线粒体相关蛋白调控突触核蛋白病理学作用研究

发布时间：2018-04-07 17:17

本文选题：PINK1　切入点：突触核蛋白　出处：《河南大学》2015年硕士论文

【摘要】：PINK1是一种线粒体丝氨酸/苏氨酸激酶,保护细胞压力应激所导致的线粒体功能障碍,调控神经元分化。突变的PINK1(PARK6)导致常染色体隐性遗传早发性帕金森病(PD)。β-synuclein(β-syn)是α-syn同系物,其错义突变β-syn P123H被发现存在于家族性路易体痴呆患者。突变蛋白β-syn P123H在细胞内形成嗜酸性包涵体,并且与LAMP-2,ATP13A2,cathepsin等溶酶体标志物共存,在胞浆聚集诱导细胞凋亡,具有加速神经退化的毒性作用。PINK1能够维持线粒体内稳态,在PD病理学变化过程起着关键作用,但是PINK1通过线粒体保护作用介导突触核蛋白病理学过程的机制还不清楚。本课题将在稳定表达Parkin的He La细胞及稳定表达β-syn P123H的B103神经母细胞瘤细胞进行,研究PINK1/Parkin通过调控线粒体自噬,促进β-syn P123H自噬降解的神经保护作用及其分子机制。本文采用PCR构建PINK1野生型(PINK1 WT)及两步PCR法定点突变技术构建PINK1 Q126P、E240K、G309D、L347P、P498L突变真核表达载体,测序验证后应用免疫印迹技术(Western Blot)鉴定其在细胞内表达情况。WST-1细胞活性检测及LDH细胞毒性实验分析PINK1 WT及突变体对细胞存活的影响。结果显示,转染PINK1突变体的细胞存活率下降,细胞毒性增加。相比对照组,转染野生型PINK1质粒的细胞无显著性差异。应用细胞免疫荧光化学染色的方法,观察PINK1 WT及其突变体细胞定位情况,以及Parkin突变体T240R、R275W的细胞分布形态。运用线粒体TMRE荧光标记技术,以FCCP组作为阳性对照,分析Parkin突变体T240R、R275W对线粒体膜电位的影响。通过脂质体转染及Western Blot方法,分析PINK1、Parkin对线粒体分裂融合动态变化的影响。免疫荧光细胞化学结果显示:PINK1突变后线粒体异常聚集。Parkin突变体在胞浆形成聚集体,并且导致线粒体膜电位降低。免疫印迹分析结果显示:PINK1、Parkin的突变则导致了线粒体分裂、融合功能降低。野生型PINK1、Parkin参与线粒体质量控制,促进β-syn P123H自噬降解。免疫荧光细胞化学实验证明β-syn P123H及Parkin T240R聚集体经自噬溶酶体途径降解,Parkin T240R则加重了β-syn P123H聚集体的形成。通过自噬诱导剂Rapamycin,抑制剂3-MA、NH4Cl作用,结合TUNEL细胞凋亡检测方法,研究自噬在β-syn P123H、Parkin T240R所诱导的细胞凋亡过程的作用。对TUNEL阳性细胞进行计数,统计分析结果显示:相对于DMSO组,3-MA、NH4Cl组细胞死亡数目明显增多,而Rapamycin组细胞死亡数目减少。总之,突变PINK1诱导线粒体断裂形成聚集体,并且导致细胞活性下降,细胞毒性增加。Parkin突变体在细胞内形成聚集体,导致线粒体膜电位降低。Parkin突变聚集体和β-syn P123H经自噬溶酶体途径降解,并且Parkin突变体加重了β-syn P123H聚集体的形成。野生型PINK1、Parkin参与线粒体质量控制,促进β-syn P123H自噬降解。在β-syn P123H诱导的突触核蛋白病理学过程中,线粒体相关蛋白PINK1/Parkin介导线粒体自噬及分裂、融合过程发挥保护作用。
[Abstract]:PINK1 is a mitochondrial serine / threonine kinase that protects mitochondria from stress stress and regulates neuronal differentiation.尾 -synuclein (尾 -synn) is a homologue of 伪 -syn. The missense mutation 尾 -syn P123H has been found in familial dementia patients.The mutant 尾 -syn P123H forms eosinophilic inclusion bodies in the cells and coexists with lysosomal markers such as LAMP-2ATP 13A2cathepsin. The mutant protein 尾 -syn P123H induces apoptosis in the cytoplasm, which can accelerate neurodegenerative toxicity. PINK1 can maintain the homeostasis of mitochondria.PINK1 plays a key role in the pathological changes of PD, but the mechanism of PINK1 mediated synaptic nuclear protein pathological process through mitochondrial protection is not clear.In this study, we studied the neuroprotective effect of PINK1/Parkin on 尾 -syn P123H and 尾 -syn P123H in He-La cells and B103 neuroblastoma cells stably expressing 尾 -syn P123H by regulating mitochondrial autophagy and promoting the degradation of 尾 -syn P123H autophagy.In this paper, PCR was used to construct PINK1 wild-type PINK1 WTs and two-step PCR site-directed mutagenesis technique to construct the eukaryotic expression vector of PINK1 Q126PnE240KG309DX L347PnP498L mutation.After