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.

A Targeted Mass Spectrometry Approach to Identify Peripheral Changes in Metabolic Pathways of Patients with Alzheimer's Disease.

Alzheimer's disease (AD), a neurodegenerative disorder, is the most common cause of dementia in the elderly population. Since its original description, there has been intense debate regarding the factors that trigger its pathology. It is becoming apparent that AD is more than a brain disease and harms the whole-body metabolism. We analyzed 630 polar and apolar metabolites in the blood of 20 patients with AD and 20 healthy individuals, to determine whether the composition of plasma metabolites could offer additional indicators to evaluate any alterations in the metabolic pathways related to the illness. Multivariate statistical analysis showed that there were at least 25 significantly dysregulated metabolites in patients with AD compared with the controls. Two membrane lipid components, glycerophospholipids and ceramide, were upregulated, whereas glutamic acid, other phospholipids, and sphingolipids were downregulated. The data were analyzed using metabolite set enrichment analysis and pathway analysis using the KEGG library. The results showed that at least five pathways involved in the metabolism of polar compounds were dysregulated in patients with AD. Conversely, the lipid pathways did not show significant alterations. These results support the possibility of using metabolome analysis to understand alterations in the metabolic pathways related to AD pathophysiology.

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.

Ncx3-Induced Mitochondrial Dysfunction in Midbrain Leads to Neuroinflammation in Striatum of A53t-α-Synuclein Transgenic Old Mice.

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca2+-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson's disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging.

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.

Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in neuronal growth factor (NGF)-induced neuronal differentiation through Ca2+-dependent Akt phosphorylation.

NGF induces neuronal differentiation by modulating [Ca(2+)]i. However, the role of the three isoforms of the main Ca(2+)-extruding system, the Na(+)/Ca(2+) exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca(2+)]i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca(2+) content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na(+) currents and 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-detected [Na(+)]i significantly increased in cells overexpressing NCX1.4 as well as ER Ca(2+) content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca(2+)]i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca(2+) content and PI3K signaling.

Glial Na(+) -dependent ion transporters in pathophysiological conditions.

Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na(+) signaling influences and regulates important glial activities, and plays a role in neuron-glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na(+) pumps and Na(+) -dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na(+) homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na(+) -dependent ion transporters, including Na(+) /K(+) ATPase, Na(+) /Ca(2+) exchangers, Na(+) /H(+) exchangers, Na(+) -K(+) -Cl(-) cotransporters, and Na(+) - HCO3- cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na(+) -dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na(+) dynamics in different neurological disorders. GLIA 2016;64:1677-1697.

Sumoylation of LYS590 of NCX3 f-Loop by SUMO1 Participates in Brain Neuroprotection Induced by Ischemic Preconditioning.

BACKGROUND AND PURPOSE: The small ubiquitin-like modifier (SUMO), a ubiquitin-like protein involved in posttranslational protein modifications, is activated by several conditions, such as heat stress, hypoxia, and hibernation and confers neuroprotection. Sumoylation enzymes and substrates are expressed also at the plasma membrane level. Among the numerous plasma membrane proteins controlling ionic homeostasis during cerebral ischemia, 1 of the 3 brain sodium/calcium exchangers (NCX3), exerts a protective role during ischemic preconditioning. In this study, we evaluated whether NCX3 is a target for sumoylation and whether this posttranslational modification participates in ischemic preconditioning-induced neuroprotection. To test these hypotheses, we analyzed (1) SUMO1 conjugation pattern after ischemic preconditioning; (2) the effect of SUMO1 knockdown on the ischemic damage after transient middle cerebral artery occlusion and ischemic preconditioning, (3) the possible interaction between SUMO1 and NCX3 and (4) the molecular determinants of NCX3 sequence responsible for sumoylation. METHODS: Focal brain ischemia and ischemic preconditioning were induced in rats by middle cerebral artery occlusion. SUMOylation was evaluated by western blot and immunohistochemistry. SUMO1 and NCX3 interaction was analyzed by site-directed mutagenesis and immunoprecipitation assay. RESULTS: We found that (1) SUMO1 knockdown worsened ischemic damage and reduced the protective effect of preconditioning; (2) SUMO1 bound to NCX3 at lysine residue 590, and its silencing increased NCX3 degradation; and (3) NCX3 sumoylation participates in SUMO1 protective role during ischemic preconditioning. Thus, our results demonstrate that NCX3 sumoylation confers additional neuroprotection in ischemic preconditioning. CONCLUSIONS: Finally, this study suggests that NCX3 sumoylation might be a new target to enhance ischemic preconditioning-induced neuroprotection.

