Molecular Mechanisms of Prion Protein Amyloid Formation

传染性海绵状脑病（TSEs或朊病毒病）是一组罕见的神经退行性疾病，包括绵羊瘙痒症、牛海绵状脑病（BSE）以及黑尾鹿和麋鹿的慢性消耗性疾病（CWD）。 在人类中，最常见的朊病毒病类型是克雅氏病（CJD），它可以以几种形式发生。 散发性CJD（sCJD）占大多数病例，在全世界以每百万人1-2例的发病率随机发生。 医源性CJD（iCJD）与暴露于朊病毒污染的医疗器械或产品有关，而家族性CJD（fCJD）与朊病毒蛋白基因突变有关。TSE疾病的感染因子称为朊病毒，主要由正常的蛋白酶敏感朊病毒蛋白PrPC的异常重折叠的蛋白酶抗性形式（PrPSc）组成。 PrPSc可以作为弥散性淀粉样蛋白阴性沉积物或作为致密的淀粉样蛋白阳性沉积物沉积在脑中。 淀粉样蛋白形式的朊病毒疾病似乎比非淀粉样蛋白形式的传染性更低。 此外，尚不清楚PrPSc主要作为淀粉样蛋白沉积的朊病毒疾病是否遵循与PrPSc主要作为非淀粉样蛋白沉积的朊病毒疾病相同的致病过程。 我们有兴趣了解PrP淀粉样蛋白形成的分子机制，并已开始使用体外和体内模型系统来解决这个问题。 该项目的重点是：1）了解PrP淀粉样蛋白形成的途径，2）研究PrP突变如何影响家族性朊病毒病中PrPSc淀粉样蛋白的形成。 使用LC-MS/MS Nanospray离子阱质谱法，在2015年，我们已经生成了从20多例CJD病例中分离的PrPSc的蛋白质谱，其中PrPSc以弥漫性淀粉样蛋白阴性沉积物和/或淀粉样蛋白斑块的形式沉积。 我们的研究结果表明，蛋白质谱不同，这取决于是否PrPSc是来自sCJD或iCJD，这两种不同的疾病类型之间的致病性差异。 描述这项工作的手稿正在编写中。 在2015年，我们继续进行长期的体内研究工作，在表达突变型或野生型人类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朊病毒疾病的发病机制。 多种不同的CJD分离株已被接种到这些小鼠中，在某些情况下，来自同一患者的多个脑区也被接种。 虽然我们在2015年完成了其中一些分离株的传播数据，但接种了多种分离株的小鼠仍在监测疾病。 本实验代表了另一种方法来描绘淀粉样蛋白和非淀粉样蛋白形式的朊病毒疾病的机制。这些研究将需要几年时间才能完成，但将在人类朊病毒疾病的几个知之甚少的领域提供重要信息，包括1）宿主对朊病毒株对不同体内疾病表型的贡献，和2）淀粉样蛋白对非淀粉样蛋白朊病毒形成的机制。 在2015年，我们继续进行体外fietization研究，以研究与遗传性人类朊病毒病相关的不同PrPC突变如何决定PrP淀粉样蛋白的形成。 特别是，我们已经研究了蛋白水解加工如何影响PrP原纤维的形成。 这些研究将帮助我们了解PrP纤维形成所需的细胞环境，并深入了解为什么淀粉样蛋白或纤维状形式的人PrPSc往往与低朊病毒感染性相关。 因此，它们直接解决了错误折叠的蛋白质具有感染性所需的问题。

Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. In humans, the most common type of prion disease is Creutzfeldt-Jakob disease (CJD) which can occur in several forms. Sporadic CJD (sCJD) makes up the majority of the cases and occurs randomly at an incidence of 1-2 per million worldwide. Iatrogenic CJD (iCJD) is associated with exposure to prion contaminated medical instruments or products while familial CJD (fCJD) is associated with mutations in the prion protein gene. 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, in 2015 we have generated the protein profiles of PrPSc isolated from over 20 cases of CJD where PrPSc is deposited as diffuse, amyloid negative deposits and/or as amyloid plaques. Our results show that the protein profiles differ depending upon whether PrPSc is derived from sCJD or iCJD suggesting pathogenic differences between the two different disease types. A manuscript describing this work is in preparation. In 2015 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. Multiple different CJD isolates have been inoculated into these mice and, in some cases, multiple brain regions from the same patient have also been inoculated. While we completed transmission data for some of these isolates in 2015, mice inoculated with multiple isolates are still being monitored for disease. This experiment represents another approach to delineate the mechanisms underlying amyloid and non-amyloid forms of prion disease. These studies will 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 2015, we continued 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. In particular, we have looked at how proteolytic processing may influence PrP fibril formation. These studies will help us to understand the cellular environments required for PrP fibril formation and provide insight into why amyloid or fibrillar forms of human PrPSc tend to be associated with low prion infectivity. Thus, they directly address the issue of what is required for a misfolded protein to be infectious.