【作者】 兰丹梅；

【导师】 蒋雨平； 王坚； 邬剑军； 任惠民； 陈嬿；

【作者基本信息】 复旦大学 ， 神经病学， 2009， 博士

【摘要】 帕金森病(Parkinson’s disease，PD)是常见的神经退行性疾病，但迄今该病病因和发病机制未明。路易小体(Lewy body)是PD患者脑内特征性的病理学标志，异常聚集的α突触核蛋白(α-synuclein)是家族遗传性和散发性PD患者脑内路易小体的主要成份。α突触核蛋白的病理性积聚是PD临床症状发生、发展及受累脑区变性的重要原因。如何阻断α-synuclein的异常沉积或者促进聚集的α-synuclein降解是当前PD研究中的重要课题。近年来，相关研究发现海藻糖通过与蛋白质分子间的相互作用，可抑制中枢神经系统变性病中相关蛋白的异常聚集。海藻糖还可促进强力霉素诱导的转染人A53T突变型α-synuclein基因的PC12细胞自噬，并且能促使突变的α-synuclein降解。我们利用慢病毒表达载体，建立长期稳定过表达人野生型和A53T突变型α-synuclein的PC12细胞株，利用蛋白酶体抑制剂(MG132)抑制α-synuclein降解，建立α-synuclein异常聚集的PD细胞模型，观察海藻糖是否对未转染的PC12细胞、过表达人野生型和A53T突变型α-synuclein的PC12细胞有相似的促自噬作用，并且能否使异常聚集的野生型和A53T突变型α-synuclein降解；另外，自噬的增强对细胞的存活是否有影响。第一部分人野生型和A53T突变型α-突触核蛋白慢病毒表达载体的构建及在PC12细胞中的转染目的构建长期稳定过表达人野生型和A53T突变型α-突触核蛋白的PC12细胞株。材料和方法首先制备慢病毒表达载体，经测序鉴定正确后转染PC12细胞，然后通过细胞的免疫荧光染色检测α-synuclein-V5融合蛋白，PCR法扩增转基因PC12细胞的SNCA片段后进行测序检测突变基因，Western Blot法检测PC12细胞α-synuclein的表达从而鉴定转基因细胞能否稳定表达目的基因，MTT法测细胞活力，绘制生长曲线。结果经测序，pLenti6／V5-SNCA-WT和pLenti6／V5-SNCA-A53T表达质粒构建成功；免疫荧光染色示基因转染后，大于95％的PC12细胞中有α-synuclein-V5融合蛋白表达；转基因细胞的SNCA片段测序结果显示，慢病毒表达载体成功整合入PC12细胞基因组；Western Blot法检测结果示转基因PC12细胞能够过表达α-synuclein蛋白。3种细胞生长曲线无明显差异。结论我们已成功构建了人野生型和A53T突变型α-突触核蛋白慢病毒表达载体，并且成功建立了稳定过表达人野生型和A53T突变型α-突触核蛋白的PC12细胞株，过表达人野生型和A53T突变型α-突触核蛋白对细胞活力无明显影响。第二部分海藻糖促进未转染和已转染人野生型或A53T突变型α突触核蛋白基因的PC12细胞自噬目的研究海藻糖对未转染和已转染人野生型或A53T突变型α-synuclein基因的PC12细胞的促自噬作用。材料和方法用不同浓度(10mM、50mM和100mM)的海藻糖干预细胞24h，然后用免疫荧光检测自噬泡及自噬溶酶体，用透射电镜观察细胞内自噬泡数量的变化，用免疫印迹检测LC3II和α-synuclein的表达，观察海藻糖对细胞是否有促自噬作用，能否促进过表达的人野生型和A53T突变型α-synuclein降解；用MTT法检测细胞活力，观察海藻糖对细胞有无损伤或保护作用。结果50mM和100mM的海藻糖能使细胞内自噬泡和自噬溶酶体显著增多，并且使LC3II的表达量显著上调(P＜0.01)，同时促进A53T突变型α-synuclein的降解(50mM海藻糖组，P＜0.05；100mM海藻糖组，P＜0.01)，而对WT型α-synuclein无明显影响(P＞0.05)；给予2mM 3-MA预孵育3小时可以阻断海藻糖的促自噬作用；50mM和100mM海藻糖均能降低PC12细胞和A53T转基因细胞的活力(P＜0.01)，仅100mM海藻糖能降低WT转基因细胞活力(P＜0.05)。海藻糖对WT转基因细胞的损伤作用较其他两种细胞弱(P＜0.05)。结论海藻糖能促进未转染和已转染人野生型或A53T突变型α-synuclein基因的PC12细胞自噬，并且能促进A53T突变型α-synuclein降解，但对野生型α-synuclein无明显作用。大剂量海藻糖能降低细胞活力，对WT转基因细胞的活力影响较轻。第三部分海藻糖对蛋白酶体抑制剂介导的PD细胞模型的作用目的研究海藻糖对蛋白酶体抑制剂介导的α-synuclein异常聚集模型的促自噬作用。材料和方法给予500nM MG132、500nM MG132同时加不同浓度(10mM、50mM、100mM)海藻糖进行干预，继续培养24h后，进行免疫荧光检测细胞内的自噬泡、溶酶体和自噬溶酶体，用免疫印迹检测LC3II和α-synuclein的表达，观察海藻糖对MG132介导的细胞模型是否有促自噬作用，是否能促进异常聚集的人野生型和A53T突变型α-synuclein降解；用MTT法检测细胞活力，观察海藻糖对MG132介导的细胞损伤有无保护作用。结果500nM MG132能显著抑制α-synuclein降解(P＜0.05)，并且代偿型增多细胞内自噬泡、溶酶体和自噬溶酶体的数量，海藻糖能增强MG132介导的细胞自噬现象，使LC3II的表达量比500nM MG132单独作用时明显上调(P＜0.05)；海藻糖能促进细胞内异常聚集的A53T突变型α-synuclein降解(50mM海藻糖组，P＜0.05；100mM海藻糖组，P＜0.01)，但对野生型α-synuclein无明显作用；海藻糖能明显加重MG132介导的细胞损伤(P＜0.05)。结论蛋白酶体抑制剂MG132能导致细胞内α—synuclein异常聚集，同时增强细胞自噬。海藻糖可显著增强MG132介导的细胞自噬，并且能促进异常聚集的A53T突变型α-synuclein降解，但对野生型α-synuclein无明显作用。海藻糖加重MG132介导的细胞损伤。结论1．成功建立长期、稳定过表达人野生型和A53T突变型α—synuclein的PC12细胞系。2．海藻糖增强细胞的自噬降解活性，但仅能降解A53T突变型α—synuclein蛋白，对野生型α—synuclein作用不明显。3．在营养状况正常的情况下，大剂量海藻糖使细胞活力下降。4．蛋白酶体抑制剂MG132能导致细胞α—synuclein异常聚集，同时增强细胞自噬，可能为细胞的代偿性反应。5．海藻糖能显著增强蛋白酶体抑制剂MG132介导的细胞自噬。6．海藻糖加重蛋白酶体抑制剂MG132介导的细胞损伤。更多还原

【Abstract】 Parkinson’s disease (PD) is a common neurodegenerative disease, but its exactpathogenesis is still unknown so far.α-synuclein, a main component of Lewy bodyof the familial and sporadic PD patient’s brain, has been demonstrated to beimplicated in formation of Lewy body and pathogenesis of PD. The aggregation ofα-synuclein is being considered as one of the main cause to the apoptosis indopaminergic neurons, leading to the manifestation of bradykinesia, tremor, andrigidity. How to inhibit the formation of aggregatedα-synuclein or enhance itsdegradation is an issue with significant potential for clinical application. During thepast several years, trehalose has been found to be able to prevent the aggregation ofthe key proteins responsible for other neurodegenerative diseases includingAlzheimer’s disease and