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.

Volumetric Absorptive Microsampling (VAMS) for Targeted LC-MS/MS Determination of Tryptophan-Related Biomarkers.

L-Tryptophan (TRP) metabolites and related biomarkers play crucial roles in physiological functions, and their imbalances are implicated in central nervous system pathologies and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, schizophrenia and depression. The measurement of TRP metabolites and related biomarkers possesses great potential to elucidate the disease mechanisms, aid preclinical drug development, highlight potential therapeutic targets and evaluate the outcomes of therapeutic interventions. An effective, straightforward, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 24 TRP-related compounds in miniaturised murine whole blood samples. Sampling and sample pretreatment miniaturisation were achieved thanks to the development of a volumetric dried blood microsampling approach. Volumetric absorptive microsampling (VAMS) allows the accurate sampling of microvolumes of blood with advantages including, but not limited to, minimal sampling invasiveness, logistical improvements, method sustainability in terms of solvents and energy consumption, and improvement of animal studies in the framework of the 3Rs (Replacement, Reduction and Refinement) principles on animal welfare. The VAMS-LC-MS/MS method exhibited good selectivity, and correlation coefficient values for the calibration curves of each analyte were >0.9987. The limits of quantitation ranged from 0.1 to 25 ng/mL. The intra- and inter-day precisions in terms of RSD were <9.6%. All analytes were stable in whole blood VAMS samples stored at room temperature for at least 30 days with analyte losses < 14%. The developed method was successfully applied to the analysis of biological samples from mice, leading to the unambiguous determination of all the considered target analytes. This method can therefore be applied to analyse TRP metabolites and related biomarkers levels to monitor disease states, perform mechanistic studies and investigate the outcomes of therapeutic interventions.

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.

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.

Sp3/REST/HDAC1/HDAC2 Complex Represses and Sp1/HIF-1/p300 Complex Activates ncx1 Gene Transcription, in Brain Ischemia and in Ischemic Brain Preconditioning, by Epigenetic Mechanism.

The Na(+)-Ca(2+) exchanger 1 (NCX1) is reduced in stroke by the RE1-silencing transcription factor (REST), whereas it is increased in ischemic brain preconditioning (PC) by hypoxia-inducible factor 1 (HIF-1). Because ncx1 brain promoter (ncx1-Br) has five putative consensus sequences, named Sp1A-E, for the specificity protein (Sp) family of transcription factors (Sp1-4), we investigated the role of this family in regulating ncx1 transcription in rat cortical neurons. Here we found that Sp1 is a transcriptional activator, whereas Sp3 is a transcriptional repressor of ncx1, and that both bind ncx1-Br in a sequence-specific manner, modulating ncx1 transcription through the Sp1 sites C-E. Furthermore, by transient middle cerebral artery occlusion (tMCAO) in rats, the transcriptional repressors Sp3 and REST colocalized with the two histone-deacetylases (HDACs) HDAC1 and HDAC2 on the ncx1-Br, with a consequent hypoacetylation. Contrarily, in PC+tMCAO the transcriptional activators Sp1 and HIF-1 colocalized with histone acetyltransferase p300 on ncx1-Br with a consequent hyperacetylation. In addition, in neurons silenced with siRNA of NCX1 and subjected to oxygen and glucose deprivation (OGD) (3 h) plus reoxygenation (RX) (24 h), the neuroprotection of Class I HDAC inhibitor MS-275 was counteracted, whereas in neurons overexpressing NCX1 and subjected to ischemic preconditioning (PC+OGD/RX), the neurotoxic effect of p300 inhibitor C646 was prevented. Collectively, these results demonstrate that NCX1 expression is regulated by the Sp3/REST/HDAC1/HDAC2 complex in tMCAO and by the Sp1/HIF-1/p300 complex in PC+tMCAO and that epigenetic intervention, by modulating the acetylation of ncx1-Br, may be a strategy for the development of innovative therapeutic intervention in stroke.

