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

doi:10.1128/JVI.01368-17

. 2017 Dec 14;92(1):e01368-17.

doi: 10.1128/JVI.01368-17. Print 2018 Jan 1.

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

Affiliations

¹ Division of Molecular Neurobiology, The Institute for Enzyme Research (KOSOKEN), Tokushima University, Kuramoto, Tokushima, Japan.
² Animal Research Center, School of Medicine, University of Occupational and Environmental Health, Yahatanishi, Kitakyushu, Japan.
³ Student Laboratory, Faculty of Medicine, Tokushima University, Kuramoto, Tokushima, Japan.
⁴ Laboratory of Integrative Biological Science, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
⁵ National Institute of Animal Health (NIAH), National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan.
⁶ Division of Molecular Neurobiology, The Institute for Enzyme Research (KOSOKEN), Tokushima University, Kuramoto, Tokushima, Japan sakaguchi@tokushima-u.ac.jp.

^# Contributed equally.

PMID: 29046443
PMCID: PMC5730770
DOI: 10.1128/JVI.01368-17

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

Hideyuki Hara et al. J Virol. 2017.

. 2017 Dec 14;92(1):e01368-17.

doi: 10.1128/JVI.01368-17. Print 2018 Jan 1.

Authors

Affiliations

¹ Division of Molecular Neurobiology, The Institute for Enzyme Research (KOSOKEN), Tokushima University, Kuramoto, Tokushima, Japan.
² Animal Research Center, School of Medicine, University of Occupational and Environmental Health, Yahatanishi, Kitakyushu, Japan.
³ Student Laboratory, Faculty of Medicine, Tokushima University, Kuramoto, Tokushima, Japan.
⁴ Laboratory of Integrative Biological Science, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
⁵ National Institute of Animal Health (NIAH), National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan.
⁶ Division of Molecular Neurobiology, The Institute for Enzyme Research (KOSOKEN), Tokushima University, Kuramoto, Tokushima, Japan sakaguchi@tokushima-u.ac.jp.

^# Contributed equally.

PMID: 29046443
PMCID: PMC5730770
DOI: 10.1128/JVI.01368-17

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)/Prnp⁰^/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)/Prnp⁰^/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)/Prnp⁰^/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)/Prnp⁰^/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)/Prnp⁰^/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.

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Figures

**FIG 1**
PrPΔOR expression in the brains of Tg(PrPΔOR)/*Prnp⁰*^/0 and Tg(PrPΔOR-3608)/*Prnp⁰*^/0 mice. (A, left panel) Western blotting with 6D11 anti-PrP Ab of the brains of WT (n = 3), Tg(PrPΔOR)/*Prnp⁰*^/0 (n = 3), and *Prnp⁰*^/0 (n = 3) mice. (Right panel) Expression levels of PrPΔOR in Tg(PrPΔOR)/*Prnp⁰*^/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)/*Prnp⁰*^/0 (n = 3), and *Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/0 mice infected with BSE prions. (A) Brain homogenates from WT (n = 3) and Tg(PrPΔOR)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/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)/*Prnp⁰*^/0 brain homogenates.

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References

1. Prusiner SB. 1998. Prions. Proc Natl Acad Sci U S A 95:13363–13383. doi:10.1073/pnas.95.23.13363. - DOI - PMC - PubMed
1. Stahl N, Borchelt DR, Hsiao K, Prusiner SB. 1987. Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51:229–240. doi:10.1016/0092-8674(87)90150-4. - DOI - PubMed
1. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C. 1993. Mice devoid of PrP are resistant to scrapie. Cell 73:1339–1347. doi:10.1016/0092-8674(93)90360-3. - DOI - PubMed
1. Prusiner SB, Groth D, Serban A, Koehler R, Foster D, Torchia M, Burton D, Yang SL, DeArmond SJ. 1993. Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc Natl Acad Sci U S A 90:10608–10612. doi:10.1073/pnas.90.22.10608. - DOI - PMC - PubMed
1. Manson JC, Clarke AR, McBride PA, McConnell I, Hope J. 1994. PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology. Neurodegeneration 3:331–340. - PubMed

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[1] Prusiner SB. 1998. Prions. Proc Natl Acad Sci U S A 95:13363–13383. doi:10.1073/pnas.95.23.13363. - DOI - PMC - PubMed

[2] Prusiner SB. 1998. Prions. Proc Natl Acad Sci U S A 95:13363–13383. doi:10.1073/pnas.95.23.13363. - DOI - PMC - PubMed

[3] Stahl N, Borchelt DR, Hsiao K, Prusiner SB. 1987. Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51:229–240. doi:10.1016/0092-8674(87)90150-4. - DOI - PubMed

[4] Stahl N, Borchelt DR, Hsiao K, Prusiner SB. 1987. Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51:229–240. doi:10.1016/0092-8674(87)90150-4. - DOI - PubMed

[5] Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C. 1993. Mice devoid of PrP are resistant to scrapie. Cell 73:1339–1347. doi:10.1016/0092-8674(93)90360-3. - DOI - PubMed

[6] Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C. 1993. Mice devoid of PrP are resistant to scrapie. Cell 73:1339–1347. doi:10.1016/0092-8674(93)90360-3. - DOI - PubMed

[7] Prusiner SB, Groth D, Serban A, Koehler R, Foster D, Torchia M, Burton D, Yang SL, DeArmond SJ. 1993. Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc Natl Acad Sci U S A 90:10608–10612. doi:10.1073/pnas.90.22.10608. - DOI - PMC - PubMed

[8] Prusiner SB, Groth D, Serban A, Koehler R, Foster D, Torchia M, Burton D, Yang SL, DeArmond SJ. 1993. Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc Natl Acad Sci U S A 90:10608–10612. doi:10.1073/pnas.90.22.10608. - DOI - PMC - PubMed

[9] Manson JC, Clarke AR, McBride PA, McConnell I, Hope J. 1994. PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology. Neurodegeneration 3:331–340. - PubMed

[10] Manson JC, Clarke AR, McBride PA, McConnell I, Hope J. 1994. PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology. Neurodegeneration 3:331–340. - PubMed

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

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

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