Prion Protein Devoid of the Octapeptide Repeat Region Delays Bovine Spongiform Encephalopathy Pathogenesis in Mice

Abstract

Conformational conversion of the cellular isoform of prion protein, PrP^C, into the abnormally folded, amyloidogenic isoform, PrP^Sc, is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. We previously reported that the octapeptide repeat (OR) region could be dispensable for converting PrP^C into PrP^Sc after infection with RML prions. We demonstrated that mice transgenically expressing mouse PrP with deletion of the OR region on the PrP knockout background, designated Tg(PrPΔOR)/Prnp^0/0 mice, did not show reduced susceptibility to RML scrapie prions, with abundant accumulation of PrP^ScΔOR in their brains. We show here that Tg(PrPΔOR)/Prnp^0/0 mice were highly resistant to BSE prions, developing the disease with markedly elongated incubation times after infection with BSE prions. The conversion of PrPΔOR into PrP^ScΔOR was markedly delayed in their brains. These results suggest that the OR region may have a crucial role in the conversion of PrP^C into PrP^Sc after infection with BSE prions. However, Tg(PrPΔOR)/Prnp^0/0 mice remained susceptible to RML and 22L scrapie prions, developing the disease without elongated incubation times after infection with RML and 22L prions. PrP^ScΔOR accumulated only slightly less in the brains of RML- or 22L-infected Tg(PrPΔOR)/Prnp^0/0 mice than PrP^Sc in control wild-type mice. Taken together, these results indicate that the OR region of PrP^C could play a differential role in the pathogenesis of BSE prions and RML or 22L scrapie prions.IMPORTANCE Structure-function relationship studies of PrP^C conformational conversion into PrP^Sc are worthwhile to understand the mechanism of the conversion of PrP^C into PrP^Sc We show here that, by inoculating Tg(PrPΔOR)/Prnp^0/0 mice with the three different strains of RML, 22L, and BSE prions, the OR region could play a differential role in the conversion of PrP^C into PrP^Sc after infection with RML or 22L scrapie prions and BSE prions. PrPΔOR was efficiently converted into PrP^ScΔOR after infection with RML and 22L prions. However, the conversion of PrPΔOR into PrP^ScΔOR was markedly delayed after infection with BSE prions. Further investigation into the role of the OR region in the conversion of PrP^C into PrP^Sc after infection with BSE prions might be helpful for understanding the pathogenesis of BSE prions.

Keywords: BSE; bovine spongiform encephalopathy; octapeptide repeat; prion; prion protein; scrapie.

FIG 1

PrPΔOR expression in the brains of Tg(PrPΔOR)/Prnp^0/0 and Tg(PrPΔOR-3608)/Prnp^0/0 mice. (A, left panel) Western blotting with 6D11 anti-PrP Ab of the brains of WT (n = 3), Tg(PrPΔOR)/Prnp^0/0 (n = 3), and Prnp^0/0 (n = 3) mice. (Right panel) Expression levels of PrPΔOR in Tg(PrPΔOR)/Prnp^0/0 mice compared to PrP^C in WT mice. (B, left panel) Western blotting of the brains of WT (n = 3), Prnp^+/0 (n = 3), Tg(PrPΔOR-3608)/Prnp^0/0 (n = 3), and Prnp^0/0 (n = 3) mice with 6D11 anti-PrP Ab. (Right panel) Expression levels of PrP^C in Prnp^+/0 mice and PrPΔOR in Tg(PrPΔOR-3608)/Prnp^0/0 mice compared to PrP^C in WT mice. AU, arbitrary units.

FIG 2

Different levels of PrP^ScΔOR accumulated in the brains of Tg(PrPΔOR)/Prnp^0/0 mice infected with RML, 22L, and BSE prions at terminal stages. Western blotting with 6D11 anti-PrP Ab of the brains of terminally ill WT (n = 3) and Tg(PrPΔOR)/Prnp^0/0 mice (n = 3) infected with RML (A), 22L (B), and BSE (C) prions after treatment with (+) or without (−) PK. The right panels show levels of PrP^ScΔOR in Tg(PrPΔOR)/Prnp^0/0 mice compared to PrP^Sc in WT mice. AU, arbitrary units; ns, not significant; *, P < 0.05; **, P < 0.01.

