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1.
Proc Natl Acad Sci U S A ; 115(41): E9620-E9629, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30249665

RESUMO

Mammals develop age-associated clonal expansion of somatic mtDNA mutations resulting in severe respiratory chain deficiency in a subset of cells in a variety of tissues. Both mathematical modeling based on descriptive data from humans and experimental data from mtDNA mutator mice suggest that the somatic mutations are formed early in life and then undergo mitotic segregation during adult life to reach very high levels in certain cells. To address whether mtDNA mutations have a universal effect on aging metazoans, we investigated their role in physiology and aging of fruit flies. To this end, we utilized genetically engineered flies expressing mutant versions of the catalytic subunit of mitochondrial DNA polymerase (DmPOLγA) as a means to introduce mtDNA mutations. We report here that lifespan and health in fruit flies are remarkably tolerant to mtDNA mutations. Our results show that the short lifespan and wide genetic bottleneck of fruit flies are limiting the extent of clonal expansion of mtDNA mutations both in individuals and between generations. However, an increase of mtDNA mutations to very high levels caused sensitivity to mechanical and starvation stress, intestinal stem cell dysfunction, and reduced lifespan under standard conditions. In addition, the effects of dietary restriction, widely considered beneficial for organismal health, were attenuated in flies with very high levels of mtDNA mutations.


Assuntos
DNA Mitocondrial , Longevidade/genética , Mutação , Animais , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Drosophila melanogaster
2.
Nucleic Acids Res ; 46(13): 6642-6669, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29860357

RESUMO

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.


Assuntos
Reparo do DNA , DNA Mitocondrial/química , Estresse Oxidativo , Mutação Puntual , Animais , Núcleo Celular/enzimologia , DNA Glicosilases/metabolismo , Replicação do DNA , Proteínas Ferro-Enxofre/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Proteômica , Superóxido Dismutase/genética , Transcrição Gênica
3.
Hum Mol Genet ; 26(13): 2515-2525, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28430993

RESUMO

Mutations in the mitochondrial DNA polymerase, POLG, are associated with a variety of clinical presentations, ranging from early onset fatal brain disease in Alpers syndrome to chronic progressive external ophthalmoplegia. The majority of mutations are linked with disturbances of mitochondrial DNA (mtDNA) integrity and maintenance. On a molecular level, depending on their location within the enzyme, mutations either lead to mtDNA depletion or the accumulation of multiple mtDNA deletions, and in some cases these molecular changes can be correlated to the clinical presentation. We identified a patient with a dominant p.Y955H mutation in POLG, presenting with a severe, early-onset multi-systemic mitochondrial disease with bilateral sensorineural hearing loss, cataract, myopathy, and liver failure. Using a combination of disease models of Drosophila melanogaster and in vitro biochemistry analysis, we compare the molecular consequences of the p.Y955H mutation to the well-documented p.Y955C mutation. We demonstrate that both mutations affect mtDNA replication and display a dominant negative effect, with the p.Y955H allele resulting in a more severe polymerase dysfunction.


Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Adulto , Sequência de Aminoácidos , Animais , DNA Polimerase gama , Replicação do DNA/genética , DNA Mitocondrial/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Feminino , Humanos , Lactente , Mitocôndrias/genética , Mutação/genética , Oftalmoplegia Externa Progressiva Crônica/enzimologia , Linhagem , Fenótipo
4.
Cell Metab ; 25(1): 57-71, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28094012

RESUMO

Mitochondria were first postulated to contribute to aging more than 40 years ago. During the following decades, multiple lines of evidence in model organisms and humans showed that impaired mitochondrial function can contribute to age-associated disease phenotypes and aging. However, in contrast to the original theory favoring oxidative damage as a cause for mtDNA mutations, there are now strong data arguing that most mammalian mtDNA mutations originate as replication errors made by the mtDNA polymerase. Currently, a substantial amount of mitochondrial research is focused on finding ways to either remove or counteract the effects of mtDNA mutations with the hope of extending the human health- and lifespan. This review summarizes the current knowledge regarding the formation of mtDNA mutations and their impact on mitochondrial function. We also critically discuss proposed pathways interlinked with mammalian mtDNA mutations and suggest future research strategies to elucidate the role of mtDNA mutations in aging.


Assuntos
Envelhecimento/metabolismo , Mamíferos/metabolismo , Mitocôndrias/metabolismo , Animais , DNA Mitocondrial/genética , Doença/genética , Humanos , Espécies Reativas de Oxigênio/metabolismo
5.
Nat Commun ; 6: 8808, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26554610

RESUMO

Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineer the tamas locus, encoding fly POLγA, and introduce alleles expressing exonuclease- (exo(-)) and polymerase-deficient (pol(-)) POLγA versions. The exo(-) mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol(-) mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease.


Assuntos
DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/metabolismo , Drosophila/genética , Exodesoxirribonucleases/metabolismo , Engenharia Genética , Animais , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Mutagênese Sítio-Dirigida , Mutação , Subunidades Proteicas
6.
Nucleic Acids Res ; 42(22): 13920-38, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25428350

RESUMO

Members of the pentatricopeptide repeat domain (PPR) protein family bind RNA and are important for post-transcriptional control of organelle gene expression in unicellular eukaryotes, metazoans and plants. They also have a role in human pathology, as mutations in the leucine-rich PPR-containing (LRPPRC) gene cause severe neurodegeneration. We have previously shown that the mammalian LRPPRC protein and its Drosophila melanogaster homolog DmLRPPRC1 (also known as bicoid stability factor) are necessary for mitochondrial translation by controlling stability and polyadenylation of mRNAs. We here report characterization of DmLRPPRC2, a second fruit fly homolog of LRPPRC, and show that it has a predominant mitochondrial localization and interacts with a stem-loop interacting RNA binding protein (DmSLIRP2). Ubiquitous downregulation of DmLrpprc2 expression causes respiratory chain dysfunction, developmental delay and shortened lifespan. Unexpectedly, decreased DmLRPPRC2 expression does not globally affect steady-state levels or polyadenylation of mitochondrial transcripts. However, some mitochondrial transcripts abnormally associate with the mitochondrial ribosomes and some products are dramatically overproduced and other ones decreased, which, in turn, results in severe deficiency of respiratory chain complexes. The function of DmLRPPRC2 thus seems to be to ensure that mitochondrial transcripts are presented to the mitochondrial ribosomes in an orderly fashion to avoid poorly coordinated translation.


Assuntos
Proteínas de Drosophila/fisiologia , Mitocôndrias/genética , Proteínas Mitocondriais/fisiologia , Biossíntese de Proteínas , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Transporte de Elétrons , Longevidade , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Poliadenilação , RNA/metabolismo , Interferência de RNA , RNA Mitocondrial , Transcrição Gênica
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