Human mesenchymal stromal cell transplantation modulates neuroinflammatory milieu in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs), after intraparenchymal, intrathecal and endovenous administration, have been previously tested for cell therapy in amyotrophic lateral sclerosis in the SOD1 (superoxide dismutase 1) mouse. However, every administration route has specific pros and cons. METHODS: We administrated human MSCs (hMSCs) in the cisterna lumbaris, which is easily accessible and could be used in outpatient surgery, in the SOD1 G93A mouse, at the earliest onset of symptoms. Control animals received saline injections. Motor behavior was checked starting from 2 months of age until the mice were killed. Animals were killed 2 weeks after transplantation; lumbar motoneurons were stereologically counted, astrocytes and microglia were analyzed and quantified after immunohistochemistry and cytokine expression was assayed by means of real-time polymerase chain reaction. RESULTS: We provide evidence that this route of administration can exert strongly positive effects. Motoneuron death and motor decay were delayed, astrogliosis was reduced and microglial activation was modulated. In addition, hMSC transplantation prevented the downregulation of the anti-inflammatory interleukin-10, as well as that of vascular endothelial growth factor observed in saline-treated transgenic mice compared with wild type, and resulted in a dramatic increase in the expression of the anti-inflammatory interleukin-13. CONCLUSIONS: Our results suggest that hMSCs, when intracisternally administered, can exert their paracrine potential, influencing the inflammatory response of the host.

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.

NCX1 is a new rest target gene: role in cerebral ischemia.

The Na(+)-Ca(2+) exchanger 1 (NCX1), a bidirectional transporter that mediates the electrogenic exchange of one calcium ion for three sodium ions across the plasma membrane, is known to be involved in brain ischemia. Since the RE1-silencing transcription factor (REST) is a key modulator of neuronal gene expression in several neurological conditions, we studied the possible involvement of REST in regulating NCX1 gene expression and activity in stroke. We found that: (1) REST binds in a sequence specific manner and represses through H4 deacetylation, ncx1 gene in neuronal cells by recruting CoREST, but not mSin3A. (2) In neurons and in SH-SY5Y cells REST silencing by siRNA and site-direct mutagenesis of REST consensus sequence on NCX1 brain promoter determined an increase in NCX1 promoter activity. (3) By contrast, REST overexpression caused a reduction in NCX1 protein expression and activity. (4) Interestingly, in rats subjected to transient middle cerebral artery occlusion (tMCAO) and in organotypic hippocampal slices or SH-SY5Y cells exposed to oxygen and glucose deprivation (OGD) plus reoxygenation (RX), the increase in REST was associated with a decrease in NCX1. However, this reduction was reverted by REST silencing. (5) REST knocking down, along with the deriving NCX1 overexpression in the deep V and VIb cortical layers caused a marked reduction in infarct volume after tMCAO. Double silencing of REST and NCX1 completely abolished neuroprotection induced by siREST administration. Collectively, these results demonstrate that REST, by regulating NCX1 expression, may represent a potential druggable target for the treatment of brain ischemia.

Transcriptional regulation of ncx1 gene in the brain.

The ubiquitous sodium-calcium exchanger isoform 1 (NCX1) is a -bidirectional transporter that plays a relevant role under physiological and pathophysiological conditions including brain ischemia by regulating intraneuronal Ca(2+) and Na(+) homeostasis. Although changes in ncx1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still largely unexplored. Here, we reviewed our recent findings on several transcription factors including cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-κB), and hypoxia-inducible factor-1 (HIF-1) in the control of the ncx1 gene expression in neuronal cells.

SMN deficiency perturbs monoamine neurotransmitter metabolism in spinal muscular atrophy.

