Type I interferon protects neurons from prions in in vivo models

Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Satoh, Katsuya; Mori, Tsuyoshi; Atarashi, Ryuichiro; Nishida, Noriyuki

Murine neuroblastoma cells (N2a) and fibroblast cells (NIH3T3) were obtained from the American Type Culture Collection. To create an in vitro model using cells persistently infected by prions, N2a-58 cells overexpressing PrP C , which were established from N2a cells integrating mouse Prnp gene in N2a cells, were subjected to prion infection with a mouseadapted 22 L strain from scrapie as previously described (Nishida et al., 2000; Ishibashi et al., 2012a) . As packaging cells, HEK293T cells for lentiviral vector system with SIN virus (provided by Dr Miyoshi in RIKEN BRC) and NIH3T3-derived RetroPack PT67 cells (Clontech) expressing 10A1 viral envelope protein for MSCV retroviral expressing system were obtained from commercial sources. All cells were grown at 37 C in 5% CO 2 in Dulbecco's-modified Eagle medium (DMEM, Wako) containing 4500 mg/l glucose, 10% heat-inactivated foetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin (Nacalai Tesque). Prion-infected and non-infected cells were transfected with Ifnb gene plasmids using Fugene Õ 6 (Roche) as per the manufacturer's protocol, and grown in 6-well plates for 2 days. In the antiprion treatment, 20 mg/ml PPS (Caughey and Raymond, 1993) , 10 mg/ml anti-PrP antibody (3S9) (Miyamoto et al., 2005) or 0.5 to 500 mM RO8191 was added to the prion-infected cells and incubated for 48 h. In stable line construction, non-tagged IFN-b-overexpressing cells were established and in vitro 22L scrapie infection experiments were performed using their clonal cells. To establish cell lines stably expressing target proteins, pcDNA3.1 plasmids containing target genes were transfected, using Fugene Õ 6 (Roche), into N2a-58 cells, the cells were then selected by 350 to 500 mg/ml HygroGold TM (Invivogen) treatment, and drug-resistant colonies were isolated.