小腦脊髓運動失調症第三型(spinocerebellar ataxia type 3, SCA3)又稱為Machdo-Joseph Disease (MJD)，是體染色體顯性遺傳、漸進式的神經退化性疾病。其致病原因是在第十四條染色體的長臂(14q24.3-q32.2)有三核苷酸CAG倍增突變，進而轉譯出帶有大量麩胺酸的ataxin-3蛋白堆積，目前尚無有效的治療方法。本研究主要探討利用穩定轉染(Stable transfection)的方法把突變的Q78-ataxin-3基因轉染到SK-N-SH細胞內並與另外SK-N-SH (wide-type和轉染正常Q26-ataxin-3基因的MJD26作為internal control進行比較分析，並藉由透過饑餓誘導細胞自噬(autophagy)機制啟動減輕ataxin-3蛋白的堆積所造成的傷害。實驗結果顯示帶有突變ataxin-3蛋白的 MJD78細胞株其粒線體膜電位較SK-N-SH低，在細胞週期分析也易停滯於sub-G1期，促使細胞走向凋亡，其活性氧分子(reactive oxygen species, ROS)含量亦有較高的表現量。結論說明突變的ataxin-3蛋白的堆積能容易引起氧化壓力增高令粒線體膜電位下降從而改變其通透性而釋放凋亡因子造成細胞死亡。而饑餓誘導的細胞自噬發現在MJD78細胞有較高的表達，顯示細胞自噬的啟動對於突變的ataxin-3蛋白堆積有緩解的作用，總體而言，本論文提供了一個良好表徵的MJD78細胞資源用於神經變性疾病研究，並有助於了解突變體ataxin-3生物學及其分子方面的研究。Spinocerebellar ataxia type 3 (SCA3), also known as Machdo Joseph Disease (MJD), is an autosomal dominant inherited cerebellar ataxia, late-onset neurodegenerative disorder. SCA3 is a caused by a CAG trinucleotide repeat expansion in the ATXN3 gene on chromosome 14q24.3-q32.2, which results in an abnormally polyglutamine in the ataxin-3 protein. No treatment able to modify the disease progression is available. In this study, neuroblastoma cells (SK-N-SH) was transduced to express either non-pathogenic ataxin-3-26Q (MJD26 cells) or pathogenic ataxin-3-78Q proteins (MJD78 cells). The results showed that accumulation of ataxin-3 protein were causes the mitochondrial membrane potential of MJD78 cell decrease than SK-N-SH. Data from cell cycle analysis revealed that MJD78 were arrested in subG1 phase and also had a high level reactive oxygen species (ROS), compare with SK-N-SH. In conclusion, the mutation of ataxin-3 protein has a potential to induce oxidative stress which lead to down regulation of mitochondrial membrane potential, in the results the changing of the permeability of mitochondrial membrane lead to cellular apoptosis. In our research, we observed MJD78 can up-regulate autophagy which indicated that the activation of autophagy play a rule in alleviation of the accumulation of mutated ataxin-3 protein. Overall, our results provide a well-characterized MJD78 cell resource for neurodegenerative disease research and contribute for the understanding of mutant ataxin-3 biology and its molecular consequences.
