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ABSTRACT Background: Exocytosis-related gene variants have been suggested to be associated with externalizing behaviors. Objective: This study aimed to examine VAMP2 26 bp Ins/Del, synaptotagmin XI (Syt11) rs3820594 and 33-bp promoter, Syntaxin 1A (Syn-1A) rs1569061 and SNAP-25 rs1051312 and rs3746544 polymorphisms, their serum levels and their relationship with impulsivity, temperament in individuals with alcohol dependence (AD) and healthy controls (HC). Methods: The study included 107 individuals with AD and 104 HCs. Single-nucleotide polymorphisms (SNPs) were studied with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and serum levels with ELISA. Michigan Alcohol Screening Test (MAST), Barratt Impulsiveness Scale-11 (BIS-11) and Temperament Evaluation of Memphis, Pisa, Paris and San Diego Autoquestionnaire (TEMPS-A) were applied. Results: Syn-1A rs1569061 C allele polymorphism was significantly higher in AD group. Syn-1A rs1569061 C allele was associated with 1.5 times increased risk of AD. All serum levels were significantly higher in the HC group. There was a relationship between Syn-1A rs1569061 polymorphism and BIS-11 motor impulsiveness in the AD group; Syt11 rs3820594 polymorphism and BIS-11 total, TEMPS-A depressive, hyperthymia in the HC group. Discussion: In our study, gene variants and serum levels of synaptic vesicle and presynaptic plasma membrane proteins were related to AD, impulsivity and temperament.
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Objective: Berberine, a cationic alkaloid first isolated in 1917, has been approved by the China Drug Administration for decades. Accumulating evidence demonstrated its antidepressant-like activities in vivo. Our previous study has shown that chronic stress leads to the upregulation of miR-34a in the hippocampus of mice. This study aims to evaluate the underlying miR-34a mediated mechanism of berberine in chronic stress-induced depression in mice. Methods: In the present study, mice were administered with berberine during chronic stress. Levels of miR-34a, dendritic density, mitochondrial morphology, and neurogenesis were assessed in the hippocampus. Subsequently, miR-34a agomir was used as a pharmacological intervention for the investigation of berberine. Results: The results showed that berberine reversed the decrease in sucrose preference and the increase in latency to feed without altering total food consumption. Furthermore, chronic stress-induced overexpression of miR-34a decreased synaptotagmin-1 and Bcl-2 levels, thereby impairing spinal morphology, mitochondria and neurogenesis. Berberine inhibited miR-34a expression, in turn restored synaptotagmin-1 and Bcl-2 levels, and thus improved spinal morphology, mitochondria and neurogenesis in the hippocampus. However, the improvements induced by berberine were totally blocked by the pretreatment of miR-34a agomir, which caused the elevation of miR-34a levels in the hippocampus. Conclusion: This finding demonstrated that miR-34a downregulation was involved in the antidepressant-like effects of berberine in mice exposed to chronic stress.
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@#Objective To investigate the dynamics of cortical granules exocytosis (CGE), and the role of Synaptotagmin1 (Syt1) in mouse fertilization. Methods The dynamics of mouse CGE were filmed on Perkin Elmer precisely Ulta VIEW VOX confocal Imaging System. The Syt1 functions on mouse fertilization and expression in zygotes were analyzed by immunofluorescence, Western blot assy and qRT-PCR. Results The dynamic process of mouse CGE was observed after oocyte activation, and the exact pot seems to be close to the polar body. The Syt1 expression was gradually increased after fertilization. The mouse fertilization rate after Syt1 knockdown was significantly lower than that of the control group (P＜ 0.05). Conclusion The dynamic process of CGE is studied. It is found that Syt1 is involved in mouse fertilization process.
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Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.
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Animals , Female , Humans , Mice , Rats , Amino Acid Sequence , Exocytosis , HEK293 Cells , Molecular Sequence Data , Mutant Proteins/metabolism , Phosphorylation , Phosphothreonine/metabolism , Protein Binding , Protein Interaction Mapping , Protein Structure, Tertiary , Protein Serine-Threonine Kinases/metabolism , Qa-SNARE Proteins/metabolism , Rats, Sprague-Dawley , Synaptosomal-Associated Protein 25/metabolism , Synaptotagmins/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , Vesicular Transport Proteins/chemistryABSTRACT
Objective To observe the expression of synaptotagmin I(syt I)protein in the prefrontal cortex of adult-onset hypothyroidism rats and the effects of replicated therapy in different doses of thyroid hormone on the syt I protein.Methods All 44 aduh male Sprague-Dawley rats were divided into 4 groups randomly according to their body mass:hypothyroidism group,routine dosage thyroxine treatment group,high dosage thyroxine treatment group and control group.The adult male Sprague-Dawley rats were replicated to the adult-onset hypothyroidism and treatment models with propyhhiouracil(PTU).The levels of serum T3,T4 were assayed by the radioimmunoassay method and the level of the syt I protein in the molecular layer,external granular layer,external pyramidal layer,internal granular layer and internal pyramidal layer in prefrontal cortex was analyzed by immunohistochemistry.Results In the hypothyroidism group,the levels of serum T3 and T4[(0.34±0.04),(43.01±2.95)nmol/L]were significantly lower than those in the control group[(0.65±0.15), (55.20±3.56)nmol/L, F value: 6.026,5.940,4.503,P<0.05 or <0.01 ], the levels of the syt I protein in the molecular layer(0.018±0.010), external granular layer (0.020±0.007), external pyramidal layer(0.013±0.008), internal granular layer(0.011±0.005), internal pyramidal layer(0.024±0.013) of prefrontal lobe were significantly lower compared to the control group[(0.028±0.010,0.031 ± 0.010,0.028 ± 0.010,0.022 ± 0.008,0.038 ± 0.013), F value: 5.697,8.965,14.668,13.597,6.807,P<0.05 or <0.01 ]. In the routine dosage of the thyroxine treatment group, the levels of serum T3,T4 [(0.63 ±0.05), (55.04 ± 3.77)nmol/L] were not significantly different compared to the control group(F value: 3.162,0.367,all P>0.05), and the level of the syt I protein in the molecular layer, external granular layer, external pyramidal layer, internal granular layer and internal pyramidal layer in prefrontal cortex showed a significant improvement of the syt I protein(0.027 ± 0.013,0.025 ± 0.009,0.022 ± 0.008,0.020 ± 0.010,0.033 ± 0.010), which were similar to that of the control group(F value: 0.094,2.208,2.467,0.350,0.693, all P>0.05). In the high dosage thyroxine thyroid hormone treatment group, the levels of serum T3 and T4[ (1.11 ± 0.10), (96.68 ± 6.42)nmoL/L] were higher than the control group(F value: 6.291,12.031, all P<0.01), the expression of the syt I protein(0.028 ± 0.008,0.031 ±0.011,0.026 ± 0.012,0.023 ± 0.011,0.038 ± 0.010) were not significantly different compare to the control group (F value: 0.001,0.019,0.111,0.061,0.001, all P>0.05). Conclusions The expression of the syt I protein in the prefrontal cortex of adult-onset hypothyroidism can be decreased, which can be reversed by routine dosage of thyroxine treatment.
