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. 2013 Mar 6;32(5):756-69.
doi: 10.1038/emboj.2013.6. Epub 2013 Feb 8.

Post-translational changes to PrP alter transmissible spongiform encephalopathy strain properties

Enrico Cancellotti  1 , Sukhvir P Mahal, Robert Somerville, Abigail Diack, Deborah Brown, Pedro Piccardo, Charles Weissmann, Jean C Manson
Affiliations

Post-translational changes to PrP alter transmissible spongiform encephalopathy strain properties

Enrico Cancellotti et al. EMBO J. .

Abstract

The agents responsible for transmissible spongiform encephalopathies (TSEs), or prion diseases, contain as a major component PrP(Sc), an abnormal conformer of the host glycoprotein PrP(C). TSE agents are distinguished by differences in phenotypic properties in the host, which nevertheless can contain PrP(Sc) with the same amino-acid sequence. If PrP alone carries information defining strain properties, these must be encoded by post-translational events. Here we investigated whether the glycosylation status of host PrP affects TSE strain characteristics. We inoculated wild-type mice with three TSE strains passaged through transgenic mice with PrP devoid of glycans at the first, second or both N-glycosylation sites. We compared the infectious properties of the emerging isolates with TSE strains passaged in wild-type mice by in vivo strain typing and by the standard scrapie cell assay in vitro. Strain-specific characteristics of the 79A TSE strain changed when PrP(Sc) was devoid of one or both glycans. Thus infectious properties of a TSE strain can be altered by post-translational changes to PrP which we propose result in the selection of mutant TSE strains.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Western blot analysis of differentially glycosylated PrPSc in the inocula. Example of PrPSc from 79A infected brains after passage through 129Ola wild-type, G1, G2 and G3 mice, and a diagram of the glycosylation pattern of PrP in the four mouse genotypes.(A) Wild-type brain after PK digestion showing PrPSc that comprises di, mono and unglycosylated isoforms because both glycosylation sites at codons 180 and 196 are preserved. (B) G1 inoculum expressing PrPSc lacking the diglycosylated isoform because the 180 glycosylation site has been abolished. (C) G2 inoculum with PrPSc lacking the diglycosylated isoform because the 196 glycosylation site has been abolished. (D) G3 inoculum expressing totally unglycosylated PrPSc because both 180 and 196 glycosylation sites have been modified.
Figure 2
Figure 2
Incubation period analyses of passages of 79A through G1, G2 and G3 mice. The 79A TSE strain was passaged in glycosylation-deficient mice (first pass). Passages were established from one 129 mouse (passage from 129) or two G1, G2 or G3 mice (pass from G1/G2/G3) in C57, VM or CVF1 mice or repassaged in glycosylation-deficient mice. The graphs show the incubation period for each mouse strain for each passage. (A) Passage through G1 mice, (B) G2 passage through G2 mice and (C) passage through G3 mice. The small yellow triangle and arrow indicate the single positive case in VM mice in the G3-79A(a) passage.
Figure 3
Figure 3
Lesion profile analyses of passages of 79A through G1, G2 and G3 mice. Lesion profiles are shown for each passage of 79A in individual mouse strains (G1-79A, A–E; G2-79A, F–J, and G3-79A, K–O). Spongiform degeneration was scored in nine grey matter areas: G1, dorsal medulla; G2, cerebellar cortex; G3, superior colliculus; G4, hypothalamus; G5, medial thalamus; G6, hippocampus; G7, septum; G8, cerebral cortex; and G9, forebrain cortex, and in three white matter areas: W1, cerebellar white matter; W2, mesencephalic tegmentum; and W3, pyramidal tract (x axis) of mice scored clinically positive.
Figure 4
Figure 4
