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Mitochondrial DNA (mtDNA) mutations accumulate in both mitotic and post-mitotic somatic tissues of normal individuals with age. They clonally expand within individual cells and cause mitochondrial dysfunction. In contrast, in patients with inherited disease-causing mtDNA mutations the mutation load decreases in mitotic tissues over time, whereas the mutations load in post-mitotic tissues remains relatively stable. The mechanisms underlying this decrease in mitotic tissues, and whether mitochondrial function is restored at the tissue level are unknown. Here, using a combination of homogenate tissue and single crypt/muscle fibre pyrosequencing we have shown a decrease in the mutation load of the germline heteroplasmic m.5024C>T mutation in multiple mitotic tissues of a mouse model of inherited mitochondrial disease (C5024T mice). We have then usedin silicopredictions to model the cellular dynamics of mtDNA mutation load in mitotic and post mitotic tissues. We demonstrate that: (1) the rate of m.5024C>T decrease correlates with the rate of tissue turnover; (2) the mutation load decrease is not associated with changes in overall cellular proliferation and apoptosis within the mitotic colonic epithelium; instead, it could be due to an upper limit of m.5024C>T load in stem cell populations; (3) the m.5024C>T mutation load is maintained in post-mitotic tissues over time with a consistent load amongst individual muscle fibres; (4)in silicomodelling supports a scenario where genetic drift is accelerated in mitotic tissues by high levels of mtDNA replication coupled with mtDNA segregation at cell division. This study has advanced our understanding of the dynamics of mtDNA mutations and phenotype development in patients with mtDNA disease.Author SummaryHealthy individuals randomly accumulate pathogenic mtDNA mutations with age in dividing cells, causing mitochondrial dysfunction. Interestingly, patients with mitochondrial disease show a relative decrease in the loads of inherited mtDNA mutations in some dividing cells over time. The mechanisms underlying this decrease are unknown. Here we show a decrease in the load of the germline heteroplasmic m.5024C>T mutation in dividing cells and tissues of a mouse model of mitochondrial disease. In contrast, the mutation load in non-dividing cells and tissue remains stable. Our data are consistent with the hypothesis that a higher frequency of mtDNA replication in dividing cells, coupled with stem cells having an upper tolerance limit for m.5024C>T, causes an overall decrease in m.5024C>T load at the tissue level.

Alterations in mitochondrial metabolism have been described as one of the major hallmarks of both ageing cells and cancer. Age is the biggest risk factor for the development of a significant number of cancer types and this therefore raises the question of whether there is a link between age-related mitochondrial dysfunction and the advantageous changes in mitochondrial metabolism prevalent in cancer cells. A common underlying feature of both ageing and cancer cells is the presence of somatic mutations of the mitochondrial genome (mtDNA) which we postulate may drive compensatory alterations in mitochondrial metabolism that are advantageous for tumour growth. In this review, we discuss basic mitochondrial functions, mechanisms of mtDNA mutagenesis and their metabolic consequences, and review the evidence for and against a role for mtDNA mutations in cancer development.

Precision cut liver slices (PCLSs) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono- or co-cultures. The aim of this study was to develop a bioreactor system to increase the healthy life span of PCLSs and model fibrogenesis. PCLSs were generated from normal rat or human liver, or fibrotic rat liver, and cultured in our bioreactor. PCLS function was quantified by albumin enzyme-linked immunosorbent assay (ELISA). Fibrosis was induced in PCLSs by transforming growth factor beta 1 (TGFβ1) and platelet-derived growth factor (PDGFββ) stimulation ± therapy. Fibrosis was assessed by gene expression, picrosirius red, and α-smooth muscle actin staining, hydroxyproline assay, and soluble ELISAs. Bioreactor-cultured PCLSs are viable, maintaining tissue structure, metabolic activity, and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell-cultured PCLSs rapidly deteriorate, and albumin secretion is significantly impaired by 48 hours. TGFβ1/PDGFββ stimulation of rat or human PCLSs induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts, and histological fibrosis. Fibrogenesis slowly progresses over 6 days in cultured fibrotic rat PCLSs without exogenous challenge. Activin receptor-like kinase 5 (Alk5) inhibitor (Alk5i), nintedanib, and obeticholic acid therapy limited fibrogenesis in TGFβ1/PDGFββ-stimulated PCLSs, and Alk5i blunted progression of fibrosis in fibrotic PCLS. Conclusion: We describe a bioreactor technology that maintains functional PCLS cultures for 6 days. Bioreactor-cultured PCLSs can be successfully used to model fibrogenesis and demonstrate efficacy of antifibrotic therapies. ispartof: HEPATOLOGY vol:70 issue:4 pages:1377-1391 ispartof: location:United States status: published

