3.1. Age-dependent increase in basal levels of mitochondrial and nuclear DNA damage in C57BL/6 mouse striatum
To first get an assessment of age-dependent increase in DNA damage we measured basal levels of mitochondrial and nuclear DNA damage in the striatum from 4-, 17-, and 24-month-old mice and determined that there is an age-dependent decrease in the relative amplification of a 10 kb mtDNA fragment (, panels A and D). The QPCR assay is based on the principle that lesions that block the thermostable DNA polymerase on the DNA template will lead to a decrease in amplification of the fragment of interest. Because the DNA polymerase amplifies only undamaged templates, amplification is inversely proportional to the presence of damage. Damage to the mtDNA is observed in the striatum from 17-month-old mice as a significant 20.4% decrease in the relative amplification of the mitochondrial fragment as compared to the striatum from the 4-month-old mice. The maximum decrease in relative amplification is detected in the striatum from 24-month-old mice, with a highly significant 35% decrease in amplification as compared to striatum from the 4-month-old mice. The frequency of mtDNA lesions in the striatum from 17- and 24-month-old mice are 0.23 lesions/10 kb and 0.43 lesions/10 kb, respectively (, panel D). To exclude the possibility that the decrease in the amplification of the 10 kb mtDNA fragment resulted from the loss of mtDNA molecules, we amplify a small (91 bp) mtDNA fragment. Because the probability of introducing a lesion in a small fragment is low, the amplification of a small mtDNA fragment is independent of the presence of lesions and provides an accurate determination of the steady-state levels of mtDNA molecules. We show that amplification of a 91 bp mtDNA fragment is similar between samples and shows that the age-dependent decrease in the amplification of the 10.0 kb mtDNA fragment results from the presence of lesions in the mtDNA (, panel B). Therefore, these data indicate that there is an extensive accumulation of basal mtDNA lesions in the 24-month-old mouse striatum.
Fig. 1 Mitochondrial and nuclear DNA damage in mouse striatum increases with age. Total DNA was isolated from striatum obtained from 4-, 17-, and 24-month-old C57BL/6 mice for QPCR analysis. (A) Representative gel showing the amplification of a 10 kb mtDNA fragment. (more ...)
We sought to test the hypothesis that, similarly to mtDNA, nDNA from aged mice already contains persistent levels of damage. We measured the formation of age-induced nDNA lesions in the HPRT gene of striatum from 4-, 17-, and 24-month-old mice using QPCR. , panel C shows an age-dependent decrease in the amplification of a 6.9 kb nDNA fragment of 10.1% and 25% in the striatum from 17- and 24-month-old mice, respectively, as compared to the 4-month-old mice. The number of lesions in the nDNA fragment per 10 kb was 0.16 and 0.41 in the 17- and 24-month-old mice, respectively (, panel D). Taken together these data show that there are age-dependent increases of damage in both the mitochondrial and nuclear genomes.
3.2. Age-dependent decrease in the repair of 3-NPA-induced mitochondrial DNA lesions in C57BL/6 mouse striatum
Systemic administration of 3-NPA results in a neurodegenerative phenotype that replicates HD and thus 3-NPA has been extensively used as an experimental model of HD [30
]. By using the 3-NPA model of HD, we sought to test the hypothesis that repair of mtDNA damage is relevant to the pathogenesis of HD. We exposed 5- and 24-month-old C57BL/6 mice to 100 mg/kg 3-NPA, followed by DNA isolation at 6, 12, 24, and 48 h after treatment and DNA damage and repair was determined using QPCR. 3-NPA has been shown to induce oxidative damage in vivo
] and to induce the generation of mitochondrial superoxide in cells [32
]. Since oxidants such as hydrogen peroxide cause a large spectrum of oxidative DNA lesions of which ~50% are adducts that represent strong blocks to the thermostable polymerase, most likely the QPCR assay is detecting most of the oxidative lesions produced by 3-NPA. In addition, others have extensively used the QPCR assay to measure oxidative DNA damage [33
, panel A shows that DNA from striatum of 5-month-old mice exhibit a time-dependent decrease in the amplification of a 10 kb mtDNA fragment up to 24 h, indicating the induction of DNA lesions by 3-NPA. The relative amount of amplification shows a significant 14.4%, 30.2%, and 32% reduction after 6, 12, and 24 h, respectively, of 3-NPA treatment (, panel C). Our results also show that at 48 h after 3-NPA, the amplification of the mtDNA fragment in striatum from 5-month-old mice is back to levels similar to the controls. Thus, the recovery in the amplification of the 10 kb fragment to levels comparable to the DNA from untreated mice represents DNA repair activity. We conclude that the decrease in the relative amplification of the 10 kb mtDNA fragment is due to the presence of mtDNA damage since we find no changes in the amplification of a small (91 bp) mtDNA fragment, whose amplification is independent of the presence of lesions and represents steady-state levels of mtDNA molecules (, panel B). 3-NPA induced damage to mtDNA in the 5-month-old mice which peaked at 24 h and was almost completely repaired by and 48 h.
