透過TDP-43變異體及抗體闡明阿茲海默症中TDP-43與乙型類澱粉胜肽間的分子相互作用

Abstract

阿茲海默症（AD）是最常見的失智症類型，其特徵是由乙型類澱粉胜肽（amyloid-β，簡稱 Aβ）組成的澱粉樣斑塊以及由過度磷酸化的 tau 蛋白所形成的神經纖維纏結（neurofibrillary tangles，簡稱 NFT）。臨床研究發現 TDP-43 會在 AD 患者的大腦中累積。先前的研究表明，TAR 去氧核糖核酸結合酶-43 (簡稱 TDP-43)會透過維持 Aβ 處於寡聚體狀態來抑制 Aβ 纖維化的過程。TDP-43 和 Aβ 在 AD 患者海馬迴的同定位顯示它們在 AD 病理中存在可能的相互作用。然而，TDP-43 與 Aβ 之間的具體結合界面尚不清楚，此外，TDP-43 誘導的 Aβ 寡聚體的相對毒性亦尚未確定。在本研究中，我系統性地對模擬複合物模型中 TDP-43 的潛在相互作用位點進行了點突變，並利用生化和生物物理方法驗證了這些點突變對 Aβ 結合的影響。結果顯示，突變某個特定帶電胺基酸殘基後，結合能力顯著下降，表明該胺基酸殘基在與 Aβ 的相互作用中扮演關鍵角色。細胞毒性測定顯示，Aβ 寡聚體在不同的纖維化狀態下是最具毒性的物種。此外，我們還使用了幾種自製針對特定區域的 TDP-43 抗體來阻斷 TDP-43 與 Aβ 之間的相互作用。本研究實驗結果顯示，這一關鍵相互作用可能會加劇細胞毒性的增加，且抗體篩選結果有助於發展針對 TDP-43 在 AD 中所引起的有害影響的治療方向。

Alzheimer's disease (AD) is the most prevalent cause of dementia, characterized by senile plaques composed of amyloid-β (Aβ) and neurofibrillary tangles (NFT) comprising hyperphosphorylated tau. Notably, TAR DNA-binding protein-43 (TDP-43) accumulation has been observed in the brain of AD patients. Our previous publication revealed the role of TDP-43 in mediating Aβ fibrillization by maintaining Aβ in the oligomeric state. The co-localization of TDP-43 and Aβ in the hippocampus of AD patients indicates their potential interaction in AD pathology. However, the precise binding interface and sites between TDP-43 and Aβ remains unknown. Furthermore, the relative toxicity of TDP-43-induced Aβ oligomers remains unknown. In this study, I systematically mutated the residues identified as potential interaction sites in a simulated complex model of TDP-43 RRM and Aβ. Then, I validated their impact on Aβ binding in TDP-43 variants by biochemical and biophysical methods including ELISA, biolayer interferometry (BLI), and surface plasmon resonance (SPR) assays. Results demonstrated a significant decrease in binding following mutating a specific charged residue, suggesting the crucial role of this residue as an interaction site with Aβ. Cytotoxicity assays revealed Aβ oligomers are the more toxic species across various fibrillization states. Moreover, we employed a few in-house TDP-43 antibodies to block the interaction and also investigate the minimal epitope of the antibodies. These findings reveal that the critical interaction potentially contributes to the increased cytotoxicity. The finding in antibody screening promotes the therapeutic development for the detrimental effect attributed to TDP-43 in AD.