The DNA-PK catalytic subunit regulates Bax-mediated excitotoxic cell death by Ku70 phosphorylation

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.