The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that D-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo Ϫ/Ϫ ), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of D-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo Ϫ/Ϫ brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo Ϫ/Ϫ brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate. Key words: aging; D-amino acids; DNA methylation; neurodegeneration; NMDA receptor Significance StatementThe enzyme D-aspartate oxidase (DDO) catalyzes the degradation of the NMDA receptor agonist, D-aspartate. In the brain, DDO is expressed only during postnatal life, thus reducing the embryonic storage of D-aspartate and keeping this D-amino acid at low levels during adulthood. Although the presence of DDO in mammals is long established, its biological role in the brain and the mechanism regulating its expression are still unclear. Here, we found that Ddo promoter demethylation enables the postnatal expression of Ddo. Moreover, persistent suppression of Ddo expression leads to persistent spillover of extracellular D-aspartate and produces precocious cell death in the mouse brain, thus suggesting a key role for DDO in preventing early neurodegeneration triggered by excessive NMDA receptor stimulation.

Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury.

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 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 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.

SummaryThe mitochondrial influx and efflux of Ca 2+ play a relevant role in cytosolic and mitochondrial Ca 2+ homeostasis, and contribute to the regulation of mitochondrial functions in neurons. The mitochondrial Na + /Ca 2+ 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 + /Ca 2+ exchanger extruding Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ efflux mode at the level of the OMM. Collectively, these results suggest that, in neurons, NCX3 regulates mitochondrial Ca 2+ handling from the OMM through an AKAP121-anchored signaling complex, thus promoting cell survival during hypoxia.