title: Prion replication elicits cytopathic changes in differentiated neurosphere cultures.
abstract: The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level.
authors.affiliation: Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Iwamaru
authors.forename: Takato
authors.initials: T
authors.lastname: Takenouchi
authors.forename: Morikazu
authors.initials: M
authors.lastname: Imamura
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Kohtaro
authors.initials: K
authors.lastname: Miyazawa
authors.forename: Shirou
authors.initials: S
authors.lastname: Mohri
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokoyama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kitani
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D016923:Cell Death; D002470:Cell Survival; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000166:cytology / Q000473:pathology* / Q000502:physiology; D011328:Prions / Q000378:metabolism* / Q000472:pathogenicity*
chemical_list: D011328:Prions; D007770:L-Lactate Dehydrogenase