Flora Boroni

NF-κB factor c-Rel mediates neuroprotection elicited by mGlu5 receptor agonists against amyloid β -peptide toxicity

Opposite effects of nuclear factor-κB (NF-κB) on neuron survival rely on activation of diverse NF-κB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1β. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-β (Aβ), a peptide implicated in Alzheimers disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Aβ toxicity depends on c-Rel activation. Aβ peptide induced NF-κB factors p50 and p65. The mGlu5 agonists activated c-Rel, besides p50 and p65, and the expression of manganese superoxide dismutase (MnSOD) and Bcl-XL. Targeting c-Rel expression by RNA interference suppressed the induction of both antiapoptotic genes. Targeting c-Rel or Bcl-XL prevented the prosurvival effect of mGlu5 agonists. Conversely, c-Rel overexpression or TAT-Bcl-XL addition rescued neurons from Aβ toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Rel-dependent antiapoptotic pathway responsible for neuroprotection against Aβ peptide.

NF‐κB p50/RelA and c‐Rel‐containing dimers: opposite regulators of neuron vulnerability to ischaemia

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.

Prevention of neuron and oligodendrocyte degeneration by interleukin-6 (IL-6) and IL-6 receptor/IL-6 fusion protein in organotypic hippocampal slices

We investigated the effects of IL-6 and a chimeric derivative of IL-6 and soluble IL-6 receptor (IL6RIL6 chimera) on excitotoxic injury in rat organotypic hippocampal slices. Brief application of N-methyl-d-aspartate (NMDA) induced astrocyte reactivity, neuron cell death, and oligodendrocyte degeneration, the latter caused by secondary activation of AMPA/kainate receptors. Both these cytokines rescued neurons and oligodendrocytes, albeit the chimeric compound was much more potent and efficient than IL-6. No change was produced on reactive astrocytosis. The cytokines preserved myelin basic protein (MBP) production in slices exposed to excitotoxic insult, and when applied singularly for a week, they also enhanced both MBP and proteolipid protein expression. These effects occurred through activating the signal transducer gp130 and were associated with stimulation of transcription factors STAT1 and STAT3. Our results suggest that IL-6 and IL6RIL6 may prove to be valuable in treating neurodegenerative and demyelinating diseases.

Regulation of Nuclear Factor κB in the Hippocampus by Group I Metabotropic Glutamate Receptors

An increasing amount of evidence suggests that the family of nuclear factor κB (NF-κB) transcription factors plays an important role in synaptic plasticity and long-term memory formation. The present study investigated the regulation of NF-κB family members p50, p65/RelA, and c-Rel in the hippocampus in response to metabotropic glutamate receptor (mGluR) signaling. Activation of group I metabotropic glutamate receptors (GpI-mGluRs) with the agonist (S)-3,5-dihydroxyphenylglycine (DHPG) resulted in a time-dependent increase in DNA binding activity of p50, p65, and c-Rel in area CA1 of the hippocampus. An antagonist of mGluR5, 2-Methyl-6-(phenylethynyl)pyridine, inhibited the DHPG-induced activation of NF-κB, whereas an antagonist of mGluR1, (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid, did not. Using a series of inhibitors, we investigated the signaling pathways necessary for DHPG-induced activation of NF-κB and found that they included the phosphatidyl inositol 3-kinase, protein kinase C, mitogen-activated protein kinase kinase, and p38-mitogen-activated protein kinase pathways. To determine the functional significance of mGluR-induced regulation of NF-κB, we measured long-term depression (LTD) of Schaffer-collateral synapses in the hippocampus of c-Rel knock-out mice. Early phase LTD was normal in c-rel−/− mice. However, late-phase LTD (>90 min) was impaired in c-rel−/− mice. The observations of this deficit in hippocampal synaptic plasticity prompted us to further investigate long-term memory formation in c-rel−/− mice. c-rel−/− mice exhibited impaired performance in a long-term passive avoidance task, providing additional evidence for c-Rel in long-term memory formation. These results demonstrate that the NF-κB transcription factor family is regulated by GpI-mGluRs in the hippocampus and that the c-Rel transcription factor is necessary for long-term maintenance of LTD and formation of long-term memory.

