Molecular Neurobiology

, Volume 56, Issue 9, pp 6501–6511 | Cite as

A Single Amino Acid Substitution, Found in Mammals with Low Susceptibility to Prion Diseases, Delays Propagation of Two Prion Strains in Highly Susceptible Transgenic Mouse Models

  • Alicia Otero
  • Carlos Hedman
  • Natalia Fernández-Borges
  • Hasier Eraña
  • Belén Marín
  • Marta Monzón
  • Manuel A. Sánchez-Martín
  • Romolo Nonno
  • Juan José Badiola
  • Rosa BoleaEmail author
  • Joaquín CastillaEmail author


Specific variations in the amino acid sequence of prion protein (PrP) are key determinants of susceptibility to prion diseases. We previously showed that an amino acid substitution specific to canids confers resistance to prion diseases when expressed in mice and demonstrated its dominant-negative protective effect against a variety of infectious prion strains of different origins and characteristics. Here, we show that expression of this single amino acid change significantly increases survival time in transgenic mice expressing bank vole cellular prion protein (PrPC), which is inherently prone to misfolding, following inoculation with two distinct prion strains (the CWD-vole strain and an atypical strain of spontaneous origin). This amino acid substitution hinders the propagation of both prion strains, even when expressed in the context of a PrPC uniquely susceptible to a wide range of prion isolates. Non-inoculated mice expressing this substitution experience spontaneous prion formation, but showing an increase in survival time comparable to that observed in mutant mice inoculated with the atypical strain. Our results underscore the importance of this PrP variant in the search for molecules with therapeutic potential against prion diseases.


Prions Prion propagation Transmissible spongiform encephalopathies Canine PrP Bank vole PrP 



We thank MINECO for the Severo Ochoa Excellence Accreditation (SEV-2016-0644). The authors would like to thank the following for their support: the IKERBasque Foundation, the staff at the CIC bioGUNE animal facility and Patricia Piñeiro and Dr. Jan Langeveld from Central Veterinary Institute, Wageningen for kindly providing the 12B2 antibody.

Authors’ Contribution

JC and RB conceived the study; AO, CH, NFB, HE, BM, and MM performed most of the experiments; MASM and RN collaborated in the creation of the transgenic lines used; AO, HE, JJB, RB, and JC evaluated the results; AO, RB, JJB, HE, and JC wrote and reviewed the manuscript.

Funding Information

This work was supported financially by the Spanish (AGL2015-65046-C2-1-R, AGL2015-65560-R) (MINECO/FEDER) and Interreg (POCTEFA EFA148/16) grants. Alicia Otero was supported by a research grant from the Government of Aragón (C020/2014) co-financed by the European Social Fund.

Compliance with Ethical Standards

Ethics Statement

All procedures involving animals were approved by the University of Zaragoza’s Ethics Committee for Animal Experiments (permit number PI32/13) and were performed in accordance with recommendations for the care and use of experimental animals and with Spanish law (R.D. 1201/05).

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12035_2019_1535_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1080 kb)


