LRRK2突变体诱导人多巴胺能神经元凋亡的蛋白组学的初步研究

【作者】 陈洁；

【导师】 陈俊；

【作者基本信息】 复旦大学 ， 神经生物学， 2009， 硕士

【摘要】 背景：帕金森病是第二大神经系统退行性疾病,其发病率仅次于老年痴呆症,在50岁人群中其发病率约为0.3%,而在85岁人群中则高达4.3%。目前对帕金森病的发病机制还不完全清楚,近年来对家族性帕金森病的研究发现,LRRK2 (Leucine-rich repeat kinase2)基因的突变与常染色体显性帕金森病密切相关。在散发性病人中LRRK2的突变率约为3%,而在家族性帕金森病人中突变率却高达13%,并且这些病人的临床表现与自发性帕金森病无法区别,因此对LRRK2突变体的毒性研究将有助于加深对帕金森病发病机制的理解。方法：我们利用脂质体Lipofectamine2000把GFP质粒和LRRK2质粒共转染到SH-SY5Y细胞中建立凋亡模型,并以绿色荧光为信号用流式细胞仪进行细胞分选,收集阳性转染细胞。然后对分选得到的细胞进行2-D蛋白电泳和MS-MS质谱分析,找出可能出现差异表达的蛋白,并利用western blot免疫印迹法对这些蛋白进行验证。最后,我们对表达确实发生变化的蛋白进行了初步的功能研究。结果：本课题建立了LRRK2突变体G2019S-LRRK2诱导SH-SY5Y细胞株凋亡的细胞模型,并通过蛋白组学研究方法初步探讨了该突变体过表达后细胞蛋白表达谱的变化。与空白对照组和野生型组相比,在G2019S-LRRK2过表达的细胞中以下6种蛋白的表达发生了变化:protein disulfide isomerase、EEF1G (eukaryotic translation enlongation factor 1 gamma)、lactate dehydrogenase B、GSTP1 (glutathione S-transferase P1)、GDI alpha (GDP dissociation inhibitor alpha)和YWHAZ蛋白。其中G2019S-LRRK2过表达组与空白对照组和野生型组相比,GSTP1蛋白的表达显著降低,进一步研究发现GSTP1蛋白的高表达可以降低G2019S-LRRK2过表达引起的细胞毒性,对细胞具有保护作用。结论：我们的研究结果表明：一、与空白对照组相比,Wt-LRRK2和G2019S-LRRK2的过表达都能诱导SH-SY5Y细胞的凋亡。同时,突变型LRRK2诱导的细胞凋亡显著高于野生型,提示突变型LRRK2的细胞毒性显著增强。二、与空白对照组和野生型组相比,G2019S-LRRK2过表达组中以下6个蛋白的表达发生了变化：protein disulfide isomerase、EEF1G、lactate dehydrogenase B、GSTP1、GDI alpha和YWHAZ蛋白。三、过表达GSTP1可以降低G2019S-LRRK2过表达引起的细胞毒性,对细胞具有保护作用,提示G2019S-LRRK2过表达引起的细胞毒性可能与氧化应激反应相关。更多还原

【Abstract】 Background and purpose:PD(parkinson’s disease) is considered as the second neurodegenerative disease, affecting estimated 0.3% at age of 50, and 4.3% by 85. This syndrome is characterized clinically with resting tremor, rigidity, bradykinesia and postural instability, while pathologically progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of intracellular Lewy bodies in surviving neurons of the brainstem. So far the etiology of PD remains unclear, genetic studies on familiar PD resulted in discovery of sevaral pathogenic genes, including Parkin, PINK1, DJ-1,α-Synuclein, UCHL1 and LRRK2. Among them, LRRK2 has recently captured a lot of attention for its association with indistinguishable symptoms from typical late-onset PD. Anatomy studies on PD autopsies with LRRK2 mutants displayed all the pathological phenotypes that have been observed in parkinsonism including:ubiquitin intranuclear inclusions (Marinesco bodies), a-synucleinopathy and tauopathy, except for nigral loss alone. Therefore, studies on LRRK2 will provide a great opportunity to further understand the etiology of PD.Methords:we transfected SH-SY5Y cells with either wildtype or G2019S-mutant LRRK2 plasmid to examine the toxicity of LRRK2 mutant. Using 2-D electrophoresis combind with MS-MS, we explored the proteomic variation resulted from the toxicity.Results:Using 2-D electrophoresis and MS-MS, we identified six proteins with different expression between wildtype-LRRK2 and G2019S-LRRK2 overexpressed cells including:GID alpha, GSTP1, EEF1G, protein disulfide isomerase, Lactate dehydrogenase B and YWHAZ protein. Further study revealed that total GSTP1 activity was diminished and overexpression of GSTP1 was found protective.Conclusion:These results demonstrate that either Wt or G2019S LRRK2 overexpression is toxic to cell, while the mutant LRRK2 displayed more severe toxicity. Also, the proteomics results demonstrate six differentially expressed proteins in G2019S LRRK2 overexpressed cells including: GID alpha, GSTP1, EEF1G, protein disulfide isomerase, Lactate dehydrogenase B and YWHAZ protein. Futhermore, we found that overexpression of GSTP1 protected against LRRK2-mutant induced apoptosis, indicating that oxidative stress may be implicated in G2019S-LRRK2 toxicity.更多还原