Liu Jia, Naegele Janice R, Lin Stanley L
Program in Neuroscience and Behavior, Department of Biology, Wesleyan University, Middletown, CT 06459-0170, USA.
Brain Res. 2009 Nov 3;1296:164-75. doi: 10.1016/j.brainres.2009.07.101. Epub 2009 Aug 4.
DNA repair deficiency results in neurodegenerative disease and increased susceptibility to excitotoxic cell death, suggesting a critical but undefined role for DNA damage in neurodegeneration. We compared DNA damage, Ku70-Bax interaction, and Bax-dependent excitotoxic cell death in kainic acid-treated primary cortical neurons derived from both wild-type mice and mice deficient in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) encoded by the Prkdc gene. In both wild-type and Prkdc(-/-) neurons, kainic acid treatment resulted in rapid induction of DNA damage (53BP1 foci formation) followed by nuclear pyknosis. Bax deficiency, by either Bax shRNA-mediated knockdown or gene deletion, protected wild-type and heterozygous but not Prkdc(-/-) neurons from kainate-induced excitotoxicity. Cotransfection of DNA-PKcs with Bax shRNA restored Bax shRNA-mediated neuroprotection in Prkdc(-/-) neurons, suggesting that DNA-PKcs is required for kainate-induced activation of the pro-apoptotic Bax pathway. Immunoprecipitation studies revealed that the DNA-PKcs-nonphosphorylatable Ku70 (S6A/S51A) bound 3- to 4-fold greater Bax than wild-type Ku70, suggesting that DNA-PKcs-mediated Ku70 phosphorylation causes release of Bax from Ku70. In support of this, kainic acid induced translocation of a Bax-EGFP fusion protein to the mitochondria in the presence of a cotransfected wild-type, but not mutant Ku70 (S6A/S51A) gene when examined at 4 and 8 h following kainate addition. We conclude that DNA-PKcs links DNA damage to Bax-dependent excitotoxic cell death, by phosphorylating Ku70 on serines 6 and/or 51, to initiate Bax translocation to the mitochondria and directly activate a pro-apoptotic Bax-dependent death cascade.
DNA修复缺陷会导致神经退行性疾病，并增加对兴奋性毒性细胞死亡的易感性，这表明DNA损伤在神经退行性变中起着关键但尚不明确的作用。我们比较了野生型小鼠和Prkdc基因编码的DNA依赖性蛋白激酶催化亚基（DNA-PKcs）缺陷小鼠来源的经海藻酸处理的原代皮质神经元中的DNA损伤、Ku70与Bax的相互作用以及Bax依赖性兴奋性毒性细胞死亡。在野生型和Prkdc（-/-）神经元中，海藻酸处理均导致DNA损伤迅速诱导（53BP1灶形成），随后出现核固缩。通过Bax shRNA介导的敲低或基因缺失导致的Bax缺陷，可保护野生型和杂合型神经元，但不能保护Prkdc（-/-）神经元免受海藻酸盐诱导的兴奋性毒性。将DNA-PKcs与Bax shRNA共转染可恢复Prkdc（-/-）神经元中Bax shRNA介导的神经保护作用，这表明DNA-PKcs是海藻酸盐诱导促凋亡Bax途径激活所必需的。免疫沉淀研究表明，DNA-PKcs不可磷酸化的Ku70（S6A/S51A）与Bax的结合量比野生型Ku70高3至4倍，这表明DNA-PKcs介导的Ku70磷酸化导致Bax从Ku70释放。支持这一观点的是，在添加海藻酸盐后4小时和8小时进行检测时，在共转染野生型但非突变型Ku70（S6A/S51A）基因的情况下海藻酸诱导了Bax-EGFP融合蛋白向线粒体的转位。我们得出结论，DNA-PKcs通过在丝氨酸6和/或51上磷酸化Ku70，将DNA损伤与Bax依赖性兴奋性毒性细胞死亡联系起来，从而启动Bax向线粒体的转位并直接激活促凋亡的Bax依赖性死亡级联反应。
