Murine Transgenic Models of Prion Diseases

朊病毒病的小鼠转基因模型

• 批准号: 8237710
• 负责人: DAVID A HARRIS
• 金额: $ 50.6万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8237710
• 关键词: Address; Amino Acids; Animals; Applications Grants; Axon; Biological Process; Blood; Brain; Cell-Free System; Cells; Cessation of life; Charge; Chemicals; Clinical; Cultured Cells; Development; Disease; Familial Creutzfeldt-Jakob Disease; Food Safety; Funding; Genetic; Genetically Engineered Mouse; Glutamate Receptor; Goals; Grant; Human; Infection; Inherited; Ion Channel; Knowledge; Laboratories; Ligands; Link; Liposomes; Mediating; Molecular; Mus; Mutation; N-terminal; Nature; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neurons; Organ; Patch-Clamp Techniques; Pathogenesis; Pathology; Pathway interactions; Phenotype; Play; Point Mutation; PrP; PrP gene; PrPC function; PrPSc Proteins; Prion Diseases; Prions; Property; Protein Region; Proteins; Public Health; Role; Slice; Staging; Symptoms; Synapses; Testing; Therapeutic Effect; Transgenic Mice; Transgenic Model; Work; base; cell injury; early onset; in vitro Assay; inhibitor/antagonist; killings; mouse model; mutant; neuroblastoma cell; neuropathology; neurotoxic; neurotoxicity; novel therapeutic intervention; protein aggregation; protein misfolding; response; wild-type PrP

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.

项目描述（由申请人提供）：该项目的总体目标是利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。在上一个资助期内，我们研究了三种不同的遗传机制（PrPC正常功能的丧失、获得和颠覆）如何在两种不同的转基因模型中促进神经退行性变：Tg（PG14）小鼠，其表达易于聚集的PrP，其中含有与家族性克雅氏病相关的9八肽插入突变；和Tg（CR）小鼠，它们表达一种高度神经毒性形式的PrP，在蛋白质的保守中心区域缺失21个氨基酸（残基105-125）。在我们之前的工作过程中，我们有三个关键的发现，形成了这个更新申请的基础。首先，我们发现PrP中心区域的突变，包括CR等缺失，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，可以通过片段夹紧技术观察到。其次，我们已经确定了一组抑制突变型PrP离子通道活性的PrP配体。有趣的是，在无细胞系统或小鼠中，这些配体中的大多数也抑制PrPSc的形成，这表明共同的结构域控制着PrP的功能活性及其向PrPSc的转化。第三，我们发现在PrP分子的通道形成和神经保护特性中，n端一个带正电的9个氨基酸结构域起着至关重要的作用。综上所述，这些新发现提出了一种假设，即一些遗传性朊病毒疾病的发病机制，以及可能的传染性获得性病例，可能是由于prp相关通道的活性。此外，这些致病作用可能是由PrP初级序列的一个令人惊讶的短结构域决定的，该结构域控制着离子通道活性和蛋白质的构象错误折叠。为了实现这些观察结果，我们计划(1)分析与“通道诱导”PrP突变相关的家族朊病毒疾病的转基因模型；(2)确定PrPSc是否诱导依赖于prpc的离子通道激活；(3)检测prp相关离子通道活性抑制剂对家族性和感染性朊病毒疾病小鼠模型是否有治疗作用；(4)研究关键神经毒性决定因素PrP的n端多碱基结构域的功能。该项目解决了关于朊病毒神经毒性机制的主要知识缺口。它探索了可能被朊病毒激活的特定分子和细胞途径，并可能在其致病作用中发挥作用。该建议将朊病毒疾病与其他由于离子通道异常活动而导致的神经退行性疾病联系起来，并为通过阻断特定的神经毒性途径治疗朊病毒疾病奠定了基础。