Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

Abstract

bThe 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. P rion diseases comprise a class of transmissible, fatal, neurodegenerative diseases, and they include Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker syndrome (GSS) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, and chronic wasting disease in deer. The neuropathological hallmarks of prion diseases are neuronal loss, vacuolation, synaptic alterations, astrogliosis, microglial activation, and the progressive accumulation of a misfolded and protease-resistant isoform (PrPres) of host-encoded proteasesensitive prion protein (protease-sensitive PrP [PrPsen]). The conversion of PrPsen into PrPres and its accumulation are implicated in the pathogenesis of prion diseases (1, 2); however, the molecular basis of neurodegeneration in prion diseases is largely unclear.Several lines of evidence have revealed that animals can harbor high levels of infectivity before or without developing clinical signs (3, 4), indicating the decoupling of prion infectivity from toxicity. One possible explanation for the dissociation is that toxic species of PrPres distinct from the infectious particles are produced after reaching a plateau of infectivity (4). Investigations of the function of putative toxic forms of PrP and PrP in toxic signaling are crucial fo...

The RNA hairpin binder TRIM71 modulates alternative splicing by repressing MBNL1

“…mESCs were differentiated to embryoid bodies (EBs) and treated with retinoic acid (RA) as described previously (Bibel et al 2007). The culture of neuroprogenitors as neurospheres from RA-treated EBs was performed as described previously (Iwamaru et al 2013). For generation of neurons, neurospheres were dissociated and plated on laminin/poly-DL-ornithine-coated adherent plates as described previously (Bibel et al 2007).…”

Susceptibility of GT1–7 cells to mouse-passaged field scrapie isolates with a long incubation

“…16,17,29,30 Rodent-passaged laboratory scrapie strains (Chandler, 22L and ME7) were maintained by serial-passage into wild-type mice (ICR; Japan SLC, Hamamatsu, Japan). 38 All experiments involving animals were approved by the Animal Ethical Committee and the Animal Care and Use Committee of the National Institute of Animal Health (authorization 11-008 and 11-012).…”

Chronic wasting disease prion infection of differentiated neurospheres

“…We previously reported that the neurosphere cultures from transgenic mice expressing murine PrP were susceptible to prion infection after differentiation by serum-free media supplemented with N-2 factor and EGF (dNP20/ EGF cultures). 12 In the current study, despite applying the same differentiation condition as dNP20/EGF cultures, the dNP5037/EGF cultures were not susceptible to prion infection. The inconsistency in prion susceptibility between mouse PrP dNP20/EGF and cervid PrP dNP5037/EGF cultures could be related to the different transgenic mouse lines (Tga20 vs Tg5037) used for these studies, as it is known that transgene expression is influenced by genome insertion position and multipllcity.…”

“…Neurospheres were cultured from the brains of neonatal Tg5037 mice expressing the elk PrP amino acid sequence (GenBank accession no.ABS87888.1) and in PrP-deficient (PrP 0/0 ) mice harvested on the day of birth (identified as NP5037 and NP0, respectively), as described previously. 12 The neurosphere cultures were passaged in serum-free media [Dulbecco's modified Eagle's medium-nutrient F12 Ham supplemented with N-2 factors, 50 ng/ml epidermal growth factor (EGF), 50 ng/ml basic fibroblast growth factor (bFGF) and penicillin and streptomycin] more than 8 times. The single-cell suspensions of neurospheres were cultured as monolayers in 6 well plates coated with fibronectin for 4 d. The cultures were differentiated by serum-free media supplemented with N-2 factor and any one of the following growth factors; i) EGF, ii) bFGF or iii) 5% fetal bovine serum (FBS).…”

“…[3][4][5][6][7][8] One possible strategy to develop more diverse cell culture systems permissive to infection with naturally occurring prions in humans and animals is the use of neural stem cells/neurospheres from transgenic (Tg) mouse lines expressing prion protein (PrP) of the native host species, since undifferentiated and differentiated neurospheres have been demonstrated to be permissive to mouse prion infection. [9][10][11][12] Although several transgenic mice lines expressing PrPC from various species have been reported, no neurosphere model susceptible to infection with natural non-murine adapted prions has been reported.…”