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

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.