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Cell bodies of trigeminal nerves, which are located in the trigeminal ganglion, are completely surrounded by satellite glial cells and together form a functional unit that regulates neuronal excitability. The goals of this study were to investigate the cellular organization of the rat trigeminal ganglia during postnatal development and correlate those findings with expression of proteins implicated in neuron–glia interactions. During postnatal development there was an increase in the volume of the neuronal cell body, which correlated with a steady increase in the number of glial cells associated with an individual neuron from an average of 2.16 at birth to 7.35 on day 56 in young adults. Interestingly, while the levels of the inwardly rectifying K+ channel Kir4.1 were barely detectable during the first week, its expression in satellite glial cells increased by day 9 and correlated with initial formation of functional units. Similarly, expression of the vesicle docking protein SNAP-25 and neuropeptide calcitonin gene-related peptide was readily detected beginning on day 9 and remained elevated throughout postnatal development. Based on our findings, we propose that the expression of proteins involved in facilitating neuron–glia interactions temporally correlates with the formation of mature functional units during postnatal development of trigeminal ganglion. [ABSTRACT FROM PUBLISHER]

Abstract: Evidence indicates that accumulation of excitotoxic mediators, such as glutamate, contributes to neuronal damage after an ischaemic insult. It is not clear, however, whether this accumulation is due to excess synaptic release or to impaired uptake. To test a role for synaptic release, here we investigated the neuroprotective potential of the synaptic blocker botulinum neurotoxin E (BoNT/E), that prevents vesicle fusion via the cleavage of the SNARE (soluble NSF-attachment receptor) protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Focal ischaemia was induced in vivo by infusing the potent vasoconstricting peptide endothelin-1 (ET-1) into the CA1 area of the hippocampus in adult rats; BoNT/E or vehicle were administered into the same site 20 min later. Injection of ET-1 was found to produce a transient and massive increase in glutamate release that was potently antagonized by BoNT/E. To assess whether blocking transmitter release translates into neuroprotection, the extent of the ischaemic damage was determined 24 h and 6 weeks after the insult. We found that BoNT/E administration consistently reduced the loss of CA1 pyramidal neurons at 24 h. The neuroprotective effect of BoNT/E, however, was no longer significant at 6 weeks. These data provide evidence that blockade of synaptic transmitter release delays neuronal cell death following focal brain ischaemia, and underline the importance of assessing long-term neuroprotection in experimental stroke studies. [Copyright &y& Elsevier]

The SNARE-complex consisting of synaptobrevin-2/VAMP-2, SNAP-25 and syntaxin-1 is essential for evoked neurotransmission and also involved in spontaneous release. Here, we used cultured autaptic hippocampal neurons from Snap-25 null mice rescued with mutants challenging the C-terminal, N-terminal and middle domains of the SNARE-bundle to dissect out the involvement of these domains in neurotransmission. We report that the stabilities of two different sub-domains of the SNARE-bundle have opposing functions in setting the probability for both spontaneous and evoked neurotransmission. Destabilizing the C-terminal end of the SNARE-bundle abolishes spontaneous neurotransmitter release and reduces evoked release probability, indicating that the C-terminal end promotes both modes of release. In contrast, destabilizing the middle or deleting the N-terminal end of the SNARE-bundle increases both spontaneous and evoked release probabilities. In both cases, spontaneous release was affected more than evoked neurotransmission. In addition, the N-terminal deletion delays vesicle priming after a high-frequency train. We propose that the stability of N-terminal two-thirds of the SNARE-bundle has a function for vesicle priming and limiting spontaneous release. [ABSTRACT FROM AUTHOR]

Abstract: 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), α-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 α-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. [Copyright &y& Elsevier]

J. Neurochem. (2010) 114, 171–181. The double C2 (Doc2) family is characterized by an N-terminal Munc13-1-interacting domain and C-terminal tandem C2 domains, and it comprises three isoforms, Doc2α, Doc2β, and Doc2γ, in humans and mice. Doc2α, the best-characterized, brain-specific isoform, exhibits Ca2+-dependent phospholipid-binding activity through its C2A domain, and the Ca2+-binding activity is thought to be important for the regulation of Ca2+-dependent exocytosis. In contrast to the C2A domain, however, nothing is known about the physiological functions of the C2B domain in regulated exocytosis. In this study, we demonstrated by a mutation analysis that the polybasic sequence in the C2B domain of Doc2α (306 KKSKHKTCVKKK 317) is required for binding of syntaxin-1a/synaptosome-associated protein of 25 kDa (SNAP-25) heterodimer. We also investigated the effect of Lys-to-Gln (named KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in PC12 cells. A Doc2α(KQ) mutant, which lacks binding activity toward syntaxin-1a/SNAP-25 heterodimer, significantly decreased the number of plasma membrane-docked vesicles before stimulation and strongly inhibited high-KCl-induced exocytosis from the plasma membrane-docked vesicles. These results indicate that the polybasic sequence in the C2B domain functions as a binding site for syntaxin-1a/SNAP-25 heterodimer and controls the number of ‘readily releasable’ vesicles in neuroendocrine cells. [ABSTRACT FROM AUTHOR]

Bipolar disorder (BD) is one of the most common and persistent psychiatric disorders. Early-onset BD has been shown to be the most severe and familial form. We recently carried out a whole-genome linkage analysis on sibpairs affected by early-onset BD and showed that the 20p12 region was more frequently shared in our families than expected by chance. The synaptosomal-associated protein SNAP25 is a presynaptic plasma membrane protein essential for the triggering of vesicular fusion and neurotransmitter release, and for which abnormal protein levels have been reported in postmortem studies of bipolar patients. We hypothesised that variations in the gene encoding SNAP25, located on chromosome 20p12, might influence the susceptibility to early-onset BD. We screened SNAP25 for mutations and performed a case–control association study in 197 patients with early-onset BD, 202 patients with late-onset BD and 136 unaffected subjects. In addition, we analysed the expression level of the two SNAP25 isoforms in 60 brains. We showed that one variant, located in the promoter region, was associated with early-onset BD but not with the late-onset subgroup. In addition, individuals homozygous for this variant showed a significant higher SNAP25b expression level in prefrontal cortex. These results show that variations in SNAP25, associated with an increased gene expression level in prefrontal cortex, might predispose to early-onset BD. Further analyses of this gene, as well as analysis of genes encoding for the SNAP25 protein partners, are required to understand the impact of such molecular mechanisms in BD. [ABSTRACT FROM AUTHOR]

