Abstract

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.