SNAP-25 mRNA Research Articles

SNAP-25, but not SNAP-23, is essential for photoreceptor development, survival, and function in mice

SNARE-mediated vesicular transport is thought to play roles in photoreceptor glutamate exocytosis and photopigment delivery. However, the functions of Synaptosomal-associated protein (SNAP) isoforms in photoreceptors are unknown. Here, we revisit the expression of SNAP-23 and SNAP-25 and generate photoreceptor-specific knockout mice to investigate their roles. Although we find that SNAP-23 shows weak mRNA expression in photoreceptors, SNAP-23 removal does not affect retinal morphology or vision. SNAP-25 mRNA is developmentally regulated and undergoes mRNA trafficking to photoreceptor inner segments at postnatal day 9 (P9). SNAP-25 knockout photoreceptors develop normally until P9 but degenerate by P14 resulting in severe retinal thinning. Photoreceptor loss in SNAP-25 knockout mice is associated with abolished electroretinograms and vision loss. We find mistrafficked photopigments, enlarged synaptic vesicles, and abnormal synaptic ribbons which potentially underlie photoreceptor degeneration. Our results conclude that SNAP-25, but not SNAP-23, mediates photopigment delivery and synaptic functioning required for photoreceptor development, survival, and function.

MiR-23a-3p and miR-181a-5p modulate SNAP-25 expression.

SNAP-25 protein is a key protein of the SNARE complex that is involved in synaptic vesicles fusion with plasma membranes and neurotransmitter release, playing a fundamental role in neural plasticity. Recently the concentration of three specific miRNAs-miR-27b-3p, miR-181a-5p and miR-23a-3p -was found to be associated with a specific SNAP-25 polymorphism (rs363050). in silico analysis showed that all the three miRNAs target SNAP-25, but the effect of the interaction between these miRNAs and the 3'UTR of SNAP-25 mRNA is currently unknown. For this reason, we verified in vitro whether miR-27b-3p, miR-181a-5p and miR-23a-3p modulate SNAP-25 gene and protein expression. Initial experiments using miRNAs-co-transfected Vero cells and SNAP-25 3'UTR luciferase reporter plasmids showed that miR-181a-5p (p≤0.01) and miR-23a-3p (p<0.05), but not miR-27b-3p, modulate the luciferase signal, indicating that these two miRNAs bind the SNAP-25 3'UTR. Results obtained using human oligodendroglial cell line (MO3.13) transfected with miR-181a-5p or miR-27b-3p confirmed that miR-181a-5p and miR-23a-3p regulate SNAP-25 gene and protein expression. Interestingly, the two miRNAs modulate in an opposite way SNAP-25, as miR-181a-5p significantly increases (p<0.0005), whereas miR-23a-3p decreases (p<0.0005) its expression. These results for the first time describe the ability of miR-181a-5p and miR-23a-3p to modulate SNAP-25 expression, suggesting their possible use as biomarkers or as therapeutical targets for diseases in which SNAP-25 expression is altered.

Caspase-2 mRNA levels are not elevated in mild cognitive impairment, Alzheimer's disease, Huntington's disease, or Lewy Body dementia.

Caspase-2 is a member of the caspase family that exhibits both apoptotic and non-apoptotic properties, and has been shown to mediate synaptic deficits in models of several neurological conditions, including Alzheimer's disease (AD), Huntington's disease (HD), and Lewy Body dementia (LBD). Our lab previously reported that caspase-2 protein levels are elevated in these diseases, leading us to hypothesize that elevated caspase-2 protein levels are due to increased transcription of caspase-2 mRNA. There are two major isoforms of caspase-2 mRNA, caspase-2L and caspase-2S. We tested our hypothesis by measuring the levels of these mRNA isoforms normalized to levels of RPL13 mRNA, a reference gene that showed no disease-associated changes. Here, we report no increases in caspase-2L mRNA levels in any of the three diseases studied, AD (with mild cognitive impairment (MCI)), HD and LBD, disproving our hypothesis. Caspase-2S mRNA showed a non-significant downward trend in AD. We also analyzed expression levels of SNAP25 and βIII-tubulin mRNA. SNAP25 mRNA was significantly lower in AD and there were downward trends in MCI, LBD, and HD. βIII-tubulin mRNA expression remained unchanged between disease groups and controls. These findings indicate that factors besides transcriptional regulation cause increases in caspase-2 protein levels. The reduction of SNAP25 mRNA expression suggests that presynaptic dysfunction contributes to cognitive deficits in neurodegeneration.

