探討Mitogen-activated Protein Kinase訊息傳遞路徑在第三型小腦脊髓運動失調症中所扮演的角色

第三型小腦脊髓運動失調症 (Spinocerebellar ataxia type3, SCA3)，是體染色體顯性遺傳的罕見疾病，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對染色體上的ataxin-3基因的CAG核苷酸異常大量擴增，進而轉譯出帶有大量麩胺酸的ataxin-3蛋白堆積，導致神經細胞死亡。本論文，我們主要以分析正常人與SCA3病人之間細胞的差異，近一步釐清病人突變蛋白ataxin-3的致病機轉。為了瞭解這些突變蛋白為何會不正常堆積，我們利用熱刺激實驗，觀察病人細胞中的熱休克蛋白Hsp70蛋白功能與表現量是否異於正常人。實驗結果顯示，病人細胞的Hsp70能夠有效地受到熱的刺激而表現量上升，然而在病人內生性的Hsp70蛋白或mRNA表現量上卻明顯低於正常人。此結果意味著病人細胞中的Hsp70仍具有能被正常誘導的功能，但表現量卻受到了抑制。另一方面，我們想了解細胞因受到突變蛋白的刺激，而促使細胞走向凋亡(apoptosis)的訊息傳遞路徑是否經由mitogen-activated protein kinase (MAPK)這條訊息路徑所調控。我們發現，不論是磷酸化的p38或是磷酸化的ERK1/2蛋白，都會高度的表達於病人的細胞中；並且當利用SB203580去抑制p38蛋白的磷酸化，同時也能有效地促使細胞中Hsp70蛋白表現量的上升，而此現象在病人細胞中更為顯著。除此之外，在病人細胞中大量表現的磷酸化的ERK1/2，以及細胞自噬(autophagy)作用的標記蛋白–活化態的Atg8，會特異性地堆積於粒線體內，但正常人的細胞則不會有此現象。最後，在針對模擬細胞受到氧化性刺激的實驗中，發現病人的細胞較易受到刺激而死亡；但在氧化性刺激前先給予茶多酚(epigallocatechin gallate, EGCG)時，則能有效改善正常人與病人細胞之間對於氧化性刺激耐受性的差異。總結本篇研究，作者懷疑病人發病的原因，可能是細胞受到不正常堆積的突變蛋白產生的氧化性刺激，造成Hsp70、磷酸化的p38及ERK1/2的蛋白表現異常所致。值得注意的是，有良好抗氧化能力的茶多酚，或許能有效地減緩在病人細胞中異常的情形，深具後續研發的潛力。

Spinocerebellar ataxia type 3 (SCA3), an autosomal dominant neurodegenerative disease, is caused by an increased number of CAG trinuclear expansion repeats in coding regain of disease-associated gene ataxin3 and thus results in a defective protein with a long polyQ track. In this study, two lymphoblastoid cell lines (LCLs) isolated from SCA3 patients were utilized to compare with the wild-type lymphoblastoid cells. To investigate whether the mutant ataxin-3 accumulation is caused by molecular chaperone dysfunction, heat shock induction strategy was employed and then analyzed by western blot. Our results revealed that although a normal heat shock response was still present in the cell expressing mutant ataxin-3, a significant decrease of Hsp70 protein and mRNA expression in patients’ LCLs was observed. It suggests that the induction of Hsp70 protein was functional; however, the expression was suppressed. In the second experiment, we wished to determine whether the apoptotic pathway involved in SCA3 is mediated by mutant ataxin-3 through mitogen-activated protein kinase (MAPK) signaling. Our data indicated that both p-p38 and p-ERK1/2 were significantly upregulated in the cells isolated from patients. Surprisingly, when treating with SB203580 to suppress p38 phosphorylation, it was found that the Hsp70 protein level was also up-regulated, especially in the cells expressing mutant ataxin-3. Interestingly, both p-ERK1/2 and activated Atg8 (LC3 class II), an autophagy marker, were found specifically accumulated in the mitochondria in patients’ LCLs, but not in wild-type LCL. Finally, we probed the resistance of wild-type and patients’ LCLs to ROS in terms of ID50. The results demonstrated that patients’ LCLs were much more sensitive to ROS than wild-type cells; however, these effects could be effectively attenuated by pre-treatment with epigallocatechin gallate (EGCG). In conclusion, our results displayed that the abnormal expression of Hsp70, p-p38, and p-ERK1/2 regulated by ROS, released from aggregated mutant proteins, might play a critical role in the pathogenic process, and EGCG might possess an inherent capability to attenuate these effects.