Neurotoxicity Research

, Volume 23, Issue 4, pp 301–314 | Cite as

Excitotoxicity Through NMDA Receptors Mediates Cerebellar Granule Neuron Apoptosis Induced by Prion Protein 90-231 Fragment

  • Stefano Thellung
  • Elena Gatta
  • Francesca Pellistri
  • Alessandro Corsaro
  • Valentina Villa
  • Massimo Vassalli
  • Mauro Robello
  • Tullio FlorioEmail author
Original Article


Prion diseases recognize, as a unique molecular trait, the misfolding of CNS-enriched prion protein (PrPC) into an aberrant isoform (PrPSc). In this work, we characterize the in vitro toxicity of amino-terminally truncated recombinant PrP fragment (amino acids 90-231, PrP90-231), on rat cerebellar granule neurons (CGN), focusing on glutamatergic receptor activation and Ca2+ homeostasis impairment. This recombinant fragment assumes a toxic conformation (PrP90-231TOX) after controlled thermal denaturation (1 h at 53 °C) acquiring structural characteristics identified in PrPSc (enrichment in β-structures, increased hydrophobicity, partial resistance to proteinase K, and aggregation in amyloid fibrils). By annexin-V binding assay, and evaluation of the percentage of fragmented and condensed nuclei, we show that treatment with PrP90-231TOX, used in pre-fibrillar aggregation state, induces CGN apoptosis. This effect was associated with a delayed, but sustained elevation of [Ca2+]i. Both CGN apoptosis and [Ca2+]i increase were not observed using PrP90-231 in PrPC-like conformation. PrP90-231TOX effects were significantly reduced in the presence of ionotropic glutamate receptor antagonists. In particular, CGN apoptosis and [Ca2+]i increase were largely reduced, although not fully abolished, by pre-treatment with the NMDA antagonists APV and memantine, while the AMPA antagonist CNQX produced a lower, although still significant, effect. In conclusion, we report that CGN apoptosis induced by PrP90-231TOX correlates with a sustained elevation of [Ca2+]i mediated by the activation of NMDA and AMPA receptors.


Cerebellar neurons Prion PrP90-231 Apoptosis Calcium NMDA receptor 



This study has been supported by grants from Italian Ministry of University and Research (MIUR-PRIN 2008, and Accordi di Programma FIRB, Project No. RBAP11HSZS, 2011).


  1. Aguzzi A, Polymenidou M (2004) Mammalian prion biology: one century of evolving concepts. Cell 116(2):313–327PubMedCrossRefGoogle Scholar
  2. Alberdi E, Sanchez-Gomez MV, Cavaliere F, Perez-Samartin A, Zugaza JL, Trullas R, Domercq M, Matute C (2010) Amyloid beta oligomers induce Ca2+ dysregulation and neuronal death through activation of ionotropic glutamate receptors. Cell Calcium 47(3):264–272PubMedCrossRefGoogle Scholar
  3. Bajetto A, Bonavia R, Barbero S, Piccioli P, Costa A, Florio T, Schettini G (1999) Glial and neuronal cells express functional chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor 1. J Neurochem 73(6):2348–2357PubMedCrossRefGoogle Scholar
  4. Baskakov IV, Legname G, Baldwin MA, Prusiner SB, Cohen FE (2002) Pathway complexity of prion protein assembly into amyloid. J Biol Chem 277(24):21140–21148PubMedCrossRefGoogle Scholar
  5. Bate C, Reid S, Williams A (2001) Killing of prion-damaged neurones by microglia. Neuroreport 12(11):2589–2594PubMedCrossRefGoogle Scholar
  6. Bate C, Salmona M, Diomede L, Williams A (2004) Squalestatin cures prion-infected neurons and protects against prion neurotoxicity. J Biol Chem 279(15):14983–14990PubMedCrossRefGoogle Scholar
  7. Bezprozvanny I, Mattson MP (2008) Neuronal calcium mishandling and the pathogenesis of Alzheimer’s disease. Trends Neurosci 31(9):454–463PubMedCrossRefGoogle Scholar
  8. Bi X, Gall CM, Zhou J, Lynch G (2002) Uptake and pathogenic effects of amyloid beta peptide 1–42 are enhanced by integrin antagonists and blocked by NMDA receptor antagonists. Neuroscience 112(4):827–840PubMedCrossRefGoogle Scholar