  1. 1.
    Colby DW, Prusiner SB (2011) Prions. Cold Spring Harb Perspect Biol 3(1):a006833. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Collins SJ, Lawson VA, Masters CL (2004) Transmissible spongiform encephalopathies. Lancet 363(9402):51–61CrossRefPubMedGoogle Scholar
  3. 3.
    Prusiner SB (1998) The prion diseases. Brain Pathol 8(3):499–513CrossRefPubMedGoogle Scholar
  4. 4.
    Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216(4542):136–144CrossRefPubMedGoogle Scholar
  5. 5.
    Stewart P, Campbell L, Skogtvedt S, Griffin KA, Arnemo JM, Tryland M, Girling S, Miller MW et al (2012) Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region. PLoS One 7(12):e50623. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Fernandez-Borges N, Parra B, Vidal E, Erana H, Sanchez-Martin MA, de Castro J, Elezgarai SR, Pumarola M et al (2017) Unraveling the key to the resistance of canids to prion diseases. PLoS Pathog 13(11):e1006716. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Vidal E, Fernandez-Borges N, Pintado B, Ordonez M, Marquez M, Fondevila D, Torres JM, Pumarola M et al (2013) Bovine spongiform encephalopathy induces misfolding of alleged prion-resistant species cellular prion protein without altering its pathobiological features. J Neurosci 33(18):7778–7786. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Polymenidou M, Trusheim H, Stallmach L, Moos R, Julius C, Miele G, Lenz-Bauer C, Aguzzi A (2008) Canine MDCK cell lines are refractory to infection with human and mouse prions. Vaccine 26(21):2601–2614. CrossRefPubMedGoogle Scholar
  9. 9.
    Khan MQ, Sweeting B, Mulligan VK, Arslan PE, Cashman NR, Pai EF, Chakrabartty A (2010) Prion disease susceptibility is affected by beta-structure folding propensity and local side-chain interactions in PrP. Proc Natl Acad Sci U S A 107(46):19808–19813. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Chianini F, Fernandez-Borges N, Vidal E, Gibbard L, Pintado B, de Castro J, Priola SA, Hamilton S et al (2012) Rabbits are not resistant to prion infection. Proc Natl Acad Sci U S A 109(13):5080–5085. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bian J, Khaychuk V, Angers RC, Fernandez-Borges N, Vidal E, Meyerett-Reid C, Kim S, Calvi CL et al (2017) Prion replication without host adaptation during interspecies transmissions. Proc Natl Acad Sci U S A 114(5):1141–1146. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Nonno R, Di Bari MA, Cardone F, Vaccari G, Fazzi P, Dell'Omo G, Cartoni C, Ingrosso L et al (2006) Efficient transmission and characterization of Creutzfeldt-Jakob disease strains in bank voles. PLoS Pathog 2(2):e12. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Agrimi U, Nonno R, Dell'Omo G, Di Bari MA, Conte M, Chiappini B, Esposito E, Di Guardo G et al (2008) Prion protein amino acid determinants of differential susceptibility and molecular feature of prion strains in mice and voles. PLoS Pathog 4(7):e1000113. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pirisinu L, Di Bari MA, D'Agostino C, Marcon S, Riccardi G, Poleggi A, Cohen ML, Appleby BS et al (2016) Gerstmann-Straussler-Scheinker disease subtypes efficiently transmit in bank voles as genuine prion diseases. Sci Rep 6:20443. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Watts JC, Giles K, Patel S, Oehler A, DeArmond SJ, Prusiner SB (2014) Evidence that bank vole PrP is a universal acceptor for prions. PLoS Pathog 10(4):e1003990. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Di Bari MA, Nonno R, Castilla J, D'Agostino C, Pirisinu L, Riccardi G, Conte M, Richt J et al (2013) Chronic wasting disease in bank voles: Characterisation of the shortest incubation time model for prion diseases. PLoS Pathog 9(3):e1003219. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Cartoni C, Schinina ME, Maras B, Nonno R, Vaccari G, Di Baria MA, Conte M, Liu QG et al (2005) Identification of the pathological prion protein allotypes in scrapie-infected heterozygous bank voles (Clethrionomys glareolus) by high-performance liquid chromatography-mass spectrometry. J Chromatogr A 1081(1):122–126CrossRefPubMedGoogle Scholar
  18. 18.
    Watts JC, Giles K, Stohr J, Oehler A, Bhardwaj S, Grillo SK, Patel S, DeArmond SJ et al (2012) Spontaneous generation of rapidly transmissible prions in transgenic mice expressing wild-type bank vole prion protein. Proc Natl Acad Sci U S A 109(9):3498–3503. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Will RG, Ironside JW (2017) Sporadic and infectious human prion diseases. Cold Spring Harb Perspect Med 7(1).
