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PubMed2000

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.

Animals↗

PubMed1998

Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons.

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

Animals↗

PubMed1994

Association of N-ethylmaleimide-sensitive factor with synaptic vesicles.

N-Ethylmaleimide-sensitive factor (NSF) mediates docking and/or fusion of transport vesicles in the multi-pathways of vesicular transport. NSF was highly expressed in brain and adrenal gland. Immunostaining of cerebellum with an anti-NSF monoclonal antibody showed that NSF is predominantly localized in the molecular layers and the glomeruli of the granule cell layers. This distribution coincided well with that of synaptophysin, a marker protein of synaptic vesicles. Purification and immunoprecipitation revealed that NSF is associated with brain synaptic vesicles. The present results suggest that NSF is associated with synaptic vesicles without Ca2+ influx.

Animals↗

PubMed1996

Synaptic vesicle proteins in cells of the sympathoadrenal lineage.

The cells of sympathoadrenal lineage display different characteristics after differentiation, although they stem from the same neural crest precursor during embryonic development. In the present study we compared the distribution patterns of several synaptic vesicle proteins in the superior cervical ganglion (SCG) and the adrenal medulla. Using indirect immunofluorescence combined with confocal laser scanning microscopy, it was observed that antisera against integral synaptic vesicle membrane proteins (SV2, synaptotagmin I, synaptobrevin II and synaptophysin) induced strong immunoreactivities in these cells, but anti-synaptobrevin I caused only a faint fluorescence. Immunoreactivities of the synaptic vesicle-associated proteins Rab3a and SNAP25 were also observed in the cells. Synapsin-Ia-reactive material appeared absent from chromaffin cells but present in small amounts in sympathetic neurons in the SCG and iris terminals. On the other hand, synapsin IIa immunoreactive material was strong in most SCG neurons and in adrenergic iris terminals. The neural specific clatrin light chain was detected in the SCG cells and in ganglion cells of the adrenal, but only weak traces could be observed in chromaffin cells. One of the vesicular monoamine transmitter transporters, VMAT2, which is expressed in catecholamine neurons in the brain stem, was observed in most cells in the SCG and also in groups of cells in the adrenal medulla, where the VMAT2-positive small chromaffin cells were PNMT-negative. SIF cells in the SCG contained most of the synaptic vesicle proteins investigated. The results show that after differentiation, sympathetic neurons, SIF cells and adrenal chromaffin cells still share many vesicle proteins even though their physiology is different.

Adrenal Glands↗

PubMed1991

Protein kinase C and Ca2+/calmodulin-dependent protein kinase II phosphorylate a novel 58-kDa protein in synaptic vesicles.

A monoclonal antibody was made using the spleen cells of a mouse immunized with chick synaptic membranes and designated as mAb 1D12. It immunoprecipitated 25% of the omega-conotoxin binding protein but no dihydropyridine binding protein solubilized from chick brain membranes. By immunoblotting, a polypeptide of 58-kDa was identified as the antigen of this antibody in chick, rat, rabbit and guinea pig brain. Immunohistochemical observation indicated the immunoreactivity of mAb 1D12 to be localized in the synaptic regions of central and peripheral neurons. In peripheral organs, there was additional staining in the distal portions of nerve fibers. Immunoelectron microscopy showed immunoreactivity to be located in synaptic vesicle and presynaptic plasma membranes. In the subcellular fractionation of rat brain, 58-kDa protein was recovered in the fractions of synaptic vesicles and plasma membranes but not soluble proteins. This protein could be extracted from membranes by Triton X-100 but treatment with EDTA, acid, base or high salt failed to have such effect. Solubilized 58-kDa protein of rat brain was purified by immunoaffinity chromatography using mAb 1D12. Both protein kinase C and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated purified 58-kDa protein, and maxima of 0.47 and 0.94 mol of phosphates, respectively, were incorporated per mol of 58-kDa protein. 58-kDa protein was not phosphorylated by either cAMP-dependent or cGMP-dependent protein kinase. When present in membranes, it was also phosphorylated by protein kinase C and CaM kinase II. Possible involvement of 58-kDa protein in the protein kinase C and CaM kinase II-mediated regulation of synaptic transmission in central and peripheral neurons is discussed.

Animals↗

PubMed1986

Brain protein kinase in synaptic vesicles is inhibited by a factor in sera from systemic lupus erythematosus patients with central nervous system manifestations.

Sera from systemic lupus erythematosus patients with clinical central nervous system manifestations had a high mean percent of brain synaptic vesicle protein kinase inhibition (62 +/- 9.3, P less than 0.001). This conclusion was derived from screening sera of a total of 287 patients with heterogeneous diseases and from 12 healthy controls. Low levels of synaptic vesicle protein kinase inhibition also were detected in the sera of patients with certain autoimmune, inflammatory, neurological and/or psychiatric symptoms. Because 87.5% of the patients whose sera showed strong synaptic vesicle protein kinase inhibition (over 50%) had neuropsychiatric manifestations, we postulate this relationship may be due to the presence of an inhibitor factor, of which the etiology and molecular characteristics were investigated.

