The molecular mechanisms responsible for increasing iron and neurodegeneration in brain ischemia are an interesting area of research which could open new therapeutic approaches. Previous evidence has shown that activation of nuclear factor kappa B (NF-κB) through RelA acetylation on Lys310 is the prerequisite for p50/RelA-mediated apoptosis in cellular and animal models of brain ischemia. We hypothesized that the increase of iron through a NF-κB-regulated 1B isoform of the divalent metal transporter-1 (1B/DMT1) might contribute to post-ischemic neuronal damage. Both in mice subjected to transient middle cerebral artery occlusion (MCAO) and in neuronally differentiated SK-N-SH cells exposed to oxygen-glucose-deprivation (OGD), 1A/DMT1 was only barely expressed while the 1B/DMT1 without iron-response-element (−IRE) protein and mRNA were early up-regulated. Either OGD or over-expression of 1B/(−)IRE DMT1 isoform significantly increased iron uptake, as detected by total reflection X-ray fluorescence, and iron-dependent cell death. Iron chelation by deferoxamine treatment or (−)IRE DMT1 RNA silencing displayed significant neuroprotection against OGD which concomitantly decreased intracellular iron levels. We found evidence that 1B/(−)IRE DMT1 was a target gene for RelA activation and acetylation on Lys310 residue during ischemia. Chromatin immunoprecipitation analysis of the 1B/DMT1 promoter showed there was increased interaction with RelA and acetylation of H3 histone during OGD exposure of cortical neurons. Over-expression of wild-type RelA increased 1B/DMT1 promoter-luciferase activity, the (−)IRE DMT1 protein, as well as neuronal death. Expression of the acetylation-resistant RelA-K310R construct, which carried a mutation from lysine 310 to arginine, but not the acetyl-mimic mutant RelA-K310Q, down-regulated the 1B/DMT1 promoter, consequently offering neuroprotection. Our data showed that 1B/(−)IRE DMT1 expression and intracellular iron influx are early downstream responses to NF-κB/RelA activation and acetylation during brain ischemia and contribute to the pathogenesis of stroke-induced neuronal damage.
Nuclear factor‐kappaB (NF‐κB) has been proposed to serve a dual function as a regulator of neuron survival in pathological conditions associated with neurodegeneration. NF‐κB is a transcription family of factors comprising five different proteins, namely p50, RelA/p65, c‐Rel, RelB and p52, which can combine differently to form active dimers in response to external stimuli. Recent research shows that diverse NF‐κB dimers lead to cell death or cell survival in neurons exposed to ischemic injury. While the p50/p65 dimer participates in the pathogenesis of post‐ischemic injury by inducing pro‐apoptotic gene expression, c‐Rel‐containing dimers increase neuron resistance to ischemia by inducing anti‐apoptotic gene transcription. We present, in this report, the latest findings and consider the therapeutic potential of targeting different NF‐κB dimers to limit ischemia‐associated neurodegeneration.
Abnormal amyloid-β (Aβ) production and deposition is believed to represent one of the main causes of Alzheimer's disease (AD). γ-Secretase is the enzymatic complex responsible for Aβ generation from its precursor protein. Inhibition or modulation of γ-secretase represents an attractive therapeutic approach. CHF5074 is a new γ-secretase modulator that has been shown to inhibit brain plaque deposition and to attenuate memory deficit in adult AD transgenic mice after chronic treatment. To date, it is not known whether the positive behavioral effects of this compound also occur in young transgenic mice without plaque deposition. Here, we evaluated the effects of acute and subchronic treatment with CHF5074 on contextual and recognition memory and on hippocampal synaptic plasticity in plaque-free Tg2576 mice. We found that at 5 months of age, contextual memory impairment was significantly attenuated after acute subcutaneous administration of 30 mg/kg CHF5074. At 6 months of age, recognition memory impairment was fully reversed after a 4-week oral treatment in the diet (≈60 mg/kg/day). These cognitive effects were associated with a reversal of long-term potentiation (LTP) impairment in the hippocampus. A significant reduction in brain intraneuronal AβPP/Aβ levels and hyperphosphorylated tau, but no change in soluble or oligomeric Aβ levels was detected in Tg2576 mice showing functional recovery following CHF5074 treatment. We conclude that the beneficial effects of CHF5074 treatment in young transgenic mice occurred at a stage that precedes plaque formation and were associated with a reduction in intraneuronal AβPP/Aβ and hyperphosphorylated tau.