Inhibition of neuroexocytosis by tetanus neurotoxin (TeNT) involves VAMP-2/synaptobrevin-2 cleavage. However, deletion of the TeNT activity does not completely abolish its inhibitory action. TeNT is a potent activator of the cross-linking enzyme transglutaminase 2 (TGase 2) in vitro. The role of the latter mechanism in TeNT poisoning was investigated in isolated nerve terminals and intact neurons. TeNT-induced inhibition of glutamate release from rat cortical synaptosomes was associated with a simultaneous activation of neuronal transglutaminase (TGase) activity. The TeNT-induced blockade of neuroexocytosis was strongly attenuated by pretreatment of either live Aplysia neurons or isolated nerve terminals with specific TGase inhibitors or neutralizing antibodies. The same treatments completely abolished the residual blockade of neuroexocytosis of a non-proteolytic mutant of TeNT light chain. Electrophysiological studies indicated that TGase activation occurs at an early step of TeNT poisoning and contributes to the inhibition of transmitter release. Bioinformatics and biochemical analyses identified synapsin I and SNAP-25 as potential presynaptic TGase substrates in isolated nerve terminals, which are potentially involved in the inhibitory action of TeNT. The results suggest that neuronal TGase activity plays an important role in the regulation of neuroexocytosis and is one of the intracellular targets of TeNT in neurons. [ABSTRACT FROM AUTHOR]

Abstract: Background: Botulinum toxin type A (BoNTA) has been successfully used in the treatment of refractory detrusor overactivity. The toxin is internalized after binding a high-affinity receptor, synaptic vesicle protein 2 (SV2), which is exposed in the cell membrane during the exocytosis process. In the cytoplasm, BoNTA cleaves specific sites of synaptosomal-associated protein 25 (SNAP-25), preventing the assembly of the synaptic fusion complex SNARE and blocking exocytosis. Objective: In the present work, the distribution of SV2 and SNAP-25 was first investigated in human bladders. The neurochemistry of BoNTA-sensitive structures was then investigated using markers for parasympathetic, sympathetic, and sensory fibers. Design, setting, and participants: Human bladders were obtained from cadaveric organ donors (age range: 19–74 yr). Measurements: Bladder sections were processed for single or dual immunofluorescence staining with antibodies against SV2, SNAP-25, β-3 tubulin, vesicular acetylcholine transporter, tyrosine hydroxilase, and calcitonin gene-related peptide. Results and limitations: SV2 and SNAP-25 immunoreactive fibers were distributed throughout the suburothelium and muscular layer. Double labeling showed extensive colocalization of both proteins in nerve fibers. SV2 is more expressed in parasympathetic fibers than in sympathetic or sensory fibers. No expression was found in urothelial or muscular cells. Because only normal bladders were used, this distribution should be applied with caution to pathologic bladders. Conclusions: SV2 and SNAP-25 colocalize abundantly throughout the urinary bladder. SV2 is more abundant in cholinergic, parasympathetic fibers. These nerves are suggested to be the main target for BoNTA action in the human urinary bladder. [Copyright &y& Elsevier]

Abstract: SNAP-25 is a SNARE protein implicated in exocytosis and in the negative modulation of voltage-gated calcium channels. We have previously shown that GABAergic synapses, which express SNAP-25 at much lower levels relative to glutamatergic ones, are characterized by a higher calcium responsiveness to depolarization and are largely resistant to botulinum toxin A. We show here that silencing of SNAP-25 in glutamatergic neurons, a procedure which increases KCl-induced calcium elevations, confers these synapses with toxin resistance. Since it is known that calcium reverts the efficacy of botulinum A, we investigated whether the lower effectiveness of the toxin in inhibiting GABAergic vesicle cycling might be attributable to higher evoked calcium transients of inhibitory neurons. We demonstrate that either expression of SNAP-251–197 or BAPTA/AM treatment, both inhibiting calcium dynamics, facilitate block of GABAergic vesicle exocytosis upon toxin treatment. These data indicate that intrinsic calcium dynamics control botulinum A susceptibility in distinct neuronal populations. [Copyright &y& Elsevier]

Abnormalities of amount and function of presynaptic terminals may have an important role in the mechanism of illness in schizophrenia. The SNARE proteins (SNAP-25, syntaxin, and VAMP) are enriched in presynaptic terminals, where they interact to form a functional complex to facilitate vesicle fusion. SNARE protein amounts are altered in the cortical regions in schizophrenia, but studies of protein–protein interactions are limited. We extended these investigations to the striatal regions (such as the nucleus accumbens, ventromedial caudate (VMC), and dorsal caudate) relevant to disease symptoms. In addition to measuring SNARE protein levels, we studied SNARE protein–protein interactions using a novel ELISA method. The possible effect of antipsychotic treatment was investigated in parallel in the striatum of rodents that were administered haloperidol and clozapine. In schizophrenia samples, compared with controls, SNAP-25 was 32% lower (P=0.015) and syntaxin was 26% lower (P=0.006) in the VMC. In contrast, in the same region, SNARE protein–protein interactions were higher in schizophrenia (P=0.008). Confocal microscopy of schizophrenia and control VMC showed qualitatively similar SNARE protein immunostaining. Haloperidol treatment of rats increased levels of SNAP-25 (mean 24%, P=0.003), syntaxin (mean 18%, P=0.010), and VAMP (mean 16%, P=0.001), whereas clozapine increased only the VAMP level (mean 13%, P=0.004). Neither drug altered SNARE protein–protein interactions. These results indicate abnormalities of amount and interactions of proteins directly related to presynaptic function in the VMC in schizophrenia. SNARE proteins and their interactions may be a novel target for the development of therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) polymerase chain reaction (PCR) and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina. [Copyright &y& Elsevier]

The article presents a study which explores the relation between changes in protein expression and development of motor coordination using rats with cerebral infarction. The study compares the rat groups with treadmill training and without training. The motor performance was measured through proteome analysis and rotarod test. It shows that glial fibrillary acidic protein and 25-kDa synaptosomal-associated protein were increased in rats' cerebellum with training than in rats without training.