  9. Biasini E, Turnbaugh JA, Unterberger U, Harris DA (2012) Prion protein at the crossroads of physiology and disease. Trends Neurosci 35(2):92–103PubMedCrossRefGoogle Scholar
  10. Bounhar Y, Zhang Y, Goodyer CG, LeBlanc A (2001) Prion protein protects human neurons against Bax-mediated apoptosis. J Biol Chem 276(42):39145–39149PubMedCrossRefGoogle Scholar
  11. Bracalello A, Santopietro V, Vassalli M, Marletta G, Del Gaudio R, Bochicchio B, Pepe A (2011) Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide. Biomacromolecules 12(8):2957–2965PubMedCrossRefGoogle Scholar
  12. Brown DR, Schulz-Schaeffer WJ, Schmidt B, Kretzschmar HA (1997) Prion protein-deficient cells show altered response to oxidative stress due to decreased SOD-1 activity. Exp Neurol 146(1):104–112PubMedCrossRefGoogle Scholar
  13. Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, Stefani M (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416(6880):507–511PubMedCrossRefGoogle Scholar
  14. Bucciantini M, Calloni G, Chiti F, Formigli L, Nosi D, Dobson CM, Stefani M (2004) Prefibrillar amyloid protein aggregates share common features of cytotoxicity. J Biol Chem 279(30):31374–31382PubMedCrossRefGoogle Scholar
  15. Castilla J, Hetz C, Soto C (2004) Molecular mechanisms of neurotoxicity of pathological prion protein. Curr Mol Med 4(4):397–403PubMedCrossRefGoogle Scholar
  16. Caudle WM, Zhang J (2009) Glutamate, excitotoxicity, and programmed cell death in Parkinson disease. Exp Neurol 220(2):230–233PubMedCrossRefGoogle Scholar
  17. Chabry J, Ratsimanohatra C, Sponne I, Elena PP, Vincent JP, Pillot T (2003) In vivo and in vitro neurotoxicity of the human prion protein (PrP) fragment P118–135 independently of PrP expression. J Neurosci 23(2):462–469PubMedGoogle Scholar
  18. Chen SG, Teplow DB, Parchi P, Teller JK, Gambetti P, Autilio-Gambetti L (1995) Truncated forms of the human prion protein in normal brain and in prion diseases. J Biol Chem 270(32):19173–19180PubMedCrossRefGoogle Scholar
  19. Chiesa R, Harris DA (2001) Prion diseases: what is the neurotoxic molecule? Neurobiol Dis 8(5):743–763PubMedCrossRefGoogle Scholar
  20. Chiovitti K, Corsaro A, Thellung S, Villa V, Paludi D, D’Arrigo C, Russo C, Perico A, Ianieri A, Di Cola D, Vergara A, Aceto A, Florio T (2007) Intracellular accumulation of a mild-denatured monomer of the human PrP fragment 90–231, as possible mechanism of its neurotoxic effects. J Neurochem 103(6):2597–2609PubMedGoogle Scholar
  21. Ciccotosto GD, Cappai R, White AR (2008) Neurotoxicity of prion peptides on cultured cerebellar neurons. Methods Mol Biol 459:83–96PubMedCrossRefGoogle Scholar
  22. 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):295–297PubMedCrossRefGoogle Scholar
  23. Corsaro A, Thellung S, Russo C, Villa V, Arena S, D’Adamo MC, Paludi D, Rossi Principe D, Damonte G, Benatti U, Aceto A, Tagliavini F, Schettini G, Florio T (2002) Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein. Neurochem Int 41(1):55–63PubMedCrossRefGoogle Scholar
  24. Corsaro A, Paludi D, Villa V, D’Arrigo C, Chiovitti K, Thellung S, Russo C, Di Cola D, Ballerini P, Patrone E, Schettini G, Aceto A, Florio T (2006) Conformation dependent pro-apoptotic activity of the recombinant human prion protein fragment 90–231. Int J Immunopathol Pharmacol 19(2):339–356PubMedGoogle Scholar
  25. Corsaro A, Thellung S, Chiovitti K, Villa V, Simi A, Raggi F, Paludi D, Russo C, Aceto A, Florio T (2009) Dual modulation of ERK1/2 and p38 MAP kinase activities induced by minocycline reverses the neurotoxic effects of the prion protein fragment 90–231. Neurotox Res 15(2):138–154PubMedCrossRefGoogle Scholar