Serum miRNAs Expression and SNAP-25 Genotype in Alzheimer's Disease.

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by binding their 3′ untranslated region (3′UTR) region; these molecules play a fundamental role in several pathologies, including Alzheimer’s disease (AD). Synaptosomal-associated protein of 25 kDa (SNAP-25) is a vesicular protein of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in neural plasticity and in the exocytosis of neurotransmitters, processes that are altered in AD. Recent results showed that a reduction of SNAP-25 is associated with dementia, and that the rs363050 SNAP-25 polymorphism correlates with cognitive decline and brain atrophy, as well as with the outcome of multistructured rehabilitation in AD patients. We verified the presence of possible correlations between the serum concentration of miRNAs that bind the SNAP-25 3′UTR region and AD. Six different microRNAs (miR-181a-5p, miR-361-3p, miR-23a-3p, miR-15b-3p, 130a-3p and miR-27b-3p) that bind the SNAP-25 3′UTR region were measured by qPCR in serum of AD patients (n = 22), mild cognitive impairment (MCI) subjects (n = 22) and age- and sex-matched controls (n = 22); analysis of results was done stratified for the rs363050 SNAP-25 genotype. Results showed that miR-27b-3p, miR-23a-3p and miR181a-5p serum concentration was significantly reduced in rs363050 SNAP-25 GG homozygous AD patients. Notably, concentration of these miRNAs was comparable in rs363050 AA homozygous AD patients, MCI and healthy controls (HCs). Data herein suggest that miRNAs that bind the SNAP-25 3′UTR region interact with SNAP-25 polymorphisms to influence the neural plasticity typical of AD brains, possibly as a consequence of modulatory activity on SNAP-25 mRNA and/or protein.

MicroRNA-210-5p Contributes to Cognitive Impairment in Early Vascular Dementia Rat Model Through Targeting Snap25

Vascular dementia (VD) is the most common form of dementia in elderly people. However, little is understood about the role of microRNAs (miRNAs) involved in cognitive impairment in early VD. Here, a VD model induced by chronic cerebral ischemia and fetal bovine serum (FBS)-free cell model that detects synapse formation was established to investigate the function of miRNAs in early VD. The microarray analysis and real-time reverse transcription polymerase chain reaction (RT-PCR) showed that miR-210-5p increased significantly in the hippocampus of rats with 4 weeks of ischemia. The VD model rats also displayed significant cognitive deficits and synaptic loss. The overexpression of miR-210-5p decreased the synaptic number in primary hippocampal neurons, whereas specific suppression of miR-210-5p resulted in the formation of more synapses. Additionally, intracerebroventricular (ICV) injection of miR-210-5p agomir to VD rats aggravated phenotypes of cognitive impairment and synaptic loss. These VD-induced phenotypes were effectively attenuated by miR-210-5p antagomir. Moreover, bioinformatic prediction revealed that synaptosomal-associated protein of 25 KDa (Snap25) mRNA is targeted by miR-210-5p. The miR-210-5p decreased the luciferase activities of 3’ untranslated region (3’UTR) of Snap25 mRNA. Mutation of predicted miR-210-5p binding sites in the 3’ UTR of Snap25 mRNA abolished the miR-210-5p-induced decrease in luciferase activity. Western blot and immunofluorescence staining confirmed that miR-210-5p targets Snap25. Finally, RT-quantitative PCR (qPCR) and immunofluorescence staining detected that miR-210-5p agomir downregulated Snap25 expression in the cornu ammonis1 (CA1) region of hippocampi in VD rats, whereas miR-210-5p antagomir upregulated Snap25 expression. Altogether, miR-210-5p contributes to cognitive impairment in chronic ischemia-induced VD model through the regulation of Snap25 expression, which potentially provides an opportunity to develop a new therapeutic strategy for VD.