  20. 20.
    Otero A, Bolea R, Hedman C, Fernández-Borges N, Marín B, López-Pérez O, Barrio T, Eraña H et al (2017) An amino acid substitution found in animals with low susceptibility to prion diseases confers a protective dominant-negative effect in prion-infected transgenic mice. Mol Neurobiol 55:6182–6192. CrossRefPubMedGoogle Scholar
  21. 21.
    Castilla J, Gutierrez-Adan A, Brun A, Doyle D, Pintado B, Ramirez MA, Salguero FJ, Parra B et al (2004) Subclinical bovine spongiform encephalopathy infection in transgenic mice expressing porcine prion protein. J Neurosci 24(21):5063–5069. CrossRefPubMedGoogle Scholar
  22. 22.
    Fernandez-Borges N, Di Bari MA, Erana H, Sanchez-Martin M, Pirisinu L, Parra B, Elezgarai SR, Vanni I et al (2018) Cofactors influence the biological properties of infectious recombinant prions. Acta Neuropathol 135(2):179–199. CrossRefPubMedGoogle Scholar
  23. 23.
    Fraser H, Dickinson AG (1968) The sequential development of the brain lesion of scrapie in three strains of mice. J Comp Pathol 78(3):301–311CrossRefPubMedGoogle Scholar
  24. 24.
    Schulz-Schaeffer WJ, Tschoke S, Kranefuss N, Drose W, Hause-Reitner D, Giese A, Groschup MH, Kretzschmar HA (2000) The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases. Am J Pathol 156(1):51–56. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Andreoletti O, Simon S, Lacroux C, Morel N, Tabouret G, Chabert A, Lugan S, Corbiere F et al (2004) PrPSc accumulation in myocytes from sheep incubating natural scrapie. Nat Med 10(6):591–593. CrossRefPubMedGoogle Scholar
  26. 26.
    Monleon E, Monzon M, Hortells P, Bolea R, Acin C, Vargas F, Badiola JJ (2005) Approaches to scrapie diagnosis by applying immunohistochemistry and rapid tests on central nervous and lymphoreticular systems. J Virol Methods 125(2):165–171. CrossRefPubMedGoogle Scholar
  27. 27.
    Castilla J, Saa P, Hetz C, Soto C (2005) In vitro generation of infectious scrapie prions. Cell 121(2):195–206. CrossRefPubMedGoogle Scholar
  28. 28.
    Pirisinu L, Marcon S, Di Bari MA, D'Agostino C, Agrimi U, Nonno R (2013) Biochemical characterization of prion strains in bank voles. Pathogens 2(3):446–456. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Westaway D, Zuliani V, Cooper CM, Da Costa M, Neuman S, Jenny AL, Detwiler L, Prusiner SB (1994) Homozygosity for prion protein alleles encoding glutamine-171 renders sheep susceptible to natural scrapie. Genes Dev 8(8):959–969CrossRefPubMedGoogle Scholar
  30. 30.
    Clouscard C, Beaudry P, Elsen JM, Milan D, Dussaucy M, Bounneau C, Schelcher F, Chatelain J et al (1995) Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie. J Gen Virol 76(Pt 8):2097–2101CrossRefPubMedGoogle Scholar
  31. 31.
    Kaneko K, Zulianello L, Scott M, Cooper CM, Wallace AC, James TL, Cohen FE, Prusiner SB (1997) Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proc Natl Acad Sci U S A 94(19):10069–10074CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Zulianello L, Kaneko K, Scott M, Erpel S, Han D, Cohen FE, Prusiner SB (2000) Dominant-negative inhibition of prion formation diminished by deletion mutagenesis of the prion protein. J Virol 74(9):4351–4360CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Perrier V, Kaneko K, Safar J, Vergara J, Tremblay P, DeArmond SJ, Cohen FE, Prusiner SB et al (2002) Dominant-negative inhibition of prion replication in transgenic mice. Proc Natl Acad Sci U S A 99(20):13079–13084. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Geoghegan JC, Miller MB, Kwak AH, Harris BT, Supattapone S (2009) Trans-dominant inhibition of prion propagation in vitro is not mediated by an accessory cofactor. PLoS Pathog 5(7):e1000535. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Palmer MS, Dryden AJ, Hughes JT, Collinge J (1991) Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease. Nature 352(6333):340–342. CrossRefPubMedGoogle Scholar
  36. 36.
    Shibuya S, Higuchi J, Shin RW, Tateishi J, Kitamoto T (1998) Codon 219 Lys allele of PRNP is not found in sporadic Creutzfeldt-Jakob disease. Ann Neurol 43(6):826–828. CrossRefPubMedGoogle Scholar
  37. 37.
