Genetics of Prion Susceptibility in vitro

朊病毒病是由朊病毒蛋白（PrP）错误折叠引起的人类和动物的神经退行性疾病。由朊病毒引起的疾病可以传染给实验动物，无论其起源是遗传性的、传染性的还是散发性的。朊病毒病通过摄入牛海绵状脑病（BSE）朊病毒而传播给人类已有文献记载。慢性消耗病（CWD）通过北美鹿群和麋鹿的传播是一个严重的问题，人类对CWD朊病毒的易感性尚不清楚。所有朊病毒疾病都涉及PrP从其良性细胞同种型PrPc到疾病特异性同种型PrPSc的构象变化。PrPSc足以传播疾病，并且其构象加密朊病毒株特性。对PrP生物学意义的认识来自于 在正常和转基因小鼠中的孵育时间研究。然而，除了朊病毒蛋白基因（Prnp）之外的基因在朊病毒复制和疾病易感性中也是重要的，但是由于在小鼠中孵育时间研究的长度和费用，它们的鉴定已被证明是异常困难的。类似地，从E.大肠杆菌在转基因（Tg）小鼠中引起疾病，但潜伏期通常超过500天。只有少数细胞系可以被感染 朊病毒，提供有限的遗传多样性和敏感性，只有一个或两个朊病毒株。CNS干细胞可以从正常或Tg小鼠的成年或胎儿脑中分离并在培养物中生长。这些细胞的聚集体，称为神经球，可以感染朊病毒，并为朊病毒研究提供了一种令人兴奋的新技术。感染的效率、从细胞到细胞的传播和朊病毒复制的速率可以在神经球培养物中区分，并且构象依赖性表位允许鉴定具有以下特征的单个细胞： 胞内PrPSc。该计划项目申请的四名高级研究人员将应用这种新的体外模型来开发快速和灵敏的朊病毒生物测定法，包括人类和有蹄类动物的朊病毒。 感染性朊病毒的构象稳定性对朊病毒繁殖速率的影响在培养物中比在小鼠中更有效。从任何菌株或Tg系中分离神经球系的能力提供了在培养物中对朊病毒疾病进行遗传分析的机会，以及筛选含有朊病毒孵育时间修饰基因座的染色体区域内的基因的候选者的系统。 神经球线也提供了一个完善的系统方法，以确定基因网络干扰的朊病毒复制和致病过程。CNS干细胞可定向分化为神经元和神经胶质。将在神经球和“成熟脑球”中评价感染对分化的影响和感染对分化细胞的影响，强调Notch-Hes途径。

Prion diseases are neurodegenerative disorders of humans and animals caused by misfolding of prion protein (PrP). Diseases caused by prions can be transmitted to experimental animals whether their origin is genetic, infectious, or sporadic. The transmission of prion disease to humans by ingestion of bovine spongiform encephalopathy (BSE) prions has been documented. The spread of chronic wasting disease (CWD) through herds of deer and elk in North America is a serious concern and the susceptibility of humans to CWD prions is unknown. All prion diseases involve changes in the conformation of PrP from its benign cellular isoform, PrPc, to a disease-specific isoform, PrPSc. PrPSc is sufficient to transmit disease and its conformation enciphers prion strain properties. Appreciation of the biological significance of PrP came from incubation time studies in normal and transgenic mice. However, genes in addition to the prion protein gene (Prnp) also are important in prion replication and disease susceptibility but their identification has proven exceptionally difficult due to the length and expense of incubation time studies in mice. Similarly, synthetic prions created from recombinant PrP produced in E. coli cause disease in transgenic (Tg) mice but incubation times often exceed 500 days. Only a few cell lines were available that could be infected with prions, offering limited genetic diversity and sensitivity to only one or two prion strains. CNS stem cells can be isolated from adult or fetal brains of normal or Tg mice and grown in culture. Aggregates of these cells, called neurospheres, can be infected with prions and offer an exciting new technology for prion research. Efficiency of infection, spread from cell to cell, and rate of prion replication can be discriminated in neurosphere cultures and conformation dependent epitopes allow identification of individual cells with intracellular PrPSc. The four senior investigators on this Program Project application will apply this novel in vitro model to develop rapid and sensitive bioassays for prions, including those of humans and ungulates. The influence of conformational stability of infectious prions on the rate of prion propagation can be addressed more efficiently in culture than in mice. The ability to isolate neurosphere lines from any strain or Tg line offers the opportunity for genetic analysis of prion disease in culture and a system to screen candidates for the genes within chromosomal regions containing prion incubation time modifier loci. Neurosphere lines also offer a refinement over mice for the systems approach to identify gene networks perturbed by prion replication and pathogenic processes. CNS stem cells can be directed to differentiate to neurons and glia. Effects of infection on differentiation and effects of infection on differentiated cells will be evaluated in neurospheres and in "matured brain spheres", emphasizing the Notch-Hes pathway.