【作者】 马开利；

【导师】 陈乃宏；

【作者基本信息】 北京协和医学院 ， 药理学， 2011， 博士

【摘要】 帕金森氏病(PD, parkinson’s disease)是临床最常见的神经系统退行性疾病之一,65岁以上人群的发病率为1%,我国大约有170万帕金森氏病患者,随着人口的老龄化的加剧,发病人数还将呈现出逐年上升的趋势。到目前为止,在大多数情况下帕金森氏病的病因仍然不是很清楚。1913年Frederick Lewy首次证明路易氏小体(LB, Lewy body)是帕金森氏病病人所具备的病理特征。路易氏小体的主要成分是一种以非折叠形式存在并且具有高度聚集倾向性的蛋白,即a-synuclein。有很多研究表明,a-synuclein存在于神经元的胞核内。尽管如此,直至今日仍然没有a-synuclein在细胞核内确切功能的报道。是否核定位是a-synuclein的一种普遍特性,还是PD的病理结果,到目前为止仍然是个迷。在本研究中,我们试图解决以下三个方面的问题：①对a-synuclein核输入起决定性作用的序列；②α-synuclein进入细胞核的方式；③a-synuclein在细胞核内对细胞功能的影响。首先,我们应用现代生物信息学的方法对a-synuclein的核定位信号以及核输入相关的片段进行预测,并应用PCR (polymerase chain reaction)的方法获得一系列与a-synuclein核输入相关的片段,并构建入pEGFP-N1真核表达载体系统,转染PC12细胞后在细胞水平寻找对a-synuclein核输入起决定性作用的序列片段。其次,在确定核输入方式上,我们采用了核输入抑制与RNA干扰(RNAi, RNA interference)相结合的方法,同时应用免疫荧光、免疫沉淀、实时荧光定量PCR (Real Time PCR, real-time quantitative polymerase chain reaction)以及WB (western blot)对a-synuclein的核输入方式进行验证。最后,为了探讨a-synuclein在细胞核中所发挥的作用,我们分别构建了a-synuclein与一个核定位序列(NLS, nuclear localization signal)以及a-synuclein与一个核输出序列(NES, nuclear export signal)融合表达的载体。转染PC12细胞并经G418稳定筛选后获得了a-synuclein分别在细胞核或细胞浆集中分布的稳定细胞株,分别从生长特性、形态学分析、细胞周期分布、对神经毒性剂敏感性等方面研究a-synuclein在细胞核集中分布所发挥的作用。同时,我们利用酵母表达纯化了a-synuclein-GFP融合蛋白,并应用圆二色谱研究a-synuclein对DNA的影响。结果表明,a-synuclein的N端和C端都在其核输入过程中发挥一定作用,并且其进入细胞核需要importin a的介导并在Ran水解GTP提供能量的情况下才能完成。a-synuclein进入细胞核后对细胞形态几乎没有影响,但会导致细胞生长速度减慢以及细胞周期的延迟,并进一步增加细胞对神经毒性剂MPP+(1-methyl-4-phenylpyridinium)的敏感性。另外,圆二色谱结果表明,两个α-synuclein分子能够与一个类GC box样单核苷酸序列结合。综上所述,α-synuclein可能在其N端和C端片段共同介导下,首先与importinα结合,并进一步与importinβ结合后形成核输入复合物,在Ran水解GTP提供能量的情况下,通过核孔复合物进入到细胞核内。α-synuclein进入细胞核后,能与转录因子SP1 (specificity protein 1)竞争性结合启动子区上富含GC的区域,从而导致转录因子SPl所介导的E2F家族蛋白与启动子区上富含GC的DNA序列相结合并进一步形成cyclins和cdks复合物的过程受阻,从而引起细胞周期调控的异常,进一步导致其对神经毒性剂MPP+的敏感性增加。更多还原

【Abstract】 PD (parkinson’s disease) is one of the most common neurodegenerative diseases, which affect 1%of the people over 65 years old. There are 170 million PD patients in our country, the figure will increase year by year with the aggravating of aging. So far, the pathogen of PD is unclear in most case. The LB, which was first described by Frederick Lewy in 1913, is present in essentially all cases of PD. The major protein constituent of LBs is alpha-synuclein (a-synuclein), a natively unfolded protein having high propensity for aggregation. Several studies have shown the presence of a-synuclein in the neuronal nuclei. However, no definite nuclear functions have been attributed to a-synuclein to date. It is not known if nuclear localization is the common property of a-synuclein or it is cause/consequence of PD pathology.In this study, we sought to address the following three aspects:the protein domains responsible for the nuclear import of a-synuclein; the way of a-synuclein import into the nuclear; the effect on cell function of a-synuclein in the nuclear.First of all, the bioinformatics were used to predict both the NLS in a-synuclein and the protein domains responsible for the nuclear localization of a-synuclein. Using deletion mutant forms of a-synuclein, we identified protein domains that are responsible for the observed nuclear import of a-synuclein in PC 12 cells.Secondly, the nuclear import inhibition、RNAi (RNA interference)-. Immunofluorescence、Immunoprecipitatio、Real Time PCR (real-time quantitative polymerase chain reaction) and WB (western blot), were used to explore the way of a-synuclein import into the nuclei.Finally, in order to examine the ability of a-synuclein in the nucleus, we created constructs of a-synuclein tagged with either a nuclear localization sequence (NLS) or a nuclear export sequence (NES). We used these constructs to transfect PC12 cells, and screened with G418, then the stable cell lines which express a-synuclein are located in the nucleus or cytoplasm were acquired. The growth curve morphological analysis、cell cycle sensitivity to neurotoxic agents (MPP+) of the stable cell lines were analyzed. The a-synuclein-GFP fusion protein was expressed and purified with yeast, then the circular dichroism (CD) was used to study the effect of a-synuclein on DNA.The results showed that, both the N terminal and the C terminal were responsible for the nuclear import of a-synuclein. a-synuclein could import into the nuclei through nuclear pore complex (NPC), mediated by importin a and provided energy by hydrolysis of GTP. There was no effect on cell morphological whenα-synuclein import into the nuclei, but both the growth curve and the cell cycle were delayed. And the PC 12 cells with a-synuclein localization in nuclei were sensitivity to neurotoxic agents (MPP+). Moreover, the results of the circular dichroism showed that twoα-synuclein molecules were prone to combine with one GC box-like single nucleotide sequence.In summary, mediated by both the N terminal and the C terminal,α-synuclein could combine with importin a and importinβ, then import into the nuclei through nuclear pore complex (NPC), provided energy by hydrolysis of GTP.α-synuclein could compete with the transcription factor SP1 for the same GC rich region in promoter, then the process of both the E2F family protein interaction with the GC rich region mediated by the transcription factor SP1 and the cyclins/cdks complex formation were inhibited, so the cell cycle was disturbed, it led to the PC 12 cells with a-synuclein localization in nuclei more sensitivity to neurotoxic agents (MPP+).更多还原