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Membrane composition of adrenergic large and small dense cored vesicles and of synaptic vesicles: consequences for their biogenesis.

The membrane proteins of adrenergic large dense cored vesicles, in particular those of chromaffin granules, have been characterized in detail. With the exception of the nucleotide carrier all major peptides have been cloned. There has been a controversy whether these vesicles contain antigens like synaptophysin, synaptotagmin and VAMP or synaptobrevin found in high concentration in synaptic vesicles. One can now conclude that large dense core vesicles also contain these peptides although in lower concentrations. The biosynthesis of large dense core vesicles is analogous to that of other peptide secreting vesicles of the regulated pathway. One cannot yet definitely define the biosynthesis of small dense core vesicles which apparently have a very similar membrane composition to that of large dense core vesicles. They may form directly from large dense core vesicles when their membranes have been retrieved after exocytosis. These membranes may become sorted in an endosomal compartment where peptides may be deleted or added. Such an addition could be derived from synaptophysin-rich vesicles present in adrenergic axons. However small dense core vesicle peptides may also be transported axonally independent of large dense core vesicles. For proving one of these possibilities some crucial experiments have been suggested.

Animals↗

SV2C is a synaptic vesicle protein with an unusually restricted localization: anatomy of a synaptic vesicle protein family.

We describe here the identification and molecular characterization of a new brain protein that we named SV2C because it is homologous to the synaptic vesicle proteins SV2A and SV2B, and because it is also recognized by the monoclonal SV2 antibody that led to the initial discovery of SV2A and SV2B. SV2C is more closely related to SV2A (62% identity) than to SV2B (57% identity), and contains 12 transmembrane regions similar to these proteins. To characterize SV2C and compare its properties and localization with those of SV2A and SV2B, we raised an SV2C-specific antibody. Using this antibody, we show that SV2C is an N-glycosylated protein that is concentrated on small synaptic vesicles; in addition, it is found on microvesicles in adrenal chromaffin cells. We evaluated the relative localization of the three SV2 isoforms by staining rat brain sections with antibodies specific for SV2A, SV2B and SV2C. Analysis of the resulting staining patterns confirmed previous conclusions that SV2A is ubiquitously expressed in virtually all synapses. SV2B, although more restricted in distribution, was also found in a wide variety of synapses throughout the brain. In striking contrast to this general localization and to similarly wide distributions of other synaptic vesicle proteins, SV2C was observed only in few brain areas. High levels of SV2C were found primarily in phylogenetically old brain regions such as the pallidum, the substantia nigra, the midbrain, the brainstem and the olfactory bulb. SV2C was undetectable in the cerebral cortex and the hippocampus, and found at low levels in the cerebellar cortex. Our data suggest that closely related members of a synaptic vesicle protein family can either have very general (SV2A) or restricted distributions (SV2C), possibly in order to allow specialization in the regulation of the expression or of the function of these abundant synaptic vesicle proteins.

Amino Acid Sequence↗

Properties of the bovine striatal synaptic vesicles ATPase.

Synaptic vesicles prepared from bovine corpus striatum exhibit an ATPase activity that is insensitive to ouabain and specific inhibitors of mitochondrial ATPase, but that is stimulated by proton ionophores and inhibited by sulfhydryl reagents. Low concentrations of orthovanadate, DCCD and tributyl tin are also ineffective as inhibitors of the vesicle-associated activity. The properties of the synaptic vesicle enzyme suggest that this ATPase may be similar to that of clathrin-coated vesicles, and to one of the activities described in preparations of adrenal chromaffin granule membranes.

Adenosine Triphosphatases↗

Synaptic vesicle transport and synaptic membrane transporter sites in excitatory amino acid nerve terminals in Alzheimer disease.

The apparent l-[3H]glutamate uptake rate (v') was measured in synaptic vesicles isolated from cerebral cortex synaptosomes prepared from autopsied Alzheimer and non-Alzheimer dementia cases, and age-matched controls. The initial synaptosome preparations exhibited similar densities of d-[3H]aspartate membrane binding sites (BMAX values) in the three groups. In control brain the temporal cortex d-[3H]aspartate BMAX was 132% of that in motor cortex, parallel with the l-[3H]glutamate v' values (temporal=139% of motor; NS). Unlike d-[3H]aspartate BMAX values, l-[3H]glutamate v' values were markedly and selectively lower in Alzheimer brain preparations than in controls, particularly in temporal cortex. The difference could not be attributed to differential effects of autopsy interval or age at death. Non-Alzheimer dementia cases resembled controls. The selective loss of vesicular glutamate transport is consistent with a dysfunction in the recycling of transmitter glutamate.

