Prions Strongly Reduce NMDA Receptor S-Nitrosylation Levels at Pre-symptomatic and Terminal Stages of Prion Diseases

  • Elisa Meneghetti
  • Lisa Gasperini
  • Tommaso Virgilio
  • Fabio Moda
  • Fabrizio Tagliavini
  • Federico Benetti
  • Giuseppe LegnameEmail author


Prion diseases are fatal neurodegenerative disorders characterized by the cellular prion protein (PrPC) conversion into a misfolded and infectious isoform termed prion or PrPSc. The neuropathological mechanism underlying prion toxicity is still unclear, and the debate on prion protein gain- or loss-of-function is still open. PrPC participates to a plethora of physiological mechanisms. For instance, PrPC and copper cooperatively modulate N-methyl-D-aspartate receptor (NMDAR) activity by mediating S-nitrosylation, an inhibitory post-translational modification, hence protecting neurons from excitotoxicity. Here, NMDAR S-nitrosylation levels were biochemically investigated at pre- and post-symptomatic stages of mice intracerebrally inoculated with RML, 139A, and ME7 prion strains. Neuropathological aspects of prion disease were studied by histological analysis and proteinase K digestion. We report that hippocampal NMDAR S-nitrosylation is greatly reduced in all three prion strain infections in both pre-symptomatic and terminal stages of mouse disease. Indeed, we show that NMDAR S-nitrosylation dysregulation affecting prion-inoculated animals precedes the appearance of clinical signs of disease and visible neuropathological changes, such as PrPSc accumulation and deposition. The pre-symptomatic reduction of NMDAR S-nitrosylation in prion-infected mice may be a possible cause of neuronal death in prion pathology, and it might contribute to the pathology progression opening new therapeutic strategies against prion disorders.


Prions NMDA receptor S-Nitrosylation Nitric oxide Copper Excitotoxicity 


Funding Information

This work was supported/partially supported by the Italian Ministry of Health (GR-2013-02355724 and RC) to FM, Italian Ministry of Health to FT, SISSA Intramural Funding to GL, and SISSA grant to FB (Young SISSA Scientists Research Projects 2011–2012 Scheme). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with Ethical Standards

Animal procedures were performed in accordance with European regulations (Directive 2010/63/EU) and with Italian Legislative Decree 26/2014 and were approved by the local authority veterinary service and by the Italian Ministry of Health, Directorate General for Animal Health. All efforts were made to minimize animal suffering and to reduce the number of animals used. Animal facility is licensed and inspected by the Italian Ministry of Health.

Conflict of Interests

The authors declare that they have no competing interests.

Supplementary material

12035_2019_1505_MOESM1_ESM.pdf (232 kb)
ESM 1 (PDF 231 kb)


