Pasquale Molinaro

Targeted Disruption of Na+/Ca2+ Exchanger 3 (NCX3) Gene Leads to a Worsening of Ischemic Brain Damage

Na+/Ca2+ exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in the brain and is involved in the maintenance of intracellular Na+ and Ca2+ homeostasis. Interestingly, whereas the function of NCX3 under physiological conditions has been determined, its role under anoxia is still unknown. To assess NCX3 role in cerebral ischemia, we exposed ncx3−/− mice to transient middle cerebral artery occlusion followed by reperfusion. In addition, to evaluate the effect of ncx3 ablation on neuronal survival, organotypic hippocampal cultures and primary cortical neurons from ncx3−/− mice were subjected to oxygen glucose deprivation (OGD) plus reoxygenation. Here we report that ncx3 gene suppression leads to a worsening of brain damage after focal ischemia and to a massive neuronal death in all the hippocampal fields of organotypic cultures as well as in cortical neurons from ncx3−/− mice exposed to OGD plus reoxygenation. In addition, in ncx3−/− cortical neurons exposed to hypoxia, NCX currents, recorded in the reverse mode of operation, were significantly lower than those detected in ncx3+/+. From these results, NCX3 protein emerges as a new molecular target that may have a potential therapeutic value in modulating cerebral ischemia.

HIF‐1α reveals a binding activity to the promoter of iNOS gene after permanent middle cerebral artery occlusion

Hypoxia inducible factor (HIF‐1)‐1α is a specific, oxygen‐sensitive protein that regulates the activity of HIF‐1, a transcriptional factor that increases after cerebral ischemia and may either promote or prevent neuronal survival. In this study to determine whether the inducible nitric oxide synthase (iNOS) gene containing the sequence of the hypoxia‐responsive enhancer (HRE) was an HIF‐1 target after cerebral ischemia induced by permanent middle cerebral artery occlusion (pMCAO), electrophoretic mobility shift assay (EMSA) and iNOS western blot analysis were performed in the ischemic core, in the area surrounding the infarct and in the hippocampus ipsilateral and contralateral to the lesion. In addition, both HIF‐1α mRNA and protein expression were examined in the ischemic core, in the area surrounding the ischemic core and in the hippocampus ipsilateral to the insult. Our results revealed that pMCAO up‐regulates iNOS protein in the ischemic core, in the area surrounding the ischemic core and in the hippocampus ipsilateral to the lesion, and that the activation of iNOS expression is mediated by HIF‐1. Moreover, HIF‐1α mRNA and protein levels increased in the ischemic core and in the hippocampus ipsilateral to the lesion compared with the levels obtained in the corresponding areas of sham‐operated controls or in the contralateral hemisphere. Particularly in the area surrounding the ischemic core, HIF‐1α protein accumulated during pMCAO although mRNA did not increase. Our study suggests that the activation of HIF‐1 might be involved in the mechanisms whereby iNOS promotes cell survival and/or death after cerebral ischemia.

The two isoforms of the Na+/Ca2+ exchanger, NCX1 and NCX3, constitute novel additional targets for the prosurvival action of Akt/protein kinase B pathway.

The proteins NCX1, NCX2, and NCX3 expressed on the plasma membrane of neurons play a crucial role in ionic regulation because they are the major bidirectional system promoting the efflux and influx of Na+ and Ca2+ ions. Here, we demonstrate that NCX1 and NCX3 proteins are novel additional targets for the survival action of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Indeed, the doxycycline-dependent overexpression of constitutively active Akt1 in tetracycline (Tet)-Off PC-12 positive mutants and the exposure of Tet-Off PC-12 wild type to nerve growth factor induced an up-regulation of NCX1 and NCX3 proteins. NCX1 up-regulation induced by Akt1 activation occurred at the transcriptional level because NCX1 mRNA increased, and it was counteracted by cAMP response element-binding protein 1 inhibition through small interfering RNA strategy. In contrast, Akt1-induced NCX3 up-regulation recognized a post-transcriptional mechanism occurring at the proteasome level because 1) NCX3 transcript did not increase and 2) the proteasome inhibitor N-benzyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) did not further enhance NCX3 protein levels in Akt1 active mutants as it would be expected if the ubiquitin-proteasome complex was not already blocked by Akt1 pathway. As expected, in PC-12 Tet-Off wild-type cells MG-132 enhanced NCX3 protein levels. This up-regulation produced an increased activity of NCX function. Furthermore, NCX1 and NCX3 up-regulation contributed to the survival action of Akt1 during chemical hypoxia because both the silencing of NCX1 or NCX3 and the pharmacological paninhibition of NCX isoforms reduced the prosurvival property of Akt1. Together, these results indicated that NCX1 and NCX3 represent novel additional molecular targets for the prosurvival action of PI3-K/Akt pathway.

