【作者】 刘康永；

【导师】 刘春风；

【作者基本信息】 苏州大学 ， 神经病学， 2008， 博士

【摘要】 第一部分突变型α-synuclein的自噬性降解途径及可能机制目的观察PC12细胞中突变型α-synuclein与自噬之间的关系和可能的降解途径,探讨其在帕金森病发病机制中的作用。方法采用RT-PCR方法从人胎脑中克隆野生型α-synuclein基因,采取T-A克隆基因并测定其序列,在此基础上利用单核苷酸差异引物定点诱变构建α-synuclein同义突变基因和致病突变A30P的α-synuclein基因并进行测序;对转染了α-synuclein(A30P)的PC12细胞进行药物干预,检测细胞的增殖活性和CAT、SOD的水平,还采用透射电镜观察细胞超微结构改变以及自噬的特征行改变,同时检测α-synuclein、LC3的蛋白的表达。结果突变型α-synuclein激活了自噬途径,并加剧了MPP~+的毒性作用,对于转染突变型α-synuclein(A30P)的PC12细胞,形态学上明确有自噬现象的发生,自噬确实参与了变异型α-synuclein(A30P)的降解,从分子生物学角度也检测到LC3-Ⅱ在转染突变型α-synuclein(A30P)的PC12细胞中的表达。自噬抑制剂Wortmannin可加剧α-synuclein积聚和导致细胞死亡, LC3-Ⅱ的表达也受到抑制,而自噬诱导剂Rapamycin则可以促进α-synuclein的降解和细胞生长,LC3-Ⅱ的表达显著增强。结论转染突变型α-synuclein(A30P)的PC12细胞在形态学上明确有自噬现象的发生,在MPP+作用下,突变型α-synuclein的表达水平明显增高,α-synuclein的异常积聚导致PC12细胞的死亡,而促进自噬有助于突变型α-synuclein的降解,对细胞具有保护作用。第二部分突变型α-synuclein(A30P)在氧化应激状态下的降解途径和可能机制目的观察突变型α-synuclein(A30P)在氧化应激状态下的降解途径,探讨其在帕金森病发病机制中的作用和可能机制。方法利用MPP~+对转染了α-synuclein(A30P)的PC12细胞进行干预,模拟在氧化应激状态下的PD细胞模型,在此基础上,分别给予自噬诱导剂—Rapamycin和抑制剂—Wortmannin,检测细胞的增殖活性,还采用透射电镜观察细胞超微结构改变以及自噬的特征行改变,同时检测α-synuclein、LC3蛋白的表达和细胞培养基中CAT、SOD的水平。结果在氧化应急状态下(MPP~+),突变型α-synuclein的表达明显增加,自噬水平显著减低;细胞超微结构显示:线粒体肿胀、空泡化,嵴少,内质网扩张,细胞核皱缩,染色质有边集,细胞有凋亡的倾向。而联合使用自噬抑制剂-Wortmannin处理过细胞,细胞培养液中CAT和SOD的水平较单独应用MPP~+的细胞有显著降低,细胞增殖活性明显降低,细胞死亡明显增加;α-synuclein的表达明显增加,自噬水平显著减低;细胞超微结构显示:线粒体肿胀,嵴少,内质网扩张,细胞核皱缩,染色质有边集,并可见凋亡小体。相反,联合使用自噬诱导剂-Rapamycin提高了细胞培养液中CAT和SOD水平,促进了细胞的增殖和分化;α-synuclein的表达明显减低,自噬水平显著增高;细胞超微结构显示:胞质中可见多个具有特征性双层膜结构的自噬小体,胞核、线粒体正常。结论在氧化应激状态下,突变型α-synuclein(A30P)对外源性毒素的敏感性明显增高,α-synuclein聚集更为显著,自噬水平明显减低,从而导致自噬应激;提高自噬水平有助于突变型α-synuclein的降解,对细胞具有保护作用。第三部分Rapamycin治疗帕金森病小鼠模型的实验研究及可能机制目的探讨自噬诱导剂—Rapamycin对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的帕金森病(PD)小鼠模型是否具有治疗作用及可能机制。方法Rapamycin采用静脉直接给药对MPTP诱导的PD小鼠模型进行治疗,而后采用免疫组织化学及蛋白印迹分析(Western blot)的方法对PD相关标志物酪氨酸羟化酶(TH)以及α-synuclein的可能改变进行检测,并采用高效液相色谱-电化学检测仪(HPLC-ECD)检测纹状体单胺类递质的可能变化;还采用透射电镜观察细胞超微结构改变以及自噬的特征行改变。为进一步研究自噬在异常积聚的α-synuclein降解过程中的作用,我们对其中一个关键的自噬基因-微管相关蛋白轻链3(microtubule-associated protein light chain 3, LC3)进行检测分析。结果(1)黑质免疫组化染色显示:NS治疗组较正常对照组TH阳性细胞数明显减少(p=0.003), Rapamycin治疗组可逐渐恢复TH阳性细胞损失;(2)纹状体Western blot结果显示:Rapamycin治疗组较NS治疗组TH表达明显增多;Rapamycin治疗组与NS治疗组比较α-synuclein明显下降(p=0.004);Rapamycin治疗组较NS治疗组LC3明显升高(p=0.003)。(3) HPLC-ECD显示DA及其代谢产物DOPAC在NS治疗组明显下降,而其他单胺类递质则无明显改变;治疗组DOPAC含量较NS治疗组明显升高(p=0.001),与正常对照组无明显差异。(4) NS治疗组较正常对照组DAT和VMAT-2表达明显减少,而Rapamycin治疗组较NS治疗组DAT和VMAT-2表达明显增多,但是通过对比,我们可以发现:Rapamycin治疗组较NS治疗组DAT和VMAT-2升高的程度有明显的差异,与正常对照组比较,Rapamycin治疗组DAT的表达仍低于对照组,而VMAT-2表达却明显高于对照组。DAT和VMAT-2对MPTP的敏感性不同,在经过MPTP后,二者对Rapamycin的反应也存在明显的差异。结论静脉注射Rapamycin可以对MPTP诱导的小鼠PD模型产生明显的治疗作用; Rapamycin不但可以减少DA能神经元死亡,还能够恢复DA能神经元的功能,从而增加了DOPAC的含量;Rapamycin的治疗作用可能与促进异常积聚的蛋白α-synuclein的降解有关。更多还原

