ABSTRACT
The activation of nuclear factor kappa B (NF-κB) p50/RelA is a key event in ischemic neuronal injury, as well as in brain ischemic tolerance. We tested whether epigenetic mechanisms affecting the acetylation state of RelA might discriminate between neuroprotective and neurotoxic activation of NF-κB during ischemia. NF-κB activation and RelA acetylation were investigated in cortices of mice subjected to preconditioning brain ischemia or lethal middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to preconditioning or lethal oxygen-glucose deprivation (OGD). In mice subjected to MCAO and in cortical neurons exposed to lethal OGD, activated RelA displayed a high level of Lys310 acetylation in spite of reduced total acetylation. Also, acetylated RelA on Lys310 interacted strongly with the CREB-binding protein (CBP). Conversely, RelA activated during preconditioning ischemia appeared deacetylated on Lys310. Overexpressing RelA increased Bim promoter activity and neuronal cell death both induced by lethal OGD, whereas overexpressing the acetylation-resistant RelA-K310R, carrying a mutation from Lys310 to arginine, prevented both responses. Pharmacological manipulation of Lys310 acetylation by the sirtuin 1 activator resveratrol repressed the activity of the Bim promoter and reduced the neuronal cell loss. We conclude that the acetylation of RelA in Lys310 dictates NF-κB-dependent pro-apoptotic responses and represents a suitable target to dissect pathological from neuroprotective NF-κB activation in brain ischemia.
Subject(s)
Brain Ischemia/metabolism , Lysine/metabolism , NF-kappa B p50 Subunit/metabolism , Transcription Factor RelA/metabolism , Acetylation , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Brain Ischemia/pathology , CREB-Binding Protein/metabolism , Infarction, Middle Cerebral Artery/metabolism , Infarction, Middle Cerebral Artery/pathology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Sirtuin 1/chemistry , Sirtuin 1/metabolismABSTRACT
Among the diverse mechanisms involved in the pathophysiology of post-ischemic and post-traumatic injuries, excitotoxicity and nuclear factor-kappaB (NF-kappaB) activation through induction of IkappaB kinase (IKK) complex have a primary role. We investigated the effects of the selective inhibitor of the IKK2 subunit, the anilinopyrimidine derivative AS602868, on excitotoxic injury produced in rat organotypic hippocampal slices and cerebellar primary neurons. Brief exposure to N-methyl-D-aspartate (NMDA) induces astrocyte reactivity, neuron cell death and oligodendrocyte degeneration in hippocampal slices. Application of AS602868 elicited a long-lasting protection of both neurons and oligodendrocytes. Maximal effect was observed with prolonged application of the compound after NMDA exposure. Neuroprotection was also evident in primary cultures of cerebellar granule cells starting from 20 nM concentration. AS602868-elicited neuroprotection correlated with inhibition of NF-kappaB activity. Our results suggest that AS602868 may prove to be a valuable approach in treating neurodegeneration and demyelination associated with cerebral trauma and ischemia.
Subject(s)
I-kappa B Kinase/metabolism , Neurons/drug effects , Neuroprotective Agents/pharmacology , Oligodendroglia/drug effects , Pyrimidines/pharmacology , Analysis of Variance , Animals , Animals, Newborn , Dose-Response Relationship, Drug , Electrophoretic Mobility Shift Assay/methods , Excitatory Amino Acid Agonists/toxicity , Gene Expression Regulation/drug effects , Hippocampus/cytology , In Vitro Techniques , Myelin Basic Protein/metabolism , N-Methylaspartate/toxicity , Rats , Rats, Wistar , Time FactorsABSTRACT
Nuclear factor-kappaB (NF-kappaB) is a transcriptional regulator of neuron survival eliciting diverse effects according to the specific composition of its active dimer. While p50/p65 mediates neurodegenerative events, c-Rel-containing dimers promote cell survival. Stimulation of metabotropic glutamate receptors type 5 (mGlu5) reduces neuron vulnerability to amyloid-beta through activation of anti-apoptotic, c-Rel-dependent transcription of Bcl-X(L) pathway. We here evaluated the protective activity of mGlu5 agonists in dopaminergic SK-N-SH cells exposed to 1-methyl-4-phenylpyridinium (MPP(+)), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causing parkinsonism in experimental animals. MPP(+) produced a concentration-dependent cell loss. Activation of mGlu5 receptors by CHPG (1 mM) and 3HPG (50 microM) abolished the toxic effect produced by 3 microM MPP(+). The neuroprotection was associated with activation of NF-kappaB p65/c-Rel dimer and reduction of p50/p65. These effects were prevented by the mGlu5 receptor antagonist MPEP (5 microM). It is suggested that mGlu5 receptor agonists through activation of a c-Rel-dependent anti-apoptotic pathway can rescue dopaminergic cell from mitochondrial toxicity.
