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.

Anti-miRNA103/107 encapsulated in transferrin-conjugated lipid nanoparticles crosses blood-brain barrier and reduces brain ischemic damage.

MicroRNA (miRNA), by post-transcriptionally regulating the expression of genes involved in stroke response, represents important effectors in stroke pathophysiology. Recently, the 103/107 miRNA family emerged as a possible therapeutic target in stroke, as it controls the expression of sodium calcium exchanger 1, a plasma membrane transporter that plays a fundamental role in stroke pathophysiology. Although the neuroprotective properties of this and other miRNAs are promising, several pharmacokinetic drawbacks remain to be faced for the development of a translatable therapy based on small RNAs in CNS diseases. In the present study, to overcome these limitations, the anti-miRNA103/107 was encapsulated in specific preparations of lipid nanoparticles (LNPs), and their effectiveness was evaluated both in an in vitro model of hypoxia represented by primary neuronal cortical cultures exposed to oxygen and glucose deprivation followed by reoxygenation, and in an in vivo model of stroke obtained in rats exposed to transient occlusion of the middle cerebral artery. The results of the present study demonstrated that the encapsulation of anti-miRNA103/107 in transferrin-conjugated PEG-stabilized LNPs allowed the blood-brain barrier crossing and significantly reduced brain ischemic damage. The present achievements pave the way for the exploitation of a systemic intravenous miRNA delivery strategy in stroke therapy.

Correction: Iannuzzi et al. Might Fibroblasts from Patients with Alzheimer's Disease Reflect the Brain Pathology? A Focus on the Increased Phosphorylation of Amyloid Precursor Protein Tyr682 Residue. Brain Sci. 2021, 11, 103.

In the original article [...].

Chronic exposure to l-BMAA cyanotoxin induces cytoplasmic TDP-43 accumulation and glial activation, reproducing an amyotrophic lateral sclerosis-like phenotype in mice.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive and often fatal neurodegenerative disease characterized by the loss of Motor Neurons (MNs) in spinal cord, motor cortex and brainstem. Despite significant efforts in the field, the exact pathogenetic mechanisms underlying both familial and sporadic forms of ALS have not been fully elucidated, and the therapeutic possibilities are still very limited. Here we investigate the molecular mechanisms of neurodegeneration induced by chronic exposure to the environmental cyanotoxin L-BMAA, which causes a form of ALS/Parkinson's disease (PD) in several populations consuming food and/or water containing high amounts of this compound. METHODS: In this effort, mice were chronically exposed to L-BMAA and analyzed at different time points to evaluate cellular and molecular alterations and behavioral deficits, performing MTT assay, immunoblot, immunofluorescence and immunohistochemistry analysis, and behavioral tests. RESULTS: We found that cyanotoxin L-BMAA determines apoptotic cell death and a marked astrogliosis in spinal cord and motor cortex, and induces neurotoxicity by favoring TDP-43 cytoplasmic accumulation. CONCLUSIONS: Overall, our results characterize a new versatile neurotoxic animal model of ALS that may be useful for the identification of new druggable targets to develop innovative therapeutic strategies for this disease.

Emerging Role of DREAM in Healthy Brain and Neurological Diseases.

The downstream regulatory element antagonist modulator (DREAM) is a multifunctional Ca2+-sensitive protein exerting a dual mechanism of action to regulate several Ca2+-dependent processes. Upon sumoylation, DREAM enters in nucleus where it downregulates the expression of several genes provided with a consensus sequence named dream regulatory element (DRE). On the other hand, DREAM could also directly modulate the activity or the localization of several cytosolic and plasma membrane proteins. In this review, we summarize recent advances in the knowledge of DREAM dysregulation and DREAM-dependent epigenetic remodeling as a central mechanism in the progression of several diseases affecting central nervous system, including stroke, Alzheimer's and Huntington's diseases, amyotrophic lateral sclerosis, and neuropathic pain. Interestingly, DREAM seems to exert a common detrimental role in these diseases by inhibiting the transcription of several neuroprotective genes, including the sodium/calcium exchanger isoform 3 (NCX3), brain-derived neurotrophic factor (BDNF), pro-dynorphin, and c-fos. These findings lead to the concept that DREAM might represent a pharmacological target to ameliorate symptoms and reduce neurodegenerative processes in several pathological conditions affecting central nervous system.

