以二維螢光影像及螢光相關光譜探討Aβ胜肽在膜中之動態行為

Abstract

阿茲海默症是一種慢性的神經退化疾病，也是最常見的失智症型態。目前普遍認為造成阿茲海默症的最主要原因是類澱粉蛋白 β (Aβ) 在大腦的堆積。Aβ 是前類澱粉蛋白的代謝產物, 在類澱粉蛋白生成途徑中經由 β- 和 γ-secretase 的剪切而產生。在 γ-secretase 剪切後，產生的 Aβ 會被釋出的細胞外，並堆積形成寡聚體或纖維。過去有許多研究專注於探討 Aβ 纖維的形成機制，但是較少有研究關注在 γ-secretase 剪切後的 Aβ 胜肽在細胞膜上的動態行為，包含如何被釋出以及如何聚集。此外，曾有研究指出細胞膜的組成會影響膜的流動性，進而改變 Aβ 的生成和堆積。然而 Aβ 胜肽在細胞膜中的動態行為是否會受到胞膜成分的影響，目前仍是未知。 本研究主要目的為以螢光相關光譜技術建立一個可觀察 Aβ 胜肽在膜中的動態行為的方法。我們已成功地合成並純化與前類澱粉蛋白穿膜區相符的胜肽序列，並加上螢光標定，也成功地在序列中的 γ-secretase 剪切位中插入一個光敏感化合物，以利用光解的方式模仿 γ-secretase 剪切。我們也建立了一個製造胜肽鑲嵌脂質體以及胜肽鑲嵌脂雙層的方法，並且初步地測試了適合的螢光相關光譜條件。在未來，我們可進一步將此系統結合已合成的螢光標定光敏感胜肽，觀察光解後前類澱粉蛋白氨基端部分在膜中移動之動態行為，以及動態行為與細胞膜組成成分之關係。

Alzheimer’s disease (AD) is a slowly progressing neurodegenerative disease and the most common type of dementia. The accumulation of amyloid-β (Aβ) in brain is generally considered to be the major culprit of AD. Aβ is a catabolic product of amyloid precursor protein (APP), generated in the amyloidogenic pathway by β- and γ-secretase cleavage. After γ-secretase cleavage, Aβ peptides will be released into extracellular spaces and aggregate into oligomers and fibrils. Despite numerous researches of fibrillogenesis mechanism, there are very few studies exploring how Aβ peptides are released from membrane and associate together after the γ-secretase cleavage. In addition, it has been reported that membrane fluidity, which is affected by lipid composition, can influence Aβ generation and aggregation. Whether the dynamic behavior of Aβ peptides in the membrane is affected by lipid composition or not is rarely discussed. In this study, we aimed to develop a method to observe the dynamic behavior of Aβ peptide in membrane with fluorescence correlation spectroscopy (FCS). We have successfully synthesized the fluorophore-labeled peptide corresponding to APP transmembrane region, as well as the one with a photolabile linker to mimic the γ-secretase cleavage on APP. We have also built a method to prepare peptide-inserted liposomes and lipid bilayers, and preliminarily set up a suitable condition for FCS measurements. This system, combined with the fluorescent photolabile linker introduced peptide we produced, can allow us to further investigate the movement of N-terminus of APP after photolysis in membrane with different lipid composition.