第八型脊髓小腦運動失調症之果蠅模式建立與致病機制研究

Abstract

壹、中文摘要 脊髓小腦運動失調症（SCAs）為一群退化性神經疾病，其特徵為小腦功能異常，但也常伴隨中樞神經系統其他部位神經性異常，第八型脊髓小腦運動失調症（SCA8）與染色體13q21上的SCA8基因3’端非轉譯區CTG三核酸重複序列擴增相關。本研究嘗試建立SCA8 果蠅模式以供致病機制之研究，將CTG重複序列表現於果蠅複眼時，會造成感光細胞退化，顯見重複序列為致病的重要因子，以原位雜合顯示細胞含重複序列的核酸分子，會聚集於核內形成核酸聚點(RNA foci)；此外先前研究由基因序列分析顯示SCA8不含顯著的開放譯讀區(ORF) ，推論SCA8不會有蛋白質產生，然而以綠螢光蛋白基因接在SCA8尾端轉殖果蠅，發現綠螢光蛋白可因SCA8的轉錄而表現，顯示SCA8基因可能合成蛋白質產物；將SCA8含重複序列的ORF表現時，亦會造成感光細胞退化，且含重複序列核酸會於核內形成核酸聚點，無法輸送至細胞質轉譯成有毒的聚白胺基酸蛋白。由上我們得知重複序列在核酸階層即可造成細胞毒害，文獻指出含CUG重複序列可藉與muscle-blind及PKAAP等核酸結合蛋白形成核酸聚點，降低這類核酸結合蛋白的表現量，而增強CUG重複序列對細胞的毒害；將SCA8果蠅模式置於這些基因缺失的背景下，我們的確發現複眼性狀受到影響。此外我們也發現熱休克蛋白(Hsp70)可減緩果蠅模式的性狀，由此結果推論蛋白質不正常摺疊可能為SCA8致病機制之一。由於SCA8與KLHL1基因（actin結合蛋白）的5’端共用一段DNA序列，且均表現於腦部，我們也試圖了解KLHL1在SCA8的致病機制中是否扮演一定角色；單獨表現KLHL1蛋白於果蠅複眼並未造成明顯性狀，將SCA8重複序列與KLHL1共同表現於感光細胞，也未增加複眼之退化，初步認為KLHL1並非導致SCA8的主因。

Abstract The spinocerebellar ataxias (SCAs) are a group of neurodegenerative disorders characterized by cerebellar dysfunction alone or in combination with other neurological abnormalities. SCA type 8 (SCA8) has been attributed to the expanded CTG repeat at 3’ end of SCA8 gene on chromosome 13q21. To unravel the pathogenic mechanisms underlying SCA8 we tried to establish SCA8 Drosophila models in this study. The photoreceptor cells were degenerated when SCA8 gene with expanded CTG repeats were expressed in the transgenic flies, suggesting the repeat sequence exhibits pathogenic effect. The repeat containing transcripts were found accumulated in nuclei as RNA foci using in situ hybridization. As sequence analysis of SCA8 gene did not reveal that SCA8 encodes any significant ORFs in previous study, suggesting that SCA8 will not encode any protein product. Nevertheless, expression constructs with EGFP fused at 3’ end of SCA8 were able to express in the eye discs of transgenic flies, suggesting that SCA8 may contain translatable ORF. Expressing the repeat containing ORF in transgenic flies cause severe degenerative phenotype. However the transcripts were aggregated in RNA foci and can not be transported to cytosol to be translated into polyLeu containing polypeptide. From these results we have learned that SCA8 can exert its cytotoxcity effect at RNA level. Previous studies demonstrated that RNA binding proteins, such as muslcle-blind and PKAAP, were associated with CUG containing transcripts to form RNA foci, which sequesters the expression level of these RNA binding proteins and enhance the pathogenic effect. We found our SCA8 fly model displayed different degenerative phenotype when placed at mbl, and PKAAP mutant background. Interestingly, the chaperone protein, Hsp70, was found alleviated SCA8 disease presentation. It is very likely that mis-folded proteins may also play a role in SCA8 pathogenesis. Since 5’ end of SCA8 is overlapped with a nearby gene KLHL1, we would like to know whether KLHL1 is also play a role in SCA8 pathogenesis. Retinal expression of KLHL1 did not cause obviously eye degeneration. Co-expression of both SCA8 and KLHL1 did not enhance the disease phenotype. This has demonstrated that KLHL1 may not participate in the pathogenesis of SCA8.