A Study and Review of Effects of Botulinum Toxins on Mast Cell Dependent and Independent Pruritus.

Pruriceptive itch originates following activation of peripheral sensory nerve terminals when pruritogens come in contact with the skin. The ability of botulinum neurotoxins (BoNTs) to attenuate transmitter release from afferent terminals provides a rationale for studying its effect on pruritus. This study investigated the effects of BoNT/A1 and BoNT/B1 on mast cell dependent (Compound 48/80:48/80) and independent (Chloroquine:CQ) scratching. C57Bl/6 male mice received intradermal injection of 1.5 U of BoNT/A1, BoNT/B1 or saline 2, 7, 14 and 21 days prior to ipsilateral 48/80 or CQ at the nape of the neck. Ipsilateral hind paw scratching was determined using an automated recording device. The effect of BoNTs on 48/80 mediated mast cell degranulation was analyzed in human and murine mast cells and the presence of SNAREs was determined using qPCR, immunostaining and Western blot. Pre-treatment with BoNT/A1 and BoNT/B1 reduced 48/80 and CQ induced scratching behavior starting on day 2 with reversal by day 21. Both serotypes inhibited 48/80 induced mast cell degranulation. qPCR and immunostaining detected SNAP-25 mRNA and protein, respectively, in mast cells, however, Western blots did not. This study demonstrates the long-lasting anti-pruritic effects of two BoNT serotypes, in a murine pruritus model using two different mechanistically driven pruritogens. These data also indicate that BoNTs may have a direct effect upon mast cell degranulation.

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.

MiR-124a expression contributes to the monophasic pattern of insulin secretion in islets from pregnant rats submitted to a low-protein diet

To evaluate the role of miR-124a in the regulation of genes involved in insulin exocytosis and its effects on the kinetics of insulin secretion in pancreatic islets from pregnant rats submitted to a low-protein diet. Adult control non-pregnant (CNP) and control pregnant (CP) rats were fed a normal protein diet (17%), whereas low-protein non-pregnant (LPNP) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) from days 1 to 15 of pregnancy. Kinetics of the glucose-induced insulin release and measurement of [Ca2+]i in pancreatic islets were assessed by standard protocols. The miR-124a expression and gene transcriptions from pancreatic islets were determined by real-time polymerase chain reaction. In islets from LPP rats, the first phase of insulin release was abrogated. The AUC [Ca2+]i from the LPP group was lower compared with the other groups. miR-124a expression was reduced by a low-protein diet. SNAP-25 mRNA, protein expression, and Rab3A protein content were lower in the LPP rats than in CP rats. Syntaxin 1A and Kir6.2 mRNA levels were decreased in islets from low-protein rats compared with control rats, whereas their protein content was reduced in islets from pregnant rats. Loss of biphasic insulin secretion in islets from LPP rats appears to have resulted from reduced [Ca2+]i due, at least in part, to Kir6.2 underexpression and from the changes in exocytotic elements that are influenced either directly or indirectly by miR-124a.

Select putative neurodevelopmental toxins modify SNAP-25 expression in primary cultures of rat cerebellar granule cells.

A presynaptic protein SNAP-25 belonging to SNARE complex which is instrumental in intracellular vesicular trafficking and exocytosis, has been implicated in hyperactivity and cognitive abilities in some neuropsychiatric disorders. The unclear etiology of the behavior disrupting neurodevelopmental disabilities in addition to genetic causes most likely involves environmental factors. The aim of this in vitro study was to test if various suspected developmental neurotoxins can alter SNAP-25 mRNA and protein expression in neurons. Real-time PCR and Western blotting analyses were used to assess SNAP-25 mRNA and protein levels in primary cultures of rat cerebellar granule cells (CGCs). The test substances: tetrabromobisphenol-A (TBBPA), thimerosal (TH), silver nanoparticles (NAg), valproic acid (VPA) and thalidomide (THAL), were administered to CGC cultures at subtoxic concentrations for 24h. The results demonstrated that SNAP-25 mRNA levels were increased by 49 and 66% by TBBPA and THAL, respectively, whereas VPA and NAg reduced these levels to 48 and 64% of the control, respectively. The SNAP-25 protein content in CGCs was increased by 79% by TBBPA, 25% by THAL and 21% by NAg; VPA and TH reduced these levels to 73 and 69% of the control, respectively. The variety of changes in SNAP-25 expression on mRNA and protein level suggests the diversity of the mechanism of action of the test substances. This initial study provided no data on concentration-effect relations and on functional changes in CGCs. However it is the first to demonstrate the effect of different compounds that are suspected of causing neurodevelopmental disabilities on SNAP-25 expression. These results suggest that this protein may be a common target for not only inherited but also environmental modifications linked to behavioral deficits in neurodevelopmental disabilities.

