探討以逆相微胞製備之 TDP-43 胜肽寡聚物的特性

Abstract

部分神經退化性疾病致病原因爲蛋⽩質或胜肽的不正常堆疊，形成有神經毒性的類澱粉樣聚集物。在 2006 年，TAR DNA-binding Protein 43 (TDP-43) 被發現為造成肌萎縮性脊髓側索硬化症 (amyotrophic lateral sclerosis, ALS) 和額顳葉退化症 (frontotemporal lobar degeneration, FTLD) 病理堆積物的主要成分。⽽近年來有 TDP-43 蛋⽩質 C 端⽚段 (TDP-43 C-terminal fragment) 被發現有極⾼的傾向形成澱粉樣纖維。⽬前已有許多⽂獻指出，寡聚物的神經毒性⽐單體與纖維都來得⾼。然⽽，這些寡聚物較不穩定且胜肽聚合過程呈多態性 (polymorphism)，因此較難直接研究寡聚物的特性。 在本實驗中，我們嘗試利⽤逆相微胞去限制胜肽的聚集化過程。使⽤Aerosol-OT (sodium bis (2-ethylhexyl) sulfosuccinate, AOT) 界⾯活性劑來製備逆相微胞，並選擇粒徑⼤⼩為 33 nm ([H2O]/[AOT surfactant] = 70) 的逆相微胞來包覆單體，微胞內的⽔相環境可使胜肽單體碰撞聚合，並產⽣胜肽寡聚物。在逆相微胞內的寡聚物⽣成後，會因為系統內有限的空間與單體數⽬，⽽無法形成纖維。本研究將利⽤ TDP-43 蛋⽩質 C 端胜肽⽚段作為研究聚合過程的澱粉樣胜肽，並在除去微胞並純化寡聚物後，利⽤ ThT (thiofalvin T) 螢光，研究聚集情形；同時以圓⼆⾊光譜儀及電⼦顯微鏡，研究寡聚物之⼆級結構變化與形貌。最後藉由引晶效應 (seeding effect) 來證實寡聚物能作為核種 (seed)，並加速 TDP-43 蛋⽩質聚集化現象。

Misfolded proteins or peptides forming neurotoxic amyloid aggregates are hallmarks of many neurodegenerative diseases. In 2006, TAR DNA-binding protein (TDP-43) was identified as major protein components of inclusion bodies derived from amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients. It is now known that TDP-43 C-terminal fragments are not only the major component of the inclusions but also show the propensity to form fibrils. According to recent research, oligomeric intermediates are more relevant to pathological progression than species such as monomers or fibrillar aggregates. Unfortunately, these oligomeric intermediates are always unstable and short-lived. Herein, we developed a system applying reversed micelles (RMs) to constrain limited peptide molecules inside each vesicle. After forming oligomers inside RMs, limited molecules disallow them from further developing into fibril structure as final state. In this study, peptide fragments derived from TDP-43 C-terminal were prepared by RMs method. By stabilizing the unstable peptidic oligomeric intermediates, it allows us to study the biophysical properties by steady-state techniques, such as transmission electron microscopy (TEM), circular dichroism (CD) and thioflavin T (ThT).Aerosol-OT (AOT, sodium bis(2-ethylhexyl) sulfosuccinate), an anionic surfactant, was used to prepare RMs. In water-in-oil microemulsion systems, water droplets containing pre-disaggregated peptides were encapsulated by AOT and dispersed in isooctane. The average size of RMs was related to water loading parameter (w0), which is the molar ratio of water to surfactant molecules (w0 = [H2O] / [AOT]). Amyloid peptides derived from TDP-43 in RMs (w0 = 70) resulted in vesicles with 33 nm diameters measured by dynamic light scattering (DLS). In addition, we also disrupt reverse micelle to obtain the oligomers and keep tracking their dramatically changed conformations by using ThT fluorescence. As time goes on, freshly extracted oligomers will form fibrils/ films. Moreover, oligomeric intermediates would induce TDP-43 protein aggregation by showing seeding effect compared to monomers or fibrils.