title: Multiple domains in the C-terminus of NMDA receptor GluN2B subunit contribute to neuronal death following in vitro ischemia.
abstract: Global cerebral ischemia induces selective degeneration of specific subsets of neurons throughout the brain, particularly in the hippocampus and cortex. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca(2+) overload and ultimately neuronal demise. N-methyl-d-aspartate receptors (NMDARs) are considered to be largely responsible for excitotoxic injury due to their high Ca(2+) permeability. In the hippocampus and cortex, these receptors are most prominently composed of combinations of two GluN1 subunits and two GluN2A and/or GluN2B subunits. Due to the controversy regarding the differential role of GluN2A and GluN2B subunits in excitotoxic cell death, we investigated the role of GluN2B in the activation of pro-death signaling following an in vitro model of global ischemia, oxygen and glucose deprivation (OGD). For this purpose, we used GluN2B(-/-) mouse cortical cultures and observed that OGD-induced damage was reduced in these neurons, and partially prevented in wild-type rat neurons by a selective GluN2B antagonist. Notably, we found a crucial role of the C-terminal domain of the GluN2B subunit in triggering excitotoxic signaling. Indeed, expression of YFP-GluN2B C-terminus mutants for the binding sites to post-synaptic density protein 95 (PSD95), Ca(2+)-calmodulin kinase IIα (CaMKIIα) or clathrin adaptor protein 2 (AP2) failed to mediate neuronal death in OGD conditions. We focused on the GluN2B-CaMKIIα interaction and found a determinant role of this interaction in OGD-induced death. Inhibition or knock-down of CaMKIIα exerted a neuroprotective effect against OGD-induced death, whereas overexpression of this kinase had a detrimental effect. Importantly, in comparison with neurons overexpressing wild-type CaMKIIα, neurons overexpressing a mutant form of the kinase (CaMKII-I205K), unable to interact with GluN2B, were partially protected against OGD-induced damage. Taken together, our results identify crucial determinants in the C-terminal domain of GluN2B subunits in promoting neuronal death in ischemic conditions. These mechanisms underlie the divergent roles of the GluN2A- and GluN2B-NMDARs in determining neuronal fate in cerebral ischemia.
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
authors.forename: Marta M
authors.initials: MM
authors.lastname: Vieira
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; PDBEB - Doctoral Program in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal.
authors.forename: Jeannette
authors.initials: J
authors.lastname: Schmidt
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
authors.forename: Joana S
authors.initials: JS
authors.lastname: Ferreira
authors.affiliation: Brain Research Centre and Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Kevin
authors.initials: K
authors.lastname: She
authors.affiliation: Brain Research Centre and Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Oku
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
authors.forename: Miranda
authors.initials: M
authors.lastname: Mele
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal.
authors.forename: Armanda E
authors.initials: AE
authors.lastname: Santos
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Department of Life Sciences, University of Coimbra, Coimbra, Portugal.
authors.forename: Carlos B
authors.initials: CB
authors.lastname: Duarte
authors.affiliation: Brain Research Centre and Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Ann Marie
authors.initials: AM
authors.lastname: Craig
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Department of Life Sciences, University of Coimbra, Coimbra, Portugal. Electronic address: alc@cnc.uc.pt.
authors.forename: Ana Luísa
authors.initials: AL
authors.lastname: Carvalho
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C121001:NR2B NMDA receptor; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2
keywords: AP2; CaMKIIα; Cerebral ischemia; GluN2B; NMDA receptors; Oxygen-glucose deprivation; PSD95