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PubMed1993

Synaptic vesicle phosphoproteins and regulation of synaptic function.

Complex brain functions, such as learning and memory, are believed to involve changes in the efficiency of communication between nerve cells. Therefore, the elucidation of the molecular mechanisms that regulate synaptic transmission, the process of intercellular communication, is an essential step toward understanding nervous system function. Several proteins associated with synaptic vesicles, the organelles that store neurotransmitters, are targets for protein phosphorylation and dephosphorylation. One of these phosphoproteins, synapsin I, by means of changes in its state of phosphorylation, appears to control the fraction of synaptic vesicles available for release and thereby to regulate the efficiency of neurotransmitter release. This article describes current understanding of the mechanism by which synapsin I modulates communication between nerve cells and reviews the properties and putative functions of other phosphoproteins associated with synaptic vesicles.

Animals↗
PubMed1996

Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence.

In nerve terminals, synaptic vesicles form large clusters anchored to the presynaptic plasmalemma. Recently, FM1-43 photobleaching experiments carried out a frog motor end-plates demonstrated lack of lateral intermixing of synaptic vesicles within clusters, even during sustained nerve terminal stimulation (Henkel and Betz, 1995; Henkel et al., 1996b). We now have investigated the mobility of synaptic vesicle membranes during the endocytic limb of their exo-endocytic cycle. To this aim, we have carried out photobleaching experiments on nerve terminals of hippocampal neurons prelabeled with CY3-conjugated antibodies directed against lumenal epitopes of synaptotagmin I. This conjugate is taken up specifically by synaptic vesicle membranes during endocytosis and then is recovered in newly formed synaptic vesicles. Using this method, we show that synaptic vesicle membranes intermix after endocytosis. Staurosporine, which at hippocampal synapses partially inhibits unloading of FM1-43, but does not block uptake of antibody probes, prevents this intermixing. Our results indicate that synaptic vesicle docking and/or fusion with the plasmalemma correlate with the release of their membranes from a restraining matrix that hinders their lateral mobility. They suggest that membrane intermediates involved in synaptic vesicle reformation interact with a distinct, highly dynamic cytoskeleton and that newly formed synaptic vesicles are recaptured at random within vesicle clusters. Staurosporine, by inhibiting mobility within the terminal, may favor recapture of new vesicles near sites of endocytosis.

Animals↗
PubMed1996

Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal.

After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.

Amino Acid Sequence↗
PubMed1995

A targeting signal in VAMP regulating transport to synaptic vesicles.

VAMP is a synaptic vesicle membrane protein required for fusion. Synaptic vesicle targeting was measured for mutants of an epitope-tagged form of VAMP in transfected PC12 cells. A signal within a predicted amphipathic alpha helix is essential for targeting to synaptic vesicles. Cellubrevin, a nonneural VAMP homolog, contains this signal and is also targeted to synaptic vesicles. Amino acid substitutions within the synaptic vesicle targeting signal either enhance or inhibit sorting of VAMP to synaptic vesicles, but do not affect the ability of VAMP to form complexes with syntaxin and SNAP-25.

Amino Acid Sequence↗
PubMed1994

Vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle.

The synaptic vesicle membrane protein VAMP (vesicle-associated membrane protein or synaptobrevin) has been implicated in synaptic vesicle docking and fusion. Synaptophsin (p38), also a synaptic vesicle membrane protein, has four transmembrane domains and may function as a gap junction-like pore or channel. Here we report evidence for a direct interaction between VAMP and synaptophysin using chemical cross-linking followed by the identification of immunoreactive protein complexes. A prominent complex of 56 kDa was found to consist of VAMP and synaptophysin. Furthermore, we demonstrate that this VAMP-synaptophysin complex is enriched in the synaptic vesicle fraction of rat brain, is independent of detergent solubilization, and is present in PC12 cells subjected to in vivo cross-linking.

Animals↗
PubMed2005

Regulation of synaptic vesicle recycling by calcineurin in different vesicle pools.

