【作者】 杨昉；

【导师】 刘春风；

【作者基本信息】 苏州大学 ， 神经病学， 2009， 博士

【摘要】 第一部分自噬和蛋白酶体降解途径在过表达人α-synuclein细胞凋亡中的作用目的利用建立好的转染过表达野生型(WT)及突变型A53T、A30Pα-synuclein的大鼠嗜铬细胞瘤(PC12)细胞株,探讨自噬和泛素-蛋白酶体通路在细胞凋亡途径中的具体作用。方法以蛋白酶体抑制剂环氧酶素(epoxomicin,EPO)和/或大自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)、大自噬诱导剂雷帕霉素(rapamycin,RAP)分别处理各株细胞,然后通过MTT法检测细胞活力,光镜观察活细胞一般形态学变化,流式细胞仪检测各组细胞凋亡率,并测定caspase 3蛋白表达,采用透射电镜方法观察细胞超微结构改变。结果(1)过表达WT、A30P、A53Tα-synuclein PC12细胞经3-MA、环氧酶素、环氧酶素+3-MA和环氧酶素+雷帕霉素处理24小时后细胞活力降低,雷帕霉素组处理细胞24小时后,细胞活力显著高于其余用药组;(2)3-MA、环氧酶素、环氧酶素+3-MA作用24小时后细胞的凋亡百分率显著增高,而雷帕霉素可降低环氧酶素导致的凋亡率增加;(3)Caspase 3活性片段蛋白检测发现3-MA组、环氧酶素组和环氧酶素+3-MA组条带较为明显;(4)各组细胞超微结构显示环氧酶素处理WT、A30P、A53Tα-突触核蛋白PC12细胞的细胞内自噬体和凋亡现象增多;3-MA处理后,WT、A30P、A53T组细胞可见各种凋亡改变,并且自噬囊泡减少;雷帕霉素可使细胞自噬增加,无明显凋亡改变。结论过表达WT、A30P、A53Tα-synuclein PC12细胞内大自噬和蛋白酶体途径障碍促进了凋亡发生,诱导自噬可抑制凋亡的发生。第二部分过表达人α-synuclein细胞中不同自噬途径间及其和蛋白酶体途径之间的交互联系目的研究过表达人野生型及致病突变A53T、A30P的α-synuclein的PC12细胞中不同自噬形式和蛋白酶体途径间的关系,并了解它们在α-synuclein代谢中的作用。方法Western blot检测α-synuclein、LC3、lamp-2a、E1和E2酶蛋白表达水平;激光共聚焦显微镜下观察各组PC12细胞株α-synuclein与LC3的免疫荧光表达;多功能酶标仪检测药物干预后各组细胞蛋白酶体活性。结果蛋白酶体抑制剂环氧酶素(epoxomicin)和大自噬抑制剂3-MA都可导致α-突触核蛋白的降解减少,大自噬刺激剂雷帕霉素(rapamycin)和环氧酶素合用可缓解环氧酶素导致的α-synuclein降解减少;蛋白酶体抑制剂环氧酶素增加了细胞大自噬特异标志物微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)的表达;同时3-MA、环氧酶素和雷帕霉素均可增加分子伴侣介导的自噬中的关键限速酶溶酶体相关膜蛋白-2a(lysosome-associated membrane protein-2a,lamp-2a)的表达。大自噬抑制剂3-MA降低了糜蛋白酶样蛋白酶体活性,而雷帕霉素却有使糜蛋白酶样蛋白酶体活性增加的趋势。检测蛋白酶体途径中E1活化酶、E2结合酶的表达提示3-MA和环氧酶素可使E2酶的表达增加。结论抑制蛋白酶体和大自噬途径都可导致α-synuclein的代谢异常,同时抑制蛋白酶体可激活大自噬和CMA途径,而抑制大自噬途径可导致CMA的激活但却下调了蛋白酶体活性,诱导大自噬同时可上调CMA并可能增加蛋白酶体活性。第三部分蛋白酶体和自噬途径缺陷时过表达A30P、A53T和WTα-synuclein PC12细胞氧化应激状态目的观察蛋白酶体功能和自噬缺陷时过表达人野生型及致病突变A30P、A53Tα-synuclein的PC12细胞的氧化应激反应。方法选择特异性蛋白酶体抑制剂和大自噬抑制剂及诱导剂作用于稳定转染的野生型(WT)、A30P、A53T细胞株,并以检测试剂盒测定细胞培养液中一氧化氮(Nitric oxide,NO)、超氧化物歧化酶(superoxide dismutase,SOD)的活力和Western blot法检测细胞内热休克蛋白70(heat shock protein 70,hsp70)及过氧化氢酶(catalase,CAT)蛋白表达。结果抑制蛋白酶体途径可增加A30P、A53T细胞细胞培养液中NO水平;同时抑制蛋白酶体途径和诱导大自噬增加了三株细胞的细胞培养液中超氧化物歧化酶(SOD)的活力;而大自噬抑制剂处理后细胞SOD活力低于对照组。Hsp70和CAT的蛋白表达研究亦显示抑制蛋白酶体途径导致hsp70和CAT蛋白含量明显高于对照组。结论与自噬途径相比,蛋白酶体途径和氧化应激的关系更紧密,也就是说干预蛋白酶体通路比干预自噬对氧化应激状态的影响更大,作为PD发病机制之一的氧化应激在蛋白酶体途径障碍时的作用比自噬缺陷时显得更为重要。更多还原

