Role of prion protein in neuronal survival

朊病毒蛋白在神经元存活中的作用

• 批准号: 6847473
• 负责人: ANDREA C LEBLANC
• 金额: $ 20.52万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-15 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6847473
• 关键词: Bax gene /protein; apoptosis; confocal scanning microscopy; disease /disorder model; gene mutation; genetically modified animals; human tissue; immunoelectron microscopy; immunoprecipitation; intermolecular interaction; laboratory mouse; microinjections; molecular pathology; molecular site; neural degeneration; neurogenetics; neurons; neuropathology; neuroprotectants; prions; protein localization; protein structure function; stoichiometry; tissue /cell culture

DESCRIPTION (Adapted from applicant's abstract): The long-term goal of this application is to identify the function of prion protein (PrP) in the central nervous system. Four octapeptide repeats in the N-terminus of PrP are homologous to the BH2 domain of Bcl2 family of proteins. Based on preliminary observations that PrP interacts with pro-apoptotic Bax proteins and protects yeast and primary human neurons against Bax-mediated cell death, the investigator proposes the hypothesis that interaction between PrP and Bax mediates neuronal survival, and that this mechanism may be implicated in prion disease associated neuronal loss. This hypothesis will be tested in the following Specific Aims. Aim 1 will determine if PrP interaction with Bax is necessary for the neuroprotective function of PrP. PrP mutants will be generated that prevent PrP-Bax interaction and determining if co-expression of these mutants abolishes PrP's ability to inhibit Bax-mediated cell death. The function of these mutants will be tested in yeast by using galactose inducible constructs and micro-injection of eukaryotic expression constructs in human neurons. Aim 2 will determine if human PrP mutations associated with prion diseases dysregulate PrP-Bax interaction and PrP neuroprotective function. Mutations of PrP that are known to cause disease will be tested for their ability to interact with Bax and protect yeast or neurons against Bax-mediated cell death. Aim 3 will determine the location of neuroprotective PrP. The functional location of PrP and Bax will be determined by using mutant PrP and protein trafficking blocks. Primary cultures of human neurons will be used to examine protein localization by subcellular fractionation, beta-galactosidase complementation, pulse-chase experiments, immunofluorescence and confocal microscopy, and immuno-EM. Aim 4 will determine the molecular mechanism of neuronal protection by PrP. Specific Aim 5 will determine if PrP interacts with other pro-apoptotic proteins. In Aim 6, the neuroprotective role of PrP and the possible elimination of this function in PrP mutations associated with familial prion diseases will be assessed in mice models of familial prion diseases. The result of these experiments will clearly define the neuroprotective role of PrP against Bax mediated cell death.

描述（根据申请人的摘要改编）：这是长期目标 应用是识别中央prion蛋白（PRP）的功能 神经系统。 PRP的N末端中的四个八肽重复是 与BCl2蛋白质家族的BH2域同源。基于初步 观察到PRP与促凋亡的Bax蛋白相互作用并保护 酵母和原发性人神经元抗白细胞介导的细胞死亡， 研究者提出了一个假设，即PRP与Bax之间的相互作用 介导神经元的生存率，并且这种机制可能与Prion有关 疾病相关的神经元丧失。该假设将在 遵循特定目标。 AIM 1将确定PRP与Bax的相互作用是否为 PRP的神经保护功能所必需的。 PRP突变体将是 产生的可以防止PRP-BAX相互作用并确定是否共表达 这些突变体废除了PRP抑制Bax介导的细胞死亡的能力。这 这些突变体的功能将在酵母中使用半乳糖诱导进行测试 人类真核表达构建体的构造和微注射 神经元。 AIM 2将确定人类PRP突变是否与Prion相关 疾病失调的PRP-BAX相互作用和PRP神经保护功能。 已知引起疾病的PRP突变将被测试 能够与Bax相互作用并保护酵母或神经元免受BAX介导的能力 细胞死亡。 AIM 3将确定神经保护性PRP的位置。这 PRP和BAX的功能位置将通过使用突变体PRP和 蛋白质运输块。人类神经元的主要培养物将用于 通过亚细胞分馏，β-半乳糖苷酶检查蛋白质定位 互补，脉搏追踪实验，免疫荧光和共焦 显微镜和免疫EM。 AIM 4将确定分子机制 PRP的神经元保护。特定目标5将确定PRP是否与 其他促凋亡蛋白。在AIM 6中，PRP的神经保护作用 可能消除与家族性相关的PRP突变中的该功能 将在家族性prion疾病的小鼠模型中评估病毒疾病。这 这些实验的结果将清楚地定义PRP的神经保护作用 反对Bax介导的细胞死亡。