Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

Abstract

Mitochondrial DNA (mtDNA) mutations become more prevalent with age and are postulated to contribute to the ageing process. Point mutations of mtDNA have been suggested to originate from two main sources, i.e. replicative errors and oxidative damage, but the contribution of each of these processes is much discussed. To elucidate the origin of mtDNA mutations, we measured point mutation load in mice with deficient mitochondrial base-excision repair (BER) caused by knockout alleles preventing mitochondrial import of the DNA repair glycosylases OGG1 and MUTYH (Ogg1 dMTS, Mutyh dMTS). Surprisingly, we detected no increase in the mtDNA mutation load in old Ogg1 dMTS mice. As DNA repair is especially important in the germ line, we bred the BER deficient mice for five consecutive generations but found no increase in the mtDNA mutation load in these maternal lineages. To increase reactive oxygen species (ROS) levels and oxidative damage, we bred the Ogg1 dMTS mice with tissue specific Sod2 knockout mice. Although increased superoxide levels caused a plethora of changes in mitochondrial function, we did not detect any changes in the mutation load of mtDNA or mtRNA. Our results show that the importance of oxidative damage as a contributor of mtDNA mutations should be re-evaluated.

Figure 1.

Evaluating mitochondrial targeting of OGG1 and MUTYH. (A) Subcellular localization of OGG1 with and without the predicted sequence encoding for the mitochondrial targeting sequence (dMTS). HeLa cells were transiently transfected with Ogg1-FLAG constructs (NM_010957.4, OGG1-FLAG, OGG1 dMTS-FLAG, ΔL2-W23) and target proteins were visualized by immunocytochemistry. Nuclear staining (DAPI, blue), mitochondrial signal (TOM20, red), OGG1 (green, FLAG). Scale bar represents 25 μm. The subcellular localization of FLAG signal was quantified by counting it from 100 cells. (B) Subcellular localization of mouse and human MUTYH with and without the predicted sequence encoding for the mitochondrial targeting sequence (dMTS). HeLa cells were transiently transfected with Mutyh-FLAG constructs and target proteins were visualized by immunocytochemistry. Nuclear staining (blue, DAPI), mitochondrial signal (red, TOM20), MUTYH (green, FLAG). Mouse MUTYH variant b/2 (NM_133250.2). Human MUTYH alpha3 variant (NM_001048171.1) and gamma3 variant (NM_001048173.1). Scale bar represents 25 μm.

Figure 2.

Excluding OGG1 and MUTYH from mitochondria does not lead to decrease in mtDNA copy number. (A) Targeting strategy of Mutyh dMTS mice to remove sequence encoding the predicted mitochondrial targeting sequence (dMTS) of endogenous MUTYH (ΔK2-P33). (B) Targeting strategy of Ogg1 dMTS mice to remove the sequence encoding the predicted mitochondrial targeting sequence (dMTS) of endogenous OGG1 (ΔL2-W23). (C) Splice variants of Mutyh dMTS and PCR amplification from cDNA to verify the presence of all the splice variants and correct length of the modified transcripts. Transcript variants a, b and c are also known as variants 1, 2 and 3, respectively. (D) PCR amplification of Ogg1 dMTS transcripts from cDNA to verify the correct length of the modified transcript from various genotypes. (E) 8-oxo-dG glycosylase/AP lyase assay to verify that Ogg1 dMTS animals lack OGG1 8-oxo-dG glycosylase activity. Total and mitochondrial lysates were incubated with 8-oxo-dG containing double-stranded oligonucleotide and reaction products were resolved on a denaturing acrylamide gel. WT n = 4, Ogg1 dMTS n = 5 Recombinant OGG1 was used as positive control (+) and in negative control no protein lysate was added. For longer exposure see Supplementary Figure S1A. (F) Relative mtDNA copy number of Ogg1 dMTS mice assessed from liver with qPCR. MtDNA levels were analyzed with a ND1 probe and nuclear DNA with a 18S probe. White circles indicate samples from wild-type controls (++, n = 7, 95–109 week old) and gray circles indicate samples from homozygous Ogg1 dMTS mice (dd, n = 6, 88–107 week old). Horizontal lines represent means, error bars represent SD, *P < 0.05, Student's t-test. For relative copy number analysis with Southern blot see Supplementary Figure S2A. (G) Relative mtDNA copy number of Mutyh dMTS × Ogg1 dMTS mice assessed from liver with qPCR. MtDNA levels were analyzed with a ND1 probe and nuclear DNA with a 18S probe. White circles indicate samples from wild-type controls (++, n = 7, 40–51 week old) and gray circles indicate samples from homozygous Mutyh dMTS × Ogg1 dMTS mice (dd, n = 6, 39–50 week old). Horizontal lines represent means, error bars represent SD, *P < 0.05, Student's t-test, Welch-corrected. For relative copy number analysis with Southern blot see Supplementary Figure S2B.