  26. Corsaro A, Thellung S, Bucciarelli T, Scotti L, Chiovitti K, Villa V, D’Arrigo C, Aceto A, Florio T (2011) High hydrophobic amino acid exposure is responsible of the neurotoxic effects induced by E200 K or D202 N disease-related mutations of the human prion protein. Int J Biochem Cell Biol 43(3):372–382PubMedCrossRefGoogle Scholar
  27. Corsaro A, Thellung S, Villa V, Nizzari M, Aceto A, Florio T (2012) Recombinant human prion protein fragment 90–231, a useful model to study prion neurotoxicity. OMICS 16(1–2):50–59PubMedCrossRefGoogle Scholar
  28. Demuro A, Mina E, Kayed R, Milton SC, Parker I, Glabe CG (2005) Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. J Biol Chem 280(17):17294–17300PubMedCrossRefGoogle Scholar
  29. Faucheux BA, Privat N, Brandel JP, Sazdovitch V, Laplanche JL, Maurage CA, Hauw JJ, Haik S (2009) Loss of cerebellar granule neurons is associated with punctate but not with large focal deposits of prion protein in Creutzfeldt–Jakob disease. J Neuropathol Exp Neurol 68(8):892–901PubMedCrossRefGoogle Scholar
  30. Florio T, Grimaldi M, Scorziello A, Salmona M, Bugiani O, Tagliavini F, Forloni G, Schettini G (1996) Intracellular calcium rise through L-type calcium channels, as molecular mechanism for prion protein fragment 106–126-induced astroglial proliferation. Biochem Biophys Res Commun 228(2):397–405PubMedCrossRefGoogle Scholar
  31. Florio T, Thellung S, Amico C, Robello M, Salmona M, Bugiani O, Tagliavini F, Forloni G, Schettini G (1998) Prion protein fragment 106–126 induces apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in the GH3 cell line. J Neurosci Res 54(3):341–352PubMedCrossRefGoogle Scholar
  32. Florio T, Paludi D, Villa V, Principe DR, Corsaro A, Millo E, Damonte G, D’Arrigo C, Russo C, Schettini G, Aceto A (2003) Contribution of two conserved glycine residues to fibrillogenesis of the 106–126 prion protein fragment. Evidence that a soluble variant of the 106–126 peptide is neurotoxic. J Neurochem 85(1):62–72PubMedCrossRefGoogle Scholar
  33. Forloni G, Angeretti N, Chiesa R, Monzani E, Salmona M, Bugiani O, Tagliavini F (1993) Neurotoxicity of a prion protein fragment. Nature 362(6420):543–546PubMedCrossRefGoogle Scholar
  34. Gatta E, Cupello A, Pellistri F, Robello M (2009) GABA(A) receptors of cerebellar granule cells in culture: explanation of overall insensitivity to ethanol. Neuroscience 162(4):1187–1191PubMedCrossRefGoogle Scholar
  35. Giese A, Brown DR, Groschup MH, Feldmann C, Haist I, Kretzschmar HA (1998) Role of microglia in neuronal cell death in prion disease. Brain Pathol (Zurich, Switzerland) 8(3):449–457CrossRefGoogle Scholar
  36. Hafiz FB, Brown DR (2000) A model for the mechanism of astrogliosis in prion disease. Mol Cell Neurosci 16(3):221–232PubMedCrossRefGoogle Scholar
  37. James TL, Liu H, Ulyanov NB, Farr-Jones S, Zhang H, Donne DG, Kaneko K, Groth D, Mehlhorn I, Prusiner SB, Cohen FE (1997) Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform. Proc Natl Acad Sci USA 94(19):10086–10091PubMedCrossRefGoogle Scholar
  38. Kelly BL, Ferreira A (2006) Beta-amyloid-induced dynamin 1 degradation is mediated by N-methyl-d-aspartate receptors in hippocampal neurons. J Biol Chem 281(38):28079–28089PubMedCrossRefGoogle Scholar
  39. Kourie JI, Culverson A (2000) Prion peptide fragment PrP[106-126] forms distinct cation channel types. J Neurosci Res 62(1):120–133PubMedCrossRefGoogle Scholar
  40. Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB (2004) Synthetic mammalian prions. Science (New York, NY) 305(5684):673–676CrossRefGoogle Scholar
  41. Lin MC, Mirzabekov T, Kagan BL (1997) Channel formation by a neurotoxic prion protein fragment. J Biol Chem 272(1):44–47PubMedCrossRefGoogle Scholar
  42. 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 (New York, NY) 302(5646):871–874CrossRefGoogle Scholar
  43. Marella M, Chabry J (2004) Neurons and astrocytes respond to prion infection by inducing microglia recruitment. J Neurosci 24(3):620–627PubMedCrossRefGoogle Scholar
