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

In ex vivo prion infection in cell culture, the cells were infected with 22 L scrapie strain-infected brain homogenate prepared from mice terminally sick with the 22 L strain (final concentration 2 Â 10 À3 % brain homogenate for neuronal cells; 2 Â 10 À3 , 2 Â 10 À2 % for NIH3T3 cells; 6 Â 10 À3 , 3 Â 10 À2 , 1.5 Â 10 À1 % brain homogenate for MEF cells) in a 6-well culture plate for 48 h, and subsequently grown and scaled up to a 75 cm 2 flask. Once confluent, the subcultures were diluted 5-or 10-fold in fibroblast or neuronal cells. In experiments of the inhibitory effect against prion infection at early phase, I-IFNs and RO8191 (0.5-500 mM) were treated in the cells and incubated for 24 h before prion infection simultaneously with recombinant mouse I-IFNs, and cultured until the fifth passage (#1 to #5) after scale-up. Poly I:C (0.2 to 2 mg/well) stimuli were transfected by Lipofectamine TM LTX and incubated for 8 h before prion infection. In in vivo prion infection, 4-weekold male mice (wild-type and Ifnar1 À/À mice of the same C57BL/6-derived genetic background) were inoculated via the intracerebral (i.c.) and intraperitoneal (i.p.) route with 22 Lbrain homogenate (i.c.: 20 ml of a 10 À1 and 10 À2 dilution of brain homogenate; i.p.: 100 ml of a 10 À2 and 10 À3 dilution of brain homogenate). Mice were monitored every other day until the terminal stage of disease. Clinical onset in prion disease has been defined as the presence of two or more of the following signs: greasy and/or yellowish hair, hunchback, weight loss, yellow pubic hair, ataxic gait, and non-parallel hind limbs. The prion-infected mice were sacrificed at preclinical onset [100 days post-inoculation (dpi)] and terminal stage, and the brains and spleens were removed. The right hemisphere of the brains and a part of the spleens were immediately frozen and homogenized at 20% and 10% (weight/volume) in phosphate buffered saline, for immunoblotting analysis, and total proteins were extracted by mixing the same amount of 2Â Lysis Buffer as described below. Remaining tissues were fixed in 4% paraformaldehyde and pathological analysis was performed. As a negative control (mock), age-and strainmatched mice were inoculated intracerebrally or intraperitoneally with normal mouse brain homogenate. In a bioassay with RO8191, the drug was dissolved in PBS including 20% Kolliphor HS 15 (BASF), which is one of the solvents used for pharmaceutical applications. Four-week-old ddY male mice purchased from SLC (Hamamatsu) were intracerebrally inoculated with 20 ml of a 10 À1 dilution of brain homogenate containing strain 22 L. Sequentially, the mice were intraperitoneally administered 2.0 mg drug/kg/day every other day. As a control, age-and strain-matched mice were intraperitoneally inoculated with the solvents without the drug. Vehicle and RO8191-treated mice were weighed three times a week from the moment before onset to the terminal stage of the disease.