Rossana Sirabella

Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions

Reactive oxygen species (ROS) have been implicated in the pathophysiology of many neurologic disorders and brain dysfunction. In the same pathological settings evidence has been provided in favour of a participation of intracellular Ca(2+) concentration altered homeostasis in the chain of events leading to neuronal apoptosis. In the present review literature reports and experimental data on the relationship between caspase activation and alteration of intracellular calcium concentrations in the mechanisms triggering neuronal apoptosis are discussed. The data gathered support the conclusion that during oxidative stress in neuronal cells the production of ROS triggers a mechanism that, through the release of cytochrome c from mitochondria and caspase-3 activation, leads to apoptosis; the concomitant ROS-mediated elevation of intracellular Ca(2+) concentration triggers caspase-2 activation but both events do not seem to be involved in cell death.

Two Sodium/Calcium Exchanger Gene Products, NCX1 and NCX3, Play a Major Role in the Development of Permanent Focal Cerebral Ischemia

Background and Purpose— The Na+/Ca2+ exchanger, by mediating Ca2+ and Na+ fluxes in a bidirectional way across the synaptic plasma membrane, may play a pivotal role in the events leading to anoxic damage. In the brain, there are 3 different genes coding for 3 different proteins: NCX1, NCX2, and NCX3. The aim of this study was to determine whether NCX1, NCX2, and NCX3 might play a differential role in the development of cerebral injury induced by permanent middle cerebral artery occlusion (pMCAO). Methods— By means of Western blotting, NCX1, NCX2, and NCX3 protein expression was evaluated in the ischemic core and in the remaining nonischemic area of the slice at different time intervals starting from ischemia induction. The role of each isoform was also assessed with antisense oligodeoxynucleotides (ODNs) targeted for each isoform. These ODNs were continuously intracerebroventricularly infused with an osmotic minipump (1 &mgr;L/h) for 48 hours, 24 hours before pMCAO. Results— The results showed that after pMCAO all 3 NCX proteins were downregulated in ischemic core; NCX3 decreased in periinfarctual area whereas NCX1 and NCX2 were unchanged. The ODNs for NCX1 and NCX3 gene products were capable of inducing an increase in the ischemic lesion and to worsen neurological scores. Conclusions— The results of this study suggest that in the neuroprotective effect exerted by NCX during ischemic injury, the major role is prevalently exerted by NCX1 and NCX3 gene products.

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.

Up-Regulation and Increased Activity of KV3.4 Channels and Their Accessory Subunit MinK-Related Peptide 2 Induced by Amyloid Peptide Are Involved in Apoptotic Neuronal Death

The aim of the present study was to investigate whether KV3.4 channel subunits are involved in neuronal death induced by neurotoxic β-amyloid peptides (Aβ). In particular, to test this hypothesis, three main questions were addressed: 1) whether the Aβ peptide can up-regulate both the transcription/translation and activity of KV3.4 channel subunit and its accessory subunit, MinK-related peptide 2 (MIRP2); 2) whether the increase in KV3.4 expression and activity can be mediated by the nuclear factor-κB (NF-κB) family of transcriptional factors; and 3) whether the specific inhibition of KV3.4 channel subunit reverts the Aβ peptide-induced neurodegeneration in hippocampal neurons and nerve growth factor (NGF)-differentiated PC-12 cells. We found that Aβ1–42 treatment induced an increase in KV3.4 and MIRP2 transcripts and proteins, detected by reverse transcription-polymerase chain reaction and Western blot analysis, respectively, in NGF-differentiated PC-12 cells and hippocampal neurons. Patch-clamp experiments performed in whole-cell configuration revealed that the Aβ peptide caused an increase in IA current amplitude carried by KV3.4 channel subunits, as revealed by their specific blockade with blood depressing substance-I (BDS-I) in both hippocampal neurons and NGF-differentiated PC-12 cells. The inhibition of NF-κB nuclear translocation with the cell membrane-permeable peptide SN-50 prevented the increase in KV3.4 protein and transcript expression. In addition, the SN-50 peptide was able to block Aβ1–42-induced increase in KV3.4 K+ currents and to prevent cell death caused by Aβ1–42 exposure. Finally, BDS-I produced a similar neuroprotective effect by inhibiting the increase in KV3.4 expression. As a whole, our data indicate that KV3.4 channels could be a novel target for Alzheimers disease pharmacological therapy.