Beyond motor neuron degeneration, homozygous mutations in the survival motor neuron 1 (SMN1) gene cause multiorgan and metabolic defects in patients with spinal muscular atrophy (SMA). However, the precise biochemical features of these alterations and the age of onset in the brain and peripheral organs remain unclear. Using untargeted NMR-based metabolomics in SMA mice, we identify cerebral and hepatic abnormalities related to energy homeostasis pathways and amino acid metabolism, emerging already at postnatal day 3 (P3) in the liver. Through HPLC, we find that SMN deficiency induces a drop in cerebral norepinephrine levels in overt symptomatic SMA mice at P11, affecting the mRNA and protein expression of key genes regulating monoamine metabolism, including aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DßH) and monoamine oxidase A (MAO-A). In support of the translational value of our preclinical observations, we also discovered that SMN upregulation increases cerebrospinal fluid norepinephrine concentration in Nusinersen-treated SMA1 patients. Our findings highlight a previously unrecognized harmful influence of low SMN levels on the expression of critical enzymes involved in monoamine metabolism, suggesting that SMN-inducing therapies may modulate catecholamine neurotransmission. These results may also be relevant for setting therapeutic approaches to counteract peripheral metabolic defects in SMA.

Role of JNK isoforms in the development of neuropathic pain following sciatic nerve transection in the mouse.

BACKGROUND: Current tools for analgesia are often only partially successful, thus investigations of new targets for pain therapy stimulate great interest. Consequent to peripheral nerve injury, c-Jun N-terminal kinase (JNK) activity in cells of the dorsal root ganglia (DRGs) and spinal cord is involved in triggering neuropathic pain. However, the relative contribution of distinct JNK isoforms is unclear. Using knockout mice for single isoforms, and blockade of JNK activity by a peptide inhibitor, we have used behavioral tests to analyze the contribution of JNK in the development of neuropathic pain after unilateral sciatic nerve transection. In addition, immunohistochemical labelling for the growth associated protein (GAP)-43 and Calcitonin Gene Related Peptide (CGRP) in DRGs was used to relate injury related compensatory growth to altered sensory function. RESULTS: Peripheral nerve injury produced pain-related behavior on the ipsilateral hindpaw, accompanied by an increase in the percentage of GAP43-immunoreactive (IR) neurons and a decrease in the percentage of CGRP-IR neurons in the lumbar DRGs. The JNK inhibitor, D-JNKI-1, successfully modulated the effects of the sciatic nerve transection. The onset of neuropathic pain was not prevented by the deletion of a single JNK isoform, leading us to conclude that all JNK isoforms collectively contribute to maintain neuropathy. Autotomy behavior, typically induced by sciatic nerve axotomy, was absent in both the JNK1 and JNK3 knockout mice. CONCLUSIONS: JNK signaling plays an important role in regulating pain threshold: the inhibition of all of the JNK isoforms prevents the onset of neuropathic pain, while the deletion of a single splice JNK isoform mitigates established sensory abnormalities. JNK inactivation also has an effect on axonal sprouting following peripheral nerve injury.

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.

Chromatographic measurement of 3-hydroxyanthranilate 3,4-dioxygenase activity reveals that edaravone can mitigate the formation of quinolinic acid through a direct enzyme inhibition.

