Fukui, H. & Moraes, C. T. Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons. Hum. Mol. Genet. 18, 1028-1036

Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.
Human Molecular Genetics (Impact Factor: 6.39). 01/2009; 18(6):1028-36. DOI: 10.1093/hmg/ddn437
Source: PubMed


Age-dependent accumulation of partially deleted mitochondrial DNA (DeltamtDNA) has been suggested to contribute to aging and the development of age-associated diseases including Parkinson's disease. However, the molecular mechanisms underlying the generation and accumulation of DeltamtDNA have not been addressed in vivo. In this study, we have developed a mouse model expressing an inducible mitochondria-targeted restriction endonuclease (PstI). Using this system, we could trigger mtDNA double-strand breaks (DSBs) in adult neurons. We found that this transient event leads to the generation of a family of DeltamtDNA with features that closely resemble naturally-occurring mtDNA deletions. The formation of these deleted species is likely to be mediated by yet uncharacterized DNA repairing machineries that participate in homologous recombination and non-homologous end-joining. Furthermore, we obtained in vivo evidence that DeltamtDNAs with larger deletions accumulate faster than those with smaller deletions, implying a replicative advantage of smaller mtDNAs. These findings identify DSB, DNA repair systems and replicative advantage as likely mechanisms underlying the generation and age-associated accumulation of DeltamtDNA in mammalian neurons.

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    • "To study the molecular mechanisms underlying the generation and accumulation of mtDNA deletions in neurons in vivo, mice expressing neuron-specific mito-PstI were created by crossing the TRE-mito-PstI mouse with a model carrying the tTA gene under the control of the Cam- KIIa promoter (Fukui & Moraes, 2009). In the absence of Dox, CamKIIαtTA drives the expression of mito-PstI in forebrain neurons. "
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    ABSTRACT: For more than a decade, mitochondria-targeted nucleases have been used to promote double-strand breaks in the mitochondrial genome. This was done in mitochondrial DNA (mtDNA) homoplasmic systems, where all mtDNA molecules can be affected, to create models of mitochondrial deficiencies. Alternatively, they were also used in a heteroplasmic model, where only a subset of the mtDNA molecules were substrates for cleavage. The latter approach showed that mitochondrial-targeted nucleases can reduce mtDNA haplotype loads in affected tissues, with clear implications for the treatment of patients with mitochondrial diseases. In the last few years, designer nucleases, such as ZFN and TALEN, have been adapted to cleave mtDNA, greatly expanding the potential therapeutic use. This chapter describes the techniques and approaches used to test these designer enzymes.
    Methods in enzymology 11/2014; 547:373-97. DOI:10.1016/B978-0-12-801415-8.00018-7 · 2.09 Impact Factor
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    • "We predict that mutations affecting cluster 1 will reduce primarily pol activity, and these have been associated with increased replication fork stalling in vivo [32] [52] [53]. The stalling phenotype is characterized by slow progression of or suspended replication forks and causes an accumulation of replication intermediates that can lead to doublestranded DNA breaks, mtDNA deletions, and base substitution mutations [39] [40] [41]. It is thus unlikely that a compound heterozygote with two cluster 1 mutations would be viable unless the defects are very mild. "
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    ABSTRACT: We establish the genotype-phenotype correlations for the complete spectrum of POLG syndromes, by refining our previously described protocol for mapping pathogenic mutations in the human POLG gene to functional clusters in the catalytic core of the mitochondrial replicase, Pol γ (1). We assigned 136 mutations to five clusters and identify segments of primary sequence that can be used to delimit the boundaries of each cluster. We report that compound heterozygotes with two mutations from different clusters manifested more severe, earlier onset POLG syndromes, whereas two mutations from the same cluster are less common and generally are associated with less severe, later onset POLG syndromes. We also show that specific cluster combinations are more severe than others, and have a higher likelihood to manifest at an earlier age. Our clustering method provides a powerful tool to predict the pathogenic potential and predicted disease phenotype of novel variants and mutations in POLG, the most common nuclear gene underlying mitochondrial disorders. We propose that such a prediction tool would be useful for routine diagnostics for mitochondrial disorders. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.
    Biochimica et Biophysica Acta 07/2014; 1837(7). DOI:10.1016/j.bbabio.2014.01.021 · 4.66 Impact Factor
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    • "We believe that most plausible is a 'mixed' scenario, where expansions are fast in some cells and slow in other ('fast' and 'slow expanders', correspondingly), probably with the whole spectrum of expansion rates in between. Expansion rates may differ between cell types or between cells of the same type differing in individual activity, stress, levels of ROS, length of deletion (Fukui & Moraes, 2009), etc. An example of such a difference is given by myoblasts, which, unlike their descendant myofibers, support only very slow, if any, expansion of mtDNA deletions (Moraes et al., 1989). "
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    ABSTRACT: Somatic mtDNA mutations and deletions in particular are known to clonally expand within cells, eventually reaching detrimental intracellular concentrations. The possibility that clonal expansion is a slow process taking a lifetime had prompted an idea that founder mutations of mutant clones that cause mitochondrial dysfunction in the aged tissue might have originated early in life. If, conversely, expansion was fast, founder mutations should predominantly originate later in life. This distinction is important: indeed, from which mutations should we protect ourselves – those of early development/childhood or those happening at old age? Recently, high-resolution data describing the distribution of mtDNA deletions have been obtained using a novel, highly efficient method (Taylor et al., ). These data have been interpreted as supporting predominantly early origin of founder mutations. Re-analysis of the data implies that the data actually better fit mostly late origin of founders, although more research is clearly needed to resolve the controversy.
    Aging cell 05/2014; 13(4). DOI:10.1111/acel.12231 · 6.34 Impact Factor
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