K+-Dependent Na+/Ca2+ Exchanger Isoform 2, Nckx2, Takes Part in the Neuroprotection Elicited by Ischemic Preconditioning in Brain Ischemia

Sodium/Calcium exchangers are neuronal plasma membrane antiporters which, by coupling Ca2+ and Na+ fluxes across neuronal membranes, play a relevant role in brain ischemia. The most brain-expressed isoform among the members of the K+-dependent Na+/Ca2+ exchanger family, NCKX2, is involved in the progression of the ischemic lesion, since both its knocking-down and its knocking-out worsens ischemic damage. The aim of this study was to elucidate whether NCKX2 functions as an effector in the neuroprotection evoked by ischemic preconditioning. For this purpose, we investigated: (1) brain NCKX2 expression after preconditioning and preconditioning + ischemia; (2) the contribution of AKT and calpain to modulating NCKX2 expression during preconditioning; and (3) the effect of NCKX2 knocking-out on the neuroprotection mediated by ischemic preconditioning. Our results showed that NCKX2 expression increased in those brain regions protected by ischemic preconditioning. These changes were p-AKT-mediated since its inhibition prevented NCKX2 up-regulation. More interestingly, NCKX2 knocking-out significantly prevented the protection exerted by ischemic preconditioning. Overall, our results suggest that NCKX2 plays a fundamental role in the neuroprotective effect mediated by ischemic preconditioning and support the idea that the enhancement of its expression and activity might represent a reasonable strategy to reduce infarct extension after stroke

Models and methods for conditioning the ischemic brain

Abstract Background In the last decades the need to find new neuroprotective targets has addressed the researchers to investigate the endogenous molecular mechanisms that brain activates when exposed to a conditioning stimulus. Indeed, conditioning is an adaptive biological process activated by those interventions able to confer resistance to a deleterious brain event through the exposure to a sub-threshold insult. Specifically, preconditioning and postconditioning are realized when the conditioning stimulus is applied before or after, respectively, the harmul ischemia. Aims and Results The present review will describe the most common methods to induce brain conditioning, with particular regards to surgical, physical exercise, temperature-induced and pharmacological approaches. It has been well recognized that when the subliminal stimulus is delivered after the ischemic insult, the achieved neuroprotection is comparable to that observed in models of ischemic preconditioning. In addition, subjecting the brain to both preconditioning as well as postconditioning did not cause greater protection than each treatment alone. Conclusions The last decades have provided fascinating insights into the mechanisms and potential application of strategies to induce brain conditioning. Since the identification of intrinsic cell‐survival pathways should provide more direct opportunities for translational neuroprotection trials, an accurate examination of the different models of preconditioning and postconditioning is mandatory before starting any new project

Ionic homeostasis in brain conditioning

Most of the current focus on developing neuroprotective therapies is aimed at preventing neuronal death. However, these approaches have not been successful despite many years of clinical trials mainly because the numerous side effects observed in humans and absent in animals used at preclinical level. Recently, the research in this field aims to overcome this problem by developing strategies which induce, mimic, or boost endogenous protective responses and thus do not interfere with physiological neurotransmission. Preconditioning is a protective strategy in which a subliminal stimulus is applied before a subsequent harmful stimulus, thus inducing a state of tolerance in which the injury inflicted by the challenge is mitigated. Tolerance may be observed in ischemia, seizure, and infection. Since it requires protein synthesis, it confers delayed and temporary neuroprotection, taking hours to develop, with a pick at 1-3 days. A new promising approach for neuroprotection derives from post-conditioning, in which neuroprotection is achieved by a modified reperfusion subsequent to a prolonged ischemic episode. Many pathways have been proposed as plausible mechanisms to explain the neuroprotection offered by preconditioning and post-conditioning. Although the mechanisms through which these two endogenous protective strategies exert their effects are not yet fully understood, recent evidence highlights that the maintenance of ionic homeostasis plays a key role in propagating these neuroprotective phenomena. The present article will review the role of protein transporters and ionic channels involved in the control of ionic homeostasis in the neuroprotective effect of ischemic preconditioning and post-conditioning in adult brain, with particular regards to the Na(+)/Ca2(+) exchangers (NCX), the plasma membrane Ca2(+)-ATPase (PMCA), the Na(+)/H(+) exchange (NHE), the Na(+)/K(+)/2Cl(-) cotransport (NKCC) and the acid-sensing cation channels (ASIC). Ischemic stroke is the third leading cause of death and disability. Up until now, all clinical trials testing potential stroke neuroprotectants failed. For this reason attention of researchers has been focusing on the identification of brain endogenous neuroprotective mechanisms activated after cerebral ischemia. In this context, ischemic preconditioning and ischemic post-conditioning represent two neuroprotecive strategies to investigate in order to identify new molecular target to reduce the ischemic damage

