Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex.

BACKGROUND: REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na+/Ca2+exchanger-1 (Ncx1) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence (Ht-RE1) that participates in neuronal Ncx1 transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in Ncx1 transcriptional regulation after stroke. METHODS AND RESULTS: Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's t test was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P<0.05. CONCLUSIONS: If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke 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.

Identification and characterization of the promoter and transcription factors regulating the expression of cerebral sodium/calcium exchanger 2 (NCX2) gene.

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.

The Transcriptional Complex Sp1/KMT2A by Up-Regulating Restrictive Element 1 Silencing Transcription Factor Accelerates Methylmercury-Induced Cell Death in Motor Neuron-Like NSC34 Cells Overexpressing SOD1-G93A.

Methylmercury (MeHg) exposure has been related to amyotrophic lateral sclerosis (ALS) pathogenesis and molecular mechanisms of its neurotoxicity has been associated to an overexpression of the Restrictive Element 1 Silencing Transcription factor (REST). Herein, we evaluated the possibility that MeHg could accelerate neuronal death of the motor neuron-like NSC34 cells transiently overexpressing the human Cu2+/Zn2+superoxide dismutase 1 (SOD1) gene mutated at glycine 93 (SOD1-G93A). Indeed, SOD1-G93A cells exposed to 100 nM MeHg for 24 h showed a reduction in cell viability, as compared to cells transfected with empty vector or with unmutated SOD1 construct. Interestingly, cell survival reduction in SOD1-G93A cells was associated with an increase of REST mRNA and protein levels. Furthermore, MeHg increased the expression of the transcriptional factor Sp1 and promoted its binding to REST gene promoter sequence. Notably, Sp1 knockdown reverted MeHg-induced REST increase. Co-immunoprecipitation experiments demonstrated that Sp1 physically interacted with the epigenetic writer Lysine-Methyltransferase-2A (KMT2A). Moreover, knocking-down of KMT2A reduced MeHg-induced REST mRNA and protein increase in SOD1-G93A cells. Finally, we found that MeHg-induced REST up-regulation triggered necropoptotic cell death, monitored by RIPK1 increased protein expression. Interestingly, REST knockdown or treatment with the necroptosis inhibitor Necrostatin-1 (Nec) decelerated MeH-induced cell death in SOD1-G93A cells. Collectively, this study demonstrated that MeHg hastens necroptotic cell death in SOD1-G93A cells via Sp1/KMT2A complex, that by epigenetic mechanisms increases REST gene expression.

GATA3 (GATA-Binding Protein 3)/KMT2A (Lysine-Methyltransferase-2A) Complex by Increasing H3K4-3me (Trimethylated Lysine-4 of Histone-3) Upregulates NCX3 (Na+-Ca2+ Exchanger 3) Transcription and Contributes to Ischemic Preconditioning Neuroprotection.

Background and Purpose: 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. 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 occlusion­induced 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.

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

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.

Transcriptional and epigenetic regulation of ncx1 and ncx3 in the brain.

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.

miR-206 Reduces the Severity of Motor Neuron Degeneration in the Facial Nuclei of the Brainstem in a Mouse Model of SMA.

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.

HDAC4 and HDAC5 form a complex with DREAM that epigenetically down-regulates NCX3 gene and its pharmacological inhibition reduces neuronal stroke damage.

