MAB333 Sigma-AldrichAnti-Synaptobrevin Antibody, clone SP10

Given the ability of mood stabilizers and antipsychotics to promote cell proliferation, we wanted to determine the effects of these drugs on neuronal markers previously reported to be altered in subjects with psychiatric disorders.Male Sprauge-Dawley rats were treated with vehicle (ethanol), lithium (25.5 mg per day), haloperidol (0.1 mg/kg), olanzapine (1.0 mg/kg) or a combination of lithium and either of the antipsychotic drugs for 28 days. Levels of cortical synaptic (synaptosomal associated protein-25, synaptophysin, vesicle associated protein and syntaxin) and structural (neural cell adhesion molecule and alpha-synuclein) proteins were determined in each treatment group using Western blots.Compared to the vehicle treated group; animals treated with haloperidol had greater levels of synaptosomal associated protein-25 (pless than 0.01) and neural cell adhesion molecule (pless than 0.05), those treated with olanzapine had greater levels of synaptophysin (pless than 0.01) and syntaxin (pless than 0.01). Treatment with lithium alone did not affect the levels of any of the proteins. Combining lithium and haloperidol resulted in greater levels of synaptophysin (pless than 0.01), synaptosomal associated protein-25 (pless than 0.01) and neural cell adhesion molecule (pless than 0.01). The combination of lithium and olanzapine produced greater levels of synaptophysin (pless than 0.01) and alpha-synuclein (pless than 0.05).Lithium alone had no effect on the neuronal markers. However, haloperidol and olanzapine affected different presynaptic markers. Combining lithium with olanzapine additionally increased alpha-synuclein. These drug effects need to be taken into account by future studies examining presynaptic and neuronal markers in tissue from subjects with psychiatric disorders.

Upon wounding, syndecan-4 detects the appearance of fibronectin in the wound bed and mediates regulation of the small GTPases, Rac1, RhoA and RhoG. Cohesive regulation of these molecules results in cycles of membrane protrusion and cytoskeletal contraction, and triggers the endocytosis of α 5β 1-integrin, which collectively lead to immigration of fibroblasts into the wound bed. In this manuscript we identify the regulation of a fourth GTPase, Arf6 that is responsible for α 5β 1-integrin recycling and thereby completes the cycle of syndecan-4-regulated integrin trafficking. We demonstrate that each of the GTPase signals can be regulated by syndecan-4, but that they are independent of one another. By doing so we identify syndecan-4 as the coordinating center of pro-migratory signals.

BACKGROUND: Volatile general anesthetics (VAs) have a number of synaptic actions, one of which is to inhibit excitatory neurotransmitter release; however, no presynaptic VA binding proteins have been identified. Genetic data in Caenorhabditis elegans have led to the hypothesis that a protein that interacts with the presynaptic protein syntaxin 1A is a VA target. Motivated by this hypothesis, the authors measured the ability of syntaxin 1A and proteins that interact with syntaxin to bind to halothane and isoflurane. METHODS: Recombinant rat syntaxin 1A, SNAP-25B, VAMP2, and the ternary SNARE complex that they form were tested. Binding of VAs to these proteins was detected by F-nuclear magnetic resonance relaxation measurements. Structural alterations in the proteins were examined by circular dichroism and ability to form complexes. RESULTS: Volatile anesthetics did not bind to VAMP2. At concentrations in the clinical range, VAs did bind to SNAP-25B; however, binding was detected only in preparations containing SNAP-25B homomultimers. VAs also bound at clinical concentrations to both syntaxin and the SNARE complex. Addition of an N-terminal His6 tag to syntaxin abolished its ability to bind VAs despite normal secondary structure and ability to form SNARE complexes; thrombin cleavage of the tag restored VA binding. Thus, the VA binding site(s) has structural requirements and is not simply any alpha-helical bundle. VAs at supraclinical concentrations produced an increase in helicity of the SNARE complex; otherwise, VA binding produced no gross alteration in the stability or secondary structure of the SNARE complex. CONCLUSION: SNARE proteins are potential synaptic targets of volatile anesthetics.