Herein it is reported the development and application of two chromatographic assays for the measurement of the activity of 3-Hydroxyanthranilate-3,4-dioxygenase (3HAO). Such an enzyme converts 3-Hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconic semialdehyde (ACMS), which undergo a spontaneous, non-enzymatic cyclization to produce quinolinic acid (QUIN). The enzyme activity was measured by quantitation of the substrate consumption over time either with spectrophotometric (UV) or mass spectrometric (MS) detection upon reversed-phase chromatographic separation. MS detection resulted more selective and sensitive, but less accurate and precise. However, both methods have sufficient sensitivity to allow the measurement of enzyme activity with consistent results compared to literature data. Since MS detection allowed less sample consumption it was used to calculate the kinetics parameters (i.e., Vmax and Kd) of recombinant 3HAO. Another MS-based method was then developed to measure the amount of QUIN produced, revealing an incomplete conversion of 3HAA to QUIN. As suggested by previous studies, the enzyme activity was apparently sensitive to the redox state of the enzyme thiols. In fact, thiol reducing agents such as dithiothreitol (DTT) and glutathione (GSH), can alter the enzyme activity although the investigation on the exact mechanism involved in such effect was beyond the scope of the research. Interestingly, edaravone (EDA) induced an in vitro suppression of QUIN production through direct, competitive 3HAO inhibition. EDA is a molecule approved for the treatment of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease associated with an increase of QUIN concentrations in both serum and cerebrospinal fluid. Although EDA was reported to mitigate ALS progression its mode of action is still largely unknown. Some studies reported antioxidant and radical scavenger properties of EDA, but none confirm a direct activity as 3HAO enzyme inhibitor. Since QUIN is reported to be a neurotoxic metabolite, 3HAO inhibition can contribute to the beneficial effect of EDA in ALS, although such a mechanism must be then confirmed in vivo. However, EDA might be a convenient scaffold for the design of selective 3HAO inhibitors with potential applications in ALS treatment.

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.

Nusinersen Induces Disease-Severity-Specific Neurometabolic Effects in Spinal Muscular Atrophy.

Intrathecal delivery of Nusinersen-an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction-is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients.

NCX1 is a novel target gene for hypoxia-inducible factor-1 in ischemic brain preconditioning.

BACKGROUND AND PURPOSE: The sodium-calcium exchanger-1 (NCX1) represents a key mediator for maintaining [Na(+)](i) and [Ca(2+)](i) homeostasis. Although changes in NCX1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still unknown. Thus far, however, there is evidence that hypoxia-inducible factor-1 (HIF-1) is a nuclear factor required for transcriptional activation of several genes implicated in stroke. The main objective of this study was to investigate whether NCX1 gene might be a novel target of HIF-1 in the brain. METHODS: Here we report that: (1) in neuronal cells, NCX1 increased expression after oxygen and glucose deprivation or cobalt-induced HIF-1 activation was prevented by silencing HIF-1; (2) the brain NCX1 promoter cloned upstream of the firefly-luciferase gene contained 2 regions of HIF-1 target genes called hypoxia-responsive elements that are sensitive to oxygen and glucose deprivation or cobalt chloride; (3) HIF-1 specifically bound hypoxia-responsive elements on brain NCX1, as demonstrated by band-shift and chromatin immunoprecipitation assays; (4) HIF-1α silencing prevented NCX1 upregulation and neuroprotection induced by ischemic preconditioning; and (5) NCX1 silencing partially reverted the preconditioning-induced neuroprotection in rats. CONCLUSIONS: NCX1 gene is a novel HIF-1 target, and HIF-1 exerts its prosurvival role through NCX1 upregulation during brain preconditioning.

The hypoxia sensitive metal transcription factor MTF-1 activates NCX1 brain promoter and participates in remote postconditioning neuroprotection in stroke.

Remote limb ischemic postconditioning (RLIP) is an experimental strategy in which short femoral artery ischemia reduces brain damage induced by a previous harmful ischemic insult. Ionic homeostasis maintenance in the CNS seems to play a relevant role in mediating RLIP neuroprotection and among the effectors, the sodium-calcium exchanger 1 (NCX1) may give an important contribution, being expressed in all CNS cells involved in brain ischemic pathophysiology. The aim of this work was to investigate whether the metal responsive transcription factor 1 (MTF-1), an important hypoxia sensitive transcription factor, may (i) interact and regulate NCX1, and (ii) play a role in the neuroprotective effect mediated by RLIP through NCX1 activation. Here we demonstrated that in brain ischemia induced by transient middle cerebral occlusion (tMCAO), MTF-1 is triggered by a subsequent temporary femoral artery occlusion (FAO) and represents a mediator of endogenous neuroprotection. More importantly, we showed that MTF-1 translocates to the nucleus where it binds the metal responsive element (MRE) located at -23/-17 bp of Ncx1 brain promoter thus activating its transcription and inducing an upregulation of NCX1 that has been demonstrated to be neuroprotective. Furthermore, RLIP restored MTF-1 and NCX1 protein levels in the ischemic rat brain cortex and the silencing of MTF-1 prevented the increase of NCX1 observed in RLIP protected rats, thus demonstrating a direct regulation of NCX1 by MTF-1 in the ischemic cortex of rat exposed to tMCAO followed by FAO. Moreover, silencing of MTF-1 significantly reduced the neuroprotective effect elicited by RLIP as demonstrated by the enlargement of brain infarct volume observed in rats subjected to RLIP and treated with MTF-1 silencing. Overall, MTF-dependent activation of NCX1 and their upregulation elicited by RLIP, besides unraveling a new molecular pathway of neuroprotection during brain ischemia, might represent an additional mechanism to intervene in stroke pathophysiology.

