MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.
References
[1]
Spinazzola A, Zeviani M (2009) Disorders from perturbations of nuclear-mitochondrial intergenomic cross-talk. J Intern Med 265: 174–192. doi: 10.1111/j.1365-2796.2008.02059.x. pmid:19192035
[2]
Mandel H, Szargel R, Labay V, Elpeleg O, Saada A, et al. (2001) The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. Nat Genet 29: 337–341. pmid:11687800 doi: 10.1038/ng746
[3]
Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S, et al. (2001) Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat Genet 29: 342–344. pmid:11687801 doi: 10.1038/ng751
[4]
Bourdon A, Minai L, Serre V, Jais JP, Sarzi E, et al. (2007) Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 39: 776–780. pmid:17486094 doi: 10.1038/ng2040
[5]
Naviaux RK, Nguyen KV (2004) POLG mutations associated with Alpers' syndrome and mitochondrial DNA depletion. Ann Neurol 55: 706–712. pmid:15122711 doi: 10.1002/ana.20079
[6]
Alston CL, Schaefer AM, Raman P, Solaroli N, Krishnan KJ, et al. (2013) Late-onset respiratory failure due to TK2 mutations causing multiple mtDNA deletions. Neurology 81: 2051–2053. doi: 10.1212/01.wnl.0000436931.94291.e6. pmid:24198295
[7]
Nishino I, Spinazzola A, Hirano M (1999) Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science 283: 689–692. pmid:9924029 doi: 10.1126/science.283.5402.689
[8]
Ronchi D, Garone C, Bordoni A, Gutierrez Rios P, Calvo SE, et al. (2012) Next-generation sequencing reveals DGUOK mutations in adult patients with mitochondrial DNA multiple deletions. Brain 135: 3404–3415. doi: 10.1093/brain/aws258. pmid:23043144
[9]
Spelbrink JN, Li FY, Tiranti V, Nikali K, Yuan QP, et al. (2001) Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat Genet 28: 223–231. pmid:11431692
[10]
Tyynismaa H, Sun R, Ahola-Erkkila S, Almusa H, Poyhonen R, et al. (2012) Thymidine kinase 2 mutations in autosomal recessive progressive external ophthalmoplegia with multiple mitochondrial DNA deletions. Hum Mol Genet 21: 66–75. doi: 10.1093/hmg/ddr438. pmid:21937588
[11]
Van Goethem G, Dermaut B, Lofgren A, Martin JJ, Van Broeckhoven C (2001) Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat Genet 28: 211–212. pmid:11431686 doi: 10.1038/90034
[12]
Tyynismaa H, Ylikallio E, Patel M, Molnar MJ, Haller RG, et al. (2009) A heterozygous truncating mutation in RRM2B causes autosomal-dominant progressive external ophthalmoplegia with multiple mtDNA deletions. Am J Hum Genet 85: 290–295. doi: 10.1016/j.ajhg.2009.07.009. pmid:19664747
[13]
Sarzi E, Goffart S, Serre V, Chretien D, Slama A, et al. (2007) Twinkle helicase (PEO1) gene mutation causes mitochondrial DNA depletion. Ann Neurol 62: 579–587. pmid:17722119 doi: 10.1002/ana.21207
[14]
Spinazzola A, Viscomi C, Fernandez-Vizarra E, Carrara F, D'Adamo P, et al. (2006) MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nat Genet 38: 570–575. pmid:16582910 doi: 10.1038/ng1765
[15]
Blakely EL, Butterworth A, Hadden RD, Bodi I, He L, et al. (2012) MPV17 mutation causes neuropathy and leukoencephalopathy with multiple mtDNA deletions in muscle. Neuromuscul Disord 22: 587–591. doi: 10.1016/j.nmd.2012.03.006. pmid:22508010
[16]
Garone C, Rubio JC, Calvo SE, Naini A, Tanji K, et al. (2012) MPV17 Mutations Causing Adult-Onset Multisystemic Disorder With Multiple Mitochondrial DNA Deletions. Arch Neurol 69: 1648–1651. doi: 10.1001/archneurol.2012.405. pmid:22964873
[17]
Pontarin G, Ferraro P, Hakansson P, Thelander L, Reichard P, et al. (2007) p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage. J Biol Chem 282: 16820–16828. pmid:17416930 doi: 10.1074/jbc.m701310200
[18]
