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

Abstract

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.