The synaptic vesicles keep recycling by the processes of endocytosis and exocytosis to maintain the normal synaptic transmission. The synaptic vesicles are classified as the readily releasable pool (RRP) and the reserve pool (RP). In the endocytosis process, calcineurin (CaN), a Ca2+/calmodulin-dependent protein phosphatase, has been shown to play important roles. However, it is unclear about its roles in different vesicle pools. Here, we investigated the role of CaN in the regulation of vesicle recycling in the RRP and RP. Vesicle recycling was monitored by using fluorescent dyes FM1-43 and FM4-64 in the primary cultures of hippocampal neurons. Inhibition of CaN by FK506 and cyclosporin A suppressed the endocytosis in the RP, but not in the RRP. Inhibition of CaN also restrained the exocytic process triggered by 10 Hz stimulation, but had no effect on 3-5 Hz stimulation-induced exocytosis. FK506 also reduced the total vesicle pool size in the synaptic terminals. A synthesized CaN inhibitory peptide showed the similar effects as FK506 and cyclosporin A. These results revealed a novel mechanism that CaN plays critical roles in the distinct vesicle recycling processes.

Animals↗
PubMed1995

The vesicular monoamine transporter 2 is present in small synaptic vesicles and preferentially localizes to large dense core vesicles in rat solitary tract nuclei.

In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.

Animals↗
PubMed2000

Mechanisms of synaptic vesicle exocytosis.

Chemical synaptic transmission serves as the main form of cell to cell communication in the nervous system. Neurotransmitter release occurs through the process of regulated exocytosis, in which a synaptic vesicle releases its contents in response to an increase in calcium. The use of genetic, biochemical, structural, and functional studies has led to the identification of factors important in the synaptic vesicle life cycle. Here we focus on the prominent role of SNARE (soluble NSF attachment protein receptor) proteins during membrane fusion and the regulation of SNARE function by Rab3a, nSec1, and NSF. Many of the proteins important for transmitter release have homologs involved in intracellular vesicle transport, and all forms of vesicle trafficking share common basic principles. Finally, modifications to the synaptic exocytosis pathway are very likely to underlie certain forms of synaptic plasticity and therefore contribute to learning and memory.

Animals↗
PubMed1994

Specificity and regulation of a synaptic vesicle docking complex.

Synaptic vesicles are proposed to dock at the presynaptic plasma membrane through the interaction of two integral membrane proteins of synaptic vesicles, VAMP and synaptotagmin, and two plasma membrane proteins, syntaxin and SNAP-25. We have characterized the binding properties of these proteins and observed SNAP-25 potentiation of VAMP 2 binding to syntaxins 1a and 4 but not syntaxins 2 or 3. n-sec1, a neuron-specific syntaxin-binding protein, bound syntaxin with nanomolar affinity, forming a complex that is distinct from the previously identified 7S and 20S syntaxin-containing complexes. This suggests that syntaxin exists in at least three states: bound to n-sec1, in a 7S particle, and in a 20S particle. Recombinant n-sec1 inhibited VAMP or SNAP-25 binding to syntaxin. We propose that the specific associations of VAMP, SNAP-25, and syntaxin mediate vesicle docking and that a syntaxin/n-sec1 complex precedes and/or regulates formation of these complexes.

Amino Acid Sequence↗
PubMed2006

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling.

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.

Acyltransferases↗
PubMed2006

UNC-13 and UNC-10/rim localize synaptic vesicles to specific membrane domains.

Synaptic vesicles undergo a maturation step, termed priming, in which they become competent to fuse with the plasma membrane. To morphologically define the site of vesicle priming and identify fusion-competent synaptic vesicles, we combined a rapid physical-fixation technique with immunogold staining and high-resolution morphometric analysis at Caenorhabditis elegans neuromuscular junctions. In these presynaptic terminals, a subset of synaptic vesicles contact the plasma membrane within approximately 100 nm of a presynaptic dense projection. UNC-13, a protein required for vesicle priming, localizes to this same region of the plasma membrane. In an unc-13 null mutant, few synaptic vesicles contact the plasma membrane, suggesting that membrane-contacting synaptic vesicles represent the morphological correlates of primed vesicles. Interestingly, a subpopulation of membrane-contacting vesicles, located within 30 nm of a dense projection, are unperturbed in unc-13 mutants. We show that UNC-10/Rim, a protein implicated in presynaptic plasticity, localizes to dense projections and that loss of UNC-10/Rim causes an UNC-13-independent reduction in membrane-contacting synaptic vesicles within 30 nm of the dense projections. Our data together identify a discrete domain for vesicle priming within 100 nm of dense projections and further suggest that UNC-10/Rim and UNC-13 separately contribute to the membrane localization of synaptic vesicles within this domain.

