No evidence of mitochondrial respiratory dysfunction in OGG1-null mice deficient in removal of 8-oxodeoxyguanine from mitochondrial DNA

Accumulation of high levels of mutagenic oxidative mitochondrial DNA (mtDNA) lesions like 8-oxodeoxyguanine (8-oxodG) is thought to be involved in the development of mitochondrial dysfunction in aging and in disorders associated with aging. Mice null for oxoguanine DNA glycosylase (OGG1) are deficient in 8-oxodG removal and accumulate 8-oxodG in mtDNA to levels 20-fold higher than in wild-type mice (N.C. Souza-Pinto et al., 2001, Cancer Res. 61, 5378-5381). We have used these animals to investigate the effects on mitochondrial function of accumulating this particular oxidative base modification. Despite the presence of high levels of 8-oxodG, mitochondria isolated from livers and hearts of Ogg1-/- mice were functionally normal. No differences were detected in maximal (chemically uncoupled) respiration rates, ADP phosphorylating respiration rates, or nonphosphorylating rates with glutamate/malate or with succinate/rotenone. Similarly, maximal activities of respiratory complexes I and IV from liver and heart were not different between wild-type and Ogg1-/- mice. In addition, there was no indication of increased oxidative stress in mitochondria from Ogg1-/- mice, as measured by mitochondrial protein carbonyl content. We conclude, therefore, that highly elevated levels of 8-oxodG in mtDNA do not cause mitochondrial respiratory dysfunction in mice.     

Clonal expansion of mitochondrial DNA deletions is a private mechanism of aging in long‐lived animals

Disruption of mitochondrial metabolism and loss of mitochondrial DNA (mtDNA) integrity are widely considered as evolutionarily conserved (public) mechanisms of aging (López‐Otín et al., Cell, 153, 2013 and 1194). Human aging is associated with loss in skeletal muscle mass and function (Sarcopenia), contributing significantly to morbidity and mortality. Muscle aging is associated with loss of mtDNA integrity. In humans, clonally expanded mtDNA deletions colocalize with sites of fiber breakage and atrophy in skeletal muscle. mtDNA deletions may therefore play an important, possibly causal role in sarcopenia. The nematode Caenorhabditis elegans also exhibits age‐dependent decline in mitochondrial function and a form of sarcopenia. However, it is unclear if mtDNA deletions play a role in C. elegans aging. Here, we report identification of 266 novel mtDNA deletions in aging nematodes. Analysis of the mtDNA mutation spectrum and quantification of mutation burden indicates that (a) mtDNA deletions in nematode are extremely rare, (b) there is no significant age‐dependent increase in mtDNA deletions, and (c) there is little evidence for clonal expansion driving mtDNA deletion dynamics. Thus, mtDNA deletions are unlikely to drive the age‐dependent functional decline commonly observed in C. elegans. Computational modeling of mtDNA dynamics in C. elegans indicates that the lifespan of short‐lived animals such as C. elegans is likely too short to allow for significant clonal expansion of mtDNA deletions. Together, these findings suggest that clonal expansion of mtDNA deletions is likely a private mechanism of aging predominantly relevant in long‐lived animals such as humans and rhesus monkey and possibly in rodents.     

Defects of intergenomic communication: autosomal disorders that cause multiple deletions and depletion of mitochondrial DNA

Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a growing number of autosomal diseases that have been classified as defects of intergenomic communication. MNGIE, an autosomal recessive disorder associated with mtDNA alterations is due to mutations in thymidine phosphorylase that may cause imbalance of the mitochondrial nucleotide pool. Subsequently, mutations in the mitochondrial proteins adenine nucleotide translocator 1, Twinkle, and polymerase gamma have been found to cause autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA. Uncovering the molecular bases of intergenomic communication defects will enhance our understanding of the mechanisms responsible for maintaining mtDNA integrity.     