  1. 1.
    Prusiner SB (2001) Neurodegenerative diseases and prions. N Engl J Med 344(20):1516–1526. CrossRefPubMedGoogle Scholar
  2. 2.
    Aguzzi A, Heikenwalder M, Miele G (2004) Progress and problems in the biology, diagnostics, and therapeutics of prion diseases. J Clin Invest 114(2):153–160. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216(4542):136–144CrossRefGoogle Scholar
  4. 4.
    Oesch B, Westaway D, Walchli M, McKinley MP, Kent SB, Aebersold R, Barry RA, Tempst P, Teplow DB, Hood LE, et al. (1985) A cellular gene encodes scrapie PrP 27-30 protein. Cell 40 (4):735–746.Google Scholar
  5. 5.
    Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS (1991) Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry 30(31):7672–7680CrossRefGoogle Scholar
  6. 6.
    Gasset M, Baldwin MA, Fletterick RJ, Prusiner SB (1993) Perturbation of the secondary structure of the scrapie prion protein under conditions that alter infectivity. Proc Natl Acad Sci U S A 90(1):1–5CrossRefGoogle Scholar
  7. 7.
    Bieschke J, Weber P, Sarafoff N, Beekes M, Giese A, Kretzschmar H (2004) Autocatalytic self-propagation of misfolded prion protein. Proc Natl Acad Sci U S A 101(33):12207–12211. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Soto C (2003) Unfolding the role of protein misfolding in neurodegenerative diseases. Nat Rev Neurosci 4(1):49–60. CrossRefPubMedGoogle Scholar
  9. 9.
    Soto C, Satani N (2011) The intricate mechanisms of neurodegeneration in prion diseases. Trends Mol Med 17(1):14–24. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Budka H (2003) Neuropathology of prion diseases. Br Med Bull 66:121–130CrossRefGoogle Scholar
  11. 11.
    Westergard L, Christensen HM, Harris DA (2007) The cellular prion protein (PrP(C)): its physiological function and role in disease. Biochim Biophys Acta 1772(6):629–644. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Halliday M, Radford H, Mallucci GR (2014) Prions: generation and spread versus neurotoxicity. J Biol Chem 289(29):19862–19868. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Brown DR (2001) Prion and prejudice: normal protein and the synapse. Trends Neurosci 24(2):85–90CrossRefGoogle Scholar
  14. 14.
    Aguzzi A, Baumann F, Bremer J (2008) The prion’s elusive reason for being. Annu Rev Neurosci 31:439–477. CrossRefPubMedGoogle Scholar
  15. 15.
    Caughey B, Baron GS (2006) Prions and their partners in crime. Nature 443(7113):803–810. CrossRefPubMedGoogle Scholar
  16. 16.
    Collinge J, Whittington MA, Sidle KC, Smith CJ, Palmer MS, Clarke AR, Jefferys JG (1994) Prion protein is necessary for normal synaptic function. Nature 370(6487):295–297. CrossRefPubMedGoogle Scholar
  17. 17.
    Manson JC, Hope J, Clarke AR, Johnston A, Black C, MacLeod N (1995) PrP gene dosage and long term potentiation. Neurodegeneration 4(1):113–114CrossRefGoogle Scholar
  18. 18.
    Colling SB, Collinge J, Jefferys JG (1996) Hippocampal slices from prion protein null mice: disrupted Ca(2+)-activated K+ currents. Neurosci Lett 209(1):49–52CrossRefGoogle Scholar
  19. 19.
    Carleton A, Tremblay P, Vincent JD, Lledo PM (2001) Dose-dependent, prion protein (PrP)-mediated facilitation of excitatory synaptic transmission in the mouse hippocampus. Pflugers Arch 442(2):223–229CrossRefGoogle Scholar
  20. 20.
    Herms JW, Tings T, Dunker S, Kretzschmar HA (2001) Prion protein affects Ca2+-activated K+ currents in cerebellar Purkinje cells. Neurobiol Dis 8(2):324–330. CrossRefPubMedGoogle Scholar
  21. 21.
    Mallucci GR, Ratte S, Asante EA, Linehan J, Gowland I, Jefferys JG, Collinge J (2002) Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration. EMBO J 21(3):202–210. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Maglio LE, Perez MF, Martins VR, Brentani RR, Ramirez OA (2004) Hippocampal synaptic plasticity in mice devoid of cellular prion protein. Brain Res Mol Brain Res 131(1–2):58–64. CrossRefPubMedGoogle Scholar
  23. 23.
    Fuhrmann M, Bittner T, Mitteregger G, Haider N, Moosmang S, Kretzschmar H, Herms J (2006) Loss of the cellular prion protein affects the Ca2+ homeostasis in hippocampal CA1 neurons. J Neurochem 98(6):1876–1885. CrossRefPubMedGoogle Scholar
