研究TDP-43對阿茲海默症中乙型類澱粉胜肽纖維化作用的影響

Abstract

阿茲海默症是一種慢性的神經退化性疾病，影響全球數千萬65歲以上的老年人口。根據類澱粉胜肽假說，乙型類澱粉胜肽的聚集對阿茲海默症發病機轉至關重要，而其中乙型類澱粉胜肽的寡聚體被認為是主要的毒性來源及致病形式。前類澱粉蛋白質被乙型分泌酶及丙型分泌酶切割後產生乙型類澱粉胜肽，以含有40個胺基酸及42個胺基酸的產物為大宗，了解乙型類澱粉胜肽將有助於促進阿茲海默症治療方法的進展。近年來有研究顯示，在阿茲海默症患者的腦中也發現了肌萎縮側索硬化症相關蛋白TDP-43的聚集，實驗室先前的研究亦顯示TDP-43的寡聚體會抑制乙型類澱粉胜肽的聚集，因此我們認為TDP-43在阿茲海默症的發病機制中可能扮演了重要的角色。在我們的研究中利用生化技術檢視了全長TDP-43對乙型類澱粉胜肽聚集過程的影響，發現其會減緩乙型類澱粉胜肽聚集，形成纖維的量也會減少，透過圓二色性光譜，我們也觀察到在全長TDP-43存在的情況下，乙型類澱粉胜肽停留在隨機螺旋(random coil)時間較長。為了瞭解TDP-43是如何抑制乙型類澱粉胜肽聚集，我們也製備數個較短的TDP-43蛋白，包含N端區域、與核糖核酸的結合區域及兩者合併。我們發現TDP-43的N端區域可能是抑制乙型類澱粉胜肽聚集的主要構造，但核糖核酸結合區域可能也有協助的功能。此外，我們透過酵素結合免疫吸附分析法和生物膜干涉技術測試了TDP-43和乙型類澱粉胜肽之間的相互作用。我們的發現更進一步了解TDP-43抑制乙型類澱粉胜肽聚集的機制，將有助於釐清TDP-43在阿茲海默症中所扮演的角色。

Alzheimer‘s disease (AD) is a chronic neurodegenerative disease that affects tens of million people over 65 years old. According to the amyloid cascade hypothesis, amyloid β (Aβ) aggregation is critical to AD pathogenesis and Aβ oligomers are the primary pathogenic form. Aβ is generated from amyloid precursor protein (APP) proteolytic digested by β-secretase and γ-secretase and has two major isoforms, Aβ40 and Aβ42. Understanding the process of Aβ aggregation is helpful to facilitate therapeutic development for AD. Recently, aggregation of amyotrophic-lateral-sclerosis related protein, TDP-43, is also found in brain of AD patients. Our previous study showed that the oligomeric form of TDP-43, prevent Aβ40 forming amyloid fibrils. In addition, TDP-43 oligomers exhibited neurotoxicity. The configuration and function of TDP-43 oligomers are different from the normal TDP-43. These results indicate that TDP-43 may also play an important role in AD pathogenesis. In this study, we monitored the conformational change of Aβ40 when TDP-43 oligomers are present. Besides, several TDP-43 truncated proteins including N-terminal region, RRM1/RRM2 region, and C-terminal truncation are prepared. We demonstrated that the recombinant full-length human TDP-43 effectively inhibits Aβ40 fibrillization process at the initial stage and oligomeric stage. N-terminal domain of TDP-43 is the dominant structural region to inhibit Aβ40 fibrillization. In the milder shaking condition, the recombinant full-length human TDP-43 also had impact on the inhibition of Aβ42 fibrillization. Additionally, we studied the effect of TDP-43 in Aβ aggregation and interaction by ELISA and biolayer interferometry analysis. Our results provided the detail of Aβ fibrillization affected by TDP-43.