In Vitro Dissection of Genetic Susceptibility to Prion Disease

朊病毒病遗传易感性的体外剖析

• 批准号: 8085696
• 负责人: George A. Carlson
• 金额: $ 45.96万
• 依托单位: MC LAUGHLIN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8085696
• 关键词: Affinity; Alleles; Antibodies; Biochemical; Biological Assay; Biological Markers; Biology; Brain; Candidate Disease Gene; Cattle; Cell Culture System; Cell Survival; Cells; Collaborations; Dissection; Epitopes; Gene-Modified; Genes; Genetic; Genetic Predisposition to Disease; Genome; Goals; Human; In Vitro; Individual; Infection; Learning; Modeling; Molecular Conformation; Mouse Strains; Mus; Mutation; Neuronal Dysfunction; Pathway interactions; PrP; PrPSc Proteins; Predisposition; Prion Diseases; Prions; Production; Quantitative Trait Loci; RNA Interference; Research; Scrapie; Stem cells; Testing; Time; Time Study; Transgenic Mice; Work; base; cell type; cervid; in vitro Bioassay; novel; novel strategies; overexpression; stem cell differentiation

The goals of this project are to dissect the biochemical networks that impinge on prion replication and to learn how prion replication causes neuronal dysfunction. The identification and characterization of genes in addition to Prnp that modify prion replication and susceptibility to prion disease has proven exceptionally difficult due to the need for incubation time studies in mice. Even the mechanisms by which alternative alleles of Prnp determine scrapie incubation time are unresolved. CNS stem cell neurosphere cultures can be produced from any strain or stock of mice, as well as from other species, and can be infected with prions. Neurosphere cultures provide a new approach to study prion biology and will be developed as a sensitive bioassay. Efficiency of infection, spread from cell to cell, and rate of prion replication can be discriminated in neurosphere cultures. Conformation dependent epitopes allow identification of individual cells with intracellular PrPSc; quantitative assays for infection, spread, and rate of replication will be refined. These parameters will be compared in prion strain-mouse strain combinations that vary in prion susceptibility or incubation time. Neurospheres that recapitulate the susceptibility of the mice of origin will form the substrates for dissection of the mechanisms and genes that are involved. Based on the hypothesis that alternative alleles of many quantitative trait loci reflect either differences in level or expression or different affinities for interacting molecules, RNAi will be used to evaluate effects of candidate genes on infection and production of PrPSc. The ability to produce neurosphere lines from any strain of mice permits testing modifiers using the same genetic backgroundon which they were detected. CNS stem cells can be induced to differentiate along several pathways. The cell types produced under specific conditions from infected and non-infected neurosphere cultures will be compared, as will cell survival. Brain homogenates from mice infected with the RML prion strain diluted 10-8 can infect neurospheres from transgenic mice overexpressing PrP, and the limits of sensitivity have not yet been reached. Shortening the assay time from weeks to days will employ antibody epitopes present on PrP of the neurospheres, but not in PrPSc in the inoculum. This genetically tractable cell culture system has the potential to revolutionize prion research.

该项目的目标是剖析影响朊病毒复制的生化网络并 了解朊病毒复制如何导致神经元功能障碍。基因的鉴定和表征 除 Prnp 外，它还可以改变朊病毒复制和对朊病毒疾病的易感性，已被证明具有特殊作用 由于需要在小鼠中进行孵化时间研究，因此很困难。甚至替代方案的机制 Prnp 等位基因决定瘙痒病潜伏时间的问题尚未解决。中枢神经系统干细胞神经球培养物可以 可以从任何品系或种群的小鼠以及其他物种中产生，并且可以被朊病毒感染。 神经球培养物为研究朊病毒生物学提供了一种新方法，并将被开发为敏感的 生物测定。感染效率、从一个细胞到另一个细胞的传播以及朊病毒复制的速率可以通过以下因素来区分： 神经球培养物。构象依赖性表位允许识别单个细胞 细胞内PrPSc；感染、传播和复制率的定量分析将得到完善。这些 将在朊病毒菌株-小鼠菌株组合中比较参数，这些组合的朊病毒敏感性或 孵化时间。概括原始小鼠易感性的神经球将形成 用于解剖所涉及的机制和基因的底物。基于以下假设 许多数量性状基因座的替代等位基因反映了水平或表达的差异或不同 由于相互作用分子的亲和力，RNAi 将用于评估候选基因对感染和感染的影响 PrPSc 的生产。从任何品系的小鼠中产生神经球系的能力允许进行测试 使用与检测到它们相同的遗传背景的修饰符。可诱导中枢神经系统干细胞 沿着多种途径进行区分。在特定条件下由感染和感染产生的细胞类型 将比较未感染的神经球培养物以及细胞存活率。小鼠脑匀浆 感染稀释10-8的RML朊病毒株可以感染过度表达的转基因小鼠的神经球 PrP，尚未达到灵敏度极限。将检测时间从几周缩短至几天 将采用神经球 PrP 上存在的抗体表位，但接种物中 PrPSc 中不存在的抗体表位。这 遗传易处理的细胞培养系统有可能彻底改变朊病毒研究。