Figure 3.

Mitochondrial BER deficient mice do not accumulate point mutations to mtDNA after five generations of consecutive breeding. (A) Breeding scheme to accumulate mutations into mtDNA and study germ line mutations. Homozygous Mutyh dMTS × Ogg1 dMTS female mice were bred with homo- or heterozygous Mutyh dMTS × Ogg1 dMTS male mice for five consecutive generations. To minimize the nuclear effects, heterozygote male mice were also used in the breedings. N1–N5 indicates the generations of breeding. (B) Mutation load of mtDNA with Illumina sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. The sequencing was carried out from purified mtDNA from liver. Data is quality filtered and minimum variant allele frequency is set to 0.5%. In unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. In total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (++ n = 6, pp n = 2, 10–13 week old) and gray circles indicate samples from homozygous Mutyh dMTS × Ogg1 dMTS mice (dd dd, n = 8, 10–15 week old). Horizontal lines represent means. C. Mutation profile of mtDNA with Illumina sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. Samples as in B. Horizontal lines represent means. For only quality filtered data see Supplementary Figure S3.

Figure 4.

Heart Sod2 knockout mice display severe dilated cardiomyopathy. (A) Western blot analysis of SOD2 protein levels from purified mitochondria of control (pp) and Sod2 loxP × Ckmm cre mice (pp, cre) (9–11 week old). ATP5A was used as a loading control. (B) Vertical sections through the midpoint of paraffin embedded hearts stained with hematoxylin and eosin staining. Control (pp, 11-week old) and Sod2 loxP × Ckmm cre (pp, cre, 10-week old). Scale bar represents 1 mm. (C) Heart weight of control (pp), heterozygous Sod2 loxP x Ckmm cre and homozygous Sod2 loxP x Ckmm cre (pp, cre) mice. White box indicates control mice (pp female n = 22, male n = 35, 9–11 week old), light gray box indicates heterozygous Sod2 loxP x Ckmm cre mice (male +p, cre, n = 2, 9-week old) and dark gray box indicates homozygous Sod2 loxP x Ckmm cre mice (pp, cre, female n = 28, male n = 15, 9–10 week old). D. Body weight of control (pp), heterozygous Sod2 loxP x Ckmm cre and homozygous Sod2 loxP x Ckmm cre (pp, cre) mice. White box indicates control mice (pp female n = 26, male n = 36, 9–11 week old), light gray box indicates heterozygous Sod2 loxP x Ckmm cre mice (male +p, cre, n = 3, 9-week old) and dark gray box indicates homozygous Sod2 loxP x Ckmm cre mice (pp, cre, female n = 30, male n = 19, 9–10 week old). Whiskers represent min and max values, horizontal lines medians; ****P< 0.0001, females Student's t-test, Welch corrected. ***P< 0.001, males one-way ANOVA, Dunnett's multiple comparison test.

Figure 5.

[4Fe–4S] cluster proteins are severely affected in heart Sod2 knockout mice indicating strong increase in superoxide levels. (A) Aconitase activity from purified mitochondria from control (pp) and Sod2 loxP × Ckmm cre mice (pp, cre). White bar indicates activity in control samples (n = 6, 9–10 week old) and gray bar in Sod2 loxP x Ckmm cre samples (n = 6, 9–12 week old). Activity is normalized to control. (B) Western blot analysis of ACO 2 (aconitase) protein levels from purified mitochondria of control (pp) and Sod2 loxP x Ckmm cre mice (pp, cre) (9–10 week old). ATP5A and Coomassie-stained membrane were used as loading controls. (C) Oxygen consumption rate of isolated heart mitochondria from control (pp, white bars, n = 9, 9–11 week old) and Sod2 loxP x Ckmm cre mice (pp, cre, gray bars, n = 9, 9–12 week old). Isolated mitochondria were incubated with complex I (PMG) or complex II (SUCC) substrates. Each set of substrates was successively combined with ADP (to assess the phosphorylating respiration, PMG3, SUCC3), oligomycin (to assess the non-phosphorylating respiration PMG4, SUCC4) and CCCP (to assess uncoupled respiration PMGc, SUCCc). (D) Activity of the respiratory chain complexes I (CI), II (CII), IV (CIV) and the activity from complex II to III (CII-III) of heart mitochondria from control (pp, write bars, n = 3, 11-week old) and Sod2 loxP x Ckmm cre mice (pp, cre, gray bars n = 3, 11–12 week old). Citrate synthase activity (CS) was used as a control. Error bars represent SD. *P< 0.05, **P< 0.005, ****P< 0.0001, Student's t-test, Welch corrected. (E) Western blot analysis of OXPHOS proteins from purified heart mitochondria from control (pp) and Sod2 loxP × Ckmm cre (pp, cre) mice (9–11 week old).