The Inhibitory Effect of Platelet-Rich Plasma on Botulinum Toxin Type-A: An Experimental Study in Rabbits.

Combination treatments of botulinum toxin type-A and other rejuvenation agents or instruments are gradually becoming more popular. After observing a high incidence of therapy failure following simultaneous applications of botulinum toxin type-A and platelet-rich plasma mesotherapy, we aimed to investigate whether PRP has an inhibitory effect on botulinum toxin type-A. Twenty-four New Zealand white rabbits were divided into 4 groups, and the anterior auricular muscle and overlying skin were used for injections. Groups I and II both received onabotulinumtoxinA intramuscular injections. In addition, autologous platelet-rich plasma mesotherapy was performed in Group I while Group II received saline mesotherapy. Group III was designed as the in vitro mixture group in which onabotulinumtoxinA and platelet-rich plasma were mixed and then administered intramuscularly. Group IV received saline within the mixture instead of platelet-rich plasma. The contralateral ears of all the rabbits served as control and were only treated with onabotulinumtoxinA. Visual evaluation of ear positions and electroneuromyographic studies were done prior to all procedures and at day 14. Anterior auricular muscles were harvested at day 14 and were evaluated with quantitative real-time PCR. Visual and electroneuromyographic studies revealed less onabotulinumtoxinA activity in Groups I and III. When platelet-rich plasma was administered through skin mesotherapy, onabotulinumtoxinA activity failure was more severe in comparison with direct contact. No significant difference in SNAP-25 mRNA expression through quantitative real-time PCR was observed between groups. Although we could not explain the exact mechanism underlying this interaction, platelet-rich plasma applications result in less onabotulinumtoxinA muscle paralysis activity.

Expression and regulation of SNAP-25 and synaptotagmin VII in developing mouse ovarian follicles via the FSH receptor

Soluble-NSF attachment protein receptor (SNARE) proteins play a role in vesicle fusion, exocytosis, and intracellular trafficking in neuronal cells as well as in fertilization and embryogenesis. We investigated the expression patterns of two SNARE proteins, SNAP-25 and synaptotagmin VII (SytVII), and their regulation by pregnant mare serum gonadotropin (PMSG) during mouse ovarian follicular development. Ovaries were obtained at 0, 12, 24, 36, and 48h post-PMSG injection of immature mice. SNAP-25 and SytVII mRNA expression levels increased gradually in a time-dependant manner. However, protein levels revealed different patterns of expression, suggesting different translational regulation following PMSG stimulation. SNAP-25 and SytVII expression was closely associated with thickening of the granulosa cell (GC) layer and follicle morphological changes from a flattened to a cuboidal shape. To explore follicle stimulating hormone receptor (FSHR)-mediated regulation of their expression, GCs from preantral follicles were cultured to examine the effects of FSHR siRNA knockdown. FSHR siRNA abolished upregulation of the SNAREs in both PMSG and FSH-stimulated GCs. This abolished gene expression was rescued by adding dibutyryl cyclic AMP to the cultures. These results suggest that SNAP-25 and SytVII expression is regulated via the FSHR-cAMP pathway during follicular development.

