朊病毒感染激活自噬的分子机制研究

Prion diseases are known as a group of fatal neurodegenerative disorders, which affect both human and mammalian animals. It is widely accepted that prion diseases are caused by mifolding of a host-encoded glycoprotein PrPC into PrPSc as well as the mutation happened on PRNP gene. Autophagy is a critical cellular process involves degradation and digestion of cytoplasmic content, organelles and misfolded proteins by lysosomes. We have reported that autophagic flux was activated in 263K-infected hamsters and human genetic prion diseases, and the down-regulation of mTOR was mainly responsible for it. In this study, we aim to research if prion infection could activate other autophagy regulation pathways, like AMPK-ULK1, DAPK signaling pathway and NF1-regualted pathways. We will detect the protein expression levels of the molecules in these pathways and their phosphorylated forms in brain tissues from hamsters, mice and human infected with prion diseases. We also will test their expression level in chronic prion-infected cell lines. The dynamic changes of these molecules and their active forms, as well as the neuropathologic damages will be observed in the incubation period. Then we will reverse the expression of the key factor of each pathway by knock-down or over-expression to see if autophagy could be prohibited. All together, we could understand the role of these pathways in prion infection and neuropahtologic damages. It will be very helpful to explore the potential therapeutic method to treat prion diseases.

朊病毒病是一类感染人及多种动物的致死性神经退行性疾病，由朊蛋白的错误折叠或突变引起。细胞自噬是真核细胞用于清除胞内聚集物、受损细胞器、适应营养缺乏、环境胁迫等维持细胞稳态平衡的一种蛋白质降解过程。本课题组前期研究的结果显示朊病毒感染后自噬活化，且是通过mTOR下调引起。本研究旨在从体内、体外两个层面研究朊病毒感染后其他自噬调节通路：AMPK直接调节ULK1引发自噬的通路、DAPK调节的信号通路以及NF1调节的自噬通路上各分子及其活性的磷酸化形式蛋白表达水平的变化，然后通过在感染动物潜伏期内的不同时间点动态观察这些分子的变化过程，并与PrPSc沉积、神经元丢失等病理变化相结合，最后通过在细胞模型上敲降或过表达关键分子，检测细胞自噬情况，来反向证实这些信号通路在朊病毒感染中的作用，进一步揭示感染后自噬激活的分子机制，为在更广阔范围内寻找治疗朊病毒病的潜在治疗方法奠定基础。

朊病毒病是一类感染人及多种动物的致死性神经退行性疾病，由朊蛋白的错误折叠引起。细胞自噬是真核细胞清除错误折叠蛋白的重要途径。本课题组前期研究发现朊病毒感染后通过抑制mTOR信号通路激活大自噬，在本项目中，通过朊病毒感染的动物模型及稳定感染的细胞模型研究发现朊病毒感染可通过AMPK-ULK1信号通路激活自噬，不依赖于mTOR。PLK3在朊病毒感染过程中，除了通过介导神经细胞重新进入细胞周期，阻滞后凋亡，还可通过分子伴侣介导的自噬降解朊病毒。通过挖掘前期朊病毒病人脑组织基因表达数据，发现BDNF（Brain-derived neurotrophic factor）在终末期下调明显，通过在动物模型上蛋白水平的验证，发现BDNF-TrkB通路上多个分子在朊病毒感染后下调，参与神经元凋亡及丢失。结合课题组之前的研究，在自噬方面较为完整地揭示朊病毒致病分子机制：感染后激活自噬，降解错误折叠的朊病毒，当错误折叠蛋白堆积过多后不能通过自噬完全降解，在疾病终末期触发多条信号通路介导的神经细胞凋亡。.此外，本项目还对一例复杂染色体重排患者多系统异常的病因学进行研究，找到致病原因，通过SNP微阵列芯片分析早期胎停育分子机制，发现眼睛、表皮、脑等发育的信号通路失调是导致停育关键原因。此外，发现与NT（胎儿颈部透明层）增厚与TTTs（双胎输血综合征）两种胎儿发育异常相关的SNP位点。