乙型類澱粉樣蛋白40與42片段間交互引晶作用之研究

乙型澱粉樣蛋白 (β-amyloid peptide, Aβ) 是阿茲海默症的致病因子之一，其具有序列 (Aβ40, Aβ42)、形貌和結構多樣性。在阿茲海默症病人腦中的纖維沉積主要由 Aβ42 組成，但共存於腦中的 Aβ40 總含量卻遠高於 Aβ42，兩者之間是否具有交互作用是此領域關注的焦點之一。近年研究也指出處在纖維化中間態的寡聚物 (oligomer) 具有更高的細胞毒性，尤其是 Aβ42 寡聚物，其在阿茲海默症形成初期可能具有關鍵的病理意義，但受限於寡聚物結構的不穩定性、聚集型態的多樣性，對於寡聚物的結構、其誘發其他蛋白聚集的機制 (引晶效應) 及與最終纖維結構的關聯性等至今仍是尚待釐清的謎。為提升寡聚物同質性(homogeneity)，本研究以 AOT/isooctane/H2O 逆相微胞系統作為物理性限制空間，使 Aβ 單體在逆相微胞碰撞時經物質交換過程逐漸聚集並維持在寡聚物 (RMAβ) 階段，於 30 ℃ 培養 7 天後，結合 6E10 抗體及穿透式電子顯微鏡確認反萃後之 RMAβ 仍維持在粒徑為 37 nm 寡聚物狀態，經暗場 TEM 影像粗估其分子量在 200–400 kDa；Thioflavin-T 螢光分析及圓偏光二色光譜 (CD) 結果指出已具 β-sheet 二級結構的 RMAβ 脫離逆相微胞後仍會繼續聚集成纖維 (on-pathway)，且由固態核磁共振光譜判定其結構並非單一構型。為探討on-path way 寡聚物對單體的引晶效應，嘗試將不同組成的 RMAβ 寡聚物與 Aβ40 單體共培養，竟意外地發現 RMAb42 寡聚物具有加速 Aβ40 聚集的特性，這與其他文獻提出的結論──Ab42 纖維對 Aβ40 不具有引晶能力──是截然不同的，所以在本研究中分別透過 RMAβ40 自聚集及以 RMAβ40 和 RMAβ42 作為核種對 Aβ40 單體進行引晶兩種方式培養澱粉樣纖維，以電子顯微鏡追蹤聚集物的形貌變化，並利用低溫電子顯微鏡 (cryo-EM) 重建主要纖維的結構，希望能進一步說明不同核種對於引晶機制及纖維結構的影響。

關鍵字

乙型類澱粉樣蛋白

並列摘要

Beta-amyloid peptides (Aβ) have been regarded as the key factor in the pathology of Alzheimer’s disease (AD) for years. Amyloid-β peptides of variable length coexist in the central nervous system and possess strong propensity to aggregate. There are distinct Aβ aggregates forming during the fibrillization process and Aβ oligomers are considered to be a more relevant therapeutic target than other species such as fibril in recent research. In this study, we have developed H2O/sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane reverse micelles (RMs) system to provide a physically confined space to restrict the growth of Aβ aggregates. After incubated at 30 ℃ for 7 days, Aβ40 peptides were in the oligomeric state (RMAβ40) with 34 nm average diameter as confirmed by TEM images and 6E10 dot-blot assay. The size of the oligomers was estimated by intensity ratio between β-amyloid fibril and tobacco mosaic virus (TMV) standard in dark-field TEM images to be about 200–400 kDa. The result of Thioflavin-T (ThT) assay and circular dichroism (CD) spectra revealed that the extracted RMAβ40 with partial β-sheet secondary structure grew rapidly into protofibril without lag-phase (on-pathway). Although the solid-state NMR data indicated that the structure of RMAβ40 oligomers was polymorphic, the RMs system is still a clean and simple plateform for the preparation of Aβ oligomers. Furthermore, the interaction between Aβ40 and Aβ42 have been investigated, because the neurotoxicity of Aβ42 oligomers and the cross-seeding between Aβ42 and Aβ40 peptides are critical issues for the pathology of AD. We tried to prepare RMAβ40, RMAβ42, and RMAβ42/40 oligomers as seeds to promote Aβ40 fibrillization. The aggregation kinetics was tracked by ThT assay and the fibril morphology by transmission electron microscope (TEM). Surprisingly, the data showed a pronounced seeding effect of RMAβ42 oligomers to Aβ40 monomers, which is in stark contrast to the results when using Aβ42 fibril fragments as seeds in other studies. To unravel whether Aβ42 oligomers also affect the structure of Aβ40 monomer during the seeding process, we attempted to use cryogenic electron microscopy (cryo-EM) to build 3D structure of the corresponding fibrils.