Lead impairs ability of learning and memory and affects expression of synaptosomal-associated protein-25 in hippocampus of offspring

To find the effects of lead taken by pregnant mice on learning and memory and the expression of synaptosomal-associated protein (SNAP)-25 mRNA and protein, in order to reveal the mechanism of neurotoxicity induced by lead. Lead exposure was conducted through freely drinking the corresponding lead acetate solutions with dosages of 0.3, 1.0, 3.0 g/L respectively. Each group was composed of 10 mice. 7, 14 and 21 days after their birth. The lead contents in blood and hippocampus of the offspring were determined. At the 21st day the expression of SNAP-25 mRNA and protein in hippocampus of all the offspring in various dosages groups were determined by RT-PCR and immunohistochemistry assay. The lead contents in blood and hippocampus of various lead exposed groups were significantly higher than those of the control group (P < 0.05). The lead levels in blood and hippocampus changed accordingly to the days of growth. In Water Morris Maze experiment, the result of 0.3 g/L group was not significantly different from that of the control group (P > 0.05), however, the results of 1.0, 3.0 g/L groups (5.89 ± 0.54, 9.53 ± 1.03) were significantly different from those of the control group (1.73 ± 0.07) (P < 0.05, P < 0.01). The expression of SNAP-25 mRNA and protein was lower in lead exposed groups than that of the control group (P < 0.05). Maternal lead exposure may induce the damage in the ability of learning and memory of the offspring. The neurotoxicity of lead may be induced by decreasing the expression of SNAP-25 mRNA and protein so as to affect the release of neurotransmitter from presynaptic terminal resulted in nerve damages.

Platelet Reactivity Is Associated with VAMP8 Expression and a VAMP8 3′UTR Polymorphism

Variations in platelet reactivity can promote either a hemorrhagic or thrombotic phenotype, and platelet granule secretion is essential for platelet activation and normal hemostasis. We have earlier rigorously phenotyped platelet reactivity in 337 healthy subjects and found excellent reproducibility for secretion-dependent platelet functions. Vesicle associated membrane protein (VAMP)-8, a v-SNARE, is the primary regulator of platelet secretion. Our goals were to determine whether the expression of 9 key molecules involved in the platelet secretory pathway – SNAP23, SNAP25, VAMP3, VAMP8, STX2, STX4, STX7, STXBP3, and RAB27B are associated with the well-known variability in platelet reactivity and investigate potential mechanisms. Leukocyte-depleted platelets were prepared from 10 subjects with hyperreactive platelets and 6 subjects with hyporeactive platelets. No significant variations were observed for SNAP25, STX2, STX4 and RAB27B mRNA levels. Although small variations were observed for other genes examined VAMP8 mRNA levels showed the highest variation. VAMP8 mRNA levels were 4.8-fold higher in hyperreactive platelets than hyporeactive platelets (P=0.0023). Immunoblotting showed that VAMP8 proteins were also significantly elevated by 2.5-fold in the hyperresponsive subjects (P = 0.04). An A-to-G transition in the 3′UTR region of the VAMP8 gene (rs1010) has been consistently shown to be associated with a high risk of myocardial infarction (MI) in European Americans [EA]. We genotyped all 337 subjects (182 EA and 155 African Americans [AA]) in our cohort for this rs1010 SNP. The ‘G’ allele frequency was 0.44 and 0.62 in EA and AA, respectively. The ‘G’ allele was associated with enhanced aggregation to epinephrine (P=0.001) and ADP (P=0.001) in an age dependent manner. The 3′ UTR of VAMP8 has at least 10 predicted microRNAs (miRNAs), 8 of which we have identified in platelets and 5 of which encompass the rs1010 SNP. In summary, variable VAMP8 expression levels appear to be a major regulator of platelet reactivity, and a common VAMP8 SNP known to be associated with MI is associated with the platelet aggregation response to epinephrine and ADP. Studies are currently on-going to assess whether this SNP modifies miRNA binding and whether this miRNA regulates VAMP8 expression.

Thyroid hormone regulates the expression of SNAP-25 during rat brain development

Thyroid hormones are major regulators of postnatal brain development. Thyroid hormones act through nuclear receptors to modulate the expression of specific genes in the brain. We have used microarray analysis to identify novel responsive genes in 14-day-old hypothyroid rat brains, and discovered that synaptosomal-associated protein of 25 kDa (SNAP-25) was one of the thyroid hormone-responsive genes. SNAP-25 is a presynaptic plasma membrane protein and an integral component of the vesicle docking and fusion machinery mediating secretion of neurotransmitters and is required for neuritic outgrowth and synaptogenesis. Using microarray analysis we have shown that SNAP-25 was down-regulated in the hypothyroid rat brain compared with the age-matched controls. Real-time RT-PCR and western blotting analysis confirmed that SNAP-25 mRNA and protein levels decreased significantly in the developing hypothyroid rat brain. Our data suggest that in the developing rat brain, SNAP-25 expression is regulated by thyroid hormone, and thyroid hormone deficiency can cause decreased expression of SNAP-25 and this may on some level account for the impaired brain development seen in hypothyroidism.

