氧化壓力誘導Musashi-1 斷裂造成視網膜色素上皮 細胞變性

Abstract

老年性黃斑部病變 (Age-related macular degeneration (AMD) 與年齡有關，是老年人視力嚴重喪失主要原因。而AMD發生率的增加也和現代發達國家的生活方式有關。在此疾病的進展中，視網膜色素上皮層（Retinal Pigment Epithelium）的退化導致感光細胞死亡，造成中央視覺喪失。基因突變與AMD之間具有低相關性，使AMD的發生機制需要進一步地闡明。此外，老化是AMD最重要的因素，目前主流的研究指出AMD的中間型態，亦稱為乾型的AMD是由於光誘導型自由基導致細胞萎縮所造成。另外，Musashi-1 (MSI-1)是RNA結合蛋白，同時也是神經幹細胞中的標記。MSI-1在成熟的感光細胞中和RPE幹細胞中均有表現。曾經有報告指出MSI-1基因缺陷的小鼠其感光細胞會退化並缺乏視覺循環。本研究的目的是證明假說，AMD的成因是RPE細胞系 (ARPE-19) 在氧化壓力下，MSI-1 調控AMD蛋白質的錯誤摺疊。當細胞在氧化壓力條件下MSI-1 蛋白會轉移至細胞核中。同時，我們建構了MSI-1 區域交換，並建立了穩定表達此構造的細胞。我們發現正常型和C端切除的Musashi-1（MSI-1ΔC）細胞在氧化壓力的狀況下顯示出相同的表現型態，而在TUNEL 檢測下MSI-1ΔC 有較高的細胞凋亡信號。不論如何，MSI-1 蛋白在未施予過氧化氫的控制組中和施予過氧化氫的實驗組中的表達上有所不同。綜合上述實驗結果，我們目前的結果顯示出在自由基的刺激下，MSI-1ΔC 的表現可能使ARPE 細胞衰退。因此我們建立了一個AMD致病機轉：MSI-1可以透過依賴蛋白質不正常折疊途徑調控AMD。

Age-related macular degeneration (AMD), is the leading cause of severe vision loss in aged individuals and the incidence of AMD is also increasing in the developed countries due to modern lifestyles. In this progressive disease, the degeneration of the retinal pigment epithelium (RPE) results in the death of photoreceptor, leading to a loss of central vision. The correlation between genomic mutation and AMD are poor, and the mechanism of AMD pathogenesis needs further elucidations. Besides, aging is the most determinative factor for AMD, it is widely accepted that build up of photo-inducible free radical overtime will lead to atrophy which the intermediate state of dry AMD. Furthermore, Musashi-1 (MSI-1), an RNA binding protein which is a neural stem cell marker and it is an expression in mature photoreceptor and RPE stem cells can be detected. It has been reported that MSI-1 knock-out mice results in degeneration of photoreceptor and lack of visual cycle had been reported. The aim of this study is to prove the hypothesis of proteopathy of AMD progression mediated by MSI-1 under oxidative stress condition in RPE cell line (ARPE-19). We found that MSI-1 protein translocated into the nucleus in cells treated with oxidative stress as compared to control cells that only expressed in the cytoplasm. Furthermore, we constructed MSI-1 domain swap and established stable cell expressing these construct. We found that wild-type and C-terminal Truncated Musashi-1 (MSI-1ΔC) cell showed the same phenotype in oxidative stress-treated cells and MSI-1ΔC results in higher apoptotic signals revealed by TUNEL assay. However expression profile of MSI-1 protein in control and treated cell with hydrogen peroxide are different. Taken together, our current result demonstrates that expression of MSI-1ΔC might deteriorate ARPE-19 cells when stimulated by free radicals. It is probable that MSI-1 is one of the critical factors that mediated AMD by proteopathy dependent pathway.