The histone deacetylases (HDACs)-dependent mechanisms regulating gene transcription of the Na+/Ca+ exchanger isoform 3 (ncx3) after stroke are still unknown. Overexpression or knocking-down of HDAC4/HDAC5 down-regulates or increases, respectively, NCX3 mRNA and protein. Likewise, MC1568 (class IIa HDACs inhibitor), but not MS-275 (class I HDACs inhibitor) increased NCX3 promoter activity, gene and protein expression. Furthermore, HDAC4 and HDAC5 physically interacted with the transcription factor downstream regulatory element antagonist modulator (DREAM). As MC1568, DREAM knocking-down prevented HDAC4 and HDAC5 recruitment to the ncx3 promoter. Importantly, DREAM, HDAC4, and HDAC5 recruitment to the ncx3 gene was increased in the temporoparietal cortex of rats subjected to transient middle cerebral artery occlusion (tMCAO), with a consequent histone-deacetylation of ncx3 promoter. Conversely, the tMCAO-induced NCX3 reduction was prevented by intracerebroventricular injection of siDREAM, siHDAC4, and siHDAC5. Notably, MC1568 prevented oxygen glucose deprivation plus reoxygenation and tMCAO-induced neuronal damage, whereas its neuroprotective effect was abolished by ncx3 knockdown. Collectively, we found that: (1) DREAM/HDAC4/HDAC5 complex epigenetically down-regulates ncx3 gene transcription after stroke, and (2) pharmacological inhibition of class IIa HDACs reduces stroke-induced neurodetrimental effects.

Anti-miR-223-5p Ameliorates Ischemic Damage and Improves Neurological Function by Preventing NCKX2 Downregulation after Ischemia in Rats.

It has been demonstrated that the K+-dependent Na+/Ca2+ exchanger, NCKX2, is a new promising stroke neuroprotective target. However, because no pharmacological activator of NCKX2 is still available, microRNA (miRNA) may represent an alternative method to modulate NCKX2 expression. In particular, by bioinformatics analysis, miR-223-5p emerged as a possible modulator of NCKX2 expression. In the light of these premises, the aims of the present study were: (1) to evaluate miR-223-5p and NCKX2 expression in the temporoparietal cortex and striatum of rats subjected to transient middle cerebral artery occlusion; (2) to evaluate whether miR-223-5p targets the 3' UTR of the NCKX2 transcript; and (3) to evaluate the effect of miR-223-5p modulation on brain ischemic volume and neurological deficits. Our results showed that miR-223-5p expression increased in a time-dependent manner in the striatum of ischemic rats in parallel with NCKX2 downregulation, and that the transfection of cortical neurons with miR-223-5p induced a reduction of NCKX2 expression. Moreover, a luciferase assay showed that miR-223-5p specifically interacts with the NCKX2 3' UTR subregion (+7037 to +8697), thus repressing NCKX2 translation. More interestingly, intracerebroventricular infusion of anti-miR-223-5p prevented NCKX2 downregulation after ischemia, thus promoting neuroprotection. The present findings support the idea that blocking miR-223-5p by antimiRNA is a reasonable strategy to reduce the neurodetrimental effect induced by NCKX2 downregulation during brain ischemia.

Erratum: Author Correction: Na+/Ca2+ exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca2+ regulation during neuronal differentiation.

[This corrects the article DOI: 10.1038/s41420-017-0018-1.].

D-Aspartate treatment attenuates myelin damage and stimulates myelin repair.

Glutamate signaling may orchestrate oligodendrocyte precursor cell (OPC) development and myelin regeneration through the activation of glutamate receptors at OPC-neuron synapses. D-Aspartate is a D-amino acid exerting modulatory actions at glutamatergic synapses. Chronic administration of D-Aspartate has been proposed as therapeutic treatment in diseases related to myelin dysfunction and NMDA receptors hypofunction, including schizophrenia and cognitive deficits. Here, we show, by using an in vivo remyelination model, that administration of D-Aspartate during remyelination improved motor coordination, accelerated myelin recovery, and significantly increased the number of small-diameter myelinated axons. Chronically administered during demyelination, D-Aspartate also attenuated myelin loss and inflammation. Interestingly, D-Aspartate exposure stimulated OPC maturation and accelerated developmental myelination in organotypic cerebellar slices. D-Aspartate promoting effects on OPC maturation involved the activation of glutamate transporters, AMPA and NMDA receptors, and the Na+/Ca2+ exchanger NCX3. While blocking NMDA or NCX3 significantly prevented D-Aspartate-induced [Ca2+]i oscillations, blocking AMPA and glutamate transporters prevented both the initial and oscillatory [Ca2+]i response as well as D-Aspartate-induced inward currents in OPC Our findings reveal that D-Aspartate treatment may represent a novel strategy for promoting myelin recovery.