Anoxia-Induced NF-kB-Dependent Upregulation of NCX1 Contributes to Ca2+ Refilling Into Endoplasmic Reticulum in Cortical Neurons

Background and Purpose— The 3 gene products of the Na+/Ca2+ exchanger (NCX), viz, NCX1, NCX2, and NCX3, may play a pivotal role in the pathophysiology of brain ischemia. The aim of this study was to investigate the transductional and posttranslational mechanisms involved in the expression of these isoforms during oxygen and glucose deprivation and their role in endoplasmic reticulum Ca2+ refilling in cortical neurons. Methods— NCX1, NCX2, and NCX3 transcript and protein expression was evaluated in primary cortical neurons by reverse transcriptase–polymerase chain reaction and Western blot. NCX currents (INCX) and cytosolic Ca2+ concentrations ([Ca2+]i) were monitored by means of patch-clamp in whole-cell configuration and Fura-2AM single-cell video imaging, respectively. Results— Exposure of cortical neurons to 3 hours of oxygen and glucose deprivation yielded dissimilar effects on the 3 isoforms. First, it induced an upregulation in NCX1 transcript and protein expression. This change was exerted at the transcriptional level because the inhibition of nuclear factor kappa B translocation by small interfering RNA against p65 and SN-50 prevented oxygen and glucose deprivation-induced NCX1 upregulation. Second, it elicited a downregulation of NCX3 protein expression. This change, unlike NCX1, was exerted at the posttranscriptional level because it was prevented by the proteasome inhibitor MG-132. Finally, we found that it significantly increased INCX both in the forward and reverse modes of operation and promoted an increase in ER Ca2+ accumulation. Interestingly, such accumulation was prevented by the silencing of NCX1 and the NCX inhibitor CB-DMB that triggered caspase-12 activation. Conclusions— These results suggest that nuclear factor kappa B-dependent NCX1 upregulation may play a fundamental role in Ca2+ refilling in the endoplasmic reticulum, thus helping neurons to prevent endoplasmic reticulum stress during oxygen and glucose deprivation.

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.

NO‐induced neuroprotection in ischemic preconditioning stimulates mitochondrial Mn‐SOD activity and expression via RAS/ERK1/2 pathway

To identify the transductional mechanisms responsible for the neuroprotective effect of nitric oxide (NO) during ischemic preconditioning (IPC), we investigated the effects of this gaseous mediator on mitochondrial Mn‐superoxide dismutase (Mn‐SOD) expression and activity. In addition, the possible involvement of Ras/extracellular‐regulated kinase (ERK) ERK1/2 pathway in preserving cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation was also examined. Ischemic preconditioning was obtained by exposing neurons to a 30‐min sublethal OGD (95% N2 and 5% CO2). Then, after a 24‐h interval, neurons were exposed to 3 h of OGD followed by 24 h of reoxygenation (OGD/Rx). Our results revealed that IPC reduced cytochrome c (cyt c) release into the cytosol, improved mitochondrial function, and decreased free radical production. Moreover, it induced an increase in nNOS expression and NO production and promoted ERK1/2 activation. These effects were paralleled by an increase in Mn‐SOD expression and activity that persisted throughout the following OGD phase. When the neurons were treated with L‐NAME, a well known NOS inhibitor, the increase in Mn‐SOD expression occurring during IPC was reduced and, as a result, IPC‐induced neuroprotection was prevented. Similarly, when ERK1/2 was inhibited by its selective inhibitor PD98059, the increase in Mn‐SOD expression observed during IPC was almost completely abolished. As a result, its neuroprotective effect on cellular survival was thwarted. The present findings indicate that during IPC the increase in Mn‐SOD expression and activity are paralleled by NO production. This suggests that NO neuroprotective role occurs through the stimulation of Mn‐SOD expression and activity. In particular, NO via Ras activation stimulates downstream ERK1/2 cascade. This pathway, in turn, post‐transcriptionally activates Mn‐SOD expression and activity, thus promoting neuroprotection during preconditioning.

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 [Ca2+]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 and Results— 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-1&agr; 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 NCX3 isoform of the Na+/Ca2+ exchanger contributes to neuroprotection elicited by ischemic postconditioning

It has been recently shown that a short sublethal brain ischemia subsequent to a prolonged harmful ischemic episode may confer ischemic neuroprotection, a phenomenon termed ischemic postconditioning. Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are plasma membrane ionic transporters widely distributed in the brain and involved in the control of Na+ and Ca2+ homeostasis and in the progression of stroke damage. The objective of this study was to evaluate the role of these three proteins in the postconditioning-induced neuroprotection. The NCX protein and mRNA expression was evaluated at different time points in the ischemic temporoparietal cortex of rats subjected to tMCAO alone or to tMCAO plus ischemic postconditioning. The results of this study showed that NCX3 protein and ncx3 mRNA were upregulated in those brain regions protected by postconditioning treatment. These changes in NCX3 expression were mediated by the phosphorylated form of the ubiquitously expressed serine/threonine protein kinase p-AKT, as the p-AKT inhibition prevented NCX3 upregulation. The relevant role of NCX3 during postconditioning was further confirmed by results showing that NCX3 silencing, induced by intracerebroventricular infusion of small interfering RNA (siRNA), partially reverted the postconditioning-induced neuroprotection. The results of this study support the idea that the enhancement of NCX3 expression and activity might represent a reasonable strategy to reduce the infarct extension after stroke.

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.