Design and construction of diverse mammalian prion strains

Abstract

Prions are infectious proteins that encipher biological information within their conformations; variations in these conformations dictate different prion strains. Toward elucidating the molecular language of prion protein (PrP) conformations, we produced an array of recombinant PrP amyloids with varying conformational stabilities. In mice, the most stable amyloids produced the most stable prion strains that exhibited the longest incubation times, whereas more labile amyloids generated less stable strains and shorter incubation times. The direct relationship between stability and incubation time of prion strains suggests that labile prions are more fit, in that they accumulate more rapidly and thus kill the host faster. Although incubation times can be changed by altering the PrP expression level, PrP sequence, prion dose, or route of inoculation, we report here the ability to modify the incubation time predictably in mice by modulating the prion conformation.

Fig. 1.

Characterization of diverse PrP amyloid conformations. Formation of Amyloid 5 (blue), Amyloid 6 (green), Amyloid 7 (orange), and Amyloid 8 (red) was monitored by ThT fluorescence (A), followed by measurement of conformational stability by ELISA coupled with denaturation with increasing concentrations of GdnHCl (B). Electron micrographs (C) show morphology of Amyloids 5–8, as indicated. (Scale bars, 100 nm.)

Fig. 2.

Prion disease results from inoculation of Amyloids 5, 6, and 7 into Tg4053 mice. Amyloids 5 (blue), 6 (green), 7 (orange), and 8 (red) were inoculated into Tg4053 mice (n = 8–12 per inoculum). Monomeric, α-helical recPrP (purple) was inoculated as a control. Amyloids 5, 6, and 7 resulted in prion disease (A), which was confirmed by Western blotting for protease-resistant PrP^Sc (B) and neuropathology (D). The resulting isolates were designated “MoSP5,” “MoSP6,” and “MoSP7,” respectively. Amyloid 8 and monomeric recPrP did not result in prion disease; additional control experiments are shown in Fig. S4. The conformational stability of MoSP5 (blue), MoSP6 (green), and MoSP7 (orange) was measured by titration with GdnHCl followed by PK digestion (C). •, mouse with symptomatic prion disease; ▲, mouse with asymptomatic prion disease; ×, deceased mouse with no indication of prions. In B, a mouse infected with RML prions is shown as a control for PK-resistant PrP^Sc. Molecular masses based on the migration of protein standards are shown in kDa. Neuropathology included PrP deposits (dark brown, top row in D) and vacuolation (white holes, bottom row in D). cc, corpus callosum; P, pyramidal cell layer. (Scale bar, 30 μm.)

Fig. 3.

New prion strains are serially transmissible and have distinct incubation periods and conformational stabilities. Serial transmission (A, B) and GdnHCl denaturation (C, D) of MoSP5 (blue), MoSP6 (green), and MoSP7 (orange) indicate 3 unique prion strains. (Primary data are shown in Fig. S7.) All 3 prion isolates were transmitted to Tg4053 mice (A, C); MoSP6 and MoSP7 were transmitted to wild-type FVB mice (B, D).

Fig. 4.

Distinguishing neuropathology of MoSP5, MoSP6, and MoSP7. (A) PrP immunohistochemistry (Top row) shows finely granular PrP^Sc deposits in the hippocampal gray matter with MoSP5, coarsely granular, nonamyloid deposits of PrP^Sc primarily in the subcallosal-corpus callosum with MoSP6, and multiple, PrP-immunopositive amyloid plaques in the subcallosal region as well as abundant finely granular PrP^Sc deposits with MoSP7. H&E staining (Bottom row) shows large numbers of vacuoles with MoSP5, moderate numbers of vacuoles with MoSP6, and sparse numbers of vacuoles and severe nerve cell loss in the CA1 region with MoSP7. The control is a mock-inoculated Tg4053 mouse. (B) Neuropathology resulting from MoSP6 and MoSP7 initially transmitted in Tg4053 mice and then passaged to FVB mice shows a phenotype for MoSP6 and MoSP7 very similar to that found in Tg4053 mice. cc, corpus callosum; P, pyramidal cell layer. (Scale bars, 50 μm.)

Fig. 5.

The properties of new synthetic prion strains are modulated by the conformational stability of amyloids used to generate them. GdnHCl_1/2 values of synthetic prion strains directly correlated to the GdnHCl_1/2 values of the respective amyloid preparations (A: R = 0.94, n = 9, P = 0.0002), as well as to incubation periods (B: dashed line from ref. 25) in Tg4053 mice (○; R = 0.993, n = 6, P < 0.0001) and FVB mice (□; R = 0.994, n = 3, P = 0.07). MoSP5 (blue), MoSP6 (green), MoSP7 (orange), MoSP9 (yellow), MoSP12 (lime), MoSP13 (purple). Additional data for amyloids and prion isolates are shown in Tables S1, S3, and S5. Amyloids and synthetic prions retain their relative order of conformational stability during first (1° Tx) and second (2° Tx) transmission (C). ^aAmyloid conformational stability was measured by epitope exposure in ELISA; ^bMoSP conformational stability was measured by susceptibility to PK digestion.