NF‐κB pathway: a target for preventing β‐amyloid (Aβ)‐induced neuronal damage and Aβ42 production

Beta‐amyloid (Aβ) peptides are key proteins in the pathophysiology of Alzheimers disease (AD). While Aβ42 aggregates very rapidly to form early diffuse plaques, supplemental Aβ40 deposition is required to form mature neuritic plaques. We here investigated the role of nuclear factor‐κB (NF‐κB) pathway in Aβ40‐mediated neuronal damage and amyloid pathology. In rat primary neurons and human postmitotic neuronal cells, the Aβ 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 Aβ42 oligomers. Moreover, Aβ40 activated the NF‐κB 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‐κB pathway, we tested the capability of a double‐stranded κB decoy oligonucleotide, the anti‐inflammatory drug aspirin and the selective IκB kinase 2 inhibitor, AS602868, to modify the Aβ40‐mediated effects. These treatments, transiently applied before Aβ exposure, completely inhibited p50/p65 nuclear translocation and neuronal damage. The κB decoy also inhibited the Aβ‐induced release of cytochrome c, restored the levels of Bcl‐XL, and prevented intraneuronal accumulation and secretion of Aβ42. These results open up interesting perspectives on the development of novel strategies targeting out NF‐κB p50/p65 dimers for pharmacological intervention in AD.

Leptin Is Induced in the Ischemic Cerebral Cortex and Exerts Neuroprotection Through NF-κB/c-Rel–Dependent Transcription

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 &kgr;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-&kgr;B/c-Rel−/− mice exposed to oxygen–glucose deprivation. Wild-type, c-Rel−/− 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 μg/g) intraperitoneally at the onset of ischemia. Results— Leptin treatment activated the nuclear translocation of nuclear transcription factors &kgr;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−/− 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−/− mice. Conclusions— Leptin acts as an endogenous mediator of neuroprotection during cerebral ischemia. Exogenous leptin administration protects against ischemic neuronal injury in vitro and in vivo in a c-Rel-dependent manner.

NF-kappaB pathway: a target for preventing beta-amyloid (Abeta)-induced neuronal damage and Abeta42 production

Beta‐amyloid (Aβ) peptides are key proteins in the pathophysiology of Alzheimers disease (AD). While Aβ42 aggregates very rapidly to form early diffuse plaques, supplemental Aβ40 deposition is required to form mature neuritic plaques. We here investigated the role of nuclear factor‐κB (NF‐κB) pathway in Aβ40‐mediated neuronal damage and amyloid pathology. In rat primary neurons and human postmitotic neuronal cells, the Aβ 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 Aβ42 oligomers. Moreover, Aβ40 activated the NF‐κB 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‐κB pathway, we tested the capability of a double‐stranded κB decoy oligonucleotide, the anti‐inflammatory drug aspirin and the selective IκB kinase 2 inhibitor, AS602868, to modify the Aβ40‐mediated effects. These treatments, transiently applied before Aβ exposure, completely inhibited p50/p65 nuclear translocation and neuronal damage. The κB decoy also inhibited the Aβ‐induced release of cytochrome c, restored the levels of Bcl‐XL, and prevented intraneuronal accumulation and secretion of Aβ42. These results open up interesting perspectives on the development of novel strategies targeting out NF‐κB p50/p65 dimers for pharmacological intervention in AD.

Expression of functional NR1/NR2B‐type NMDA receptors in neuronally differentiated SK‐N‐SH human cell line

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.

Neuroprotection by metabotropic glutamate receptor agonists on kainate-induced degeneration of motor neurons in spinal cord slices from adult rat

Research has provided evidence about the role of excitotoxicity in the pathophysiology of sporadic amyotrophic lateral sclerosis and suggests that AMPA/kainate receptor activation contributes greatly in mediating glutamate injury to motor neurons. The recent finding of variable expression of metabotropic glutamate (mGlu) receptor subtypes in adult rat spinal cord has prompted us to investigate their contribution to the excitotoxic process. We report here that stimulation of mGlu receptors efficiently prevents motor neuron degeneration induced by kainate. The application of kainate to lumbar spinal cord slices from adult rats induced a massive degeneration of motor neurons which became shrunken, dark and TUNEL-positive. On the contrary, no significant neurotoxicity was observed after NMDA application. A blockade of ionotropic non-NMDA receptors by CNQX, and mGlu receptor stimulation, efficiently counteracted kainate-mediated cell death. Among the various agonists for mGlu receptors, we tested 3-hydroxyphenylglycine (3HPG), which selectively stimulates group I mGlu receptors. In addition, we tested 2-(carboxycyclopropyl)glycine (L-CCG-I) and 4-carboxy-3-hydroxyphenylglycine (4C3HPG), two selective agonists for group II receptors, as well as L-amino-4-phosphonobutyrate (L-AP4), a preferential agonist for group III. The results suggest that all three groups of mGlu receptors are involved in inhibiting excitotoxic phenomena mediated by kainate on spinal cord motor neurons. This was despite being localized differently and, possibly, activating different neuroprotective pathways.

The acetylation of RelA in Lys310 dictates the NF-κB-dependent response in post-ischemic injury.

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.