BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein

Abstract

Variant Creutzfeldt-Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrP(Sc) type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.

Fig. 1. Immunohistochemistry of cerebral cortex and hippocampal regions of transgenic mouse brain showing abnormal PrP immunoreactivity, including PrP-positive florid plaques (enlarged in insets). (A) vCJD-inoculated 129MM Tg35 mouse. (B) BSE-inoculated 129MM Tg35 mouse. (C) vCJD-inoculated 129MM Tg45 mouse. (D) BSE-inoculated 129MM Tg45 mouse. (E–G) Histological analysis showing the thalamus of a BSE-inoculated 129MM Tg35 mouse propagating type 2 human PrP^Sc with widespread vacuolation (E; H&E), extensive gliosis (F; GFAP), but no specific PrP immunoreactive deposits (G; ICSM 35). Scale bar: (A–D) = 100 µm; (E), (F) and (G) = 50 µm.

Fig. 2. Western blots of proteinase K (PK)-treated brain homogenates from transgenic mice, human cases of variant and sporadic CJD, and lines of wild-type mice. (A) Lane 1, vCJD; lane 2, vCJD-inoculated 129MM Tg35 mouse. (B) Lane 1, vCJD-inoculated 129MM Tg35 mouse; lane 2, BSE-inoculated 129MM Tg35 mouse propagating type 2 PrP^Sc; lane 3, vCJD. (C) Lanes 1 and 2, BSE-inoculated 129MM Tg35 mouse propagating either type 2 PrP^Sc (lane 1) or type 4 PrP^Sc (lane 2). (D) Lane 1, BSE-inoculated 129MM Tg35 mouse propagating type 2 PrP^Sc; lane 2, human sporadic CJD type 2 PrP^Sc (PRNP genotype 129MM). (E) Lanes 1–3, human sporadic CJD type 2 PrP^Sc (PRNP genotype 129MM); lanes 4–6, BSE-inoculated 129MM Tg35 mouse propagating type 2 PrP^Sc. Samples were PK digested in the absence (lanes 1, 3, 4 and 6) or presence (lanes 2 and 5) of 25 mM EDTA. *Following proteolysis, samples in lanes 3 and 6 were boiled in SDS sample buffer and subsequently adjusted to 25 mM EDTA before electrophoresis. (F) Transmission of vCJD and BSE to 129MM Tg45 mice. Lane 1, vCJD; lane 2, vCJD-inoculated 129MM Tg45 mouse; lane 3, BSE- inoculated 129MM Tg45 mouse. (G) Primary transmission of vCJD and BSE to wild-type inbred mice. Lane 1, BSE-inoculated FVB mouse; lane 2, vCJD-inoculated FVB mouse; lane 3, BSE-inoculated C57BL/6 mouse; lane 4, BSE-inoculated SJL mouse; lane 5, vCJD-inoculated SJL mouse; lane 6, BSE-inoculated RIIIS mouse. (H) Secondary transmission of vCJD and BSE in wild-type inbred mice. Lanes 1–4, BSE was passaged twice in C57BL/6 mice and then passaged in different wild-type mice: lane 1, C57BL/6 mouse; lane 2, FVB mouse; lane 3, SJL mouse; lane 4, RIIIS mouse. Lanes 5 and 6, second passage of SJL-passaged BSE in further SJL mice (lane 5) or FVB mice (lane 6). Western blots were analysed by high-sensitivity ECL using biotinylated anti-PrP monoclonal antibody ICSM 35 (A–D, F–H) or 3F4 (E).

Fig. 3. Scattergraph of proportions of protease-resistant PrP in higher molecular mass (diglycosylated) and low molecular mass (monoglycosylated) glycoforms seen in brain tissue from sporadic CJD, vCJD, BSE and in transgenic mice following challenge with CJD, vCJD and BSE. Data points are plotted as mean ± SEM. Human cases, indicated as circles: sporadic CJD type 1 PrP^Sc, light grey (n = 12); sporadic CJD type 2 PrP^Sc, mid-grey (n = 49); sporadic CJD type 3 PrP^Sc, dark grey (n = 22); vCJD type 4 PrP^Sc, yellow (n = 16). Cattle BSE, black square (n = 3). Transmissions to 129MM Tg35 mice, upward triangles: sporadic CJD type 1 PrP^Sc-inoculated mice, blue (n = 7); vCJD type 4 PrP^Sc-inoculated mice, green (n = 10); BSE-inoculated mice, red (n = 9; n = 1). Transmissions to 129MM Tg45 mice, inverted triangles: sporadic CJD type 2 PrP^Sc-inoculated mice, blue (n = 3); vCJD type 4 PrP^Sc-inoculated mice, green (n = 4); BSE-inoculated mice, red (n = 4).

Fig. 4. Scattergraph of proportions of protease-resistant PrP in higher molecular mass (diglycosylated) and low molecular mass (monoglycosylated) glycoforms seen in sporadic CJD, vCJD, BSE and in wild-type mice following challenge with vCJD and BSE. Data points are plotted as mean ± SEM. (A–C) Human cases indicated as circles: sporadic CJD type 1 PrP^Sc, light grey (n = 12); sporadic CJD type 2 PrP^Sc, mid-grey (n = 49); sporadic CJD type 3 PrP^Sc, dark grey (n = 22); vCJD type-4 PrP^Sc, yellow (n = 16). Cattle BSE, black square (n = 3). (A) Primary transmission of vCJD and BSE to wild-type mice: vCJD-inoculated FVB mice, green diamond (n = 19); vCJD-inoculated SJL mice, green triangle (n = 4); BSE-inoculated FVB mice, red diamond (n = 12); BSE-inoculated SJL mice, red triangle (n = 7); BSE-inoculated RIIIS mice, red star (n = 4); BSE-inoculated C57BL/6 mice, inverted red triangle (n = 3). (B) Transmission of SJL-passaged BSE to further wild-type mice: SJL-passaged-BSE-inoculated FVB mice, blue diamond (n = 4); BSE passaged twice in SJL mice, blue triangle (n = 3); BSE passaged three times in SJL mice, open triangle (n = 3). (C) Transmission of BSE passaged twice in C57BL/6 mice to further wild-type mice: C57BL/6-passaged BSE to FVB mice, orange diamond (n = 3); C57BL/6-passaged BSE to SJL mice, orange triangle (n = 4); C57BL/6-passaged BSE to RIIIS mice, orange star (n = 3); C57BL/6-passaged BSE to C57BL/6 mice, inverted orange triangle (n = 3).