SNAP-25 Protein Expression Research Articles

Excessive Iron Induces Oxidative Stress Promoting Cellular Perturbations and Insulin Secretory Dysfunction in MIN6 Beta Cells.

Exposure to high levels of glucose and iron are co-related to reactive oxygen species (ROS) generation and dysregulation of insulin synthesis and secretion, although the precise mechanisms are not well clarified. The focus of this study was to examine the consequences of exposure to high iron levels on MIN6 β-cells. MIN6 pseudoislets were exposed to 20 µM (control) or 100 µM (high) iron at predefined glucose levels (5.5 mM and 11 mM) at various time points (3, 24, 48, and 72 h). Total iron content was estimated by a colourimetric FerroZine™ assay in presence or absence of transferrin-bound iron. Cell viability was assessed by a resazurin dye-based assay, and ROS-mediated cellular oxidative stress was assessed by estimating malondialdehyde levels. β-cell iron absorption was determined by a ferritin immunoassay. Cellular insulin release and content was measured by an insulin immunoassay. Expression of SNAP-25, a key protein in the core SNARE complex that modulates vesicle exocytosis, was measured by immunoblotting. Our results demonstrate that exposure to high iron levels resulted in a 15-fold (48 h) and 4-fold (72 h) increase in cellular iron accumulation. These observations were consistent with data from oxidative stress analysis which demonstrated 2.7-fold higher levels of lipid peroxidation. Furthermore, exposure to supraphysiological (11 mM) levels of glucose and high iron (100 µM) at 72 h exerted the most detrimental effect on the MIN6 β-cell viability. The effect of high iron exposure on total cellular iron content was identical in the presence or absence of transferrin. High iron exposure (100 µM) resulted in a decrease of MIN6 insulin secretion (64% reduction) as well as cellular insulin content (10% reduction). Finally, a significant reduction in MIN6 β-cell SNAP-25 protein expression was evident at 48 h upon exposure to 100 µM iron. Our data suggest that exposure to high iron and glucose concentrations results in cellular oxidative damage and may initiate insulin secretory dysfunction in pancreatic β-cells by modulation of the exocytotic machinery.

Overexpression of miR-1 in the heart attenuates hippocampal synaptic vesicle exocytosis by the posttranscriptional regulation of SNAP-25 through the transportation of exosomes

BackgroundThe link between cardiac diseases and cognitive deterioration has been accepted from the concept of “cardiogenic dementia”, which was proposed in the late 1970s. However, the molecular mechanism is unclarified.MethodsThe two animal models used in this study were cardiac-specific overexpression of microRNA-1-2 transgenic (Tg) mice and a myocardial infarction mouse model generated by left coronary artery ligation (LCA). First, we observed the microRNA-1 (miR-1) level and synaptic vesicles (SV) distribution in the hippocampus using in situ hybridization and transmission electron microscopy (TEM) and evaluated the expression of vesicle exocytosis related proteins by western blotting. Second, we used dual luciferase reporter assay as well as antagonist and miRNA-masking techniques to identify the posttranscriptional regulatory effect of miR-1 on the Snap25 gene. Third, FM1–43 staining was performed to investigate the effect of miR-1 on synaptic vesicle exocytosis. Lastly, we used GW4869 to inhibit the biogenesis and secretion of exosomes to determine the transportation effect of exosomes for miR-1 from the heart to the brain.ResultsCompared with the levels in age-matched WT mice, miR-1 levels were increased in both the hearts and hippocampi of Tg mice, accompanied by the redistribution of SVs and the reduction in SV exocytosis-related protein SNAP-25 expression. In vitro studies showed that SNAP-25 protein expression was down- or upregulated by miR-1 overexpression or inhibition, respectively, however, unchanged by miRNA-masking the 3’UTR of the Snap25 gene. SV exocytosis was inhibited by miR-1 overexpression, which could be prevented by co-transfection with an anti-miR-1 oligonucleotide fragment (AMO-1). The knockdown of miR-1 by hippocampal stereotaxic injection of AMO-1 carried by a lentivirus vector (lenti-pre-AMO-1) led to the upregulation of SNAP-25 expression and prevented SV concentration in the synapses in the hippocampi of Tg mice. The application of GW4869 significantly reversed the increased miR-1 level in the blood and hippocampi as well as reduced the SNAP-25 protein levels in the hippocampi of both Tg and LCA mice.ConclusionThe overexpression of miR-1 in the heart attenuated SV exocytosis in the hippocampus by posttranscriptionally regulating SNAP-25 through the transportation of exosomes. This study contributes to the understanding of the relationship between cardiovascular disease and brain dysfunction.

