以chaperone、proteasome為目標的多麩醯胺小腦萎縮症治療策略

Abstract

在多麩醯胺居間的疾病中，轉譯CAG三核苷酸重複的擴增造成各自的蛋白中包含一長的多麩醯胺鏈，在神經細胞之細胞核和細胞質中形成聚集。熱休克蛋白可防止蛋白錯誤摺疊及聚集。泛素蛋白酶體系統的活化亦會幫助不正常摺疊蛋白的降解。本研究目標在建立細胞系統，用來鑑定可增強熱休克蛋白/蛋白酶體功能的化合物/中草藥，來治療多麩醯胺疾病。利用螢光報告基因的細胞檢測，本研究發現NH014-1及NC001-8化合物可增強HSF1、HSPA8和HSPA1A表現，NH005和NH006中草藥可增強蛋白酶體功能。另外，本研究建立誘導表現SCA17 TBP/Q36~79的Flp-In SH-SY5Y細胞株，螢光顯微鏡檢視及Metamorph軟體分析顯示，retinoic acid誘導分化後，表現的TBP/Q79蛋白形成聚集，並伴隨神經纖維生長減緩(包含突出、分支數)。化合物/中草藥處理表現TBP/Q79及誘導神經分化的SH-SY5Y細胞後，聚集抑制的情形顯示NC001-8、NH005、NH006為有潛能治療策略。

In polyQ-mediated disorders, the expansions of translated CAG repeats in the disease genes result in long polyQ tracts in the respective proteins, leading to intranuclear and cytoplasmic accumulation of aggregated polyQ proteins inside neurons. The molecular chaperones act in preventing protein misfolding and aggregation. Induction of ubiquitin proteasome also enhances the clearance of aggregate-prone proteins. This study set up cell systems to identify compounds/herbs enhancing chaperone/proteasome function for effective treatment of polyQ diseases. Using fluorescent reporter cell-based assay, pure compounds NH014-1 and NC001-8 were shown to enhance HSF1, HSPA8 and HSPA1A expression and Chinese herbs NH005 and NH006 were demonstrated to enhance proteasome function. In addition, Flp-In SH-SY5Y cells with SCA17 TBP/Q36~79-GFP expression in an inducible fashion were established. In retinoic acid-induced differentiated SH-SY5Y cells, fluorescent microscopy examination revealed that the expressed TBP/Q79-GFP formed aggregates, accompanying with reducing neurite outgrowth (including processes and branches) assessed by Metamorph software. With assessing aggregate suppression, the potential therapeutic strategies can also be demonstrated upon treatment of TBP/Q79-expressing differentiated SH-SY5Y cells with NC001-8, NH005 and NH006.