  24. 24.
    Prestori F, Rossi P, Bearzatto B, Laine J, Necchi D, Diwakar S, Schiffmann SN, Axelrad H, D'Angelo E (2008) Altered neuron excitability and synaptic plasticity in the cerebellar granular layer of juvenile prion protein knock-out mice with impaired motor control. J Neurosci 28 (28):7091–7103. doi:
  25. 25.
    Lazzari C, Peggion C, Stella R, Massimino ML, Lim D, Bertoli A, Sorgato MC (2011) Cellular prion protein is implicated in the regulation of local Ca2+ movements in cerebellar granule neurons. J Neurochem 116(5):881–890. CrossRefPubMedGoogle Scholar
  26. 26.
    Khosravani H, Zhang Y, Tsutsui S, Hameed S, Altier C, Hamid J, Chen L, Villemaire M et al (2008) Prion protein attenuates excitotoxicity by inhibiting NMDA receptors. J Cell Biol 181(3):551–565. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    You H, Tsutsui S, Hameed S, Kannanayakal TJ, Chen L, Xia P, Engbers JD, Lipton SA et al (2012) Abeta neurotoxicity depends on interactions between copper ions, prion protein, and N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A 109(5):1737–1742. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Spudich A, Frigg R, Kilic E, Kilic U, Oesch B, Raeber A, Bassetti CL, Hermann DM (2005) Aggravation of ischemic brain injury by prion protein deficiency: role of ERK-1/-2 and STAT-1. Neurobiol Dis 20 (2):442–449. doi:10.1016/j.nbd.2005.04.002Google Scholar
  29. 29.
    Rangel A, Burgaya F, Gavin R, Soriano E, Aguzzi A, Del Rio JA (2007) Enhanced susceptibility of Prnp-deficient mice to kainate-induced seizures, neuronal apoptosis, and death: role of AMPA/kainate receptors. J Neurosci Res 85(12):2741–2755. CrossRefPubMedGoogle Scholar
  30. 30.
    Gasperini L, Meneghetti E, Pastore B, Benetti F, Legname G (2015) Prion protein and copper cooperatively protect neurons by modulating NMDA receptor through S-nitrosylation. Antioxid Redox Signal 22(9):772–784. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Choi YB, Tenneti L, Le DA, Ortiz J, Bai G, Chen HS, Lipton SA (2000) Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation. Nat Neurosci 3(1):15–21. CrossRefPubMedGoogle Scholar
  32. 32.
    Lipton SA, Choi YB, Takahashi H, Zhang D, Li W, Godzik A, Bankston LA (2002) Cysteine regulation of protein function—as exemplified by NMDA-receptor modulation. Trends Neurosci 25(9):474–480CrossRefGoogle Scholar
  33. 33.
    Nakamura T, Lipton SA (2011) Redox modulation by S-nitrosylation contributes to protein misfolding, mitochondrial dynamics, and neuronal synaptic damage in neurodegenerative diseases. Cell Death Differ 18(9):1478–1486. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Kone BC, Kuncewicz T, Zhang W, Yu ZY (2003) Protein interactions with nitric oxide synthases: controlling the right time, the right place, and the right amount of nitric oxide. American Journal of Physiology Renal Physiology 285(2):F178–F190. CrossRefPubMedGoogle Scholar
  35. 35.
    Forstermann U, Sessa WC (2012) Nitric oxide synthases: regulation and function. European Heart Journal 33(7):829–837, 837a-837d. CrossRefPubMedGoogle Scholar
  36. 36.
    Delint-Ramirez I, Fernandez E, Bayes A, Kicsi E, Komiyama NH, Grant SG (2010) In vivo composition of NMDA receptor signaling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93. J Neurosci 30(24):8162–8170. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Naslavsky N, Stein R, Yanai A, Friedlander G, Taraboulos A (1997) Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform. J Biol Chem 272(10):6324–6331CrossRefGoogle Scholar
  38. 38.
    Bonomo RP, Pappalardo G, Rizzarelli E, Tabbi G, Vagliasindi LI (2008) Studies of nitric oxide interaction with mono- and dinuclear copper(II) complexes of prion protein bis-octarepeat fragments. Dalton Trans (29):3805–3816.
  39. 39.
    Stockel J, Safar J, Wallace AC, Cohen FE, Prusiner SB (1998) Prion protein selectively binds copper(II) ions. Biochemistry 37(20):7185–7193.[pii]Google Scholar
  40. 40.