A possible postsynaptic role for SNAP-25 in hippocampal synapses.

The SNARE protein SNAP-25 is well documented as regulator of presynaptic vesicle exocytosis. Increasing evidence suggests roles for SNARE proteins in postsynaptic trafficking of glutamate receptors as a basic mechanism in synaptic plasticity. Despite these indications, detailed quantitative subsynaptic localization studies of SNAP-25 have never been performed. Here, we provide novel electron microscopic data of SNAP-25 localization in postsynaptic spines. In addition to its expected presynaptic localization, we show that the protein is also present in the postsynaptic density (PSD), the postsynaptic lateral membrane and on small vesicles in the postsynaptic cytoplasm. We further investigated possible changes in synaptic SNAP-25 protein expression after hippocampal long-term potentiation (LTP). Quantitative analysis of immunogold-labeled electron microscopy sections did not show statistically significant changes of SNAP-25 gold particle densities 1h after LTP induction, indicating that local trafficking of SNAP-25 does not play a role in the early phases of LTP. However, the strong expression of SNAP-25 in postsynaptic plasma membranes suggests a function of the protein in postsynaptic vesicle exocytosis and a possible role in hippocampal synaptic plasticity.

Impact of Botox-A SNAP-25 protein expression and the mechanism of inhibitory neurotransmitter imbalance in chronic sciatic nerve pain rat model.

The Botox-A impact on the expression of SNAP-25 protein in rat chronic sciatic nerve pain model was assessed and the mechanism of inhibitory neurotransmitter imbalance was studied. A chronic constriction injury (CCI) model consisted of 30 healthy male rats. The rats were randomly divided into the sham-operated group, CCI group and BoNT/A intervention group, and during 1, 7 and 14 days we conducted mechanical withdrawal threshold (MWT) test and thermal withdrawal latency (TWL) test before and after operation. After 14 days, the animals were sacrificed. SNAP-25 protein expression level, mRNA subunit NR2B within excitatory neurotransmitter glutamate GLT and protein expression level, as well as GAT mRNA, the inhibitory GABA neurotransmitter transporter and protein expression level were studied by RT-polymerase chain reaction and western blot analysis. The difference between MWT and TWL at each point in time before and after operation showed no statistical significance (P>0.05) in the sham-operated group. For the CCI group at each time point, MWT and TWL were obviously lower than the sham-operated group and the difference was statistically significant (P<0.05) while the internal difference at each time point showed no statistical significance (P>0.05). The expression level of protein of SNAP-25 and NR2B mRNA in the CCI group was clearly higher than sham-operated group. Additionally, the expression level of GAT-1 mRNA and protein in CCI group was apparently lower than the sham-operated group. In conclusion, Botox-A helped reduce SNAP-25 within rat chronic sciatic nerve pain model thereby relieving pain.

Reduced SNAP‐25 alters short‐term plasticity at developing glutamatergic synapses

SNAP-25 is a key component of the synaptic-vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP-25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP-25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP-25 levels at 13-14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage-gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13-14 DIV neurons with reduced SNAP-25 expression show paired-pulse depression as opposed to paired-pulse facilitation occurring in their wild-type counterparts. This phenotype disappears with synapse maturation. As alterations in short-term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP-25.