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.

L-Ornithine L-Aspartate Restores Mitochondrial Function and Modulates Intracellular Calcium Homeostasis in Parkinson's Disease Models.

The altered crosstalk between mitochondrial dysfunction, intracellular Ca2+ homeostasis, and oxidative stress has a central role in the dopaminergic neurodegeneration. In the present study, we investigated the hypothesis that pharmacological strategies able to improve mitochondrial functions might prevent neuronal dysfunction in in vitro models of Parkinson's disease. To this aim, the attention was focused on the amino acid ornithine due to its ability to cross the blood-brain barrier, to selectively reach and penetrate the mitochondria through the ornithine transporter 1, and to control mitochondrial function. To pursue this issue, experiments were performed in human neuroblastoma cells SH-SY5Y treated with rotenone and 6-hydroxydopamine to investigate the pharmacological profile of the compound L-Ornithine-L-Aspartate (LOLA) as a new potential therapeutic strategy to prevent dopaminergic neurons' death. In these models, confocal microscopy experiments with fluorescent dyes measuring mitochondrial calcium content, mitochondrial membrane potential, and mitochondrial ROS production, demonstrated that LOLA improved mitochondrial functions. Moreover, by increasing NCXs expression and activity, LOLA also reduced cytosolic [Ca2+] thanks to its ability to modulate NO production. Collectively, these results indicate that LOLA, by interfering with those mitochondrial mechanisms related to ROS and RNS production, promotes mitochondrial functional recovery, thus confirming the tight relationship existing between cytosolic ionic homeostasis and cellular metabolism depending on the type of insult applied.

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

INTRODUCTION AND AIMS: 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. METHODS: RGWE effects on ischemic damage and neurological function were evaluated in adult rats subjected to transient occlusion of the Middle Cerebral Artery (tMCAO), receiving two intraperitoneal injections of RGWE, 100 and 300 min after the induction of ischemia. In addition, astroglial and microglial activation was measured as GFAP and IBA-1 expression by immunofluorescence and confocal microscopy analysis. RESULTS: Treatment with RGWE containing 10 mg/kg of Rutin, the major component, ameliorates the ischemic damage and improves neurological performances. Interestingly, the pro-inflammatory states of astrocytes and microglia, respectively detected by using C3 and iNOS markers, were significantly reduced in ipsilateral cortical and striatal areas in ischemic RGWE-treated rats. CONCLUSIONS: RGWE shows a neuroprotective effect on brain infarct volume extent in a transient focal cerebral ischemia model and this effect was paralleled by the prevention of pro-inflammatory astroglial and microglial activation. Collectively, our findings support the idea that natural compounds may represent potential therapeutic opportunities against ischemic stroke.

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.

IN BRAIN POST-ISCHEMIC PLASTICITY, Na+/Ca2+ EXCHANGER 1 AND Ascl1 INTERVENE IN MICROGLIA-DEPENDENT CONVERSION OF ASTROCYTES INTO NEURONAL LINEAGE.

The intricate glia interaction occurring after stroke strongly depend on the maintenance of intraglial ionic homeostasis. Among the several ionic channels and transporters, the plasmamembrane Na+/Ca2+ exchanger (NCX) represents a key player in maintaining astroglial Na+ and Ca2+ homeostasis. Here, using a combined in vitro, in vivo and ex vivo experimental strategy we evaluated whether microglia responding to ischemic injury may influence the morphological and the transcriptional plasticity of post-ischemic astrocytes. Astrocyte plasticity was monitored by the expression of the transcription factor Acheate-scute like 1 (Ascl1), which plays a central role in the commitment of astrocytes towards the neuronal lineage. Furthermore, we explored the implication of NCX1 expression and activity in mediating Ascl1-dependent post-ischemic astrocyte remodeling. We demonstrated that: (a) in astrocytes co-cultured with microglia the exposure to oxygen and glucose deprivation followed by 7 days of reoxygenation induced a prevalence of bipolar astrocytes overexpressing Ascl1 and NCX1, whereas this did not occur in monocultured astrocytes; (b) the reoxygenation of anoxic astrocytes with the conditioned medium derived from IL-4 stimulated microglia strongly elicited the astrocytic co-expression of Ascl1 and NCX1; (c) Ascl1 expression in anoxic astrocytes was dependenton NCX1 since its silencing prevented Ascl1 expression both in in vitro and in post-ischemic ex vivo experimental conditions. Collectively, the results of our study support the idea that, after brain ischemia, astrocyte-microglia crosstalk can influence astrocytic morphology and its Ascl1 expression. This phenomenon is strictly dependent on ischemia-induced increase of NCX1 which in turn induces Ascl1 overexpression possibly through astrocytic Ca2+ elevation.