Stroke by inducing HDAC9-dependent deacetylation of HIF-1 and Sp1, promotes TfR1 transcription and GPX4 reduction, thus determining ferroptotic neuronal death

: Background: The inhibition of histone deacetylase 9 (HDAC9) represents a promising druggable target for stroke intervention. Indeed, HDAC9 is overexpressed in neurons after brain ischemia where exerts a neurodetrimental role. However, mechanisms of HDAC9-dependent neuronal cell death are not yet well established. Methods: Brain ischemia was obtained in vitro by primary cortical neurons exposed to glucose deprivation plus reoxygenation (OGD/Rx) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcript and protein levels. Chromatin immunoprecipitation was used to evaluate the binding of transcription factors to the promoter of target genes. Cell viability was measured by MTT and LDH assays. Ferroptosis was evaluated by iron overload and 4-hydroxynonenal (4-HNE) release. Results: Our results showed that HDAC9 binds to hypoxia-inducible factor 1 (HIF-1) and specificity protein 1 (Sp1), two transcription activators of transferrin 1 receptor (TfR1) and glutathione peroxidase 4 (GPX4) genes, respectively, in neuronal cells exposed to OGD/Rx. Consequently, HDAC9 induced: (1) an increase in protein level of HIF-1 by deacetylation and deubiquitination, thus promoting the transcription of the pro-ferroptotic TfR1 gene; and (2) a reduction in Sp1 protein levels by deacetylation and ubiquitination, thus resulting in a down-regulation of the anti-ferroptotic GPX4 gene. Supporting these results, the silencing of HDAC9 partially prevented either HIF-1 increase and Sp1 reduction after OGD/Rx. Interestingly, silencing of the neurodetrimental factors, HDAC9, HIF-1, or TfR1 or the overexpression of the prosurvival factors Sp1 or GPX4 significantly reduced a well-known marker of ferroptosis 4-HNE after OGD/Rx. More important, in vivo, intracerebroventricular injection of siHDAC9 reduced 4-HNE levels after stroke by preventing: (1) HIF-1 and TfR1 increase and thus the augmented intracellular iron overload; and (2) a reduction of Sp1 and its target gene GPX4. Conclusions: Collectively, results obtained suggest that HDAC9 mediates post-traslational modifications of HIF-1 and Sp1 that, in turn, increases TfR1 and decreases GPX4 expression, thus promoting neuronal ferroptosis in in vitro and in vivo models of stroke

Synergistic association of valproate and resveratrol reduces brain injury in ischemic stroke

Histone deacetylation, together with altered acetylation of NF-κB/RelA, encompassing the K310 residue acetylation, occur during brain ischemia. By restoring the normal acetylation condition, we previously reported that sub-threshold doses of resveratrol and entinostat (MS-275), respectively, an activator of the AMP-activated kinase (AMPK)-sirtuin 1 pathway and an inhibitor of class I histone deacetylases (HDACs), synergistically elicited neuroprotection in a mouse model of ischemic stroke. To improve the translational power of this approach, we investigated the efficacy of MS-275 replacement with valproate, the antiepileptic drug also reported to be a class I HDAC blocker. In cortical neurons previously exposed to oxygen glucose deprivation (OGD), valproate elicited neuroprotection at 100 nmol/mL concentration when used alone and at 1 nmol/mL concentration when associated with resveratrol (3 nmol/mL). Resveratrol and valproate restored the acetylation of histone H3 (K9/18), and they reduced the RelA(K310) acetylation and the Bim level in neurons exposed to OGD. Chromatin immunoprecipitation analysis showed that the synergistic drug association impaired the RelA binding to the Bim promoter, as well as the promoter-specific H3 (K9/18) acetylation. In mice subjected to 60 min of middle cerebral artery occlusion (MCAO), the association of resveratrol 680 µg/kg and valproate 200 µg/kg significantly reduced the infarct volume as well as the neurological deficits. The present study suggests that valproate and resveratrol may represent a promising ready-to-use strategy to treat post-ischemic brain damage

