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The maternal mtDNA mutation load heavily influences phenotypes in mtDNA mutator mice
578
Abstracts
mitochondria. Through site directed mutagenesis we identified the probable RNA interacting domains of the mRPS6 protein, in agreement with structures determined for the corresponding prokaryotic small ribosomal subunit. The highly conserved RPS6 protein is found in prokaryotes as well as eukaryotes. Curiously, a truncated protein in which the highly conserved mammalian-specific 29C-terminal amino-acids are deleted could also fully restore ribosome function.
78
doi:10.1016/j.mito.2012.07.070
The mtDNA mutator mice express only a proof-reading-deficient version of mitochondrial DNA polymerase, which creates high levels of mtDNA point mutations, leading to ageing-like phenotypes and premature death. Wildtype littermates have been used as controls in most mtDNA mutator studies. However, these wildtype mice are offspring of females heterozygous for the PolgAmut allele. Since the heterozygous females are also inducing mtDNA mutations, it is possible that their developing oocytes will carry mtDNA mutations to the resulting offspring. If clonally expanded, these germline transmitted mutations may constitute an important source of mtDNA mutations in the mutator mice and apparent wild-type littermate controls. We tested this hypothesis by breeding heterzoygous knockout females (PolgA+/KO), that do not have elevated levels of mtDNA mutations in the germline, to heterozygous mtDNA mutator males (PolgA+/mut), thus creating hemizygous mtDNA mutator mice of the genotype PolgAKO/mut. By comparing hemizygous (PolgAKO/ mut) and standard (PolgAmut/mut) mtDNA mutators, as well as other genotypes from these crosses, we were able to investigate the amount of mtDNA mutations contributed by heterozygous mothers (PolgA+/mut) to their offspring. We did observed clonal expansion of mtDNA molecules in offspring to the heterozygous mothers. The physiological consequences of these mutations were assessed by comparing the standard mtDNA mutator mice to the hemizygous mtDNA mutators. The absence of maternally transmitted mutations in the hemizygous mtDNA mutators led to a delayed onset of respiratory chain deficiency, which, in turn, resulted in a delayed onset of weight loss and alleviation of male sterility. This suppression of characteristic symptoms in mtDNA mutator mice is reminiscent of recent results obtained by using PGC1-α overexpression or exercise as an intervention in mtDNA mutator mice. In contrast, the anemia and blood abnormalities were consistent between these two types of mtDNA mutator mice, suggesting a direct role of somatic mutagenesis in creating stem cell phenotypes. Our results demonstrate the importance of using control animals that are free of inherited mtDNA mutations in studies comparing e.g. life span or physiological phenotypes. Our results suggest that subset of the mutations derived from the female germline will be prone to clonal expansion, and therefore may drift to high levels in certain tissues, as observed in ageing humans. Thus, it will be critically important for our understanding of the role of mtDNA mutations in the ageing process, to disentangle the relative contribution of inherited mtDNA mutations versus the ongoing mutation load supplied by the PolgAmut polymerase.
