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PrP-sen的异常重折叠的蛋白酶抗性形式（PrP-res或PrPSc）组成。感染易感性可受PrP-sen和PrP-res之间氨基酸同源性的影响，而PrP-res分子之间的结构差异被认为是编码菌株表型的原因。我的研究涉及朊病毒疾病在分子水平和致病水平上的许多不同方面。特别是，我的实验室专注于：1）识别朊病毒感染期间发生的最早事件，2）精确定义PrP-res形成发生的不同细胞区室，3）确定朊病毒株的分子基础，4）开发有效的朊病毒疗法。 在大多数形式的朊病毒疾病中，传染性主要存在于中枢神经系统或免疫系统器官，如脾和淋巴结。 然而，对小鼠和非人类灵长类动物的研究表明，朊病毒也可以作为淀粉样蛋白沉积物存在于心脏组织中。 感染性朊病毒在人类心脏组织中以淀粉样蛋白的形式沉积将是一个重大的公共卫生问题。 2013年，我们完成并发表了研究，以确定朊病毒感染性是否可以在人类心脏组织中发现。 我们将两名sCJD患者的甲醛固定脑和心脏组织以及两名淀粉样心脏病患者的朊病毒蛋白阳性固定心脏组织接种到人朊病毒病的小鼠模型中。 我们的研究结果表明：1）朊病毒感染性不可能存在于sCJD患者的心脏组织中，2）朊病毒感染性与淀粉样心脏病患者的朊病毒蛋白阳性心脏组织无关，3）朊病毒感染性可以在存档的脑组织学标本中存活数十年。 这项工作于2013年发表在Journal of Virology上。 朊病毒的细胞摄取是细胞暴露于外源性朊病毒后最早发生的事件之一，目前尚不清楚PrP-res的生物化学性质如何影响其摄取，尽管大小被认为是重要的。2013年，我们完成并发表了实验，描述了在小鼠朊病毒感染的初始阶段PrP-res与细胞的相互作用。 我们表明，对于两种不同的朊病毒株，绝大多数的PrP-RES所采取的细胞仅限于一个特定的聚集部分。 然而，细胞分解PrP-res聚集体的速率取决于朊病毒菌株，不能感染细胞的菌株分解得更快。 这项工作在《病毒学杂志》上发表。 2013年，我们开始了体内研究，以观察颅内接种后朊病毒感染期间的早期事件。 这些研究将检查PrP-res的早期形成以及朊病毒感染急性期宿主PrP-sen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PrP-res以及PrP-sen如何受到影响。 2013年，我们还启动了体外研究工作，研究不同的人类朊病毒株如何与人类细胞系相互作用，并开始开发体外系统，研究人类PrP-res的急性形成。这些研究将有助于我们了解人类朊病毒如何与不同类型的细胞相互作用，引发感染。 最后，在2013年，我们开始与苏格兰爱丁堡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。

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 (PrP-res or PrPSc) of the normal, protease-sensitive prion protein, PrP-sen. Susceptibility to infection can be influenced by amino acid homology between PrP-sen and PrP-res while differences in structure between PrP-res molecules are believed to encode strain phenotypes. My studies address many different aspects of prion diseases at both the molecular and pathogenic level. In particular, my laboratory focuses on: 1) identifying the earliest events which occur during prion infection, 2) precisely defining the different cellular compartments where PrP-res formation occurs, 3) determining the molecular basis of prion strains and, 4) development of effective prion therapeutics. In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, studies in mice and non-human primates have shown that prions can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. In 2013, we completed and published studies to determine whether or not prion infectivity can be found in human heart tissue. We inoculated archival formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into a mouse model of human prion disease. Our results showed that: 1) prion infectivity is not likely present in cardiac tissue from sCJD patients, 2) prion infectivity is not associated with prion protein positive cardiac tissue from amyloid heart disease patients and, 3) prion infectivity can survive in archival brain histology specimens for decades. This work was published in 2013 in the Journal of Virology. One of the earliest events occurring in cells following exposure to an exogenous source of prions is the cellular uptake of PrP-res. It is unclear how the biochemical properties of PrP-res influence its uptake, although size is thought to be important. In 2013, we completed and published experiments characterizing the interaction of PrP-res with the cell during the initial stages of mouse prion infection. We show that, for two different prion strains, the vast majority of PrP-res taken up by the cell is limited to a specific aggregated fraction. However, the rate at which the cell breaks up the PrP-res aggregates is dependent upon the prion strain, with the strain that is unable to infect cells being broken up faster. This work is in press at the Journal of Virology. In 2013, we initiated in vivo work to look at early events during prion infection following intracranial inoculation. These studies will examine the early formation of PrP-res as well as changes in the expression of the host PrP-sen 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 PrP-res occurs and how PrP-sen is affected. In 2013, we also initiated in vitro work to study how different human prion strains interact with human cell lines and began developing in vitro systems to study acute formation of human PrP-res. These studies will help us to understand how human prions interact with different cell types to trigger infection. Finally, in 2013 we initiated a 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.