907 EFFECT OF INTRAVESICAL BOTULINUM TOXIN A DELIVERY (USING DMSO) IN RAT OVERACTIVE BLADDER MODEL

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Kidney & Bladder1 Apr 2012907 EFFECT OF INTRAVESICAL BOTULINUM TOXIN A DELIVERY (USING DMSO) IN RAT OVERACTIVE BLADDER MODEL Shogo Shimizu, Marcia Wheeler, Motoaki Saito, Robert Weiss, and Adam Hittelman Shogo ShimizuShogo Shimizu New Haven, CT More articles by this author , Marcia WheelerMarcia Wheeler New Haven, CT More articles by this author , Motoaki SaitoMotoaki Saito Yonago, Japan More articles by this author , Robert WeissRobert Weiss New Haven, CT More articles by this author , and Adam HittelmanAdam Hittelman New Haven, CT More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1003AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cystoscopic guided bladder injection of Botulinum Toxin A (BoNT) is becoming an important treatment modality for neurogenic and overactive bladder. Intravesical instillation of BoNT may provide high local drug concentrations with a lower risk of side effects as compared to intramuscular injection into the detrusor. However, BoNT does not easily pass through the urothelial barrier to reach the smooth muscle. DMSO has been shown to enhance bladder absorption. We tested the effects of BoNT instillation into control and an overactive rat bladder model using DMSO as a vehicle. Bladder overactivity was induced by intravesical instillation of acetic acid (AA). METHODS Bladders of 8 weeks old Sprague-Dawley female rats were initially intravesically instilled (2 hr) with 750 ul of the following: Saline; 25% DMSO; 10U BoNT in saline: or 10U BoNT in 25% DMSO. One day prior to sacrifice (6 and 29 days after the initial intravesical instillation), bladders were instilled with saline or with 0.25% AA for 1 hour. Bladders were harvested 24 hours after the second instillation (7 and 30 days). Histology, SNAP-25 and calcitonin gene-related peptide (CGRP) expression were measured by immunohistochemistry (IHC), western blot, and qPCR, respectively. RESULTS Intravesical instillation of BoNT-DMSO, but not BoNT alone, into normal rat bladder decreased SNAP25 protein expression at 7 days (57±3.0% SEM). This effect was not preserved at 30 days. CGRP mRNA expression was increased 4 fold, 7 days after the drug instillation as compared to saline controls. Rats treated with AA had increased bladder inflammation, as demonstrated by a large increase in the number of neutrophils, edema, and thickening of bladder mucosa on H&E slides. BoNT-DMSO decreased inflammatory reaction induced by AA in rat model at 7 days, but not at 30 days. Moreover, BoNT-DMSO treatment decreased SNAP25 immunoreactivity and increased CGRP immunoreactivity by IHC at 7days in AA overactive bladder model, as compared to control AA treated rats. This effect was not observed at 30 days post treatment. CONCLUSIONS Intravesical instillation of BoNT-DMSO reduces inflammation to a greater extent than BoNT alone at 7 days. We speculate that BoNT-DMSO modulates the inflammatory response by inducing CGRP expression in a rat model of overactive bladder. These results also confirm the benefit of DMSO as a delivery vehicle to enhance BoNT absorption. Our data further support the role of BoNT in the management of overactive bladder. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetailsCited byTyagi P, Kashyap M, Yoshimura N, Chancellor M and Chermansky C (2018) Past, Present and Future of Chemodenervation with Botulinum Toxin in the Treatment of Overactive BladderJournal of Urology, VOL. 197, NO. 4, (982-990), Online publication date: 1-Apr-2017. Volume 187Issue 4SApril 2012Page: e370 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shogo Shimizu New Haven, CT More articles by this author Marcia Wheeler New Haven, CT More articles by this author Motoaki Saito Yonago, Japan More articles by this author Robert Weiss New Haven, CT More articles by this author Adam Hittelman New Haven, CT More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Role for Reelin in Neurotransmitter Release

The extracellular matrix molecule Reelin is known to control neuronal migration during development. Recent evidence suggests that it also plays a role in the maturation of postsynaptic dendrites and spines as well as in synaptic plasticity. Here, we aimed to address the question whether Reelin plays a role in presynaptic structural organization and function. Quantitative electron microscopic analysis of the number of presynaptic boutons in the stratum radiatum of hippocampal region CA1 did not reveal differences between wild-type animals and Reelin-deficient reeler mutant mice. However, additional detailed analysis showed that the number of presynaptic vesicles was significantly increased in CA1 synapses of reeler mutants. To test the hypothesis that vesicle fusion is altered in reeler, we studied proteins known to control transmitter release. SNAP25, a protein of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, was found to be significantly reduced in reeler mutants, whereas other SNARE complex proteins remained unaltered. Addition of recombinant Reelin to organotypic slice cultures of reeler hippocampi substantially rescued not only SNAP25 protein expression levels but also the number of vesicles per bouton area indicating a role for Reelin in presynaptic functions. Next, we analyzed paired-pulse facilitation, a presynaptic mechanism associated with transmitter release, and observed a significant decrease at CA1 synapses of reeler mutants when compared with wild-type animals. Together, these novel findings suggest a role for Reelin in modulating presynaptic release mechanisms.