A New Concept: Aβ1–42 Generates a Hyperfunctional Proteolytic NCX3 Fragment That Delays Caspase-12 Activation and Neuronal Death

Although the amyloid-β1–42 (Aβ1–42) peptide involved in Alzheimers disease is known to cause a dysregulation of intracellular Ca2+ homeostasis, its molecular mechanisms still remain unclear. We report that the extracellular-dependent early increase (30 min) in intracellular calcium concentration ([Ca2+]i), following Aβ1–42 exposure, caused the activation of calpain that in turn elicited a cleavage of the Na+/Ca2+ exchanger isoform NCX3. This cleavage generated a hyperfunctional form of the antiporter and increased NCX currents (INCX) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the Aβ1–42-dependent INCX 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 Ca2+ 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 INCX and Ca2+ content in ER stores, suggesting that NCX3 was involved in the increase of ER Ca2+ content stimulated by Aβ1–42. By contrast, in the late phase (72 h), when the NCX3 proteolytic cleavage abruptly ceased, the occurrence of a parallel reduction in ER Ca2+ content triggered ER stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca2+]i coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of Aβ1–42-induced caspase-12. Indeed, in NCX3-silenced neurons, Aβ1–42 exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that Aβ1–42, through Ca2+-dependent calpain activation, generates a hyperfunctional form of NCX3 that, by increasing Ca2+ content into ER, delays caspase-12 activation and thus neuronal death.

ncx1, ncx2, and ncx3 Gene Product Expression and Function in Neuronal Anoxia and Brain Ischemia

Abstract:  Over the last few years, although extensive studies have focused on the relevant function played by the sodium–calcium exchanger (NCX) during focal ischemia, a thorough understanding of its role still remains a controversial issue. We explored the consequences of the pharmacological inhibition of this antiporter with conventional pharmacological approach, with the synthetic inhibitory peptide, XIP, or with an antisense strategy on the extent of brain damage induced by the permanent occlusion of middle cerebral artery (pMCAO) in rats. Collectively, the results of these studies suggest that ncx1 and ncx3 genes could be play a major role to limit the severity of ischemic damage probably as they act to dampen [Na+]i and [Ca2+]i overload. This mechanism seems to be normally activated in the ischemic brain as we found a selective upregulation of NCX1 and NCX3 mRNA levels in regions of the brain surviving to an ischemic insult. Despite this transcript increase, NCX1, NCX2, and NCX3 proteins undergo an extensive proteolytic degradation in the ipsilateral cerebral hemisphere. All together these results suggest that a rescue program centered on an increase NCX function and expression could halt the progression of the ischemic damage. On the basis of this evidence we directed our attention to the understanding of the transductional and transcriptional pathways responsible for NCX upregulation. To this aim, we are studying whether the brain isoform of Akt, Akt1, which is a downstream effector of neurotrophic factors, such as NGF can, in addition to affecting the other prosurvival cascades, also exert its neuroprotective effect by modulating the expression and activity of ncx1, ncx2, and ncx3 gene products.

NCX3 regulates mitochondrial Ca2+ handling through the AKAP121-anchored signaling complex and prevents hypoxia-induced neuronal death

