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PubMed1998

A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells.

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

Amino Acid Sequence↗
PubMed1996

Differential distribution of syntaxin isoforms 1A and 1B in the rat central nervous system.

Syntaxin 1 binds to several proteins of the synaptic terminal and is a central component in the pathway of protein-protein interactions that underlies docking and fusion of synaptic vesicles. Molecular studies revealed the occurrence of two isoforms, syntaxin 1A and syntaxin 1B, which coexpress in neural tissues. However, they display differential expression patterns in endocrine cell types. We generated isoform-specific antibodies that were used in Western blotting and immunocytochemical studies. First, we confirmed the sole presence of syntaxin 1A in endocrine pituitary cells. Second, we found distinctive immunolabelling patterns of each isoform in the rat olfactory system, hippocampus, striatum, thalamus and spinal cord. In addition, the principal white matter commissures displayed distinct immunoreactivity for each isoform. This report shows, for the first time, major differences between the distributions of syntaxin 1A and syntaxin 1B isoforms in the rat central nervous system.

Animals↗
PubMed2005

Transcriptome analysis of frontal cortex in alcohol-preferring and nonpreferring rats.

Although it is widely accepted that alcohol abuse and alcoholism have a significant genetic component of risk, the identities of the genes themselves remain obscure. To illuminate such potential genetic contributions, DNA macroarrays were used to probe for differences in normative cortical gene expression between rat strains genetically selected for alcohol self-administration preference, AA (Alko, alcohol) and P (Indiana, preferring), or avoidance, ANA (Alko, nonalcohol) and NP (Indiana, nonpreferring). Among 1,176 genes studied, six demonstrated confirmable, differential expression following comparison of ethanol-naive AA and ANA rats. Specifically, the mRNA level for metabotropic glutamate receptor 3 (mGluR3) was down-regulated in the AA vs. ANA lines. In contrast, calcium channel subunit alpha2delta1 (cacna2d1), vesicle-associated membrane protein 2 (VAMP2), syntaxin 1 (both syntaxin 1a and 1b; STX1a and STX1b), and syntaxin binding protein (MUNC-18) mRNAs were found to be increased in frontal cortex following comparison of AA with ANA animals. Bioinformatic analysis of these molecular targets showed that mGluR3 and cacna2d1 fall within chromosomal locations reported to be alcohol-related by the Collaborative Study on the Genetics of Alcoholism (COGA) as well as quantitative trait loci (QTL) studies. To determine further whether these differences were strain specific, the above-mentioned genes were compared in ethanol-preferring (P) and -nonpreferring (NP) selected lines. VAMP2 was the only gene that displayed statistically different mRNA levels in a comparison of P and NP rats. In conclusion, the altered cortical gene expression illuminated here would have the effect of altering neurotransmitter release in AA rats (compared with ANA rats). Such alterations, however, might not be a universal characteristic of all animal models of alcohol abuse and will also require further investigation in post-mortem human samples.

Alcohol Drinking↗
PubMed1997

Regulated secretion in platelets: identification of elements of the platelet exocytosis machinery.

To further characterize the molecular mechanisms of platelet function, we have sought to identify some of the proteins that mediate the secretory events of the platelet release reaction. We report that platelets contain the general elements of the membrane transport apparatus: N-ethylmaleimide sensitive fusion protein (NSF), p115/transcytosis-associated protein (p115/TAP), and the soluble NSF attachment proteins (alpha- and, gamma-SNAP). The cDNAs encoding two of these proteins, alpha- and gamma-SNAP, have been cloned from a human platelet-derived cDNA library. Platelet membrane extracts possess SNAP receptor (SNARE) activity, suggesting that the class of proteins (SNAREs) proposed to provide the specificity for vesicle docking and membrane fusion are present in platelets. To identify these proteins, we have used specific antibodies against known SNAREs to probe platelet extracts. Syntaxin 2 and 4 can be readily detected in platelet membrane preparations and are shown to participate in 20 S complex formation. Syntaxin 1, 3, and 5 could not be detected. Other known SNARE and SNARE-associated proteins such as vesicle-associated membrane protein (VAMP)/synaptobrevin 2, SNAP-25, synaptophysin, or synaptotagmin I could not be immunochemically detected in platelet membrane preparations. The presence of both the general transport proteins (NSF and SNAPs) and specific transport proteins (syntaxin 2 and 4) indicates that platelet exocytosis uses a molecular mechanism similar to other secretory cells such as neurons. However, the subcellular concentrations of these proteins suggest that, unlike neuronal secretion, granule-to plasma membrane docking may be the limiting step in platelet exocytosis.

