Mitochondrial DNA damage is associated with reduced mitochondrial bioenergetics in Huntington's disease

Abstract

Oxidative stress and mitochondrial dysfunction have been implicated in the pathology of HD; however, the precise mechanisms by which mutant huntingtin modulates levels of oxidative damage in turn resulting in mitochondrial dysfunction are not known. We hypothesize that mutant huntingtin increases oxidative mtDNA damage leading to mitochondrial dysfunction. We measured nuclear and mitochondrial DNA lesions and mitochondrial bioenergetics in the STHdhQ7 and STHdhQ111 in vitro striatal model of HD. Striatal cells expressing mutant huntingtin show higher basal levels of mitochondrial-generated ROS and mtDNA lesions and a lower spare respiratory capacity. Silencing of APE1, the major mammalian apurinic/apyrimidinic (AP) endonuclease that participates in the base excision repair (BER) pathway, caused further reductions of spare respiratory capacity in the mutant huntingtin-expressing cells. Localization experiments show that APE1 increases in the mitochondria of wild-type Q7 cells but not in the mutant huntingtin Q111 cells after treatment with hydrogen peroxide. Moreover, these results are recapitulated in human HD striata and HD skin fibroblasts that show significant mtDNA damage (increased lesion frequency and mtDNA depletion) and significant decreases in spare respiratory capacity, respectively. These data suggest that mtDNA is a major target of mutant huntingtin-associated oxidative stress and may contribute to subsequent mitochondrial dysfunction and that APE1 (and, by extension, BER) is an important target in the maintenance of mitochondrial function in HD.

Fig. 1

Mitochondrial superoxide production is increased in Q111 cells compared to wild type Q7 cells. Cells were cultured as described in Methods and live imaging was performed using a Zeiss LSM 510 confocal microscope. (A) Superoxide production was measured using MitoSOX™ red (2.5 µM) and mitochondria were detected using MitoTracker® green (50 nM). (B) The bar graph shows quantification of intensity of the fluorescence generated by MitoSOX™ after normalization using MitoTracker® fluorescence to correct for differences in mitochondrial abundance. The relative intensity of the fluorescence was determined using NIH Image J. *p=0.03 and n=2 independent experiments. (C) MitoTracker® green fluorescence was normalized for changes in cell numbers; **p=0.02. (D) Quantification of the expression levels of the mitochondrial protein VDAC by Western blot analysis (n=2 independent experiments; *p<0.05). Data are expressed relative to Q7 cells.

Fig. 1

Mitochondrial superoxide production is increased in Q111 cells compared to wild type Q7 cells. Cells were cultured as described in Methods and live imaging was performed using a Zeiss LSM 510 confocal microscope. (A) Superoxide production was measured using MitoSOX™ red (2.5 µM) and mitochondria were detected using MitoTracker® green (50 nM). (B) The bar graph shows quantification of intensity of the fluorescence generated by MitoSOX™ after normalization using MitoTracker® fluorescence to correct for differences in mitochondrial abundance. The relative intensity of the fluorescence was determined using NIH Image J. *p=0.03 and n=2 independent experiments. (C) MitoTracker® green fluorescence was normalized for changes in cell numbers; **p=0.02. (D) Quantification of the expression levels of the mitochondrial protein VDAC by Western blot analysis (n=2 independent experiments; *p<0.05). Data are expressed relative to Q7 cells.

Fig. 1

Mitochondrial superoxide production is increased in Q111 cells compared to wild type Q7 cells. Cells were cultured as described in Methods and live imaging was performed using a Zeiss LSM 510 confocal microscope. (A) Superoxide production was measured using MitoSOX™ red (2.5 µM) and mitochondria were detected using MitoTracker® green (50 nM). (B) The bar graph shows quantification of intensity of the fluorescence generated by MitoSOX™ after normalization using MitoTracker® fluorescence to correct for differences in mitochondrial abundance. The relative intensity of the fluorescence was determined using NIH Image J. *p=0.03 and n=2 independent experiments. (C) MitoTracker® green fluorescence was normalized for changes in cell numbers; **p=0.02. (D) Quantification of the expression levels of the mitochondrial protein VDAC by Western blot analysis (n=2 independent experiments; *p<0.05). Data are expressed relative to Q7 cells.