    Jackson GS, Murray I, Hosszu LL, Gibbs N, Waltho JP, Clarke AR, Collinge J (2001) Location and properties of metal-binding sites on the human prion protein. Proc Natl Acad Sci USA 98(15):8531–8535.[pii]Google Scholar
  41. 41.
    Singh N, Das D, Singh A, Mohan ML (2010) Prion protein and metal interaction: physiological and pathological implications. Curr Issues Mol Biol 12(2):99–107PubMedGoogle Scholar
  42. 42.
    Liu L, Jiang D, McDonald A, Hao Y, Millhauser GL, Zhou F (2011) Copper redox cycling in the prion protein depends critically on binding mode. J Am Chem Soc 133(31):12229–12237. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Benvegnu S, Poggiolini I, Legname G (2010) Neurodevelopmental expression and localization of the cellular prion protein in the central nervous system of the mouse. J Comp Neurol 518(11):1879–1891. CrossRefPubMedGoogle Scholar
  44. 44.
    Marmiroli P, Cavaletti G (2012) The glutamatergic neurotransmission in the central nervous system. Curr Med Chem 19(9):1269–1276CrossRefGoogle Scholar
  45. 45.
    Prusiner SB, Groth DF, Bolton DC, Kent SB, Hood LE (1984) Purification and structural studies of a major scrapie prion protein. Cell 38(1):127–134CrossRefGoogle Scholar
  46. 46.
    Chiesa R, Harris DA (2001) Prion diseases: what is the neurotoxic molecule? Neurobiol Dis 8(5):743–763. CrossRefGoogle Scholar
  47. 47.
    Bueler H, Fischer M, Lang Y, Bluethmann H, Lipp HP, DeArmond SJ, Prusiner SB, Aguet M et al (1992) Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356(6370):577–582. CrossRefPubMedGoogle Scholar
  48. 48.
    Manson JC, Clarke AR, Hooper ML, Aitchison L, McConnell I, Hope J (1994) 129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal. Mol Neurobiol 8(2–3):121–127. CrossRefPubMedGoogle Scholar
  49. 49.
    Steele AD, Lindquist S, Aguzzi A (2007) The prion protein knockout mouse: a phenotype under challenge. Prion 1(2):83–93CrossRefGoogle Scholar
  50. 50.
    Chiesa R, Harris DA (2009) Fishing for prion protein function. PLoS Biol 7(3):e75. CrossRefPubMedGoogle Scholar
  51. 51.
    Linden R, Martins VR, Prado MA, Cammarota M, Izquierdo I, Brentani RR (2008) Physiology of the prion protein. Physiol Rev 88(2):673–728. CrossRefPubMedGoogle Scholar
  52. 52.
    Lewerenz J, Maher P (2015) Chronic glutamate toxicity in neurodegenerative diseases—what is the evidence? Front Neurosci 9:469. CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Telling GC, Parchi P, DeArmond SJ, Cortelli P, Montagna P, Gabizon R, Mastrianni J, Lugaresi E et al (1996) Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity. Science 274(5295):2079–2082CrossRefGoogle Scholar
  54. 54.
    Solforosi L, Milani M, Mancini N, Clementi M, Burioni R (2013) A closer look at prion strains: characterization and important implications. Prion 7(2):99–108. CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Jendroska K, Heinzel FP, Torchia M, Stowring L, Kretzschmar HA, Kon A, Stern A, Prusiner SB et al (1991) Proteinase-resistant prion protein accumulation in Syrian hamster brain correlates with regional pathology and scrapie infectivity. Neurology 41(9):1482–1490CrossRefGoogle Scholar
  56. 56.
    Williams A, Lucassen PJ, Ritchie D, Bruce M (1997) PrP deposition, microglial activation, and neuronal apoptosis in murine scrapie. Exp Neurol 144(2):433–438. CrossRefGoogle Scholar
  57. 57.
    Jeffrey M, Martin S, Barr J, Chong A, Fraser JR (2001) Onset of accumulation of PrPres in murine ME7 scrapie in relation to pathological and PrP immunohistochemical changes. J Comp Pathol 124(1):20–28. CrossRefPubMedGoogle Scholar
  58. 58.
    Hill AF, Collinge J (2003) Subclinical prion infection in humans and animals. Br Med Bull 66(1):161–170. CrossRefPubMedGoogle Scholar
  59. 59.
    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(7):1339–1347CrossRefGoogle Scholar
  60. 60.
    Sonati T, Reimann RR, Falsig J, Baral PK, O'Connor T, Hornemann S, Yaganoglu S, Li B et al (2013) The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein. Nature 501(7465):102–106. CrossRefPubMedGoogle Scholar
  61. 61.
