Genetics of Prion Susceptibility in vitro

描述（由申请人提供）：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。由于需要在小鼠中进行孵育时间研究，除 Prnp 之外改变朊病毒复制和对朊病毒疾病易感性的基因的鉴定和表征异常困难。甚至 Prnp 的替代等位基因决定痒病潜伏时间的机制也尚未解决。中枢神经系统干细胞神经球培养物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。神经球培养物提供了一种研究朊病毒生物学的新方法，并将被开发为一种灵敏的生物测定法。在神经球培养物中可以区分感染效率、细胞间传播以及朊病毒复制速率。构象依赖性表位允许通过细胞内 PrPSc 来识别单个细胞；感染、传播和复制率的定量分析将得到完善。这些参数将在朊病毒株-小鼠株组合中进行比较，这些组合的朊病毒敏感性或孵育时间不同。概括原始小鼠易感性的神经球将形成解剖所涉及的机制和基因的基质。基于许多数量性状基因座的替代等位基因反映水平或表达差异或相互作用分子的不同亲和力的假设，RNAi将用于评估候选基因对感染和PrPSc产生的影响。从任何品系的小鼠中产生神经球系的能力允许使用与检测它们相同的遗传背景来测试修饰物。中枢神经系统干细胞可以被诱导沿着多种途径分化。将比较在特定条件下从感染和未感染的神经球培养物产生的细胞类型以及细胞存活率。感染 RML 朊病毒株的小鼠脑匀浆稀释 10-8 后可感染过度表达 PrP 的转基因小鼠的神经球，且尚未达到敏感性极限。将测定时间从几周缩短到几天将采用神经球 PrP 上存在的抗体表位，但接种物中 PrPSc 中不存在的抗体表位。这种遗传上易于处理的细胞培养系统有可能彻底改变朊病毒研究。

Description (provided by applicant): 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 background on 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.