摘要
目的探讨上调淀粉样前体蛋白(APP)剪切产物C99对线粒体相关内质网膜(MAM)及线粒体自噬的影响。方法制作N2a/APP695细胞的AD模型,分为正常对照组及γ-分泌酶抑制剂组(DAPT组,10μmol/L DAPT完全培养基培养20 h);采用蛋白免疫印迹(Western blot)法检测2组细胞中C99、胆固醇酰基转移酶(ACAT1)、线粒体融合蛋白1和2(MFN2/1)、线粒体分裂蛋白1(FIS1)、线粒体自噬相关蛋白PTEN诱导的推定激酶1(PINK1)、E3泛素-蛋白连接酶(Parkin)、微管相关蛋白1-轻链3(LC3)、P62蛋白及细胞抗凋亡蛋白B淋巴细胞瘤-2蛋白(BCL-2)的表达;用JC-1染色法检测线粒体膜电位变化情况,激光共聚焦显微镜检测线粒体与内质网偶联情况。结果经过DAPT处理20 h后DAPT组N2a/APP695细胞中C99蛋白表达明显增加(P<0.01);与正常对照组比较,DAPT组细胞中ACAT1、MFN2、MFN1、FIS1、PINK1、Parkin及LC3蛋白表达水平上调,但P62、BCL-2蛋白表达下调,差异有统计学意义(P<0.05);JC-1染色结果显示,DAPT组细胞中线粒体膜电位较正常对照组明显降低(P<0.01),而内质网与线粒体偶联增加。结论上调C99水平可以使N2a/APP695细胞中MAM的活性增加,影响线粒体分裂融合后导致线粒体自噬异常。
Objective To investigate the effect of upregulation of amyloid beta-precursor protein(APP)proteolytic product C99 on mitochondria-associated endoplasmic reticulum membrane(MAM)and mitophagy.Methods N2a/APP695 cells were used as an AD model and divided into normal control group andγ-secretase inhibitor group(DAPT group,cultured in complete medium supplemented with 10μmol/L DAPT for 20 h).Western blot was applied to detect the expression levels of C99,cholesterol acyltransferase(ACAT1),mitochondrial fusion proteins 1 and 2(MFN2/1),mitochondrial fission protein 1(FIS1),mitophagy-related protein PTEN-induced putative kinase 1(PINK1),E3 ubiquitin-protein linkage Enzyme(Parkin),microtubule-associated protein 1-light chain 3(LC3),P62,and anti-apoptotic protein B-lymphoma-2 protein(BCL-2)in two groups.JC-1 staining was performed to measure mitochondrial membrane potential changes.Confocal laser microscopy was used to detect the coupling between mitochondria and endoplasmic reticulum.Results When compared to the normal control group,the protein expression levels of ACAT1,MFN2,MFN1,FIS1,PINK1,Parkin,and LC3 in were upregulated in DAPT group,but the protein expression levels of P62 and BCL-2 were downregulated(P<0.05).JC-1 staining showed that mitochondrial membrane potential in DAPT group was significantly lower than that in normal control group(P<0.01).Coupling between the endoplasmic reticulum and mitochondria was increased.Conclusion Upregulating C99 can increase MAM activity in N2a/APP695 cells,influence mitochondrial fission and fusion,leading to abnormal mitophagy.
作者
付燕林
张文萍
龙培艳
郑菊
高霄
齐晓岚
肖雁
FU Yanlin;ZHANG Wenping;LONG Peiyan;ZHENG Ju;GAO Xiao;QI Xiaolan;XIAO Yan(Key Laboratory of Endemic and Minority Diseases of Ministry of Education&Guizhou Provincial Key Laboratory of Medical Molecular Biology,Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2022年第5期504-510,共7页
Journal of Guizhou Medical University
基金
国家自然科学基金(81660207)
贵州省科技厅基础重点项目(黔科合基础〔2019〕1440)
贵州医科大学国家自然科学基金培育项目(20NSP069)。
关键词
阿尔茨海默病
C99
线粒体相关内质膜
线粒体自噬
线粒体分裂融合
alzheimer's disease(AD)
C99
mitochondrial associated endoplasmic membrane
mitophagy
mitochondrial division and fusion