The core complex, formed by the SNARE proteins synaptobrevin 2, syntaxin 1 and SNAP-25, is an important component of the synaptic fusion machinery and shows remarkable in vitro stability, as exemplified by its SDS-resistance. In western blots, antibodies against one of these SNARE proteins reveal the existence of not only an SDS-resistant ternary complex but also as many as five bands between 60 and >200 kDa. Structural conformation as well as possible functions of these various complexes remained elusive. In western blots of protein extracts from PC12 cell membranes, an antibody against SNAP-25 detected two heat-sensitive SDS-resistant bands with apparent molecular weights of 100 and 230 kDa. A syntaxin antibody recognized only the 230 kDa band and required heat-treatment of the blotting membrane to detect the 100 kDa band. Various antibodies against synaptobrevin failed to detect SNARE complexes in conventional western blots and detected either the 100 kDa band or the 230 kDa band on heat-treated blotting membranes. When PC12 cells were exposed to various extracellular K(+)-concentrations (to evoke depolarization-induced Ca(2+) influx) or permeabilized in the presence of basal or elevated free Ca(2+), levels of these SNARE complexes were altered differentially: moderate Ca(2+) rises (</=1 microM) caused an increase, whereas Ca(2+) elevations of more than 1 microM led to a decrease in the 230 kDa band. Under both conditions the 100 kDa band was either increased or remained unchanged. Our data show that various SDS-resistant complexes occur in living cells and indicate that they represent SNARE complexes with different structures and diverging functions. The distinct behavior of these complexes under release-promoting conditions indicates that these SNARE structures have different roles in exocytosis.

Certain excitatory pathways in the rat hippocampus can release aspartate along with glutamate. This study utilized rat hippocampal synaptosomes to characterize the mechanism of aspartate release and to compare it with glutamate release. Releases of aspartate and glutamate from the same tissue samples were quantitated simultaneously. Both amino acids were released by 25 mM K(+), 300 microM 4-aminopyridine (4-AP) and 0.5 and 1 microM ionomycin in a predominantly Ca(2+)-dependent manner. For a roughly equivalent quantity of glutamate released, aspartate release was significantly greater during exposure to elevated [K(+)] than to 4-AP and during exposure to 0.5 than to 1 microM ionomycin. Aspartate release was inefficiently coupled to P/Q-type voltage-dependent Ca(2+) channels and was reduced by KB-R7943, an inhibitor of reversed Na(+)/Ca(2+) exchange. In contrast, glutamate release depended primarily on Ca(2+) influx through P/Q-type channels and was not significantly affected by KB-R7943. Pretreatment of the synaptosomes with tetanus toxin and botulinum neurotoxins C and F reduced glutamate release, but not aspartate release. Aspartate release was also resistant to bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase, whereas glutamate release was markedly reduced. (+/-) -Threo-3-methylglutamate, a non-transportable competitive inhibitor of excitatory amino acid transport, did not reduce aspartate release. Niflumic acid, a blocker of Ca(2+)-dependent anion channels, did not alter the release of either amino acid. Exogenous aspartate and aspartate recently synthesized from glutamate accessed the releasable pool of aspartate as readily as exogenous glutamate and glutamate recently synthesized from aspartate accessed the releasable glutamate pool. These results are compatible with release of aspartate from either a vesicular pool by a non-classical form of exocytosis or directly from the cytoplasm by an as-yet-undescribed Ca(2+)-dependent mechanism. In either case, they suggest aspartate is released mainly outside the presynaptic active zones and may therefore serve as the predominant agonist for extrasynaptic N-methyl-D-aspartate receptors.

Dysfunction of the prefrontal cortex in schizophrenia may be associated with abnormalities in synaptic structure and/or function and reflected in altered concentrations of proteins in presynaptic terminals and involved in synaptic plasticity (synaptobrevin/ vesicle-associated membrane protein (VAMP), synaptosomal-associated protein-25 (SNAP-25), syntaxin, synaptophysin and growth-associated protein-43 (GAP-43)). We examined the immunoreactivity of these synapse-associated proteins via quantitative immunoblotting in the prefrontal cortex of patients with schizophrenia (n=18) and in normal controls (n=23). We also tested the stability of these proteins across successive post-mortem intervals in rat brains (at 0, 3, 12, 24, 48, and 70 h). To investigate whether experimental manipulation of prefrontal cortical development in the rat alters prefrontal synaptic protein levels, we lesioned the ventral hippocampus of rats on postnatal day 7 and measured immunoreactivity of presynaptic proteins in the prefrontal cortex on postnatal day 70. VAMP immunoreactivity was lower in the schizophrenic patients by 22% (Pless than 0.03). There were no differences in the immunoreactivity of any other proteins measured in schizophrenic patients as compared to the matched controls. Proteins were fairly stable up to 24 h and thereafter the abundance of most proteins examined was significantly reduced (falling to as low as 20% of baseline levels at 48-70 h). VAMP immunoreactivity was higher in the lesioned rats as compared to sham controls by 22% (P&less than 0.03). There were no significant differences between the lesioned rats and sham animals in any other presynaptic protein. These data suggest that apparently profound prefrontal cortical dysfunction in schizophrenia, as well as in an animal model of schizophrenia, may exist without gross changes in the abundance of many synaptic proteins but discrete changes in selected presynaptic molecules may be present.