【Abstract】 Part I Autophagic pathway and probable mechanism in the degradation of mutantα-synuclein in PC12 cellsOBJECTIVE: The accumulation of mutantα-synuclein plays a major role and morphologic evidence of autophagy has been reported in Parkinson disease. We aim to observe the effect of mutantα-synuclein (A30P) in autophagic programmed cell death by transfected PC12 cells and explored its probable role and pathway in PD.METHODS: We constructed definite PC12 cells which were transfected mutantα-synuclein (A30P) at first and administrate MPP+, Rapamycin and Wortmanin to transfected PC12 cells with mutantα-synuclein. We not only detected proliferative activity of cells with MTT method but also observed the ultrastructure changes of cells and expression ofα-synuclein in different circumstance by Transmission electron microscopy (TEM), Western Blotting and and the level of SOD.RESULTS: Mutantα-synuclein (A30P) leading to PC12 cells death involvesα-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutantα-synuclein (A30P) mediates the toxicity of MPP+. Rapamycin, an inducer of autophagy, reduces the aggregation ofα-synuclein in transfected cells. Meanwhile, Wortmanin, an inhibitor of autophagy, promotes the aggregation ofα-synuclein in transfected cells and induces cells to die.CONCLUSION: The abnormal aggregation ofα-synuclein induces autophagic programmed cell death in PC12 cells and mutantα-synuclein (A30P) mediates the toxicity of MPP~+. Meanwhile, Rapamycin may reduce the aggregation ofα-synuclein in transfected cells by activation of autophagic pathway. Part II Mutant -synuclein (A30P)are degraded by autophagic pathway and mechanism under oxidative stressObjective The accumulation of mutantα-synuclein plays a major role and morphologic evidence of autophagy has been reported in Parkinson disease. We aim to observe the effect of MPP~+ to mutantα-synuclein (A30P) in autophagic programmed cell death by transfected PC12 cells and explored its probable role and degradative pathway in PD. Method We constructed definite PC12 cells which were transfected mutantα-synuclein (A30P) at first and administrate MPP~+ to establish the the model of PD with cells under oxidative stress. Rapamycin and Wortmanin were administered to transfected PC12 cells with mutantα-synuclein respectively . We not only detected proliferative activity of cells with MTT method but also observed the ultrastructure changes of cells by Transmission electron microscopy (TEM). Meanwhile expression ofα-synuclein and LC3 in different circumstance was examined by Western Blotting. Moreover, the levels of CAT and SOD in cell cultures were also compared in order to discuss the relation between oxidative stress and autophagy. Results MPP~+ to mutantα-synuclein (A30P) leads to PC12 cells death by means of autophagy involvesα-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutantα-synuclein (A30P) mediates the toxicity and enhances the sensitivity of MPP~+ . Rapamycin, an inducer of autophagy, reduces the aggregation ofα-synuclein in transfected cells. Meanwhile, Wortmanin, an inhibitor