Infectivity-associated PrP(Sc) and disease duration-associated PrP(Sc) of mouse BSE prions

Abstract

Disease-related prion protein (PrP(Sc)), which is a structural isoform of the host-encoded cellular prion protein, is thought to be a causative agent of transmissible spongiform encephalopathies. However, the specific role of PrP(Sc) in prion pathogenesis and its relationship to infectivity remain controversial. A time-course study of prion-affected mice was conducted, which showed that the prion infectivity was not simply proportional to the amount of PrP(Sc) in the brain. Centrifugation (20,000 ×g) of the brain homogenate showed that most of the PrP(Sc) was precipitated into the pellet, and the supernatant contained only a slight amount of PrP(Sc). Interestingly, mice inoculated with the obtained supernatant showed incubation periods that were approximately 15 d longer than those of mice inoculated with the crude homogenate even though both inocula contained almost the same infectivity. Our results suggest that a small population of fine PrP(Sc) may be responsible for prion infectivity and that large, aggregated PrP(Sc) may contribute to determining prion disease duration.

Keywords: PK-digestion; PrPSc aggregation; endpoint titration; incubation period; prion infectivity.

FIGURE 1.

Comparison of PrP^Sc in brains digested with various PK concentrations. PrP^Sc was detected with anti-PrP mAb T2. The PK concentrations are shown on the top of each lane. The % PrP^Sc values are indicated at the bottom of each lane. Molecular marker sizes are indicated on the left.

FIGURE 2.

Pathological changes in the brain occurring during mBSE disease progression. Groups of mice were sacrificed at the defined time points indicated on the left side of the panels. Fixed brain sections from each culling time were subjected to hematoxylin and eosin staining (B, D, F, H, J, and L) together with immunohistochemistry for PrP^Sc (A, C, E, G, I, and K). PrP^Sc immunoreactivities were detected with the anti-PrP monoclonal antibody SAF84. Micrographs show the dorsal medulla (A, B, E, F, I, and J) and cerebral cortex (C, D, G, H, K, and L).

FIGURE 3.

Detection of PrP^Sc in the brains of mice by western blot. PrP^Sc was detected with anti-PrP mAb T2. The defined time points (days post-inoculation, dpi) are indicated on the top of the blot. Mo and Nl indicate mouse scrapie Obihiro and the uninfected mouse brain, respectively. The relative protein load in each lane is indicated on the bottom; 1x indicates 0.25 mg brain equivalent. Molecular markers are indicated on the left side of the blot.

FIGURE 4.

Kinetics of mBSE prion replication and PrP^Sc accumulation in the mouse brain during disease progression. Black circles and blue squares show the infectious units and PrP^Sc accumulation in the mouse brains. The infectious dose of mBSE prion at each time point was estimated by the incubation period bioassay method. ELISA was used for measuring the amount of PrP^Sc at the same time points, and the relative values (%) are indicated against the amount of PrP^Sc in the brain at the terminal stage. The kinetics of mBSE prion replication and PrP^Sc accumulation are shown as a semi-logarithmic plot. An exponential correlation was observed between the incubation time and mBSE prion infectivity from 5 to 100 dpi (y = 33557* e^(0.082279x) R² = 0.94593, where y is the infectious unit and x is the survival days). A similar correlation was observed between the incubation time and PrP^Sc accumulation from 40 to 140 dpi (y = 0.024371* e^(0.056019x) R² = 0.90149, where y is the relative value (%) compared to the PrP^Sc values of 140-dpi brains and x is the survival days).

FIGURE 5.

Comparison of PrP^Sc and prion infectivity between the crude homogenate and 20K-sup. (A) Comparison of PrP^Sc amounts between the crude homogenate and 20K-sup. The relative protein load in each lane is indicated on the top. Nl indicates the uninfected mouse brain. PrP was detected with anti-PrP mAb SAF84; 1x indicates 0.25 mg brain equivalent. Molecular markers are indicated on the left side of the blot. (B) The relationship between incubation time (mean days ± standard error) and infectious units (log LD₅₀/gram of the brain) in mice inoculated with serial dilutions of the crude homogenate (black circles and solid line; y = 16.642 – 0.061312x, where y is log LD₅₀ and x is the incubation period) and the 20K-sup (blue squares and dotted line y = 17.474 – 0.060916x).