Mitochondrial DNA depletion can be prevented by dGMP and dAMP supplementation in a resting culture of deoxyguanosine kinase-deficient fibroblasts.
(49/884)
Deoxyguanosine kinase is a constitutively expressed, mitochondrial enzyme of the deoxyribonucleoside salvage pathway. Deficiency of deoxyguanosine kinase causes early-onset, hepatocerebral mitochondrial DNA (mtDNA) depletion syndrome. To clarify the molecular mechanism of the disease, a skin fibroblast culture was studied from a patient carrying a homozygous nonsense mutation in the gene for deoxyguanosine kinase. In situ examination of DNA synthesis demonstrated that, although mtDNA synthesis is cell cycle independent in control fibroblasts, mtDNA synthesis occurs mainly during the S-phase in deoxyguanosine kinase-deficient cells. Consistent with this observation, it was found that the mtDNA content of exponentially growing, deoxyguanosine kinase-deficient cells is only mildly affected. When cycling is inhibited by serum-deprivation and cells are in a resting state, however, the mtDNA content drops considerably in deoxyguanosine kinase-deficient cells, yet remains stable in control fibroblasts. The decline in mtDNA content in resting, deoxyguanosine kinase-deficient cells can be prevented by dGMP and dAMP supplementation, providing conclusive evidence that substrate limitation triggers mtDNA depletion in deoxyguanosine kinase-deficient cells. (+info)
Quantification of mitochondrial DNA deletion, depletion, and overreplication: application to diagnosis.
(50/884)
BACKGROUND: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations. METHODS: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal (16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis. We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication. RESULTS: Both methods quantified 5-76% of deleted mtDNA in muscle, 59-97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r(2)) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis. CONCLUSIONS: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis. (+info)
POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA.
(51/884)
In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD(+), with a twofold preference for NADH. Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA 50-fold, as measured by the arg8(m) reversion assay, with no increase in nuclear mutations. Also, a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed. POS5 was previously described as a gene required for resistance to hydrogen peroxide. Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper. Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction. Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species. These results predict a role for NADH kinase in human mitochondrial diseases. (+info)
Investigation of a pathogenic mtDNA microdeletion reveals a translation-dependent deadenylation decay pathway in human mitochondria.
(52/884)
Human mtDNA is transcribed from both strands, producing polycistronic RNA species that are immediately processed. Discrete RNA units are matured by the addition of nucleotides at their 3' termini: -CCA trinucleotide is added to mt-tRNAs, whilst mt-rRNAs and mt-mRNAs are oligo- or polyadenylated, respectively. The cis-acting elements, enzymes and indeed the mechanisms involved in these processes are still largely uncharacterized. Further, the function of polyadenylation in promoting stability, translation or decay of human mt-mRNA is unclear. A microdeletion has been identified in a patient presenting with mtDNA disease. Loss of these two residues removes the termination codon for MTATP6 and sets MTCO3 immediately in frame. Accurate processing at this site still occurs, but there is a markedly decreased steady-state level of RNA14, the ATPase 8- and 6-encoding bi-cistronic mRNA unit, establishing that an mtDNA mutation can cause dysregulation of mRNA stability. Analysis of the polyadenylation profile of the processed RNA14 at steady state revealed substantial abnormalities. The majority of mutated RNA14 terminated with short poly (A) extensions and a second, partially truncated population, was also present. Initial maturation of mutated RNA14 was unaffected, but deadenylation occurred rapidly. Inhibition of mitochondrial protein synthesis showed that the deadenylation was dependent on translation. Finally, deadenylation was shown to enhance mRNA decay, explaining the decrease in steady-state RNA14. An hypothesis is presented to describe how an mtDNA mutation that results in the loss of a termination codon causes enhanced mt-mRNA decay by translation-dependent deadenylation. (+info)
Disorders of mitochondrial protein synthesis.
