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Background and Purpose: NCX3 (Na + -Ca 2+ exchanger 3) plays a relevant role in stroke; indeed its pharmacological blockade or its genetic ablation exacerbates brain ischemic damage, whereas its upregulation takes part in the neuroprotection elicited by ischemic preconditioning. To identify an effective strategy to induce an overexpression of NCX3, we examined transcription factors and epigenetic mechanisms potentially involved in NCX3 gene regulation. Methods: Brain ischemia and ischemic preconditioning were induced in vitro by exposure of cortical neurons to oxygen and glucose deprivation plus reoxygenation (OGD/Reoxy) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcripts and proteins of GATA3 (GATA-binding protein 3), KMT2A (lysine-methyltransferase-2A), and NCX3. GATA3 and KMT2A binding on NCX3 gene was evaluated by chromatin immunoprecipitation and Rechromatin immunoprecipitation experiments. Results: Among the putative transcription factors sharing a consensus sequence on the ncx3 brain promoter region, GATA3 was the only able to up-regulate ncx3. Interestingly, GATA3 physically interacted with KMT2A, and their overexpression or knocking-down increased or downregulated NCX3 mRNA and protein, respectively. Notably, site-direct mutagenesis of GATA site on ncx3 brain promoter region counteracted GATA3 and KMT2A binding on NCX3 gene. More importantly, we found that in the perischemic cortical regions of preconditioned rats GATA3 recruited KMT2A and the complex H3K4-3me (trimethylated lysine-4 of histone-3) on ncx3 brain promoter region, thus reducing transient middle cerebral artery occlusion–induced damage. Consistently, in vivo silencing of either GATA3 or KMT2A prevented NCX3 upregulation and consequently the neuroprotective effect of preconditioning stimulus. The involvement of GATA3/KMT2A complex in neuroprotection elicited by ischemic preconditioning was further confirmed by in vitro experiments in which the knocking-down of GATA3 and KMT2A reverted the neuroprotection induced by NCX3 overexpression in cortical neurons exposed to anoxic preconditioning followed by oxygen and glucose deprivation plus reoxygenation. Conclusions: Collectively, our results revealed that GATA3/KMT2A complex epigenetically activates NCX3 gene transcription during ischemic preconditioning.

Spinal muscular atrophy (SMA) is a severe neuromuscular disease affecting infants caused by alterations of the survival motor neuron gene, which results in progressive degeneration of motor neurons (MNs). Although an effective treatment for SMA patients has been recently developed, the molecular pathway involved in selective MN degeneration has not been yet elucidated. In particular, miR-206 has been demonstrated to play a relevant role in the regeneration of neuromuscular junction in several MN diseases, and particularly it is upregulated in the quadriceps, tibialis anterior, spinal cord, and serum of SMA mice. In the present paper, we demonstrated that miR-206 was transiently upregulated also in the brainstem of the mouse model of SMA, SMAΔ7, in the early phase of the disease paralleling MN degeneration and was down-regulated in the late symptomatic phase. To prevent this downregulation, we intracerebroventricularly injected miR-206 in SMA pups, demonstrating that miR-206 reduced the severity of SMA pathology, slowing down disease progression, increasing survival rate, and improving behavioral performance of mice. Interestingly, exogenous miRNA-206-induced upregulation caused a reduction of the predicted target sodium calcium exchanger isoform 2, NCX2, one of the main regulators of intracellular [Ca2+] and [Na+]. Therefore, we hypothesized that miR-206 might exert part of its neuroprotective effect modulating NCX2 expression in SMA disease. Motor neuron degeneration occurs very early in the facial nuclei of SMA mice, paralleling a transient upregulation of miR-206. The prolonged increase of miR-206 induced by i.c.v. administration reduces motor neuron loss, ameliorates behavioral performance, and increases mouse lifespan, also through the modulation of the plasma membrane Na+/Ca2+ exchanger 2, NCX2.

