Gregory C. Kujoth

Mitochondrial point mutations do not limit the natural lifespan of mice

Whether mitochondrial mutations cause mammalian aging, or are merely correlated with it, is an area of intense debate. Here, we use a new, highly sensitive assay to redefine the relationship between mitochondrial mutations and age. We measured the in vivo rate of change of the mitochondrial genome at a single–base pair level in mice, and we demonstrate that the mutation frequency in mouse mitochondria is more than ten times lower than previously reported. Although we observed an 11-fold increase in mitochondrial point mutations with age, we report that a mitochondrial mutator mouse was able to sustain a 500-fold higher mutation burden than normal mice, without any obvious features of rapidly accelerated aging. Thus, our results strongly indicate that mitochondrial mutations do not limit the lifespan of wild-type mice.

DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice.

Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in many age-related pathologies. Here we identify mtDNA deletions as a driving force behind the premature aging phenotype of mitochondrial mutator mice, and provide evidence for a homology-directed DNA repair mechanism in mitochondria that is directly linked to the formation of mtDNA deletions. In addition, our results demonstrate that the rate at which mtDNA mutations reach phenotypic expression differs markedly among tissues, which may be an important factor in determining the tolerance of a tissue to random mitochondrial mutagenesis.

Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities.

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction, Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. Methodology/Principal Findings We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψm). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. Conclusions/Significance These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

The Role of Mitochondrial DNA Mutations in Mammalian Aging

Mitochondrial DNA (mtDNA) accumulates both base-substitution mutations and deletions with aging in several tissues in mammals. Here, we examine the evidence supporting a causative role for mtDNA mutations in mammalian aging. We describe and compare human diseases and mouse models associated with mitochondrial genome instability. We also discuss potential mechanisms for the generation of these mutations and the means by which they may mediate their pathological consequences. Strategies for slowing the accumulation and attenuating the effects of mtDNA mutations are discussed.

Erythroid dysplasia, megaloblastic anemia, and impaired lymphopoiesis arising from mitochondrial dysfunction.

Recent reports describe hematopoietic abnormalities in mice with targeted instability of the mitochondrial genome. However, these abnormalities have not been fully described. We demonstrate that mutant animals develop an age-dependent, macrocytic anemia with abnormal erythroid maturation and megaloblastic changes, as well as profound defects in lymphopoiesis. Mice die of severe fatal anemia at 15 months of age. Bone-marrow transplantation studies demonstrate that these abnormalities are intrinsic to the hematopoietic compartment and dependent upon the age of donor hematopoietic stem cells. These abnormalities are phenotypically similar to those found in patients with refractory anemia, suggesting that, in some cases, the myelodysplastic syndromes are caused by abnormalities of mitochondrial function.

Dysregulation of Mitochondrial Quality Control Processes Contribute to Sarcopenia in a Mouse Model of Premature Aging

Mitochondrial DNA (mtDNA) mutations lead to decrements in mitochondrial function and accelerated rates of these mutations has been linked to skeletal muscle loss (sarcopenia). The purpose of this study was to investigate the effect of mtDNA mutations on mitochondrial quality control processes in skeletal muscle from animals (young; 3–6 months and older; 8–15 months) expressing a proofreading-deficient version of mtDNA polymerase gamma (PolG). This progeroid aging model exhibits elevated mtDNA mutation rates, mitochondrial dysfunction, and a premature aging phenotype that includes sarcopenia. We found increased expression of the mitochondrial biogenesis regulator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its target proteins, nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (Tfam) in PolG animals compared to wild-type (WT) (P<0.05). Muscle from older PolG animals displayed higher mitochondrial fission protein 1 (Fis1) concurrent with greater induction of autophagy, as indicated by changes in Atg5 and p62 protein content (P<0.05). Additionally, levels of the Tom22 import protein were higher in PolG animals when compared to WT (P<0.05). In contrast, muscle from normally-aged animals exhibited a distinctly different expression profile compared to PolG animals. Older WT animals appeared to have higher fusion (greater Mfn1/Mfn2, and lower Fis1) and lower autophagy (Beclin-1 and p62) compared to young WT suggesting that autophagy is impaired in aging muscle. In conclusion, muscle from mtDNA mutator mice display higher mitochondrial fission and autophagy levels that likely contribute to the sarcopenic phenotype observed in premature aging and this differs from the response observed in normally-aged muscle.

The role of mtDNA mutations in the pathogenesis of age-related hearing loss in mice carrying a mutator DNA polymerase γ

Mitochondrial DNA (mtDNA) mutations may contribute to aging and age-related diseases. Previously, we reported that accumulation of mtDNA mutations is associated with age-related hearing loss in mice carrying a mutator allele of the mitochondrial Polg DNA polymerase. To elucidate the role of mtDNA mutations in the pathogenesis of age-related hearing loss or presbycusis, we performed large scale gene expression analysis to identify mtDNA mutation-responsive genes and biological process categories associated with mtDNA mutations by comparing the gene expression patterns of cochlear tissues from 9-month-old mitochondrial mutator and control mice. mtDNA mutations were associated with transcriptional alterations consistent with impairment of energy metabolism, induction of apoptosis, cytoskeletal dysfunction, and hearing dysfunction in the cochlea of aged mitochondrial mutator mice. TUNEL staining and caspase-3 immunostaining analysis demonstrated that the levels of apoptotic markers were significantly increased in the cochleae of mitochondrial mutator mice compared to age-matched controls. These observations support a new model of how mtDNA mutations impact cochlear function whereby accumulation of mtDNA mutations lead to mitochondrial dysfunction, an associated impairment of energy metabolism, and the induction of an apoptotic program. The data presented here provide the first global assessment at the molecular level of the pathogenesis of age-related disease in mitochondrial mutator mice and reveal previously unrecognized biological pathways associated with mtDNA mutations.

Mitochondrial DNA polymerase editing mutation, PolgD257A, disturbs stem-progenitor cell cycling in the small intestine and restricts excess fat absorption

Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg(D257A), that exhibit accelerated aging phenotype. As early as 3-7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.

Behavioral and metabolic characterization of heterozygous and homozygous POLG mutator mice.

The mitochondrial DNA (mtDNA) polymerase γ (POLG) mutator mice provide the first experimental evidence that high levels of somatic mtDNA mutations can be functionally significant. Here we report that older homozygous, but not heterozygous, POLG mice show significant reductions in striatal dopaminergic terminals as well as deficits in motor function. However, resting oxygen consumption, heat production, mtDNA content and mitochondrial electron transport chain activities are significantly decreased at older ages in both homozygous and heterozygous mice. These results indicate that high levels of somatic mtDNA mutations can contribute to dopaminergic dysfunction and to behavioral and metabolic deficits.