MAB336 Sigma-AldrichAnti-Syntaxin Antibody, clone SP8

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.

Aberrant regulation of synaptic function is thought to play a role in the aetiology of psychiatric disorders, including schizophrenia and bipolar disorder. Normal neurotransmitter release is dependent on a complex group of presynaptic proteins that regulate synaptic vesicle docking, membrane fusion and fission, including synaptophysin, syntaxin, synaptosomal-associated protein-25 (SNAP-25), vesicle-associated membrane protein (VAMP), alpha-synuclein and dynamin I. In addition, structural and signalling proteins such as neural cell adhesion molecule (NCAM) maintain the integrity of the synapse. We have assessed the levels of these important synaptic proteins using Western blots, in three cortical regions (BA10, 40 and 46) obtained post-mortem from subjects with bipolar 1 disorder, schizophrenia or no history of a psychiatric disorder. In bipolar 1 disorder cortex (parietal; BA40), we found a significant increase in the expression of SNAP-25, and a significant reduction in alpha-synuclein compared with controls. These changes in presynaptic protein expression are proposed to inhibit synaptic function in bipolar 1 disorder. In schizophrenia, a significant reduction in the ratio of the two major membrane-bound forms of NCAM (180 and 140) was observed in BA10. The distinct functions of these two NCAM forms suggest that changes in the comparative levels of these proteins could lead to a destabilisation of synaptic signalling. Our data support the notion that there are complex and region-specific alterations in presynaptic proteins that may lead to alterations in synaptic activity in both schizophrenia and bipolar disorder.

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.

Gastrointestinal stromal tumors (GISTs) are thought to originate from the interstitial cells of Cajal, which share many properties with neurons of the gastrointestinal tract. Recently, we demonstrated expression of the hormone ghrelin in GIST. The aim of the present study was therefore to evaluate a possible neuroendocrine phenotype of GIST. Specimens from 41 GISTs were examined for the expression of 12 different synaptic vesicle proteins. Expression of synaptic-like microvesicle proteins, e.g., Synaptic vesicle protein 2 (SV2), synaptobrevin, synapsin 1, and amphiphysin was demonstrated in a majority of GISTs by immunohistochemistry, western blotting, and quantitative reversetranscriptase PCR. One-third of the tumors also expressed the large dense core vesicle protein vesicular monoamine transporter 1. Presence of microvesicles and dense core vesicles in GIST was confirmed by electron microscopy. The expression of synaptic-like microvesicle proteins in GIST was not related to risk profile or to KIT/platelet derived growth factor alpha (PDGFRA) mutational status. Thus, GISTs regularly express a subset of synaptic-like microvesicle proteins necessary for the regulated secretion of neurotransmitters and hormones. Expression of synaptic-like micro-vesicle proteins, ghrelin and peptide hormone receptors in GIST indicate a neuroendocrine phenotype and suggest novel possibilities to treat therapy-resistant GIST.

In order to identify whether the mechanisms associated with neurotransmitter release are involved in the pathologies of bipolar disorder and schizophrenia, levels of presynaptic [synaptosomal-associated protein-25 (SNAP-25), syntaxin, synaptophysin, vesicle-associated membrane protein, dynamin I] and structural (neuronal cell adhesion molecule and alpha-synuclein) neuronal markers were measured in Brodmann's area 9 obtained postmortem from eight subjects with bipolar I disorder (BPDI), 20 with schizophrenia and 20 controls.Determinations of protein levels were carried out using Western blot techniques with specific antibodies. Levels of mRNA were measured using real-time polymerase chain reaction.In BPDI, levels of SNAP-25 (p < 0.01) and synaptophysin (p < 0.05) increased. There were no changes in schizophrenia or any other changes in BPDI. Levels of mRNA for SNAP-25 were decreased in BPDI (p < 0.05).Changes in SNAP-25 and synaptophysin in BPDI suggest that changes in specific neuronal functions could be linked to the pathology of the disorder.

The spontaneous autosomal recessive mouse mutant for hydrocephaly with hop gait (hyh) exhibits dramatic cystic dilation of the ventricles at birth and invariably develops hopping gait. We show that the gene for soluble N-ethylmaleimide sensitive factor attachment protein alpha, also known as alpha-SNAP, is mutated in hyh mice. alpha-SNAP plays a key role in a wide variety of membrane fusion events in eukaryotic cells, including the regulated exocytosis of neurotransmitters. Homozygous mutant mice harbor a missense mutation M105I in a conserved residue in one of the alpha-helical domains. We demonstrate that the hyh mutant is not a null allele and is expressed; however, the mutant protein is 40% less abundant in hyh mice. The hyh mutant provides a valuable in vivo model to study vesicle/membrane trafficking and provides insight into the potential roles of alpha-SNAP in embryogenesis and brain development.

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.

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.

Cysteine-string protein is a vesicle-associated protein that plays a vital function in neurotransmitter release. We have studied its expression and regulation during cochlear maturation. Both the mRNA and the protein were found in primary auditory neurons and the sensory inner hair cells. More importantly, cysteine-string protein was localized on synaptic vesicles associated with the synaptic ribbon in inner hair cells and with presynaptic differentiations in lateral and medial olivocochlear terminals -- the cell bodies of which lie in the auditory brainstem. No cysteine-string protein was expressed by the sensory outer hair cells suggesting that the distinct functions of the two cochlear hair cell types imply different mechanisms of neurotransmitter release. In developmental studies in the rat, we observed that cysteine-string protein was present beneath the inner hair cells at birth and beneath outer hair cells by postnatal day 2 only. We found no expression in the inner hair cells before about postnatal day 12, which corresponds to the period during which the first cochlear action potentials could be recorded. In conclusion, the close association of cysteine-string protein with synaptic vesicles tethered to synaptic ribbons in inner hair cells and its synchronized expression with the appearance and maturation of the cochlear potentials strongly suggest that this protein plays a fundamental role in sound-evoked glutamate release by inner hair cells. This also suggests that this role may be common to ribbon synapses and conventional central nervous system synapses.

Small molecules present during brain tissue homogenization are known to be entrapped within subsequently isolated synaptosomes. We have revisited this technique in view of its systematic utilization to incorporate into nerve endings impermeant probes of large size. Rat neocortical synaptosomes were prepared in the absence or in the presence of each of the following compounds: 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), tetanus toxin (TeTx) or its light chain (TeTx-LC), pertussis toxin (PTx), anti-syntaxin, or anti-SNAP25 monoclonal antibodies. Release of endogenous GABA and glutamate was then evoked by high K+ depolarization. GABA and glutamate overflows were inhibited by entrapped BAPTA and in synaptosomes prepared by homogenization in the presence of varying concentrations of TeTx or TeTx-LC. When synaptobrevin cleavage in synaptosomes entrapped with TeTx was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by western blotting, the extent of proteolysis was found to correspond quantitatively to that of release inhibition. GABA and glutamate overflows were increased by entrapped PTx; moreover, (-)-baclofen inhibited amino acid overflow more potently in standard than in PTx-containing synaptosomes. The overflows of GABA and glutamate were similarly decreased following incorporation of anti-syntaxin or anti-SNAP25 antibodies. Synaptosomal entrapping may be routinely used to internalize membrane-impermeant agents of different size in studies of presynaptic mechanisms.