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

An imidazonaphthyridine with the structural formula 8-(1, 3, 4-oxadiazol-2-yl)-2, 4-bis (trifluoromethyl) imidazo [1, 2a] [1, 8] naphthyridine (RO4948191, referred to as RO8191) is able to block hepatitis C virus infection to several cells including cancer cell lines and human primary hepatocytes. The reported functional mechanism of RO8191 is selective binding to the I-IFN receptor and the sequential induction of many interferon-stimulated genes, followed by the activation of I-IFN signalling (Tga20) inducing Ifnb with lentivirus system. Panels show localization of lentivirus in thalamus 20 days after stereotaxic injection to Tga20 mice brain (Venus: green; nuclei: blue) (E). Scale bar = 50 mm. PrP Sc levels in brain of 22 Lprion-infected mice in the terminal stage brain pre-injected stereotaxically (s.t.) and intracerebrally (i.c.) with LV-venus and -IFN (F). Graphs show quantifications of PrP Sc band intensities in brain (n = 3 mice per each lentiviral injection group). Statistical significance was determined using unpaired Student's t-test (C and F) and one-way ANOVA, followed by Dunnett's test in multiple comparisons (A, B and D). *P 5 0.05, **P 5 0.01 and ***P 5 0.001. Data are presented as mean AE SEM. Western blot results represent at least three independent experiments. (Konishi et al., 2012) . To determine whether IFN-dependent signalling mediated I-IFN receptor-regulated prion infection, we investigated PrP Sc levels in persistently prion-infected cells after RO8191 treatment for 48 h. PrP Sc levels in N2a-22 L cells treated with RO8191 were significantly reduced in a dose-dependent manner (0.5, 5, 50, 250 and 500 mM RO8191) (Fig. 5A) . However, PrP C levels in N2a-58 cells treated with RO8191 showed no change (Fig. 5B) . In both cells, the level of 2 0 -5 0 oligoadenylate synthetase 1a (OAS1a), encoded by an interferon-stimulated gene, was dose-dependently increased by RO8191 treatment. Moreover, we investigated the potential anti-prion effect of RO8191 in cell culture models. RO8191 pretreated cells became resistant to 22 L-prion primary infection with the increase in expression of OAS1a in a dose-dependent manner (Fig. 5C) , suggesting that RO8191, which induces interferon-stimulated genes via I-IFN signalling during the innate immune response, might prevent the establishment of prion infection without affecting PrP C expression. Next, to evaluate the efficacy of RO8191 against prion propagation in a prion infectious animal model, we performed a bioassay using prion-infected mice. The ddY mice intracerebrally inoculated with 10% 22 L prion brain homogenate were intraperitoneally administered 2 mg/kg/day RO8191 three times a week from 2 days post-inoculation until sacrifice. The survival periods of RO8191-treated mice were significantly longer (165 AE 7 days, n = 8; P = 0.0123, Log-rank test) than those of the vehicle mice (156 AE 4 days, n = 7) (Fig. 6A) . Changes in the body weight of each group were consistent with prolonged survival times. The changes in body weight of the RO8191 group gently decreased compared with those of vehicle group, and this was delayed by about 2 weeks. RO8191-treated mice were observed for toxicity due to chronic administration and the symptoms of prion disease were similar to those of the vehicle mice ( Supplementary Fig. 4A ). We analysed PrP Sc and histological changes in the brains of mice at 100 dpi, which was the time point before disease onset. PrP Sc levels in the RO8191-treated brains