Vesicle associated membrane protein (VAMP; also known as synaptobrevin) is a key component of the core complex needed for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Recent work indicates that the precise complement of presynaptic proteins associated with transmitter release and their isoforms vary among synapses, presumably conferring specific functional release properties. The retina contains two types of vesicular synapses with distinct morphologic, functional, and biochemical characteristics: ribbon and conventional synapses. Although the precise complement of presynaptic proteins is known to differ between conventional and ribbon synapses and among conventional synapses, the distribution of VAMP isoforms among retinal synapses has not been determined. The expression and localization of VAMP isoforms in the salamander retina, a major model system for studies of retinal circuitry, was examined by using immunocytochemical and immunoblotting methods. Both methods indicated that at least two VAMP isoforms were expressed in salamander retina. One isoform, recognized by an immunoglobulin M antibody that recognizes both mammalian VAMP-1 and VAMP-2, was associated with photoreceptor and bipolar cell terminals as well as many conventional synapses, and probably corresponds to mammalian VAMP-2. A different VAMP isoform associated with a subset of amacrine cells, was recognized only by antibodies directed against the N-terminus of mammalian VAMP-2. An antiserum directed against the N-terminus of mammalian VAMP-1 did not specifically recognize any salamander VAMPs in either immunocytochemical or immunoblotting experiments. Heterogeneous distribution of VAMP isoforms among conventional retinal synapses was confirmed by double labeling for synapsin I, a marker for conventional synapses. These studies indicate that VAMP isoforms are expressed heterogeneously among retinal synapses but cannot account for the differences in transmitter release characteristics at ribbon and conventional synapses. These results also corroborate previous studies in Xenopus indicating that the N-terminus of nonmammalian VAMP isoforms differs from their mammalian counterparts.

Little is known about presynaptic assembly during central nervous system synaptogenesis. Here we used time-lapse fluorescence imaging, immunocytochemistry and electron microscopy to study hippocampal neuronal cultures transfected with a fusion construct of the presynaptic vesicle protein VAMP and green fluorescent protein. Our results suggest that major cytoplasmic and membrane-associated protein precursors of the presynaptic active zone are transported along developing axons together as discrete packets. Retrospective electron microscopy demonstrated varied vesicular and tubulovesicular membrane structures. Packets containing these heterogeneous structures were stabilized specifically at new sites of dendrite- and axon-initiated cell-cell contact; within less than one hour, evoked vesicle recycling was observed at these putative nascent synapses. These observations suggest that substantial membrane remodeling may be necessary to produce the uniform vesicles typical of the mature active zone, and that many presynaptic proteins may be united early in their biogenesis and sorting pathways.

Synaptic pathology is likely to be an important feature of a number of neuropsychiatric illnesses. An antibody called EP10 was used previously to demonstrate a regional reduction in a 38 kDa synaptophysin-like protein in Alzheimer's disease. The SP antibodies were developed for further study of this and other synaptic proteins in human brain. Human brain proteins immunoprecipitated with EP10 were used as the immunogen. Hybridoma screening was carried out with a sequential ELISA-immunocytochemical approach. Sixteen antibodies were obtained, the antigens clustered into five groups. Five antibodies were reactive with a 38 kDa synaptophysin-like protein. Another two antibodies were reactive with a 16 kDa antigen which may be synaptobrevin. Immunocytochemical studies indicated these two antigens appeared to be co-localized in human brain. Four antibodies were reactive with a distinct, 34-36 kDa antigen. In the cerebellum, this antigen was restricted to terminals in the molecular layer, putatively in the parallel fibre synapses. Two antibodies were reactive with a 26-27 kDa antigen. In the cerebellum, this antigen localized to a subset of terminals which included the axo-axonal contacts of the Basket and Purkinje cells. The final group of three antibodies detected a complex group of 38 kDa. 40 kDa and higher molecular weight antigens. The results suggest that heterogeneity among synapses can be defined through antibodies directed against distinct proteins. The SP antibodies may be useful probes for studies of human synaptic proteins, and for studies of pathological conditions which disrupt these molecules.