Mitochondrial DNA repair: a critical player in the response of cells of the CNS to genotoxic insults.

Cells of the CNS are constantly exposed to agents which damage DNA. Although much attention has been paid to the effects of this damage on nuclear DNA, the nucleus is not the only organelle containing DNA. Within each cell, there are hundreds to thousands of mitochondria. Within each mitochondrion are multiple copies of the mitochondrial genome. These genomes are extremely vulnerable to insult and mutations in mitochondrial DNA (mtDNA) have been linked to several neurodegenerative diseases, as well as the normal process of aging. The principal mechanism utilized by cells to avoid DNA mutations is DNA repair. Multiple pathways of DNA repair have been elucidated for nuclear DNA. However, it appears that only base excision repair is functioning in mitochondria. This repair pathway is responsible for the removal of most endogenous damage including alkylation damage, depurination reactions and oxidative damage. Within the rat CNS, there are cell-specific differences mtDNA repair. Astrocytes exhibit efficient repair, whereas, other glial cell types and neuronal cells exhibit a reduced ability to remove lesions from mtDNA. Additionally, a correlation was observed between those cells with reduced mtDNA repair and an increase in the induction of apoptosis. To demonstrate a causative relationship, a strategy of targeting DNA repair proteins to mitochondria to enhance mtDNA repair capacity was employed. Enhancement of mtDNA repair in oligodendrocytes provided protection from reactive oxygen species- and cytokine-induced apoptosis. These experiments provide a novel strategy for protecting sensitive CNS cells from genotoxic insults and thus provide new treatment options for neurodegenerative diseases.

Glial cell type-specific responses to menadione-induced oxidative stress.

Glial cell types in the central nervous system are continuously exposed to reactive oxygen species (ROS) due to their high oxygen metabolism and demonstrate differential susceptibility to certain pathological conditions believed to involve oxidative stress. The purpose of the current studies was to test the hypothesis that mtDNA damage could contribute to the differential susceptibility of glial cell types to apoptosis induced by oxidative stress. Primary cultures of rat astrocytes, oligodendrocytes, and microglia were utilized, and menadione was used to produce the oxidative stress. Apoptosis was detected and quantitated in menadione-treated oligodendrocytes and microglia (but not astrocytes) using either positive annexin-V staining or positive staining for 3'-OH groups in DNA. The apoptotic pathway that was activated involved the release of cytochrome c from the intermitochondrial space and activation of caspase 9. Caspase 8 was not activated after exposure to menadione in any of the cells. Using equimolar concentrations of menadione, more initial damage was observed in mtDNA from oligodendrocytes and microglia. Additionally, using concentrations of menadione that resulted in comparable initial mtDNA damage, more efficient repair was observed in astrocytes compared to either oligodendrocytes or microglia. The differential susceptibility of glial cell types to oxidative damage and apoptosis did not appear related to cellular antioxidant capacity, because under the current culture conditions astrocytes had lower total glutathione content and superoxide dismutase activity than oligodendrocytes and microglia. These results show that the differential susceptibility of glial cell types to menadione-induced oxidative stress and apoptosis appears to correlate with increased oxidative mtDNA damage and support the hypothesis that mtDNA damage could participate in the initiation of apoptosis through the enhanced release of cytochrome c and the activation of caspase 9.