FIG 3

Indistinguishable distribution of PrP^Sc and PrP^ScΔOR accumulated in the brains of terminally ill WT and Tg(PrPΔOR)/Prnp^0/0 mice. Brain slices from uninfected (A) and RML (B)-, 22L (C)-, and BSE (D)-infected terminally ill WT (n = 3 in each mouse group) and Tg(PrPΔOR)/Prnp^0/0 (n = 3 in each mouse group) mice were immunohistochemically stained for PrP^Sc and PrP^ScΔOR by 6D11 anti-PrP Ab using the HCl autoclaving method. Three sections from each mouse brain were subjected to investigation of PrP^Sc and PrP^ScΔOR distribution. Cx, cerebral cortex; Hp, hippocampus; Th, thalamus; Cb, cerebellum. Size bars, 100 μm.

FIG 4

Similar vacuolation in the brains of terminally ill WT and Tg(PrPΔOR)/Prnp^0/0 mice. Brain slices from uninfected (A) and RML (B)-, 22L (C)-, and BSE (D)-infected terminally ill WT (n = 3 in each mouse group) and Tg(PrPΔOR)/Prnp^0/0 (n = 3 in each mouse group) mice were subjected to hematoxylin-eosin staining, and vacuoles in 0.1-mm² areas were counted in various brain regions, including the cerebral cortex, hippocampus, thalamus, and cerebellum, respectively. Three sections from each mouse brain were subjected to counting of vacuoles.

FIG 5

Similar pathologies in the brains of terminally ill WT and Tg(PrPΔOR)/Prnp^0/0 mice infected with RML, 22L, or BSE prions. Brain slices from uninfected (A) and RML (B)-, 22L (C)-, and BSE (D)-infected terminally ill WT (n = 3 in each mouse group) and Tg(PrPΔOR)/Prnp^0/0 (n = 3 in each mouse group) mice were subjected to hematoxylin-eosin staining. Three sections from each mouse brain were used for the pathological examinations. Cx, cerebral cortex; Hp, hippocampus; Th, thalamus; Cb, cerebellum. Size bars, 100 μm.

FIG 6

Delayed accumulation of PrP^ScΔOR in the brains of Tg(PrPΔOR)/Prnp^0/0 mice infected with BSE prions. (A) Brain homogenates from WT (n = 3) and Tg(PrPΔOR)/Prnp^0/0 mice (n = 3) sacrificed at 190 dpi with BSE prions were treated with (+) or without (−) PK and then subjected to Western blotting with the 6D11 anti-PrP Ab. (B) PrP^Sc and PrP^ScΔOR levels in the lower panels of panel A. AU, arbitrary units; ***, P < 0.001.

FIG 7

The pre-OR region of PrP^ScΔOR is PK resistant, but not in WT PrP^Sc. Brain homogenates from terminally ill WT (n = 3) and Tg(PrPΔOR)/Prnp^0/0 mice (n = 3) infected with RML (A), 22L (B), and BSE (C) prions were treated with (+) or without (−) PK and then subjected to Western blotting with IBL-N anti-PrP Abs.

FIG 8

Biochemical characterization of PrP^Sc produced in WT mice after inoculation with full-length PrP^Sc and PrP^ScΔOR prions. (A) Western blotting with 6D11 anti-PrP Ab of the brains of terminally ill WT mice inoculated with RML-, 22L-, or BSE-infected WT and Tg(PrPΔOR)/Prnp^0/0 brain homogenates. (B) Western blotting with 6D11 anti-PrP Ab of the brains of terminally ill WT mice inoculated with RML-, 22L-, or BSE-infected WT and Tg(PrPΔOR)/Prnp^0/0 brain homogenates after treatment with PNGase F. (C) Percentage of the diglycosylated, monoglycosylated, and unglycosylated forms of PrP^Sc in the brains of terminally ill WT mice inoculated with RML-, 22L-, or BSE-infected WT and Tg(PrPΔOR)/Prnp^0/0 brain homogenates.