Cell death by glutamate excitotoxicity, mediated by N-methyl-d-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

Cell death by glutamate excitotoxicity, mediated by N-methyl-D-aspartate (NMDA) receptors, negatively impacts brain function affecting hippocampal, i.e. sensitive neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence and luciferase reporter assays, we found that NMDA stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e. resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous posttranslational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to excitotoxicity. Introduction.Neuronal death by glutamate excitotoxicity is implicated in the pathogenesis of several neurological disorders, ranging from neurodegeneration to epilepsy, stroke and traumatic brain injury (37, 52). The overstimulation of glutamate receptors leads to massive calcium influx, mainly through N-methyl-D-aspartate receptors (NMDA-Rs), triggering several intracellular pro-death signaling pathways (54). Endogenous/homeostatic protective mechanisms in response to glutamate, are incompletely known. In that line, the nuclear factor kappa B (NF-κB) family of transcription factors has been implicated in excitotoxicity in the retina, the striatum, cerebral cortex and hippocampus (29,44,53). This is associated with induction of pro-apoptotic and pro-inflammatory genes, including IL-1β. The canonic activation of the ubiquitous transcription factor NF-κB depends on phosphorylation and degradation of IκB proteins, leading to release and nuclear translocation of NF-κB, a dimer composed most frequently by a p65 and a p50 subunit (10,22). Its transcriptional activity in the nucleus is regulated by several post-translational modifications, such as phosphorylation and S-nitrosylation (i.e. the reversible coupling of nitric oxide (NO) to cysteine residues). In particular, S-nitrosylation of the p65 cysteine 38 inhibits its transcriptional activity in diverse cell types (24,41,46). However, the contribution of this mechanism to excitotoxicity is unknown and might imply a homeostatic regulation of the NF-κB activity. The main source of NO in the brain are nitric oxide synthases, i.e. the neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) enzymes (5,9,15). Considering the novel finding that eNOS is present in neuronal cultures and in glutamatergic synapses (6), we examined whether eNOS is involved in p65 S-nitrosylation and thus, in the regulation of its transcriptional activit...