【作者】 范骏；

【导师】 王光辉；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2008， 博士

【摘要】 在全球范围内,帕金森病(Parkinson’s Disease PD)是发病率仅次于阿尔茨海姆病的神经退行性疾病,60岁以上人群发病率约为1%。在我国,随着社会老龄化的进程,帕金森病已成为最主要的神经退行性疾病之一,严重损害老年人群的生活质量,并给家庭,社会带来沉重的负担。帕金森病的主要病理特征包括位于中脑黑质致密部的多巴胺能神经元的丧失及在残存的神经元中发现的称之为“路易小体”的病理性聚集。该病能导致患者出现肌强直,运动徐缓,静止性振颤等临床症状。帕金森病的确切病因目前还不完全清楚,但诱因主要包括环境因素和遗传因素。从遗传学的角度,帕金森病可分为家族遗传型帕金森病和散发型帕金森病。在家族遗传型帕金森病中,致病基因的突变引起蛋白功能的改变而导致发病。虽然家族遗传型帕金森病的发病率比散发型帕金森病低,但对帕金森病相关致病基因及其蛋白的研究为理解帕金森病的病因病理提供了重要启示,这些研究显示氧化应激、线粒体功能损伤、蛋白酶体系统障碍和细胞凋亡等在帕金森病发生发展过程中发挥着重要的作用。DJ-1最初是作为原癌基因产物被发现的,在荷兰和意大利的两个遗传型帕金森病家族中发现DJ-1的突变与常染色体隐性遗传性帕金森病发病相关。虽然众多的研究显示DJ-1是一个功能多样的蛋白,能够抗氧化应激,调节转录并在一定条件下具有分子伴侣样作用。但到目前为止,DJ-1的功能以及其在帕金森病发病过程中所发挥的作用还不完全清楚。SUMO化是一种重要的翻译后修饰,可以通过调节底物蛋白的稳定性,生物学活性和亚细胞定位而实现调控细胞功能。在阿尔茨海姆病及多聚谷氨酰胺病等神经退行性疾病的病理过程中,SUMO化也扮演着重要的角色。目前已知DJ-1可以在K130位点发生SUMO化修饰,但DJ-1的SUMO化修饰对其正常功能的影响以及在帕金森病的发生发展中的意义还有待研究。DJ-1是作为原癌基因产物被发现,而大多原癌基因产物都具有抗凋亡作用。鉴于此,本论文主要探讨DJ-1是否同样具有抗凋亡功能及DJ-1发挥抗凋亡作用的机制。此外,DJ-1的SUMO化修饰对DJ-1功能的调节,DJ-1的SUMO化修饰在帕金森病病理过程中的意义也是本研究关注的重点。我们目前主要研究结果如下:1.在凋亡诱导剂的刺激下,过表达DJ-1的细胞能够在细胞形态上保持完整并抑制凋亡下游因子半胱天冬酶(Caspase)-3和Caspase-9的激活。进一步研究发现,凋亡通路上的重要因子Bax蛋白在过表达DJ-1细胞中的表达水平减少,而这一现象在含有p53蛋白的细胞中可以很好的重现,但在p53缺失型的细胞中丧失,提示DJ-1能够下调Bax水平并且这种能力与p53密切相关。我们又用基因敲减技术,下调DJ-1的表达。结果我们观察到随着DJ-1的下调,Bax表达也随之上升,而且这种现象也依赖于p53的存在。利用体外Pulldown实验和体内的免疫共沉淀实验,我们发现DJ-1能够在细胞核内与p53的DNA结合区域及C末端相互结合。接下来的报告基因实验结果显示DJ-1能够抑制p53针对Bax的转录活性。这些结果提示,DJ-1在细胞核内通过与p53结合,可能影响到p53与Bax启动子的结合,干扰了p53对Bax的转录,从而抑制了下游凋亡通路的发展。2.在我们的实验系统中,我们也发现DJ-1能够被SUMO在K130位点上修饰。丧失SUMO化位点的突变体,DJ-1(K130R),从细胞核内转位到细胞质,并丧失了下调Bax、阻断半胱天冬酶激活及抑制p53转录活性等抗凋亡的能力。这些结果提示,SUMO化修饰是DJ-1执行抗凋亡及抑制p53功能的必要前提条件。DJ-1的细胞核定位在其抗凋亡机制也具有重要的意义。综合以上结果,我们可以看出,SUMO化的DJ-1能够进入细胞核,与p53结合后,可能干扰了p53与Bax启动子的结合,抑制了p53对Bax的转录,表现为Bax蛋白水平的降低和下游凋亡通路的阻断。一旦DJ-1不能被正常SUMO化,则被滞留在细胞质中无法进入细胞核去和p53结合,不能发挥对Bax的转录抑制作用,继而丧失了抗凋亡的能力。总之,正常SUMO化的DJ-1通过对Bax的转录抑制阻断凋亡通路而实现对细胞的保护作用。这一发现可能为我们更好地了解帕金森病的发病机制,寻求治疗帕金森病的方法提供一些帮助。更多还原