Hakansson P, Hofer A, Thelander L (2006) Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells. J Biol Chem 281: 7834–7841. pmid:16436374 doi: 10.1074/jbc.m512894200
[19]
Tanaka H, Arakawa H, Yamaguchi T, Shiraishi K, Fukuda S, et al. (2000) A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 404: 42–49. pmid:10716435
[20]
Pontarin G, Ferraro P, Bee L, Reichard P, Bianchi V (2012) Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells. Proc Natl Acad Sci U S A 109: 13302–13307. doi: 10.1073/pnas.1211289109. pmid:22847445
[21]
Besse A, Wu P, Bruni F, Donti T, Graham BH, et al. (2015) The GABA transaminase, ABAT, is essential for mitochondrial nucleoside metabolism. Cell Metab 21: 417–427. doi: 10.1016/j.cmet.2015.02.008. pmid:25738457
[22]
Spinazzola A, Marti R, Nishino I, Andreu AL, Naini A, et al. (2002) Altered thymidine metabolism due to defects of thymidine phosphorylase. J Biol Chem 277: 4128–4133. pmid:11733540 doi: 10.1074/jbc.m111028200
[23]
Nishigaki Y, Marti R, Copeland WC, Hirano M (2003) Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency. J Clin Invest 111: 1913–1921. pmid:12813027 doi: 10.1172/jci17828
[24]
Gonzalez-Vioque E, Torres-Torronteras J, Andreu AL, Marti R (2011) Limited dCTP availability accounts for mitochondrial DNA depletion in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). PLoS Genet 7: e1002035. doi: 10.1371/journal.pgen.1002035. pmid:21483760
[25]
Song S, Wheeler LJ, Mathews CK (2003) Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA. J Biol Chem 278: 43893–43896. pmid:13679382 doi: 10.1074/jbc.c300401200
[26]
Gandhi VV, Samuels DC (2011) Enzyme kinetics of the mitochondrial deoxyribonucleoside salvage pathway are not sufficient to support rapid mtDNA replication. PLoS Comput Biol 7: e1002078. doi: 10.1371/journal.pcbi.1002078. pmid:21829339
[27]
Young P, Leeds JM, Slabaugh MB, Mathews CK (1994) Ribonucleotide reductase: evidence for specific association with HeLa cell mitochondria. Biochem Biophys Res Commun 203: 46–52. pmid:8074691 doi: 10.1006/bbrc.1994.2146
[28]
Chimploy K, Song S, Wheeler LJ, Mathews CK (2013) Ribonucleotide reductase association with mammalian liver mitochondria. J Biol Chem 288: 13145–13155. doi: 10.1074/jbc.M113.461111. pmid:23504325
[29]
Anderson DD, Quintero CM, Stover PJ (2011) Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria. Proc Natl Acad Sci U S A 108: 15163–15168. doi: 10.1073/pnas.1103623108. pmid:21876188
[30]
Carrozzo R, Wittig I, Santorelli FM, Bertini E, Hofmann S, et al. (2006) Subcomplexes of human ATP synthase mark mitochondrial biosynthesis disorders. Ann Neurol 59: 265–275. pmid:16365880 doi: 10.1002/ana.20729
[31]
Viscomi C, Spinazzola A, Maggioni M, Fernandez-Vizarra E, Massa V, et al. (2009) Early-onset liver mtDNA depletion and late-onset proteinuric nephropathy in Mpv17 knockout mice. Hum Mol Genet 18: 12–26. doi: 10.1093/hmg/ddn309. pmid:18818194
[32]
Weiher H, Noda T, Gray DA, Sharpe AH, Jaenisch R (1990) Transgenic mouse model of kidney disease: insertional inactivation of ubiquitously expressed gene leads to nephrotic syndrome. Cell 62: 425–434. pmid:1696177 doi: 10.1016/0092-8674(90)90008-3
[33]
Ashley N, Adams S, Slama A, Zeviani M, Suomalainen A, et al. (2007) Defects in maintenance of mitochondrial DNA are associated with intramitochondrial nucleotide imbalances. Hum Mol Genet 16: 1400–1411. pmid:17483096 doi: 10.1093/hmg/ddm090
[34]
Han Z, Stachow C (1994) Analysis of Schizosaccharomyces pombe mitochondrial DNA replication by two dimensional gel electrophoresis. Chromosoma 103: 162–170. pmid:7924618 doi: 10.1007/bf00368008
[35]
Reyes A, Kazak L, Wood SR, Yasukawa T, Jacobs HT, et al. (2013) Mitochondrial DNA replication proceeds via a 'bootlace' mechanism involving the incorporation of processed transcripts. Nucleic Acids Res 41: 5837–5850. doi: 10.1093/nar/gkt196. pmid:23595151