Amino Acid Sequence↗
PubMed1995

Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.

There is strong evidence to indicate that proteins of the synaptobrevin family play a key role in exocytosis. Synaptobrevin 1 and 2 are expressed at high concentration in brain where they are localized on synaptic vesicles. Cellubrevin, a very similar protein, has a widespread tissue distribution and in fibroblasts is localized on endosome-derived, transferin receptor-positive vesicles. Since brain cellubrevin is not detectable in synaptic vesicles, we investigated whether cellubrevin and the synaptobrevins are differentially targeted when co-expressed in the same cell. We report that in the nervous system cellubrevin is expressed at significant levels only by glia and vascular cells. However, cellubrevin is coexpressed with the two synaptobrevins in PC12 cells, a neuroendocrine cell line which contains synaptic vesicle-like microvesicles. In PC12 cells, cellubrevin has a distribution very similar to that of synaptobrevin 1 and 2. The three proteins are targeted to neurites which exclude the transferrin receptor and are enriched in synaptic-like microvesicles and dense-core granules. They are recovered in the synaptic-like microvesicle peak of glycerol velocity gradients, have a similar distribution in isopycnic fractionation and are coprecipitated by anti-synaptobrevin 2 immunobeads. Finally, cellubrevin, like the synaptobrevins, interact with the neuronal t-SNAREs syntaxin 1 and SNAP-25. These results suggest that cellubrevin and the synaptobrevins have similar function and do not play a specialized role in constitutive and regulated exocytosis, respectively.

Amino Acid Sequence↗
PubMed2005

Antidepressants differentially affect expression of complexin I and II RNA in rat hippocampus.

Disturbance of synaptic transmission is currently viewed as an important pathophysiological mechanism and therapeutic target of mood disorders. Amongst other lines of evidence this theory is based on human post-mortem investigations showing differential expression of complexins. In order to discriminate between molecular correlates of the disease itself and effects of psychotropic drugs given to patients, we performed an animal trial using subchronic antidepressant treatment. Cohorts of adult male Sprague-Dawley rats were treated over a period of 14 days with intraperitoneal injections of either saline (0.9%, n=8), desipramine (15 mg/kg, n=7), fluoxetine (10 mg/kg, n=8), or tranylcypromine (10 mg/kg, n=5). Brain slices were used for in situ hybridizations with 35S labelled RNA probes of the genes complexin I, complexin II and syntaxin 1 A, the SNARE complex protein interacting with the complexins, and assessed semi-quantitatively for region-specific expression levels. Expression of complexin I was induced only in habenular nuclei after treatment with fluoxetine. In contrast, complexin II was significantly induced by desipramine and tranylcypromine, but not fluoxetine, in several brain regions. All treatment groups, but most significantly fluoxetine-treated animals, showed higher expression levels of syntaxin 1A. Antidepressants differentially affect expression levels of complexin I and more prominently complexin II and syntaxin 1A. The induction of complexin II and syntaxin 1A might strengthen the synaptic transmission at axo-dendritic or axo-axonal synapses. Previous post-mortem findings reporting on downregulation of complexins cannot be explained as mere effects of psychotropic drug treatment.

Adaptor Proteins, Vesicular Transport↗
PubMed2006

Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature.

Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.

Animals↗
PubMed1998

Interaction of cysteine string proteins with the alpha1A subunit of the P/Q-type calcium channel.

Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.

Animals↗
PubMed2005

SNARE complexes prepare for membrane fusion.

According to a popular theory, the three presynaptic SNARE proteins (syntaxin 1, synaptobrevin 2 and SNAP-25) drive neuroexocytosis by forming a complex that forces vesicle and plasma membranes together. However, individual reactions in this process have been difficult to resolve. Now, two groups have succeeded in studying a very early and a very late step in the fusion process.

Animals↗
PubMed2004

Comparative analysis of tandem C2 domains from the mammalian synaptotagmin family.