Fig. 2 Repair of mitochondrial DNA lesions induced by 3-NPA in striatum from young and aged C57BL/6 mice. Total DNA was isolated from striatum of 5- and 24-month-old mice and analyzed by QPCR. (A) Left and right panels, representative gels showing the amplification (more ...)
We also determined the relative amplification of the 10 kb mtDNA fragment in striatum from 24-month-old mice after 3-NPA treatment. We observe a 10, 30, 32, and 33% decrease in amplification of the mtDNA fragment 6, 12, 24 and 48 h after exposure to 3-NPA, respectively (, panel C). In contrast to the 5-month-old mice, the mtDNA damage induced by 3-NPA was not repaired in the 24-month-old animals, as shown by a 30% decrease in the relative amplification of the mtDNA fragment 48 h after 3-NPA. mtDNA damage in the striatum of 24-month-old mice reached a peak by 24 h and unlike 5-month-old mice, the damage continued to persist at high levels at 48 h (, panel C). It is important to note that the 3-NPA-induced increment in the number of mtDNA lesions in the aged striatum represents an increase beyond the age-associated lesions present in the striatum (). These data indicate that 3-NPA treatment leads to persistent mtDNA lesions in the striatum of 24-month-old mice.
3.3. Repair of 3-NPA-induced damage of a nuclear fragment in C57BL/6 mice
To determine levels of damage and the repair kinetics in a nDNA fragment, we amplified a 6.9 kb fragment of the HPRT gene from 5- and 24-month-old mice treated with 3-NPA. Treatment of young mice with 3-NPA did not result in the induction of lesions to nDNA (, panels A and B), therefore, in the 5-month-old mice, mtDNA was more susceptible to the effects of 3-NPA than nDNA. 3-NPA-induced lesions are only detected in the aged mice as shown by a 12%, 24.1%, 26.4%, and 31.4% reduction in the amplifiction of a 6.9 kb nDNA fragment at 12, 24, and 48 h after 3-NPA exposure, respectively (, panel B). Similar to mtDNA, the 3-NPA-induced damage was not repaired in nDNA from aged mice. Taken together these data suggest that in striatum from 5-month-old mice mtDNA is more sensitive to 3-NPA-induced lesions than nDNA but that striatum from old mice exhibited persistent DNA lesions in both the mitochondrial and nuclear genomes.
Fig. 3 Repair of nuclear DNA lesions induced by 3-NPA in striatum from young and aged C57BL/6 mice. Total DNA was isolated from striatum of 5- and 24-month-old mice and analyzed by QPCR. (A) Left and right panels, representative gels showing the amplification (more ...)