The Na+/Ca2+ exchanger 1 (NCX1) participates in the maintenance of neuronal Na+ and Ca2+ homeostasis, and it is highly expressed at synapse level of some brain areas involved in learning and memory processes, including the hippocampus, cortex, and amygdala. Furthermore, NCX1 increases Akt1 phosphorylation and enhances glutamate-mediated Ca2+ influx during depolarization in hippocampal and cortical neurons, two processes involved in learning and memory mechanisms. We investigated whether the modulation of NCX1 expression/activity might influence learning and memory processes. To this aim, we used a knock-in mouse overexpressing NCX1 in hippocampal, cortical, and amygdala neurons (ncx1.4over) and a newly synthesized selective NCX1 stimulating compound, named CN-PYB2. Both ncx1.4over and CN-PYB2-treated mice showed an amelioration in spatial learning performance in Barnes maze task, and in context-dependent memory consolidation after trace fear conditioning. On the other hand, these mice showed no improvement in novel object recognition task which is mainly dependent on non-spatial memory and displayed an increase in the active phosphorylated CaMKIIα levels in the hippocampus. Interestingly, both of these mice showed an increased level of context-dependent anxiety. Altogether, these results demonstrate that neuronal NCX1 participates in spatial-dependent hippocampal learning and memory processes.

NCX3 (Na+-Ca2+ exchanger 3) plays a relevant role in stroke; indeed its pharmacological blockade or its genetic ablation exacerbates brain ischemic damage, whereas its upregulation takes part in the neuroprotection elicited by ischemic preconditioning. To identify an effective strategy to induce an overexpression of NCX3, we examined transcription factors and epigenetic mechanisms potentially involved in NCX3 gene regulation.Brain ischemia and ischemic preconditioning were induced in vitro by exposure of cortical neurons to oxygen and glucose deprivation plus reoxygenation (OGD/Reoxy) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcripts and proteins of GATA3 (GATA-binding protein 3), KMT2A (lysine-methyltransferase-2A), and NCX3. GATA3 and KMT2A binding on NCX3 gene was evaluated by chromatin immunoprecipitation and Rechromatin immunoprecipitation experiments.Among the putative transcription factors sharing a consensus sequence on the ncx3 brain promoter region, GATA3 was the only able to up-regulate ncx3. Interestingly, GATA3 physically interacted with KMT2A, and their overexpression or knocking-down increased or downregulated NCX3 mRNA and protein, respectively. Notably, site-direct mutagenesis of GATA site on ncx3 brain promoter region counteracted GATA3 and KMT2A binding on NCX3 gene. More importantly, we found that in the perischemic cortical regions of preconditioned rats GATA3 recruited KMT2A and the complex H3K4-3me (trimethylated lysine-4 of histone-3) on ncx3 brain promoter region, thus reducing transient middle cerebral artery occlusion–induced damage. Consistently, in vivo silencing of either GATA3 or KMT2A prevented NCX3 upregulation and consequently the neuroprotective effect of preconditioning stimulus. The involvement of GATA3/KMT2A complex in neuroprotection elicited by ischemic preconditioning was further confirmed by in vitro experiments in which the knocking-down of GATA3 and KMT2A reverted the neuroprotection induced by NCX3 overexpression in cortical neurons exposed to anoxic preconditioning followed by oxygen and glucose deprivation plus reoxygenation.Collectively, our results revealed that GATA3/KMT2A complex epigenetically activates NCX3 gene transcription during ischemic preconditioning.

The isoform 2 of sodium-calcium exchanger family (NCX2) is selectively expressed in neuronal and glial cells where it participates in Ca2+-clearance following neuronal depolarization, synaptic plasticity, hippocampal-dependent learning and memory consolidation processes. On the other hand, NCX2 is also involved in a neuroprotective effect following stroke. Despite the relevance of this antiporter under physiological and pathophysiological conditions, no studies have been reported on its genetic/epigenetic regulation. Therefore, we identified, cloned, and characterized a transcriptional regulatory region (R3) of rat Slc8a2 gene encoding for NCX2. In particular, R3 sequence displayed a promoter activity in PC12, SH-SY5Y and U87MG cell lines consistent with their endogenous NCX2 expression levels. On the other hand, R3 failed to induce detectable luciferase activity in BHK cell line that does not express NCX2 under control conditions. These data support the hypothesis that R3 represents the promoter region of NCX2. Moreover, among several conserved binding sequences for transcription factors identified by in-silico analysis, we evaluated the transcriptional regulation and the binding sites of Sp1, Sp4, NFkB1, GATA2 and CREB1 on R3 sequence by using site-direct mutagenesis and ChIP assays. In particular, transfection of Sp1, Sp4, and CREB1 enhanced both R3 promoter activity and NCX2 transcription in PC12 cell line. More important, CREB1 transfection also enhanced NCX2 protein levels and NCX reverse mode activity in PC12 cells. Altogether, these data suggested that: (i) the identified region contained the regulatory promoter of the antiporter; (ii) NCX2 might represent a downstream effector of transcription factors involved in synaptic plasticity and neuronal survival.

