摘要
目的探讨Sirt1对突触囊泡蛋白表达的影响和其在突触前位点的功能。方法采用CRISPRa技术激活SH-SY5Y细胞内源Sirt1表达,通过转染aSirt1质粒将细胞分为aSirt1#1、aSirt1#2、aSirt1#3、aSirt1#NC组并设置空白对照(Mock)组。采用乳酸脱氢酶检测细胞毒性,通过Western blot检测突触囊泡蛋白及自噬相关蛋白表达水平。结果aSirt1#1组和aSirt1#3组细胞毒性低于aSirt1#NC组,差异有统计学意义(P<0.05)。aSirt1#1组细胞的Sirt1、突触素、囊泡相关膜蛋白2(VAMP2)、α-突触核蛋白(α-Syn)表达水平高于Mock组,差异有统计学意义(P<0.05)。aSirt1#1组SH-SY5Y细胞中自噬相关蛋白Atg13、Becln1表达水平高于Mock组,自噬底物SQSTM1表达水平低于Mock组,差异均有统计学意义(均P<0.05)。aSirt1#1组SH-SY5Y细胞中P-AMPK/AMPK、P-ULK1/ULK1比值高于Mock组,差异有统计学意义(P<0.05)。结论Sirt1可以通过激活AMPK-ULK1通路的来调节自噬,影响突触囊泡蛋白的表达,有利于突触囊泡循环,影响突触前位点的功能。
Objective To explore the effect of Sirt1 on the expression of synaptic vesicle protein and its function at presynaptic sites.Methods Endogenous Sirt1 expression in SH-SY5Y cells was activated using CRISPRa technology.The cells were divided into aSirt#1,aSirt1#2,aSirt1#3,and aSirt1#NC groups by transfection with aSirt1 plasmid,and a blank control(Mock)group was set up.Lactate dehydrogenase was used to detect cytotoxicity,and Western blot was used to detect the expression levels of synaptic vesicle protein and autophagy related proteins.Results The cytotoxicity of the aSirt1#1 and aSirt1#3 groups was lower than that of the aSirt1#NC group,and the difference was statistically significant(P<0.05).The expression levels of Sirt1,synaptophysin,vesicle associated membrane protein 2(VAMP2),and alpha-synuclein(α-Syn)in cells of aSirt1#1 group were higher than those of the Mock group,and the difference was statistically significant(P<0.05).The expression levels of autophagy related proteins Atg13 and Becln1 in SH-SY5Y cells of aSirt1#1 group were higher than those in the Mock group,and the expression levels of autophagy substrate SQSTM1 were lower than those in the Mock group,with statistical differences(all P<0.05).The P-AMPK/AMPK and P-ULK1/ULK1 ratio in SH-SY5Y cells of aSirt1#1 group were higher than those in the Mock group,and the differences were statistically significant(P<0.05).Conclusions Sirt1 can regulate autophagy by activating the AMPK-ULK1 pathway,affecting the expression of synaptic vesicle proteins,promoting synaptic vesicle circulation,and affecting the function of presynaptic sites.
作者
何静
王蓉
He Jing;Wang Rong(Central Laboratory,Xuanwu Hospital,Capital Medical University,Beijing 100053,China;Institute of Alzheimer's Disease,Beijing Institute of Brain Disorders,Beijing 100053,China;Beijing Research Center for Geriatrics,Beijing 100053,China)
出处
《神经疾病与精神卫生》
2024年第4期243-249,共7页
Journal of Neuroscience and Mental Health
基金
国家重点研发计划(2018YFA0108503,2022YFC2403500)。
