PrP-scrapie transport across intestinal & BBB

描述(申请人提供)：变异克雅氏病(VCJD)通过被牛海绵状脑病(BSE)污染的肉类传播给人类，以及通过静脉接种外周血液将BSE传播给实验动物提出了两个重要的问题：1)食物中的Prion是如何通过肠道上皮屏障运输的，2)外周血中的Prion如何穿过内皮血脑屏障(BBB)。随着人们意识到近100万头感染疯牛病的奶牛可能已进入人类食物链，这些问题变得越来越重要。一个新出现的问题是慢性消耗性疾病(CWD)的传播，CWD是美国某些地区鹿和麋鹿的一种普恩病毒疾病，以及关于其向牲畜和人类传播的不确定性。尽管存在这些担忧，但令人惊讶的是，人们对传染性PrP-scrapie(PrPsc)的机制知之甚少(S)，PrPsc是一种27-30 kDa的蛋白质，通过它从肠道或外周血液运输到中枢神经系统。 来自我的实验室的初步数据表明，散发性CJD(SCJD)脑匀浆中的PrPsc与铁蛋白一起跨上皮细胞转运。当考虑到其他数据表明sCJD病例脑实质中氧化还原活性铁导致大脑铁蛋白水平上调时，这一观察结果提出了重要的问题。我们假设PrPsc的跨上皮和内皮细胞屏障的运输是由像铁蛋白这样具有明确的跨细胞路线的蛋白质促进的，并且大脑铁稳态的失衡直接导致了某些Pron疾病的发病，并通过铁蛋白促进感染性而间接地参与了这一过程。因此，这项建议的中心目标是研究PRPS相关蛋白包括铁蛋白和转铁蛋白在促进其跨肠上皮和血脑屏障转运中的作用，并评估氧化还原活性铁在PrP疾病发病机制中的作用。拟议的研究将在三个具体目标下进行。在目标1中，将评估铁蛋白、转铁蛋白和其他PrPsc相关蛋白在PrPsc穿越体外模型的人肠上皮细胞屏障和血脑屏障中的作用。在目标2中，从目标1中的体外模型获得的结果将在体内被表达人PrP的转基因小鼠所证实。在目标3中，将利用铁蛋白过表达和H-铁蛋白缺失转基因小鼠来研究氧化还原活性铁在先天疾病发病机制中的作用。这些研究将有助于评估人类人群感染BSE、vCJD和CWD的风险，并有助于了解Pron病的发病机制。

DESCRIPTION (provided by applicant): The transmission of variant Creutzfeldt-Jakob disease (vCJD) to humans from bovine-spongiform encephalopathy (BSE)-contaminated meat, and the transmission of BSE by intra-venous inoculation of peripheral blood to experimental animals raises two important questions: 1) how are prions from food transported across the intestinal epithelial barrier, and 2) how do prions in the peripheral blood cross the endothelial blood brain barrier (BBB). These questions have gained increasing importance with the realization that close to one million BSE infected cows may have entered the human food chain. An emerging concern is the spread of Chronic Wasting Disease (CWD), a prion disease of the deer and elk in certain parts of USA, and the uncertainties regarding its transmission to livestock and humans. Despite these concerns, surprisingly little is known about the mechanism(s) by which the infectious prion or PrP-scrapie (PrPsc), a protein of 27-30kDa, is transported from the intestine or peripheral blood to the central nervous system. Preliminary data from my laboratory demonstrate that PrPsc in sporadic CJD (sCJD) brain homogenates is transported across epithelial cells in association with ferritin. When considered in context with additional data indicating an upregulation of brain ferritin levels in response to redox active iron in the brain parenchyma of sCJD cases, this observation raises important questions. We hypothesize that the transport of PrPsc across epithelial and endothelial cell barriers is facilitated by proteins like ferritin that have a defined transcytotic route, and that imbalance of brain iron homeostasis contributes directly to the pathogenesis of certain prion disorders, and indirectly by promoting infectivity through ferritin. Thus, the central goal of this proposal is to investigate the role of PrPs-associated proteins including ferritin and transferrin in facilitating its transport across the intestinal epithelium and the BBB, and to evaluate the role of redox active iron in the pathogenesis of prion disorders. The proposed studies will be carried out in three specific aims. In aim 1, the role of ferritin, transferrin, and other PrPsc-associated proteins in the transport of PrPsc across in vitro models of human intestinal epithelial cell barrier and the BBB will be evaluated. In aim 2, the results obtained from in vitro models in aim 1 will be confirmed in vivo in transgenic mice expressing human PrP. In aim 3, the role of redox active iron in the pathogenesis of priori disorders will be investigated using ferritin over-expressing and H-ferritin deletion transgenic mice. These studies will help in evaluating the risk of human population to BSE, vCJD, and CWD infection, and help in understanding the mechanism of prion disease pathogenesis.