An immunohistochemical study of the olfactory bulb (OB) in human fetuses was performed. Immunohistochemical markers for nervous system-specific protein complexes S-100 and SNAP-25 were used. At 20–22 weeks of development, the development of cells expressing protein S-100 was greater in the OB than in the neocortex (the areas of the gyrus rectus and the gyrus orbitalis longitudinalis internus). These quantitative differences indicate heterochronicity in glial differentiation in the OB and cerebral cortex. Immunopositive reactions for SNAP-25 were for the first time detected at the periphery of the OB and in glomeruli in human fetuses at 15–16 and 20–22 weeks of development. Immunohistochemical staining of the OB with antibodies to SNAP-25 indicated that the human olfactory system cannot function prior to 20–22 weeks of development. In the OB of fetuses at 28–29 weeks of development, the intensity of the immunohistochemical reaction showed topological differences. The reaction with antibodies to SNAP-25 in the OB of full-term fetuses was similar to the reaction in the OB of adult humans. The results of these immunohistochemical studies using the reaction for the protein complex SNAP-25 in fetuses of different ages suggest the that the primary olfactory center in humans starts to function no earlier than the 30th week of development. [ABSTRACT FROM AUTHOR]

Abstract: Low doses of psychostimulants produce beneficial behavioral effects in ADHD patients but the mechanisms underlying the response are not understood. Here we use the hyperactive mouse mutant coloboma to identify D2-like dopamine receptor subtypes that mediate the hyperactivity and response to amphetamine; we have previously demonstrated that D1-like dopamine receptors are not involved. Targeted deletion of the D2, but not the D3 or the D4, dopamine receptor in coloboma mice eliminated the hyperactivity; depleting D2 dopamine receptors also restored the excess dopamine overflow that may drive the hyperactivity to normal concentrations. Similar to its effects on ADHD patients, amphetamine reduced the hyperactivity of coloboma mice. The D2 dopamine receptor-selective antagonist L-741,626, but not D3 or D4 dopamine receptor-selective antagonists, blocked the amphetamine-induced reduction in locomotor activity. Thus, the D2 dopamine receptor subtype mediates both the hyperactivity and response to amphetamine, suggesting a specific target for novel therapeutics in ADHD. [Copyright &y& Elsevier]

Abstract: The cellular molecular motor kinesin-1 mediates the microtubule-dependent transport of a range of cargo. We have previously identified an interaction between the cargo-binding domain of kinesin-1 heavy chain KIF5B and the membrane-associated SNARE proteins SNAP-25 and SNAP-23. In this study we further defined the minimal SNAP-25 binding domain in KIF5B to residues 874–894. Overexpression of a fragment of KIF5B (residues 594–910) resulted in significant colocalization with SNAP-25 with resulting blockage of the trafficking of SNAP-25 to the periphery of cells. This indicates that kinesin-1 facilitates the transport of SNAP-25 containing vesicles as a prerequisite to SNAP-25 driven membrane fusion events. [Copyright &y& Elsevier]

Abstract: BOTOX® is a botulinum toxin type A product from Allergan that is approved in more than 70 countries, where it addresses unmet patient needs across a variety of indications. BOTOX® is a well-characterized and highly purified biological product that is not interchangeable with any other botulinum neurotoxin. The pharmacology, efficacy and safety profile of BOTOX® has been established in numerous preclinical and clinical studies in addition to meta-analyses. BOTOX® exhibits a predictable response, with a concomitant low rate of neutralizing antibody formation. Allergan is committed to the development of new indications and novel biologics that are designed to benefit individuals with unmet medical needs. [Copyright &y& Elsevier]

Mesial temporal lobe epilepsy (MTLE) is one of the most common forms of human epilepsy, and it is often resistant to conventional antiepileptic drug (AED) therapy. Here we tested whether a single intrahippocampal administration of the synaptic blocker botulinum neurotoxin E (BoNT/E) is effective in reducing spontaneous recurrent seizures (SRS) in a mouse model of MTLE. Unilateral intrahippocampal injection of kainic acid (KA) in mice was used as a model of MTLE. Electroencephalography (EEG) recordings of SRS were performed during the chronic phase of epilepsy, before and after administration of either BoNT/E or vehicle. Frequency of SRS was significantly decreased for at least 5 days following BoNT/E, but not vehicle, infusion. Our findings demonstrate that BoNT/E can effectively reduce seizure incidence in a mouse model of MTLE. [ABSTRACT FROM AUTHOR]

Chimeras of botulinum neurotoxin (BoNT) serotype A (/A) combined with /E protease might possess improved analgesic properties relative to either parent, due to inheriting the sensory neurotropism of the former with more extensive disabling of SNAP-25 from the latter. Hence, fusions of /E protease light chain (LC) to whole BoNT/A (LC/E-BoNT/A), and of the LC plus translocation domain (HN) of /E with the neuronal acceptor binding moiety (HC) of /A (BoNT/EA), created previously by gene recombination and expression in E. coli., were used. LC/E-BoNT/A (75 units/kg) injected into the whisker pad of rats seemed devoid of systemic toxicity, as reflected by an absence of weight loss, but inhibited the nocifensive behavior (grooming, freezing, and reduced mobility) induced by activating TRPV1 with capsaicin, injected at various days thereafter. No sex-related differences were observed. c-Fos expression was increased five-fold in the trigeminal nucleus caudalis ipsi-lateral to capsaicin injection, relative to the contra-lateral side and vehicle-treated controls, and this increase was virtually prevented by LC/E-BoNT/A. In vitro, LC/E-BoNT/A or /EA diminished CGRP exocytosis from rat neonate trigeminal ganglionic neurons stimulated with up to 1 µM capsaicin, whereas BoNT/A only substantially reduced the release in response to 0.1 µM or less of the stimulant, in accordance with the /E protease being known to prevent fusion of exocytotic vesicles. [ABSTRACT FROM AUTHOR]

Abstract: Voltage-gated outward K+ currents from pancreatic islet β-cells are known to repolarize the action potential during a glucose stimulus, and consequently to modulate Ca2+ entry and insulin secretion. The voltage gated K+ (Kv) channel, Kv2.1, which is expressed in rat islet β-cells, mediates over 60% of the Kv outward K+ currents. A novel peptidyl inhibitor of Kv2.1/Kv2.2 channels, guangxitoxin (GxTX)-1, has been shown to enhance glucose-stimulated insulin secretion. Here, we show that SNAP-251–180 (S180), an N-terminal SNAP-25 domain, but not SNAP-251–206 (S206), inhibits Kv current and enhances glucose-dependent insulin secretion from rat pancreatic islet β-cells, and furthermore, this enhancement was induced by the blockade of the Kv2.1 current. This study indicates that the Kv2.1 channel is a potential target for novel therapeutic agent design for the treatment of type 2 diabetes. This target may possess advantages over currently-used therapies, which modulate insulin secretion in a glucose-independent manner. [Copyright &y& Elsevier]