Huntington’s disease. Trehalose was also reported that itcould enhance the autophagy of the doxycycline-inducible PC12 cell lines transfectedwith A53T mutant SNCA and increase the degradation of A53T mutantα-synuclein.In the current study, we constructed PC12 cells lines stably expressing wild type andA53T mutantα-synuclein by using the lentivirus expression vectors ofpLenti6/V5-SNCA-WT and pLenti6/V5-SNCA-A53T and establish an in vitro modelfor PD by applying proteasome inhibitor(500nM MG132), to investigate whethertrehalose can enhance autophagy of untransfected and transfected PC12 cells withhuman WT or A53T mutant SNCA gene, and whether trehalose can accelerate thedegradation of WT and A53T mutantα-synuclein. Furthermore, we also investigatedthe possible effects of the enhanced autophagy induced by trehalose on the cellviability. PartⅠConstruction of The Lentivirus Expression Vector of Wild Type andA53T Mutant Alpha-synuclein and Its Transfection in PC12 CellsAim: To construct PC12 cells which can stably express human wild type andA53T mutantα-synuclein. Methods: The lentivirus containing wild type and A53Tmutantα-synuclein was developed and confirmed by DNA sequencing. Then, it wastransfected into PC12 cells. To identify whether the transfected PC12 cells can stablyexpress theα-synuclein, we detectedα-synuclein-V5 merged protein by using cellularimmunofluorescence staining, confirmed mutant gene sequence by cloning SNCAgene in transfected PC12 cells and sequencing it, measured the level ofα-synucleinexpression in PC12 cells by performing Western Blot, and estimated cell viability byMTT. Results: The lentivirus of pLenti6/V5-SNCA-WT andpLenti6/V5-SNCA-A53T were successfully constructed,α-synuclein-V5 mergedprotein were detected in more than 95% gene-transfected PC12 cells, and thesequence of SNCA-WT and SNCA-A53T gene were confirmed in gene-transfectedPC12 cells by DNA sequencing, the over-expression of SNCA gene were detected ingene-transfected PC12 cells by using Western Blot, and no significant differenceswere detected among the growth curves of three kinds of PC12 cells. Conclusion: Wenot only constructed the lentivirus expression vectors of pLenti6/V5-SNCA-WT andpLenti6/V5-SNCA-A53T, created PC12 cells lines stably expressing wild type andA53T mutantα-synuclein, furthermore, we proved that over-expression of humanWT or A53T mutantα-synuclein had no obvious effects on the cell viability of PC12cells, which lays a good foundation for studying its role in pathogenesis of PD in thefurther research.PartⅡTrehalose Enhances Autophagy of Untransfected and Transfected PC12Cells with Human WT or A53T Mutant SNCA GeneAim: To study whether trehalose can enhance autophagy of untransfected andtransfected PC12cells with human WT or A53T mutant SNCA gene. Methods: Wetreated the cells with trehalose at different concentrations (10mM, 50mM and 100mM)for 24 hours, then detected the change of the number of autophagosome andautolysosome by immunofluorescence and transmission electron microscopy, andanalyzed the expression of LC3Ⅱto investigate whether trehalose could facilitate the autophagy. The expression ofα-synuclein was also analyzed by Western blotting toevaluate whether trehalose could enhance the degradation of WT and A53T mutantα-synuclein. The effects of trehalose on cell viability were judged by MTT assay.Results: Both 50mM and 100mM trehalose significantly increased the number ofautophagosome and autolysosome, up-regulated the expression of LC3Ⅱ(P＜0.01) ,and enhanced the degradation of A53T mutantα-synuclein (50mM trehalose group,P＜0.05; 100mM trehalose group, P＜0.01 ) , but had no obvious effects on theexpression of