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.

Na+/Ca2+ exchanger isoform 1 takes part to the Ca2+-related prosurvival pathway of SOD1 in primary motor neurons exposed to beta-methylamino-L-alanine.

BACKGROUND: The cycad neurotoxin beta-methylamino-L-alanine (L-BMAA), one of the environmental trigger factor for amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC), may cause neurodegeneration by disrupting organellar Ca2+ homeostasis. Through the activation of Akt/ERK1/2 pathway, the Cu,Zn-superoxide dismutase (SOD1) and its non-metallated form, ApoSOD1, prevent endoplasmic reticulum (ER) stress-induced cell death in motor neurons exposed to L-BMAA. This occurs through the rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in part flowing from the extracellular compartment and in part released from ER. However, the molecular components of this mechanism remain uncharacterized. METHODS: By an integrated approach consisting on the use of siRNA strategy, Western blotting, confocal double- labeling immunofluorescence, patch-clamp electrophysiology, and Fura 2-/SBFI-single-cell imaging, we explored in rat motor neuron-enriched cultures the involvement of the plasma membrane proteins Na+/Ca2+ exchanger (NCX) and purinergic P2X7 receptor as well as that of the intracellular cADP-ribose (cADPR) pathway, in the neuroprotective mechanism of SOD1. RESULTS: We showed that SOD1-induced [Ca2+]i rise was prevented neither by A430879, a P2X7 receptor specific antagonist or 8-bromo-cADPR, a cell permeant antagonist of cADP-ribose, but only by the pan inhibitor of NCX, CB-DMB. The same occurred for the ApoSOD1. Confocal double labeling immunofluorescence showed a huge expression of plasmalemmal NCX1 and intracellular NCX3 isoforms. Furthermore, we identified NCX1 reverse mode as the main mechanism responsible for the neuroprotective ER Ca2+ refilling elicited by SOD1 and ApoSOD1 through which they promoted translocation of active Akt in the nuclei of a subset of primary motor neurons. Finally, the activation of NCX1 by the specific agonist CN-PYB2 protected motor neurons from L-BMAA-induced cell death, mimicking the effect of SOD1. CONCLUSION: Collectively, our data indicate that SOD1 and ApoSOD1 exert their neuroprotective effect by modulating ER Ca2+ content through the activation of NCX1 reverse mode and Akt nuclear translocation in a subset of primary motor neurons. Video Abstract.

Na+/Ca2+ exchanger isoform 1 (NCX1) and canonical transient receptor potential channel 6 (TRPC6) are recruited by STIM1 to mediate Store-Operated Calcium Entry in primary cortical neurons.

Excessive calcium (Ca2+) release from the endoplasmic reticulum (ER) represents an important hallmark of several neurodegenerative diseases. ER is recharged from Ca2+ through the so-called Store-Operated Calcium Entry (SOCE) thus providing Ca2+ signals to regulate critical cell functions. Single transmembrane-spanning domain protein stromal interacting molecule 1 (STIM1), mainly residing in the ER, and plasmalemmal channel Orai1 represent the SOCE key components at neuronal level. However, many other proteins participate to ER Ca2+ refilling including the Na+/Ca2+ exchanger isoform 1 (NCX1), whose regulation by ER remains unknown. In this study, we tested the possibility that neuronal NCX1 may take part to SOCE through the interaction with STIM1. In rat primary cortical neurons and in nerve growth factor (NGF)-differentiated PC12 cells NCX1 knocking down by siRNA strategy significantly prevented SOCE as well as SOCE pharmacological inhibition by SKF-96365 and 2-APB. A significant reduction of SOCE was recorded also in synaptosomes from ncx1-/- mice brain compared with ncx1+/+ mice. Double labeling confocal experiments showed a large co-localization between NCX1 and STIM1 in rat primary cortical neurons. Accordingly, NCX1 and STIM1 co-immunoprecipitated and functionally interacted each other during ischemic preconditioning, a phenomenon inducing ischemic tolerance. However, STIM1 knocking down reduced NCX1 activity recorded by either patch-clamp electrophysiology or Fura-2 single-cell microfluorimetry. Furthermore, canonical transient receptor potential channel 6 (TRPC6) was identified as the mechanism mediating local increase of sodium (Na+) useful to drive NCX1 reverse mode and, therefore, NCX1-mediated Ca2+ refilling. In fact, TRPC6 not only interacted with STIM1, as shown by the co-localization and co-immunoprecipitation with the ER Ca2+ sensor, but it also mediated 1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester (SBFI)-monitored Na+ increase elicited by thapsigargin in primary cortical neurons. Accordingly, efficient TRPC6 knockdown prevented thapsigargin-induced intracellular Na+ elevation and SOCE. Collectively, we identify NCX1 as a new partner of STIM1 in mediating SOCE, whose activation in the reverse mode may be facilitated by the local increase of Na+ concentration due to the interaction between STIM1 and TRPC6 in primary cortical neurons.