Vitamin E at high doses improves survival, neurological performance, and brain mitochondrial function in aging male mice

Male mice receiving vitamin E (5.0 g alpha-tocopherol acetate/kg of food) from 28 wk of age showed a 40% increased median life span, from 61 +/- 4 wk to 85 +/- 4 wk, and 17% increased maximal life span, whereas female mice equally supplemented exhibited only 14% increased median life span. The alpha-tocopherol content of brain and liver was 2.5-times and 7-times increased in male mice, respectively. Vitamin E-supplemented male mice showed a better performance in the tight-rope (neuromuscular function) and the T-maze (exploratory activity) tests with improvements of 9-24% at 52 wk and of 28-45% at 78 wk. The rates of electron transfer in brain mitochondria, determined as state 3 oxygen uptake and as NADH-cytochrome c reductase and cytochrome oxidase activities, were 16-25% and 35-38% diminished at 52-78 wk. These losses of mitochondrial function were ameliorated by vitamin E supplementation by 37-56% and by 60-66% at the two time points considered. The activities of mitochondrial nitric oxide synthase and Mn-SOD decreased 28-67% upon aging and these effects were partially (41-68%) prevented by vitamin E treatment. Liver mitochondrial activities showed similar effects of aging and of vitamin E supplementation, although less marked. Brain mitochondrial enzymatic activities correlated negatively with the mitochondrial content of protein and lipid oxidation products (r2 = 0.58-0.99, P < 0.01), and the rates of respiration and of complex I and IV activities correlated positively (r2 = 0.74-0.80, P < 0.01) with success in the behavioral tests and with maximal life span.     

Increase in mitochondrial DNA mutations impairs retinal function and renders the retina vulnerable to injury

Mouse models that accumulate high levels of mitochondrial DNA (mtDNA) mutations owing to impairments in mitochondrial polymerase γ (PolG) proofreading function have been shown to develop phenotypes consistent with accelerated aging. As increase in mtDNA mutations and aging are risk factors for neurodegenerative diseases, we sought to determine whether increase in mtDNA mutations renders neurons more vulnerable to injury. We therefore examined the in vivo functional activity of retinal neurons and their ability to cope with stress in transgenic mice harboring a neural‐targeted mutant PolG gene with an impaired proofreading capability (Kasahara, et al. (2006) Mol Psychiatry 11 (6):577–93, 523). We confirmed that the retina of these transgenic mice have increased mtDNA deletions and point mutations and decreased expression of mitochondrial oxidative phosphorylation enzymes. Associated with these changes, the PolG transgenic mice demonstrated accelerated age‐related loss in retinal function as measured by dark‐adapted electroretinogram, particularly in the inner and middle retina. Furthermore, the retinal ganglion cell–dominant inner retinal function in PolG transgenic mice showed greater vulnerability to injury induced by raised intraocular pressure, an insult known to produce mechanical, metabolic, and oxidative stress in the retina. These findings indicate that an accumulation of mtDNA mutations is associated with impairment in neural function and reduced capacity of neurons to resist external stress in vivo, suggesting a potential mechanism whereby aging central nervous system can become more vulnerable to neurodegeneration.     

Replicating animal mitochondrial DNA

The field of mitochondrial DNA (mtDNA) replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s) used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark) has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading- and lagging-strand synthesis (resembling bacterial genome replication) and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS). The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase γ, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase). Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.     

Ethanol feeding enhances age-related deterioration of the rat hepatic mitochondrion

Chronic ethanol feeding damages the hepatic mitochondrion by increasing mitochondrial DNA (mtDNA) oxidation, lowering mtDNA yields and impairing mitochondrial respiration. These effects are also seen during aging. By employing a 21-day chronic feeding regimen, we investigated the effects of ethanol consumption on mtDNA content and mitochondrial respiration in 2-, 12-, and 24-mo-old male rats. Aging resulted in decreased mtDNA content, increased mtDNA damage (as indicated by inhibition of Taq polymerase progression), and a decline in state 3 respiration; effects that were further exacerbated by ethanol feeding. Additionally, ethanol consumption caused an increase in the levels of citrate synthase while not impacting mitochondrial protein content. In conclusion, ethanol and aging combine to cause deterioration in the structural and functional integrity of the hepatic mitochondrion. The additive effects of aging and ethanol feeding may have serious consequences for hepatic energy metabolism in aged animals, and their detrimental combination may serve as one of the molecular mechanisms underlying the progression of alcoholic liver disease.     

Accumulation of Mitochondrial DNA Mutations Disrupts Cardiac Progenitor Cell Function and Reduces Survival

Transfer of cardiac progenitor cells (CPCs) improves cardiac function in heart failure patients. However, CPC function is reduced with age, limiting their regenerative potential. Aging is associated with numerous changes in cells including accumulation of mitochondrial DNA (mtDNA) mutations, but it is unknown how this impacts CPC function. Here, we demonstrate that acquisition of mtDNA mutations disrupts mitochondrial function, enhances mitophagy, and reduces the replicative and regenerative capacities of the CPCs. We show that activation of differentiation in CPCs is associated with expansion of the mitochondrial network and increased mitochondrial oxidative phosphorylation. Interestingly, mutant CPCs are deficient in mitochondrial respiration and rely on glycolysis for energy. In response to differentiation, these cells fail to activate mitochondrial respiration. This inability to meet the increased energy demand leads to activation of cell death. These findings demonstrate the consequences of accumulating mtDNA mutations and the importance of mtDNA integrity in CPC homeostasis and regenerative potential.     

