Spontaneous generation of infectious prion disease in transgenic mice

Abstract

We generated transgenic mice expressing bovine cellular prion protein (PrP(C)) with a leucine substitution at codon 113 (113L). This protein is homologous to human protein with mutation 102L, and its genetic link with Gerstmann-Sträussler-Scheinker syndrome has been established. This mutation in bovine PrP(C) causes a fully penetrant, lethal, spongiform encephalopathy. This genetic disease was transmitted by intracerebral inoculation of brain homogenate from ill mice expressing mutant bovine PrP to mice expressing wild-type bovine PrP, which indicated de novo generation of infectious prions. Our findings demonstrate that a single amino acid change in the PrP(C) sequence can induce spontaneous generation of an infectious prion disease that differs from all others identified in hosts expressing the same PrP(C) sequence. These observations support the view that a variety of infectious prion strains might spontaneously emerge in hosts displaying random genetic PrP(C) mutations.

Keywords: BSE; Gerstmann–Sträussler–Scheinker syndrome; TSE; bovine PrP; bovine prion protein; bovine spongiform encephalopathy; prions and related diseases; spontaneous generation; transgenic mice; transmissible spongiform encephalopathy.

Figure 1

Immunoblots of brain extracts from hemizygous 113LBoPrP-Tg037^+/− 113LBoPrP-Tg009^+/− mouse lines compared with those of cow brain extract and BoPrP-Tg110 mouse brain extract. Brain homogenates were analyzed by Western blotting with monoclonal antibody 2A11 (30). Lane 1, 113LBoPrPTg-009; lane 2, 113LBoPrPTg-037; lane 3, Cow; lane 4, BoPrPTg-110. Equivalent amounts of total protein were loaded into each lane. 113L, leucine substitution at codon 113; BoPrP, bovine prion protein. Values on the right are molecular masses in kilodaltons.

Figure 2

Comparison between homozygous bovine prion protein (BoPrP)-Tg110^+/+ control mice (panels A–D) and hemizygous 113LBoPrP-Tg037^+/− mice with end-stage disease (panels E–H) in parietal cortex (panels A and E), CA1 region of the hipocampus (panels B and F), dentate gyrus (panels C and G), and medial thalamus (panels D and H). Severe spongiosis is seen in the cerebral cortex, hilus ofdentate gyrus, and medial thalamus, but not in the CA1 area of the hippocampus and granule cell layer of the dentate gyrus. 113L, leucine substitution at codon 113. Paraffin-embedded sections were stained with hematoxylin and eosin. Scale bar in panel H = 25 μm.

Figure 3

Host cellular prion protein (PrP^C) solubility and proteinase K (PK) resistance studies in homozygous 113LBoPrP-Tg037, 113LBoPrP-Tg009, and control BoPrP-Tg110 mice. Western blot analysis with monoclonal antibody 2A11 of soluble (S) and insoluble (P) fractions obtained from mouse brain extracts (5% sarkosyl in phosphate-buffered saline, pH 7.4, previously cleared by centrifugation at 2,000 × g) after ultracentrifugation at 100,000 × g for 1 h. P fractions were treated with 5 μg/mL of PK (PK+) at 37°C for 60 min or left untreated (PK–). Panels A and B, show brain extracts from diseased 113LBoPrP-Tg037 mice or from 500-day-old 113LBoPrP-Tg009 and BoPrP-Tg110 mice. Panel C shows brain extracts from 30-day-old mice. In panels A and C, equivalent amounts of brain material were solubilized, centrifuged, and loaded onto the gel. In panel B, an 8-fold (8×) equivalent brain tissue mass was loaded to obtain equivalent PrP^C signals for the other 2 mouse lines. 113L, leucine substitution at codon 113; BoPrP, bovine prion protein. Values on the right are molecular masses in kilodaltons.

Figure 4

Comparative Western blot analyses of brain prion protein resistant to proteinase K digestion (PrP^res) from BoPrP-Tg110 mice infected with bovine spongiform encephalopathy (BSE)-C, 113L-BSE, BSE-L, and BSE-H prions. Mice infected with newly generated 113L-BSE prion at first (P1), second (P2), and third (P3) passages are compared with mice infected with BSE-C (P1) (lane 4); BSE-L (P1) (lane 5); and BSE-H (P1) (lane 6) prions. Each panel was identified by using the monoclonal antibody (mAb) listed at the bottom left. The same quantities of PrP^res were loaded in all lanes. BoPrP, bovine prion protein; 113L, leucine substitution at codon 113. Values on the right are molecular masses in kilodaltons.

Figure 5

Immunochemical analysis of paraffin-embedded tissue blots of representative coronal sections of the hippocampus, showing deposition patterns of abnormal isoform of host-encoded prion protein in brains from BoPrP-Tg110 mice infected with bovine spongiform encephalopathy (BSE)-C (A), 113L-BSE (B), BSE-H (C), and BSE-L (D) prions. BoPrP, bovine prion protein; 113L, leucine substitution at codon 113. Monoclonal antibody Sha31 stained by using the procedure of Andréoletti et al. (29). Original magnifications ×20.

Figure 6

Vacuolar lesion profile in brains from BoPrP-Tg110 mice inoculated with bovine spongiform encephalopathy (BSE)-C (black circles, n = 6 animals), BSE-H (black triangles, n = 6 animals), BSE-L (black squares, n = 5 animals), 113L-BSE second passage (black squares, n = 5 animals), and 113L-BSE third passage (open diamonds, n = 5 animals) prions. Lesion scoring was conducted for 9 areas of gray matter (G) and 3 areas of white matter (W) in mouse brains. G1, dorsal medulla; G2, cerebellar cortex; G3, superior colliculus; G4, hypothalamus; G5, medial thalamus; G6, hippocampus; G7, septum; G8, medial cerebral cortex at the level of the thalamus; G9, medial cerebral cortex at the level of the septum (G9); W1, cerebellum; W2, mesencephalic tegmentum; W3, pyramidel tract. BoPrP, bovine prion protein; 113L, leucine substitution at codon 113. Error bars indicate SE.