Aged↗

Adenosine triphosphate. A constituent of cholinergic synaptic vesicles.

1. Synaptic vesicles separated by density-gradient centrifugation from extracts of the cholinergic nerve terminals of the electric organ of Torpedo marmorata were found to contain appreciable amounts of ATP as well as acetylcholine. 2. Vesicular ATP was stable in the presence of concentrations of apyrase and myokinase that rapidly destroyed equivalent amounts of endogenous or added free ATP; pre-treatment of cytoplasmic extracts of electric tissue with these enzymes destroyed endogenous free ATP, but did not affect the vesicular ATP. 3. When [U-(14)C]ATP was added to electric tissue at the time of comminution and extraction of the vesicles, all the radioactivity was associated with soluble components in the subsequent fractionation: none was associated with vesicles or membrane fragments; thus it is unlikely that vesicular ATP can be accounted for by the sequestration of endogenous free ATP within any vesicles formed during comminution and extraction of the tissue. 4. When synaptic vesicles were passed through iso-osmotic columns of Bio-Gel A-5m, which separates vesicles from soluble proteins and small molecules, all the recovered ATP and acetylcholine passed through together in the void volume. 5. Regression analysis showed that vesicular ATP content was highly correlated with vesicular acetylcholine content in different experiments, the molar ratio acetylcholine/ATP being 5.32+/-(s.e.m.) 0.45 (21 expts.) for the peak density-gradient fraction. The ratio varied, however, somewhat across the density-gradient peak suggesting some degree of chemical heterogeneity in the vesicle population.

Acetylcholine↗

[Molecular mechanisms underlying the synaptic vesicle cycle].

Synaptic vesicle cycle in nerve endings is a processes of multiple steps including docking, exocytosis, endocytosis and vesicle recruitment. It is mediated by protein-protein cascade reactions of vesicles, axoplasm and presynaptic membrane. The molecular model for the key steps of synaptic vesicle cycle is presented and can be used as the basis for furthering our knowledge about the activities of central nervous system.

Animals↗

Topological organization of proteins in an intracellular secretory organelle: the synaptic vesicle.

Intact synaptic vesicles prepared from the electric organ of the marine elasmobranch Narcine brasiliensis have eight major polypeptides demonstrable on sodium dodecyl sulfate gels. Six of these copurify with the synaptic vesicles during isolation of vesicles by chromatography on CPG-3000 and, by this criterion, are specific to vesicles. The other two are either shared by many membrane or are contaminants. One of these proteins comigrates with actin. Three different approaches were used to determine which proteins were exposed on the external, cytoplasmic surface of the vesicle and which were internal. The first was susceptibility to the proteases trypsin, Streptomyces griseus protease, and Pronase; the second was labeling by the membrane-impermeable reagent diazotized [125I]iodosulfanilic acid; and the third was iodination catalyzed by lactoperoxidase. In general, the three approaches give the same result: six of the eight proteins are on the external, cytoplasmic surface and two are accessible only after the vesicles are lysed by freezing and thawing or by detergents. Five of the vesicle-specific proteins are external and one is internal. The actin-like protein is internal. Proteins involved in the interaction of vesicles with the presynaptic membrane during exocytosis might be expected to be vesicle specific and external.

Acetylcholine↗

Calmodulin stimulation of 45Ca2+ transport and protein phosphorylation in cholinergic synaptic vesicles.

Cholinergic synaptic vesicles isolated from the electric organ of Torpedo californica exhibit ATP-dependent uptake of 45Ca2+ that is stimulated by exogenous calmodulin. ATP-independent uptake also occurs, but it is only weakly stimulated by calmodulin. Saturating calmodulin decreased the Michaelis constant for ATP-dependent 45Ca2+ uptake from 52 +/- 0.4 to 12 +/- 0.2 microM and increased the maximal velocity from 3.4 +/- 0.3 to 5.2 +/- 0.5 nmol/mg of protein per min. The dose-response curve for calmodulin-dependent stimulation showed a maximal increase of 3.5-fold in the uptake rate; 0.2 microM calmodulin gave half-maximal stimulation. The activity of the vesicle-associated ATPase was unaffected. Incubation of vesicles with [gamma-32P]ATP and Ca2+ resulted in phosphorylation of four polypeptides of molecular weights about 64,000, 58,000, 54,000, and 41,000 when calmodulin was added. Vesicles that were previously phosphorylated and purified exhibited 2-fold enhanced ATP-independent uptake of 45Ca2+. Cyclic AMP could not substitute for calmodulin. The calcium transport system of the cholinergic synaptic vesicle is regulated by a calcicalmodulin-dependent protein kinase that is vesicle-associated.