Intracellular membrane traffic is governed by a conserved set of proteins, including Syts (synaptotagmins). The mammalian Syt family includes 15 isoforms. Syts are membrane proteins that possess tandem C2 domains (C2AB) implicated in calcium-dependent phospholipid binding. We performed a pair-wise amino acid sequence comparison, together with functional studies of rat Syt C2ABs, to examine common and divergent properties within the mammalian family. Sequence analysis indicates three different C2AB classes, the members of which share a high degree of sequence similarity. All the other C2ABs are highly divergent in sequence. Nearly half of the Syt family does not exhibit calcium/phospholipid binding in comparison to Syt I, the major brain isoform. Syts do, however, possess a more conserved function, namely calcium-independent binding to target SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) heterodimers. All tested isoforms, except Syt XII and Syt XIII, bound the target SNARE heterodimer comprising syntaxin 1 and SNAP-25 (25 kDa synaptosome-associated protein). Our present study suggests that many Syt isoforms can function in membrane trafficking to interact with the target SNARE heterodimer on the pathway to calcium-triggered membrane fusion.

Amino Acid Sequence↗
PubMed2000

SNAP-25 regulation during adrenal gland development: comparison with differentiation markers and other SNAREs.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is one of a limited number of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) that play a major role in membrane docking of synaptic vesicles and secretory granules during regulated exocytosis. We have previously shown that SNAP-25 levels differ between noradrenergic and adrenergic chromaffin cell populations of the adult adrenal gland. We examine SNAP-25 expression by immunofluoresence in cells of the sympathoadrenal lineage in the rat during late embryonic and postnatal development. In parallel, tyrosine hydroxylase was used to identify sympathoadrenal cells, phenylethanolamine N-methyltransferase to distinguish adrenergic from noradrenergic chromaffin cells, and chromogranin A to define the presence of secretory granules. In addition, SNAP-25 protein and mRNA levels were followed in adrenal gland extracts by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Protein levels were compared with those of other molecules also implicated in organelle trafficking, including syntaxin 1 and vesicle-associated membrane protein (VAMP-2) and the nonneuronal analogues SNAP-23 and cellubrevin. This study provides evidence that SNAP-25 is expressed early during development in sympathoadrenal neurons and migrating cells. It is detected in intra-adrenal chromoblasts as soon as they enter the adrenal primordium. Its differential expression between catecholamine chromaffin cell phenotypes is already evident from the 17th embryonic day, future noradrenergic cells appearing to express higher levels than adrenergic cells. The granule maturation marker chromogranin A is expressed in chromaffin cells later than SNAP-25. Both SNAP-25 protein and mRNA increased rapidly in the adrenal gland in the perinatal period to peak during the first postnatal week, after which levels dropped dramatically to adult values. In contrast, levels of both syntaxin and SNAP-23 appeared to remain fairly constant throughout adrenal gland development. VAMP-2 expression increased gradually around birth to reach maximal levels during the first two postnatal weeks, and then decreased slightly. Cellubrevin levels also appeared to increase gradually until adult values were attained by the end of the second postnatal week. The threefold increase of SNAP-25 mRNA shortly after birth compared to the low adult levels suggests that during this period SNAP-25 is implicated in additional functions than regulated secretion, possibly associated with cellular growth or maturation.

Adrenal Glands↗
PubMed2001

Calcium-dependent translocation of synaptotagmin to the plasma membrane in the dendrites of developing neurones.

In neurones, the morphological complexity of the dendritic tree requires regulated growth and the appropriate targeting of membrane components. Accurate delivery of specific supplies depends on the translocation and fusion of transport vesicles. Vesicle SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and target membrane SNAREs play a central role in the correct execution of fusion events, and mediate interactions with molecules that endow the system with appropriate regulation. Synaptotagmins, a family of Ca(2+)-sensor proteins that includes neurone-specific members involved in regulating neurotransmitter exocytosis, are among the molecules that can tune the fusion mechanism. Using immunocytochemistry, confocal and electron microscopy, the localisation of synaptotagmin I in the dendrites of cultured rat hypothalamic neurones was demonstrated. Synaptotagmin labelling is concentrated at dendritic branch points, and in microprocesses. Following depolarisation, the N-terminal domain of synaptotagmin was detected at the extracellular surface of the dendritic plasma membrane. The insertion of synaptotagmin in the plasma membrane was elicited by L-type Ca(2+) channel activation and by mobilisation of the internal ryanodine-sensitive Ca(2+)stores. Furthermore, the localisation of L-type Ca(2+) channels and of ryanodine receptors, relative to the localisation of synaptotagmin in dendrites, suggests that both Ca(2+) entry and intracellular Ca(2+) stores may contribute to the fusion of dendritic transport vesicles with the membrane. Fusion is likely to involve SNAP-25 and syntaxin 1 as both proteins were also identified in dendrites. Taken together these results suggest a putative regulatory role of synaptotagmins in the membrane fusion events that contribute to shaping the dendritic tree during development.

