Mitochondrial DNA Breaks Activate an Integrated Stress Response to Reestablish Homeostasis

Abstract

Double-strand breaks in mitochondrial DNA (mtDSBs) lead to the degradation of the circular genomes and a reduction in copy number. However, it is unclear how mtDSBs are sensed and what signaling pathways are activated in response to mtDNA damage. In this study, we used mitochondrial-targeted restriction enzymes to investigate the cellular response to mtDSBs. Our results showed that a subset of cells with mtDSBs exhibited defects in mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy revealed compromised mitochondrial membrane and cristae ultrastructure. We also found that mtDSBs activated the integrated stress response (ISR) through the phosphorylation of eIF2 by DELE1 and HRI. Notably, inhibition of the ISR exacerbated the mitochondrial import defect and delayed the recovery of mtDNA copy number following break formation. These findings suggest that the ISR plays a role in mitigating mitochondrial dysfunction following mtDNA damage and is critical to promoting mtDNA repopulation. Last, we used proteomics to survey the proteins present in the nucleoids shortly after mtDSBs and identified ATAD3A, a membrane-anchored protein interacting with nucleoids, as a potential factor in transmitting the signal from damaged genomes to the inner mitochondrial membrane. In summary, our study reveals the sequence of events linking damaged mitochondrial genomes with the cytoplasm and highlights the unexpected role of the ISR in reestablishing homeostasis in response to mitochondrial genome instability.