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

Although the amyloid-beta(1-42) (A beta(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 A beta(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 A beta(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 A beta(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 A beta(1-42)-induced caspase-12. Indeed, in NCX3-silenced neurons, A beta(1-42) exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that A beta(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.