(53/884)
Mitochondrial tRNA gene mutations, including heteroplasmic deletions that eliminate one or more tRNAs, as well as point mutations that may be either hetero- or homoplasmic, are associated with a wide spectrum of human diseases. These range from rare syndromic disorders to cases of commoner conditions such as sensorineural deafness or cardiomyopathy. The disease spectrum of mutations in a given gene, or even a single mutation, may vary, but some patterns are evident, for example the prominence of cardiomyopathy resulting from tRNAIle defects, or of MERFF-like disease from tRNALys defects. Molecular studies of many laboratories have reached a consensus on molecular mechanisms associated with these mutations. Although precise details vary, loss of translational function of the affected tRNA(s) seems to be the final outcome, whether by impaired pre-tRNA processing, half-life, base-modification or aminoacylation. However, a mechanistic understanding of the consequences of this for the assembly and function of the mitochondrial OXPHOS complexes and for the physiological functions of the affected tissues is still a distant prospect. This review presents some views of possible downstream consequences of specific tRNA deficiencies. (+info)
Mitochondria.
(54/884)
Following the discovery in the early 1960s that mitochondria contain their own DNA (mtDNA), there were two major advances, both in the 1980s: the human mtDNA sequence was published in 1981, and in 1988 the first pathogenic mtDNA mutations were identified. The floodgates were opened, and the 1990s became the decade of the mitochondrial genome. There has been a change of emphasis in the first few years of the new millennium, away from the "magic circle" of mtDNA and back to the nuclear genome. Various nuclear genes have been identified that are fundamentally important for mitochondrial homeostasis, and when these genes are disrupted, they cause autosomally inherited mitochondrial disease. Moreover, mitochondrial dysfunction plays an important role in the pathophysiology of several well established nuclear genetic disorders, such as dominant optic atrophy (mutations in OPA1), Friedreich's ataxia (FRDA), hereditary spastic paraplegia (SPG7), and Wilson's disease (ATP7B). The next major challenge is to define the more subtle interactions between nuclear and mitochondrial genes in health and disease. (+info)
Turnover of adipose components and mitochondrial DNA in humans: kinetic biomarkers for human immunodeficiency virus-associated lipodystrophy and mitochondrial toxicity?
(55/884)
Lipoatrophy (LA)/lipodystrophy and nucleoside reverse-transcriptase inhibitor (NRTI)-associated syndrome are of central importance in human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) care. Neither of these conditions has had a clear pathogenesis or biomarker defined for early detection, prevention research, or patient management. I describe the recent development of kinetic biomarkers for LA and mitochondrial toxicity that involve the measurement of biosynthetic fluxes rather than static concentrations of molecules. The turnover of adipose-tissue components (lipids and cells) and tissue mitochondrial DNA is measured by the incorporation of deuterium from heavy water, using mass spectrometry. Preliminary results in animal models and humans, including the effects of NRTIs on mitochondrial DNA synthesis in rats and adipose-tissue lipid kinetics in HIV-associated LA, are reviewed. The results suggest that the kinetics of adipose-tissue components and mitochondrial DNA are measurable in vivo and that these measurements may prove useful as clinical biomarkers in patients with HIV/AIDS. (+info)
Oxidative alpha-ketoglutarate dehydrogenase inhibition via subtle elevations in monoamine oxidase B levels results in loss of spare respiratory capacity: implications for Parkinson's disease.
(56/884)
Age-related increases in brain monoamine oxidase B (MAO-B) and its ability to produce reactive oxygen species as a by-product of catalysis could contribute to neurodegeneration associated with Parkinson's disease. This may be via increased oxidative stress and/or mitochondrial dysfunction either on its own or through its interaction with endogenous or exogenous neurotoxic species. We have created genetically engineered dopaminergic PC12 cell lines with subtly increased levels of MAO-B mimicking those observed during normal aging. In our cells, increased MAO-B activity was found to result in increased H2O2 production. This was found to correlate with a decrease in mitochondrial complex I activity which may involve both direct oxidative damage to the complex itself as well as oxidative effects on the tricarboxylic acid cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) which provides substrate for the complex. Both complex I and KGDH activities have been reported to be decreased in the Parkinsonian brain. These in vitro events are reversible by catalase addition. Importantly, MAO-B elevation was found to abolish the spare KGDH threshold capacity, which can normally be significantly inhibited before it affects maximal mitochondrial oxygen consumption rates. Our data suggest that H2O2 production via subtle elevations in MAO-B levels can result in oxidative effects on KGDH that can compromise the ability of dopaminergic neurons to cope with increased energetic stress. (+info)