CHF5074 is a new gamma-secretase modulator in clinical development for the treatment of Alzheimer's disease (AD). Recent studies have shown that CHF5074 can induce astrocyte stellation independently from gamma-secretase modulation (JAD 2010; 22: 1135-55). These results prompt us to investigate on neuroprotective activity of CHF5074 in experimental models unrelated to A-beta accumulation. Much research is actually focused on epigenetic mechanism regulating histone acetylation and involved in process of chromatin remodeling, microtubule dynamics, metabolism and ageing with relevance in different complex, late-onset neurodegenerative diseases. Histone deacetylase inhibitors have proved to be neuroprotective in animal models of AD and brain ischemia. The aim of our study was to investigate the effect of CHF5074 in mouse primary cortical neurons exposed to oxygen glucose deprivation (OGD), a cell-based model of brain ischemia. The effect of CHF5074 was compared to that elicited by ibuprofen and other neuroprotective agents, the sirtuin activator resveratrol and the HDAC inhibitor MS-275. Fifteen days embryonic C57BL/6 mice were harvested with caesarean section from anaesthetized pregnant dams. Cerebral cortices were isolated and dissociated by manual dispersion. The cells were plated in Neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine and 50 U/mL penicillin/streptomicin. At 10 days in vitro near pure cortical neurons were exposed to OGD at 37° C for 3 hours. At the end of the OGD period, cortical neurons were allowed to recover for 24 hours in Neurobasal medium containing 0.4% B27 supplement, under normoxic conditions and with or without the test drugs. We analyzed the effect of CHF5074 in the concentration range eliciting anti-amyloidogenic activity, from 1-30 μM, ibuprofen at 500 μM, resveratrol at 30 μM and MS275 at 1 μM. Neuronal injury and neuroprotection were evaluated by measuring the amount of lactate dehydrogenase (LDH) released into the culture medium. To test histone acetylation, nuclear extracts were prepared 6 hours after the OGD exposure and nuclear proteins were processed for Western blot analysis by using antibody specific for histone H3 acetylated on K9/18 residues. Near pure cortical neurons were exposed to CHF5074 from 1 to 30 ÂμM during the OGD period and the following 24 hour recovery. In another set of experiments, CHF5074 was added only after the OGD exposure. When compared to neurons exposed to OGD, cells treated with CHF5074 displayed higher survival at all the concentrations tested, i.e 1, 3, 10, 30 μM. Maximal neuroprotection (75 % reduction of LDH release) was evident at 10 μM concentration. If added only after the OGD period, CHF5074 was still neuroprotective, though with lower potency. Likewise 3 μM CHF5074, 30 μM resveratrol and 1 μM MS-275 added in the post-OGD period also elicited neuroprotection while 500 ÂμM ibuprofen exacerbated the neuronal cell death. Analysis of histone acetylation, showed a decreased H3 acetylation on the K9/18 residues at 6 hours after OGD. The acetylation state of histone H3 recovered in cells treated in the post-OGD period with the HDAC inhibitor MS-275 or with CHF5074, but not with the sirtuin activator resveratrol. These data indicate that CHF5074, but not ibuprofen, elicits neuroprotection in cortical neurons previously exposed to OGD. CHF5074 effects were associated with the increase of histone H3 acetylation. In spite of observed similar neuroprotection, CHF5074 effect on histone acetylation was reproduced by MS-275 but not by resveratrol. This leaves open the possibility that CHF5074 may directly or indirectly modulate the HDAC activity. The possible role of HDAC activity modulation in the beneficial effects of CHF5074 in AD deserves further investigations.
CSP-1103 (formerly CHF5074) has been shown to reverse memory impairment and reduce amyloid plaque as well as inflammatory microglia activation in preclinical models of Alzheimer's disease. Moreover, it was found to improve cognition and reduce brain inflammation in patients with mild cognitive impairment. Recent evidence suggests that CSP-1103 acts through a single molecular target, the amyloid precursor protein intracellular domain (AICD), a transcriptional regulator implicated in inflammation and apoptosis. We here tested the possible anti-apoptotic and neuroprotective activity of CSP-1103 in a cell-based model of post-ischemic injury, wherein the primary mouse cortical neurons were exposed to oxygen-glucose deprivation (OGD). When added after OGD, CSP-1103 prevented the apoptosis cascade by reducing cytochrome c release and caspase-3 activation and the secondary necrosis. Additionally, CSP-1103 limited earlier activation of p38 and nuclear factor κB (NF-κB) pathways. These results demonstrate that CSP-1103 is neuroprotective in a model of post-ischemic brain injury and provide further mechanistic insights as regards its ability to reduce apoptosis and potential production of pro-inflammatory cytokines. In conclusion, these findings suggest a potential use of CSP-1103 for the treatment of brain ischemia.
