Molecular Genetics Of Scrapie Pathogenesis

传染性海绵状脑病（Transmissible spongiform encephalopathies，TSEs）是一组罕见的神经退行性疾病，包括人类的克雅氏病（Creutzfeldt-Jakob disease，CJD）、绵羊的瘙痒病、牛海绵状脑病（bovine spongiform encephalopathies，BSE）和骡鹿和麋鹿的慢性消耗病（chronic wasting diseases，CWD）。TSE传染性可跨越物种屏障。BSE在英国感染人类的事实和CWD在美国可能具有类似作用的担忧强调了了解TSE发病机制和开发有效治疗方法的重要性。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。对感染的易感性可以受到PrPC和PrPSc之间的氨基酸同源性的影响，而PrPSc分子之间的结构差异被认为是编码菌株表型。我的实验室致力于朊病毒疾病在分子和致病水平上的许多不同方面。特别是，我的研究集中在：1）确定朊病毒感染期间发生的最早事件，2）精确定义PrPSc形成发生的不同细胞区室，3）确定朊病毒株的分子基础，4）开发有效的朊病毒治疗方法。 暴露于外源性朊病毒源后细胞中最早发生的事件之一是PrPSc的细胞摄取，但尚不清楚PrPSc的不同生化特性如何影响其摄取。历史上，人们一直认为感染性朊病毒颗粒主要由高度蛋白酶抗性的PrPSc组成。 然而，最近，很明显，并不是所有的感染性PrPSc是高度蛋白酶耐药。存在相对蛋白酶敏感的PrPSc形式，其以与蛋白酶抗性PrPSc化学计量的量存在。 这种蛋白酶敏感与蛋白酶抗性PrPSc的比例可以根据朊病毒分离株而变化，其中一些感染性分离株主要由蛋白酶敏感的PrPSc组成。 蛋白酶敏感的PrPSc分子被认为有助于体内朊病毒发病机制的不同方面，如疾病潜伏期，尽管机制尚不清楚。 特别是，没有研究蛋白酶敏感的PrPSc与蛋白酶耐药的PrPSc相比如何与细胞相互作用。 2014年，我们完成并发表了实验，描述了在人类朊病毒感染的初始阶段，蛋白酶敏感型与蛋白酶抗性型PrPSc与人类星形胶质细胞的相互作用。 我们发现，对于4种不同类型的人朊病毒，蛋白酶敏感的PrPSc被内化和降解的细胞类似于蛋白酶耐药的PrPSc。 这项工作在美国病理学杂志上出版。 2014年，我们继续进行体内研究，以观察颅内接种后朊病毒感染期间的早期事件。 这些研究的目的是检查PrPSc的早期形成以及在朊病毒感染的急性期宿主PrPC分子表达的变化。 我们的工作将提供有关暴露于朊病毒后最初几个小时内发生的事件的关键信息，包括何时形成新的PrPSc以及PrPC如何受到影响。 2014年，我们开始使用新开发的体外系统研究PrPC氨基酸多态性对人PrPSc形成的影响。 我们还利用无细胞和基于细胞的系统来评估人类PrPSc的形成以及不同的人类朊病毒株如何与不同的细胞类型相互作用以引发感染。 2014年，我们继续与苏格兰爱丁堡CJD监测部门的Mark Head博士和James Ironside博士合作。 这项合作涉及使用蛋白质组学研究不同的人类朊病毒株。 这些研究将为不同人类朊病毒疾病的发病机制和分类提供深入了解。 最后，在2014年，我们启动了初步研究，以测试神经炎症抑制剂是否也可以抑制或延迟朊病毒疾病。

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. TSE infectivity can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding TSE pathogenesis and developing effective therapeutics. 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. Susceptibility to infection can be influenced by amino acid homology between PrPC and PrPSc while differences in structure between PrPSc molecules are believed to encode strain phenotypes. My laboratory addresses many different aspects of prion diseases at both the molecular and pathogenic level. In particular, my studies focus on: 1) identifying the earliest events which occur during prion infection, 2) precisely defining the different cellular compartments where PrPSc formation occurs, 3) determining the molecular basis of prion strains and, 4) development of effective prion therapeutics. One of the earliest events occurring in cells following exposure to an exogenous source of prions is the cellular uptake of PrPSc, but it is unclear how the different biochemical properties of PrPSc influence its uptake. Historically, it has always been believed that the infectious prion particle consisted mainly of highly protease-resistant PrPSc. Recently, however, it has become apparent that not all infectious PrPSc is highly protease-resistant. There are forms of PrPSc which are relatively protease-sensitive that are present in stoichiometric amounts with protease-resistant PrPSc. This ratio of protease-sensitive to protease-resistant PrPSc can vary depending upon the prion isolate, with some infectious isolates consisting of mostly protease-sensitive PrPSc. Protease-sensitive PrPSc molecules are thought to contribute to different aspects of prion pathogenesis in vivo such as disease incubation times, although the mechanisms are unclear. In particular, there are no studies looking at how protease-sensitive PrPSc interacts with cells when compared to protease-resistant PrPSc. In 2014, we completed and published experiments characterizing the interaction of protease-sensitive versus protease-resistant forms of PrPSc with human astrocyte cells during the initial stages of human prion infection. We showed that, for 4 different types of human prions, protease-sensitive PrPSc was internalized and degraded by the cell similarly to protease-resistant PrPSc. This work is in press at The American Journal of Pathology. In 2014, we continued in vivo work to look at early events during prion infection following intracranial inoculation. These studies are designed to examine the early formation of PrPSc as well as changes in the expression of the host PrPC molecule during the acute stage of prion infection. Our work will provide critical information about the events that occur during the first few hours following exposure to prions, including when formation of new PrPSc occurs and how PrPC is affected. In 2014, we have begun using a newly developed in vitro system to study the effect of PrPC amino acid polymorphisms on human PrPSc formation. We are also utilizing both cell-free and cell-based systems to assess human PrPSc formation and how different human prion strains interact with different cell types to trigger infection. In 2014 we continued our collaboration with Dr. Mark Head and Dr. James Ironside at the CJD Surveillance unit in Edinburgh, Scotland. This collaboration involves using proteomics to study different human prion strains. These studies will provide insight into the pathogenesis and classification of different human prion diseases. Finally, in 2014 we have initiated preliminary studies to test whether or not inhibitors of neuroinflammation can also inhibit or delay prion disease.