Murine Transgenic Models of Prion Diseases

描述（由申请人提供）：本项目的总体目标是使用转基因小鼠模型研究朊病毒疾病的发病机制。我们的目标是了解朊病毒杀死神经元和损害中枢神经系统的分子和细胞机制。在上一个资助期间，我们研究了三种不同的遗传机制在两种不同的转基因模型中，PrPC正常功能的丧失、获得和破坏导致神经变性：Tg（PG 14）小鼠，其表达具有与家族性克雅氏病相关的九肽插入突变的聚集倾向PrP;和Tg（CR）小鼠，其表达PrP的高度神经毒性形式，在蛋白质的保守中心区域具有21个氨基酸缺失（残基105-125）。 在我们之前的工作中，我们有三个关键发现，这些发现构成了这次更新申请的基础。首先，我们发现PrP中心区域的突变，包括缺失如CR，以及与人类家族性朊病毒疾病相关的点突变，诱导了强大的离子通道活性，可以通过膜片钳技术观察到。其次，我们已经确定了一组PrP配体，抑制突变体PrP的离子通道活性。有趣的是，这些配体中的大多数也抑制PrPSc的形成，在无细胞系统或小鼠，这表明共同的结构域控制PrP的功能活性及其转化为PrPSc。第三，我们观察到一个带正电荷的，在N-末端的9个氨基酸的结构域在PrP分子的通道形成和神经保护特性中起着至关重要的作用。总之，这些新的发现表明，一些遗传性朊病毒疾病的发病机制，以及可能的感染性获得的情况下，可能是由于PrP相关的通道活动的假设。此外，这些致病作用可能是由一个令人惊讶的短结构域的PrP一级序列，控制离子通道活性和构象错误折叠的蛋白质。 为了进一步观察这些结果，我们计划（1）分析与“通道诱导”PrP突变相关的家族性朊病毒疾病的转基因模型;（2）确定PrPSc是否诱导PrPC依赖的离子通道激活;（3）测试PrP相关离子通道活性的抑制剂是否在家族性和感染性朊病毒疾病的小鼠模型中具有治疗效果;（4）探讨PrP N端多碱基结构域作为神经毒性决定因子的功能。 这个项目解决了关于朊病毒神经毒性机制的知识上的一个主要空白。它探讨了可能被朊病毒激活的特定分子和细胞途径，这可能在其致病作用中起作用。该提案将朊病毒疾病与其他由于离子通道异常活动引起的神经退行性疾病联系起来，并且它为通过阻断除朊病毒传播之外的特定神经毒性途径来治疗朊病毒疾病奠定了基础。 公共卫生相关性：朊病毒病是人类和动物的致命性神经退行性疾病，对公共卫生构成严重威胁，并危及食品、血液和器官供应的安全。这项拨款申请利用基因工程小鼠来探索朊病毒杀死神经细胞和损害大脑的机制。该项目为开发基于阻断特定神经毒性通路的新型治疗方法奠定了基础。

DESCRIPTION (provided by applicant): The overall goal of this project is to investigate the pathogenesis of prion diseases using transgenic mouse models. Our objective is to understand the molecular and cellular mechanisms by which prions kill neurons and damage the central nervous system. During the last funding period, we investigated how three, distinct genetic mechanisms (loss, gain, and subversion of the normal function of PrPC) contribute to neurodegeneration in two different transgenic models: Tg(PG14) mice, which express an aggregation-prone PrP harboring a nine-octapeptide insertional mutation linked to familial Creutzfeldt-Jakob disease; and Tg(CR) mice, which express a highly neurotoxic form of PrP with a 21-amino acid deletion (residues 105-125) in the conserved, central region of the protein. In the course of our previous work, we made three key discoveries that form the basis for this renewal application. First, we found that mutations in the central region of PrP, including deletions such as CR, as well as point mutations associated with familial prion diseases of humans, induce a powerful ion channel activity that can be observed by patch-clamping techniques. Second, we have identified a group of PrP ligands that inhibit the ion channel activity of mutant PrP. Interestingly, most of these ligands also inhibit the formation of PrPSc, in cell-free systems or mice, suggesting that common structural domains control both the functional activity of PrP and its conversion to PrPSc. Third, we observed that a positively-charged, nine amino acid domain at the N-terminus plays an essential role in the channel-forming and neuroprotective properties of PrP molecules. Taken together, these new findings suggest the hypothesis that the pathogenesis of some inherited prion diseases, and possibly of infectiously acquired cases as well, may be due to PrP-related channel activity. Moreover, these pathogenic effects may be determined by a surprisingly short domain of the PrP primary sequence that controls both ion channel activity and conformational misfolding of the protein. To pursue these observations, we plan to (1) Analyze transgenic models of familial prion diseases associated with a "channel-inducing" PrP mutations; (2) Determine whether PrPSc induces PrPC-dependent, ion channel activation; (3) Test whether inhibitors of PrP-related ion channel activity have therapeutic effect in mouse models of familial and infectious prion diseases; and (4) Investigate the function of the N-terminal polybasic domain of PrP a critical neurotoxicity determinant. This project addresses a major gap in knowledge concerning the mechanisms by which prions are neurotoxic. It explores specific molecular and cellular pathways that may be activated by prions, and that may a role in their pathogenic effects. The proposal ties prion diseases to other neurodegenerative conditions due to abnormal activity of ion channels, and it sets the stage for treating prion diseases by blocking specific neurotoxic pathways in addition to prion propagation. PUBLIC HEALTH RELEVANCE: Prion diseases are fatal neurodegenerative disorders of humans and animals that pose a grave threat to public health, and endanger the safety of the food, blood and organ supplies. This grant application utilizes genetically engineered mice to explore the mechanisms by which prions kill nerve cells and damage the brain. The project sets the stage for development of novel therapeutic approaches based on blocking specific neurotoxic pathways.