Adult↗

PubMed2000

Fast synaptic fatigue in shibire mutants reveals a rapid requirement for dynamin in synaptic vesicle membrane trafficking.

The GTPase dynamin is involved in endocytosis in many cell types, as first revealed by temperature-sensitive paralytic mutations in the Drosophila dynamin gene, shibire (shi), which disrupt synaptic vesicle endocytosis and deplete synaptic terminals of vesicles. Here we report that shi synapses exhibit a fast synaptic fatigue phenotype within 20 ms of repetitive stimulation, which cannot be explained by vesicle depletion, as we confirmed by electron microscopy. These results suggest that, in addition to its well-characterized role in synaptic vesicle recycling, dynamin may be required for short-term maintenance of the readily releasable pool of synaptic vesicles.

Animals↗

PubMed1994

Localization of Rabphilin-3A on the synaptic vesicle.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. Rabphilin-3A is expressed mainly in brain, but its subcellular localization remains to be clarified. Immunohistochemical analysis has revealed that Rabphilin-3A is most abundant in the synaptic area of the rat cerebellum, retina, and neuromuscular junction. Ultrastructural analysis of the neuromuscular junction using the immunogold method indicates that Rabphilin-3A is localized on the synaptic vesicle. Subcellular fractionation analysis of rat brain has shown that Rabphilin-3A is most highly concentrated in the purified synaptic vesicle fraction. These results indicate that Rabphilin-3A is localized on the synaptic vesicle in the presynapse.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Developmental changes in the localization of the synaptic vesicle protein rab3A in rat brain.

Rab3A is a protein associated with the membrane of synaptic vesicles and is involved in the control of the targeting or docking of these vesicles at the presynaptic membrane for the release of neurotransmitters. Here, we have examined the expression and localization of this protein during the development of the rat brain. Relative to total protein, the concentration of rab3A greatly increased during brain development. Both the intracellular localization of the protein and its cerebral distribution showed an age-dependent shift. In contrast to other synaptic vesicle proteins, rab3A was heavily concentrated in cell bodies when immature neurons were migrating and during early differentiation. Later, the protein disappeared from perikarya and had a diffuse distribution in the neuropil, indicating a redistribution to nerve terminals, its exclusive localization in the adult. In the developing somatosensory cortex, rab3A delimited the modular organization of the barrels well after the afferents have arrived but just around the time that mature synaptic activity has been observed. In the hippocampus, rab3A defined a novel "blob-like" organization of the mossy fibre terminals and its appearance in terminal fields closely preceded the known onset of long-term potentiation. The appearance of rab3A in specific terminal fields during the period of increased physiological activity suggests that this small GTP-binding protein may be an important late element in the establishment of the mature characteristics of the presynaptic terminal.

Aging↗

PubMed2006

Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling.

During recycling of synaptic vesicles (SVs), the retrieval machinery faces the challenge of recapturing SV proteins in a timely and precise manner. The significant dilution factor that would result from equilibration of vesicle proteins with the much larger cell surface would make recapture by diffusional encounter with the endocytic retrieval machinery unlikely. If SV proteins exchanged with counterparts residing at steady state on the cell surface, the dilution problem would be largely avoided. In this scenario, during electrical activity, endocytosis would be driven by the concentration of a pre-existing pool of SVs residing on the axonal or synaptic surface rather than the heavily diluted postfusion vesicular pool. Using both live cell imaging of endogenous synaptotagmin Ia (sytIa) as well as pHluorin-tagged sytIa and VAMP-2, we show here that synaptic vesicle proteins interchange with a large pool on the cell axonal surface whose concentration is approximately 10-fold lower than that in SVs.

Animals↗

PubMed1986

[3H]Dihydrotetrabenazine binding to bovine striatal synaptic vesicles.

[2-3H]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-11bH-benzo[a]quinolizine) bound to a single class of binding sites in synaptic vesicles isolated from bovine corpus striatum, with an apparent dissociation constant (Kd) of 2.3 nM and a Bmax of 15.1 pmol/mg of protein determined at equilibrium. Kinetic determination of the equilibrium dissociation constant yielded a value of 5.4 nM. ATP had no effect on the apparent Kd or Bmax, nor did it alter the kinetics of association or dissociation. Dopamine, serotonin, and other substrates for transport into synaptic vesicles inhibited binding at concentrations that were several orders of magnitude higher than their Km values for transport in the presence of ATP. The potent uptake blocker reserpine inhibited with a Ki of 340 nM in the absence of ATP, but biphasic inhibition, with Ki values of 3.2 and 345 nM, was observed in the presence of ATP. With incubation times of 24 hr, the potency of reserpine as an inhibitor of binding in the absence of ATP is increased by 1 to 2 orders of magnitude, implying a slow association rate for reserpine in the absence of nucleotide. These results suggest that dihydrotetrabenazine interacts with the catecholamine/serotonin porter in synaptic vesicles, although the binding site is probably not identical to that involved in active transport of substrate.

Adenosine Triphosphate↗

PubMed1999

Essential role of phosphoinositide metabolism in synaptic vesicle recycling.

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.

