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Title: | 囊泡型ATPase次單元V0d1調節細胞自噬作用對類澱粉前驅蛋白恆定之影響 The Role of Vacuolar ATPase Subunit V0d1 in Modulating Autophagic Clearance of Amyloid Precursor Protein C-terminal Fragments in Alzheimer’s Disease |
Authors: | Ju-Yun Chou 周儒筠 |
Advisor: | 廖永豐 |
Keyword: | 阿茲海默氏症,β類澱粉蛋白,ATP6V0d1,細胞自噬作用,自噬作用受體蛋白p62,自噬作用標記蛋白LC3,溶?體酸鹼值, Alzheimer’s disease,Amyloid-β,ATP6V0d1,Autophagy,LC3,p62,Lysosomal pH value, |
Publication Year : | 2018 |
Degree: | 碩士 |
Abstract: | 位於大腦額葉皮層及海馬迴中的β類澱粉斑塊是阿茲海默氏症患者的典型病理特徵之一,而β類澱粉蛋白由類澱粉前驅蛋白(APP)經β-secretase和γ-secretase先後切割產生並被釋放至細胞外。當β類澱粉蛋白異常累積並聚集形成β類澱粉斑塊,會使腦內產生發炎反應並進一步引發神經纖維糾結的產生,導致神經細胞死亡、大腦萎縮等現象,最終造成阿茲海默氏症之神經退化性疾病。為了減少β類澱粉蛋白的產生,我們期望找到能調控γ-secretase酵素活性的目標蛋白質。藉由RNA干擾法篩選出三種對γ-secretase作用於amyloidogenic反應路徑相較Notch路徑有選擇性影響的γ-secretase結合蛋白,並針對與阿茲海默氏症之相關性尚未被探討過的ATP6V0d1蛋白做進一步的研究。藉由慢病毒將對ATP6V0d1有抑制作用的shRNA送入共同過量表現APP-C99和胞外片段剔除之Notch蛋白(NΔE)的細胞株中,希望能確認ATP6V0d1蛋白確實具有調控γ-secretase的功能,且會選擇性地影響γ-secretase對amyloidogenic反應路徑的作用。當ATP6V0d1基因表現被削弱後,細胞內部的APP-C99及其γ-secretase切割產物─類澱粉前驅蛋白胞內片段(AICD)和NΔE及其γ-secretase切割產物─Notch蛋白胞內片段(NICD)都會同時大量累積,且APP-C99及AICD的累積倍數明顯多於NΔE及NICD增加的趨勢,顯示ATP6V0d1的抑制對γ-secretase進行amyloidogenic反應路徑有較大影響。ATP6V0d1是囊泡型ATPase的次單元之一,且已知囊泡型ATPase會協助溶酶體建立內部酸性環境,故推測抑制ATP6V0d1的表現可能會影響溶酶體內部酸鹼值,並間接影響細胞自噬作用的進行。在我們實驗中也發現,在ATP6V0d1表現量下降時,自噬作用受體蛋白p62的量和自噬作用標記蛋白LC3-II/I的比率皆有明顯的提升,顯示細胞自噬囊泡與溶酶體融合後,可能因酸鹼值改變而無法正常將蛋白質降解。這些研究結果證實,在抑制ATP6V0d1基因表現後,可能會藉由阻斷細胞自噬作用的進行,而顯著影響γ-secretase對amyloidogenic反應路徑的催化效率。這也顯示ATP6V0d1的存在對於細胞正常生理功能十分重要,且若此蛋白發生異常可能有潛在強化amyloidogenic反應路徑的風險。 The deposition of beta-amyloid plaques in cortex and hippocampus is one of the classical pathological features of Alzheimer’s disease (AD). Amyloid precursor protein (APP) is cleaved by β-secretase and γ-secretase to produce amyloid-β (Aβ). Our RNA interference (RNAi) screening has led us to identify ATP6V0d1 as a potential γ-secretase modulator. ATP6V0d1 is a member of the γ-secretase interactome that consists of 57 distinct presenilin-1 (PS1)-binding proteins. We have demonstrated that down-regulation of ATP6V0d1 by RNAi could induce a significant reduction in γ-secretase activity. Using a HEK293-derived cell line (CCNY) that stably co-expresses yellow fluorescent protein (YFP)-tagged extracellular domain truncated Notch (NΔE) and cyan fluorescent protein (CFP)-tagged APP-C99, we found that the levels of APP-C99, APP intracellular domain (AICD), NΔE, and Notch intracellular domain (NICD) are significantly accumulated in response to downregulation of ATP6V0d1. Given that ATP6V0d1 is an integral component of the vacuolar H+-ATPase governing the maturation of autolysosomes, we examined the levels of p62 and LC3-I/II in CCNY cells that are subject to ATP6V0d1 knockdown. Our data showed that ATP6V0d1 knockdown effectively induces concomitant increases in the level of p62 and the ratio of LC3-II/LC3-I, suggesting that ATP6V0d1 knockdown could block the autophagic flux to induce concomitant accumulation of APP-C99 and NΔE. Together, the present findings suggest that ATP6V0d1 could modulate the clearance of APP-C99 through controlling the maturation of autolysosome. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7508 |
DOI: | 10.6342/NTU201802746 |
Fulltext Rights: | 同意授權(全球公開) |
metadata.dc.date.embargo-lift: | 2023-08-15 |
Appears in Collections: | 生命科學系 |
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ntu-107-1.pdf | 2.43 MB | Adobe PDF | View/Open |
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