探討細胞自噬作用在第二型和第三型小腦脊髓運動失調症中所扮演的角色

The Role of Autophagy in Pathogenesis of Spinalcerebellar Ataxia Type 2 and Type 3

黃靖樺(Hua-Ching Huang)

指導教授 ： 蔡蕙芳

中山醫學大學/醫學科技學院/醫學檢驗暨生物技術學系碩士班/碩士(2013年)

摘要

小腦脊髓運動失調症第三型 (spinocerebellar ataxia type 3, SCA3)，又稱為Machdo-Joseph Disease (MJD)，為體染色體顯性遺傳罕見疾病，屬於漸進性神經退化性疾病的一種亞型。此遺傳疾病主要是位於第14對染色體上ataxin-3基因有三核苷酸CAG異常擴增，進而轉譯出帶有大量麩胺酸的ataxin-3蛋白堆積，造成神經細胞死亡，但目前為止仍無有效治療方法。本研究主要以分析正常人與SCA3病人間細胞的差異性，並藉由誘導細胞自噬 (autophagy) 機制的啟動減輕ataxin-3蛋白堆積所造成的細胞毒性，實驗結果顯示ataxin-3蛋白堆積造成細胞生長停止於Sub-G1時期，使細胞易走向細胞凋亡，且SCA3細胞株的粒線體膜電位較正常細胞低，此外，在氧化壓力 (oxidative stress) 下其SCA3細胞活性氧分子 (reactive oxygen species, ROS) 亦有較高表現，綜合上述實驗證明突變ataxin-3蛋白堆積可造成氧化壓力並促使粒線體膜電位下降，誘導細胞凋亡前驅物質cytochrome c釋放造成細胞凋亡。細胞自噬是一種在演化上高度守護的細胞內大分子物質分解機制，透過飢餓環境、bafilomycin A1藥物誘導自噬作用後，SCA3細胞的Bcl2蛋白表現量高，顯示細胞在抗凋亡狀態且細胞自噬機制啟動能減輕ataxin-3蛋白所造成的毒性，減少細胞死亡所造成的神經退化性疾病。此外，也進一步分析小腦脊髓運動失調症第二型 (spinocerebellar ataxia type 2, SCA2)的細胞自噬作用，結果抗凋亡蛋白Bcl2表現量高，顯示細胞自噬啟動對於ataxin-2蛋白堆積所造成的細胞傷害亦有減緩作用，細胞自噬啟動或許能成為治療第二型、第三型小腦脊髓運動失調有效方針。

關鍵字

小腦脊髓運動失調症第二型 ； 小腦脊髓運動失調症第三型 ； 細胞自噬 ； 細胞凋亡

並列摘要

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), which is an autosomal dominant neurodegenerative disorders caused by expansion of CAG triplet in the coding region of ataxin-3 gene (chromosome locus 14q32.1). This CAG repeat is translated into an extended glutamine stretch in the mutant protein, which causes a gain of toxicity inducing neuronal cell death. However, the cellular mechanisms causing toxicity are still understood. In this study, two lymphoblastoid cell line (LCLs) isolated from SCA 3 patient were utilized to compare with the wide-type LCL. Our result revealed that accumulation of ataxin-3 protein were causes cell grow arrest at Sub-G1 phase, which may induce apoptosis in SCA3 cells. In the futher finding, SCA3 cells were accompanied by the loss of mitochondrial membrane potential and in relation to ROS level, there has an increased oxidative stress in SCA3 cells. In conclusion, aggregation of ataxin-3 protein can inhibited the growth of SCA3 cells via cell cycle arrest and apoptosis which may caused by MMP downregulation and increased level of ROS. Autophagy is an intracellular degradation process responsible for the clearance of most long-lived proteins and organelles. In our research used lowly concentration of bafilomycin A1 as autophagy induction. It revealed that, autophagosomal protein LC3-II (ATG8) accumulated enhances the cell viability through Bcl2 and Bax expression by western blot. The same result also shown in SCA2 cells. Inducing autophagy can decrease the toxicity which caused by accumulation of mutant ataxin-2 by detecting the proapoptotic protein, Bax expression. It suggest that up regulation of autophagy enhances the clearance of ataxin-2 and ataxin-3 proteins which may cause cell tocicity. It may be a hopeful therapeutic strategy for SCA2 and SCA3.

並列關鍵字

Spinocerebellar ataxia type 2 and 3 ； autophagy ； apoptosis

參考文獻

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