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...

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.

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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.

Opposite effects of nuclear factor-jB (NF-jB) on neuron survival rely on activation of diverse NF-jB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1b. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-b (Ab), a peptide implicated in Alzheimer's disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Ab toxicity depends on cRel activation. Ab peptide induced NF-jB 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 TATBcl-X L addition rescued neurons from Ab toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Reldependent antiapoptotic pathway responsible for neuroprotection against Ab peptide.

Background and Purpose-Leptin is an adipose hormone endowed with angiopoietic, neurotrophic, and neuroprotective properties. We tested the hypothesis that leptin might act as an endogenous mediator of recovery after ischemic stroke and investigated whether nuclear transcription factors B activation is involved in leptin-mediated neuroprotection. Methods-The antiapoptotic effects of leptin were evaluated in cultured mouse cortical neurons from wild-type or NF-B/c-Rel Ϫ/Ϫ mice exposed to oxygen-glucose deprivation. Wild-type, c-Rel Ϫ/Ϫ and leptin-deficient ob/ob mice were subjected to permanent middle cerebral artery occlusion. Leptin production was measured in brains from wild-type mice with quantitative reverse transcriptase-polymerase chain reaction and immunostaining. Mice received a leptin bolus (20 g/g) intraperitoneally at the onset of ischemia. Results-Leptin treatment activated the nuclear translocation of nuclear transcription factors B dimers containing the c-Rel subunit, induced the expression of the antiapoptotic c-Rel target gene Bcl-xL in both control and oxygen-glucose deprivation conditions, and counteracted the oxygen-glucose deprivation-mediated apoptotic death of cultured cortical neurons. Leptin-mediated Bcl-xL induction and neuroprotection against oxygen-glucose deprivation were hampered in cortical neurons from c-Rel Ϫ/Ϫ mice. Leptin mRNA was induced and the protein was detectable in microglia/macrophage cells from the ischemic penumbra of wild-type mice subjected to permanent middle cerebral artery occlusion. Ob/ob mice were more susceptible than wild-type mice to the permanent middle cerebral artery occlusion injury. Leptin injection significantly reduced the permanent middle cerebral artery occlusion-mediated cortical damage in wild-type and ob/ob mice, but not in c-Rel Ϫ/Ϫ mice. Conclusions-Leptin

The present study demonstrates that human SK-N-SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK-N-SH cells switched from non-neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT-PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA-differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N-methyl-D-aspartate (NMDA) receptors. Glutamate or NMDA application, concentration-dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK-N-SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor-mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK-N-SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron-like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors.