Diverse nuclear factor‐κB subunits mediate opposite effects of extracellular signals on neuron survival. While RelA is activated by neurotoxic agents, c‐Rel drives neuroprotective effects. In brain ischaemia RelA and p50 factors rapidly activate, but how they associate with c‐Rel to form active dimers and contribute to the changes in diverse dimer activation for neuron susceptibility is unknown. We show that in both cortical neurons exposed to oxygen glucose deprivation (OGD) and mice subjected to brain ischaemia, activation of p50/RelA was associated with inhibition of c‐Rel/RelA dimer and no change p50/c‐Rel. Targeting c‐Rel and RelA expression revealed that c‐Rel dimers reduced while p50/RelA enhanced neuronal susceptibility to anoxia. Activation of p50/RelA complex is known to induce the pro‐apoptotic Bim and Noxa genes. We now show that c‐Rel‐containing dimers, p50/c‐Rel and RelA/c‐Rel, but not p50/RelA, promoted Bcl‐xL transcription. Accordingly, the OGD exposure induced Bim, but reduced Bcl‐xL promoter activity and decreased the content of endogenous Bcl‐xL protein. These findings demonstrate that within the same neuronal cell, the balance between activation of p50/RelA and c‐Rel‐containing complexes fine tunes the threshold of neuron vulnerability to the ischaemic insult. Selective targeting of different dimers will unravel new approaches to limit ischaemia‐associated apoptosis.

The transcription factors NF-B/Rel play a key role in regulating a diverse array of genes involved in cell growth, differentiation, and adaptive responses to environmental factors that are cell-and stimulus-specific (1). In the central nervous system, NF-B/Rel proteins are ubiquitously expressed in neurons and glia (2, 3) where, in addition to regulating physiological processes, they participate in pathological events associated with neurodegeneration (3, 4). Increased NF-B/Rel levels have been observed in the dying neurons of brains exposed to trauma and ischemia (5-8) as well as in brains of patients with Alzheimer's disease and Parkinson's disease (9 -11). Whether NF-B/Rel participates in a neurodegenerative program or otherwise in a neuroprotective process by increasing neuronal resistance to various noxae is still debated. Although many studies support the antiapoptotic effects of NF-B/Rel in cultured neurons (12-15), conflicting evidence has emerged from experimental models of pathological conditions affecting adult neurons. For example, some studies showed that NF-B/Rel mediates the neuroprotection elicited by the tumor necrosis factor in hippocampal cells (16,17) and promotes neuronal resistance to excitotoxicity (18) and ␤-amyloid-induced apoptosis (19). Other studies demonstrated that the activation of NF-B/Rel triggers neuronal degeneration after cerebral ischemia (6, 8) and mediates the glutamate-activated cell death program during excitotoxic insults to central neurons (4,20,21).NF-B/Rel proteins are a family of transcription factors composed of several members, including p50, p52, p65/RelA, RelB, and c-Rel, that form homo-and heterodimers capable of transmitting receptor signals to the nucleus (3,22). In resting cells, NF-B/Rel factors are retained in the cytoplasm by association with the inhibitory IB proteins. In stimulated cells, IB is phosphorylated and degradated, thus allowing the release and nuclear translocation of NF-B dimers. Recently, a more complex regulation of NF-B/Rel activation that involves modulatory phosphorylations has been emerging. The phosphorylation of NF-B/Rel components may operate to optimize their DNA binding and transcriptional activities, as well as functional interaction with coactivators (23). The diverse phenotypes of different NF-B/Rel knockout mice suggest that each NF-B/ Rel member serves unique physiological roles in vivo, presumably via the regulation of distinct sets of target genes. Thus, the opposite regulation of neuron survival by NF-B/Rel may very well depend on the activation of a distinct combination of subunits, resulting in the differential regulation of target genes and the induction of diverse genetic programs that dictate the cell fate (24 -26).In this study, we investigated the contribution of different NF-B/Rel proteins to the cell survival of brain neurons exposed to IL-1␤ 1 and glutamate, two common activators of NF-* This work was supported by grants from the Consiglio Nazionale delle Richerche (CNR 2000), the Italian Health Ministry, the Ital...