Since the discovery of the three isoforms of the Na+/Ca2+ exchanger, NCX1, NCX2 and NCX3 in 1990s, many studies have been devoted to identifying their specific roles in different tissues under several physiological or pathophysiological conditions. In particular, several seminal experimental works laid the foundation for better understanding gene and protein structures, tissue distribution, and regulatory functions of each antiporter isoform. On the other hand, despite the efforts in the development of specific compounds selectively targeting NCX1, NCX2 or NCX3 to test their physiological or pathophysiological roles, several drawbacks hampered the achievement of these goals. In fact, at present no isoform-specific compounds have been yet identified. Moreover, these compounds, despite their potency, possess some nonspecific actions against other ion antiporters, ion channels, and channel receptors. As a result, it is difficult to discriminate direct effects of inhibition/activation of NCX isoforms from the inhibitory or stimulatory effects exerted on other antiporters, channels, receptors, or enzymes. To overcome these difficulties, some research groups used transgenic, knock-out and knock-in mice for NCX isoforms as the most straightforward and fruitful strategy to characterize the biological role exerted by each antiporter isoform. The present review will survey the techniques used to study the roles of NCXs and the current knowledge obtained from these genetic modified mice focusing on the advantages obtained with these strategies in understanding the contribution exerted by each isoform.

Methylmercury (MeHg) causes neuronal death through different pathways. Particularly, we found that in cortical neurons it increased the expression of Repressor Element-1 Silencing Transcription Factor (REST), Histone Deacetylase (HDAC)4, Specificity Protein (Sp)1, Sp4 and reduced the levels of Brain-Derived Neurotrophic Factor (BDNF). Herein, in rat cortical neurons we investigated whether microRNA (miR)206 can modulate MeHg-induced cell death by regulating REST/HDAC4/Sp1/Sp4/BDNF axis. MeHg (1µM) reduced miR206 expression after both 12 and 24 hours and miR206 transfection prevented MeHg-induced neuronal death. Furthermore, miR206 reverted MeHg-induced REST and Sp4 increase and BDNF reduction at gene and protein level, and reverted HDAC4 protein increase, but not HDAC4 mRNA up-regulation. Moreover, since no miR206 seed sequences were identified in the 3'-untranslated regions (3'-UTR) of REST and SP4, we investigated the role of JunD, that presents a consensus motif on REST, Sp4 and BDNF promoters. Indeed, MeHg increased JunD mRNA and protein levels, and JunD knockdown counteracted MeHg-induced REST, Sp4 increase, but not BDNF reduction. Furthermore, we identified a miR206 binding site in the 3'-UTR of JunD mRNA (miR206/JunD) and mutagenesis of miR206/JunD site reverted JunD luciferase activity reduction induced by miR206. Finally, miR206 prevented MeHg-increased JunD binding to REST and Sp4 promoters, and MeHg-reduced BDNF expression was determined by the increase of HDAC4 binding on BDNF promoter IV. Collectively, these results suggest that miR206 down-regulation induced by MeHg exposure determines an up-regulation of HDAC4, that in turn down-regulated BDNF, and the activation of JunD that, by binding REST and Sp4 gene promoters, increased their expression.

The Na

In humans, mutation of glycine 93 to alanine of Cu++/Zn++ superoxide dismutase type-1 (SOD1-G93 A) has been associated to some familial cases of Amyotrophic Lateral Sclerosis (ALS). Several evidence proposed the involvement of environmental pollutants that like mercury could accelerate ALS symptoms. SH-SY5Y cells stably transfected with SOD1 and G93 A mutant of SOD1 constructs were exposed to non-toxic concentrations (0.01 μM) of ethylmercury thiosalicylate (thimerosal) for 24 h. Interestingly, we found that thimerosal, in SOD1-G93 A cells, but not in SOD1 cells, reduced cell survival. Furthermore, thimerosal-induced cell death occurred in a concentration dependent-manner and was prevented by the Sirtuin 1 (SIRT1) activator Resveratrol (RSV). Moreover, thimerosal decreased the protein expression of transcription factor Downstream Regulatory Element Antagonist Modulator (DREAM), but not DREAM gene. Interestingly, DREAM reduction was blocked by co-treatment with RSV, suggesting the participation of SIRT1 in determining this effect. Immunoprecipitation experiments in SOD1-G93 A cells exposed to thimerosal demonstrated that RSV increased DREAM deacetylation and reduced its polyubiquitination. In addition, RSV counteracted thimerosal-enhanced prodynorphin (PDYN) mRNA, a DREAM target gene. Furthermore, cortical neurons transiently transfected with SOD1-G93 A construct and exposed to thimerosal (0.5 μM/24 h) showed a reduction of DREAM and an up-regulation of the prodynorphin gene. Importantly, both the treatment with RSV or the transfection of siRNA against prodynorphin significantly reduced thimerosal-induced neurotoxicity, while DREAM knocking-down potentiated thimerosal-reduced cell survival. These results demonstrate the particular vulnerability of SOD1-G93 A neuronal cells to thimerosal and that RSV via SIRT1 counteracts the neurodetrimental effect of this toxicant by preventing DREAM reduction and prodynorphin up-regulation.