The Na+/Ca2+ Exchanger 3 Is Functionally Coupled With the NaV1.6 Voltage-Gated Channel and Promotes an Endoplasmic Reticulum Ca2+ Refilling in a Transgenic Model of Alzheimer's Disease.

The remodelling of neuronal ionic homeostasis by altered channels and transporters is a critical feature of the Alzheimer's disease (AD) pathogenesis. Different reports converge on the concept that the Na+/Ca2+ exchanger (NCX), as one of the main regulators of Na+ and Ca2+ concentrations and signalling, could exert a neuroprotective role in AD. The activity of NCX has been found to be increased in AD brains, where it seemed to correlate with an increased neuronal survival. Moreover, the enhancement of the NCX3 currents (INCX) in primary neurons treated with the neurotoxic amyloid ß 1-42 (Aß1-42) oligomers prevented the endoplasmic reticulum (ER) stress and neuronal death. The present study has been designed to investigate any possible modulation of the INCX, the functional interaction between NCX and the NaV1.6 channel, and their impact on the Ca2+ homeostasis in a transgenic in vitro model of AD, the primary hippocampal neurons from the Tg2576 mouse, which overproduce the Aß1-42 peptide. Electrophysiological studies, carried in the presence of siRNA and the isoform-selective NCX inhibitor KB-R7943, showed that the activity of a specific NCX isoform, NCX3, was upregulated in its reverse, Ca2+ influx mode of operation in the Tg2576 neurons. The enhanced NCX activity contributed, in turn, to increase the ER Ca2+ content, without affecting the cytosolic Ca2+ concentrations of the Tg2576 neurons. Interestingly, our experiments have also uncovered a functional coupling between NCX3 and the voltage-gated NaV1.6 channels. In particular, the increased NaV1.6 currents appeared to be responsible for the upregulation of the reverse mode of NCX3, since both TTX and the Streptomyces griseolus antibiotic anisomycin, by reducing the NaV1.6 currents, counteracted the increase of the INCX in the Tg2576 neurons. In agreement, our immunofluorescence analyses revealed that the NCX3/NaV1.6 co-expression was increased in the Tg2576 hippocampal neurons in comparison with the WT neurons. Collectively, these findings indicate that NCX3 might intervene in the Ca2+ remodelling occurring in the Tg2576 primary neurons thus emerging as a molecular target with a neuroprotective potential, and provide a new outcome of the NaV1.6 upregulation related to the modulation of the intracellular Ca2+ concentrations in AD neurons.

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.

New Insights into the Structure-Activity Relationship and Neuroprotective Profile of Benzodiazepinone Derivatives of Neurounina-1 as Modulators of the Na+/Ca2+ Exchanger Isoforms.

