A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

Mitochondrial dysfunction impairs osteogenesis, increases osteoclast activity, and accelerates age related bone loss.

The pathogenesis of declining bone mineral density, a universal feature of ageing, is not fully understood. Somatic mitochondrial DNA (mtDNA) mutations accumulate with age in human tissues and mounting evidence suggests that they may be integral to the ageing process. To explore the potential effects of mtDNA mutations on bone biology, we compared bone microarchitecture and turnover in an ageing series of wild type mice with that of the PolgAmut/mut mitochondrial DNA 'mutator' mouse. In vivo analyses showed an age-related loss of bone in both groups of mice; however, it was significantly accelerated in the PolgAmut/mut mice. This accelerated rate of bone loss is associated with significantly reduced bone formation rate, reduced osteoblast population densities, increased osteoclast population densities, and mitochondrial respiratory chain deficiency in osteoblasts and osteoclasts in PolgAmut/mut mice compared with wild-type mice. In vitro assays demonstrated severely impaired mineralised matrix formation and increased osteoclast resorption by PolgAmut/mut cells. Finally, application of an exercise intervention to a subset of PolgAmut/mut mice showed no effect on bone mass or mineralised matrix formation in vitro. Our data demonstrate that mitochondrial dysfunction, a universal feature of human ageing, impairs osteogenesis and is associated with accelerated bone loss.

Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

Polymerase γ catalytic subunit (POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro-9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG-mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blázquez-Bermejo, C., Carreño-Gago, L., Molina-Granada, D., Aguirre, J., Ramón, J., Torres-Torronteras, J., Cabrera-Pérez, R., Martín, M. Á., Domínguez-González, C., de la Cruz, X., Lombès, A., García-Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling.

Autophagy is a highly regulated evolutionarily conserved metabolic process induced by stress and energy deprivation. Here, we show that DNA polymerase gamma (Polγ) deficiency activates a selective prosurvival autophagic response via mitochondria-mediated reactive oxygen species (ROS) signaling and the mammalian target of rapamycin complex 2 (mTORC2) activities. In keratinocytes, Polγ deficiency causes metabolic adaptation that triggers cytosolic sensing of energy demand for survival. Knockdown of Polγ causes mitochondrial stress, decreases mitochondrial energy production, increases glycolysis, increases the expression of autophagy-associated genes, and enhances AKT phosphorylation and cell proliferation. Deficiency of Polγ preferentially activates mTORC2 formation to increase autophagy and cell proliferation, and knocking down Rictor abrogates these responses. Overexpression of Rictor, but not Raptor, reactivates autophagy in Polγ-deficient cells. Importantly, inhibition of ROS by a mitochondria-selective ROS scavenger abolishes autophagy and cell proliferation. These results identify Rictor as a critical link between mitochondrial stress, ROS, and autophagy. They represent a major shift in our understanding of the prosurvival role of the mTOR complexes and highlight mitochondria-mediated ROS as a prosurvival autophagy regulator during cancer development.

Speech and swallowing abnormalities in adults with POLG associated ataxia (POLG-A).

BACKGROUND: Mutations in the nuclear-encoded mitochondrial DNA polymerase gamma (POLG) can result in a wide spectrum of neurological deficits. A common presentation is progressive ataxia (POLG-A) which includes impaired speech and swallowing. The nature, severity and impact of these deficits in POLG-A is not known. A comprehensive quantitative and qualitative characterization of dysarthria and dysphagia in this recurrent ataxia disorder will assist in diagnostics, provide insights into the underlying pathology, and establish the foundation for future therapy trials. METHODS: 14 consecutive patients with POLG (9 females, mean age=50.1y, SD=11.2) and 34 healthy controls were enrolled. Comprehensive assessments of motor speech and swallowing function, acoustic analysis of speech, videofluoroscopy and measures of quality of life were conducted. RESULTS: The speech profile of individuals with POLG-A was characterized by poor control of pitch and strain-strangled voice quality, reduced rate of speech and longer variable silences between words, and articulatory breakdown including imprecise consonants and vowel distortions. Swallowing deficits included slower initiation of the swallow reflex, poor control of bolus and late epiglottic closure. Speech and swallowing related quality of life was worse than healthy controls. CONCLUSIONS: The dysarthria and dysphagia profiles in POLG-A are largely symptomatic of impaired timing, indicating a mainly spinocerebellar deficit. Dysarthria and dysphagia contribute to a significant impairment in functional quality of life, and progress distinctly from other POLG-A dysfunctions like ataxia or cognitive impairment. Our assessments establish meaningful patient focused outcome measures that will be suitable for use in natural history studies and clinical trials.