Sodium-calcium exchanger (NCX) 1 and 3, have been demonstrated to play a relevant role in controlling the intracellular homeostasis of sodium and calcium ions in physiological and patho-physiological conditions. While NCX1 and NCX3 knocking-down have been both implicated in brain ischemia, several aspects of the epigenetic regulation of these two antiporters transcription were not yet well characterized. In response to stroke, NCX1 and NCX3 transcriptional regulation occurs from specific promoter sequences. Several evidences have shown that the expression of NCX1 and NCX3 can be determined by epigenetic modifications, consisting in changes of the histone acetylation levels on their promoter sequences. An interesting issue is that histone modifications at the NCX1 and NCX3 promoters could be linked to neurodegeneration occurring after stroke. Therefore, identifying the epigenetic regulation at the NCX1 and NCX3 promoters could permit to identify new molecular targets that can open new strategies for stroke treatment. The current review reassumes the recent knowledge of histone modifications of NCX1 and NCX3 genes in brain in physiological and patho-physiological conditions.

Our previous study showed that the environmental neurotoxicant non-dioxin-like polychlorinated biphenyl (PCB)-95 increases RE1-silencing transcription factor (REST) expression, which is related to necrosis, but not apoptosis, of neurons. Meanwhile, necroptosis is a type of a programmed necrosis that is positively regulated by receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) and negatively regulated by caspase-8. Here we evaluated whether necroptosis contributes to PCB-95-induced neuronal death through REST up-regulation. Our results demonstrated that in cortical neurons PCB-95 increased RIPK1, RIPK3, and MLKL expression and decreased caspase-8 at the gene and protein level. Furthermore, the RIPK1 inhibitor necrostatin-1 or siRNA-mediated RIPK1, RIPK3 and MLKL expression knockdown significantly reduced PCB-95-induced neuronal death. Intriguingly, PCB-95-induced increases in RIPK1, RIPK3, MLKL expression and decreases in caspase-8 expression were reversed by knockdown of REST expression with a REST-specific siRNA (siREST). Notably, in silico analysis of the rat genome identified a REST consensus sequence in the caspase-8 gene promoter (Casp8-RE1), but not the RIPK1, RIPK3 and MLKL promoters. Interestingly, in PCB-95-treated neurons, REST binding to the Casp8-RE1 sequence increased in parallel with a reduction in its promoter activity, whereas under the same experimental conditions, transfection of siREST or mutation of the Casp8-RE1 sequence blocked PCB-95-induced caspase-8 reduction. Since RIPK1, RIPK3 and MLKL rat genes showed no putative REST binding site, we assessed whether the transcription factor cAMP Responsive Element Binding Protein (CREB), which has a consensus sequence in all three genes, affected neuronal death. In neurons treated with PCB-95, CREB protein expression decreased in parallel with a reduction in binding to the RIPK1, RIPK3 and MLKL gene promoter sequence. Furthermore, CREB overexpression was associated with reduced promoter activity of the RIPK1, RIPK3 and MLKL genes. Collectively, these results indicate that PCB-95 was associated with REST-induced necroptotic cell death by increasing RIPK1, RIPK3 and MLKL expression and reducing caspase-8 levels. In addition, since REST is involved in several neurological disorders, therapies that block REST-induced necroptosis could be a new strategy to revert the neurodetrimental effects associated to its overexpression.

The present work aimed at the development and application of a lipid-based nanocarrier for targeted delivery of nucleic acids to glioblastoma (GBM). For this purpose, chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells while showing no affinity for non-neoplastic cells, was covalently coupled to liposomes encapsulating antisense oligonucleotides (asOs) or small interfering RNAs (siRNAs). The resulting targeted nanoparticles, designated CTX-coupled stable nucleic acid lipid particles (SNALPs), exhibited excellent features for in vivo application, namely small size (