Kiyotoshi Kaneko

Abbreviated incubation times for human prions in mice expressing a chimeric mouse-human prion protein transgene.

Transgenic (Tg) mouse lines that express chimeric mouse–human prion protein (PrP), designated MHu2M, are susceptible to prions from patients with sporadic Creutzfeldt–Jakob disease (sCJD). With the aim of decreasing the incubation time to fewer than 200 days, we constructed transgenes in which one or more of the nine human residues in MHu2M were changed to mouse. The construct with murine residues at positions 165 and 167 was expressed in Tg(MHu2M,M165V,E167Q) mice and resulted in shortening the incubation time to ≈110 days for prions from sCJD patients. The construct with a murine residue at position 96 resulted in lengthening the incubation time to more than 280 days for sCJD prions. When murine residues 96, 165, and 167 were expressed, the abbreviated incubation times for sCJD prions were abolished. Variant CJD prions showed prolonged incubation times between 300 and 700 days in Tg(MHu2M) mice on first passage and incubation times of ≈350 days in Tg(MHu2M,M165V,E167Q) mice. On second and third passages of variant CJD prions in Tg(MHu2M) mice, multiple strains of prions were detected based on incubation times and the sizes of the protease-resistant, deglycosylated PrPSc fragments. Our discovery of a previously undescribed chimeric transgene with abbreviated incubation times for sCJD prions should facilitate studies on the prion species barrier and human prion diversity.

Expression of unglycosylated mutated prion protein facilitates PrPSc formation in neuroblastoma cells infected with different prion strains.

Prion replication involves conversion of the normal, host-encoded prion protein PrP(C), which is a sialoglycoprotein bound to the plasma membrane by a glycophosphatidylinositol anchor, into a pathogenic isoform, PrP(Sc). In earlier studies, tunicamycin prevented glycosylation of PrP(C) in scrapie-infected mouse neuroblastoma (ScN2a) cells but it was still expressed on the cell surface and converted into PrP(Sc); mutation of PrP(C) at glycosylation consensus sites (T182A, T198A) produced low steady-state levels of PrP that were insufficient to propagate prions in transgenic mice. By mutating asparagines to glutamines at the consensus sites, we obtained expression of unglycosylated, epitope-tagged MHM2PrP(N180Q,N196Q), which was converted into PrP(Sc) in ScN2a cells. Cultures of uninfected neuroblastoma (N2a) cells transiently expressing mutated PrP were exposed to brain homogenates prepared from mice infected with the RML, Me7 or 301V prion strains. In each case, mutated PrP was converted into PrP(Sc) as judged by Western blotting. These findings raise the possibility that the N2a cell line can support replication of different strains of prions.