  44. Mattson MP, Cheng B, Davis D, Bryant K, Lieberburg I, Rydel RE (1992) Beta-amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J Neurosci 12(2):376–389PubMedGoogle Scholar
  45. 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):261–267PubMedCrossRefGoogle Scholar
  46. Novitskaya V, Bocharova OV, Bronstein I, Baskakov IV (2006) Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons. J Biol Chem 281(19):13828–13836PubMedCrossRefGoogle Scholar
  47. Paulis D, Maras B, Schinina ME, di Francesco L, Principe S, Galeno R, Abdel-Haq H, Cardone F, Florio T, Pocchiari M, Mazzanti M (2011) The pathological prion protein forms ionic conductance in lipid bilayer. Neurochem Int 59(2):168–174PubMedCrossRefGoogle Scholar
  48. Peggion C, Bertoli A, Sorgato MC (2011) Possible role for Ca2+ in the pathophysiology of the prion protein? Biofactors (Oxford, England) 37(3):241–249CrossRefGoogle Scholar
  49. Pellistri F, Bucciantini M, Relini A, Nosi D, Gliozzi A, Robello M, Stefani M (2008) Nonspecific interaction of prefibrillar amyloid aggregates with glutamatergic receptors results in Ca2+ increase in primary neuronal cells. J Biol Chem 283(44):29950–29960PubMedCrossRefGoogle Scholar
  50. Post K, Brown DR, Groschup M, Kretzschmar HA, Riesner D (2000) Neurotoxicity but not infectivity of prion proteins can be induced reversibly in vitro. Arch Virol 16:265–273Google Scholar
  51. Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95(23):13363–13383PubMedCrossRefGoogle Scholar
  52. Rambold AS, Muller V, Ron U, Ben-Tal N, Winklhofer KF, Tatzelt J (2008) Stress-protective signalling of prion protein is corrupted by scrapie prions. EMBO J 27(14):1974–1984PubMedCrossRefGoogle Scholar
  53. Sakaguchi S, Katamine S, Nishida N, Moriuchi R, Shigematsu K, Sugimoto T, Nakatani A, Kataoka Y, Houtani T, Shirabe S, Okada H, Hasegawa S, Miyamoto T, Noda T (1996) Loss of cerebellar Purkinje cells in aged mice homozygous for a disrupted PrP gene. Nature 380(6574):528–531PubMedCrossRefGoogle Scholar
  54. Salmona M, Forloni G, Diomede L, Algeri M, De Gioia L, Angeretti N, Giaccone G, Tagliavini F, Bugiani O (1997) A neurotoxic and gliotrophic fragment of the prion protein increases plasma membrane microviscosity. Neurobiol Dis 4(1):47–57PubMedCrossRefGoogle Scholar
  55. Salmona M, Morbin M, Massignan T, Colombo L, Mazzoleni G, Capobianco R, Diomede L, Thaler F, Mollica L, Musco G, Kourie JJ, Bugiani O, Sharma D, Inouye H, Kirschner DA, Forloni G, Tagliavini F (2003) Structural properties of Gerstmann–Straussler–Scheinker disease amyloid protein. J Biol Chem 278(48):48146–48153PubMedCrossRefGoogle Scholar
  56. Sandberg MK, Al-Doujaily H, Sharps B, Clarke AR, Collinge J (2011) Prion propagation and toxicity in vivo occur in two distinct mechanistic phases. Nature 470(7335):540–542PubMedCrossRefGoogle Scholar
  57. Scallet AC, Ye X (1997) Excitotoxic mechanisms of neurodegeneration in transmissible spongiform encephalopathies. Ann N Y Acad Sci 825:194–205PubMedCrossRefGoogle Scholar
  58. Scorziello A, Meucci O, Florio T, Fattore M, Forloni G, Salmona M, Schettini G (1996) Beta 25–35 alters calcium homeostasis and induces neurotoxicity in cerebellar granule cells. J Neurochem 66(5):1995–2003PubMedCrossRefGoogle Scholar
  59. Simoneau S, Rezaei H, Sales N, Kaiser-Schulz G, Lefebvre-Roque M, Vidal C, Fournier JG, Comte J, Wopfner F, Grosclaude J, Schatzl H, Lasmezas CI (2007) In vitro and in vivo neurotoxicity of prion protein oligomers. PLoS Pathog 3(8):e125PubMedCrossRefGoogle Scholar
  60. Song MS, Rauw G, Baker GB, Kar S (2008) Memantine protects rat cortical cultured neurons against beta-amyloid-induced toxicity by attenuating tau phosphorylation. Eur J Neurosci 28(10):1989–2002PubMedCrossRefGoogle Scholar
