In Vitro Dissection of Genetic Susceptibility to Prion Disease

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 除了Prnp外，还特别证明了Prnp修饰朊病毒复制和朊病毒疾病易感性 由于需要在小鼠中进行孵育时间研究，因此难以进行。即使是替代方案的机制 Prnp的等位基因决定羊瘙痒病的潜伏期。CNS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。 神经球培养为朊病毒生物学的研究提供了一种新的方法， 生物测定感染的效率，从细胞到细胞的传播，以及朊病毒复制的速度可以通过 神经圈文化构象依赖性表位允许鉴定单个细胞， 细胞内PrPSc;感染、扩散和复制率的定量测定将得到改进。这些 将在朊病毒易感性不同的朊病毒株-小鼠株组合中比较参数， 孵育时间概括小鼠起源易感性的神经球将形成 用于解剖相关机制和基因的底物。基于这样的假设， 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，或不同的 RNAi将用于评估候选基因对感染的影响， 生产PrPSc。从任何品系的小鼠中产生神经球系的能力允许测试 使用相同的遗传背景，他们被检测到的修改器。中枢神经系统干细胞可以诱导 来分化沿着几条途径。在特定条件下从受感染和 将比较未感染的神经球培养物以及细胞存活。小鼠脑匀浆 用稀释10-8的RML朊病毒株感染可以感染来自过表达RML的转基因小鼠的神经球。 PrP，灵敏度极限尚未达到。将检测时间从数周缩短至数天 将采用存在于神经球的PrP上但不存在于接种物中的PrPSc中的抗体表位。这 遗传上易处理的细胞培养系统有可能彻底改变朊病毒的研究。

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.