sequencing, Western blot technique was used to identify the expression of WST-1 in cells and the effects of PINK1 WT and mutants on cell survival were analyzed by LDH cytotoxicity test.The results showed that the survival rate and cytotoxicity of the transfected PINK1 mutants were decreased.Compared with the control group, there was no significant difference between the cells transfected with wild type PINK1 plasmid.The localization of PINK1 WT and its mutant cells and the cell distribution of Parkin mutant T240 RN R275W were observed by immunofluorescence staining.Mitochondrial TMRE fluorescence labeling technique was used to analyze the effect of Parkin mutant T240RN R275W on mitochondrial membrane potential (MMP) in FCCP group.The effects of PINK1 and Western Blot on the dynamic changes of mitochondrial mitotic fusion were analyzed by liposome transfection and Western Blot.The results of immunofluorescence cytochemistry showed that the abnormal aggregation of mitochondria. Parkin mutants formed aggregates in the cytoplasm and resulted in the decrease of mitochondrial membrane potential after 1: PINK1 mutation.Western blot analysis showed that the mutation of PINK1 Parkin resulted in mitochondrial division and decreased fusion function.Wild type PINK1 Parkin participates in mitochondrial quality control and promotes 尾 -syn P123H autophagy degradation.Immunofluorescence cytochemistry showed that 尾 -syn P123H and Parkin T240R aggregates were degraded by autophagic lysosome pathway, and the formation of 尾 -syn P123H aggregates was aggravated.The effect of autophagy on the apoptosis induced by 尾 -syn P123H ~ (2 +) Parkin T240R was studied by means of the effects of Rapamycin, an inhibitor 3-MAN NH _ 4cl, and TUNEL cell apoptosis assay.TUNEL positive cells were counted. The results of statistical analysis showed that the number of cell death in DMSO group was significantly higher than that in DMSO group, but that in Rapamycin group was decreased.In short, mutated PINK1 induced mitochondrial fragmentation to form aggregates, which led to a decrease in cellular activity and increased cytotoxicity. Parkin mutants formed aggregates in cells.The mitochondrial membrane potential decreased. Parkin mutant aggregates and 尾 -syn P123H were degraded by autophagic lysosome pathway, and the formation of 尾 -syn P123H aggregates was aggravated by Parkin mutants.Wild type PINK1 Parkin participates in mitochondrial quality control and promotes 尾 -syn P123H autophagy degradation.In the process of 尾 -syn P123H induced synaptic nuclear protein pathology, mitochondrial associated protein PINK1/Parkin mediates mitochondrial autophagy and mitosis, and the fusion process plays a protective role.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R742.5

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