Synaptosomal-Associated Protein 25 Gene Expression Is Hormonally Regulated during Ovulation and Is Involved in Cytokine/Chemokine Exocytosis from Granulosa Cells

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.

ProEGF cytoplasmic domain (proEGFcyt) is a novel regulator of cathepsin-L secretion and elastinolytic activity in human thyroid carcinoma cells

We and others have shown that the cytoplasmic domains of EGF-like ligands have important biological functions. Employing stable transfectants of the human follicular thyroid carcinoma cell line FTC-133 over-expressing the proEGF cytoplasmic domain (FTC-133-proEGFcyt) we show here that proEGFcyt causes a transcriptional up-regulation of cathepsin- (cath-) B and -D and affects the processing of cath-L. Cath-L has strong elastinolytic activity and was the only cathepsin to be secreted by FTC-133-proEGFcyt transfectants. ProEGFcyt significantly impaired the ability of FTC-133 to migrate through elastin matrices when compared with FTC-133 stably transfected with the empty plasmid and a natural proEGFsplice mutant construct. Decreased migration through elastin matrix resulted from decreased secretion of cath-L in FTC-133-proEGFcyt. A similar reduction in elastinolytic activity was observed when FTC-133 control or proEGFsplice form transfectants were incubated with a specific cath-L inhibitor suggesting that this elastinolytic activity detected was largely mediated by cath-L. Down-regulation of cath-L in FTC-133-proEGFcyt involved the upregulation of the t-SNARE component SNAP25 as determined by siRNA knock-down of SNAP25 mRNA. Incubation of FTC-133-proEGFcyt with soluble EGF reversed the effects of proEGFcyt and this antagonistic EGF action was mediated by the EGFR. In summary, we provide first molecular evidence for the suppressive role of proEGFcyt on thyroid carcinoma cell elastinolytic activity and invasiveness and identify novel opposing modular functions of soluble EGF and the cytoplasmic domain of human proEGF.

SNAP-25 in hippocampal CA3 region is required for long-term memory formation

SNAP-25 is a synaptosomal protein of 25 kDa, a key component of synaptic vesicle-docking/fusion machinery, and plays a critical role in exocytosis and neurotransmitter release. We previously reported that SNAP-25 in the hippocampal CA1 region is involved in consolidation of contextual fear memory and water-maze spatial memory (Hou et al. European J Neuroscience, 20: 1593–1603, 2004). SNAP-25 is expressed not only in the CA1 region, but also in the CA3 region, and the SNAP-25 mRNA level in the CA3 region is higher than in the CA1 region. Here, we provide evidence that SNAP-25 in the CA3 region is also involved in learning/memory. Intra-CA3 infusion of SNAP-25 antisense oligonucleotide impaired both long-term contextual fear memory and water-maze spatial memory, with short-term memory intact. Furthermore, the SNAP-25 antisense oligonucleotide suppressed the long-term potentiation (LTP) of field excitatory post-synaptic potential (fEPSP) in the mossy-fiber pathway (DG-CA3 pathway), with no effect on paired-pulse facilitation of the fEPSP. These results are consistent with the notion that SNAP-25 in the hippocampal CA3 region is required for long-term memory formation.