Animals↗
PubMed2004

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.

Action Potentials↗
PubMed2001

Studies of synaptic vesicle endocytosis in the nematode C. elegans.

After synaptic vesicle exocytosis, synaptic vesicle proteins must be retrieved from the plasma membrane, sorted away from other membrane proteins, and reconstituted into a functional synaptic vesicle. The nematode Caenorhabditis elegans is an organism well suited for a genetic analysis of this process. In particular, three types of genetic studies have contributed to our understanding of synaptic vesicle endocytosis. First, screens for mutants defective in synaptic vesicle recycling have identified new proteins that function specifically in neurons. Second, RNA interference has been used to quickly confirm the roles of known proteins in endocytosis. Third, gene targeting techniques have elucidated the roles of genes thought to play modulatory or subtle roles in synaptic vesicle recycling. We describe a molecular model for synaptic vesicle recycling and discuss how protein disruption experiments in C. elegans have contributed to this model.

Animals↗
PubMed1977

Immunochemical comparison of synaptic plasma membrane and synaptic vesicle membrane antigens.

A synaptic vesicle fraction and a synaptic plasma membrane fraction obtained after subfractionation of synaptosomes from chick forebrain have been used to produce antisera in rabbits. Immunofluorescence histology with the two antisera revealed that they reacted strongly with synaptic terminal regions present in the chick forebrain, cerebellum and spinal cord. In addition, the synaptic plasma membrane antiserum (but not the synaptic vesicle antiserum) reacted with preterminal axons in the cerebellum and spinal cord. Comparison of the two antisera by two-dimensional immunoelectrophoresis, revealed the presence of common antigens in the synaptosomal vesicle and plasma membrane fractions. Incubation of synaptosomes in vitro with the synaptosomal vesicle antiserum and complement produced a dose-dependent inhibition of synaptosome swelling up to a maximum of 55% of that obtained with the synaptosomal plasma membrane antiserum. The results of this test are consistent with the hypothesis that some synaptosomal vesicle antigens may be present also in the synaptosomal plasma membrane and imply that they face the external surface of the synaptosomes. The fate of vesicle membrane components in synaptosomal plasma membranes is not known. The possibility is discussed that they may be recycled locally by a mechanism similar to that proposed by Heuser and Reese (1973) for re-use of synaptic vesicle membranes at the neuromuscular junction.

Animals↗
PubMed1989

AH5183 and cetiedil: two potent inhibitors of acetylcholine uptake into isolated synaptic vesicles from Torpedo marmorata.

Synaptic vesicles purified on a sucrose-KCl sedimentation gradient were tested for their ability to accumulate [1-14C]acetylcholine ([1-14C]ACh) in the absence and in the presence of AH5183 and cetiedil. Kinetic studies of ACh transport showed that it was time dependent and saturable as a function of ACh concentration, with a KT of 1.2 mM. The protein-modifying agents N-ethylmaleimide and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole were powerful inhibitors of ACh uptake. In agreement with other studies, AH5183 was found to be a potent inhibitor of ACh uptake by synaptic vesicles. Inhibition was of the mixed noncompetitive type, and the inhibition constant was 45.2 +/- 3.4 nM. Cetiedil, a drug that resembles ACh, was previously shown on intact nerve endings to inhibit the translocation of newly synthesized ACh into the synaptic vesicle compartment, and we demonstrate here that cetiedil is indeed an efficient blocker of ACh uptake by isolated synaptic vesicles. It acted as a competitive inhibitor, with a Ki of 118.5 +/- 9.5 nM. Neither ATP-dependent calcium uptake nor Mg2+-ATPase activity was affected by the drugs, a finding showing their specificity toward the ACh uptake process. The binding of L-[3H]AH5183 to intact vesicles was characterized in the absence or the presence of ACh or cetiedil. Saturation experiments showed a total binding capacity of approximately 126 pmol/mg of vesicular protein and a dissociation constant of 19.9 +/- 4.1 nM under control conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1994

Detection of G-protein heterotrimers on large dense core and small synaptic vesicles of neuroendocrine and neuronal cells.