77 An evolving bioinformatics tool reveals mitochondrial pathology in diseases and aging Presenter: Morten Scheibye-Knudsen Morten Scheibye-Knudsena, Karsten Scheibye-Alsingb, Chandrika Canugovia, Deborah L. Croteaua, Vilhelm A. Bohra a Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA b Section of Genetics and Bioinformatics, IBHV, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark Bona fide mitochondrial diseases represent a heterogeneous group of genetic syndromes with a combined incidence of around 1:5000. The complex phenotype of these disorders poses a significant diagnostic challenge for clinicians and an analytical barrier for scientists. Further, mitochondrial dysfunction has been proposed to be an underlying origin of aging through free radical production. These highly volatile species will readily react with any lipid, protein and nucleic acid. The mitochondrial theory of aging states that an accumulation of damage to these macromolecules throughout the lifetime of an organism leads to cellular decay, loss of tissue homeostasis, and finally death. If mitochondrial dysfunction is causative in aging, we would expect diseases displaying accelerated aging to exhibit signs and symptoms of mitochondrial diseases. To investigate this hypothesis, we compiled an evolving online database, www.mitodb.com, of the signs and symptoms observed in mitochondrial diseases. We then developed an array of bioinformatics tools allowing us to test if a disorder can be characterized as mitochondrial. These tools can be found on www.mitodb.com and consist of various hierarchical clustering algorithms, a support vector machine, a scoring system and others. Using these applications we detected mitochondrial dysfunction in Xeroderma Pigmentosum, group A (XPA); a disease with a DNA repair defect not previously associated with mitochondrial dysfunction. We then experimentally tested whether several primary XPA patient fibroblasts displayed mitochondrial dysfunction relative to normal control primary fibroblasts. Specifically, we found increased oxygen consumption (~2-fold, p b 0.001), mitochondrial content (~20%, p = 0.007), mitochondrial membrane potential (~50%, p b 0.001) and free radical production (~50%, p = 0.02/p = 0.006) in XPA cells, relative to controls; demonstrating mitochondrial dysfunction in XPA, thus validating the usefulness of the database. Next, we tested the progerias, in addition to normal aging, using our database and found that several of the segmental progerias (Cockayne Syndrome, Ataxia-Telangiectasia and XPA) as well as aging display features of mitochondrial dysfunction. In closing, we have developed a unique tool that will significantly aid clinicians and scientists in uncovering mitochondrial dysfunction in diseases and in correlating complex disease phenotypes with mitochondrial pathology.
doi:10.1016/j.mito.2012.07.071
The maternal mtDNA mutation load heavily influences phenotypes in mtDNA mutator mice Presenter: James B. Stewart James B. Stewart, Christoph Freyer, Arnaud Mourier, Erik Hagstrom, Marie Lagouge, Anna Wredenberg, Nils-Göran Larsson Max Planck Institute for the Biology of Ageing, Gleueler Str. 50aD-50931 Cologne, Germany
doi:10.1016/j.mito.2012.07.072
79 Gene regulation of cytochrome C oxidase subunit 4 isoform 2: A tale of three factors Presenter: Lawrence Grossman Lawrence Grossmana, Siddhesh Arasa, Pak Olegb, Natascha Sommerb, Maik Hüttemanna, Norbert Weissmannb a Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, United States b University of Giessen Lung Center, Giessen, Germany
Abstracts
mitochondria. Through site directed mutagenesis we identified the probable RNA interacting domains of the mRPS6 protein, in agreement with structures determined for the corresponding prokaryotic small ribosomal subunit. The highly conserved RPS6 protein is found in prokaryotes as well as eukaryotes. Curiously, a truncated protein in which the highly conserved mammalian-specific 29C-terminal amino-acids are deleted could also fully restore ribosome function.
78
doi:10.1016/j.mito.2012.07.070
The mtDNA mutator mice express only a proof-reading-deficient version of mitochondrial DNA polymerase, which creates high levels of mtDNA point mutations, leading to ageing-like phenotypes and premature death. Wildtype littermates have been used as controls in most mtDNA mutator studies. However, these wildtype mice are offspring of females heterozygous for the PolgAmut allele. Since the heterozygous females are also inducing mtDNA mutations, it is possible that their developing oocytes will carry mtDNA mutations to the resulting offspring. If clonally expanded, these germline transmitted mutations may constitute an important source of mtDNA mutations in the mutator mice and apparent wild-type littermate controls. We tested this hypothesis by breeding heterzoygous knockout females (PolgA+/KO), that do not have elevated levels of mtDNA mutations in the germline, to heterozygous mtDNA mutator males (PolgA+/mut), thus creating hemizygous mtDNA mutator mice of the genotype PolgAKO/mut. By comparing hemizygous (PolgAKO/ mut) and standard (PolgAmut/mut) mtDNA mutators, as well as other genotypes from these crosses, we were able to investigate the amount of mtDNA mutations contributed by heterozygous mothers (PolgA+/mut) to their offspring. We did observed clonal expansion of mtDNA molecules in offspring to the heterozygous mothers. The physiological consequences of these mutations were assessed by comparing the standard mtDNA mutator mice to the hemizygous mtDNA mutators. The absence of maternally transmitted mutations in the hemizygous mtDNA mutators led to a delayed onset of respiratory chain deficiency, which, in turn, resulted in a delayed onset of weight loss and alleviation of male sterility. This suppression of characteristic symptoms in mtDNA mutator mice is reminiscent of recent results obtained by using PGC1-α overexpression or exercise as an intervention in mtDNA mutator mice. In contrast, the anemia and blood abnormalities were consistent between these two types of mtDNA mutator mice, suggesting a direct role of somatic mutagenesis in creating stem cell phenotypes. Our results demonstrate the importance of using control animals that are free of inherited mtDNA mutations in studies comparing e.g. life span or physiological phenotypes. Our results suggest that subset of the mutations derived from the female germline will be prone to clonal expansion, and therefore may drift to high levels in certain tissues, as observed in ageing humans. Thus, it will be critically important for our understanding of the role of mtDNA mutations in the ageing process, to disentangle the relative contribution of inherited mtDNA mutations versus the ongoing mutation load supplied by the PolgAmut polymerase.