A new cell-penetrating peptide that blocks the autoinhibitory XIP domain of NCX1 and enhances antiporter activity.

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) is a high-capacity ionic transporter that exchanges 3Na(+) ions for 1Ca(2+) ion. The first 20 amino acids of the f-loop, named exchanger inhibitory peptide (XIP(NCX1)), represent an autoinhibitory region involved in the Na(+)-dependent inactivation of the exchanger. Previous research has shown that an exogenous peptide having the same amino acid sequence as the XIP(NCX1) region exerts an inhibitory effect on NCX activity. In this study, we identified another regulatory peptide, named P1, which corresponds to the 562-688aa region of the exchanger. Patch-clamp analysis revealed that P1 increased the activity of the exchanger, whereas the XIP inhibited it. Furthermore, P1 colocalized with NCX1 thus suggesting a direct binding interaction. In addition, site-directed mutagenesis experiments revealed that the binding and the stimulatory effect of P1 requires a functional XIP(NCX1) domain on NCX1 thereby suggesting that P1 increases the exchanger activity by counteracting the action of this autoinhibitory sequence. Taken together, these results open a new strategy for developing peptidomimetic compounds that, by mimicking the functional pharmacophore of P1, might increase NCX1 activity and thus exert a therapeutic action in those diseases in which an increase in NCX1 activity might be helpful.

Correction: Iannuzzi et al. Fyn Tyrosine Kinase Elicits Amyloid Precursor Protein Tyr682 Phosphorylation in Neurons from Alzheimer's Disease Patients. Cells 2020, 9, 1807.

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Semaphorin 3A Increases in the Plasma of Women with Diminished Ovarian Reserve Who Respond Better to Controlled Ovarian Stimulation.

Semaphorin 3A (SEMA3A) plays a crucial role in the development, differentiation, and plasticity of specific types of neurons that secrete Gonadotropin-Releasing Hormone (GnRH) and regulates the acquisition and maintenance of reproductive competence in humans and mice. Its insufficient expression has been linked to reproductive disorders in humans, which are characterized by reduced or failed sexual competence. Various mutations, polymorphisms, and alternatively spliced variants of SEMA3A have been associated with infertility. One of the common causes of infertility in women of reproductive age is diminished ovarian reserve (DOR), characterized by a reduced ovarian follicular pool. Despite its clinical significance, there are no universally accepted diagnostic criteria or therapeutic interventions for DOR. In this study, we analyzed the SEMA3A plasma levels in 77 women and investigated their potential role in influencing fertility in patients with DOR. The results revealed that the SEMA3A levels were significantly higher in patients with DOR than in healthy volunteers. Furthermore, the SEMA3A levels were increased in patients who underwent fertility treatment and had positive Beta-Human Chorionic Gonadotropin (ßHCG) values (ß+) after controlled ovarian stimulation (COS) compared to those who had negative ßHCG values (ß-). These findings may serve as the basis for future investigations into the diagnosis of infertility and emphasize new possibilities for the SEMA3A-related treatment of sexual hormonal dysfunction that leads to infertility.

A new concept: Aß1-42 generates a hyperfunctional proteolytic NCX3 fragment that delays caspase-12 activation and neuronal death.

Although the amyloid-ß(1-42) (Aß(1-42)) peptide involved in Alzheimer's disease is known to cause a dysregulation of intracellular Ca(2+) homeostasis, its molecular mechanisms still remain unclear. We report that the extracellular-dependent early increase (30 min) in intracellular calcium concentration ([Ca(2+)](i)), following Aß(1-42) exposure, caused the activation of calpain that in turn elicited a cleavage of the Na(+)/Ca(2+) exchanger isoform NCX3. This cleavage generated a hyperfunctional form of the antiporter and increased NCX currents (I(NCX)) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the Aß(1-42)-dependent I(NCX) increase. Indeed, the calpain inhibitor calpeptin and the removal of the calpain-cleavage recognition sequence, via site-directed mutagenesis, abolished this effect. Moreover, the enhanced NCX3 activity was paralleled by an increased Ca(2+) content in the endoplasmic reticulum (ER) stores. Remarkably, the silencing in PC-12 cells or the knocking-out in mice of the ncx3 gene prevented the enhancement of both I(NCX) and Ca(2+) content in ER stores, suggesting that NCX3 was involved in the increase of ER Ca(2+) content stimulated by Aß(1-42). By contrast, in the late phase (72 h), when the NCX3 proteolytic cleavage abruptly ceased, the occurrence of a parallel reduction in ER Ca(2+) content triggered ER stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca(2+)](i) coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of Aß(1-42)-induced caspase-12. Indeed, in NCX3-silenced neurons, Aß(1-42) exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that Aß(1-42), through Ca(2+)-dependent calpain activation, generates a hyperfunctional form of NCX3 that, by increasing Ca(2+) content into ER, delays caspase-12 activation and thus neuronal death.