Animals↗

Post-transcriptional regulation of synaptic vesicle protein expression and the developmental control of synaptic vesicle formation.

The regulated expression of synaptic vesicle (SV) proteins during development and the assembly of these proteins into functional SVs are critical aspects of nervous system maturation. We have examined the expression patterns of four SV proteins in embryonic hippocampal neurons developing in culture and have found that increases in the levels of these proteins result primarily from post-transcriptional regulation. Synaptotagmin I, vamp 2, and synapsin I proteins are synthesized at nearly constant rates as the neurons develop. However, these proteins are relatively unstable at early times in culture and undergo a progressive increase in half-life with time, possibly as a result of an increase in the efficiency with which they are incorporated into SVs. In contrast, synaptophysin is synthesized at a very low rate at early times in culture, and its rate of synthesis increases dramatically with time. The increase in synaptophysin synthesis is not simply the result of an increase in mRNA level, but is largely attributable to an increase in the rate of translational initiation. Despite the nearly constant rates of synthesis of synaptotagmin I, vamp 2, and synapsin I, we show that the number of SVs in these developing neurons increases, and that SV proteins are more efficiently targeted to SVs at later times in culture. Our results suggest that SV production during development is not limited by the rates of transcription of genes encoding the component proteins, thus allowing control of this process by cytoplasmic mechanisms, without signaling to the nucleus.

Animals↗

Comparison of nerve terminal events in vivo effecting retrograde transport of vesicles containing neurotrophins or synaptic vesicle components.

Although vesicular retrograde transport of neurotrophins in vivo is well established, relatively little is known about the mechanisms that underlie vesicle endocytosis and formation before transport. We demonstrate that in vivo not all retrograde transport vesicles are alike, nor are they all formed using identical mechanisms. As characterized by density, there are at least two populations of vesicles present in the synaptic terminal that are retrogradely transported along the axon: those containing neurotrophins (NTs) and those resulting from synaptic vesicle recycling. Vesicles containing nerve growth factor (NGF), NT-3, or NT-4 had similar densities with peak values at about 1.05 g/ml. Synaptic-derived vesicles, labeled with anti-dopamine beta-hydroxylase (DBH), had densities with peak values at about 1.16 g/ml. We assayed the effects of pharmacologic agents in vivo on retrograde transport from the anterior eye chamber to the superior cervical ganglion. Inhibitors of phosphatidylinositol-3-OH (PI-3) kinase and actin function blocked transport of both anti-DBH and NGF, demonstrating an essential role for these molecules in retrograde transport of both vesicle types. Dynamin, a key element in synaptic vesicle recycling, was axonally transported in retrograde and anterograde directions, and compounds able to interfere with dynamin function had a differential effect on retrograde transport of NTs and anti-DBH. Okadaic acid significantly decreased retrograde axonal transport of anti-DBH and increased NGF retrograde transport. We conclude that there are both different and common proteins involved in endocytosis and targeting of retrograde transport of these two populations of vesicles.

Animals↗

Biogenesis of synaptic vesicles in vitro.

Synaptic vesicles are synthesized at a rapid rate in nerve terminals to compensate for their rapid loss during neurotransmitter release. Their biogenesis involves endocytosis of synaptic vesicle membrane proteins from the plasma membrane and requires two steps, the segregation of synaptic vesicle membrane proteins from other cellular proteins, and the packaging of those unique proteins into vesicles of the correct size. By labeling an epitope-tagged variant of a synaptic vesicle protein, VAMP (synaptobrevin), at the cell surface of the neuroendocrine cell line PC12, synaptic vesicle biogenesis could be followed with considerable precision, quantitatively and kinetically. Epitope-tagged VAMP was recovered in synaptic vesicles within a few minutes of leaving the cell surface. More efficient targeting was obtained by using the VAMP mutant, del 61-70. Synaptic vesicles did not form at 15 degrees C although endocytosis still occurred. Synaptic vesicles could be generated in vitro from a homogenate of cells labeled at 15 degrees C. The newly formed vesicles are identical to those formed in vivo in their sedimentation characteristics, the presence of the synaptic vesicle protein synaptophysin, and the absence of detectable transferrin receptor. Brain, but not fibroblast cytosol, allows vesicles of the correct size to form. Vesicle formation is time and temperature-dependent, requires ATP, is calcium independent, and is inhibited by GTP-gamma S. Thus, two key steps in synaptic vesicle biogenesis have been reconstituted in vitro, allowing direct analysis of the proteins involved.