Ruta graveolens water extract (RGWE) ameliorates ischemic damage and improves neurological deficits in a rat model of transient focal brain ischemia

The limited therapeutic options for ischemic stroke treatment render necessary the identification of new strategies. In recent years, it has been shown that natural compounds may represent a valid therapeutic opportunity. Therefore, the present study aimed to evaluate the protective effect of Ruta graveolens water extract (RGWE) in an in vivo experimental model of brain ischemia

Methylmercury upregulates RE-1 silencing transcription factor (REST) in SH-SY5Y cells and mouse cerebellum

Methylmercury (MeHg) is a highly neurotoxic compound that, in adequate doses, can cause damage to the brain, including developmental defects and in severe cases cell death. The RE-1-silencing transcription factor (REST) has been found to be involved in the neurotoxic effects of environmental pollutants such as polychlorinated biphenyls (PCBs). In this study, we investigated the effects of MeHg treatment on REST expression and its role in MeHg-induced neurotoxicity in neuroblastoma SH-SY5Y cells. We found that MeHg exposure caused a dose- and time- dependent apoptotic cell death, as evidenced by the appearance of apoptotic hallmarks including caspase-3 processing and annexin V uptake. Moreover, MeHg increased REST gene and gene product expression. MeHg-induced apoptotic cell death was completely abolished by REST knockdown. Interestingly, MeHg (1. μM/24. h) increased the expression of REST Corepressor (Co-REST) and its binding with REST whereas the other REST cofactor mammalian SIN3 homolog A transcription regulator (mSin3A) was not modified. In addition, we demonstrated that the acetylation of histone protein H4 was reduced after MeHg treatment and was critical for MeHg-induced apoptosis. Accordingly, the pan-histone deacetylase inhibitor trichostatin-A (TSA) prevented MeHg-induced histone protein H4 deacetylation, thereby reverting MeHg-induced neurotoxic effect. Male mice subcutaneously injected with 10 mg/kg of MeHg for 10 days showed an increase in REST expression in the granule cell layer of the cerebellum together with a decrease in histone H4 acetylation. Collectively, we demonstrated that methylmercury exposure can cause neurotoxicity by activating REST gene expression and H4 deacetylation

Prolonged NCX activation prevents SOD1 accumulation, reduces neuroinflammation, ameliorates motor behavior and prolongs survival in a ALS mouse model.

Abstract Imbalance in cellular ionic homeostasis is a hallmark of several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS). Sodium-calcium exchanger (NCX) is a membrane antiporter that, operating in a bidirectional way, couples the exchange of Ca2+ and Na + ions in neurons and glial cells, thus controlling the intracellular homeostasis of these ions. Among the three NCX genes, NCX1 and NCX2 are widely expressed within the CNS, while NCX3 is present only in skeletal muscles and at lower levels of expression in selected brain regions. ALS mice showed a reduction in the expression and activity of NCX1 and NCX2 consistent with disease progression, therefore we aimed to investigate their role in ALS pathophysiology. Notably, we demonstrated that the pharmacological activation of NCX1 and NCX2 by the prolonged treatment of SOD1G93A mice with the newly synthesized compound neurounina: (1) prevented the reduction in NCX activity observed in spinal cord; (2) preserved motor neurons survival in the ventral spinal horn of SOD1G93A mice; (3) prevented the spinal cord accumulation of misfolded SOD1; (4) reduced astroglia and microglia activation and spared the resident microglia cells in the spinal cord; (5) improved the lifespan and mitigated motor symptoms of ALS mice. The present study highlights the significant role of NCX1 and NCX2 in the pathophysiology of this neurodegenerative disorder and paves the way for the design of a new pharmacological approach for ALS