台灣族群帕金森氏症Leucine-Rich Repeat Kinase 2 (LRRK2) 基因變異的分子功能研究

Molecular Characterization of LRRK2 Variations in Taiwanese Parkinson’s Disease

張文騰(Wen-Teng Chang)

指導教授 ： 李桂楨

國立臺灣師範大學/理學院/生命科學研究所/碩士(2011年)

摘要

PARK8基因座的LRRK2 (全名leucine-rich repeat kinase 2)基因，其突變會導致體染色體顯性的帕金森氏症。LRRK2蛋白具有多個功能區域，於神經系統、大部份器官及淋巴細胞等都有表現。新穎的R767H、S885N變異及已報導過的R1441H變異，是本實驗室在臺灣帕金森氏症患者發現的突變點，R1628P和G2385R兩變異則是華人帕金森氏症的危險因子。本論文首先建構EGFP標記的野生型及R1441H、R1628P、G2385R變異的LRRK2質體(含S1647T、M2397T多型性)，送到HEK-293T細胞中表現，經共軛焦顯微鏡觀察、西方轉漬分析研究，顯示上述變異對LRRK2蛋白在細胞內的位置及合成皆無受到影響。其次選殖α-synuclein cDNA，與R767H、S885N、R1441H、G2019S (白種人常見，作為對照)突變的LRRK2質體共轉染入SK-N-SH細胞，經細胞免疫螢光染色及共軛焦顯微鏡觀察，顯示野生型LRRK2廣泛分佈在細胞質並與α-synuclein有些微連繫。突變的R767H、S885N、R1441H、G2019S亦主要分佈在細胞質，R1441H和G2019S的LRRK2容易形成不含α-synuclein且鄰近細胞核的聚集。進一步的表現上述LRRK2質體於HEK-293T細胞二至六天後，螢光顯微鏡觀察及定量統計結果顯示R1441H和G2019S較野生型的LRRK2顯著地容易誘導更多的包涵體。最後，選殖V5標記的ARHGEF7 cDNA，並建構不含S1647T、M2397T多型性的Myc-His標記的LRRK2質體，共轉染入HEK-293T細胞，進行免疫共沉澱與GTP結合能力試驗的分析，結果發現S885N、R1441H、G2019S等突變影響LRRK2蛋白與ARHGEF7的交互作用。

關鍵字

帕金森氏症 ； 神經退化性疾病 ； 震顫素

並列摘要

Mutations in PARK8 associated leucine-rich repeat kinase 2 (LRRK2) have been shown to be the leading cause of autosomal dominant Parkinson's disease (PD). The multidomain LRRK2 is expressed ubiquitously, including the central nervous system and various organs. Previously novel R767H, S885N and reported R1441H were found in Taiwanese PD patients, in addition to R1628P and G2385R risk factors in ethnic Chinese populations. In the first part of this study, EGFP-tagged wild type, R1441H, R1628P and G2385R LRRK2 constructs (with S1647T and M2397T SNPs) were prepared for transient expression in HEK-293T cells. Western blot analysis and fuorescence microscopy examination revealed that neither localization nor processing of LRRK2 was affected by R1441H, R1628P and G2385R variations. Secondly, α-synuclein cDNA was cloned and co-transfected with the EGFP-tagged wild type or mutant (R767H, S885N, R1441H, G2019S) LRRK2 constructs in SK-N-SH cells. Confocal microscopy examination revealed that wild-type LRRK2 was widespread cytoplasmic and partially in association with α-synuclein. The distribution of R767H and S885N proteins were also mainly in cytoplasm. In contrast, both R1441H and G2019S (included as an aggregation control) LRRK2 mutants formed α-synuclein-negative perinuclear aggregates in a smaller, but still appreciable, proportion of cells, in addition to cytoplasmic distribution. Fluorescent microscopy examination and quantitation of the number of cells with LRRK2-EGFP fluorescent aggregates out of the transfected HEK-293T cell population further revealed that R1441H and G2019S both induced significant more inclusions as compared to wild-type LRRK2. Finally, V5-tagged ARHGEF7 cDNA was cloned and co-expressed with the Myc-tagged wild type or mutant (R767H, S885N, R1441H, G2019S) LRRK2 constructs in HEK-293T cells. Co-immunoprecipitation assay revealed that S885N, R1441H and G2019S reduced interaction between LRRK2 and ARHGEF7.

並列關鍵字

Parkinson's disease ； neurodegenerative disease ； dardarin

參考文獻

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