Visualizing common deletion of mitochondrial DNA-augmented mitochondrial reactive oxygen species generation and apoptosis upon oxidative stress

AbstractCommon deletion (CD) 4977 bp of mitochondrial DNA (mtDNA) disrupt specifically mitochondrial complex I, IV and V on the electron transport chain (ETC) and is closely associated with wide spectrums of clinical manifestations. To quantitatively investigate how CD-induced ETC defect alters mitochondrial reactive oxygen species (mROS) generation as well as down stream apoptotic signaling, we employed an established array of human CD cytoplasmic hybrids (cybrids) harboring 0%–80% of CD. Pathological effects of CD on the mitochondria were visualized at single cell level by the application of fluorescent probes coupled with conventional and multiphoton imaging microscopy. Intriguingly, we observed CD-augmented mROS generation omitted “threshold effect”. CD-augmented mROS generation was associated with depolarized mitochondrial membrane potential (ΔΨm). Upon oxidative stress, the amount of CD-augmented mROS generation was greatly enhanced to cause pathological apoptotic deterioration including opening of the mitochondrial permeability transition, cytochrome c release, phosphatidylserine externalization and DNA fragmentation. In addition, heterogeneous mitochondrial dysfunctions were found in cybrids containing 80% of CD (D cybrids), i.e., low sensitive-D (LS-D, roughly 80%) and a super sensitive-D (SS-D, 20%). As compared to LS-D, SS-D had higher resting mROS level but slightly hyperpolarized ΔΨm. Upon H2O2 treatment, much faster generation of mROS was observed which induced a faster depolarization of ΔΨm and later apoptotic deterioration in SS-D. We proposed a dose-dependent, feed-forward and self-accelerating vicious cycle of mROS production might be initiated in CD-induced ETC defect without threshold effect. As CD-augmented mROS generation is obligated to cause an enhanced pathological apoptosis, precise detection of CD-augmented mROS generation and their degree of heterogeneity in single cells may serve as sensitive pathological indexes for early diagnosis, prognosis and treatment of CD-associated diseases