3.4. Exposure to 3-NPA induces an age-dependent increase in the formation of 8-hydroxyguanosine (8-OHG) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the nucleic acids of C57BL/6 mouse striatum
We sought to test the hypothesis that 3-NPA-induced neuronal toxicity involves the formation of 8-OHG and 8-OHdG, two oxidative lesions that accumulate in nucleic acids after oxidative stress. Since 8-OHdG represents less than 10% of the total oxidative DNA damage [37
] and it has not been tested whether it blocks the movement of the PCR thermostable polymerase, we do not know if it is detected by our QPCR assay. We therefore used immunocytochemistry to follow the levels of 8-OHG/8-OHdG after treatment with 3-NPA. We injected 5- and 24-month-old mice with 100 mg/kg of 3-NPA, a concentration known to induce mtDNA damage in striatum of 5- and 24-month-old mice (). Brain sections were obtained at 0, 6, 12, 24, and 48 h after 3-NPA injection and 8-OHG/8-OHdG immunopositive cells were counted. Our immunostaining results show an age- and time-dependent increase in the levels of 8-OHG/8-OHdG in neurons throughout the striatum (). We observe that in 5-month-old mice the number of OHG/8-OHdG positive striatal neurons increased at 6 and 12 h after 3-NPA treatment, but returns to levels similar to the untreated mice by 24 h. The number of OHG/8-OHdG positive cells in the 24-month-old mice also increases in a time-dependent fashion, however, the OHG/8-OHdG lesions accumulate even up to 48 h after treatment, suggesting that these lesions persist in the striatum of aged mice. Furthermore, the 24-month-old mice exhibit higher basal levels of OHG/8-OHdG positive cells as compared with the 5-month-old mice. These data show that 8-OHG/8-OHdG are induced in nucleic acids by 3-NPA in the striatum, an area known to be affected by the neurotoxin and support our hypothesis that persistent oxidative damage may contribute to the loss of neurons in aging and HD.
Fig. 4 Striatal cells from aged mice exhibit higher levels of 8-OHG and 8-OHdG and delayed repair kinetics. 5- and 24-month-old C57BL/6 mice were injected with 100 mg/kg of 3-NPA and brain sections were obtained at 0 (control), 6, 12, 24, and 48 h after treatment. (more ...)
3.5. Mitochondrial and nuclear DNA damage in striatum and cerebral cortex from R6/2 mice
Evidence suggests a role for oxidative stress in the pathogenesis of HD [4
], however, there is little evidence suggesting that mtDNA damage plays a critical role in the mechanisms of neuronal degeneration in HD. The purpose of this experiment was to determine the levels of mtDNA damage in striatum and cerebral cortex obtained from the R6/2 mouse model of HD. HD is characterized by a significant loss of neurons particularly in the striatum, followed by the cerebral cortex. Mice were sacrificed at 7, 10, and 12 weeks of age and DNA obtained from wild type and HD transgenic mice was analyzed by QPCR. shows that there is an age-dependent decrease in the relative amplification of a 10 kb mtDNA fragment from both striatum and cerebral cortex. Damage to mtDNA from cerebral cortex was observed in the 10- and 12-week-old mice as a significant 16% decrease in relative amplification of the mitochondrial fragment as compared to the wild type, age-matched controls (, panels A and D). The striatum of 10- and 12-week-old mice showed a 49% decrease in the amplification of the large mtDNA fragment as compared to age-matched wild type controls. The decrease in the relative amplification of the 10 kb mtDNA fragment is due to the presence of mtDNA damage and not to a decrease in the steady state levels of the mitochondrial genome, as shown by the lack of change in the amplification of a small mtDNA fragment (, panel B). We also measured levels of damage in a nDNA fragment and found that in 12-week-old mice there is a significant 8% and 20% decrease in the amplification of a 6.9 kb nDNA fragment from striatum and cerebral cortex, respectively (, panels C and E). MtDNA sustained eight-fold more lesions than the nuclear fragment in striatum of R6/2 mice (, panels D and E). Nuclear DNA damage from cerebral cortex was similar to mtDNA damage in 12-week-old mice. Taken together these results show that in the R6/2 mice mtDNA is more susceptible to damage than nDNA, and is consistent with mtDNA damage playing a role in the pathogenesis of HD.
Fig. 5 Mitochondrial and nuclear DNA damage in striatum and cerebral cortex of R6/2 mice. Total DNA was isolated from cerebral cortex and striatum of 7, 10, and 12-week-old R6/2 mice and analyzed by QPCR. Panel A, left and right panels, representative gels showing (more ...)