Nuclear factor-kappaB (NF-κB) p50/RelA is a key molecule with a dual effect in the progression of ischemic stroke. In harmful ischemia, but not in preconditioning insult, neurotoxic activation of p50/RelA is characterized by RelA-specific acetylation at Lys310 (K310) and deacetylation at other Lys residues. The derangement of RelA acetylation is associated with activation of Bim promoter. Objective: With the aim of producing neuroprotection by correcting altered acetylation of RelA in brain ischemia, we combined the pharmacological inhibition of histone deacetylase (HDAC) 1-3, the enzymes known to reduce global RelA acetylation, and the activation of sirtuin 1, endowed with a specific deacetylase activity on the K310 residue of RelA. To afford this aim, we tested the clinically used HDAC 1-3 inhibitor entinostat (MS-275) and the sirtuin 1 activator resveratrol. Methods: We used the mouse model of transient middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to oxygen glucose deprivation (OGD). Results: The combined use of MS-275 and resveratrol, by restoring normal RelA acetylation, elicited a synergistic neuroprotection in neurons exposed to OGD. This effect correlated with MS-275 capability to increase total RelA acetylation and resveratrol capability to reduce RelA K310 acetylation through the activation of an AMPactivated protein kinase-sirtuin 1 pathway. The synergistic treatment reproduced the acetylation state of RelA peculiar of preconditioning ischemia. Neurons exposed to the combined drugs totally recovered the optimal histone H3 acetylation. Neuroprotection was reproduced in mice subjected to MCAO and treated with MS-275 (20 μg/kg and 200 μg/kg) or resveratrol (6800 μg/kg) individually. However, the administration of lowest doses of MS-275 (2 μg/kg) and resveratrol (68 μg/kg) synergistically reduced infarct volume and neurological deficits. Importantly, the treatment was effective even when administered 7 h after the stroke onset. Chromatin immunoprecipitation analysis of cortices harvested from treated mice showed that the RelA binding and histone acetylation increased at the Bcl-x L promoter and decreased at the Bim promoter. Conclusion: Our study reveals that epigenetic therapy shaping acetylation of both RelA and histones may be a promising strategy to limit post-ischemic injury with an extended therapeutic window.

Beta-amyloid (Abeta) peptides are key proteins in the pathophysiology of Alzheimer's disease (AD). While Abeta42 aggregates very rapidly to form early diffuse plaques, supplemental Abeta40 deposition is required to form mature neuritic plaques. We here investigated the role of nuclear factor-kappaB (NF-kappaB) pathway in Abeta40-mediated neuronal damage and amyloid pathology. In rat primary neurons and human postmitotic neuronal cells, the Abeta peptide induced a dose-dependent neuronal death, reduced the levels of the anti-apoptotic protein Bcl-XL, enhanced the cytosolic release of cytochrome c, and elicited the intracellular accumulation and secretion of Abeta42 oligomers. Moreover, Abeta40 activated the NF-kappaB pathway by selectively inducing the nuclear translocation of p65 and p50 subunits, and promoted an apoptotic profile of gene expression. As inhibitors of the NF-kappaB pathway, we tested the capability of a double-stranded kappaB decoy oligonucleotide, the anti-inflammatory drug aspirin and the selective IkappaB kinase 2 inhibitor, AS602868, to modify the Abeta40-mediated effects. These treatments, transiently applied before Abeta exposure, completely inhibited p50/p65 nuclear translocation and neuronal damage. The kappaB decoy also inhibited the Abeta-induced release of cytochrome c, restored the levels of Bcl-XL, and prevented intraneuronal accumulation and secretion of Abeta42. These results open up interesting perspectives on the development of novel strategies targeting out NF-kappaB p50/p65 dimers for pharmacological intervention in AD.

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.