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.
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