阿茲海默氏病患者腦萃取物衍生之乙型類澱粉蛋白寡聚體擴增與結構鑑定
Amplification and Structural Characterization of Beta Amyloid Oligomers Derived from Brain Extracts of Alzheimer’s Disease Patients
計畫主持人：臺大化學系-陳振中、中研院基因體中心-陳韻如
阿茲海默症 (Alzheimer’s disease, AD) 是最常見的神經退化性疾病，也是老年化社會的重要議題之一。然而，目前尚未有公認有效的藥物或療程能治療AD。乙型類澱粉蛋白 (Amyloid β, Aβ) 以及相關致病性蛋白，如 TDP-43，Prion，Tau等在腦部的沉積物被認為是造成AD的主要原因。近年研究認為AD與可溶性的乙型類澱粉蛋白 (Amyloid β, Aβ) 寡聚物濃度高度相關。然而，Aβ寡聚物是不穩定的中間體，不論是直接取用病人檢體或是離體培養，對Aβ寡聚物進行結構鑑定都非常困難。本團隊使用逆向微胞物理空間限制的特性，成功限制Aβ聚集在寡聚物階段，並藉由固態核磁共振儀來研究Aβ寡聚物的特性。藉由逆向微胞系統，我們能使Aβ停留在大小及結構均一的Aβ寡聚物，並藉此性質針對兩項議題進行研究：(1) 放大AD病患腦組織萃取物引晶培養之Aβ寡聚物，以及 (2) 研究 TDP-43與Aβ在寡聚物階段的交互作用。逆向微胞的優勢在於能夠製備大小、結構均一的寡聚物。因此，透過多個循環的引晶培養過程，我們成功提取和放大來自腦組織萃取的Aβ寡聚物，並使其得以進行核磁共振光譜測量。對於TDP-43與Aβ的交互作用，我們透過逆向微胞製備共培養樣品，並藉由核磁共振技術，證明TDP-43和Aβ在寡聚物狀態下形成共聚物，並首先提出Aβ40中的D23和K28是TDP-43和Aβ40寡聚物相互作用的關鍵胺基酸。我們的最終目標是製備針對腦組織萃取物引晶培養之Aβ寡聚物的單株抗體，這可能會為阿茲海默症的治療帶來新的突破。
Alzheimer’s disease (AD) is the most common neurodegenerative disease and a significant challenge in aging societies. Unfortunately, there is currently no recognized effective drug or treatment for AD. Beta-amyloid (Aβ) and related pathogenic proteins such as TDP-43, Prion, Tau, are considered the main causes of AD brain deposits. Recent studies have established a highly positive correlation between soluble Aβ oligomers and AD. However, Aβ oligomers are unstable intermediates, and identifying their structures is very challenging, whether by using patient brain extracts directly or by preparing sample in vitro. Since 2018, we have exploited reverse-micelle systems to prepare oligomeric aggregates of amyloid protein. The rationale is to incubate the protein monomers in a confine space to prevent the formation of fibrillar aggregates. The advantage of this approach allows the preparation of structurally homogenous oligomers or the study of protein-protein interactions. We have leveraged these properties to study two critical issues: (1) amplifying Aβ oligomers from brain tissue extracts, and (2) studying the interaction between TDP-43 and Aβ at the oligomeric stage. Through multiple cycles of seeding and incubation, we successfully extracted and amplified the concentration of brain-derived Aβ oligomers, increasing their concentration from pico-molar to nano-molar levels, thus enabling NMR spectroscopic measurement of these challenging brain-derived oligomeric samples. To study the interaction between TDP-43 and Aβ, we co-incubated TDP-43 and Aβ in reverse micelle, and used solid state NMR to demonstrate that TDP-43 and Aβ form co-aggregates at the oligomeric stage. We also proposed that D23 and K28 in Aβ40 are key amino acids for the interaction between TDP-43 and Aβ40 oligomers. Our ultimate goal is to develop monoclonal antibodies against Aβ oligomers induced by brain tissue extracts, which may bring new breakthroughs in the treatment of Alzheimer’s disease.