nNOS and p-ERK involvement in the neuroprotection exerted by remote postconditioning in rats subjected to transient middle cerebral artery occlusion.

It has recently been hypothesized that a sub-lethal ischemic insult induced in one organ is able to protect from a harmful ischemia occurring in a different organ. The objective of this study is to identify new putative mechanisms of neuroprotection elicited by remote ischemic femoral postconditioning. A 50% reduction in the infarct volume was observed when 100min of middle cerebral artery occlusion was followed, 10min later, by the remote postconditioning stimulus represented by 20min of femoral artery occlusion. The use of in vivo silencing strategy allowed to demonstrate that NO production through nNOS mediates part of the neuroprotection. Indeed, whereas CNS nNOS expression was up-regulated by remote postconditioning, the pharmacological inhibition of nNOS or its silencing-mediated knocking-down partially prevented this neuroprotective effect. This nNOS overexpression seemed to be p-ERK dependent. In fact, p-ERK expression increased in brain cortex after remote postconditioning, and its pharmacological inhibition prevented both nNOS overexpression and remote postconditioning-mediated neuroprotection. Interestingly, neuroprotection induced by remote postconditioning was partially prevented when ganglion transmission was pharmacologically interrupted by hexamethonium, thus showing that neural factors are involved in this phenomenon. Collectively, the present study demonstrates that p-ERK and nNOS take part to the complex cascade of events triggered by ischemic remote postconditioning.

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.

NCX as a key player in the neuroprotection exerted by ischemic preconditioning and postconditioning.

Ischemic preconditioning is a neuroprotective mechanism in which a brief non-injurious episode of ischemia protects the brain from a subsequent lethal insult. Recently, it has been reported that modified reperfusion subsequent to a prolonged ischemic episode may also confer neuroprotection, a phenomenon termed postconditioning. Mitogen-activated protein kinases (MAPK) play a key role in these two neuroprotective mechanisms. The aim of this study was to evaluate whether Na(+)/Ca(2+) exchangers (NCXs), a family of ionic transporters that contribute to the maintenance of intracellular ionic homeostasis, contribute to the neuroprotection elicited by ischemic preconditioning and postconditioning.Results of this study indicated that (1) NCX1 and NCX3 are upregulated in those brain regions protected by preconditioning, while (2) postconditioning treatment induces an upregulation only in NCX3 expression. (3) NCX1 upregulation and NCX3 upregulation are mediated by p-AKT since its inhibition reverted the neuroprotective effect of preconditioning and postconditioning and prevented NCXs overexpression. (4) The involvement of NCX in preconditioning and postconditioning neuroprotection is further supported by the results of experiments showing that a partial reversion of the protective effect induced by preconditioning was obtained by silencing NCX1 or NCX3, while the silencing of NCX3 was able to mitigate the protection induced by ischemic postconditioning.Altogether, the data presented here suggest that NCX1 and NCX3 -represent two promising druggable targets for setting on new strategies in stroke therapy.

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.

Genetically modified mice as a strategy to unravel the role played by the Na(+)/Ca (2+) exchanger in brain ischemia and in spatial learning and memory deficits.