[36]
Saada A (2009) Fishing in the (deoxyribonucleotide) pool. Biochem J 422: e3–6. doi: 10.1042/BJ20091194. pmid:19698084
[37]
Eriksson S, Wang L (2008) Molecular mechanisms of mitochondrial DNA depletion diseases caused by deficiencies in enzymes in purine and pyrimidine metabolism. Nucleosides Nucleotides Nucleic Acids 27: 800–808. doi: 10.1080/15257770802146197. pmid:18600543
[38]
Saada A (2008) Mitochondrial deoxyribonucleotide pools in deoxyguanosine kinase deficiency. Mol Genet Metab 95: 169–173. doi: 10.1016/j.ymgme.2008.07.007. pmid:18723380
[39]
Pontarin G, Ferraro P, Rampazzo C, Kollberg G, Holme E, et al. (2011) Deoxyribonucleotide metabolism in cycling and resting human fibroblasts with a missense mutation in p53R2, a subunit of ribonucleotide reductase. J Biol Chem 286: 11132–11140. doi: 10.1074/jbc.M110.202283. pmid:21297166
[40]
Camara Y, Gonzalez-Vioque E, Scarpelli M, Torres-Torronteras J, Caballero A, et al. (2014) Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome. Hum Mol Genet. doi: 10.1093/hmg/ddt641
[41]
Uusimaa J, Evans J, Smith C, Butterworth A, Craig K, et al. (2014) Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene. Eur J Hum Genet 22: 184–191. doi: 10.1038/ejhg.2013.112. pmid:23714749
[42]
Wiseman A, Attardi G (1978) Reversible tenfod reduction in mitochondria DNA content of human cells treated with ethidium bromide. Mol Gen Genet 167: 51–63. pmid:739978
[43]
Song S, Pursell ZF, Copeland WC, Longley MJ, Kunkel TA, et al. (2005) DNA precursor asymmetries in mammalian tissue mitochondria and possible contribution to mutagenesis through reduced replication fidelity. Proc Natl Acad Sci U S A 102: 4990–4995. pmid:15784738 doi: 10.1073/pnas.0500253102
[44]
Mathews CK (2006) DNA precursor metabolism and genomic stability. FASEB J 20: 1300–1314. pmid:16816105 doi: 10.1096/fj.06-5730rev
[45]
Lai Y, Tse CM, Unadkat JD (2004) Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs. J Biol Chem 279: 4490–4497. pmid:14607828 doi: 10.1074/jbc.m307938200
[46]
Lee EW, Lai Y, Zhang H, Unadkat JD (2006) Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1): implications for interspecies differences in mitochondrial toxicity of fialuridine. J Biol Chem 281: 16700–16706. pmid:16595656 doi: 10.1074/jbc.m513825200
[47]
Villarroya J, de Bolos C, Meseguer A, Hirano M, Vila MR (2009) Altered gene transcription profiles in fibroblasts harboring either TK2 or DGUOK mutations indicate compensatory mechanisms. Exp Cell Res 315: 1429–1438. doi: 10.1016/j.yexcr.2009.02.018. pmid:19265691
[48]
Di Noia MA, Todisco S, Cirigliano A, Rinaldi T, Agrimi G, et al. (2014) The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters. J Biol Chem 289: 33137–33148. doi: 10.1074/jbc.M114.610808. pmid:25320081
[49]
Elpeleg O, Miller C, Hershkovitz E, Bitner-Glindzicz M, Bondi-Rubinstein G, et al. (2005) Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion. Am J Hum Genet 76: 1081–1086. pmid:15877282 doi: 10.1086/430843
[50]
Ostergaard E (2008) Disorders caused by deficiency of succinate-CoA ligase. J Inherit Metab Dis 31: 226–229. doi: 10.1007/s10545-008-0828-7. pmid:18392745
[51]
Ostergaard E, Christensen E, Kristensen E, Mogensen B, Duno M, et al. (2007) Deficiency of the alpha subunit of succinate-coenzyme A ligase causes fatal infantile lactic acidosis with mitochondrial DNA depletion. Am J Hum Genet 81: 383–387. pmid:17668387 doi: 10.1086/519222
[52]
Dallabona C, Marsano RM, Arzuffi P, Ghezzi D, Mancini P, et al. (2010) Sym1, the yeast ortholog of the MPV17 human disease protein, is a stress-induced bioenergetic and morphogenetic mitochondrial modulator. Hum Mol Genet 19: 1098–1107. doi: 10.1093/hmg/ddp581. pmid:20042463
