Mitochondrial DNA damage is a hallmark of chemically induced and the R6/2 transgenic model of Huntington's disease

Abstract

Many forms of neurodegeneration are associated with oxidative stress and mitochondrial dysfunction. Mitochondria are prominent targets of oxidative damage, however, it is not clear whether mitochondrial DNA (mtDNA) damage and/or its lack of repair are primary events in the delayed onset observed in Huntington's disease (HD). We hypothesize that an age-dependent increase in mtDNA damage contributes to mitochondrial dysfunction in HD. Two HD mouse models were studied, the 3-nitropropionic acid (3-NPA) chemically induced model and the HD transgenic mice of the R6/2 strain containing 115-150 CAG repeats in the huntingtin gene. The mitochondrial toxin 3-NPA inhibits complex II of the electron transport system and causes neurodegeneration that resembles HD in the striatum of human and experimental animals. We measured nuclear and mtDNA damage by quantitative PCR (QPCR) in striatum of 5- and 24-month-old untreated and 3-NPA treated C57BL/6 mice. Aging caused an increase in damage in both nuclear and mitochondrial genomes. 3-NPA induced 4-6 more damage in mtDNA than nuclear DNA in 5-month-old mice, and this damage was repaired by 48h in the mtDNA. In 24-month-old mice 3NPA caused equal amounts of nuclear and mitochondrial damage and this damage persistent in both genomes for 48h. QPCR analysis showed a progressive increase in the levels of mtDNA damage in the striatum and cerebral cortex of 7-12-week-old R6/2 mice. Striatum exhibited eight-fold more damage to the mtDNA compared with a nuclear gene. These data suggest that mtDNA damage is an early biomarker for HD-associated neurodegeneration and supports the hypothesis that mtDNA lesions may contribute to the pathogenesis observed in HD.

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. (B) Representative gel showing a 91 bp mtDNA fragment from 4, 17, and 24-month-old mice, respectively. (C) Representative gel showing a 6.9 kb fragment from the nuclear HPRT gene. (D) Comparison of the relative levels of amplification of the mitochondrial and nuclear DNA fragments during aging. Amplification of the 10 kb mtDNA fragment was normalized to the amplification of a 91 bp mtDNA fragment. Results are expressed as mean ± SEM values for three QPCRs performed for each DNA per age group, n = 6 mice per age groups. (*) Statistical differences as compared to 4- month-old mice; *p ≤ 0.05 were considered significant. Insets represent the mean lesion frequency per 10 kb per strand that were calculated using the Poisson equation as described under Section 2.

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 of a 10 kb mtDNA fragment from striatum of 5- and 24-month-old control mice (0 h) and striatum obtained from mice at 6, 12, 24, and 48 h after 3-NPA, respectively. (B) Left and right panels, representative gels showing a 91 bp mtDNA fragment from 5- and 24-month-old mice, respectively. (C) Relative levels of amplification of a 10 kb mtDNA fragment from 5- and 24-month-old mice, respectively, after 0 h (controls) and 6, 12, 24, and 48 h after 3-NPA treatment. Results are expressed as mean ± SEM values for six QPCRs performed for each DNA per age group. *p ≤ 0.05 were considered significant; n = 6 mice per age and treatment groups. Insets represent the mean lesion frequency per 10 kb per strand that were calculated using the Poisson equation as described under Section 2.

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 of a 6.9 kb nDNA from 5- and 24-month-old mice, respectively, after 0 h (controls) and 6, 12, 24, and 48 h after 3-NPA. (B) Relative levels of amplification of a 6.9 kb nDNA fragment. Results are expressed as mean ± SEM values for three QPCRs performed for each DNA per age group; n = 6 mice per age and treatment groups. *p ≤ 0.05 were considered significant. Insets represent the mean lesion frequency per 10 kb per strand that were calculated using the Poisson equation as described under Section 2.

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. The number of 8-OHG and 8-OHdG positive cells were determined by immunocytochemistry. Panel A represents striatal sections from 5- and 24-month-old mice at 0 (untreated/controls) and 24 h after 3-NPA. Panel B represents the number of 8-OHG and 8-OHdG positive cells at 0, 6, 12, 24, 48 h after 3-NPA; n = 2 mice per age and treatment groups.

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 the amplification of a 10.0 kb mtDNA fragment from cerebral cortex and striatum, respectively, of 12-week-old wild type and R6/2 mice. Panel B, left and right panels, representative gels showing a 91 bp mtDNA fragment from cerebral cortex and striatum, respectively, of 12-week-old wild type and R6/2 mice. Panel C, left and right panels, relative amplification of a 6.9 kb nDNA fragment from cerebral cortex and striatum, respectively, of 12-week-old wild-type and R6/2 mice. Panel D, relative levels of amplification of a 10.0 kb mitochondrial fragment after normalization to the 91 bp mtDNA fragment. Panel E, relative levels of amplification of a 6.9 kb nDNA fragment. Results are expressed as mean ± SEM values for three QPCRs performed for each DNA per age group, n = 6 per group in the 7-week-old wild type and HD mice, n = 4 per group in the 10-week-old wild type and HD mice; n = 4 per group in the 12-week-old wild type and HD mice. (*) Statistical differences between age-matched controls; (**) statistical differences between striatum and cortex. p ≤ 0.05 were considered significant. Insets represent the mean lesion frequency per 10 kb per strand that were calculated using the Poisson equation as described under Section 2.

Fig. 6

Mitochondrial DNA damage caused by 3-NPA and mutant huntingtin. 3-NPA and mutant hutingtin lead to defective mitochondria, which in turn can lead to increased generation of ROS and extensive mtDNA damage. An age-dependent increase in mtDNA damage and/or a decline in mtDNA repair capacity could lead to persistent mtDNA damage and exacerbate the chemical or huntigntin induced problems, ultimately leading to a marked decline in mitochondrial function, resulting in neuronal death.