WTα-synuclein (P＞0.05) . When the cells were pretreated with 2mM3-MA for 3 hours, the autophagy induced by trehalose was inhibited. Both 50mM and100mM trehalose significantly decreased the cell viability of untransfected andtransfected PC12cells with human A53T mutant SNCA gene (P＜0.01) , only 100mMtrehalose reduced the cell viability of transfected PC12cells with human WT SNCAgene (P＜0.05. The reduction of cell viability of transfected PC12cells with human WTSNCA gene induced by trehalose was less than that of the other two types ofPC12cells (P＜0.05) Conclusion: Trehalose could enhance the autophagy ofuntransfected and transfected PC12cells with human WT or A53T mutant SNCA gene,accelerate the degradation of A53T mutantα-synuclein and decrease the cell viabilityof all the three above types of PC12 cells. Trehalose exerted no obvious effects on theexpression of WTα-synuclein. The reduction of cell viability of transfected PC12cells with human WT SNCA gene induced by trehalose was less than that of the othertwo types of PC12 cells.PartⅢThe Effects of Trehalose on The PD cell Models Induced by ProteasomeInhibitorAim: To investigate whether trehalose can enhance the autophagy of the PD cellmodels induced by proteasome inhibitor. Methods: We treated the cells withMG132(500nM), or MG132(500nM) and trehalose of different concentration(10mM、50mM、100mM) for 24 hours, then detected the change of the number ofautophagosome and autolysosome by immunofluorescence, and analyzed theexpression of LC3Ⅱby Western blotting to investigate whether trehalose couldenhance the autophagy of the PD cell models induced by proteasome inhibitor. Theexpression ofα-synuclein was also analyzed by Western blotting to investigatewhether trehalose could facilitate the degradation of the aggregated WT and A53Tmutantα-synuclein. The effects of trehalose on the viability of the cells treated by MG132 (500nM) were evaluated by MTT assay. Results: MG132(500nM) couldsignificantly inhibit the degradation ofα-synuclein (P＜0.05), increase the number ofautophagosome, lysosome and autolysosome. Compared with MG132(500nM) group,Trehalose could significantly enhance the autophagy of the cells induced byMG132(500nM), obviously upregulate the expression of LC3Ⅱ(P＜0.05) , alsofacilitate the degradation of the aggregated A53Tmutantα-synuclein (50mM trehalosegroup, P＜0.05; 100mM trehalose group, P＜0.01) , but had no effects on thedegradation of the aggregated WTα-synuclein induced by MG132. Trehalosesignificantly decreased the viability of the cells treated by MG132 (P＜0.05)Conclusion: MG132 could facilitate the aggregation ofα-synuclein, compensatorilyenhance autophagy. Trehalose could enhance the autophagy of the cells induced byMG132 and accelerate the degradation of the aggregated A53T mutantα-synuclein,but had no effects on the degradation of the WTα-synuclein. Trehalose significantlydecreased the viability of the cells treated by MG132.Summary1. We successfully constructed PC12 cells lines stably expressing wild type and A53Tmutantα-synuclein.2. Trehalose could enhance the autophagy of untransfected and transfected PC12cellswith human WT or A53T mutant SNCA gene, facilitate the degradation of A53Tmutantα-synuclein.3. Trehalose could decrease the cell viability of all three types of PC12 cells.4. Proteasome inhibitor (MG132) could cause the aggregation ofα-synuclein,compensatorily enhance autophagy.5. Trehalose could enhance the autophagy of the cells induced by proteasomeinhibitor (MG132) and accelerate the degradation of the aggregated A53Tmutantα-synuclein6. Trehalose significantly decreased the viability of the cells treated by proteasomeinhibitor (MG132) .更多还原