Figure 1.
Characterization of prion phenotypes in mice infected with different prion strains.
(A) Survival curves of Tga20 and WT mice IC inoculated with prions indicate that strains induced terminal clinical disease after short (blue) or long (red) incubation periods. P value is derived from a log-rank test. (B) Representative brain sections immunolabelled for PrP show the typical large, dense plaques of mCWD and 87 V and the diffuse patchy aggregates and small plaques of 22 L, ME7, and RML. Only the mCWD and 87 V plaques stained with Congo red. Scale bars = 100 µm (PrP) and 50 µm (Congo red). (C) Ultrastructure of the plaques from mCWD and RML show long fibrils in the mCWD-infected brain (arrow). No fibrils were seen in the RML-infected brain, although dead cells were observed.
Figure 2.
Comparison of mice inoculated with prion strains by intracerebral (IC) and intraperitoneal (IP) routes.
(A) WT mice. Survival curves and PrPSc-immunolabelled brain sections indicate that 22 L prions cause terminal disease after either IC or IP exposure, whereas 87 V prions cause terminal disease only after IC exposure. Histoblots of spleen show PrPSc in the lymphoid follicles of 22 L, but not 87 V-inoculated mice. (B) Tga20 mice. Survival curves and PrPSc-immunolabelled brain sections similarly show that RML prions cause terminal disease after either IC or IP exposure. mCWD prions cause terminal disease after IC and in some mice after IP exposure, suggesting inefficient neuroinvasion. Histoblots of spleen show PrPSc in the lymphoid follicles in RML-, and some mCWD-inoculated mice. Scale bar = 400 µm (brain) or 1 µm (spleen).
Figure 3.
Conformational stability of prion strains.
(A) Brain homogenate was treated with increasing concentrations of GdnHCl and digested with PK, and PrPSc was detected by ELISA. The denaturation curves were plotted as PrPSc absorbance signals against the GdnHCl concentration and fitted to a sigmoidal function. The half-maximal denaturation occurred at GdnHCl concentrations that were greater for the weakly neuroinvasive strains (mCWD and 87 V) than the strongly neuroinvasive strains (22 L, ME7, RML). (B) Plot of [GdnHCl]1/2 values for WT mice infected with 87 V and 22 L. (C) Plot of [GdnHCl]1/2 values for Tga20 mice inoculated with 22 L, ME7, RML and mCWD. Error bars are present for all data points. * and *** indicate P values of <0.01 and .0001, respectively when comparing the indicated strain with mCWD.
Figure 4.
Thermal stability of strongly and weakly neuroinvasive prion strains.
(A) PK-digested brain samples were subjected to increasing temperatures followed by SDS-PAGE. (B) Monomers were quantified by band intensity analysis, plotted against temperature, and fitted to a sigmoidal function. The PrP signal measured at 99°C was considered as total PrPSc (100%). The temperature required for 50% PrPSc disassociation into monomers is lower for the more neuroinvasive prion strains (22 L, ME7, RML). A faint band at 37°C may represent residual PrPC and was not included in the signal quantification. Values represent mean ± standard error.
Figure 5.
Soluble to insoluble PrP ratio varies depending on the prion strain.
(A) Brain homogenate was ultracentrifuged, and the supernatant and pellet fractions were subjected to SDS-PAGE and immunoblotting for PrP. The strongly neuroinvasive strain 22 L has significantly higher levels of insoluble PrP as compared to the weakly neuroinvasive 87 V. (B) Plot of the percentage of insoluble PrP. S = supernatant, P = pellet. ** indicates a P value of <0.001 for 22 L as compared with 87 V.