Molecular Genetics Of Scrapie Pathogenesis

传染性海绵状脑病(TSE)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)。寨卡病毒的传染性可以跨越物种障碍。疯牛病已在英国感染人类，并担心慢性萎缩性胃病在美国可能会有类似的表现，这一事实突显了了解TSE发病机制和开发有效的抗TSE疗法的重要性。TSE疾病的感染源的确切性质尚不清楚。感染的易感性可能受到正常宿主蛋白(PrP-sen)和该蛋白的异常蛋白酶K抗性形式PrP-res之间的氨基酸同源性的影响。PrP-RES的形成与传染性密切相关，PrP-RES被认为是TSE疾病的感染源。了解这种蛋白质是如何形成的，对于我们理解TSE的发病机制和设计防止其合成的治疗策略至关重要。我的研究在分子和致病水平上解决了TSE疾病的许多不同方面。我的实验室尤其专注于：1)确定在TSE感染期间发生的最早事件，2)准确定义发生PrP-RES形成的不同细胞室，3)确定TSE菌株的分子基础，4)了解PrP淀粉样蛋白形成的途径，5)研究PrP突变如何影响TSE家族性疾病PrP-RES的形成，以及6)开发有效的治疗TSE药物。 在过去的一年里，我们研究了PrP-Res在不同的瘙痒病菌株和细胞类型中的摄取动力学。我们已经开发出一种新的、感染瘙痒病的组织培养细胞系，这将有助于我们在体外研究PrP淀粉样蛋白的形成。我们已经将PrP纤化研究扩展到包括全长重组PrP，并正在分析不同的PrP-sen区域如何调节八肽重复区域对PrP淀粉样蛋白形成的影响。 我们开发了一种组织培养系统，使我们能够跟踪TSE感染的过程，从最初将细胞暴露于TSE传染性直到建立持续感染。利用该系统，我们研究了PrP-Res在细胞内的摄取。我们的数据表明PrP-res的摄取与温度有关，并且摄取的速度因瘙痒病菌株而异。我们目前正在研究PrP-Sen和不同群体的PrP-Res对这一过程的影响。 PRP-RES可以以弥漫性淀粉样阴性沉积或致密淀粉样阳性沉积的形式沉积在大脑中。淀粉样型的TSE疾病的传播性似乎比非淀粉样型的低，这表明它们的发病机制存在根本的差异。最近的数据表明PrP-Sen上的糖磷脂酰肌醇(GPI)膜锚可能影响PrP-Res是否形成淀粉样蛋白。为了了解淀粉样蛋白感染TSE的基本分子机制，我们获得了一株不含GPI锚点的表达小鼠PrP-sen的小鼠成纤维细胞系，并用小鼠瘙痒病感染了它。这些细胞使小鼠的PrP-res没有GPI锚点。我们目前正在1)确定这些细胞产生的PrP-Res是否是淀粉样蛋白，以及2)确定没有GPI锚点的小鼠PrP-Sen如何在这些细胞中转化为PrP-Res。 遗传性TSE病与PrP基因内的突变有关。这些突变之一是在PrP中插入额外的8个氨基酸基序的拷贝(八肽重复)。八肽重复次数越多，TSE发病越早。我们已经证明，增加哺乳动物PrP-sen或PrP淀粉样蛋白形成肽(残基23-144)中的重复数显著提高PrP-RES和PrP淀粉样蛋白的形成速度。最近，我们发现PrP分子的其他区域可能调节八肽重复区域对PrP-RES的淀粉样蛋白形式的影响。这些数据不仅为八肽重复区域的额外拷贝与早期疾病发病之间的联系提供了分子基础，而且还表明PrP-Sen不同区域之间的相互作用可能有助于确定PrP-RES是否形成淀粉样蛋白。

Transmissible spongiform encephalopathies (TSE) 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 anti-TSE therapeutics. The precise nature of the infectious agent of the TSE diseases is unknown. Susceptibility to infection can be influenced by amino acid homology between a normal host protein (PrP-sen) and the abnormal proteinase K-resistant form of this protein, PrP-res. Formation of PrP-res is closely associated with infectivity and PrP-res has been hypothesized to be the infectious agent in the TSE diseases. An understanding of how this protein is made is critical for our understanding of TSE pathogenesis and for devising therapeutic strategies to prevent its synthesis. My studies address many different aspects of the TSE diseases at both the molecular and pathogenic level. In particular, my laboratory focuses on: 1) identifying the earliest events which occur during TSE infection, 2) precisely defining the different cellular compartments where PrP-res formation occurs, 3) determining the molecular basis of TSE strains, 4) understanding the pathways of PrP amyloid formation, 5) studying how mutations in PrP influence PrP-res formation in familial forms of TSE disease, and 6) development of effective therapeutic TSE agents. In the last year, we have studied the kinetics of PrP-res uptake for different scrapie strains and cell types. We have developed a new, scrapie infected tissue culture cell line that will help us to study PrP amyloid formation in vitro. We have extended our PrP fibrillization studies to include full-length recombinant PrP and are analyzing how different PrP-sen regions modulate the influence of the octapeptide repeat region on PrP amyloid formation. We have developed a tissue culture system that enables us to follow the process of TSE infection from the initial exposure of cells to TSE infectivity through to the establishment of a persistent infection. Using this system, we have studied the uptake of PrP-res into the cell. Our data show that PrP-res uptake is temperature dependent and that the rate of uptake differs depending upon the scrapie strain. We are currently investigating both the influence of PrP-sen and different populations of PrP-res on this process. PrP-res can be deposited in the brain as either diffuse, amyloid negative deposits or as dense, amyloid positive deposits. Amyloid forms of TSE disease appear to be less transmissible than non-amyloid forms, suggesting a fundamental difference in their pathogenesis. Recent data suggest that the glycophosphatidyl-inositol (GPI) membrane anchor in PrP-sen may influence whether or not PrP-res forms amyloid. In order to understand the basic molecular mechanisms underlying amyloid forms of TSE infection, we have derived a mouse fibroblast cell line expressing mouse PrP-sen without the GPI anchor and infected it with mouse scrapie. These cells make mouse PrP-res without the GPI anchor. We are currently in the process of 1) determining whether the PrP-res made by these cells is amyloid in nature and 2) determining how mouse PrP-sen without the GPI anchor is converted into PrP-res in these cells. Hereditary forms of TSE disease are associated with mutations within the PrP gene. One of these mutations is the insertion of extra copies of an eight amino acid motif (octapeptide repeat) into PrP. The greater the number of octapeptide repeats, the earlier the onset of TSE disease. We have shown that increasing the number of repeats either in mammalian PrP-sen or in a PrP amyloid-forming peptide (residues 23-144) significantly increases the rate at which PrP-res and PrP amyloid are formed. Recently, we have found that other regions of the PrP molecule may modulate the effect of the octapeptide repeat region on amyloid forms of PrP-res. These data not only provide a molecular basis for the association of extra copies of the octapeptide repeat region with early disease onset but also suggest that interactions between different regions of PrP-sen may help to determine whether or not PrP-res forms amyloid.