    Asante EA, Smidak M, Grimshaw A, Houghton R, Tomlinson A, Jeelani A, Jakubcova T, Hamdan S et al (2015) A naturally occurring variant of the human prion protein completely prevents prion disease. Nature 522(7557):478–481. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Hill AF, Joiner S, Linehan J, Desbruslais M, Lantos PL, Collinge J (2000) Species-barrier-independent prion replication in apparently resistant species. Proc Natl Acad Sci U S A 97(18):10248–10253CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Collinge J (2001) Prion diseases of humans and animals: Their causes and molecular basis. Annu Rev Neurosci 24:519–550. CrossRefPubMedGoogle Scholar
  40. 40.
    Atarashi R, Sim VL, Nishida N, Caughey B, Katamine S (2006) Prion strain-dependent differences in conversion of mutant prion proteins in cell culture. J Virol 80(16):7854–7862. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Striebel JF, Race B, Meade-White KD, LaCasse R, Chesebro B (2011) Strain specific resistance to murine scrapie associated with a naturally occurring human prion protein polymorphism at residue 171. PLoS Pathog 7(9):e1002275. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Houston F, Goldmann W, Chong A, Jeffrey M, Gonzalez L, Foster J, Parnham D, Hunter N (2003) Prion diseases: BSE in sheep bred for resistance to infection. Nature 423(6939):498. CrossRefPubMedGoogle Scholar
  43. 43.
    Di Bari MA, Chianini F, Vaccari G, Esposito E, Conte M, Eaton SL, Hamilton S, Finlayson J et al (2008) The bank vole (Myodes glareolus) as a sensitive bioassay for sheep scrapie. J Gen Virol 89(Pt 12):2975–2985. CrossRefPubMedGoogle Scholar
  44. 44.
    Asante EA, Linehan JM, Smidak M, Tomlinson A, Grimshaw A, Jeelani A, Jakubcova T, Hamdan S et al (2013) Inherited prion disease A117V is not simply a proteinopathy but produces prions transmissible to transgenic mice expressing homologous prion protein. PLoS Pathog 9(9):e1003643. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Fernandez-Borges N, Erana H, Elezgarai SR, Harrathi C, Gayosso M, Castilla J (2013) Infectivity versus seeding in neurodegenerative diseases sharing a prion-like mechanism. Int J Cell Biol 2013:583498. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Prusiner SB, Scott M, Foster D, Pan KM, Groth D, Mirenda C, Torchia M, Yang SL et al (1990) Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell 63(4):673–686CrossRefPubMedGoogle Scholar
  47. 47.
    Hope J, Morton LJ, Farquhar CF, Multhaup G, Beyreuther K, Kimberlin RH (1986) The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP). EMBO J 5(10):2591–2597CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Priola SA, Caughey B, Race RE, Chesebro B (1994) Heterologous PrP molecules interfere with accumulation of protease-resistant PrP in scrapie-infected murine neuroblastoma cells. J Virol 68(8):4873–4878PubMedPubMedCentralGoogle Scholar
  49. 49.
    Jahandideh S, Jamalan M, Faridounnia M (2015) Molecular dynamics study of the dominant-negative E219K polymorphism in human prion protein. J Biomol Struct Dyn 33(6):1315–1325. CrossRefPubMedGoogle Scholar
  50. 50.
    Horiuchi M, Priola SA, Chabry J, Caughey B (2000) Interactions between heterologous forms of prion protein: Binding, inhibition of conversion, and species barriers. Proc Natl Acad Sci U S A 97(11):5836–5841. CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Seelig DM, Goodman PA, Skinner PJ (2016) Potential approaches for heterologous prion protein treatment of prion diseases. Prion 10(1):18–24. CrossRefPubMedGoogle Scholar
  52. 52.
    Lee CI, Yang Q, Perrier V, Baskakov IV (2007) The dominant-negative effect of the Q218K variant of the prion protein does not require protein X. Protein Sci 16(10):2166–2173. CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Zhou S, Shi D, Liu X, Liu H, Yao X (2016) Protective V127 prion variant prevents prion disease by interrupting the formation of dimer and fibril from molecular dynamics simulations. Sci Rep 6:21804. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Centro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, IA2, IISUniversidad de ZaragozaZaragozaSpain
  2. 2.CIC bioGUNEParque Tecnológico de BizkaiaDerioSpain
  3. 3.Servicio de Transgénesis, NucleusUniversidad de SalamancaSalamancaSpain
  4. 4.IBSALInstituto de Investigación Biomédica de SalamancaSalamancaSpain
  5. 5.Department of Food Safety and Veterinary Public HealthIstituto Superiore di SanitàRomeItaly
  6. 6.IKERBASQUEBasque Foundation for ScienceBilbaoSpain

Personalised recommendations