mtDNA repair associated with disease states. Whether aging and disease affects activities of the major translocase complexes TOM and TIM is not known. However, it has been proposed that oxidative modiÞcation of one or more subunits of TOM could be a possible mechanism for such an mtDNA repair defect [92].

10.5Mitochondrial DNA Damage/Repair in the Retina and RPE

Despite clear evidence of mitochondrial dysfunction in age-related macular degeneration (AMD), diabetic retinopathy, and glaucoma there has been surprisingly few studies on the role of mtDNA damage and repair in the retina and the major emphasis has focused on the RPE. Barreau et al. showed increased mtDNA deletions in aged human neural retina [156] and mitochondrial DNA deletions and cytochrome c oxidase-deÞcient cones accumulate in the aging retina, particularly in the foveal region [157]. Two studies have recently examined mtDNA from the macular region of AMD patients and age-matched control donors [158, 159]. In one of the studies, an increase in mtDNA damage with aging was detected only in the common deletion region of the mitochondrial genome (a 4977 deletion extending from the ND5 gene to the ATP8 gene (Fig. 10.1) [158]). In contrast, in macular RPE from AMD eyes, mtDNA lesions increased signiÞcantly in all regions of the mitochondrial genome [158, 159]. MŸller cell mitochondria from aged guinea pigs displayed a diminished number of well-deÞned cristae, a reduced membrane potential, and a slightly reduced respiration capacity when compared with those from young adults [160]. 8-OHdG increases in aged rodent retina and choroid compared to young animals [161, 162]. Most of the damaged mtDNA in the neural retina was in the photoreceptors and retinal ganglion cells. 8-OHdG colocalized with the mitochondrial manganese superoxide dismutase (SOD2) and measurements of nDNA and mtDNA lesions indicated that DNA damage is primarily in mtDNA. The increase in oxidative damage was associated with decreased DNA damage repair enzymes such as OGG1 and mutY homolog MYH [161, 162].

10.5.1Mitochondrial DNA Damage/Repair in the RPE

Cell culture studies show that human RPE cells exposed to oxidizing or alkylating agents exhibit preferential damage to mtDNA and not nDNA [163Ð165]. While nDNA is rapidly repaired, damaged mtDNA repair in the RPE appears to be slow and relatively inefÞcient [10]. It appears that RPE cells have the ability to adapt to survive high levels of oxidative stress through elevated cellular antioxidants and a higher nDNA repair capacity [163] and that this provides them with a greater resistance to oxidative stress than other cell types [166]. Surprisingly, there was no

adaptive beneÞt for mtDNA protection or repair in response to oxidative stress suggesting that mitochondria are a weak link in the RPE cellÕs defenses against oxidative damage [163]. RPE cells lacking poly (ADP-ribose)-polymerase (PARP) activity have a signiÞcantly lower lesion repair capacity in mtDNA which culminates in reduced cell viability [165]. Damage to mtDNA is enhanced by both phagocytosis of photoreceptor outer segments presumably through the burst of ROS generated during ingestion [68] and by exposure to blue light [167]. Interestingly, lipofuscin, a potent photogenerator of ROS, preferentially damages nuclear DNA rather than mtDNA [167]. Lin et al. showed that mtDNA damage increased with aging, and more lesions occurred in RPE cells from the macular region relative to the periphery. Furthermore, mtDNA repair capacity decreased with aging, with less mtDNA repair capacity in the macular region compared with the periphery in samples from aged subjects [159]. The age-related increase in mtDNA damage is reported to occur only in the common deletion region of the mitochondrial genome [158]. In vivo studies have shown that there is increased mtDNA damage in aged RPE and choroid of rodents and that this is likely to be due to decreased DNA repair capability [161]. Thus, mtDNA damage in the RPE is a feature of aging and may be a susceptibility factor that underlies the development of AMD.

10.5.2 DNA Repair and the Adaptive Response in the RPE

The adaptive response is a biological phenomenon which involves cells reacting at a molecular level to acquire greater cellular resistance against a wide range of physiological stresses, including oxidative stress [168]. Over the past few years, considerable information has accumulated regarding the mechanisms involved in the adaptive response [169Ð171]. The adaptive response is important in antioxidant defense for RPE cells which are cells located in a highly oxidizing microenvironment [166, 172Ð174].

The existence of inducible nuclear DNA repair pathways has been reported previously [175Ð177] and the upregulation of the BER pathway represents an additional potential antioxidant compensatory response for cells to cope with an increased oxidative burden [178, 179]. An intriguing Þnding for the RPE was the inability of the mitochondria in these cells to generate a protective adaptive response [166]. Prior exposure to sublethal H2O2 stimulated an adaptive response, resulting in a greater cellular resistance to subsequent toxic exposures compared to nonadapted RPE, greater catalase, glutathione peroxidase, and CuZnSOD (SOD1) activities, and increased nDNA protection. However, there was no adaptive beneÞt for mtDNA protection or repair in response to oxidative stress, nor was there an increase in SOD2. The mtDNA from both the adapted and nonadapted cultures both suffered a high susceptibility to H2O2-induced DNA damage and demonstrated inefÞcient mtDNA repair. This observation in the RPE lends support to studies in other cell types that mtDNA is particularly vulnerable to oxidative stress.