[53]
Krauss J, Astrinidis P, Frohnhofer HG, Walderich B, Nusslein-Volhard C (2013) transparent, a gene affecting stripe formation in Zebrafish, encodes the mitochondrial protein Mpv17 that is required for iridophore survival. Biol Open 2: 703–710. doi: 10.1242/bio.20135132. pmid:23862018
[54]
Antonenkov VD, Isomursu A, Mennerich D, Vapola MH, Weiher H, et al. (2015) The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective Channel That Modulates Membrane Potential. J Biol Chem 290: 13840–13861. doi: 10.1074/jbc.M114.608083. pmid:25861990
[55]
Reinhold R, Kruger V, Meinecke M, Schulz C, Schmidt B, et al. (2012) The channel-forming Sym1 protein is transported by the TIM23 complex in a presequence-independent manner. Mol Cell Biol 32: 5009–5021. doi: 10.1128/MCB.00843-12. pmid:23045398
[56]
Da-Re C, Franzolin E, Biscontin A, Piazzesi A, Pacchioni B, et al. (2014) Functional characterization of drim2, the Drosophila melanogaster homolog of the yeast mitochondrial deoxynucleotide transporter. J Biol Chem 289: 7448–7459. doi: 10.1074/jbc.M113.543926. pmid:24469456
[57]
Van Dyck E, Jank B, Ragnini A, Schweyen RJ, Duyckaerts C, et al. (1995) Overexpression of a novel member of the mitochondrial carrier family rescues defects in both DNA and RNA metabolism in yeast mitochondria. Mol Gen Genet 246: 426–436. pmid:7891656 doi: 10.1007/bf00290446
[58]
Marobbio CM, Di Noia MA, Palmieri F (2006) Identification of a mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization. Biochem J 393: 441–446. pmid:16194150 doi: 10.1042/bj20051284
[59]
Marcelino LA, Thilly WG (1999) Mitochondrial mutagenesis in human cells and tissues. Mutat Res 434: 177–203. pmid:10486591 doi: 10.1016/s0921-8777(99)00028-2
[60]
Richter C, Park JW, Ames BN (1988) Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc Natl Acad Sci U S A 85: 6465–6467. pmid:3413108 doi: 10.1073/pnas.85.17.6465
[61]
Kazak L, Reyes A, Holt IJ (2012) Minimizing the damage: repair pathways keep mitochondrial DNA intact. Nat Rev Mol Cell Biol 13: 659–671. doi: 10.1038/nrm3439. pmid:22992591
[62]
Ferraro P, Pontarin G, Crocco L, Fabris S, Reichard P, et al. (2005) Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). J Biol Chem 280: 24472–24480. pmid:15878850 doi: 10.1074/jbc.m502869200
[63]
Garone C, Garcia-Diaz B, Emmanuele V, Lopez LC, Tadesse S, et al. (2014) Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency. EMBO Mol Med 6: 1016–1027. doi: 10.15252/emmm.201404092. pmid:24968719
[64]
Dalla Rosa I, Durigon R, Pearce SF, Rorbach J, Hirst EM, et al. (2014) MPV17L2 is required for ribosome assembly in mitochondria. Nucleic Acids Res 42: 8500–8515. doi: 10.1093/nar/gku513. pmid:24948607
[65]
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3: 1101–1108. pmid:18546601 doi: 10.1038/nprot.2008.73
[66]
Yasukawa T, Reyes A, Cluett TJ, Yang MY, Bowmaker M, et al. (2006) Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand. EMBO J 25: 5358–5371. pmid:17066082 doi: 10.1038/sj.emboj.7601392
[67]
Reyes A, Yasukawa T, Holt IJ (2007) Analysis of replicating mitochondrial DNA by two-dimensional agarose gel electrophoresis. Methods Mol Biol 372: 219–232. doi: 10.1007/978-1-59745-365-3_16. pmid:18314729
[68]
Friedman KL, Brewer BJ (1995) Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods Enzymol 262: 613–627. pmid:8594382 doi: 10.1016/0076-6879(95)62048-6
[69]
Li H (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv preprint arXiv:13033997.
[70]
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078–2079. doi: 10.1093/bioinformatics/btp352. pmid:19505943