Animals↗
PubMed2001

Localization of cellubrevin-related peptide, endobrevin, in the early endosome in pancreatic beta cells and its physiological function in exo-endocytosis of secretory granules.

Cellubrevins are integral membrane proteins expressed in a wide variety of tissues and usually localized in recycling vesicles. Here, we investigated the cellular localization of a cellubrevin-related peptide, endobrevin, in pancreatic (beta) cells and its implication in the exo-endocytosis of insulin and (gamma)-amino butyric acid (GABA). Immunocytochemistry showed that endobrevin is associated with tubulo-vesicular structures, which are colocalized with early endosomes labeled by early endosome antigen (EEA)-1 in insulinoma MIN6 cells. To determine the cellular localization of endobrevin, we appended the green fluorescent protein (GFP) to endobrevin and the fusion protein was introduced into MIN6 cells. The subcellular localization of GFP-endobrevin was visualized by confocal laser microscopy. Colocalization study based on the expressed GFP-endobrevin and endocytosed Texas-Red(Tx-R) labeled transferrin receptor and immunocytochemistry with anti-EEA1 antibody revealed that endobrevin was preferentially localized in the early endosome. Then, we examined the functional role of endobrevin in the exocytosis of insulin and GABA from pancreatic (beta) cells. Endobrevin overexpression increased the amount of GABA released from MIN6 cells; in contrast, it decreased the glucose-stimulated insulin release from rat islets, MIN6 and INS1-D cells to approximately 50% of the control levels. Both in vitro and in vivo binding studies showed that endobrevin binds to syntaxin 1. Finally, using the fluorescent probe FM4-64, it was revealed that endobrevin overexpression accelerates vesicle recycling. We conclude that (1) endobrevin is localized in the early endosome in pancreatic (beta) cells and (2) endobrevin plays a physiological role in the exo-endocytosis of insulin and GABA from pancreatic (beta) cells, probably via an interaction between endocytic vesicles and the endosome.

Animals↗
PubMed2006

Tomosyn is expressed in beta-cells and negatively regulates insulin exocytosis.

Tomosyn, a syntaxin-binding protein, is capable of dissociating mammalian homolog of the Caenorhabditis elegans unc-18 gene from syntaxin and is involved in the regulation of exocytosis. We have investigated the expression, cellular localization, and functional role of tomosyn in pancreatic beta-cells. Western blotting revealed a 130-kDa protein corresponding to tomosyn in insulin-secreting beta-cell lines. RT-PCR amplification showed that b-, m-, and s-tomosyn isoform mRNAs are expressed in beta-cell lines and rat pancreatic islets. Immunohistochemistry revealed punctate tomosyn immunoreactivity in the cytoplasm of insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing islet cells. Syntaxin 1 coimmunoprecipitated with tomosyn in extracts of insulin-secreting cells. Overexpression of m-tomosyn in mouse beta-cells significantly decreased exocytosis, whereas inhibition of tomosyn expression by small interfering RNA increased exocytosis. Hence, in the pancreatic beta-cell, tomosyn negatively regulates insulin exocytosis.

Animals↗
PubMed2002

SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs.

During neuronal exocytosis, the vesicle-bound soluble NSF attachment protein (SNAP) receptor (SNARE) synaptobrevin 2 forms complexes with the plasma membrane-bound SNAREs syntaxin 1A and SNAP25 to initiate the fusion reaction. However, it is not known whether in the native membrane SNAREs are constitutively active or whether they are unable to enter SNARE complexes unless activated before membrane fusion. Here we used binding of labeled recombinant SNAREs to inside-out carrier supported plasma membrane sheets of PC12 cells to probe for the activity of endogenous SNAREs. Binding was specific, saturable, and depended on the presence of membrane-resident SNARE partners. Our data show that virtually all of the endogenous syntaxin 1 and SNAP-25 are highly reactive and readily form SNARE complexes with exogenously added SNAREs. Furthermore, complexes between endogenous SNAREs were not detectable when the membranes are freshly prepared, but they slowly form upon prolonged incubation in vitro. We conclude that the activity of membrane-resident SNAREs is not downregulated by control proteins but is constitutively active even if not engaged in fusion events.