Fig. 2

Mutant Q111 cells exhibit higher basal levels of mtDNA damage and are more sensitive to H₂O₂ treatment than wild type Q7 cells. (A) Cells were treated with 200 µM H₂O₂ and DNA isolated 3 hours after treatment. Frequency of mtDNA and nDNA lesions per 10 kb per strand after normalization for changes in mtDNA abundance. n=3 independent experiments and 3 QPCR analyses. *p<0.001 versus Q7 control and **p<0.001 versus Q111 control. (B) Relative abundance of mtDNA molecules. *p<0.01 versus Q7. n=6 independent experiments. (C) Cells were treated with 200 µM H₂O₂ and cell viability was determined after 6, 12, and 24 hours of treatment using the trypan blue exclusion method. Results are expressed as % of control. *p=0.02 and **p<0.001 versus WT Q7 cells. n=2−3 independent experiments in duplicate.

Fig. 2

Mutant Q111 cells exhibit higher basal levels of mtDNA damage and are more sensitive to H₂O₂ treatment than wild type Q7 cells. (A) Cells were treated with 200 µM H₂O₂ and DNA isolated 3 hours after treatment. Frequency of mtDNA and nDNA lesions per 10 kb per strand after normalization for changes in mtDNA abundance. n=3 independent experiments and 3 QPCR analyses. *p<0.001 versus Q7 control and **p<0.001 versus Q111 control. (B) Relative abundance of mtDNA molecules. *p<0.01 versus Q7. n=6 independent experiments. (C) Cells were treated with 200 µM H₂O₂ and cell viability was determined after 6, 12, and 24 hours of treatment using the trypan blue exclusion method. Results are expressed as % of control. *p=0.02 and **p<0.001 versus WT Q7 cells. n=2−3 independent experiments in duplicate.

Fig. 2

Mutant Q111 cells exhibit higher basal levels of mtDNA damage and are more sensitive to H₂O₂ treatment than wild type Q7 cells. (A) Cells were treated with 200 µM H₂O₂ and DNA isolated 3 hours after treatment. Frequency of mtDNA and nDNA lesions per 10 kb per strand after normalization for changes in mtDNA abundance. n=3 independent experiments and 3 QPCR analyses. *p<0.001 versus Q7 control and **p<0.001 versus Q111 control. (B) Relative abundance of mtDNA molecules. *p<0.01 versus Q7. n=6 independent experiments. (C) Cells were treated with 200 µM H₂O₂ and cell viability was determined after 6, 12, and 24 hours of treatment using the trypan blue exclusion method. Results are expressed as % of control. *p=0.02 and **p<0.001 versus WT Q7 cells. n=2−3 independent experiments in duplicate.

Fig. 3

Striata from human postmortem grade 3 HD subjects show increased mtDNA depletion and increased levels of mtDNA and nDNA damage. DNA was isolated from caudate/putamen from control (n=4) and HD grade 3 brains (n=4). DNA lesions and mtDNA abundance were determined using QPCR. (A) Relative abundance of mtDNA molecules. *p<0.0001 versus control. (B) Frequency of mtDNA and nDNA lesions per 10 kb per strand. **p<0.05 and ***p<0.001 versus controls.

Fig. 3

Striata from human postmortem grade 3 HD subjects show increased mtDNA depletion and increased levels of mtDNA and nDNA damage. DNA was isolated from caudate/putamen from control (n=4) and HD grade 3 brains (n=4). DNA lesions and mtDNA abundance were determined using QPCR. (A) Relative abundance of mtDNA molecules. *p<0.0001 versus control. (B) Frequency of mtDNA and nDNA lesions per 10 kb per strand. **p<0.05 and ***p<0.001 versus controls.