The expression of SNAP-25 fused to green fluorescent protein (GFP) has been instrumental in demonstrating SNARE role in exocytosis. The wild-type GFP–SNAP-25 and a Δ9 form, product of botulinum neurotoxin A activity, the main ingredient in the BOTOX preparation, were employed here to study SNARE implication in vesicle mobility and fusion in cultured bovine chromaffin cells, a neuroendocrine exocytotic model. Using total internal reflection fluorescent microscopy, we have identified membrane microdomains of 500–600 nm diameter that contain both SNAP-25 and syntaxin-1 and associate with synaptobrevin-2. Interestingly, while the SNAP-25 Δ9 formed similar clusters, they displayed increased mobility both laterally and in the axis perpendicular to the plasmalemma, and this correlates with the enhanced dynamics of associated chromaffin granules. SNARE cluster-enhanced motion is reversed by elevation of the intracellular calcium level. Furthermore, single vesicle fusion was unlikely in the highly mobile vesicles present in the cells expressing SNAP-25 Δ9, which, in addition, displayed in average slower fusion kinetics. Consequently, SNARE cluster dynamics is a new aspect to consider when determining the factors contributing to the mobility of the vesicles in close vicinity to the plasma membrane and also the probability of exocytosis of this granule population. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxin (BoNT/A) is an FDA and NICE approved second-line treatment for overactive bladder (OAB) in patients either not responsive or intolerant to anti-cholinergic drugs. BoNT/A acts to weaken muscle contraction by blocking release of the neurotransmitter acetyl choline (ACh) at neuromuscular junctions. However, this biological activity does not easily explain all the observed effects in clinical and non-clinical studies. There are also conflicting reports of expression of the BoNT/A protein receptor, SV2, and intracellular target protein, SNAP-25, in the urothelium and bladder. This review presents the current evidence of BoNT/A's effect on bladder sensation, potential mechanisms by which it might exert these effects and discusses recent advances in understanding the action of BoNT in bladder tissue. [ABSTRACT FROM AUTHOR]

SNAP-25 (synaptosomal-associated protein of 25 kDa) is a plasma membrane protein that, together with syntaxin and the synaptic vesicle protein VAMP/synaptobrevin, forms the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) docking complex for regulated exocytosis. SNAP-25 also modulates different voltage-gated calcium channels, representing therefore a multifunctional protein that plays essential roles in neurotransmitter release at different steps. Recent genetic studies of human populations and of some mouse models implicate alterations in SNAP-25 gene structure, expression, and/or function in contributing directly to these distinct neuropsychiatric and neurological disorders. [ABSTRACT FROM AUTHOR]

Cholesterol-rich clusters of SNARE (soluble NSF attachment protein receptor) proteins have been implicated as being important for exocytosis. Here we demonstrate the significance of cholesterol for normal biphasic insulin secretion in mouse β cells by removal of cholesterol from the plasma membrane using methyl-β-cyclodextrin (MBCD). Maximal inhibition of insulin secretion in static incubations was achieved using 0.1 mM MBCD. In in situ pancreatic perfusion measurements, both first and second phase insulin secretions were reduced by ~50% (P<0.05). This was accompanied by a reduced number of docked large dense core vesicles (LDCVs) (~40%; P<0.01) and a reduced exocytotic response (>50%; P<0.01). Further, subcellular fractionatious demonstrated movement of the synaptosomal protein of 25 kDa (SNAP-25) from the plasma membrane to the cytosol after MBCD treatment. The inhibitory actions of MBCD were counteracted by subsequent addition of cholesterol. We hypothesize that desorption of cholesterol leads to the disturbance of a basic exocytotic mechanism partly due to migration of SNAP-25, and we conclude that insulin secretion is highly sensitive to changes in plasma membrane cholesterol. [ABSTRACT FROM AUTHOR]

Abstract: Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC50s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC50 of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays. [Copyright &y& Elsevier]

The SNAP-25 gene is an integral part of the vesicle docking and fusion machinery that controls neurotransmitter release. Several post mortem studies revealed a reduction of SNAP-25 protein in the hippocampus of patients with schizophrenia and bipolar disorder (BD). Thirty-eight patients with schizophrenia, BD or obsessive-compulsive disorder and 17 healthy controls participated in the study. Proton magnetic resonance spectroscopy in left hippocampus was performed in each individual. Three single nucleotide polymorphisms (SNP) of the SNAP-25 gene were genotyped. Individuals with the homozygous CC genotype of the DdeI SNP presented a significantly higher ratio of N-acetyl-aspartate (NAA)/choline-containing compounds (Cho) in the left hippocampus compared to the group of individuals with the homozygous TT genotype. The SNAP-25 genotype may modulate synaptic plasticity and neurogenesis in the left hippocampus, and altered NAA/Cho ratio may be an indicator for this genetic modulation of neuronal function in the hippocampus. [ABSTRACT FROM AUTHOR]

Abstract: An increase in the prevalence of type 2 diabetes has been consistently observed among residents of high arsenic exposure areas. We have previously shown that in rat pancreatic β-cells, low arsenite doses impair the secretion of insulin without altering its synthesis. To further study the mechanism by which arsenite reduces insulin secretion, we evaluated the effects of arsenite on the calcium-calpain pathway that triggers insulin exocytosis in RINm5F cells. Cell cycle and proliferation analysis were also performed to complement the characterization. Free [Ca2+]i oscillations needed for glucose-stimulated insulin secretion were abated in the presence of subchronic low arsenite doses (0.5–2 µM). The global activity of calpains increased with 2 µM arsenite. However, during the secretion of insulin stimulated with glucose (15.6 mM), 1 µM arsenite decreased the activity of calpain-10, measured as SNAP-25 proteolysis. Both proteins are needed to fuse insulin granules with the membrane to produce insulin exocytosis. Arsenite also induced a slowdown in the β cell line proliferation in a dose-dependent manner, reflected by a reduction of dividing cells and in their arrest in G2/M. Data obtained showed that one of the mechanisms by which arsenite impairs insulin secretion is by decreasing the oscillations of free [Ca2+]i, thus reducing calcium-dependent calpain-10 partial proteolysis of SNAP-25. The effects in cell division and proliferation observed with arsenite exposure can be an indirect consequence of the decrease in insulin secretion. [Copyright &y& Elsevier]

