Molecular Mechanisms of Prion Protein Amyloid Formation

传染性海绵状脑病(TSE或Prion病)是一组罕见的神经退行性疾病，包括人类的克雅氏病(CJD)、绵羊的瘙痒病、牛海绵状脑病(BSE)和马鹿、麋鹿的慢性衰弱病(CWD)。TSE疾病的感染源被称为Prion，主要由正常的、对蛋白酶敏感的Prion蛋白PrPC的异常折叠、蛋白酶抵抗形式(PrPSc)组成。PrPSc可以以弥漫的淀粉样阴性沉积或致密的淀粉样阳性沉积的形式沉积在大脑中。类淀粉样蛋白病似乎比非类淀粉样蛋白疾病的传播性要低。此外，目前尚不清楚PrPSc主要以淀粉样沉积的PrPSc病是否遵循与PrPSc主要以非淀粉样蛋白沉积的PrPSc相同的致病过程。我们对了解PrP淀粉样蛋白形成的分子机制很感兴趣，并已开始使用体外和体内模型系统来探讨这一问题。本项目的重点是：1)了解PrP淀粉样蛋白的形成途径；2)研究PrP基因突变如何影响PrPSc类淀粉样蛋白的形成。 利用LC-MS/MS纳米螺旋离子陷阱质谱仪，我们已经产生了PrPSc以淀粉样蛋白或非淀粉样蛋白形式积聚的PrPSc感染的PrPsc感染小鼠脑组织的蛋白质组。我们的结果表明，大脑对淀粉样蛋白和非淀粉样蛋白沉积有类似的一般神经炎性反应。然而，细胞死亡途径似乎不同，线粒体的凋亡途径仅与非淀粉样蛋白形式的Pron病有关。在淀粉样蛋白表型中，金属结合和突触小泡运输受到更多的干扰。这项研究已经在蛋白质组研究杂志上发表。 2014年，我们继续了长期的体内工作，在表达突变型或野生型人PrP的转基因小鼠中研究不同形式的淀粉样蛋白和非淀粉样蛋白人类PrP疾病的发病机制。这代表了另一种描述淀粉样蛋白和非淀粉样蛋白形式的Pron疾病潜在机制的方法。这些研究可能需要几年的时间才能完成，但将在人类Pron疾病的几个鲜为人知的领域提供重要信息，包括1)宿主与Pron菌株对体内不同疾病表型的贡献，以及2)淀粉样蛋白与非淀粉样蛋白Prion形成的机制。 2014年，我们启动了一系列新的体外纤维化研究，以研究与遗传性人类PrP疾病相关的不同PrPC突变如何决定PrP淀粉样蛋白的形成。这些研究将帮助我们理解为什么人类PrPSc的淀粉样蛋白或纤维状形式往往与低普鲁恩感染性有关，从而直接解决错误折叠的蛋白质具有传染性所需的条件的问题。 2014年，我们已经开始使用LC-MS/MS纳米螺旋离子陷阱质谱仪来研究人类淀粉样蛋白和非淀粉样蛋白酪蛋白疾病病例的脑组织。这项工作将帮助我们开始了解PrPSc和其他蛋白质在两种疾病状态之间的差异，并应为其发病机制提供重要的见解。

Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrPSc) of the normal, protease-sensitive prion protein, PrPC. PrPSc can be deposited in the brain as either diffuse, amyloid negative deposits or as dense, amyloid positive deposits. Amyloid forms of prion disease appear to be less transmissible than non-amyloid forms. Furthermore, it is unclear whether or not prion diseases where PrPSc is deposited primarily as amyloid follow the same pathogenic processes as prion diseases where PrPSc is primarily deposited as non-amyloid. We are interested in understanding the molecular mechanisms underlying PrP amyloid formation and have begun to approach this issue using both in vitro and in vivo model systems. This project focuses on: 1) understanding the pathways of PrP amyloid formation and, 2) studying how mutations in PrP influence PrPSc amyloid formation in familial forms of prion disease. Using LC-MS/MS Nanospray Ion Trap Mass Spectrometry, we have generated the proteomes of prion infected mouse brain tissue which had accumulated PrPSc in either amyloid or non-amyloid forms. Our results suggest that the brain has a similar, general neuroinflammatory response to deposition of amyloid and non-amyloid forms of prions. However, cell death pathways appeared to differ, with mitochondrial pathways of apoptosis implicated only in the non-amyloid form of prion disease. Metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. This work is in press at the Journal of Proteome Research. In 2014 we continued long term in vivo work to study the pathogenesis of different forms of amyloid and non-amyloid human prion disease in transgenic mice expressing either mutant or wild-type human PrP. This represents another approach to delineate the mechanisms underlying amyloid and non-amyloid forms of prion disease. These studies will likely take several years to complete but will provide important information in several poorly understood areas of human prion disease including 1) the contribution of the host versus the contribution of the prion strain to different in vivo disease phenotypes and 2) the mechanisms of amyloid versus non-amyloid prion formation. In 2014, we have initiated a new series of in vitro fibrillization studies to study how different PrPC mutations associated with inherited forms of familial human prion disease dictate the formation of PrP amyloid. These studies will help us to understand why amyloid or fibrillar forms of human PrPSc tend to be associated with low prion infectivity and thus directly address the issue of what is required for a misfolded protein to be infectious. In 2014, we have begun to use LC-MS/MS Nanospray Ion Trap Mass Spectrometry to study brain tissue from human cases of amyloid and non-amyloid forms of prion disease. This work will help us begin to understand how PrPSc and other proteins differ between the two disease states and should provide important insights into their pathogenesis.