Because no isoform-specific blocker of NCX has ever been synthesized, a more selective strategy to identify the role of each antiporter isoform in the brain was represented by the generation of knockout and knockin mice for the different isoforms of the antiporter.Experiments performed in NCX2 and NCX3 knockout mice provided evidence that these two isoforms participate in spatial learning and memory consolidation, although in an opposite manner. These new data from ncx2-/- and ncx3-/- mice may open new experimental avenues for the development of effective therapeutic compounds that, by selectively inhibiting or activating these molecular targets, could treat patients affected by cognitive impairment including Alzheimer's, Parkinson's, Huntington's diseases, and infarct dementia.More importantly, knockout and knockin mice also provided new relevant information on the role played by NCX in maintaining the intracellular Na(+) and Ca(2+) homeostasis and in protecting neurons during brain ischemia. In particular, both ncx2-/- and ncx3-/- mice showed an increased neuronal vulnerability after the ischemic insult induced by transient middle cerebral artery occlusion.As the ubiquitous deletion of NCX1 brings about to an early death of embryos because of a lack of heartbeat, this strategy could not be successfully pursued. However, information on the role of NCX1 in normal and ischemic brain could be obtained by developing conditional knockout mice lacking NCX1 in the brain. Preliminarily results obtained in these conditional mice suggest that also NCX1 protects neurons from ischemic cell death.Overall, the use of genetic-modified mice for NCX1, NCX2, and NCX3 represents a fruitful strategy to characterize the physiological role exerted by NCX in CNS and to identify the isoforms of the antiporter as potential molecular targets for therapeutic intervention in cerebral ischemia.

Na+ -Ca2+ exchanger (NCX3) knock-out mice display an impairment in hippocampal long-term potentiation and spatial learning and memory.

Long-term potentiation (LTP) depends on the coordinated regulation of an ensemble of proteins related to Ca(2+) homeostasis, including Ca(2+) transporters. One of the major players in the regulation of intracellular Ca(2+) ([Ca(2+)](i)) homeostasis in neurons is the sodium/calcium exchanger (NCX), which represents the principal mechanism of Ca(2+) clearance in the synaptic sites of hippocampal neurons. Because NCX3, one of the three brain isoforms of the NCX family, is highly expressed in the hippocampal subfields involved in LTP, we hypothesized that it might represent a potential candidate for LTP modulation. To test this hypothesis, we first examined the effect of ncx3 gene ablation on NCX currents (I(NCX)) and Ca(2+) homeostasis in hippocampal neurons. ncx3(-/-) neurons displayed a reduced I(NCX), a higher basal level of [Ca(2+)](i), and a significantly delayed clearance of [Ca(2+)](i) following depolarization. Furthermore, measurement of field EPSPs, recorded from the CA1 area, revealed that ncx3(-/-) mice had an impaired basal synaptic transmission. Moreover, hippocampal slices from ncx3(-/-) mice exhibited a worsening in LTP compared with congenic ncx3(+/+). Consistently, immunohistochemical and immunoblot analysis indicated that in the hippocampus of ncx3(-/-) mice both Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) expression and the phosphoCaMKIIα/CaMKIIα ratio were significantly reduced compared with ncx3(+/+). Interestingly, ncx3(-/-) mice displayed a reduced spatial learning and memory performance, as revealed by the novel object recognition, Barnes maze, and context-dependent fear conditioning assays. Collectively, our findings demonstrate that the deletion of the ncx3 gene in mice has detrimental consequences on basal synaptic transmission, LTP regulation, spatial learning, and memory performance.

ERK1/2, p38, and JNK regulate the expression and the activity of the three isoforms of the Na+ /Ca2+ exchanger, NCX1, NCX2, and NCX3, in neuronal PC12 cells.

We evaluated whether changes in expression and activity of the three sodium/calcium exchanger isoforms, NCX1, NCX2, and NCX3 occurred in PC12 cells when the extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) were silenced, pharmacologically blocked, or activated with nerve growth factor (NGF). Several findings suggesting that MAPKs control NCX emerged: (1) A decrease in NCX1 and NCX3 basal expression occurred when JNK or MEK1, the extracellular-signal-regulated kinases 1/2 upstream activator, were pharmacologically blocked, respectively; (2) NGF increased cAMP response element-binding 1 (CREB1) and Specificity Protein 1 (Sp1) binding to ncx1 promoter and CREB1 binding to two different sequences close to ncx2 transcription start site on genomic DNA; (3) An up-regulation of NCX1 and NCX3, abrogated upon either MEK1 or p38 blockade, and a down-regulation of NCX2, abolished upon p38 blockade, occurred upon NGF-induced MAPK activation. The NCX1 up-regulation was abolished upon either CREB1 or Sp1 silencing, whereas NCX2 down-regulation was abrogated only by CREB1 silencing. The NCX3 up-regulation was unaffected by CREB1 or Sp1 silencing and abolished upon proteasomal inhibition; (4) Whole-cell Na(+) /Ca(2+) exchange decreased when MEK1 and JNK were blocked and increased when MAPKs were activated by NGF. Collectively, these results demonstrate a MAPK-dependent regulation of NCX expression and activity which could be relevant in mediating some of the effects of MAPKs in neurons.