建立和分析過量表現PPP2R2B基因之基因轉殖小鼠

Abstract

Spinocerebellar ataxia 12 (SCA12)，它是一種自體顯性遺傳疾病，根據文獻報告，SCA12是在已知基因PPP2R2B基因5’端CAG擴增所造成,也是目前在各型SCA疾病中所僅知功能基因5’端未轉錄部分作擴增造成之結果，PPP2R2B基因會轉譯出一個專一表現在腦部的去磷酸PP2A (protein phosphotase 2A)的調控次單位Bβ，而CAG的擴增也被認為可能會影響到去磷酸酶的活性。我們分別建立由神經專一性表現之NSE 啟動子過量表現PPP2R2B之細胞株和基因轉殖小鼠。在Bβ表現之細胞株，我們發現Bβ調控次單位會使細胞在無血清之情況下之增生能力下降，因此，我們認為Bβ可能使細胞對環境壓力有較大的敏感性。而在動物方面目前成功建立的十株基因轉殖小鼠，有八株可在小鼠的腦部觀察到明顯的綠螢光蛋白訊號，也偵測到基因轉殖小鼠line-20腦部有PPP2R2B轉殖基因之蛋白產物表現。在行為表現方面，比較同胎出生的野生型小鼠後我們發現，基因轉殖小鼠line-20在平衡桿測試(beam test)和滾輪(rota-rod)測試，明顯有較差的表現，進一步在病理學方面的分析也發現到line-20基因轉殖小鼠小腦的Purkinje數量較少， 我們初步的結論是過量表現PPP2R2B會造成基因轉殖小鼠之病變，其中之分子機轉則更需進一步的分析。

SCA12 is an autosomal dominant spinal cerebellar ataxia (SCA) disease. According to previous studies, it is caused by CAG repeat expansion of the 5’UTR of gene PPP2R2B and it is the only SCA that has CAG repeat expansion on the 5’UTR of disease-causing gene. The PPP2R2B gene encodes a brain-specific regulatory subunit Bβ of PP2A (protein phosphotase 2A) and CAG expansion is thought to effect its activity. We have established PPP2R2B overexpression stable cell line and transgenic mice in which GFP-fused transgene is driven by a neuron specific enolase (NSE) promoter. We found that cells with Bβ expression show reduce proliferation compared to vector-transfected cells during serum starvation. This result suggests that Bβ renders cells to be vulnerable to environmental stress. Of the mouse model, among the 10 founder lines, 8 lines were found to have GFP signals in the brains. Line-20 was observed to have transgene product expressed. Results from behavioral characterization have shown that these mice have significantly poor performance compare to their wild-type littermates in both beam test and rota-rod. By immunohistochemical analysis, we have also identified Purkinje cell loss in the cerebella of line 20 transgenic mice. We conclude that the overexpression of PPP2R2B causes the pathogenesis in the mouse cerebellum. Molecular mechanism of the pathogenesis needs to be further characterized.