Subject(s)
1-Methyl-4-phenylpyridinium/toxicity , Excitatory Amino Acid Agonists/pharmacology , Gene Expression Regulation/drug effects , Herbicides/toxicity , NF-kappa B/metabolism , Proto-Oncogene Proteins c-rel/metabolism , Analysis of Variance , Cell Death/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Activation/drug effects , Glycine/analogs & derivatives , Glycine/pharmacology , Humans , Neuroblastoma , Phenylacetates/pharmacology , Time FactorsABSTRACT
NF-kappaB is a nuclear transcription factor involved in the control of fundamental cellular functions including regulation of cell survival. We investigated NF-kappaB activation induced by two opposing modulators of cell viability: IL-1beta and glutamate. We found that IL-1beta activated p50, p65 and c-Rel subunits of NF-kappaB, while glutamate activated only p50 and p65 proteins. Cell stimulation by glutamate, correlated with expression of the pro-apoptotic genes Caspase-3, Caspase-2L and Bax. Conversely, IL-1beta induced the expression of the short anti-apoptotic isoform of Caspase-2. Finally, we analysed the effect of the inhibition of IkappaBalpha degradation on glutamate-induced toxicity by using BAY 11-7082, a selective inhibitor of IkappaBalpha phosphorylation. Our results suggest that BAY 11-7082 preserves neuron viability from the glutamate-mediated injury.
Subject(s)
Apoptosis/physiology , Glutamic Acid/pharmacology , I-kappa B Proteins/metabolism , Interleukin-1/pharmacology , Neurons/metabolism , Animals , Apoptosis/drug effects , Cells, Cultured , Cerebellum/cytology , Cerebellum/drug effects , Cerebellum/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors , I-kappa B Proteins/antagonists & inhibitors , NF-KappaB Inhibitor alpha , NF-kappa B , Neurons/drug effects , Phosphorylation/drug effects , Rats , Rats, Sprague-DawleyABSTRACT
Opposite effects of nuclear factor-kappaB (NF-kappaB) on neuron survival rely on activation of diverse NF-kappaB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1beta. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-beta (Abeta), a peptide implicated in Alzheimer's disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Abeta toxicity depends on c-Rel activation. Abeta peptide induced NF-kappaB factors p50 and p65. The mGlu5 agonists activated c-Rel, besides p50 and p65, and the expression of manganese superoxide dismutase (MnSOD) and Bcl-X(L). Targeting c-Rel expression by RNA interference suppressed the induction of both antiapoptotic genes. Targeting c-Rel or Bcl-X(L) prevented the prosurvival effect of mGlu5 agonists. Conversely, c-Rel overexpression or TAT-Bcl-X(L) addition rescued neurons from Abeta toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Rel-dependent antiapoptotic pathway responsible for neuroprotection against Abeta peptide.
Subject(s)
Amyloid beta-Peptides/toxicity , NF-kappa B/metabolism , Neurons/drug effects , Neuroprotective Agents/pharmacology , Proto-Oncogene Proteins c-rel/metabolism , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/metabolism , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Agonists/pharmacology , Gene Deletion , Gene Silencing , Glycine/analogs & derivatives , Glycine/pharmacology , Humans , Mice , Mice, Inbred C57BL , Neurons/cytology , Neurons/metabolism , Neurons/pathology , Neuroprotective Agents/metabolism , Phenylacetates/pharmacology , Proto-Oncogene Proteins c-rel/deficiency , Proto-Oncogene Proteins c-rel/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Metabotropic Glutamate/genetics , Superoxide Dismutase/metabolismABSTRACT
NF-kappaB is a nuclear transcription factor involved in the control of fundamental cellular functions including cell survival. Among the many target genes of this factor, both pro- and anti-apoptotic genes have been described. To evaluate the contribution of NF-kappaB activation to excitotoxic insult, we analysed the effect of IkappaBalpha (IkappaBalpha) phosphorylation blockade on glutamate-induced toxicity in adult mouse hippocampal slices. By using immunocytochemical and EMSA techniques, we found that (i) acute exposure of hippocampal slices to NMDA induced nuclear translocation of NF-kappaB, (ii) NMDA-mediated activation of NF-kappaB was prevented by BAY 11-7082, an inhibitor of IkappaBalpha phosphorylation and degradation, and (iii) BAY 11-7082-mediated inhibition of NF-kappaB activation was associated with neuroprotection.