A1-42 fragment induces an up-regulation of NCX3 activity that prevents caspase-12 activation.

The molecular mechanisms responsible for A1-42-peptide induced intracellular Ca2+ homeostasis dysregulation still remain unclear. We report data obtained in mouse hippocampal neurons and NGF-differentiated PC-12 cells suggesting that the extracellular-dependent early increase(30minutes) in intracellular calcium concentration ([Ca2+]i), following A1-42 exposure, caused the activation of calpain that in turn elicited a cleavage of the Na+/Ca2+ exchanger(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 A1-42-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 ER-stores. Remarkably, the silencing or knocking-out of the ncx3 gene prevented the enhancement of both INCX and Ca2+ content in the ER-stores, suggesting that NCX3 was involved in the increase of ER Ca2+-content stimulated by A1-42. By contrast, in the late phase (72hours), 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1-42-induced caspase-12. Indeed, in NCX3 silenced neurons, A1-42 exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that A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.