Resveratrol treatment reduces the vulnerability of SH-SY5Y cells and cortical neurons overexpressing SOD1-G93A to Thimerosal toxicity through SIRT1/DREAM/PDYN pathway.

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.

Models and methods for conditioning the ischemic brain.

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 Maintenance in ALS: Focus on New Therapeutic Targets.

Amyotrophic lateral sclerosis (ALS) is one of the most threatening neurodegenerative disease since it causes muscular paralysis for the loss of Motor Neurons in the spinal cord, brainstem and motor cortex. Up until now, no effective pharmacological treatment is available. Two forms of ALS have been described so far: 90% of the cases presents the sporadic form (sALS) whereas the remaining 10% of the cases displays the familiar form (fALS). Approximately 20% of fALS is associated with inherited mutations in the Cu, Zn-superoxide dismutase 1 (SOD1) gene. In the last decade, ionic homeostasis dysregulation has been proposed as the main trigger of the pathological cascade that brings to motor-neurons loss. In the light of these premises, the present review will analyze the involvement in ALS pathophysiology of the most well studied metal ions, i.e., calcium, sodium, iron, copper and zinc, with particular focus to the role of ionic channels and transporters able to contribute in the regulation of ionic homeostasis, in order to propose new putative molecular targets for future therapeutic strategies to ameliorate the progression of this devastating neurodegenerative disease.

Na+/Ca2+ exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca2+ regulation during neuronal differentiation.

Nuclear envelope (NE) is a Ca2+-storing organelle controlling neuronal differentiation through nuclear Ca2+ concentrations ([Ca2+]n). However, how [Ca2+]n regulates this important function remains unknown. Here, we investigated the role of the nuclear form of the Na+/Ca2+ exchanger 1(nuNCX1) during the different stages of neuronal differentiation and the involvement of PTEN/PI3'K/Akt pathway. In neuronal cells, nuNCX1 was detected on the inner membrane of the NE where protein expression and activity of the exchanger increased during NGF-induced differentiation. nuNCX1 activation by Na+-free perfusion induced a time-dependent activation of nuclear-resident PI3K/Akt pathway in isolated nuclei. To discriminate the contribution of nuNCX1 from those of plasma membrane NCX, we generated a chimeric protein composed of the fluorophore EYFP, the exchanger inhibitory peptide, and the nuclear localization signal, named XIP-NLS. Fura-2 measurements on single nuclei and patch-clamp experiments in whole-cell configuration showed that XIP-NLS selectively inhibited nuNCX1. Once it reached the nuclear compartment, XIP-NLS increased the nucleoplasmic Ca2+ peak elicited by ATP and reduced Akt phosphorylation, GAP-43 and MAP-2 expression through nuclear-resident PTEN induction. Furthermore, in accordance with the prevention of the neuronal phenotype, XIP-NLS significantly reduced TTX-sensitive Na+ currents and membrane potential during neuronal differentiation. The selective inhibition of nuNCX1 by XIP-NLS increased the percentage of ß III tubulin-positive immature neurons in mature cultures of MAP-2-positive cortical neurons, thus unraveling a new function for nuNCX1 in regulating neuronal differentiation through [Ca2+]n-dependent PTEN/PI3K/Akt pathway.

The miR206-JunD Circuit Mediates the Neurotoxic Effect of Methylmercury in Cortical Neurons.