  61. Swietnicki W, Petersen R, Gambetti P, Surewicz WK (1997) pH-dependent stability and conformation of the recombinant human prion protein PrP(90–231). J Biol Chem 272(44):27517–27520PubMedCrossRefGoogle Scholar
  62. Texido L, Martin-Satue M, Alberdi E, Solsona C, Matute C (2011) Amyloid beta peptide oligomers directly activate NMDA receptors. Cell Calcium 49(3):184–190PubMedCrossRefGoogle Scholar
  63. Thellung S, Florio T, Villa V, Corsaro A, Arena S, Amico C, Robello M, Salmona M, Forloni G, Bugiani O, Tagliavini F, Schettini G (2000) Apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in rat cerebellar granule cells treated with the prion protein fragment 106–126. Neurobiol Dis 7(4):299–309PubMedCrossRefGoogle Scholar
  64. Thellung S, Villa V, Corsaro A, Arena S, Millo E, Damonte G, Benatti U, Tagliavini F, Florio T, Schettini G (2002) p38 MAP kinase mediates the cell death induced by PrP106-126 in the SH-SY5Y neuroblastoma cells. Neurobiol Dis 9(1):69–81PubMedCrossRefGoogle Scholar
  65. Thellung S, Villa V, Corsaro A, Pellistri F, Venezia V, Russo C, Aceto A, Robello M, Florio T (2007) ERK1/2 and p38 MAP kinases control prion protein fragment 90–231-induced astrocyte proliferation and microglia activation. Glia 55(14):1469–1485PubMedCrossRefGoogle Scholar
  66. Thellung S, Corsaro A, Villa V, Simi A, Vella S, Pagano A, Florio T (2011) Human PrP90-231-induced cell death is associated with intracellular accumulation of insoluble and protease-resistant macroaggregates and lysosomal dysfunction. Cell Death Dis 2:e138PubMedCrossRefGoogle Scholar
  67. Tremblay R, Chakravarthy B, Hewitt K, Tauskela J, Morley P, Atkinson T, Durkin JP (2000) Transient NMDA receptor inactivation provides long-term protection to cultured cortical neurons from a variety of death signals. J Neurosci 20(19):7183–7192PubMedGoogle Scholar
  68. Van Den Bosch L, Van Damme P, Bogaert E, Robberecht W (2006) The role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis. Biochim Biophys Acta 1762(11–12):1068–1082Google Scholar
  69. Villa V, Corsaro A, Thellung S, Paludi D, Chiovitti K, Venezia V, Nizzari M, Russo C, Schettini G, Aceto A, Florio T (2006) Characterization of the proapoptotic intracellular mechanisms induced by a toxic conformer of the recombinant human prion protein fragment 90–231. Ann N Y Acad Sci 1090:276–291PubMedCrossRefGoogle Scholar
  70. Villa V, Tonelli M, Thellung S, Corsaro A, Tasso B, Novelli F, Canu C, Pino A, Chiovitti K, Paludi D, Russo C, Sparatore A, Aceto A, Boido V, Sparatore F, Florio T (2011) Efficacy of novel acridine derivatives in the inhibition of hPrP90-231 prion protein fragment toxicity. Neurotox Res 19(4):556–574PubMedCrossRefGoogle Scholar
  71. You H, Tsutsui S, Hameed S, Kannanayakal TJ, Chen L, Xia P, Engbers JD, Lipton SA, Stys PK, Zamponi GW (2012) Abeta neurotoxicity depends on interactions between copper ions, prion protein, and N-methyl-d-aspartate receptors. Proc Natl Acad Sci USA 109(5):1737–1742PubMedCrossRefGoogle Scholar
  72. Zou WQ, Capellari S, Parchi P, Sy MS, Gambetti P, Chen SG (2003) Identification of novel proteinase K-resistant C-terminal fragments of PrP in Creutzfeldt–Jakob disease. J Biol Chem 278(42):40429–40436PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Stefano Thellung
    • 1
  • Elena Gatta
    • 2
  • Francesca Pellistri
    • 2
  • Alessandro Corsaro
    • 1
  • Valentina Villa
    • 1
  • Massimo Vassalli
    • 3
  • Mauro Robello
    • 2
  • Tullio Florio
    • 1
    Email author
  1. 1.Department of Internal Medicine, Section of Pharmacology and Centre of Excellence for Biomedical Research (CEBR) School of MedicineUniversity of GenovaGenoaItaly
  2. 2.Department of PhysicsUniversity of GenovaGenoaItaly
  3. 3.Institute of Biophysics (IBF)National Council of Research (CNR)GenoaItaly

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