Animals↗

Biogenesis of synaptic vesicles.

The basic endosomal recycling pathway can be modified to generate transcytotic vesicles, storage vesicles and synaptic vesicles. Sorting into synaptic vesicles requires specialized sorting information not present in the transcytotic and storage vesicle proteins. Using mutagenesis we have distinguished the signals for rapid endocytosis and SV targeting in synaptobrevin. Finally, we have evidence that synaptic vesicles can be generated from an endosomal compartment in vitro.

Animals↗

Biogenesis of constitutive secretory vesicles, secretory granules and synaptic vesicles.

The formation of constitutive secretory vesicles and secretory granules from the trans-Golgi network is inhibited by brefeldin A, suggesting a role for ADP ribosylation factor and other coat proteins in the budding process, and is regulated by multiple heterotrimeric G proteins. Membrane proteins are sorted to secretory granules through signals in their cytoplasmic domains and, like secretory proteins, by aggregation of their lumenal domains. Synaptic vesicle membrane proteins undergo exocytotic-endocytotic recycling before synapse formation, consistent with the proposed biogenesis of synaptic vesicles from early endosomes.

Biopolymers↗

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.

Animals↗

A morphometric analysis of synaptic vesicle distributions.

Synaptic vesicle populations have been morphometrically analyzed for size and density. Populations composed of a single size class of vesicles are represented by normal (Gaussian) or positive (log-normal) skew histograms. Populations with multiple size classes generate negative (left) skew distributions. Fixatives containing aldehydes differentially affect these distribution patterns but vesicles are able to withstand tonic effects over a wide range. Reader bias' contribute the most error in the data-collecting process. But despite this, the sizing of vesicle populations can be accomplished with great accuracy. Vesicle density computations, on the other hand, vary over a wide range and are of less value for comparative purposes.

Animals↗

Isolated p65 protein reproduces membrane binding activity of synaptic vesicles.

Purified synaptic vesicles are highly enriched with a protein which binds cell plasma membranes. The binding is selective for acidic phospholipids and sialoglycosphingolipids. In partition chromatography of vesicle proteins, the binding activity was co-eluted with a limited set of proteins. Among them the most abundant species were two vesicle-specific proteins: p65 and synaptophysin. In affinity chromatography of vesicle proteins, only p65 bound to a column of immobilized lysoganglioside. The same protein, purified by preparative electrophoresis, retained the binding activity and fully reproduced the hemagglutinating property and the selectivity for acidic lipids of whole vesicles. The results suggest that the (hemagglutinating) lipid binding properties of the vesicles are mainly if not exclusively due to p65.

Calcium-Binding Proteins↗

Sequential steps in clathrin-mediated synaptic vesicle endocytosis.

Synaptic vesicles are recycled with remarkable speed and precision in nerve terminals. A major recycling pathway involves clathrin-mediated endocytosis at endocytic zones located around sites of release. Different 'accessory' proteins linked to this pathway have been shown to alter the shape and composition of lipid membranes, to modify membrane-coat protein interactions, and to influence actin polymerization. These include the GTPase dynamin, the lysophosphatidic acid acyl transferase endophilin, and the phosphoinositide phosphatase synaptojanin. Protein perturbation studies in living nerve terminals are now beginning to link the actions of these proteins with morphologically defined steps of endocytosis.

Animals↗

Multiple GTP-binding proteins from cholinergic synaptic vesicles.

Cholinergic synaptic vesicles purified from the electric organ of the marine ray, Discopyge ommata, contain 2 different size classes of GTP-binding proteins: one or more with an apparent molecular weight (MW) between 37 and 41 kDa, and 3 major and at least 2 minor proteins with MW between 20 and 29 kDa. These GTP-binding proteins were detectable using the alpha 32P-GTP overlay technique and covalent modification with bacterial toxins. The higher MW GTP-binding proteins are ADP-ribosylated by pertussis toxin and 2 of the lower MW GTP-binding proteins are sensitive to botulinum toxin.

Adenosine Diphosphate Ribose↗