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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃人則 | zh_TW |
| dc.contributor.advisor | Joseph Jen-Tse Huang | en |
| dc.contributor.author | 迪 莎 | zh_TW |
| dc.contributor.author | Diksha Agnihotri | en |
| dc.date.accessioned | 2025-02-18T16:18:45Z | - |
| dc.date.available | 2025-02-19 | - |
| dc.date.copyright | 2025-02-18 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2025-01-20 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/96475 | - |
| dc.description.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。 | zh_TW |
| dc.description.abstract | 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. | en |
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| dc.description.provenance | Made available in DSpace on 2025-02-18T16:18:45Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | Acknowledgement I
Abstract III Table of Content VII List of Figures XI List of Supplementary Figures XIII List of Tables XIV 1. Introduction 1 1.1 Amyotrophic Lateral Sclerosis 1 1.1.1 C9ORF72 genetic mutation in ALS 3 1.1.2 Toxic loss-of-function mechanism in C9ORF72 ALS 4 1.1.3 Toxic gain-of-function mechanism in C9ORF72 ALS 5 1.1.4 Dipeptide Repeats (DPRs) toxicity mechanism in ALS 5 1.1.5 Liquid-Liquid Phase Separation (LLPS) 6 1.1.6 Disruption of LLPS by arginine-rich dipeptides 8 1.2 TAR-DNA binding protein (TDP-43) is a pathological protein in ALS which also contains LCD 9 1.2.1 TDP-43 undergoes phase-separation 11 1.2.2 Role of NEAT1 in TDP-43 LLPS 12 1.2.3 HSP70 Chaperones TDP-43 LLPS 13 1.3 Motivation of this study: TDP-43 Nuclear Condensation in C9ORF72 ALS 14 2. Materials and Methods 15 2.1 Cell culture 15 2.2 Plasmid construction 15 2.3 Cell seeding and plasmid transfection.. 16 2.4 Establishment and validation of stable N2a cell line expressing eGFP-TDP-43.. 17 2.5 Synthesis of arginine-rich dipeptides 17 2.6 Dipeptide and inhibitor treatment 18 2.7 Antibodies 19 2.8 Immunocytochemistry and Confocal imaging 19 2.9 Proteosome activity assay 20 2.10 Immunofluorescence followed by RNA fluorescence in-site hybridization (IFRNA-FISH)… 20 2.11 Fluorescence recovery after photobleaching (FRAP) analysis 21 2.12 Time-Lapse imaging 21 2.13 RNA isolation and quantitiative real-time polymerase chain reaction (qPCR) 22 2.14 Time domain fluorescence life-time imaging 23 2.15 FLIM-FRET data analysis 23 2.16 Cell viability assay 24 2.17 Filter Trap assay 25 2.18 Western blot analysis 26 2.19 Image quantification and statistical analysis 26 3. Results 28 3.1 Transient poly-PR stress induce TDP-43 nuclear condensation 28 3.2 Transient poly-PR stress fails to induce FUS and GFP-NLS nuclear condensation …………………………………………………………………………………...31 3.3 Random peptide did not induce TDP-43 NCs 32 3.4 Nucleolar localized poly-PR indirectly induce TDP-43 nuclear condensation 33 3.5 Longer length dipeptides show similar TDP-43 NCs induction 35 3.6 Nucleolar occupied poly-PR dipeptide did not induce proteasomal stress 36 3.7 Transient Poly-PR induced TDP-43 NCs colocalize with NEAT1 RNA and HSP70 Chaperone 37 3.8 HSP40 and HSP90 did not colocalize with poly-PR induced TDP-43 NCs 39 3.9 NEAT1 promotes TDP-43 nuclear condensation and HSP70 recruitment to NCs upon transient poly-PR stress……………………………………… 42 3.10 HSP70 maintains fluidity of TDP-43 NCs under transient poly-PR stress 45 3.11 Delocalization of HSP70 from TDP-43 NCS upon prolonged poly-PR stress induce phase-transition of the condensates 47 3.12 NEAT1 participate in TDP-43 NCs formation even upon prolonged poly-PR stress but no longer involved in HSP70 recruitment 50 3.13 Localization of NEAT1 and HSP70 in TDP-43 NCs is consistent in neuronal cell-line under poly-PR stress duration 52 3.14 Prolonged poly-PR stress induced gel-like TDP-43 NCs are rich in TDP-43 oligomers 54 3.15 Prolonged poly-PR stress induces TDP-43 proteinopathy and cell death 57 4. Discussion 61 5. Summary 64 6. Supplementary Figures 66 7. Supplementary Tables 69 8. References 71 | - |
| dc.language.iso | en | - |
| dc.subject | TDP-43 | zh_TW |
| dc.subject | C9ORF72 | zh_TW |
| dc.subject | 肌萎縮性側索硬化症 | zh_TW |
| dc.subject | 熱休克蛋白70 | zh_TW |
| dc.subject | NEAT1 | zh_TW |
| dc.subject | NEAT1 | en |
| dc.subject | TDP-43 | en |
| dc.subject | HSP70 | en |
| dc.subject | C9ORF72 | en |
| dc.subject | ALS | en |
| dc.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 | zh_TW |
| dc.title | The study of TDP-43 nuclear condensation under C9ORF72 ALS-associated arginine-rich dipeptides | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-1 | - |
| dc.description.degree | 博士 | - |
| dc.contributor.coadvisor | 黃憲松 | zh_TW |
| dc.contributor.coadvisor | Hsien-Sung Huang | en |
| dc.contributor.oralexamcommittee | 陳俊安;郭紘志;譚婉玉 | zh_TW |
| dc.contributor.oralexamcommittee | Jun-An Chen;Hung-Chih Kuo;Woan-Yuh Tarn | en |
| dc.subject.keyword | C9ORF72,肌萎縮性側索硬化症,NEAT1,TDP-43,熱休克蛋白70, | zh_TW |
| dc.subject.keyword | C9ORF72,ALS,NEAT1,TDP-43,HSP70, | en |
| dc.relation.page | 80 | - |
| dc.identifier.doi | 10.6342/NTU202500138 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-01-21 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 跨領域神經科學國際研究生博士學位學程 | - |
| dc.date.embargo-lift | 2025-02-19 | - |
| 顯示於系所單位： | 跨領域神經科學國際研究生博士學位學程 | |
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