Summary The mitochondrial influx and efflux of Ca2+ play a relevant role in cytosolic and mitochondrial Ca2+ homeostasis, and contribute to the regulation of mitochondrial functions in neurons. The mitochondrial Na+/Ca2+ exchanger, which was first postulated in 1974, has been primarily investigated only from a functional point of view, and its identity and localization in the mitochondria have been a matter of debate over the past three decades. Recently, a Li+-dependent Na+/Ca2+ exchanger extruding Ca2+ from the matrix has been found in the inner mitochondrial membrane of neuronal cells. However, evidence has been provided that the outer membrane is impermeable to Ca2+ efflux into the cytoplasm. In this study, we demonstrate for the first time that the nuclear-encoded NCX3 isoform (1) is located on the outer mitochondrial membrane (OMM) of neurons; (2) colocalizes and immunoprecipitates with AKAP121 (also known as AKAP1), a member of the protein kinase A anchoring proteins (AKAPs) present on the outer membrane; (3) extrudes Ca2+ from mitochondria through AKAP121 interaction in a PKA-mediated manner, both under normoxia and hypoxia; and (4) improves cell survival when it works in the Ca2+ efflux mode at the level of the OMM. Collectively, these results suggest that, in neurons, NCX3 regulates mitochondrial Ca2+ handling from the OMM through an AKAP121-anchored signaling complex, thus promoting cell survival during hypoxia.

A Critical Role for the Potassium-Dependent Sodium–Calcium Exchanger NCKX2 in Protection against Focal Ischemic Brain Damage

The superfamily of cation/Ca2+ plasma–membrane exchangers contains two branches, the K+-independent Na+–Ca2+ exchangers (NCXs) and the K+-dependent Na+–Ca2+ exchangers (NCKXs), widely expressed in mammals. NCKX2 is the major neuronally expressed isoform among NCKX members. Despite its importance in maintaining Na+, Ca2+, and K+ homeostasis in the CNS, the role of NCKX2 during cerebral ischemia, a condition characterized by an alteration of ionic concentrations, has not yet been investigated. The present study examines NCKX2 role in the development of ischemic brain damage in permanent middle cerebral artery occlusion (pMCAO) and transient middle cerebral artery occlusion. Furthermore, to evaluate the effect of nckx2 ablation on neuronal survival, nckx2−/− primary cortical neurons were subjected to oxygen glucose deprivation plus reoxygenation. NCKX2 mRNA and protein expression was evaluated in the ischemic core and surrounding ipsilesional areas, at different time points after pMCAO in rats. In ischemic core and in periinfarctual area, NCKX2 mRNA and protein expression were downregulated. In addition, NCKX2 knock-down by antisense oligodeoxynucleotide and NCKX2 knock-out by genetic disruption dramatically increased infarct volume. Accordingly, nckx2−/− primary cortical neurons displayed a higher vulnerability and a greater [Ca2+]i increase under hypoxic conditions, compared with nckx2+/+ neurons. In addition, NCKX currents both in the forward and reverse mode of operation were significantly reduced in nckx2−/− neurons compared with nckx2+/+ cells. Overall, these results indicate that NCKX2 is involved in brain ischemia, and it may represent a new potential target to be investigated in the study of the molecular mechanisms involved in cerebral ischemia.

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

J. Neurochem. (2012) 122, 911–922.

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.

Glutamate-independent calcium toxicity: introduction.

It is widely accepted that a critical factor in determining neuronal death during cerebral ischemia is the progressive accumulation of intracellular Na+ ([Na+]i) and Ca2+ ([Ca2+]i) ions, which can precipitate necrosis and apoptosis of vulnerable neurons. Whereas the detrimental action of [Na+]i increase is attributable to both cell swelling and microtubular disorganization—2 phenomena that lead to cell necrosis1—a change in [Ca2+]i has been shown to be a key factor in ischemic brain damage, for it modulates several death pathways, including oxidative and nitrosative stress, mitochondrial dysfunction, and protease activation. Since Olney’s seminal work firstly suggested that excitatory aminoacids could elicit neurotoxicity,2 a large amount of work has been accumulated showing that glutamate extracellular concentrations briskly rise during acute brain injury, thus triggering an influx of Ca2+ and Na+ ions into neurons through ionotropic glutamate receptor subtypes. This evidence has led to the elaboration of the paradigm of glutamate excitotoxicity that explained ischemic neuronal cell death as a mere consequence of Na+ and Ca2+ influx through glutamate receptors.3 Although this theory has been guiding basic research in the field of neurodegeneration for almost 3 decades, more recently it has become the object of serious criticism and reassessment. What has aroused such skepticism among researchers has been the fact that although first, second, and third generation glutamate receptor antagonists have long yielded promising results in animal models of brain ischemia, they have failed to elicit a neuroprotective action in stroke and traumatic brain injury in humans.4 Therefore, the theory of excitotoxicity, though a fascinating paradigm, can only explain some of the events occurring in the acute phase of anoxic insult but cannot be seen as a major target for …