The synaptosomal-associated protein of 25 kDa (SNAP-25) is part of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment receptor (SNARE), which mediates synaptic neurotransmission. In earlier studies a possible involvement of this protein in schizophrenia has been shown. As neurocognitive impairment is a core feature in the pathology of schizophrenia and considered to be a putative endophenotype according to genetic studies we investigated the influences of different SNAP-25 polymorphisms on neuropsychological test results before and during treatment with atypical antipsychotics. A total of 104 schizophrenic patients treated with atypical antipsychotics were genotyped for three different polymorphisms of the SNAP-25 gene ( MnlI, TaiI and DdeI in the 3′-UTR). Cognitive function was assessed at baseline, week 4 or 6 and week 8 or 12. Results of individual neuropsychological tests were assigned to six cognitive domains (reaction time and quality; executive function; working, verbal and visual memory) and a general cognitive index. The MnlI and TaiI polymorphisms showed no associations to deficits on neuropsychological test results. In contrast, we observed a significant relation between the DdeI polymorphism of the SNAP-25 gene and cognitive dysfunctions. Homozygote T/T allele carriers of the DdeI polymorphism showed significant better neuropsychological test results in cognitive domains verbal memory and executive functions than those with the combined T/C and C/C genotypes ( P < 0.01) at all three time points, but no differences in response to treatment with atypical antipsychotics. Additionally, TT carriers exhibited significantly better results in a general cognitive index ( P < 0.05). As we observed an association between the DdeI polymorphism of the SNAP-25 gene and cognitive dysfunctions of schizophrenic patients our finding suggests that the SNAP-25 gene could play a role in the pathophysiology of neurocognitive dysfunctions in schizophrenia but is not predictive for treatment response with atypical antipsychotics. [ABSTRACT FROM AUTHOR]

Abstract: Cognitive functions usually involve various synaptic proteins and neurotrophic factors in the hippocampus. However, whether treadmill exercise can improve learning and memory by upregulating some of these molecules remain unraveled. To address this question, male BALB/c mice were divided into control and exercise groups, the latter group went through 4 weeks of treadmill exercise training. At the end of exercise training period, they were either tested for passive avoidance (PA) performance or sacrificed for quantifying the hippocampal levels of brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB, the BDNF receptor), synaptotagmin (a Ca2+-dependent synaptic vesicle protein), and SNAP-25 (a presynaptic vesicular fusion protein). Our results showed that treadmill exercise training (1) increased the retention latency without affecting the fear acquisition in the PA test, (2) transiently increased the hippocampal BDNF level at 1, 2, and 4h after the completion of exercise training, and (3) persistently increased the hippocampal protein levels of full-length TrkB, phosphorylated TrkB and synaptotagmin, but not truncated TrkB or SNAP-25. Moreover, the protein expression level of full-length TrkB or synaptotagmin was positively correlated with PA performance in mice. Finally, inhibition of TrkB signaling by K252a abolished the exercise-facilitated PA performance and upregulation of TrkB and synaptotagmin. Taken together, these data suggest that the upregulation of TrkB and synaptotagmin in the hippocampus contributes to the exercise-facilitated aversive memory. [Copyright &y& Elsevier]

Abstract: In regulated exocytosis synaptosomal-associated protein of 25kDa (SNAP-25) is one of the key-players in the formation of SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) complex and membrane fusion. SNARE proteins are essentially expressed in neurons, neuroendocrine and endocrine cells. Whether parathyroid cells express these proteins is not known. In this study, we have examined the expression of the SNARE protein SNAP-25 and its cellular homologue SNAP-23, as well as syntaxin1 and VAMP (vesicle-associated membrane protein) in samples of normal parathyroid tissue, chief cell adenoma, and parathyroid carcinoma, using immunohistochemistry and Western blot analysis. SNAP-23 and VAMP were evenly expressed in all studied parathyroid tissues using immunohistochemistry and/or Western blot analysis. SNAP-25 (and Syntaxin1) was not expressed in normal parathyroid tissue, but in approximately 20% of chief cell adenomas, and in ∼45% of parathyroid carcinoma samples. It is likely that the SNARE proteins SNAP-23 and VAMP play a role in the stimulus-secretion coupling and exocytosis of parathyroid hormone as these proteins were expressed in all of the parathyroid samples we studied. In particular, preferential expression of SNAP-23 rather than SNAP-25 provides an explanation of the high level of PTH secretion that occurs under conditions of low cytoplasmic free Ca2+ concentration (around 0.1μmol/l). SNAP-25 (and Syntaxin1) appears to be a tumour-specific protein(s) in parathyroid tissues since its expression was restricted to pathological tissues. [Copyright &y& Elsevier]

Abstract: SNAP-25 has a cysteine cluster located at its linker domain. In vivo, the cysteine residues in this cluster can be palmitoylated, and the hydrophobic palmitate molecules can target SNAP-25 to the presynaptic membrane. Here, we report that the SNAP-25a expressed in Escherichia coli is also an iron–sulfur protein binding an iron–sulfur cluster using the cysteine residues in its cysteine cluster. Therefore, SNAP-25a uses the same cysteine residues to bind two different prosthetic groups (iron–sulfur cluster and palmitate). Because the binding sites of these two prosthetic groups overlap, we suggest that these two modifications occur at different times, and probably at different places in the cell. [Copyright &y& Elsevier]

Botulinum neurotoxins (designated BoNT/A-BoNT/G) are bacterial enzymes that block neurotransmitter release by cleaving essential components of the vesicle fusion machinery. BoNT/A, which cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa), is extensively exploited in clinical medicine to treat neuromuscular pathologies, facial wrinkles, and various types of pain. It is widely assumed that BoNT/A remains at the synaptic terminal and its effects are confined to the injection site. Here we demonstrate that catalytically active BoNT/A is retrogradely transported by central neurons and motoneurons and is then transcytosed to afferent synapses, in which it cleaves SNAP-25. SNAP-25 cleavage by BoNT/A was observed in the contralateral hemisphere after unilateral BoNT/A delivery to the hippocampus. Appearance of cleaved SNAP-25 resulted in blockade of hippocampal activity in the untreated hemisphere. Injections of BoNT/A into the optic tectum led to the appearance of BoNT/A-truncated SNAP-25 in synaptic terminals within the retina. Cleaved SNAP-25 also appeared in the facial nucleus after injection of the toxin into rat whisker muscles. Experiments excluded passive spread of the toxin and demonstrated axonal migration and neuronal transcytosis of BoNT/A. These findings reveal a novel pathway of BoNT/A trafficking in neurons and have important implications for the clinical uses of this neurotoxin. [ABSTRACT FROM AUTHOR]

