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      Analysis of mtDNA/nDNA ratio in mice

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          Abstract

          The mitochondrial DNA (mtDNA) lacks a protection provided by the nucleosomes in the nuclear DNA and does not have a DNA repair mechanism, making it highly susceptible to damage, which can lead to mtDNA depletion. MtDNA depletion compromises the efficient function of cells and tissues and thus impacts negatively on health. Here, we describe a brief and easy protocol to quantify mtDNA copy number by determining the mtDNA/nDNA ratio that we validated in a cohort of young and aged mice.

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          Most cited references13

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          The role of mitochondria in aging.

          Over the last decade, accumulating evidence has suggested a causative link between mitochondrial dysfunction and major phenotypes associated with aging. Somatic mitochondrial DNA (mtDNA) mutations and respiratory chain dysfunction accompany normal aging, but the first direct experimental evidence that increased mtDNA mutation levels contribute to progeroid phenotypes came from the mtDNA mutator mouse. Recent evidence suggests that increases in aging-associated mtDNA mutations are not caused by damage accumulation, but rather are due to clonal expansion of mtDNA replication errors that occur during development. Here we discuss the caveats of the traditional mitochondrial free radical theory of aging and highlight other possible mechanisms, including insulin/IGF-1 signaling (IIS) and the target of rapamycin pathways, that underlie the central role of mitochondria in the aging process.
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            Mitochondrial DNA depletion syndromes: review and updates of genetic basis, manifestations, and therapeutic options.

            Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a genetically and clinically heterogeneous group of autosomal recessive disorders that are characterized by a severe reduction in mtDNA content leading to impaired energy production in affected tissues and organs. MDS are due to defects in mtDNA maintenance caused by mutations in nuclear genes that function in either mitochondrial nucleotide synthesis (TK2, SUCLA2, SUCLG1, RRM2B, DGUOK, and TYMP) or mtDNA replication (POLG and C10orf2). MDS are phenotypically heterogeneous and usually classified as myopathic, encephalomyopathic, hepatocerebral or neurogastrointestinal. Myopathic MDS, caused by mutations in TK2, usually present before the age of 2 years with hypotonia and muscle weakness. Encephalomyopathic MDS, caused by mutations in SUCLA2, SUCLG1, or RRM2B, typically present during infancy with hypotonia and pronounced neurological features. Hepatocerebral MDS, caused by mutations in DGUOK, MPV17, POLG, or C10orf2, commonly have an early-onset liver dysfunction and neurological involvement. Finally, TYMP mutations have been associated with mitochondrial neurogastrointestinal encephalopathy (MNGIE) disease that typically presents before the age of 20 years with progressive gastrointestinal dysmotility and peripheral neuropathy. Overall, MDS are severe disorders with poor prognosis in the majority of affected individuals. No efficacious therapy is available for any of these disorders. Affected individuals should have a comprehensive evaluation to assess the degree of involvement of different systems. Treatment is directed mainly toward providing symptomatic management. Nutritional modulation and cofactor supplementation may be beneficial. Liver transplantation remains controversial. Finally, stem cell transplantation in MNGIE disease shows promising results.
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              Loss of mitochondrial protease OMA1 alters processing of the GTPase OPA1 and causes obesity and defective thermogenesis in mice.

              Mitochondria are dynamic subcellular organelles that convert nutrient intermediates into readily available energy equivalents. Optimal mitochondrial function is ensured by a highly evolved quality control system, coordinated by protein machinery that regulates a process of continual fusion and fission. In this work, we provide in vivo evidence that the ATP-independent metalloprotease OMA1 plays an essential role in the proteolytic inactivation of the dynamin-related GTPase OPA1 (optic atrophy 1). We also show that OMA1 deficiency causes a profound perturbation of the mitochondrial fusion-fission equilibrium that has important implications for metabolic homeostasis. Thus, ablation of OMA1 in mice results in marked transcriptional changes in genes of lipid and glucose metabolic pathways and substantial alterations in circulating blood parameters. Additionally, Oma1-mutant mice exhibit an increase in body weight due to increased adipose mass, hepatic steatosis, decreased energy expenditure and impaired thermogenenesis. These alterations are especially significant under metabolic stress conditions, indicating that an intact OMA1-OPA1 system is essential for developing the appropriate adaptive response to different metabolic stressors such as a high-fat diet or cold-shock. This study provides the first description of an unexpected role in energy metabolism for the metalloprotease OMA1 and reinforces the importance of mitochondrial quality control for normal metabolic function.
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                Author and article information

                Journal
                101560384
                39214
                Curr Protoc Mouse Biol
                Curr Protoc Mouse Biol
                Current protocols in mouse biology
                2161-2617
                30 November 2016
                02 March 2017
                02 March 2017
                02 March 2018
                : 7
                : 1
                : 47-54
                Affiliations
                [1 ]Laboratory for Integrative and Systems Physiology, Ecole Polytechnique Federale de Lausanne, 1015 Lausanne, Switzerland
                Author notes
                Contact information: Laboratory of Integrative and Systems Physiology (LISP), EPFL, SV, Station 15, CH-1015 Lausanne, Switzerland, admin.auwerx@ 123456epfl.ch , Phone: +41 21 693 95 22, Fax: +41 21 693 96 00
                [*]

                These authors contribute equally to this work

                Article
                PMC5335900 PMC5335900 5335900 nihpa830312
                10.1002/cpmo.21
                5335900
                28252199
                9a468a63-33af-4a12-84a3-2d2548c9dde3
                History
                Categories
                Article

                16SrRNA,ND1,mtDNA,qPCR
                16SrRNA, ND1, mtDNA, qPCR

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