divalent cations tested, only barium stimulated release of vesicular glutamate. The release was inhibited by N-ethylmaleimide. These results are characteristic of exocytotic release of monoamines and peptides observed in endocrine systems, and constitute direct evidence for the notion that calcium-dependent release of glutamate originates from the vesicular pool.

Adenosine Triphosphate↗

PubMed1992

Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons.

In mature neurons synaptic vesicles (SVs) undergo cycles of exo-endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.

Calcium-Binding Proteins↗

PubMed2026

Synaptic vesicle glycoprotein 2A PET imaging in parkinsonian α-synucleinopathies: a systematic review.

Synaptic dysfunction is increasingly recognized as an early and biologically relevant component of α-synucleinopathies. However, conventional imaging biomarkers mainly assess dopaminergic dysfunction, glucose metabolism, or structural damage rather than presynaptic density itself. Synaptic vesicle glycoprotein 2A (SV2A) PET enables in vivo assessment of presynaptic terminal integrity and may provide complementary information in Parkinson's disease (PD), Parkinson's disease dementia/dementia with Lewy bodies (PDD/DLB), and multiple system atrophy (MSA). This systematic review synthesized the available evidence on SV2A-targeted PET in parkinsonian α-synucleinopathies, focusing on regional imaging patterns, clinical associations, longitudinal findings, and methodological determinants of interpretation. Seventeen reports were included. In PD, the most recurrent finding was reduced SV2A binding in the substantia nigra, although additional involvement of brainstem, caudate, striatal, thalamic, raphe, or cortical regions was reported in selected cohorts. In PDD/DLB, abnormalities appeared broader and more cortical, with evidence of association between cortical SV2A binding and cognitive performance. In MSA, one study suggested a distinct infratentorial and cerebellar pattern with potential relevance for phenotypic stratification. SV2A PET is a promising research biomarker for biological characterization of synucleinopathies. However, the field remains limited by small cohorts, methodological heterogeneity, variable quantification strategies, limited longitudinal evidence, and potential cohort overlap. Multicentre validation and harmonized protocols are required before clinical translation.

Humans↗

PubMed1980

Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction.

Frog cutaneous pectoris muscles were treated with low doses of crude black widow spider venom (BWSV) or purified alpha-latrotoxin, and neuromuscular transmission, quantal secretion, changes in ultrastructure and uptake of horseradish peroxidase (HRP) were studied. When these agents were applied to muscles bathed in a Ca2+-free solution with 1 mM EGTA and 4 mM Mg2+, the rate of quantal secretion rose to high levels but quickly subsided; neuromuscular transmission was totally and irreversibly blocked within 1 h. The terminals became swollen and were depleted of vesicles; HRP was not taken up. When BWSV was applied to other muscles bathed in a solution with 1.8 mM Ca2+ and 4 mM Mg2+, the rate of secretion rose to high levels and then declined to intermediate levels that were sustained throughot the hour of exposure. Neuromuscular transmission was blocked in fewer than 50% of these fibers. The ultrastructure of these terminals was normal and they contained large numbers of synaptic vesicles. If HRP had been present, most of the synaptic vesicles were labeled with reaction product. These observations suggest that Ca2+ plays an important role in endocytosis at the frog neuromuscular junction.

Acetylcholine↗

PubMed2005

The C-terminal transmembrane region of synaptobrevin binds synaptophysin from adult synaptic vesicles.

Synaptophysin and synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. In mature, differentiated neurons they form the synaptophysin/synaptobrevin (Syp/Syb) complex. Synaptobrevin also interacts with the plasma membrane-associated proteins syntaxin and SNAP25, thereby forming the SNARE complex necessary for exocytotic membrane fusion. The two complexes are mutually exclusive. Synaptobrevin is a C-terminally membrane-anchored protein with one transmembrane domain. While its interaction with its SNARE partners is mediated exclusively by its N-terminal cytosolic region it has been unclear so far how binding to synaptophysin is accomplished. Here, we show that synaptobrevin can be cleaved in its synaptophysin-bound form by tetanus toxin and botulinum neurotoxin B, or by botulinum neurotoxin D, leaving shorter or longer C-terminal peptide chains bound to synaptophysin, respectively. A recombinant, C-terminally His-tagged synaptobrevin fragment bound to nickel beads specifically bound synaptophysin, syntaxin and SNAP25 from vesicular detergent extracts. After cleavage by tetanus toxin or botulinum toxin D light chain, the remaining C-terminal fragment no longer interacted with syntaxin or SNAP 25. In contrast, synaptophysin was still able to bind to the residual C-terminal synaptobrevin cleavage product. In addition, the His-tagged C-terminal synaptobrevin peptide 68-116 was also able to bind synaptophysin in detergent extracts from adult brain membranes. These data suggest that synaptophysin interacts with the C-terminal transmembrane part of synaptobrevin, thereby allowing the N-terminal cytosolic chain to interact freely with the plasma membrane-associated SNARE proteins. Thus, by binding synaptobrevin, synaptophysin may positively modulate neurotransmission.

Animals↗

PubMed1984

Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.

Animals↗

PubMed2006

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals.

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca2+ ions with other bivalent cations (Sr2+, Ba2+, Cd2+, Mg2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca2+ from intracellular stores. The effect of extracellular Ca2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.

Animals↗

PubMed1987

Time course and frequency dependence of synaptic vesicle depletion and recovery in electrically stimulated sympathetic ganglia.

The mammalian superior cervical sympathetic ganglion has been extensively used to study the kinetics of ACh metabolism and release. The present investigation examined the time course of changes in the number of synaptic vesicles and abundance of plasma membrane at preganglionic nerve terminals using stimulation protocols similar to those used in previous biochemical and electrophysiological studies. Continuous stimulation of the preganglionic trunk to the cat superior cervical ganglion in vivo produced an initially rapid fall in the number of clear synaptic vesicles followed by a subsequent plateau. Reciprocal changes in plasma membrane occurred with a similar time course. The plateau phase is interpreted as a steady-state where vesicle exocytosis is balanced by the rate of vesicle reformation from plasma membrane. During quiescent recovery, restoration of normal resting ultrastructure is initially rapid but slows with time as vesicle number and plasma membrane abundance approach pre-stimulation values, indicating that the rate of vesicle reformation at the end of stimulation is high and proportional to the number of vesicles incorporated into the plasma membrane. These results are interpreted as consistent with the 'vesicle hypothesis' of neurotransmitter release.

Acetylcholine↗