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 (1 µM) reduced miR206 expression after both 12 and 24 h 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 upregulation. Moreover, since no miR206 seed sequences were identified in the 3'-untranslated regions (3'-UTRs) 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 downregulation induced by MeHg exposure determines an upregulation of HDAC4, that in turn downregulated BDNF, and the activation of JunD that, by binding REST and Sp4 gene promoters, increased their expression.

Preconditioning, induced by sub-toxic dose of the neurotoxin L-BMAA, delays ALS progression in mice and prevents Na+/Ca2+ exchanger 3 downregulation.

Preconditioning (PC) is a phenomenon wherein a mild insult induces resistance to a later, severe injury. Although PC has been extensively studied in several neurological disorders, no studies have been performed in amyotrophic lateral sclerosis (ALS). Here we hypothesize that a sub-toxic acute exposure to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA) is able to delay ALS progression in SOD1 G93A mice and that NCX3, a membrane transporter able to handle the deregulation of ionic homeostasis occurring during ALS, takes part to this neuroprotective effect. Preconditioning effect was examined on disease onset and duration, motor functions, and motor neurons in terms of functional declines and severity of histological damage in male and female mice. Our findings demonstrate that a sub-toxic dose of L-BMAA works as preconditioning stimulus and is able to delay ALS onset and to prolong ALS mice survival. Interestingly, preconditioning prevented NCX3 downregulation in SOD1 G93A mice spinal cord, leading to an increased number of motor neurons associated to a reduced astrogliosis, and reduced the denervation of neuromuscular junctions observed in SOD1 G93A mice. These protective effects were mitigated in ncx3+/- mice. This study established for the first time an animal model of preconditioning in ALS and candidates NCX3 as a new therapeutic target.

The neurotoxicant PCB-95 by increasing the neuronal transcriptional repressor REST down-regulates caspase-8 and increases Ripk1, Ripk3 and MLKL expression determining necroptotic neuronal death.

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.

p38/Sp1/Sp4/HDAC4/BDNF Axis Is a Novel Molecular Pathway of the Neurotoxic Effect of the Methylmercury.

The molecular pathways involved in methylmercury (MeHg)-induced neurotoxicity are not fully understood. Since pan-Histone deacetylases (HDACs) inhibition has been found to revert the neurodetrimental effect of MeHg, it appeared of interest to investigate whether the pattern of HDACs isoform protein expression is modified during MeHg-induced neurotoxicity and the transcriptional/transductional mechanisms involved. SH-SY5Y neuroblastoma cells treated with MeHg 1 µM for 12 and 24 h showed a significant increase of HDAC4 protein and gene expression, whereas the HDACs isoforms 1-3, 5, and 6 were unmodified. Furthermore, MeHg-induced HDAC4 increase was reverted when cells were transfected with siRNAs against specificity protein 1 (Sp1) and Sp4, that were both increased during MeHg exposure. Next we studied the role of extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) in MeHg-induced increase of Sp1, Sp4, and HDAC4 expression. As shown by Western Blot analysis MeHg exposure increased the phosphorylation of p38, but not of ERK and JNK. Notably, when p38 was pharmacologically blocked, MeHg-induced Sp1, Sp4 protein expression, and HDAC4 protein and gene expression was reverted. In addition, MeHg exposure increased the binding of HDAC4 to the promoter IV of the Brain-derived neurotrophic factor (BDNF) gene, determining its mRNA reduction, that was significantly counteracted by HDAC4 knocking down. Furthermore, rat cortical neurons exposed to MeHg (1 µM/24 h) showed an increased phosphorylation of p38, in parallel with an up-regulation of Sp1, Sp4, and HDAC4 and a down-regulation of BDNF proteins. Importantly, transfection of siRNAs against p38, Sp1, Sp4, and HDAC4 or transfection of vector overexpressing BDNF significantly blocked MeHg-induced cell death in cortical neurons. All these results suggest that p38/Sp1-Sp4/HDAC4/BDNF may represent a new pathway involved in MeHg-induced neurotoxicity.