Endoplasmic reticulum refilling and mitochondrial calcium extrusion promoted in neurons by NCX1 and NCX3 in ischemic preconditioning are determinant for neuroprotection

Ischemic preconditioning (IPC), an important endogenous adaptive mechanism of the CNS, renders the brain more tolerant to lethal cerebral ischemia. The molecular mechanisms responsible for the induction and maintenance of ischemic tolerance in the brain are complex and still remain undefined. Considering the increased expression of the two sodium calcium exchanger (NCX) isoforms, NCX1 and NCX3, during cerebral ischemia and the relevance of nitric oxide (NO) in IPC modulation, we investigated whether the activation of the NO/PI3K/Akt pathway induced by IPC could regulate calcium homeostasis through changes in NCX1 and NCX3 expression and activity, thus contributing to ischemic tolerance. To this aim, we set up an in vitro model of IPC by exposing cortical neurons to a 30-min oxygen and glucose deprivation (OGD) followed by 3-h OGD plus reoxygenation. IPC was able to stimulate NCX activity, as revealed by Fura-2AM single-cell microfluorimetry. This effect was mediated by the NO/PI3K/Akt pathway since it was blocked by the following: (a) the NOS inhibitors L-NAME and 7-Nitroindazole, (b) the IP3K/Akt inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, wortmannin and the Akt-negative dominant, (c) the NCX1 and NCX3 siRNA. Intriguingly, this IPC-mediated upregulation of NCX1 and NCX3 activity may control calcium level within endoplasimc reticulum (ER) and mitochondria, respectively. In fact, IPC-induced NCX1 upregulation produced an increase in ER calcium refilling since this increase was prevented by siNCX1. Moreover, by increasing NCX3 activity, IPC reduced mitochondrial calcium concentration. Accordingly, the inhibition of NCX by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 reverted this effect, thus suggesting that IPC-induced NCX3-increased activity may improve mitochondrial function during OGD/reoxygenation. Collectively, these results indicate that IPC-induced neuroprotection may occur through the modulation of calcium homeostasis in ER and mitochondria through NO/PI3K/Akt-mediated NCX1 and NCX3 upregulation.Ischemic preconditioning (IPC), an important endogenous adaptive mechanism of the brain, increases neuronal tolerance to lethal cerebral ischemia. The molecular mechanisms responsible for inducing and maintaining ischemic tolerance in the brain are complex and are not yet fully understood. Among the three isoforms of the Na⁺/Ca²⁺ exchanger, NCX1 and NCX3 represent two new possible molecular effectors involved in the neuroprotective mechanisms of IPC.^{1, 2, 3} Indeed, the increased expression of these two plasma membrane proteins, which have a fundamental role in regulating and maintaining cellular calcium and sodium homeostasis in the brain^{4, 5} during IPC, has been associated with a decrease in the infarct volume following a more severe ischemic insult.¹ However, the molecular mechanisms by which NCX1 and NCX3 upregulation lead to IPC-induced brain tolerance still remain unexplored.In vitro experiments performed in cortical neurons exposed to oxygen and glucose deprivation (OGD) and subsequent reoxygenation have demonstrated that changes in NCX isoform expression during OGD are accompanied by increases in both NCX1 activity and endoplasimc reticulum (ER) Ca²⁺ refilling.⁶ Considering the increased expression of the two sodium calcium exchanger (NCX) isoforms, NCX1 and NCX3, during cerebral ischemia and the relevance of nitric oxide (NO) in IPC modulation,^{7, 8} we investigated whether the activation of the NO/PI3K/Akt pathway induced by IPC could regulate calcium homeostasis through changes in NCX1 and NCX3 expression and activity, thus contributing to ischemic tolerance.More recently, we have reported that among the three NCX isoforms, only NCX3 is expressed on the outer mitochondrial membrane, where it works mainly by extruding calcium from the matrix.⁹ In this regard, an even more compelling result is that NCX3 gene ablation induces not only the disappearance of the protein from the OMM but also the accumulation of mitochondrial calcium in cortical neurons. Interestingly, NCX3 expression decreases in cortical neurons during OGD, a finding that correlates with an increase in [Ca²⁺]_m.⁹Furthermore, preserving mitochondrial function is relevant for preconditioning-induced neuroprotection. In fact, preconditioning positively affects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia,¹⁰ prevents mitochondrial swelling, protects mitochondrial energy metabolism during cerebral ischemia by avoiding ATP consumption¹¹ and increases Mn-SOD expression and activity through the NO/Ras/ERK1-2 pathway.⁸Although mitochondria are considered to be important mediators of endogenous neuroprotection, the mechanisms by which they might integrate cytoprotective signaling of preconditioning still remain to be fully elucidated. Thus, we investigated the role played by NCX1 and NCX3 in regulating ER and mitochondrial calcium homeostasis as a novel mechanism responsible for IPC-induced neuroprotection.For this aim, cortical neurons were exposed to 30 min of OGD followed by 3-h OGD plus reoxygenation. The expression and activity of NCX1 and NCX3 were observed by means of western blot analysis, confocal microscopy and single cell microfluorimetry. The results showed that IPC-induced neuroprotection occurs through the modulation of calcium homeostasis in ER and mitochondria through NO/Akt-mediated NCX1 and NCX3 upregulation.