Immunohistochemical Localization of SNARE Proteins in Dental Pulp and Periodontal Ligament of the Rat Incisor

Distribution of three soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, syntaxin-1, synaptosomal-associated protein of 25 kDa (SNAP-25), and vesicle-associated membrane protein-2 (VAMP-2), was examined in dental pulp and periodontal ligament of the rat incisor. In the trigeminal ganglion, syntaxin-1 and SNAP-25 immunoreactivity was predominately detected in medium- to large-sized neurons. Most syntaxin-1 immunoreactive neurons expressed SNAP-25. In contrast, VAMP-2 was localized in small- to medium-sized neurons and in slender-shaped cells surrounding SNAP-25-immunopositive neurons. When the inferior alveolar nerve, one of the mandibular nerve branches innervating the dental pulp and periodontal ligament, was ligated, SNARE proteins accumulated at the site proximal to the ligation. In the incisor dental pulp, all nerve fibers displayed immunoreactivity for syntaxin-1, SNAP-25, and VAMP-2. In the periodontal ligament of the incisor, almost all nerve fibers displayed both syntaxin-1 and SNAP-25 immunoreactivity, but lacked VAMP-2 immunoreactivity. SNAP-25 protein expression was localized around the vesicle membranes at the axon terminal of the periodontal mechanoreceptors. These present data suggest that these three SNARE proteins are synthesized at the trigeminal ganglion, transported centrally and peripherally, and expressed in sensory endings where apparent synapses are not present. Because those proteins participate in docking and exocytosis of synapse vesicles in the central nervous system, they might also contribute to vesicle exocytosis at receptive fields where apparent synapses are not present.

Immunohistochemical Expression of SNAP-25 Protein in Adenomas of the Human Pituitary

SNAP-25, a synaptosome-associated exocytosis protein of 25 kd mw, plays an important role in the secretory activity of several endocrine cells. In the present study, we investigated surgically removed pituitary adenomas including 40 prolactin (PRL), 31 growth hormone, 5 adrenocorticotropic hormone, 5 thyroid-stimulating hormone, 14 follicle-stimulating hormone/luteinizing hormone/alpha-subunit-producing tumors, and 5 null cell adenomas. Among the 40 patients with PRL-producing pituitary adenoma, 16 had been preoperatively treated with the dopamine agonist bromocriptine. Similarly, of the 31 patients with GH-producing pituitary adenomas, 15 had been treated with the long-acting somatostatin analog, octreotide. All tumors were subjected to transsphenoidal surgery, formalin-fixed, routinely processed, and paraffin-embedded. Sections of 4 to 6-microm thickness were stained with hematoxylin and eosin and the periodic acid-Schiff as well as the Gordon-Sweet silver methods. Immunostaining for SNAP-25 (streptavidin-biotin peroxidase complex method) showed that 10 PRL-producing adenomas were strongly immunoreactive. Immunopositivity was mainly cell membrane in distribution but several cells showed mild cytoplasmic staining. Nuclei were immunonegative. Preoperative bromocriptine treatment markedly decreased SNAP-25 immunopositivity. Among GH-producing adenomas, SNAP-25 was seen in 5 cases; reactivity being mild-to-moderate, membrane-bound, and cytoplasmic. Octreotide caused no significant reduction in immunopositivity. Other adenoma types were virtually immunonegative. Six autopsy-derived human pituitaries and 4 surgically obtained nontumorous pituitaries were also immunonegative for SNAP-25. It is conceivable that SNAP-25 plays an important role in PRL release and is involved in the bromocriptine-induced suppression of PRL secretion from PRL-producing adenoma cells.

Multiple calcium pathways induce the expression of SNAP-25 protein in chromaffin cells.

Incubation of bovine adrenal chromaffin cells in high K+ (38 mM) during 24-48 h enhanced 2.5 to five times the expression of SNAP-25 protein and mRNA, respectively. This increase was reduced 86% by furnidipine (an L-type Ca2+ channel blocker) but was unaffected by either omega-conotoxin GVIA (an N-type Ca2+ channel blocker) or -agatoxin IVA (a P/Q-type Ca2+ channel blocker). Combined blockade of N and P/Q channels with omega-conotoxin MVIIC did, however, block by 76% the protein expression. The inhibitory effects of fumidipine were partially reversed when the external Ca2+ concentration was raised from 1.6 to 5 mM. These findings, together with the fact that nicotinic receptor activation or Ca2+ release from internal stores also enhanced SNAP-25 protein expression, suggest that an increment of cytosolic Ca2+ concentration ([Ca2+]), rather than its source or Ca2+ entry pathway, is the critical signal to induce the protein expression. The greater coupling between L-type Ca2+ channels and protein expression might be due to two facts: (a) L channels contributed 50% to the global [Ca2+]i rise induced by 38 mM K+ in indo-1-loaded chromaffin cells and (b) L channels undergo less inactivation than N or P/Q channels on sustained stimulation of these cells.