Due to the neuroprotective role of the Na+/Ca2+ exchanger (NCX) isoforms NCX1 and NCX3, we synthesized novel benzodiazepinone derivatives of the unique NCX activator Neurounina-1, named compounds 1-19. The derivatives are characterized by a benzodiazepinonic nucleus linked to five- or six-membered cyclic amines via a methylene, ethylene, or acetyl spacer. The compounds have been screened on NCX1/NCX3 isoform activities by a high-throughput screening approach, and the most promising were characterized by patch-clamp electrophysiology and Fura-2AM video imaging. We identified two novel modulators of NCX: compound 4, inhibiting NCX1 reverse mode, and compound 14, enhancing NCX1 and NCX3 activity. Compound 1 displayed neuroprotection in two preclinical models of brain ischemia. The analysis of the conformational and steric features led to the identification of the molecular volume required for selective NCX1 activation for mixed NCX1/NCX3 activation or for NCX1 inhibition, providing the first prototypal model for the design of optimized isoform modulators.

Hemorrhagic Stroke Induces a Time-Dependent Upregulation of miR-150-5p and miR-181b-5p in the Bloodstream.

To date, the only effective pharmacological treatment for ischemic stroke is limited to the clinical use of recombinant tissue plasminogen activator (rtPA), although endovascular therapy has also emerged as an effective treatment for acute ischemic stroke. Unfortunately, the benefit of this treatment is limited to a 4.5-h time window. Most importantly, the use of rtPA is contraindicated in the case of hemorrhagic stroke. Therefore, the identification of a reliable biomarker to distinguish hemorrhagic from ischemic stroke could provide several advantages, including an earlier diagnosis, a better treatment, and a faster decision on ruling out hemorrhage so that tPA may be administered earlier. microRNAs (miRNAs) are stable non-coding RNAs crucially involved in the downregulation of gene expression via mRNA cleavage or translational repression. In the present paper, taking advantage of three preclinical animal models of stroke, we compared the miRNA blood levels of animals subjected to permanent or transient middle cerebral artery occlusion (MCAO) or to collagenase-induced hemorrhagic stroke. Preliminarily, we examined the rat miRNome in the brain tissue of ischemic and sham-operated rats; then, we selected those miRNAs whose expression was significantly modulated after stroke to create a list of miRNAs potentially involved in stroke damage. These selected miRNAs were then evaluated at different time intervals in the blood of rats subjected to permanent or transient focal ischemia or to hemorrhagic stroke. We found that four miRNAs-miR-16-5p, miR-101a-3p, miR-218-5p, and miR-27b-3p-were significantly upregulated in the plasma of rats 3 h after permanent MCAO, whereas four other different miRNAs-miR-150-5p, let-7b-5p, let-7c-5p, and miR-181b-5p-were selectively upregulated by collagenase-induced hemorrhagic stroke. Collectively, our study identified some selective miRNAs expressed in the plasma of hemorrhagic rats and pointed out the importance of a precise time point measurement to render more reliable the use of miRNAs as stroke biomarkers.

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.

Rebound effects of NCX3 pharmacological inhibition: A novel strategy to accelerate myelin formation in oligodendrocytes.

The Na+/Ca2+ exchanger NCX3 is an important regulator of sodium and calcium homeostasis in oligodendrocyte lineage. To date, no information is available on the effects resulting from prolonged exposure to NCX3 blockers and subsequent drug washout in oligodendroglia. Here, we investigated, by means of biochemical, morphological and functional analyses, the pharmacological effects of the NCX3 inhibitor, the 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED), on NCXs expression and activity, as well as intracellular [Na+]i and [Ca2+]i levels, during treatment and following drug washout both in human MO3.13 oligodendrocytes and rat primary oligodendrocyte precursor cells (OPCs). BED exposure antagonized NCX activity, induced OPCs proliferation and [Na+]i accumulation. By contrast, 2 days of BED washout after 4 days of treatment significantly upregulated low molecular weight NCX3 proteins, reversed NCX activity, and increased intracellular [Ca2+]i. This BED-free effect was accompanied by an upregulation of NCX3 expression in oligodendrocyte processes and accelerated expression of myelin markers in rat primary oligodendrocytes. Collectively, our findings show that the pharmacological inhibition of the NCX3 exchanger with BED blocker maybe followed by a rebound increase in NCX3 expression and reversal activity that accelerate myelin sheet formation in oligodendrocytes. In addition, they indicate that a particular attention should be paid to the use of NCX inhibitors for possible rebound effects, and suggest that further studies will be necessary to investigate whether selective pharmacological modulation of NCX3 exchanger may be exploited to benefit demyelination and remyelination in demyelinating diseases.