Genetics of Prion Susceptibility in vitro

PrP病是由PrP错误折叠引起的人和动物的神经退行性疾病。由Prion引起的疾病可以传播给实验动物，无论它们的来源是遗传的、传染性的还是零星的。Prion病通过摄取牛海绵状脑病(BSE)Prion传播给人类已有文献记载。慢性消耗性疾病(CWD)在北美通过鹿群和麋鹿群传播是一个严重的问题，人类对CWD普恩病毒的易感性尚不清楚。所有的PrP疾病都涉及PrP的构象变化，从其良性的细胞亚型PrPc到疾病特异性的亚型PrPSc。PrPSc足以传播疾病，它的构象编码了PrP菌株的特性。对PrP生物学意义的认识来自于 正常和转基因小鼠的孵化时间研究。然而，除了Prion蛋白基因(PRNP)之外，基因在Prion复制和疾病易感性中也是重要的，但由于在小鼠身上进行的孵化时间研究的长度和费用，它们的识别被证明是异常困难的。类似地，在大肠杆菌中产生的重组PrP产生的合成PrP会导致转基因(TG)小鼠患病，但孵化时间往往超过500天。只有少数几个细胞株可以被感染 仅对一种或两种病毒株提供有限的遗传多样性和敏感性。CNS干细胞可以从正常或转基因小鼠的成年或胎儿脑中分离出来，并在培养中生长。这些细胞的聚集体被称为神经球，可以感染普恩病毒，为普恩病毒的研究提供了一项令人兴奋的新技术。在神经球培养中，可以区分感染效率、细胞间传播和Pron复制率，构象依赖表位允许识别单个细胞 胞内PrPSc。该计划项目申请的四名高级研究人员将应用这一新颖的体外模型来开发快速和灵敏的普恩生物检测方法，包括人类和有蹄类动物的普恩病毒。 在培养中比在小鼠中更有效地解决了感染普恩病毒构象稳定性对普恩病毒繁殖率的影响。从任何菌株或TG系中分离神经球系的能力为在培养中对Pron病进行遗传分析提供了机会，并提供了一种系统来筛选包含Prion孵化时间修饰基因座的染色体区域内的候选基因。 神经球系还为小鼠提供了一种精细化的系统方法，以识别被Prion复制和致病过程干扰的基因网络。中枢神经系统干细胞可以定向分化为神经元和神经胶质细胞。将在神经球和“成熟脑球”中评估感染对分化的影响和感染对分化细胞的影响，重点是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.