77 An evolving bioinformatics tool reveals mitochondrial pathology in diseases and aging Presenter: Morten Scheibye-Knudsen Morten Scheibye-Knudsena, Karsten Scheibye-Alsingb, Chandrika Canugovia, Deborah L. Croteaua, Vilhelm A. Bohra a Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA b Section of Genetics and Bioinformatics, IBHV, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark Bona fide mitochondrial diseases represent a heterogeneous group of genetic syndromes with a combined incidence of around 1:5000. The complex phenotype of these disorders poses a significant diagnostic challenge for clinicians and an analytical barrier for scientists. Further, mitochondrial dysfunction has been proposed to be an underlying origin of aging through free radical production. These highly volatile species will readily react with any lipid, protein and nucleic acid. The mitochondrial theory of aging states that an accumulation of damage to these macromolecules throughout the lifetime of an organism leads to cellular decay, loss of tissue homeostasis, and finally death. If mitochondrial dysfunction is causative in aging, we would expect diseases displaying accelerated aging to exhibit signs and symptoms of mitochondrial diseases. To investigate this hypothesis, we compiled an evolving online database, www.mitodb.com, of the signs and symptoms observed in mitochondrial diseases. We then developed an array of bioinformatics tools allowing us to test if a disorder can be characterized as mitochondrial. These tools can be found on www.mitodb.com and consist of various hierarchical clustering algorithms, a support vector machine, a scoring system and others. Using these applications we detected mitochondrial dysfunction in Xeroderma Pigmentosum, group A (XPA); a disease with a DNA repair defect not previously associated with mitochondrial dysfunction. We then experimentally tested whether several primary XPA patient fibroblasts displayed mitochondrial dysfunction relative to normal control primary fibroblasts. Specifically, we found increased oxygen consumption (~2-fold, p b 0.001), mitochondrial content (~20%, p = 0.007), mitochondrial membrane potential (~50%, p b 0.001) and free radical production (~50%, p = 0.02/p = 0.006) in XPA cells, relative to controls; demonstrating mitochondrial dysfunction in XPA, thus validating the usefulness of the database. Next, we tested the progerias, in addition to normal aging, using our database and found that several of the segmental progerias (Cockayne Syndrome, Ataxia-Telangiectasia and XPA) as well as aging display features of mitochondrial dysfunction. In closing, we have developed a unique tool that will significantly aid clinicians and scientists in uncovering mitochondrial dysfunction in diseases and in correlating complex disease phenotypes with mitochondrial pathology.
doi:10.1016/j.mito.2012.07.071
The maternal mtDNA mutation load heavily influences phenotypes in mtDNA mutator mice Presenter: James B. Stewart James B. Stewart, Christoph Freyer, Arnaud Mourier, Erik Hagstrom, Marie Lagouge, Anna Wredenberg, Nils-Göran Larsson Max Planck Institute for the Biology of Ageing, Gleueler Str. 50aD-50931 Cologne, Germany
doi:10.1016/j.mito.2012.07.072
79 Gene regulation of cytochrome C oxidase subunit 4 isoform 2: A tale of three factors Presenter: Lawrence Grossman Lawrence Grossmana, Siddhesh Arasa, Pak Olegb, Natascha Sommerb, Maik Hüttemanna, Norbert Weissmannb a Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, United States b University of Giessen Lung Center, Giessen, Germany