Activation of diacylglycerol (DAG) signaling pathways with phorbol esters dramatically enhances Ca2+-triggered exocytosis from both endocrine cells and neurons, however the relevant targets of DAG are controversial. A possible effector mechanism for this signaling pathway is phosphorylation of SNAP-25 (25 kDa synaptosome-associated protein) at Ser187 by PKC. Here, we investigated the role of Ser187 in the enhancement of exocytosis by the phorbol ester PMA (phorbol 12 -myristate 13-acetate). We used patch-clamp measurements of membrane capacitance together with photorelease of caged-Ca2+ and membrane depolarization to study exocytosis. Expression of the nonphosphorylatable S187C SNAP-25 mutant did not attenuate the enhancement of exocytosis by PMA in either bovine chromaffin cells or the INS-1 insulin-secreting cell line. To test the effects of Ser187 mutations under conditions in which the endogenous SNAP-25 is disabled, we expressed botulinum toxin serotype E to cleave SNAP-25 in INS-1 cells. Coexpression of a toxin-resistant mutant (TR), but not wild-type SNAP-25, was able to rescue PMA-modulated exocytosis. Coexpression of the toxin with the TR-S187C SNAP-25 mutant was able to completely block the enhancement of exocytosis by PMA in response to photoelevation of [Ca2+]i to low µM levels or to a depolarizing train. The phospho-mimetic S187E mutation enhanced the small, fast burst of exocytosis evoked by photelevation of Ca2+, but, like PMA, had smaller effects on exocytosis evoked by a depolarizing train. This work supports the hypothesis that phosphorylation of Ser187 of SNAP-25 by PKC is a key step in the enhancement of exocytosis by DAG. [ABSTRACT FROM AUTHOR]

A unifying principle of sensory system organization is feature extraction by modality-specific neuronal maps in which arrays of neurons show systematically varied response properties and receptive fields. Only beginning to be understood, however, are the mechanisms by which these graded systems are established. In the peripheral auditory system, we have shown previously that the intrinsic firing features of spiral ganglion neurons are influenced by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). We now show that is but a part of a coordinated package of neurotrophin actions that also includes effects on presynaptic and postsynaptic proteins, thus encompassing the input, transmission, and output functions of the spiral ganglion neurons. Using immunocytochemical methods, we determined that proteins targeted to opposite ends of the neuron were organized and regulated in a reciprocal manner. AMPA receptor subunits GluR2 and GluR3 were enriched in base neurons compared with their apex counterparts. This distribution pattern was enhanced by exposure to BDNF but reduced by NT-3. SNAP-25 and synaptophysin were distributed and regulated in the mirror image: enriched in the apex, enhanced by NT-3 and reduced by BDNF. Moreover, we used a novel coculture to identify potential endogenous sources of neurotrophins by showing that sensory receptors from different cochlear regions were capable of altering presynaptic and postsynaptic protein levels in these neurons. From these studies, we suggest that BDNF and NT-3, which are systematically distributed in complementary gradients, are responsible for orchestrating a comprehensive set of electrophysiological specializations along the frequency contour of the cochlea. [ABSTRACT FROM AUTHOR]

Frequent exposure of children to general anesthesia is common practice in modern medicine. Although previously unrecognized, recent in vitro and in vivo animal studies suggest that exposure to clinically relevant general anesthetics at the peak of brain development could be detrimental to immature mammalian neurons, as demonstrated by massive and widespread apoptotic neurodegeneration. The survival of the developing neurons presumably depends on proper and timely formation of synapses, for which synaptic proteins (e.g., synaptophysin, synaptobrevin, amphiphysin, synaptosomal-associated protein 25 [SNAP-25], and Ca2+/calmodulin-dependent protein kinase II [CaM kinase II]) are crucially important. Overinhibition of developing neurons impairs synaptic protein function and activity-induced synaptic plasticity, which could in turn result in permanent neuronal loss. To examine the effects of general anesthesia, the pharmacological agents known to cause extensive neuronal inhibition, on synaptic proteins, and neuronal survival at the peak of synaptogenesis, we exposed 7-day-old rat pups to general anesthesia (midazolam, 9 mg/kg of body weight, subcutaneously, followed by 6 h of nitrous oxide 75 vol% and isoflurane 0.75 vol%). We found that this general anesthesia causes permanent neuronal deletion in the most vulnerable brain regions—the cerebral cortex and the thalamus—while transiently modulating protein levels of synaptophysin, synaptobrevin, amphiphysin, SNAP-25, and CaM kinase II. [ABSTRACT FROM AUTHOR]

OBJECTIVE To monitor the presence and cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) by botulinum toxin type A (botox-A), in human detrusor muscle, as the effects of botox-A in the urinary bladder last significantly longer than when applied for disorders of striated muscles. PATIENTS AND METHODS Tissue samples were obtained from eight patients with end-stage neurogenic bladder at different times after injection with botox-A. The resected bladder domes were examined using biochemical and immunohistological techniques. RESULTS The presence of intact SNAP-25 in human bladder was detected, for the first time, in all samples by both Western blotting and immunofluorescence. By contrast, detection of a band potentially representing toxin-cleaved SNAP-25A required its enrichment by precipitation with a specific antibody. This putative product was present in four of six patients treated with botox-A 5 weeks to 11 months previously, but could not be detected in one patient 30 months after botox injection, and in an untreated control. Fluorescence microscopy showed no obvious effects of the toxin treatment on the presence and pattern of SNAP-25-positive neurones. CONCLUSIONS A limited amount of SNAP-25 appears to be cleaved in nerves that innervate the smooth detrusor muscle in most patients who had been injected with botox-A; its absolute identification was precluded by the sensitivity of the detection. This protein was detectable much longer after toxin treatment than published for rodent striated muscle, and thus could contribute to the clinically reported longer duration of the effectiveness of botox-A. [ABSTRACT FROM AUTHOR]