Animals↗

PubMed2003

Colocalization of synapsin and actin during synaptic vesicle recycling.

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

Actins↗

PubMed2005

Interaction of SPIN90 with dynamin I and its participation in synaptic vesicle endocytosis.

SH3 protein interacting with Nck, 90 kDa (SPIN90) is an Nck-binding protein that contains one Src homology 3 (SH3) domain, three proline-rich domains (PRDs), a serine/threonine-rich region, and a hydrophobic C-terminal region. Previously, we have shown that SPIN90 plays roles in the sarcomere assembly in cardiac muscles and in the formation of focal contacts in HeLa cells. Besides in heart, SPIN90 is also highly expressed in the brain, but its role in the neuronal system is completely unknown. Here, we found that SPIN90 is expressed in the presynaptic compartment in which it binds dynamin I, a key component of the endocytic machinery, and that it participates in synaptic vesicle endocytosis. Pull-down analysis and coimmunoprecipitation proved the associations of SPIN90 with dynamin I through SH3-PRD interaction. Overexpression of SPIN90 or knocking down SPIN90 by small interfering RNA impaired synaptic vesicle endocytosis. We further confirmed by the rescue experiments that the endocytic defects by SPIN90 expression come from its interaction with dynamin I. Exocytosis kinetics was not affected by SPIN90 expression. Together, our findings suggest that SPIN90 could modulate the interactions of dynamin I with other endocytic proteins that cooperate in the coated vesicle formation, thus regulating synaptic vesicle endocytosis.

Adaptor Proteins, Signal Transducing↗

PubMed1984

Synaptic vesicles contain an ATP-dependent proton pump and show 'knob-like' protrusions on their surface.

Synaptic vesicles from guinea pig brain were highly purified by chromatography on Sephacryl S 1000. They were associated with a Mg-ATPase which could be solubilized with dichloromethane from vesicle membranes, and which elutes upon gel filtration with a mol. wt. approximately 300 kd. Vesicles accumulate [14C]methylamine in the presence of external ATP, indicating an ATP-dependent proton pump. Electron microscopy using a quick freeze, deep etch, rotary shadowing technique showed characteristic 'knob-like' protrusions on the surface of the vesicle. We suggest that these protrusions represent part of a proton ATPase which may be necessary for packaging of neurotransmitter into synaptic vesicles.

Adenosine Triphosphatases↗

PubMed2000

Synaptophysin regulates clathrin-independent endocytosis of synaptic vesicles.

The GTPase dynamin I is required for synaptic vesicle (SV) endocytosis. Our observation that dynamin binds to the SV protein synaptophysin in a Ca(2+)-dependent fashion suggested the possibility that a dynamin/synaptophysin complex functions in SV recycling. In this paper we show that disruption of the dynamin/synaptophysin interaction by peptide injection into the squid giant synapse preterminal results in a decrease in transmitter release during high-frequency stimulation, indicating an inhibition of SV recycling. Electron microscopy of these synapses reveals a depletion of SVs, demonstrating a block of vesicle retrieval after fusion. In addition, we observed an increase in clathrin-coated vesicles, indicating that the peptide does not block clathrin-dependent endocytosis. We conclude that the dynamin/synaptophysin complex functions in a clathrin-independent mechanism of SV endocytosis that is required for efficient synaptic transmission.

Animals↗

PubMed1997

Synaptic vesicle glutamate uptake in epileptic (EL) mice.

The ATP-dependent glutamate uptake system of synaptic vesicles was investigated in epileptic (EL) mice to determine whether glutamate uptake activity correlates with seizure susceptibility or development. Given the focal seizure onset, glutamate uptake activity was measured in four separate brain regions: cerebrum (minus hippocampus), hippocampus, cerebellum, and brain stem. The EL values were compared to those of age-matched controls; DDY and ABP/LeJ (ABP) mice. The glutamate uptake specific activity for EL cerebrum was significantly higher than that for the control mice (approx. 400 days old), but was not elevated prior to seizure onset (46 days old). No difference in glutamate uptake was observed between the strains in the other brain regions. We conclude that increased synaptic vesicle glutamate uptake is brain-region specific (cerebrum) and is associated with the development or maintenance, rather than the initial cause, of seizures in the EL model of epilepsy.

Adenosine Triphosphate↗

PubMed1999

UNC-13 is required for synaptic vesicle fusion in C. elegans.

We analyzed the synaptic physiology of unc-13 mutants in the nematode C. elegans. Mutants of unc-13 had normal nervous system architecture, and the densities of synapses and postsynaptic receptors were normal at the neuromuscular junction. However, the number of synaptic vesicles at neuromuscular junctions was two- to threefold greater in unc-13 mutants than in wild-type animals. Most importantly, evoked release at both GABAergic and cholinergic synapses was almost absent in unc-13 null alleles, as determined by whole-cell, voltage-clamp techniques. Although mutant synapses had morphologically docked vesicles, these vesicles were not competent for release as assayed by spontaneous release in calcium-free solution or by the application of hyperosmotic saline. These experiments support models in which UNC-13 mediates either fusion of vesicles during exocytosis or priming of vesicles for fusion.

Alleles↗