Heterotrimeric G proteins, initially believed to be exclusively present in the plasma membrane, have also been found to be associated with intracellular membrane compartments. There they are involved in various membrane trafficking processes including regulated secretion (reviewed in Bomsel, M., K. Mostov, Mol. Biol. Cell 3, 1317-1328 (1992)). Vesicles of two distinct types enter the regulated secretory pathway, i.e. large dense core vesicles and small synaptic vesicles, which differ in their membrane composition and content. Little is known about an association of heterotrimeric G proteins with regulated secretory vesicles, that would explain some aspects of the role heterotrimeric G proteins have during secretion. By immunofluorescence microscopy and immunoreplica analysis, we provide the first demonstration of the presence of complete sets of heterotrimeric G proteins, consisting of alpha-, beta-, and gamma-subunits, on large dense core vesicles from bovine adrenal medulla (chromaffin granules) and small synaptic vesicles from rodent and bovine brain. Each of the two types of secretory vesicles contains beta-subunits (at least beta 1 and beta 2), as well as gamma-subunits (at least gamma 2 or gamma 3). Interestingly, they differ in their composition of alpha-subunits. On small synaptic vesicles, we found two G(o) alpha-subunits (alpha o1 and alpha o2) and two Gi alpha-subunits (alpha i1 and alpha i2). In contrast, on chromaffin granules so far only one alpha o-subunit but no alpha i-subunits could be detected. Functional properties such as transmitter storage and/or exocytotic membrane fusion may be modulated by the various G-protein subunits associated with chromaffin granules and small synaptic vesicles.

Adrenal Medulla↗
PubMed1993

Uptake of the neurotoxin, 4-methylphenylpyridinium, into chromaffin granules and synaptic vesicles: a proton gradient drives its uptake through monoamine transporter.

Energy dependence for uptake of 4-methyphenylpyridinium (MPP+), a neurotoxin causing Parkinsonism-like symptoms, by adrenal chromaffin granule membrane vesicles and brain synaptic vesicles was studied. The compound was actively taken up by the chromaffin vesicles dependent on hydrolysis of ATP with a Km value of 22 microM and maximum velocity of 2.9 nmol/min/mg protein. The uptake was sensitive to reserpine (1 microM) and bafilomycin (50 nM) (inhibitors of the vesicular monoamine transporter and vacuolar-type H(+)-ATPase, respectively) and substrates for monoamine transporters, but insensitive to imipramine (an inhibitor of the monoamine transporter present in the plasma membrane). The uptake was greatly reduced upon dissipation of the proton gradient by ammonium ion or nigericin with KCl, but stimulated 1.6-fold by valinomycin plus K+. Dissipation of the proton gradient also induced rapid efflux of MPP+ from the vesicles. The MPP+ (monoamine) transporter was solubilized from chromaffin vesicles and reconstituted into liposomes with purified bacterial F0F1-ATPase. MPP+ was taken up by the liposomes coupled with ATP hydrolysis by F0F1, and the uptake was sensitive to reserpine, dissipation of the proton gradient, and azide. Brain synaptic vesicles also accumulated MPP+, showing similar kinetics, inhibitor sensitivities, and energy coupling to those of chromaffin vesicles. Furthermore, MPP+ inhibited the uptake of dopamine without affecting the uptake of glutamate or gamma-aminobutyrate. These results indicated that MPP+ was taken up through the reserpine-sensitive monoamine transporter into chromaffin vesicles and synaptic vesicles and that the energy for accumulation of MPP+ was supplied as a proton gradient (acidic inside) established by H(+)-ATPase.

1-Methyl-4-phenylpyridinium↗