Abstract: Matrix metalloproteinase-7 (MMP-7) belongs to a family of zinc dependent endopeptidases that are expressed in a variety of tissues including the brain. MMPs are known to be potent mediators of pericellular proteolysis and likely mediators of dynamic remodelling of neuronal connections. While an association between proteases and the neuronal synapse is emerging, a full understanding of this relationship is lacking. Here, we show that MMP-7 alters the structure and function of presynaptic terminals without affecting neuronal survival. Bath application of recombinant MMP-7 to cultured rat neurons induced long-lasting inhibition of vesicular recycling as measured by synaptotagmin 1 antibody uptake assays and FM4-64 optical imaging. MMP-7 application resulted in reduced abundance of vesicular and active zone proteins locally within synaptic terminals although their general levels remained unaltered. Finally, chronic application of the protease resulted in synaptic atrophy, including smaller terminals and fewer synaptic vesicles, as determined by electron microscopy. Together these results suggest that MMP-7 is a potent modulator of synaptic vesicle recycling and synaptic ultrastructure and that elevated levels of the enzyme, as may occur with brain inflammation, may adversely influence neurotransmission. [Copyright &y& Elsevier]

Abstract: Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca2+ mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca2+ entry, the main mechanism for Ca2+ influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca2+ entry for full activation. Depletion of the intracellular Ca2+ stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca2+ concentration reached a critical level (around 1.5μM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca2+ mobilization in human platelets. [Copyright &y& Elsevier]

The SNAP receptor (SNARE) complex, consisting of synaptosome-associated protein of 25 kDa (SNAP-25), synaptobrevin-2, and syntaxin-1, is involved in synaptic vesicles exocytosis. In addition, SNAP-25 has been implicated in constitutive exocytosis processes required for neurite outgrowth. However, at least three isoforms of SNAP-25 have been reported from neurons: SNAP-23, which is also present in non-neuronal cells, and the two alternative splice variants SNAP-25a and SNAP-25b. Here, we studied the differential ability of these isoforms to support the functions previously broadly ascribed to "SNAP-25." We studied the rescue of snap-25 null neurons in culture with different SNAP-25 homologs. We find that deletion of SNAP-25 leads to strongly reduced neuron survival, and, in the few surviving cells, impaired arborization, reduced spontaneous release, and complete arrest of evoked release. Lentiviral expression of SNAP-25a, SNAP-25b, or SNAP-23 rescued neuronal survival, arborization, amplitude, and frequency of spontaneous events. Also evoked release was rescued by all isoforms, but synchronous release required SNAP-25a/b in both glutamatergic and GABAergic neurons. SNAP-23 supported asynchronous release only, reminiscent of synaptotagmin-1 null neurons. SNAP-25b was superior to SNAP-25a in vesicle priming, resembling the shift to larger releasable vesicle pools that accompanies synaptic maturation. These data demonstrate a differential ability of SNAP-25b, SNAP-25a, and SNAP-23 to support neuronal function. [ABSTRACT FROM AUTHOR]

Abstract: Type 2 diabetes is characterised by elevated blood glucose and fatty acid concentrations, and aberrant expression of exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Restoration of normoglycaemia is often accomplished through use of the thiazolidinedione drug rosiglitazone (RSG), although little is known of its actions on the pancreas. Here we report that high glucose resulted in 96.6±0.2% inhibition of secretagogue-stimulated insulin secretion and 44.9±6.2% reduction in β-cell insulin content. High glucose and lipid resulted in altered target-SNARE expression, syntaxin 1 becoming barely detectable whilst SNAP-25 was greatly up-regulated. RSG intervention further increased the expression of SNAP-25, but did not up-regulate syntaxin 1 expression. In summary, high glucose results in almost total attenuation of stimulated insulin secretion, partial depletion of β-cell insulin stores and dysregulation of SNARE protein expression. RSG up-regulates SNAP-25 expression, but crucially not syntaxin 1 and hence fails to enhance insulin secretion. [Copyright &y& Elsevier]

Abstract: The mechanisms underlying the effects of psychostimulants in attention deficit hyperactivity disorder (ADHD) are not well understood, but indirect evidence implicates D2 dopamine receptors. Here we dissect the components of dopaminergic neurotransmission in the hyperactive mouse mutant coloboma to identify pre- and postsynaptic elements essential for the effects of amphetamine in these mice. Amphetamine treatment reduced locomotor activity in coloboma mice, but induced a robust increase in dopamine overflow suggesting that abnormal regulation of dopamine efflux does not account for the behavioral effect. However, the D2-like dopamine receptor antagonists haloperidol and raclopride, but not the D1-like dopamine receptor antagonist SCH23390, blocked the amphetamine-induced reduction in locomotor activity in coloboma mice, providing direct evidence that D2-like dopamine receptors mediate the effect of amphetamine in these mice. With the precedent established that it is possible to directly antagonize this response, this strategy should prove useful for identifying novel therapeutics in ADHD. [Copyright &y& Elsevier]

Botulinum neurotoxins (BoNTs), proteases specific for the SNARE proteins, are used to study the molecular machinery supporting exocytosis and are used to treat human diseases characterized by cholinergic hyperactivity. The recent extension of the use of BoNTs to central nervous system (CNS) pathologies prompted the study of their traffic in central neurons. We used fluorescent BoNT/A and BoNT/E to study the penetration, the translocation and the catalytic action of these toxins in excitatory and inhibitory neurons. We show that BoNT/A and BoNT/E, besides preferentially inhibiting synaptic vesicle recycling at glutamatergic relative to Gamma-aminobutyric acid (GABA)-ergic neurons, are more efficient in impairing the release of excitatory than inhibitory neurotransmitter from brain synaptosomes. This differential effect does not result from a defective penetration of the toxin in line with the presence of the BoNT/A receptor, synaptic vesicle protein 2 (SV2), in both types of neurons. Interestingly, exogenous expression of SNAP-25 in GABAergic neurons confers sensitivity to BoNT/A. These results indicate that the expression of the toxin substrate, and not the toxin penetration, most likely accounts for the distinct effects of the two neurotoxins at the two types of terminals and support the use of BoNTs for the therapy of CNS diseases caused by the altered activity of selected neuronal populations. [ABSTRACT FROM AUTHOR]

Rabphilin is generally thought to be involved in the regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, and it has recently been hypothesized that the C2B domain of rabphilin promotes the docking of dense-core vesicles to the plasma membrane through simultaneous interaction with a vesicle protein, Rab3A/27A, and a plasma membrane protein, SNAP-25 (synaptosome-associated protein of 25 kDa). However, the physiological significance of the rabphilin–SNAP-25 interaction in the vesicle-docking step has never been elucidated. In this study we demonstrated by a mutation analysis that the polybasic sequence (587 KKAKHKTQIKKK 598) in the C2B domain of rabphilin is required for SNAP-25 binding, and that the Asp residues in the Ca2+-binding loop 3 (D628 and D630) of the C2B domain are not required. We also investigated the effect of Lys→Gln (KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in individual PC12 cells. A rabphilin(KQ) mutant that completely lacks SNAP-25-binding activity significantly decreased the number of plasma-membrane-docked vesicles and strongly inhibited high-KCl-induced dense-core vesicle exocytosis. These results indicate that the polybasic sequence in the C2B domain functions as an effector domain for SNAP-25 and controls the number of ‘releasable’ vesicles docked to the plasma membrane. [ABSTRACT FROM AUTHOR]