PrP deposition in wild-type mouse brains after infection with 79A passaged in G1 G2, G3 or 129 mice. (A–C) 129-79A, (D–F) G1-79A, (G–I) G2-79A, (J–L) G3-79A passaged through C57, VM and CVF1 mice respectively. Widespread diffuse pattern of PrP deposition was observed in all brains irrespective of the inoculum with which they had been infected, with the hippocampus consistently targeted. A patchy pattern of PrP staining was observed in the hippocampus in VM mice infected with G1-79A (E) and G3-79A (K), intense band of staining observed in the dentate gyrus. CC indicates the corpus callosum, LM: lacunosum-moleculare, DG: Dentate gyrus. Sections stained with 6H4 antibody. Scale bar=400 μm.
Figure 5
Figure 5
Characterisation of 129-79A, G1-79A, G2-79A and G3-79A by the SSCA. The SSCA was performed on PK1 cells in the absence (blue) or presence of the glycosylation inhibitor kifunensine (kifu, pink). The RI is the reciprocal of the brain homogenate dilutions giving rise to 600 spots. (A) Samples were assayed in the absence (blue) or presence (red) of kifunensine (kifu). The ratios RI−kifu/RI+kifu for 129-79A and G3-79A are similar (>370), and more than 200 times higher than for 129-139A (1.6) and G2-79A (2.7, 3.0). The difference between 129-139A and G2-79A is statistically significant (see D). (B) Samples were assayed in the absence (blue) or presence (red) of swainsonine (swa). The ratios RI−swa/RI+swa for 129-79A (9.6, 7.0, 6.7) and G3-79A (8.0) are indistinguishable, but about twice as high as for 129-139A (3.2) and G2-79A (3.5, 4.0), which are also indistinguishable. (C) Bar diagram showing the log RI ratios of PK1/PK1kifu (blue bars) and PK1/PK1swa (red bars). The ratios of the log RIs are also shown above the bars indicating two distinct groups: ratios >2.6 for 79A-like strains and <1.0 for 139-like strains. (D) Matrix of log RI ratios of PK1/PK1kifu (blue squares) and PK1/PK1swa (red squares). Statistical analysis (unpaired t-test) was performed using raw data from each well of the assay after removing background signal. * To *** represent P-values from 0.03 to <0.0001, respectively; ns, not significant. The apparent non-identity (*) between 129-79A (FL) and 129-79A(B1) is unexplained. The figure shows that 129-79A and G3-79A are indistinguishable with both kifu and swa and differ from 129-139A and G2-79A, which are similar but not identical.
Figure 6
Figure 6
Incubation period analyses of passages of ME7 and 301C through G2 mice. The ME7 TSE strain (A) and 301C, BSE-derived strain (B) were passaged in glycosylation-deficient mice (first pass). Passes were established from one 129 mouse (pass from 129) or two G2 mice (pass from G2) in C57, VM or CVF1 mice or repassaged in glycosylation-deficient mice. The graphs show the incubation period for each mouse strain for each passage.
Figure 7
Figure 7
Lesion profile analyses of ME7 and 301C passaged through G2 mice. Lesion profiles are shown for each passage of ME7 (A–E) and 301C (F–J) in individual mouse strains. Spongiform degeneration was scored in nine grey matter areas: G1, dorsal medulla; G2, cerebellar cortex; G3, superior colliculus; G4, hypothalamus; G5, medial thalamus; G6, hippocampus; G7, septum; G8, cerebral cortex; and G9, forebrain cortex, and in three white matter areas: W1, cerebellar white matter; W2, mesencephalic tegmentum; and W3, pyramidal tract (x axis) of mice scored clinically positive.
Figure 8
Figure 8
PrP deposition in wild-type mouse brains after infection with ME7 passed in G2 or 129 mice. Diffuse widespread accumulation of abnormal PrP was observed throughout the hippocampus, this distribution was observed in both wt (A–C) and G2-ME7 (D–F) inoculated animals. Small plaques where also observed in the corpus callosum (B, E, F) but this was variable. CC indicates the corpus callosum, LM: lacunosum-moleculare, DG: Dentate gyrus. Sections stained with 6H4 antibody. Scale bar=400 μm.
Figure 9
Figure 9
PrP deposition in wild-type mouse brains after infection with 301C passed in G2 or 129 mice. There was a similar pattern of PrP deposition in both groups of mice inoculated with 129-301C (A–C) and G2-301C (D–F), with a targeted pericellular form observed in the dentate gyrus and CA2 sector of the hippocampus. CC indicates the corpus callosum, LM: lacunosum-moleculare, DG: Dentate gyrus. Sections stained with BH1 antibody. Scale bar=400 μm.
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