【Abstract】 Parkinson’s disease (PD), the second common neurodegenerative diseases following Alzheimer’s disease in the world, affects 1% people over sixty years old. In China, PD has been one of the most popular neurodegenerative disorders, destroying the patients’ lives and bringing a heavy burden to the family as well as the society. PD is a progressive movement disorder that results primarily from the death of dopaminergic neurons in the substantia nigra and the presence of some kind of protein aggregates termed Lewy Body, a hallmark of PD, in surviving neurons. The loss of dopamine production in the brain causes the primary symptoms of Parkinson disease including tremor, slowness of movement, rigidity and difficulty with balance. Up to now, the etiology of PD has still been a myth. Environmental factors and genetic factors are believed to contribute to the onset of the PD. From the genetic point of view, PD can be either sporadic form or familial form. In familial PD, the inheritable mutations in the PD-related gene produce the mutant protein which alteres the normal function of its wild type. Although most cases of PD are sporadic, the study on the rare familial PD promotes our understanding of the pathogenic pathway. These studies suggest that oxidative stress, defect in mitochondria, dysfunction in proteasome system and apoptosis play a vital role in the pathology of PD.DJ-1 was originally identified as an oncogene product. Recently, loss of DJ-1 function due to the mutations and deletion in DJ-1 gene is found to be linked with autosomal recessive early onset PD in populations of various ethnic origins. Although increasing studies reveal that DJ-1 has multiple roles in various biological processes, the exact function of DJ-1 remains unclear.Sumoylation has emerged as an important posttranslational modification, which is also involved in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and polyglutamine diseases. Sumoylation regulates the substrate stability, biological activities and its subcellular localization. DJ-1 is able to be sumoylated at K130 but the effect of this modification on the normal function of DJ-1 remains to be elucidated.As most of the oncogene products are able to inhibit apoptosis, in this thesis, we tried to determine the role of DJ-1 in anti-apoptosis as well as the mechanisms in which DJ-1 protects cell from death. In addition, we also focus on the regulation of DJ-1 by sumoylation and the effect of sumoylated DJ-1 in the pathogenic pathway of PD. The main findings of our study are listed as below.1. Under stimulus, overexpression of DJ-1 is able to protect cell from death by inhibiting the cleavage of caspase-9 and caspase-3. Furthermore, Bax, an important factor in the apoptosis pathway, is down-regulated in the cells expressing DJ-1. This result is reproducible in several cell lines harboring p53 but not in p53 null cell line. In addition, knock-down of DJ-1 increase Bax level in a p53 dependent manner. These results imply that the regulation of Bax by DJ-1 is associated with p53 status. Using pull-down and immunoprecipitation experiments, we show that DJ-1 is able to bind to p53 through its DNA-binding domain and C-terminus. Moreover, the results of the report gene assay imply that DJ-1 is able to inhibit p53 transcriptional activity on Bax promoter. In summary, we demonstrate here that DJ-1 exerts its cytoprotection through a p53-Bax-caspase pathway. DJ-1 decreases the Bax protein level and therefore blocks the caspase activation by possibly repressing p53 transcriptional activity.2. In our experimental system, we also observed that DJ-1 is able to be sumoylated at K130. However, a non-sumoylatable mutant of DJ-1, DJ-1(K130R), which shifts from nucleus to cytoplasm, loses its ability to repress p53 transcriptional activity on Bax promoter as well as that of rescuing cell death. In this regard, proper sumoylation of DJ-1 is suggested to be important for its targeting nucleus to regulate p53 activity.Taken together, our findings suggest that DJ-1 rescues cell death through p53-Bax-caspase pathway. DJ-1 binds to p53 physically in the nucleus, decreases Bax expression by attenuating p53 transcriptional activities, inhibits subsequent caspase activation and finally rescues cell death. Whereas, the non-sumoylatable K.130R mutant of DJ-1, loses its cytoprotective effect due to the sequestration in cytoplasm. As loss of DJ-1 function is responsible for the onset of PD, the findings here will be of help to expand our knowledge of DJ-1 functions, as well as its possible role in PD.更多还原