Defects in mtDNA replication challenge nuclear genome stability through nucleotide depletion and provide a unifying mechanism for mouse progerias

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Abstract

Mitochondrial DNA (mtDNA) mutagenesis and nuclear DNA repair defects are considered cellular mechanisms of ageing. mtDNA mutator mice with increased mtDNA mutagenesis show signs of premature ageing. However, why patients with mitochondrial diseases, or mice with other forms of mitochondrial dysfunction, do not age prematurely remains unknown. Here, we show that cells from mutator mice display challenged nuclear genome maintenance similar to that observed in progeric cells with defects in nuclear DNA repair. Cells from mutator mice show slow nuclear DNA replication fork progression, cell cycle stalling and chronic DNA replication stress, leading to double-strand DNA breaks in proliferating progenitor or stem cells. The underlying mechanism involves increased mtDNA replication frequency, sequestering of nucleotides to mitochondria, depletion of total cellular nucleotide pools, decreased deoxynucleoside 5′-triphosphate (dNTP) availability for nuclear genome replication and compromised nuclear genome maintenance. Our data indicate that defects in mtDNA replication can challenge nuclear genome stability. We suggest that defects in nuclear genome maintenance, particularly in the stem cell compartment, represent a unified mechanism for mouse progerias. Therefore, through their destabilizing effects on the nuclear genome, mtDNA mutations are indirect contributors to organismal ageing, suggesting that the direct role of mtDNA mutations in driving ageing-like symptoms might need to be revisited.

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Fig. 1: Disturbed cell cycle, replication stress and activation of DNA damage pathways in mtDNA mutator induced pluripotent stem cells.
Fig. 2: Altered nucleotide levels and increased mtDNA replication in mutator iPSCs.
Fig. 3: Mechanisms of mitochondrial progeria: compromized nuclear DNA maintenance and a redox-dependent self-renewal defect in stem cells.

Data availability

mtDNA sequencing data are available in the NCBI SRA database; project SRP056999. RNA sequencing data are available in the NCBI GEO database; accession number GSE133259. All other data are available on request from the corresponding authors.

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Acknowledgements

We thank T. Manninen, H. Ojala, M. Innilä and A. Muranen (University of Helsinki) for technical assistance, C. Storgaard Sørensen and K. Voßgröne (University of Copenhagen) for technical advice and T. McWilliams, C. Dunn and K. Wartiovaara (University of Helsinki) for discussion and critical comments. This work was supported by the Academy of Finland (275215 to R.H.H., 307592 to A.S., 303349 A.S., 30743 to A.S.), the Sigrid Jusélius Foundation, Jane and Aatos Erkko Foundation, the University of Helsinki, the University of Eastern Finland and the European Research Council (268955 to A.S.).

Author information

R.H.H. was responsible for study conception and design, experimental work, data analysis and interpretation, and writing of the manuscript. J.C.L., K.J.A., S.R., M.O.R., S.G. and L.W. did the experimental work and data analysis. V.B., K.I. and S.H. did the bioinformatics and data analysis. M.L. was responsible for study design and data interpretation. A.S. was responsible for study conception and design, data interpretation and writing of manuscript. All authors commented on and edited the manuscript.

Correspondence to Riikka H. Hämäläinen or Anu Suomalainen.

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Extended data

Extended Data Fig. 1 Increased DNA damage in mtDNA mutator mouse embryonic fibroblasts.

Mean fluorescence intensity (MFI) of γH2AX staining per cell; quantification of FACS signals and representative histograms. WT n = 4, Mut n = 4, three independent experiments, P = 0.0287. WT, black dots; mutator, red squares. Data represented as mean ± s.e.m. and analysed with unpaired t-test. n corresponds to biological replicates.

Extended Data Fig. 2 Increased DNA breaks in primary spermatocytes in mutator testes.

Fraction of γH2AX positive nuclei in primary spermatocytes; quantification of signals and representative original and processed images from γH2AX (red) and DAPI (blue) stainings. WT n = 5, mutator n = 7, approximately 1,000 nuclei per mouse, P = 0.0376. Scale bars, 100 μm. WT, black dots; Mut, red squares. Data represented as mean ± s.e.m. and analysed with unpaired t-test. n corresponds to biological replicates.

Extended Data Fig. 3 Decreased dNTP pools in mtDNA mutator mouse embryonic fibroblasts.

a, Total cellular nucleotide pools, WT n = 3, Mut n = 3, three independent measurements. b, Quantification of individual nucleotides. WT n = 3, Mut n = 3, three independent measurements. WT, black dots; Mut, red squares. Data presented as mean ± s.e.m. and analysed with unpaired t-test. n corresponds to biological replicates.

Extended Data Fig. 4 Supplementation with nucleosides does not increase proliferation nor significantly reduce DNA breaks in mutator iPSCs.

a, Growth curves of WT and mutator cells untreated (solid lines) and supplemented with nucleosides (C-73, G-85, U-73, A-80, T-24 mg/l) (dashed lines), n = 4, three independent experiments. b, Relative mean fluorescence intensity (MFI) of γH2AX staining per cell compared to the untreated cells; quantification of FACS signals. The 40 h treatments with excess nucleosides do not have any effect on WT cells. Slight reduction on γH2AX staining in mutator cells was only suggestive. WT n = 4, Mut n = 5, three independent experiments. WT, black dots; Mut, red squares. All data represented as mean ± s.e.m. and analysed with unpaired t-test. n corresponds to biological replicates.

Extended Data Fig. 5 Relative expression of 5′nucleotidase genes.

WT n = 3, Mut n = 5. Nt5e P = 0.0518, Nt5dc1 P = 0.0176, Nt5dc3 P = 0.00258. WT, black dots; Mut, red squares. All data represented as mean ± s.e.m. and analysed with unpaired t-test. n corresponds to biological replicates.

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