Abstract: Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn2+ binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 Å that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399–2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role. [Copyright &y& Elsevier]

Abstract: Attention deficit hyperactivity disorder (ADHD) is characterized by hyperactivity, inattention, and impulsivity. The coloboma mouse model of ADHD exhibits profound hyperactivity. To determine whether coloboma mice exhibit other signs of ADHD, we assessed latent inhibition as a test of attention, and impulsivity in a delayed reinforcement paradigm. Latent inhibition was present in control mice but was disrupted in coloboma mice. Coloboma mice also exhibited impaired performance on the delayed reinforcement task and were not able to wait as long as control mice to obtain the greater reinforcer. Because norepinephrine mediates hyperactivity in coloboma mice, we examined the role of norepinephrine in disrupted latent inhibition and impulsivity. Reduction of norepinephrine with DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride) restored latent inhibition but did not ameliorate impulsivity. In summary, coloboma mice exhibit hyperactivity, inattention as determined by latent inhibition, and impulsivity, and norepinephrine mediates hyperactivity and inattention but not impulsivity in these mice. [Copyright &y& Elsevier]

Abstract: Synapse loss in Alzheimer''s disease (AD) is poorly understood but evidence suggests it is a key pathological event. In order to precisely detect stable synaptic changes, we have developed methods for flow cytometry analysis of synaptosomes prepared from cryopreserved AD samples, and have previously shown that amyloid-β (Aβ) accumulates in surviving presynaptic terminals in AD cortex. In the present experiments we have examined amyloid-containing terminals in more detail, first dual labeling synaptosomes from AD cortex for Aβ and a series of markers, and then using quadrant analysis to compare amyloid-positive and amyloid-negative terminals. Amyloid-positive synaptosomes were larger in size than amyloid-negatives (p <0.007), and significant increases were observed in mean fluorescence for the lipid raft markers cholesterol (27%; p <0.0005) and GM1 ganglioside (24%; p <0.005). SNAP-25 immunofluorescence was increased by 31% (p <0.0001) in amyloid-bearing terminals, consistent with a sprouting response to amyloid accumulation. These results suggest that Aβ accumulation in synaptic terminals may underly dysfunction prior to or independent of extracellular amyloid deposition. [Copyright &y& Elsevier]

Abstract: Botulinum neurotoxin serotype A (BoNT/A), one of seven serotypes of botulinum neurotoxin, is taken up by neurons of the peripheral nervous system. Within the neurons it catalyzes cleavage of the synaptosomal-associated protein having a mass of 25kDa, SNAP-25, thereby blocking neurotransmission. BoNT/A has been shown to interact with SV2, as well as gangliosides that are often found in lipid rafts. Lipid rafts are microdomains that can be found on the outer leaflet of the plasma membrane and are enriched in cholesterol and glycosphingolipids. To determine whether lipid rafts are needed for BoNT/A activity, those associated with the plasma membranes of murine N2a neuroblastoma cells were disrupted using either methyl-β-cyclodextrin or filipin. Disruption of cholesterol-containing lipid rafts by either reagent did not prevent the action of BoNT/A on N2a cells, in fact activity was enhanced. While our results indicate that disruption of lipid rafts enhances BoNT/A activity, disruption of clathrin-dependent endocytosis appeared to be inhibitory. [Copyright &y& Elsevier]

Abstract: Chronic restraint stress has been associated with induction of morphological changes in the hippocampus. Postsynaptically, these changes include decreased length and branching of apical dendrites from CA3 pyramidal neurons, while presynaptically, depletion and clustering of synaptic vesicles have been observed. However, the molecular correlates of these changes remain poorly defined; while some studies have identified changes in the levels of some presynaptic proteins, none have assessed the coordinate expression of components of the membrane fusion complex, including synaptobrevin, syntaxin, and synaptosomal-associated protein 25 kDa, and their major regulatory molecules synaptotagmin, synaptophysin, and synapsin. Therefore, we undertook to assess the immunoreactivity of these proteins in hippocampal slices obtained from rats subjected to either acute (one 6 h session) or chronic (21 days at 6 h per day) of restraint stress. Specifically, we observed a significant increase in synaptobrevin immunoreactivity in the inner molecular layer of the dentate gyrus (54.2%; P=0.005), the stratum radiatum in the CA1 subfield (55.5%; P=0.007), and a region including the stratum lucidum and the proximal portion of the stratum radiatum in the CA3 subfield (52.7%; P=0.002); we also observed a trend toward increased synaptophysin levels in the stratum lucidum/radiatum of the CA3 subfield (8.0%; P=0.051) following chronic, but not acute, restraint stress. In that synaptobrevin has been associated with replenishment of the “readily-releasable” pool of synaptic vesicles and the efficiency of neurotransmitter release, the present results suggest that stress-induced changes in synaptobrevin may at least in part underlie the previously observed changes in synaptic and neuronal morphology. [Copyright &y& Elsevier]

Abstract: Synaptosomal-associated protein of 25kDa (SNAP-25), a member of the SNARE proteins essential for neurotransmitter release, is phosphorylated at Ser187 in PC12 cells and in the rat brain in a protein kinase C-dependent manner. It remains unclear how the phosphorylation of SNAP-25 is regulated during development and by neuronal activity. We studied the mode of SNAP-25 phosphorylation at Ser187 in the rat brain using an anti-phosphorylated SNAP-25 antibody. Both the expression and phosphorylation of SNAP-25 increased remarkably during the early postnatal period, but their onsets were quite different. SNAP-25 expression was detected as early as embryonic Day 18, whereas the phosphorylation of SNAP-25 could not be detected until postnatal Day 4. A delay in the onset of phosphorylation was also observed in cultured rat hippocampal neurons. The phosphorylation of SNAP-25 was regulated in a neuronal activity-dependent manner and, in the rat hippocampus, decreased by introducing seizures with kainic acid. These results clearly indicated that the phosphorylation of SNAP-25 at Ser187 is regulated in development- and neuronal activity-dependent manners, and is likely to play important roles in higher brain functions. [Copyright &y& Elsevier]