脊髓小腦萎縮症第八型致病基因分析及建立脊髓小腦萎縮症第八型離體及活體模式

Abstract

脊髓小腦萎縮症第八型是一種漸進性的神經退化疾病，其致病的原因是由於染色體13q21上的SCA8基因在3’不轉譯區的CTG三核甘酸不正常擴增所造成。目前對於此疾病的詳細致病機轉尚未了解，相關文獻指出SCA8基因並不具有轉譯的功能，而可能是扮演anti-sense RNA的調控功能，去影響對應股的KLHL1基因表現功能。我們首先利用了反轉錄聚合酶鏈鎖反應以及原位雜合兩種方法去分析SCA8及KLHL1在人類及老鼠組織特異性的表現情形。我們發現兩者高度地表現在脊髓小腦萎縮症第八型病患的臨床病徵相對應的腦部區域，正好呼應了先前研究對於兩個基因的anti-sense調控的假說。為了更進一步去了解SCA8致病的分子機制，我們因而建立了脊髓小腦萎縮症第八型基因的轉殖小鼠模式。我們利用原核胚顯微注射的方法，將人類SCA8基因帶有正常範圍(23)及致病範圍(157)兩種CTG擴增的基因片段，藉由NSE啟動子異位表現在小鼠的中樞神經系統，並進一步做行為以及組織切片的分析以了解CTG擴增對小鼠造成的影響。我們同時也將相同的基因架構轉殖進大鼠的嗜鉻細胞瘤細胞株，建立離體的模式；我們發現具有較長擴增片段的SCA8細胞株較正常範圍以及vector控制組更容易遭受到氧化壓力的影響。另外實驗室也已建立利用Purkinje專一表現之啟動子帶領nitroreductase基因之DNA片段誘導模式的基因轉殖鼠，nitroreductase可將prodrug CB1954轉化成有毒衍生物以專一性傷害小腦，因此可模擬類似小腦萎縮的病症；我們發現專一性破壞的小鼠在平衡功能產生缺失，免疫組織染色也發現purkinje 細胞缺失及高氧化壓力的訊號。利用這些轉殖鼠及細胞模式，我們已得到初步的一些資訊將有利於應用在相關疾病的基因及藥物治療研究。Spinocerebellar ataxia type 8 (SCA8) was reported caused by an unstable CTG repeat expansion in the 3-untranslated region of SCA8 gene on chromosome 13q21. How the trinucleotide expansion causes the disease is not clear now. Some studies indicate that SCA8 might not encode protein and play an anti-sense regulatory role on the sense strand gene, KLHL1. We first identified the expression patterns of KLHL1 and SCA8 in both human and mouse brain tissues with RT-PCR and in-situ hybridization. The results show that the expression of sca8 in the brain regions whose functions correlate SCA8 clinical symptoms. The similar expression patterns of sca8 and klhl1 in these regions further suggest an anti-sense regulation of KLHL1 by SCA8. To further investigate the molecular mechanism of SCA8, a transgenic mouse model of SCA8 was established. The human SCA8 genes with 23 or 157 CTG repeats was in frame fused with flag-EGFP and driven by NSE promoter, which is considered as a CNS specific promoter. The founder lines were generated by pronuclear microinjection. Expressional, behavioral and histological analyses were preceded to understand the impact of the CTG repeat expansion on these mice. We also transfected the same constructs into rat pheochromocytoma (PC12) cell line to generate the in vitro model. We found that cell line with expanded 157 CTG repeats was more vulnerable under oxidative compared to cells with normal CTG expansion or vector construct-transfected. In addition to the SCA8 model, an inducible purkinje cells albation transgenic mouse model was also generated to further mimic SCA’s clinical features. We found that NTR transgenic mouse with CB1954 administration shows impairment in balance in motor coordination. Immunohistochemistry shows purkinje cells loss and higher oxidative signal. With the model, we have obtain initial effects resulted from overexpression of SCA8 in vivo and in vitro and late onset damage of cerebellum, which should further provide more information for the therapeutic study of SCA, including SCA8.