    Resenberger UK, Harmeier A, Woerner AC, Goodman JL, Muller V, Krishnan R, Vabulas RM, Kretzschmar HA et al (2011) The cellular prion protein mediates neurotoxic signalling of beta-sheet-rich conformers independent of prion replication. EMBO J 30(10):2057–2070. CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Muller WE, Ushijima H, Schroder HC, Forrest JM, Schatton WF, Rytik PG, Heffner-Lauc M (1993) Cytoprotective effect of NMDA receptor antagonists on prion protein (PrionSc)-induced toxicity in rat cortical cell cultures. Eur J Pharmacol 246(3):261–267CrossRefGoogle Scholar
  63. 63.
    Singh N, Singh A, Das D, Mohan ML (2010) Redox control of prion and disease pathogenesis. Antioxid Redox Signal 12(11):1271–1294. CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Petersen RB, Siedlak SL, Lee H-g, Kim Y-S, Nunomura A, Tagliavini F, Ghetti B, Cras P et al (2005) Redox metals and oxidative abnormalities in human prion diseases. Acta Neuropathol 110(3):232–238. CrossRefPubMedGoogle Scholar
  65. 65.
    Chen LN, Sun J, Yang XD, Xiao K, Lv Y, Zhang BY, Zhou W, Chen C et al (2016) The brain NO levels and NOS activities ascended in the early and middle stages and descended in the terminal stage in scrapie-infected animal models. Mol Neurobiol 54:1786–1796. CrossRefPubMedGoogle Scholar
  66. 66.
    Ju WK, Park KJ, Choi EK, Kim J, Carp RI, Wisniewski HM, Kim YS (1998) Expression of inducible nitric oxide synthase in the brains of scrapie-infected mice. J Neurovirol 4(4):445–450CrossRefGoogle Scholar
  67. 67.
    Cashman NR, Caughey B (2004) Prion diseases—close to effective therapy? Nat Rev Drug Discov 3(10):874–884. CrossRefPubMedGoogle Scholar
  68. 68.
    Rigter A, Langeveld JP, van Zijderveld FG, Bossers A (2010) Prion protein self-interactions: a gateway to novel therapeutic strategies? Vaccine 28(49):7810–7823. CrossRefPubMedGoogle Scholar
  69. 69.
    Aguzzi A, O'Connor T (2010) Protein aggregation diseases: pathogenicity and therapeutic perspectives. Nat Rev Drug Discov 9(3):237–248. CrossRefPubMedGoogle Scholar
  70. 70.
    Mallucci G, Dickinson A, Linehan J, Klohn PC, Brandner S, Collinge J (2003) Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science 302(5646):871–874.[pii]Google Scholar
  71. 71.
    Pfeifer A, Eigenbrod S, Al-Khadra S, Hofmann A, Mitteregger G, Moser M, Bertsch U, Kretzschmar H (2006) Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice. J Clin Invest 116(12):3204–3210. CrossRefPubMedPubMedCentralGoogle Scholar
  72. 72.
    Lipton SA (2006) Paradigm shift in neuroprotection by NMDA receptor blockade: memantine and beyond. Nat Rev Drug Discov 5(2):160–170CrossRefGoogle Scholar
  73. 73.
    Lipton SA (2007) Pathologically activated therapeutics for neuroprotection. Nat Rev Neurosci 8(10):803–808. CrossRefPubMedGoogle Scholar
  74. 74.
    Okamoto S, Pouladi MA, Talantova M, Yao D, Xia P, Ehrnhoefer DE, Zaidi R, Clemente A et al (2009) Balance between synaptic versus extrasynaptic NMDA receptor activity influences inclusions and neurotoxicity of mutant huntingtin. Nat Med 15(12):1407–1413. CrossRefPubMedPubMedCentralGoogle Scholar
  75. 75.
    Chen HS, Pellegrini JW, Aggarwal SK, Lei SZ, Warach S, Jensen FE, Lipton SA (1992) Open-channel block of N-methyl-D-aspartate (NMDA) responses by memantine: therapeutic advantage against NMDA receptor-mediated neurotoxicity. J Neurosci 12(11):4427–4436CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Elisa Meneghetti
    • 1
  • Lisa Gasperini
    • 1
  • Tommaso Virgilio
    • 2
  • Fabio Moda
    • 2
  • Fabrizio Tagliavini
    • 3
  • Federico Benetti
    • 1
    • 4
  • Giuseppe Legname
    • 1
    • 5
    Email author
  1. 1.Laboratory of Prion Biology, Department of NeuroscienceScuola Internazionale Superiore di Studi Avanzati (SISSA)TriesteItaly
  2. 2.Fondazione IRCCS Istituto Neurologico Carlo Besta, Unit of Neuropathology and Neurology-5MilanoItaly
  3. 3.Fondazione IRCCS Istituto Neurologico Carlo Besta, Scientific DirectorateMilanoItaly
  4. 4.ECSIN-European Center for the Sustainable Impact of NanotechnologyECAMRICERT SRLPadovaItaly
  5. 5.ELETTRA LaboratoryTriesteItaly

Personalised recommendations