Animals↗
PubMed2002

Structural basis for the Golgi membrane recruitment of Sly1p by Sed5p.

Cytosolic Sec1/munc18-like proteins (SM proteins) are recruited to membrane fusion sites by interaction with syntaxin-type SNARE proteins, constituting indispensable positive regulators of intracellular membrane fusion. Here we present the crystal structure of the yeast SM protein Sly1p in complex with a short N-terminal peptide derived from the Golgi-resident syntaxin Sed5p. Sly1p folds, similarly to neuronal Sec1, into a three-domain arch-shaped assembly, and Sed5p interacts in a helical conformation predominantly with domain I of Sly1p on the opposite site of the nSec1/syntaxin-1-binding site. Sequence conservation of the major interactions suggests that homologues of Sly1p as well as the paralogous Vps45p group bind their respective syntaxins in the same way. Furthermore, we present indirect evidence that nSec1 might be able to contact syntaxin 1 in a similar fashion. The observed Sly1p-Sed5p interaction mode therefore indicates how SM proteins can stay associated with the assembling fusion machinery in order to participate in late fusion steps.

Amino Acid Sequence↗
PubMed2000

The SNARE Vti1a-beta is localized to small synaptic vesicles and participates in a novel SNARE complex.

Specific soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins are required for different membrane transport steps. The SNARE Vti1a has been colocalized with Golgi markers and Vti1b with Golgi and the trans-Golgi network or endosomal markers in fibroblast cell lines. Here we study the distribution of Vti1a and Vti1b in brain. Vti1b was found in synaptic vesicles but was not enriched in this organelle. A brain-specific splice variant of Vti1a was identified that had an insertion of seven amino acid residues next to the putative SNARE-interacting helix. This Vti1a-beta was enriched in small synaptic vesicles and clathrin-coated vesicles isolated from nerve terminals. Vti1a-beta also copurified with the synaptic vesicle R-SNARE synaptobrevin during immunoisolation of synaptic vesicles and endosomes. Therefore, both synaptobrevin and Vti1a-beta are integral parts of synaptic vesicles throughout their life cycle. Vti1a-beta was part of a SNARE complex in nerve terminals, which bound N-ethylmaleimide-sensitive factor and alpha-SNAP. This SNARE complex was different from the exocytic SNARE complex because Vti1a-beta was not coimmunoprecipitated with syntaxin 1 or SNAP-25. These data suggest that Vti1a-beta does not function in exocytosis but in a separate SNARE complex in a membrane fusion step during recycling or biogenesis of synaptic vesicles.

Alternative Splicing↗
PubMed2001

Amyloid beta-peptide inhibits neuronal glucose uptake by preventing exocytosis.

Amyloid beta peptide (Abeta) is suspected as a contributing factor for decreased glucose utilization in the brain of Alzheimer's patients; however, little is known about the regulatory mechanism of neuronal glucose uptake and how Abeta affects such a mechanism. We report that membrane depolarization by 40 mM KCl increases both neuronal glucose uptake and immunolabeling of the exofacial epitope of glucose transporter isoform GLUT3, suggesting that fusion of GLUT3 vesicles with the plasma membrane increases glucose uptake. Abeta25-35 decreased neuronal glucose uptake and this decrease was prevented by exocytosis-enhancing compounds (40 mM KCl, 50 microM ruthenium red). Abeta25-35 also inhibited exocytosis of the fluorescent membrane dye FM1-43 at neuronal cell bodies; however, 40 mM KCl was effective in overcoming this Abeta inhibition. Furthermore, GLUT3 colocalized with SNARE (N-ethylmaleimide-sensitive factor attached protein receptor) complex proteins (SNAP-25 and Syntaxin 1), and cleavage of the v-SNARE, VAMP, reduced glucose uptake. Our findings suggest that neuronal glucose uptake is regulated by SNARE complex-dependent docking and fusion of GLUT3 vesicles with the plasma membrane and that Abeta decreases glucose uptake by inhibiting fusion of these vesicles.

Amyloid beta-Peptides↗