Kuwako Ken-ichiro, Nishimura Isao, Uetsuki Taichi, Saido Takaomi C, Yoshikawa Kazuaki
Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Brain Res Mol Brain Res. 2002 Nov 15;107(2):166-75. doi: 10.1016/s0169-328x(02)00489-8.
We have previously reported that overexpression of wild-type amyloid precursor protein (APP) in postmitotic neurons induces cleavage-dependent activation of caspase-3 both in vivo and in vitro. In this study, we investigated the mechanism underlying APP-induced caspase-3 activation using adenovirus-mediated gene transfer into postmitotic neurons derived from human embryonal carcinoma NT2 cells. Overexpression of wild-type APP significantly increased intracellular (45)Ca(2+) content prior to the activation of caspase-3 in NT2-derived neurons. Chelation of intracellular Ca(2+) markedly suppressed APP-induced activation of caspase-3. Furthermore, calpain, a Ca(2+)-dependent cysteine protease, was activated in neurons overexpressing APP as assessed by increased levels of calpain-cleaved alpha-fodrin and autolytic mu-calpain fragments. Neither calpain nor caspase-3 was activated in neurons expressing an APP mutant defective in the Abeta(1-20) domain. Calpain inhibitors almost completely suppressed APP-induced activation of neuronal caspase-3. E64d, a membrane permeable inhibitor of calpain, significantly suppressed APP-induced neuronal death. These results suggest that overexpression of wild-type APP activates calpain that mediates caspase-3 activation in postmitotic neurons.
我们之前报道过，有丝分裂后神经元中野生型淀粉样前体蛋白（APP）的过表达在体内和体外均能诱导依赖切割的半胱天冬酶-3激活。在本研究中，我们利用腺病毒介导的基因转移技术，将其导入源自人胚胎癌NT2细胞的有丝分裂后神经元，来研究APP诱导半胱天冬酶-3激活的机制。在NT2衍生的神经元中，野生型APP的过表达在半胱天冬酶-3激活之前显著增加了细胞内（45）Ca（2+）含量。细胞内Ca（2+）的螯合显著抑制了APP诱导的半胱天冬酶-3激活。此外，通过钙蛋白酶切割的α-血影蛋白和自溶的μ-钙蛋白酶片段水平的增加评估发现，钙蛋白酶（一种Ca（2+）依赖性半胱氨酸蛋白酶）在过表达APP的神经元中被激活。在表达Abeta（1-20）结构域缺陷的APP突变体的神经元中，钙蛋白酶和半胱天冬酶-3均未被激活。钙蛋白酶抑制剂几乎完全抑制了APP诱导的神经元半胱天冬酶-3激活。E64d（一种可透过膜的钙蛋白酶抑制剂）显著抑制了APP诱导的神经元死亡。这些结果表明，野生型APP的过表达激活了钙蛋白酶，而钙蛋白酶在有丝分裂后神经元中介导半胱天冬酶-3的激活。
