mtDNA breaks compromise mitochondrial membrane ultrastructure and trigger an integrated stress response

Mitochondrial DNA (mtDNA) breaks are deleterious lesions that lead to degradation of mitochondrial genomes and subsequent reduction in mtDNA copy number. The signaling pathways activated in response to mtDNA damage remain ill-defined. Using mitochondrial targeted restriction enzymes, we show that cells with mtDNA breaks exhibit reduced respiratory complexes, loss of membrane potential, and mitochondrial protein import defect. Furthermore, mtDNA damage activates the integrated stress response (ISR) through phosphorylation of eIF2α by the OMA1-DELE1-HRI pathway. Electron microscopy reveals concomitant defects in mitochondrial membranes and cristae ultrastructure. Notably, inhibition of the ISR exacerbates mitochondrial defects and delays the recovery of mtDNA copy number, thereby implicating this stress response in mitigating mitochondrial dysfunction following mtDNA damage. Last, we provide evidence suggesting that ATAD3A, a membrane-anchored protein that interacts with nucleoids, relays the signal from mtDNA breaks to the inner mitochondrial membrane. Altogether, our study fully delineates the sequence of events linking damaged mitochondrial genomes with the cytoplasm and uncovers an unanticipated role for the ISR in response to mitochondrial genome instability. We performed RNA-seq analysis on GFP11 tagged RRBP1 and MTA2 cells subjected to the following treatments: ApaLI* + Dox, ApaLI + Dox, ApaLI + Dox GFP- population, ApaLI + Dox GFP+ population. RNA-seq was done in 2 replicates.

线粒体DNA（mtDNA）断裂是一类可导致线粒体基因组降解、进而引起mtDNA拷贝数降低的有害损伤。目前，针对mtDNA损伤所激活的信号通路仍未明确。本研究利用线粒体靶向限制性内切酶开展实验，结果显示携带mtDNA断裂的细胞存在呼吸复合体含量降低、膜电位丧失以及线粒体蛋白导入缺陷等表型。此外，mtDNA损伤可通过OMA1-DELE1-HRI通路对eIF2α进行磷酸化，从而激活整合应激反应（ISR）。电子显微镜观察发现，线粒体膜及嵴的超微结构同时出现异常。值得注意的是，抑制整合应激反应会加剧线粒体功能缺陷，并延缓mtDNA拷贝数的恢复，由此表明该应激反应可在mtDNA损伤后缓解线粒体功能障碍。最后，本研究提供证据表明，ATAD3A——一种与线粒体类核相互作用的膜锚定蛋白——可将mtDNA断裂的信号传递至线粒体内膜。综上，本研究完整阐明了受损线粒体基因组与细胞质之间的信号传导序列，并揭示了整合应激反应在应对线粒体基因组不稳定过程中此前未被发现的作用。
我们对经以下处理的GFP11标签标记的RRBP1与MTA2细胞开展了RNA测序（RNA-seq）分析：ApaLI* + 多西环素（Dox）、ApaLI + 多西环素（Dox）、ApaLI + 多西环素（Dox）处理的GFP阴性细胞群、ApaLI + 多西环素（Dox）处理的GFP阳性细胞群。本次RNA测序共设置2次重复实验。