Evidence that insulin secretion influences SNAP-25 through proteasomal activation

Regulation of SNARE proteins by glucose in pancreatic islets is complex and insufficiently clarified. We aimed to study effects of glucose per se separate from enhancing effects on exocytosis. A 24 h culture of rat islets at elevated glucose (27 mmol/L) increased t-SNARES (SNAP-25, syntaxin) (Western blotting). Co-culture with diazoxide, which inhibits glucose-induced insulin secretion, reversed these effects. Effects on SNAP-25 were similar in human and rat islets. Effects of diazoxide were mimicked by blocking secretion with somatostatin (rat islets). Blocking secretion by cooling abolished both glucose and diazoxide effects on SNAP-25. Total SNAP-25 mRNA as well as isoforms α and β were increased by 24-h elevated glucose. Diazoxide failed to reverse the glucose effects on mRNA. However, effects of diazoxide on SNAP-25 protein were nullified by proteasome inhibitors (ALLN, MG-132, and epoxomicin) but not by lysosomal inhibition (NH 4Cl). Exocytosis per se modifies SNAREs by a process linked to proteasomal activation.

Gene expression of nAChR, SNAP-25 and GAP-43 in skeletal muscles following botulinum toxin A injection: a study in rats

Purpose: Botulinum toxin A (BoNT-A) is used to manage spasticity in cerebral palsy. BoNT-A cleaves SNAP-25 protein, blocking acetylcholine release and weakening the muscle. Nicotinic acetylcholine receptors (nAChR) including alpha, beta, delta, gamma, and epsilon subunits, and GAP-43 protein are associated with functional recovery of neuromuscular junctions (NMJ) following BoNT-A. To better understand the mechanism behind this functional recovery, this study attempted to (1) document changes in NMJ morphometry following BoNT-A, and (2) determine the gene expression of nAChR subunits, SNAP-25, and GAP-43 protein. Methods: In this rat study (46 rats), 6 units/kg body weight of BoNT-A was injected into the gastrocnimus. NMJ morphometry and the time course of gene expression of nAChR subunits, SNAP-25, and GAP-43 were evaluated up to 1 year post-injection. Results: NMJ morphometry: gutter depth was reduced vs. the control side at two months, and normalizing by 6 months following BoNT. nAChR alpha mRNA and gamma mRNA increased by 3 days, peaked at 7 days and returned to control levels; delta mRNA peaked at 3 days. Epsilon mRNA peaked by 7 days. SNAP-25 mRNA increased from 60 to 90 days, returning to control levels by 6 months. GAP-43 mRNA was unchanged. Conclusions: Specific nAChR subunit mRNA expression up-regulates and then returns to normal within two weeks, preceding changes in NMJ morphometry. Although GAP-43 participates in nerve sprouting, no increase of GAP-43 mRNA occurred following BoNT-A. Delayed up-regulation of SNAP-25 mRNA might be associated with muscle functional recovery.

Distinct protein kinases regulate SNAP-25 expression in chromaffin cells.

The contribution of distinct Ca(2+)-sensitive protein kinases to the regulation of the expression of the synaptosomal-associated protein SNAP-25 was examined in bovine chromaffin cells. Prolonged incubation with high K(+) (38 mM) or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic receptor agonist, significantly increased SNAP-25 protein and mRNA expression, as assessed by immunoblotting and semi-quantitative RT-PCR analysis. Both stimuli preferentially enhanced mRNA coding for the SNAP-25a isoform. Increase of SNAP-25 expression induced by K(+) or DMPP was inhibited over 70% by KN-62 and KN-93, two Ca(2+)/calmodulin-dependent protein kinase (CaMK) inhibitors, whereas the inactive analogue KN-92 only reduced the expression by 34%. The three compounds also inhibited the high K(+)-elicited [Ca(2+)](i) signal by 40%, suggesting that the effect of KN-62 and KN-93 was a combination of CaMK/ Ca(2+) influx inhibitory actions. Incubation of the cells with mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126 reduced protein expression elicited by high K(+) by 50%, but did not modify the response to DMPP. Interestingly, although protein kinase A (PKA) inhibition by H-89 did not affect the high K(+) or DMPP-induced SNAP-25 expression, basal protein levels were significantly modified upon activation or inhibition of this pathway. Basal expression of SNAP-25 was also modified by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, but not by Gö6976, a PKC-alpha inhibitor, suggesting that the Ca(2+)-insensitive PKC-epsilon isoform control basal expression of SNAP-25 in these cells. Taken together, these results provide the first evidence that diverse protein kinases might converge in the induction of SNAP-25 expression in chromaffin cells. The preferential contribution of one or another kinase would depend on the physiological or experimental conditions.