of autophagy, promotes the aggregation ofα-synuclein in transfected cells and induces cells to die. Conclusion Mutantα-synuclein (A30P) mediates the toxicity of MPP~+. Meanwhile, inducer of autophagy-Rapamycin may reduce the aggregation ofα-synuclein in transfected cells by activation of autophagic pathway. Part III Therapeutic effect and probable mechanisim of Rapamycin on MPTP-induced parkinsonism in miceOBJECTIVE: To investigate the effects of inducer of autophagy-Rapamycin on the functional and morphological outcome in a mice model of Parkinson s disease (PD) rendered by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).BACKGROUND: In neurodegenerative disorders such as PD, autophagy participates in the cell death process of dopaminergic neuron.α-Synuclein is one of the major components of Lewy bodies and Lewy neuritis and mutation ofα-synuclein such as a-synuclein mutants (A53T and A30P) may be involved in the development of familial PD. Our previous study demonstrated that Rapamycin may induce autophagy and decrease aggregation ofα-synuclein and cell death.METHODS: Male C57BL mice were treated with Rapamycin(3mg/kg) by i.v for 7 days after MPTP administration(30mg/kg.14d), and were compared with saline-treated PD mice and normal control group. Immunohistochemistry and Western blot were used to detect the alterations of PD biomarker including tyrosine hydroxylase (TH), and the level of autophagy was investigated by microtubule-associated protein light chain 3(LC3) andα-synuclein cleavage. In addition, monoamine neurotransmitters in the striatum of mice were measured by the high performance liquid chromatography (HPLC).RESULTS: TH immunohistochemistry indicated that the number of dopaminergic neurons in the substantia nigra was increased in Rapamycin-treated mice compared with saline-treated mice (p=0.003). Western blot demonstrated the similar TH protein expression in striatum as the number of dopaminergic neurons.α-Synuclein was markedly decreased in Rapamycin-treated mice compared with the NS-treated mice(p=0.004) and LC3 was markedly increased in Rapamycin-treated mice compared with the NS-treated mice(p=0.003). Meanwhile, DAT and VMAT-2 were markedly decreased in Rapamycin-treated and NS-treated mice compared with control group. Although the degree was different, DAT and VMAT-2 were markedly increased in Rapamycin-treated compared with NS-treated mice. The concentrations of striatal dopamine and its metabolite DOPAC compared to the normal mice were decreased significantly in other groups. Rapamycin-treated mice minimized the reduction of DOPAC compared with the NS-treated mice (p=0.001).CONCLUSIONS: These results showed that Rapamycin is able to rescue the dopaminergic neurons in different doses and reverses the loss of DOPAC through activation of autophagy-lysosome pathway, and it may be a promising therapeutic agent in PD.更多还原