title: Excitotoxicity through NMDA receptors mediates cerebellar granule neuron apoptosis induced by prion protein 90-231 fragment.
abstract: Prion diseases recognize, as a unique molecular trait, the misfolding of CNS-enriched prion protein (PrP(C)) into an aberrant isoform (PrP(Sc)). 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 Ca(2+) homeostasis impairment. This recombinant fragment assumes a toxic conformation (PrP90-231(TOX)) after controlled thermal denaturation (1 h at 53 °C) acquiring structural characteristics identified in PrP(Sc) (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 PrP90-231(TOX), used in pre-fibrillar aggregation state, induces CGN apoptosis. This effect was associated with a delayed, but sustained elevation of [Ca(2+)]i. Both CGN apoptosis and [Ca(2+)]i increase were not observed using PrP90-231 in PrP(C)-like conformation. PrP90-231(TOX) effects were significantly reduced in the presence of ionotropic glutamate receptor antagonists. In particular, CGN apoptosis and [Ca(2+)]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 PrP90-231(TOX) correlates with a sustained elevation of [Ca(2+)]i mediated by the activation of NMDA and AMPA receptors.
authors.affiliation: Department of Internal Medicine, Section of Pharmacology and Centre of Excellence for Biomedical Research (CEBR) School of Medicine, University of Genova, Viale Benedetto XV, 2, 16132, Genoa, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Thellung
authors.forename: Elena
authors.initials: E
authors.lastname: Gatta
authors.forename: Francesca
authors.initials: F
authors.lastname: Pellistri
authors.forename: Alessandro
authors.initials: A
authors.lastname: Corsaro
authors.forename: Valentina
authors.initials: V
authors.lastname: Villa
authors.forename: Massimo
authors.initials: M
authors.lastname: Vassalli
authors.forename: Mauro
authors.initials: M
authors.lastname: Robello
authors.forename: Tullio
authors.initials: T
authors.lastname: Florio
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D010446:Peptide Fragments / Q000633:toxicity*; D011328:Prions / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*
chemical_list: D010446:Peptide Fragments; D011328:Prions; D016194:Receptors, N-Methyl-D-Aspartate; C507417:prion protein (90-231); D002118:Calcium