One of the hallmarks of aging is an accumulation of cells with defects in oxidative phosphorylation (OXPHOS) due to mutations of mitochondrial DNA (mtDNA). Rapidly dividing tissues maintained by stem cells, such as the colonic epithelium, are particularly susceptible to accumulation of OXPHOS defects over time; however, the effects on the stem cells are unknown. We have crossed a mouse model in which intestinal stem cells are labelled with EGFP (Lgr5‐EGFP‐IRES‐creERT2) with a model of accelerated mtDNA mutagenesis (PolgA mut/mut) to investigate the effect of OXPHOS dysfunction on colonic stem cell proliferation. We show that a reduction in complex I protein levels is associated with an increased rate of stem cell cycle re‐entry. These changes in stem cell homeostasis could have significant implications for age‐associated intestinal pathogenesis., Age‐associated mitochondrial DNA mutations accumulate in the colonic epithelial crypts of the PolgA mut/mut and PolgA +/mut mice. This leads to a reduction in mitochondrial complex I and IV protein levels. Stem cells with complex I defects re‐enter the cell cycle 20% more frequently than those with normal complex I levels.

Oxidative phosphorylation (OXPHOS) defects caused by somatic mitochondrial DNA (mtDNA) mutations increase with age in human colorectal epithelium and are prevalent in colorectal tumours, but whether they actively contribute to tumorigenesis remains unknown. Here we demonstrate that mtDNA mutations causing OXPHOS defects are enriched during the human adenoma/carcinoma sequence, suggesting they may confer a metabolic advantage. To test this we deleted the tumour suppressor Apc in OXPHOS deficient intestinal stem cells in mice. The resulting tumours were larger than in control mice due to accelerated cell proliferation and reduced apoptosis. We show that both normal crypts and tumours undergo metabolic remodelling in response to OXPHOS deficiency by upregulating the de novo serine synthesis pathway (SSP). Moreover, normal human colonic crypts upregulate the SSP in response to OXPHOS deficiency prior to tumorigenesis. Our data show that age-associated OXPHOS deficiency causes metabolic remodelling that can functionally contribute to accelerated intestinal cancer development.

Introduction: Age-related cumulative mitochondrial mutations can cause mitochondrial dysfunction. The mitochondrial DNA mutator mouse (PolgAmut/mut) is a model of premature ageing and has been previously shown to develop impaired insulin secretion with age. Methods: Immunofluorescence was used to study mitochondrial respiratory chain protein expression (complex I and IV) and the cell composition in islets. Experiments were conducted on pancreas tissue from PolgAmut/mut mice and age-matched wild type (WT) mice at two ages: 12 weeks (young) and 44 weeks (old). Results: (1) Comparing young WT mice versus old WT mice, there was no difference in islet complex I expression, while islet size and beta cell number increased with age (both P Conclusions: In PolgA mut/mut mice, complex I deficiency was already present in the islets of young mice and persisted with age. This was associated with a proportional increase in alpha cells and decrease in beta cells in islets from old PolgA mut/mut versus the old WT mice. We conclude that complex I deficiency in PolgA mut/mut mice is linked to altered islet cell composition with increasing age. Disclosure X. Yu: None. C. Arden: None. C. Chen: None. C. Bradshaw: None. J. Whitehall: None. M.G. White: None. S.J. Anderson: None. J.A. Shaw: Advisory Panel; Self; Sanofi. Other Relationship; Self; Novo Nordisk A/S. D. Turnbull: None. L. Greaves: None. M. Walker: None.