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Objective To evaluate myocardial apoptosis with 99Tcm-C2A-GST myocardial imaging using the recombined C2A domain of Synaptotagmin Ⅰ by gene engineering. Methods ( 1 ) The C2A gene was inserted into the prokaryotic glutathione S-transferate (GST) fusion protein expression plasmid pGEX-6P-1. The recombinant plasmid was transformed into E. coli BL21. C2A-GST fusion protein was purified after BL21 was induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG). (2)The activity of fusion protein was identified by cell binding test with fluorescein-5-isothiocyanate (FITC)-C2A-GST. (3) The C2A-GST fusion protein was labeled with 99Tcm using 2-iminothiophene hydrocoride method. Radiochemical purity was determined with thin layer chromatography. (4)99Tcm-C2A-GST (7.4 MBq) was injected to ischemia-reperfusion rat models through tail vein. The image was acquired with SPECT at 1 h after injection, and then hearts were removed, rinsed with saline and dyed with triphenyl tetrazolium coride (TTC). The ischemic myocardium was separated from the viable myocardium and was weighted. Its radioactivity was measured by gamma counting. The difference of uptake of radiotracer between ischemic myocardium and normal myocardium was compared using percentage activity of injected dose per gram of tissue ( % ID/g) with standard deviation. SPSS 12.0 and t-test were used for data analysis. Results ( 1 ) C2A-GST fusion protein wassuccessfully expressed and its relative molecular weight was 3.8 × 104. (2) FITC-C2A-GST binding to apoptotic cells could be observed by fluorescent microscopy. (3) The radiochemical purity of 99Tcm-C2A-GST was (98.90 ±0.43)%. (4)The imaging studies showed that there was focal uptake of radioactivity in the ischemic myocardium. In vitro uptake of 99Tcm-C2A-GST was (2.41 ±0.32) % ID/g by the ischemic myocardium, however 99Tcm-C2A-GST-N-hydroxysuccinimide (C2A-GST-NHS) was (0. 82 ± 0. 24) % ID/g. There was statistically significant difference between those two groups (t = 10. 6, P ＜0.01 ). Conclusion The C2A domain of Synaptotagmin Ⅰ expressed by gene engineering can be used as the tracer for noninvasive detection of ischemic myocardium in the ischemia-reperfusion rat model.
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Botulinum neurotoxin(BoNT) is the most lethal biotoxin known to mankind. It inhibits acetylcholinerelease from the cholinergic nerve ending by cleavage of SNARE proteins, followed by neuromuscular blockadeand paralysis. Gangliosides are considered to act as a first receptor of BoNT with low affinity.Then the membranebound gangliosides-BoNT complex moves laterally to reach and bind the toxin specific protein receptor,synaptotagmin, with a high affinity constant. At last the gangliosides-BoNT-synaptotagmin complex undergoesreceptor-mediated endocytosis. This double-receptors theory is widely accepted. The research data are summarizedand reviewed.
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Synaptotagmin is a Ca2+ sensing protein, which triggers a fusion of synaptic vesicles in neuronal transmission. Little is known regarding the expression of Ca2+ - dependent synaptotagmin isoforms and their contribution to the release of secretory vesicles in mouse and rat parotid acinar cells. We investigated a type of Ca2+ - dependent synaptotagmin and Ca2+ signaling in both rat and mouse parotid acinar cells using RT-PCR, microfluorometry, and amylase assay. Mouse parotid acinar cells exhibited much more sensitive amylase release in response to muscarinic stimulation than did rat parotid acinar cells. However, transient [Ca2+]i increases and Ca2+ influx in response to muscarinic stimulation in both cells were identical, suggesting that the expression or activity of the Ca2+ sensing proteins is different. Seven Ca2+ - dependent synaptotagmins, from 1 to 7, were expressed in the mouse parotid acinar cells. However, in the rat parotid acinar cells, only synaptotagmins 1, 3, 4 and 7 were expressed. These results indicate that the expression of Ca2+ - dependent synaptotagmins may contribute to the release of secretory vesicles in parotid acinar cells.