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Title: | 研究C9ORF72漸凍人症中富含精胺酸雙胜肽所造成的細胞核TDP-43蛋白凝集現象 The study of TDP-43 nuclear condensation under C9ORF72 ALS-associated arginine-rich dipeptides |
Authors: | 迪 莎 Diksha Agnihotri |
Advisor: | 黃人則 Joseph Jen-Tse Huang |
Co-Advisor: | 黃憲松 Hsien-Sung Huang |
Keyword: | C9ORF72,肌萎縮性側索硬化症,NEAT1,TDP-43,熱休克蛋白70, C9ORF72,ALS,NEAT1,TDP-43,HSP70, |
Publication Year : | 2024 |
Degree: | 博士 |
Abstract: | 肌萎縮性脊髓側索硬化症(ALS)是一種致命的神經退化性疾病,運動神經元的退化最終會導致癱瘓,患者自疾病發作後的壽命僅剩 3 到 5 年。ALS 的一個重要遺傳原因是 C9ORF72 基因中的六核苷酸重複擴增。C9ORF72 基因中的 GGGGCC 序列擴增會轉譯為五種重複雙肽(DPR),包括甘胺酸-丙胺酸(poly-GA)、脯胺酸-精胺酸(poly-PR)、甘胺酸-精胺酸(poly-GR)。在這五種 DPR 中,富含精胺酸的雙肽被認為是最具毒性的。特別是poly-PR 二肽的毒性與其擾亂無膜胞器液體狀性質的能力高度相關。富含低複雜度區域(LCD)的蛋白質進行的液-液相分離(LLPS)是形成與維持無膜胞器(如核仁與側核斑)的基本機制。與 ALS 相關的蛋白質(如 TDP-43)中的 LLPS 失調被認為與 ALS 的致病機制有關。具體來說,LLPS 的改變可能會導致 TDP-43 在細胞質中凝膠化,並在延長壓力下聚集。然而,poly-PR 壓力對 TDP-43 在細胞核中 LLPS 的影響仍不清楚。
本研究中,我們探討了 poly-PR 壓力對 TDP-43 核凝聚的短期與長期影響。我們的研究結果顯示,暫時 poly-PR 壓力會誘導形成流動性降低的 TDP-43 核凝聚體 (NCs)。值得注意的是,長鏈非編碼 RNA NEAT1 與 HSP70 分子伴護蛋白在短期 poly-PR 壓力下與 TDP-43 核凝聚體顯著共位。藉由 siRNA造成減少,發現 NEAT1 其對於 TDP-43 核凝聚體(NC)形成是必要的。此外,以藥物抑制 HSP70 顯示其維持凝聚體流動性中的作用。在長期 poly-PR 壓力下,NEAT1 繼續作為 TDP-43 核凝聚體形成的支架;然而,HSP70 從這些凝聚體中脫離,導致流動性進一步降低並最終凝膠化。 利用基於螢光壽命的成像顯微鏡結合免疫螢光染色,發現在凝膠狀核凝聚物(NCs)中存在 TDP-43 寡聚物。此外,長期暴露於 poly-PR 壓力下導致了 TDP-43 蛋白病變,其特徵包括 TDP-43 磷酸化、錯位以及 C 末端 TDP-43 片段的出現,並伴隨著細胞毒性的增加。這項研究闡明了poly-PR通過干擾TDP-43核內LLPS以及典型TDP-43蛋白病變可能導致的毒性機制,並揭示了NEAT1和HSP70在這一過程中的作用。 Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron degeneration that leads to paralysis, typically resulting in a life expectancy of 3-5 years after onset. A significant genetic factor in ALS is the hexanucleotide repeat expansion (GGGGCC) in the C9ORF72 gene, which results in the translation of five dipeptide repeats (DPRs). Among these, arginine-rich dipeptides toxicity is highly associated with their ability to disrupt liquid-like properties of membraneless organelles (such as the nucleolus and paraspeckles). Liquid-liquid phase separation (LLPS) of low-complexity domain (LCD)-rich proteins is crucial for the formation of these organelles. Dysregulated LLPS in ALS-related proteins like TAR DNA-binding protein 43 (TDP-43) has been linked to ALS pathogenesis, with alterations in LLPS potentially leading to TDP-43 gelation and aggregation under stress. However, the impact of arginine-rich dipeptides on TDP-43 LLPS in the nucleus is still unclear. In this study, we investigated the transient and prolonged effects of poly-PR and poly-GR dipeptide on TDP-43 nuclear condensation. Our findings indicate that only transient poly-PR stress induces the formation of TDP-43 nuclear condensates (NCs) with decreased fluidity. Of note, the long non-coding RNA NEAT1 and the HSP70 chaperones demonstrated significant colocalization with TDP-43 NCs under transient poly-PR stress. Through siRNA-mediated depletion, NEAT1 was found necessary for TDP-43 NCs formation. In addition, pharmacological inhibition of HSP70 suggested its role in maintaining condensate fluidity. Following prolonged exposure to poly-PR stress, NEAT1 continued to function as a scaffold for TDP-43 NC formation. However, HSP70 delocalized from these condensates, resulting in further reduced fluidity and subsequent gelation. By combining fluorescence lifetime-based imaging microscopy and immunofluorescence staining, the presence of TDP-43 oligomers were detected within gel-like NCs. In addition, prolonged poly-PR stress induced TDP-43 proteinopathy including TDP-43 phosphorylation, mislocalization and C-terminal TDP-43 fragments, along with the increased cytotoxicity. This study elucidates a potential toxicity mechanism of poly-PR dipeptide via perturbation of TDP-43 nuclear LLPS along with canonical TDP-43 proteinopathy, and shedding light on the roles of NEAT1 and HSP70 in the process. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/96475 |
DOI: | 10.6342/NTU202500138 |
Fulltext Rights: | 同意授權(全球公開) |
metadata.dc.date.embargo-lift: | 2025-02-19 |
Appears in Collections: | 跨領域神經科學國際研究生博士學位學程 |
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