Remote postconditioning ameliorates stroke damage by preventing let-7a and miR-143 up-regulation.

Remote limb ischemic postconditioning (RLIP) is a well-established neuroprotective strategy able to protect the brain from a previous harmful ischemic insult through a sub-lethal occlusion of the femoral artery. Neural and humoral mechanisms have been proposed as mediators required to transmit the peripheral signal from limb to brain. Moreover, different studies suggest that protection observed at brain level is associated to a general genetic reprogramming involving also microRNAs (miRNAs) intervention. Methods: Brain ischemia was induced in male rats by transient occlusion of the middle cerebral artery (tMCAO), whereas RLIP was achieved by one cycle of temporary occlusion of the ipsilateral femoral artery after tMCAO. The expression profile of 810 miRNAs was evaluated in ischemic brain samples from rats subjected either to tMCAO or to RLIP. Among all analyzed miRNAs, there were four whose expression were upregulated after stroke and returned to basal level after RLIP, thus suggesting a possible involvement in RLIP-induced neuroprotection. These selected miRNAs were intracerebroventricularly infused in rats subjected to remote ischemic postconditioning, and their effect was evaluated in terms of brain damage, neurological deficit scores and expression of putative targets. Results: Twenty-one miRNAs, whose expression was significantly affected by tMCAO and by tMCAO plus RLIP, were selected based on microarray microfluidic profiling. Our data showed that: (1) stroke induced an up-regulation of let-7a and miR-143 (2) these two miRNAs were involved in the protective effects induced by RLIP and (3) HIF1-α contributes to their protective effect. Indeed, their expression was reduced after RLIP and the exogenous intracerebroventricularly infusion of let-7a and miR-143 mimics prevented neuroprotection and HIF1-α overexpression induced by RLIP. Conclusions: Prevention of cerebral let-7a and miR-143 overexpression induced by brain ischemia emerges as new potential strategy in stroke intervention.

Genetically modified mice to unravel physiological and pathophysiological roles played by NCX isoforms.

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.

Sumoylation of sodium/calcium exchanger in brain ischemia and ischemic preconditioning.

The small ubiquitin-like modifier (SUMO) conjugation (or SUMOylation) is a post-translational protein modification mechanism activated by different stress conditions that has been recently investigated in experimental models of cerebral ischemia. The expression of SUMOylation enzymes and substrates is not restricted to the nucleus, since they are present also in the cytoplasm and on plasma membrane and are involved in several physiological and pathological conditions. In the last decades, convincing evidence have supported the idea that the increased levels of SUMOylated proteins may induce tolerance to ischemic stress. In particular, it has been established that protein SUMOylation may confer neuroprotection during ischemic preconditioning. Considering the increasing evidence that SUMO can modify stability and expression of ion channels and transporters and the relevance of controlling ionic homeostasis in ischemic conditions, the present review will resume the main aspects of SUMO pathways related to the key molecules involved in maintenance of ionic homeostasis during cerebral ischemia and ischemic preconditioning, with a particular focus on the on Na+/Ca2+ exchangers.