eNOS-dependent S-nitrosylation of the NF-κB subunit p65 has neuroprotective effects

Abstract

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...

Redox Post-translational Modifications of Protein Thiols in Brain Aging and Neurodegenerative Conditions—Focus on S-Nitrosation

“…Redox PTMs can also regulate the activity of several transcription factors (Brigelius-Flohé and Flohé, 2011 ). For instance, oxidation, S-nitrosation or nitration of p53, activating protein 1, myocyte enhancer factor 2 or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) modulate the DNA binding and activity of these important transcription factors (Abate et al, 1990 ; Schreck et al, 1991 ; Rainwater et al, 1995 ; Klatt et al, 1999 ; Kabe et al, 2005 ; Yakovlev et al, 2007 ; Jung et al, 2008 ; Ljubuncic et al, 2008 ; Okamoto et al, 2014 ; Caviedes et al, 2020 ). Redox PTMs can also affect the activity of transcription factors through indirect mechanisms.…”

miRNA‐384‐3p alleviates sevoflurane‐induced nerve injury by inhibiting Aak1 kinase in neonatal rats

“…The homogenate was centrifuged at 17,000 × g for 5 min to obtain the cytoplasmic fraction. The pellet was washed with buffer B (150 mM NaCl; 10 mM HEPES; 1 mM EDTA), centrifuged at 17,000 × g for 1 min at 4 °C, resuspended in buffer C (25% v/v glycerol; 20 mM HEPES; 400 mM NaCl; 1.2 mM MgCl 2 ; 0.2 mM EDTA), vortexed for 30 s and incubated on ice for 10 min (five times) to finally centrifuge at 17,000 × g for 20 min to obtain the nuclear fraction (Caviedes et al., 2021). RNA expression of GAPDH, U6, miRNA‐384‐3p, and Aak1 in the nuclear and cytoplasmic fractions was detected by RT–qPCR as mentioned above.…”

Targeting AMPK/eNOS pathway and mitochondria by sonlicromanol protects myocardial cells against ischemia-reperfusion injury

“…There is no study yet indicating the possible direct effect of sonlicromanol on eNOS or NF-κB activity, but since this drug lowers oxidative stress by modulating prostaglandin-E2 pathway (Xiao et al 2021), it may modulate NF-κB activity and related inflammatory responses in ischemic disorders. Additionally, eNOS activation reduces NF-κB signaling (Caviedes et al 2021). As a result, we can deduce that the proportion of NF-κB and AMPK/eNOS in a cell determines the status of cellular and tissue inflammatory and oxidative responses.…”