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Title: | ALS相關TDP-43突變對同源嵌入小鼠ALS發病機制的影響差異 Diacritical Effects of ALS-Associated TDP-43 Mutations on ALS Pathogenesis in Homologous Knock-in Mice |
Authors: | Shih-Ling Huang 黃士玲 |
Advisor: | 沈哲鯤 |
Keyword: | TDP-43,肌肉萎縮性側索硬化症,漸凍症,運動神經元退化性疾病,Bcl-2,自噬功能, TDP-43,ALS,MND,Bcl-2,autophagy, |
Publication Year : | 2019 |
Degree: | 博士 |
Abstract: | 肌肉萎縮性側索硬化症(俗稱漸凍症)或稱盧-葛雷克氏症,是種漸進且致命的成年型退化性疾病,為延髓區域和脊髓中的上下運動神經元退化,最終導致病患無法自主隨意肌而癱瘓。超過百分之九十五漸凍症患者的脊髓運動神經元細胞質中,皆發現TDP-43的代謝異常(ALS-TDP)。雖然目前已有50多種與ALS相關的TDP-43氨基酸突變點被鑑定出來,但是這些突變如何實際影響漸凍症的發病機制尚不清楚。本文中,我們建立兩種同源基因嵌入小鼠品系,分別帶有與遺傳性漸凍症有關之TDP-43(A315T),以及突發性漸凍症突變,TDP-43(N390D)。值得注意的是,只有N390D/+小鼠,在運動行為、細胞和分子層次上,全面的發展出年齡依賴性類漸凍症發病模式。我們利用嵌入小鼠與其胚胎幹細胞誘導分化之脊髓運動神經元,來探討分子層次上TDP-43的不同突變點引起神經退化能力的差異性。結果顯示,N390D/+小鼠而非A315T/+小鼠,在發病前其小鼠脊隨中的TDP-43蛋白質隨著年紀增長開始累積; 發病後,進一步偵測到TDP-43與TDP-43之小片段(TDP-35與TDP-25)蛋白質代謝異常與自噬系統功能缺損。不同的ALS相關TDP-43突變具有關鍵的ALS引起的能力和致病途徑,可能由其遺傳背景和環境因素改變。 機制上,我們發現在ALS-TDP致病過程的早期階段,TDP-43(N390D / +)藉由調控Bcl-2 mRNA選擇性剪輯,提高Bcl-2的蛋白質表現,導致自噬功能障礙和細胞質鈣離子水平升高,而使運動神經元(N390D/+)比 (+/+)或(A315T/+) 來得脆弱。當環境中存在有壓力時,引起運動神經元(A315T/+)中TDP-43蛋白表現量的上升和運動神經元退化與死亡。證實TDP-43突變的運動神經元,對於環境壓力存在之耐受性較為低落。總結來說,TDP-43(N390D/+)與(A315T/+)嵌入小鼠模式可以提供期待已久的機會,分別暸解ALS-TDP致病的早期機制與環境因素如何調節ALS-TDP致病過程。 Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset degenerative disorder of motor neurons. The diseased spinal cord motor neurons of more than 95% of amyotrophic lateral sclerosis (ALS) patients are characterized by the mis-metabolism of the RNA/DNA-binding protein TDP-43 (ALS-TDP), in particular the presence of cytosolic aggregates of the protein. Most available mouse models for the basic or translational studies of ALS-TDP are based on transgenic overexpression of the TDP-43 protein. Here, we report the generation and characterization of mouse lines bearing homologous knock-in of familial ALS-associated mutation A315T and sporadic ALS-associated mutation N390D, respectively. Remarkably, the heterozygous TDP-43 (N390D/+) mice but not those heterozygous for the TDP-43 (A315T/+) mice develop a full spectrum of ALS-TDP-like pathologies at the molecular, cellular and behavioral levels. Comparative analysis of the mutant mice and spinal cord motor neuron (MN) derived from their embryonic stem cell demonstrates that different ALS-associated TDP-43 mutations possess critical ALS-causing capabilities and pathogenic pathways, likely modified by their genetic background and the environmental factors. Mechanistically, we identify aberrant RNA splicing of spinal cord Bcl-2 pre-mRNA and consequent increase of a negative regulator of autophagy, Bcl-2, which correlate with and are caused by a progressive increase of TDP-43 in spinal cord MN - one of the early events associated with ALS-TDP pathogenesis. The environmental stresses caused increase of TDP-43 in the (A315T/+) MN and the MN degeneration and death implying the MN carrying mutant TDP-43 is much more fragile than the (+/+) MN. Briefly, the TDP-43 (N390D/+) knock-in mouse model provides a long-awaited opportunity for basic as well as translational studies of ALS-TDP. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21756 |
DOI: | 10.6342/NTU201900592 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 分子醫學研究所 |
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