全长朊粒蛋白淀粉样纤维引发细胞毒性的机制研究

Cytotoxic Amyloid Fibrils From Full-length Human PrPC are Transmissible to Induce The Misfolding of Endogenous PrPC

目的 朊病毒病(prion disease)是一类由朊粒蛋白(PrP)发生错误折叠、聚集形成致病性的PrPSc导致的具有高致死率的神经退行性疾病。本文在细胞和动物水平开展了PrP纤维诱导内源PrP聚集和毒性机制的研究。方法 通过超速离心结合蛋白质免疫印迹实验检测PrP聚集;通过氧化压力实验,使用Annexin V-FITC/PI双染检测细胞凋亡;运用细胞超薄切片技术检测细胞线粒体形态;在动物水平,分离新生小鼠的前额叶,进行横断切片培养,在脑片上接种PrP纤维。结果 PrP纤维种子可以诱导内源PrP聚集,PrP纤维可以诱导细胞内氧化压力升高和细胞凋亡,PrP纤维可以引起线粒体损伤,PrP纤维可以诱导小鼠前额叶内源PrP聚集。结论 本文在细胞和动物水平证实体外组装的PrP淀粉样纤维具有细胞毒性和潜在的感染性。

Objective Prion diseases are infectious,lethal neurodegenerative disorders principally caused by the conformational conversion of prion protein(PrP)from its cellular form(PrPC)into a protease-resistant,aggregated form(PrPSc)in humans and various vertebrate species.We have recently reported a cryo-EM structure of an amyloid fibril formed by full-length human PrP,which features a parallel in-register intermolecularβsheet architecture.However,it is unclear whether amyloid fibrils from full-length human PrPC are cytotoxic and transmissible.Methods Sarkosyl-insoluble Western blotting and cell viability assays were used to detect PrP aggregation and cell viability,respectively.Oxidative stress detection and annexin V-FITC apoptosis detection assays were also used for the determination of ROS production and cell apoptosis,respectively.Results Human PrP fibrils are cytotoxic,and transmissible to induce the misfolding of endogenous PrPC not only in cells but also in the frontal cortices of infant mice.The PrP fibrils also induce severe mitochondrial damage in cells stably expressing PrPC.Importantly,the PrP fibrils elevate ROS production via aggravating mitochondrial stress resulting from PrP aggregation and induce severe late apoptosis in cells stably expressing PrPC.Conclusion We demonstrate that PrP fibrils prepared in vitro are cytotoxicity and have pathogenic potential.