Fig. 4

Mutant Q111 cells exhibit a lower spare respiratory capacity than wild type Q7 cells but higher extracellular acidification rate. (A) Oxygen consumption rate (OCR) was monitored after the addition of buffer only (B), oligomycin (O), FCCP (F), and a mixture of rotenone/myxothiazol (R/M). n=10 replicates per cell clone. Data are expressed as total percentage change from baseline. *p<0.03; **p<0.0001 versus Q111. (B) The extracellular acidification rate (ECAR) was monitored after the addition of buffer only, oligomycin, FCCP, and a mixture of rotenone/myxothiazol. n=10 replicates per cell clone. Data are expressed as total percentage change from baseline. *p<0.007 versus Q7.

Fig. 5

Mutant Q111 cells exhibit decreased spare respiratory capacity after treatment with H₂O₂. Cells were treated with H₂O₂ for 24 hours and oxygen consumption rate (OCR) was monitored after the addition of buffer only (B), oligomycin (O), FCCP (F), and a mixture of rotenone/myxothiazol (R/M). *p<0.05 Q111 versus Q111 H₂O₂. n=2 independent experiments and n=5 replicates per cell clone.

Fig. 6

A primary culture of HD diploid skin fibroblasts shows lower spare respiratory capacity than control skin fibroblasts. HD skin fibroblast and control fibroblasts were monitored on an XF-24 Seahorse and oxygen consumption rate was recorded as described above. *p<0.05 versus control fibroblasts after FCCP injection; n=3 experiments.

Fig. 7

APE1 intensity increases in the mitochondria of Q7 but not in Q111 cells following hydrogen peroxide treatment. Cells were cultured as described in Methods, treated with 200 µM H₂O₂ for 6 hours, fixed and stained for APE1 (green) and mitochondria (red). (A) Confocal images of untreated Q7 and Q111 cells. (B) Confocal images of H₂O₂ treated Q7 and Q111 cells. (C) APE1 intensity after line scans were performed in 3 different fields with n=3; **p< 0.01 versus Q111 after H₂O₂ treatment. (D) Representative membrane showing APE1 expression in isolated mitochondria (upper panel). Expression levels of APE1 normalized using VDAC expression (lower panel). Data are expressed relative to Q7 control cells; n=2 independent experiments. *p< 0.02 versus Q7 control; **p <0.005 versus Q7 control and Q7 H₂O₂.

Fig. 8

Silencing of Ape1 induces mitochondrial dysfunction in the mutant huntingtin-expressing Q111 cells. Cells were cultured and transfected with scrambled and Ape1 siRNAs as described in Methods. (A) Representative Western blots after transfecting cells with scrambled siRNA and Ape1 siRNA and quantification of APE1 protein expression showing that both Q7 and Q111 cells show a significant APE1 knock down compared with their respective scramble control; ***p<0.001. (B) Oxygen consumption rate (OCR) was monitored as described. *p<0.05 scrambled Q111 versus scrambled Q7; **p<0.01 scrambled Q7 versus Ape1 siRNA Q7 and Ape1 siRNA Q111; ⁺p<0.05 represents Ape1 siRNA Q111 versus scrambled Q111; n=3 independent experiments and n=5 replicates per cell clone.

Fig. 9

Silencing of Ape1 results in enhanced caspase 3/7 activation. Cells were cultured and transfected with scrambled and Ape1 siRNAs as described in Methods. Representative bar graph shows significant increase (p<0.01) in activity in Q7 Ape1 siRNA, Q111 scramble control and Q111 Ape1 siRNA versus scramble control Q7. n=3 experiments.

Fig. 10

Model for mutant huntingtin-induced mtDNA damage, deficient mtDNA repair and mitochondrial dysfunction. Mutant huntingtin induces mitochondrial ROS which cause oxidative damage to mtDNA and mtDNA depletion. Concomitantly, APE1 cannot be translocated inside the mitochondria and hence mtDNA repair is affected. Oxidative damage to the nDNA may also contribute to mitochondrial dysfunction. The net effect is a reduction in the spare respiratory capacity, which leads to mitochondrial dysfunction and cell death associated with HD neurodegeneration.