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.

Introduction: Src tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. To identify the mechanism by which Src overexpression sustains this resistance, we tested a panel of breast cancer cell lines either sensitive or resistant to lapatinib. Methods: To determine the role of Src in lapatinib resistance, we evaluated the effects of Src inhibition/silencing in vitro on survival, migration, and invasion of lapatinib-resistant cells. In vivo experiments were performed in JIMT-1 lapatinib-resistant cells orthotopically implanted in nude mice. We used artificial metastasis assays to evaluate the effect of Src inhibition on the invasiveness of lapatinib-resistant cells. Src-dependent signal transduction was investigated with Western blot and ELISA analyses. Results: Src activation was higher in lapatinib-resistant than in lapatinib-sensitive cells. The selective small-molecule Src inhibitor saracatinib combined with lapatinib synergistically inhibited the proliferation, migration, and invasion of lapatinib-resistant cells. Saracatinib combined with lapatinib significantly prolonged survival of JIMT-1-xenografted mice compared with saracatinib alone, and impaired the formation of lung metastases. Unexpectedly, in lapatinib-resistant cells, Src preferentially interacted with epidermal growth factor receptor (EGFR) rather than with HER2. Moreover, EGFR targeting and lapatinib synergistically inhibited survival, migration, and invasion of resistant cells, thereby counteracting Src-mediated resistance. These findings demonstrate that Src activation in lapatinib-resistant cells depends on EGFR-dependent rather than on HER2-dependent signaling. Conclusions: Complete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer.

Long-term potentiation (LTP) depends on the coordinated regulation of an ensemble of proteins related to Ca²⁺homeostasis, including Ca²⁺transporters. One of the major players in the regulation of intracellular Ca²⁺([Ca²⁺]_i) homeostasis in neurons is the sodium/calcium exchanger (NCX), which represents the principal mechanism of Ca²⁺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 ofncx3gene ablation on NCX currents (I_NCX) and Ca²⁺homeostasis in hippocampal neurons.ncx3^−/−neurons displayed a reducedI_NCX, a higher basal level of [Ca²⁺]_i, and a significantly delayed clearance of [Ca²⁺]_ifollowing depolarization. Furthermore, measurement of field EPSPs, recorded from the CA1 area, revealed thatncx3^−/−mice had an impaired basal synaptic transmission. Moreover, hippocampal slices fromncx3^−/−mice exhibited a worsening in LTP compared with congenicncx3^+/+. Consistently, immunohistochemical and immunoblot analysis indicated that in the hippocampus ofncx3^−/−mice both Ca²⁺/calmodulin-dependent protein kinase IIα (CaMKIIα) expression and the phosphoCaMKIIα/CaMKIIα ratio were significantly reduced compared withncx3^+/+. 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 thencx3gene in mice has detrimental consequences on basal synaptic transmission, LTP regulation, spatial learning, and memory performance.

Although the amyloid-β_1–42 (Aβ_1–42) peptide involved in Alzheimer's 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 Aβ_1–42 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 (I_NCX) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the Aβ_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 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 I_NCX and Ca²⁺ content in ER stores, suggesting that NCX3 was involved in the increase of ER Ca²⁺ content stimulated by Aβ_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 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 Aβ_1–42-induced caspase-12. Indeed, in NCX3-silenced neurons, Aβ_1–42 exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that Aβ_1–42, 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.