【Abstract】 PartⅠRole of autophagy and proteasome degradation pathways in apoptosis of PC12 cells overexpressing humanα-synucleinObjective Parkinson’s disease is a common neurodegenerative disease in the aged people. Its causative agent and mechanisms are not clearly understood by now. Ubiquitin-proteasome and autophagy pathways play important roles in the pathology of Parkinson’s disease. We aimed to study the different roles of the proteasome and autophagy pathways in apoptosis.Methods In this study, a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate pheochromocytoma (PC12) cell lines transfected with human mutant (A30P and A53T) and wild-type (WT)α-synuclein. Cell viability was measured by means of MTT methods. The apoptosis ratio was assessed by flow cytometry. Caspase 3 expression in cell culture were determined by western blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy.Results We found that the proteasome inhibitor epoxomicin and the macroautophagy inhibitor 3-MA significantly impaired cell viability. Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P, A53T and WT cells was significantly higher after treatment with inhibitors of the proteasome and macroautophagy. The results of western blots for caspase 3 expression were similar to those of flow cytometry; The hallmarks of apoptosis and autophagy in our cell lines using electron microscopy after 24 h drug treatment also showed that the proteasome inhibitor epoxomicin increased apoptosis and autophagy and the macroautophagy inhibitor also increased apoptosis.Conclusion These findings show that inhibition of the proteasome and autophagy promotes apoptosis, and the macroautophagy stimulator rapamycin reduces the apoptosis ratio.PartⅡCrosstalk between proteasome system and autophagy in PC12 cells overexpressing humanα-synucleinObjective Parkinson’s disease (PD) is a common degenerative disorder of the central nervous system; the pathology includes the loss and degeneration of dopaminergic neurons and the formation of Lewy bodies in neurons. Recent studies found thatα-synuclein is the main component of Lewy bodies. Ubiquitin-proteasome and autophagy pathways play important roles in the degradation ofα-synuclein. So we aimed to study the different roles of the proteasome and autophagy pathways in the degradation ofα-synuclein, and the crosstalk between these proteasome and autophagy pathways.Methods In this study, PC12 cells, in which exogenous human wild-type or A30P, A53Tα-synuclein were overexpressed, were treated with a proteasome inhibitor and/or a autophagy inhibitor or a stimulator.Results We measured an increase inα-synuclein levels after treatment with the proteasome or autophagy inhibitors in all cell lines. The combination of epoxomicin with rapamycin decreased theα-synuclein accumulation induced by epoxomicin alone. In the presence of proteasome inhibitors, levels of LC3-II in cells were higher. We confirmed that the increase in lamp-2a was related to the inhibition of proteasome or macroautophagy. Interestingly, compared with the control, cells grown in medium with rapamycin also had an increased lamp-2a/β-actin ratio. The macroautophagy inhibitor 3-MA decreased chymotrypsin-like proteasome activity compared with control, and rapamycin tended to increase chymotrypsin-like proteasome activity. E1 enzyme expression did not change in both types of cells. 3-MA and epoxomicin increased E2 enzyme expression, especially when used together.Conclusion Proteasomal inhibitor and macroautophagy inhibitor decreaseα-synuclein clearance, and proteasomal inhibition results in compensatory activation of macroautophagy and chaperone-mediated autophagy(CMA). Our findings also suggest that macroautophagy inhibitor increase the expression of lamp-2a, then CMA is likely activated by inhibition of macroautophagy. In addition, in cells with macroautophagy inhibited, proteasome activity was down-regulated. Our results demonstrate the existence of crosstalk between different forms of autophagy and between autophagy and the proteasome pathway.PartⅢOxidative stress in PC12 cells overexpressing human wild type, A30P and A53Tα-synucleinObjective To study the changes of oxidative stress in cells subjected to impairment of the proteasome pathway and macroautophagy.Methods At 24h after drug treatment, the supernatant was collected and NO and SOD activity measured with a NO or SOD determination kits. And, western blot determined hsp70 and catalase expression.Results We found that the proteasome inhibitor epoxomicin increased NO levels in A30P and A53T cells, but the levels in WT cells did not changed with proteasome and autophagy drugs. And we also found that the proteasome inhibitor epoxomicin increased SOD levels in A30P, A53T and WT cells. High levels of hsp70 protein expression occurred in groups of cells treated with the proteasome inhibitor epoxomicin for 24 h. And the proteasome inhibitor epoxomicin increased the levels of CAT in cells. We did not find a significant change of CAT and hsp70 in cells after treatment with a macroautophagy inhibitor and a stimulator, but the effect of macroautophagy inhibition and stimulation slightly differed with the type of cell.Conclusion All these data indicate that the proteasome system is more tightly related to oxidative stress than autophagy. Oxidative stress as a mechanisms of PD during inhibition of the proteasome was more important than it during inhibition of autophagy.更多还原