Division of Pharmacology, Department of Neuroscience, National Institute of Neuroscience, School of Medicine, "Federico II" University of Naples, 80131 Naples, Italy.
J Neurosci. 2012 Aug 1;32(31):10609-17. doi: 10.1523/JNEUROSCI.6429-11.2012.
Although the amyloid-β(1-42) (Aβ(1-42)) peptide involved in Alzheimer's disease is known to cause a dysregulation of intracellular Ca(2+) homeostasis, its molecular mechanisms still remain unclear. We report that the extracellular-dependent early increase (30 min) in intracellular calcium concentration (Ca(2+)), following Aβ(1-42) exposure, caused the activation of calpain that in turn elicited a cleavage of the Na(+)/Ca(2+) 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 Ca(2+) 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 Ca(2+) content in ER stores, suggesting that NCX3 was involved in the increase of ER Ca(2+) 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 Ca(2+) content triggered ER stress, as revealed by caspase-12 activation. Concomitantly, the late increase in Ca(2+) 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 Ca(2+)-dependent calpain activation, generates a hyperfunctional form of NCX3 that, by increasing Ca(2+) content into ER, delays caspase-12 activation and thus neuronal death.
虽然与阿尔茨海默病相关的淀粉样β肽（1-42）（Aβ（1-42））已知会导致细胞内 Ca（2+）稳态失调，但其分子机制仍不清楚。我们报告称，Aβ（1-42）暴露后，细胞外依赖性的早期（30 分钟）细胞内钙离子浓度（[Ca（2+）]（i））增加会导致钙蛋白酶的激活，进而引发 Na（+）/Ca（2+）交换体同工型 NCX3 的切割。这种切割产生了一种具有超功能的转运体形式，并增加了反向操作模式下的 NCX 电流（I（NCX））。有趣的是，这种 NCX3 钙蛋白酶依赖性切割对于 Aβ（1-42）依赖性 I（NCX）增加是必不可少的。事实上，钙蛋白酶抑制剂 calpeptin 和通过定点突变去除钙蛋白酶切割识别序列，消除了这种作用。此外，增强的 NCX3 活性伴随着内质网（ER）储存中 Ca（2+）含量的增加。值得注意的是，PC-12 细胞中的沉默或敲除 ncx3 基因可防止 I（NCX）和 ER 储存中 Ca（2+）含量的增加，这表明 NCX3 参与了由 Aβ（1-42）刺激的 ER Ca（2+）含量的增加。相比之下，在晚期（72 小时），当 NCX3 蛋白水解切割突然停止时，ER Ca（2+）含量的平行减少触发了 ER 应激，这是通过半胱天冬酶-12 的激活揭示的。同时，晚期 [Ca（2+）]（i）的增加与神经元死亡同时发生。有趣的是，NCX3 沉默导致 Aβ（1-42）诱导的半胱天冬酶-12 的更早激活。事实上，在 NCX3 沉默的神经元中，Aβ（1-42）暴露加速了半胱天冬酶依赖性细胞凋亡，从而增强了神经元细胞死亡。这些结果表明，Aβ（1-42）通过 Ca（2+）依赖性钙蛋白酶激活，产生一种超功能形式的 NCX3，通过增加 ER 中的 Ca（2+）含量，延迟半胱天冬酶-12 的激活，从而延迟神经元死亡。