Figure 6.

Heart Sod2 knockout mice show global decrease in complex I proteins and indications of general mitochondrial stress in label-free quantitative proteomics. Heat map of selected proteins from label-free quantitative proteomic analysis of Percoll gradient purified heart mitochondria from controls (pp, 8–9 week old) and Sod2 loxP × Ckmm cre mice (pp, cre, 9–10 week old). Changes in the protein steady-state levels are blotted as Z-scores. Blue indicates decreased and red increased steady-state level from the global mean across all samples. Abundances of all the presented proteins were significantly changed, with Benjamini–Hochberg adjusted P-values of <0.05.

Figure 7.

Mitochodrial BER deficient mice do not accumulate point mutations to mtDNA even in the presence of increased oxidative stress. (A) Mutation load of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Data is quality filtered and minimum variant allele frequency is set to 0.5%. For the unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (pp n = 4 or ++ n = 3, 8–12 week old), light gray circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4 or +p dd n = 2 or +p cre+ dd n = 1, 8–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 7, 9–10 week old). Horizontal lines represent means, one-way ANOVA, Tukey's multiple comparison test. (B) Mutation profile of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Samples as in A. Horizontal lines represent mean. For only quality-filtered data see Supplementary Figure S4.

Figure 8.

Mitochondrial BER deficient mice do not accumulate point mutations of mtRNA even in the presence of increased oxidative stress. (A) Mutation load of mtRNA from Sod2 loxP x Ckmm cre mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. For the unique mutation load each specific mutation is counted only once, reflecting how many times a mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre mice from heart. White circles indicate samples from controls (+p n = 1 pp n = 2, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre n = 3, 10–11 week old). (B) Mutation load of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. White circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 4, 9–10 week old). *P< 0.05, **P< 0.005, Student's t-test. For quality-filtered data with minimum variant allele frequency set to 0.5% see Supplementary Figure S5.

Figure 9.

Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. (A) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. (B) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). (C) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, *P< 0.05, Student's t-test.

Figure 10.

De novo replication is affected in heart Sod2 knockout mice. (A) Representative experiment of in organello replication assay in heart Sod2 loxP x Ckmm cre mice. The mtDNA was radioactively labeled in isolated mitochondria, purified and half of it was boiled to release newly synthesized 7S DNA. Samples were separated on an agarose gel and transferred to a membrane. Small aliquot representing the input was analyzed with Coomassie staining after the labeling. (B) Quantification of de novo replication, the relative incorporation of radioactivity into mtDNA and newly synthesized 7S DNA in Sod2 loxP x Ckmm cre mice. The incorporation was normalized to steady-state level of mtDNA that was probed from the same membrane after the de novo signal could not be detected anymore. White circles indicate samples from controls (pp, n = 9, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 9, 9–10 week old). (C) Representative experiment of in organello transcription assay from Sod2 loxP x Ckmm cre mice. The mtRNA was radioactively labeled in isolated mitochondria and half of the sample was purified (pulse). Another half was incubated with non-radioactive UTP for 2 h (chase) and purified, and then samples were analyzed with northern blotting. Small aliquot representing the input was analyzed with Coomassie staining after the labeling. (D) Quantification of de novo transcription, the relative incorporation of radioactivity into mtRNA in Sod2 loxP x Ckmm cre mice. The incorporation was normalized to steady-state level of CytB that was probed from the same membrane after the de novo signal could not be detected anymore. See Supplementary Figure S7, which shows that CytB levels do not change in Sod2 loxP x Ckmm cre mice. White circles indicate samples from controls (pp, n = 12, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 12, 9–10 week old). Horizontal lines represent means, error bars represent SD, ****P< 0.0001, Student's t-test.

Figure 11.

POLγ steady-state levels are decreased in heart Sod2 knockout mice while POLRMT levels are increased. Western blot analysis of proteins involved in replication and transcription from purified heart mitochondria from control (pp) and Sod2 loxP x Ckmm cre (pp, cre) mice (9–10 week old). ATP5A and Coomassie-stained membrane were used as loading controls.