已知在轉譯區擴增的CAG重複序列會產生polyglutamine是造成很多人類神經退化性疾病的致病因子。然而有些疾病，雖然沒有polyglutamine胺基酸的產生，但是卻有類似的臨床特徵。先前我們實驗室在線蟲及老鼠的動物模式上發現，在GFP基因的3’-端非轉譯區接上CAG或CTG重複序列擴增，表現在肌肉組織上會造成異常的表型。而且在Hela及C2C12細胞上也發現不管CAG，CTG重複序列位於轉譯區或3’-端非轉譯區都有觀察到RNA foci和MBNL蛋白colocalize 的情形。在myotonic dystrophy疾病的研究上，已證實RNA foci和MBNL蛋白colocalize的情形是在RNA層次上致病的重要證據。基於這個理由推測CAG重複序列疾病可能同時具有在RNA及蛋白層次上經由toxic gain-of-function的致病機轉。為了證明這個假說，我們建立了在神經細胞中，以EGFP基因的3’-端非轉譯區或轉譯區表現200次CAG重複序列的轉殖基因鼠模式來探討這個問題。在轉譯區表達200次CAG重複序列的轉殖基因鼠會產生EGFP-polyglutamine的融合蛋白，其中ㄧ個品系在六個月大時會產生運動失調、神經退化、白內障的病徵。但是直到目前為止，在3’-端非轉譯區表達200次CAG重複序列的轉殖基因鼠，還沒有發現任何生理上的異常。這結果與我們當初的預期有很大的出入，推測原因至少可能有二個，一是由於所送入的轉殖基因表現量太少；二是組織特異性因子可能扮演重要的角色。之後我們利用CMV promoter在Neuro-2a細胞大量表現CAG重複序列，有觀察到RNA foci，但並沒有和MBNL蛋白colocalize。從這個結果看來RNA要產生toxic gain-of-function致病的機轉，組織特異性的分子可能扮演重要的角色，所以後續工作將著重於尋找這些組織特異性分子，及分析跟RNA層次產生致病的機轉相關的可能性。
Expanded CAG repeats encoding long polyglutamine tract are the widely accepted pathogenic agents in a variety of human neurodegenerative disorders. However, some of these disorders do not have polyglutamine expansion yet they display very similar clinical presentations. Previously, we showed that expanded CTG and CAG repeats in the 3’-UTR of GFP both had pathogenic effects in muscle of transgenic C.elegans and mice. And we found that RNA foci were formed both in Hela and C2C12 cells expressing coding and noncoding CAG expansions, as well as in those expressing noncoding CTG expansions. Moreover, the muscleblind proteins which mediate RNA pathogenesis in myotonic dystrophy are colocalized to these foci. It suggests that CAG repeats associated diseases may involve toxic gain-of- function mechanisms at both the protein and RNA levels. To test this hypothesis, we have established transgenic mice expressing neuro-specific transcripts with 200 CAG repeats in the coding region or 3’-UTR. Both transgenes are stable in parent to offspring transmissions. One of the transgenic lines expressing EGFP-polyglutamine fusion protein developed ataxia, Purkinje cell degeneration, and cataracts by 6 months of age. However, we have not observed phenotypic changes in the transgenic animals with noncoding CAG repeats so far. These results are different from original predictions. There are two possible explanations, one is expression that expression levels of transgenes are too low, and the other is the tissue specific factors play important roles. We then used CMV promoter to drive the expression of CAG repeats in the coding region or 3’-UTR in the Neuro-2a cells. We found RNA foci were formed in both cells, but the muscleblind proteins are not colocalized to these foci. These results suggest that tissue-specific factors play important roles for the RNA gain-of-function pathogenesis of CAG repeat diseases.