Pentacyclic triterpene oleanolic acid protects against cardiac aging through regulation of mitophagy and mitochondrial integrity

Advanced aging exhibits altered cardiac geometry and function involving mitochondrial anomaly. Natural compounds display promises in the regulation of cardiac homeostasis via governance of mitochondrial integrity in aging. This study examined the effect of oleanolic acid (OA), a natural pentacyclic triterpenoid with free radical scavenging and P450 cyclooxygenase-regulating properties, on cardiac aging and mechanisms involved with a focus on mitophagy. Young (4–5 month-old) and old (22–24 month-old) mice were treated with OA for 6 weeks prior to assessment of cardiac function, morphology, ultrastructure, mitochondrial integrity, cell death and autophagy. Our data revealed that OA treatment alleviated aging-induced changes in myocardial remodeling (increased heart weight, chamber size, cardiomyocyte area and interstitial fibrosis), contractile function and intracellular Ca²⁺ handling, apoptosis, necroptosis, inflammation, autophagy and mitophagy (LC3B, p62, TOM20 and FUNDC1 but not BNIP3 and Parkin). OA treatment rescued aging-induced anomalies in mitochondrial ultrastructure (loss of myofilament alignment, swollen mitochondria, increased circularity), mitochondrial biogenesis and O₂^? production without any notable effect at young age. Interestingly, OA-offered benefit against cardiomyocyte aging was nullified by deletion of the mitophagy receptor FUNDC1 using FUNDC1 knockout mice, denoting an obligatory role for FUNDC1 in OA-elicited preservation of mitophagy. OA reconciled aging-induced changes in E3 ligase MARCH5 but not FBXL2, and failed to affect aging-induced rises in IP3R3. Taken together, our data indicated a beneficial role for OA in attenuating cardiac remodeling and contractile dysfunction in aging through a FUNDC1-mediated mechanism.     

Endogenous mitochondrial oxidative stress in MnSOD-deficient mouse embryonic fibroblasts promotes mitochondrial DNA glycation

The accumulation of somatic mutations in mitochondrial DNA (mtDNA) induced by reactive oxygen species (ROS) is regarded as a major contributor to aging and age-related degenerative diseases. ROS have also been shown to facilitate the formation of certain advanced glycation end-products (AGEs) in proteins and DNA and N(2)-carboxyethyl-2'-deoxyguanosine (CEdG) has been identified as a major DNA-bound AGE. Therefore, the influence of mitochondrial ROS on the glycation of mtDNA was investigated in primary embryonic fibroblasts derived from mutant mice (Sod2(-/+)) deficient in the mitochondrial antioxidant enzyme manganese superoxide dismutase. In Sod2(-/+) fibroblasts vs wild-type fibroblasts, the CEdG content of mtDNA was increased from 1.90 ± 1.39 to 17.14 ± 6.60 pg/μg DNA (p<0.001). On the other hand, the CEdG content of nuclear DNA did not differ between Sod2(+/+) and Sod2(-/+) cells. Similarly, cytosolic proteins did not show any difference in advanced glycation end-products or protein carbonyl contents between Sod2(+/+) and Sod2(-/+). Taken together, the data suggest that mitochondrial oxidative stress specifically promotes glycation of mtDNA and does not affect nuclear DNA or cytosolic proteins. Because DNA glycation can change DNA integrity and gene functions, glycation of mtDNA may play an important role in the decline of mitochondrial functions.     

Persistent damage induces mitochondrial DNA degradation

Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by 6 h after induction of mutant uracil-N-glycosylase and by 12 h after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144 h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and “foaming” of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage.     

Mitochondrial ND5 gene variation associated with encephalomyopathy and mitochondrial ATP consumption

Mitochondrial encephalomyopathy and lactic acidosis with strokelike episodes (MELAS) is a severe young onset stroke disorder without effective treatment. We have identified a MELAS patient harboring a 13528A-->G mitochondrial DNA (mtDNA) mutation in the Complex I ND5 gene. This mutation was homoplasmic in mtDNA from patient muscle and nearly homoplasmic (99.9%) in blood. Fibroblasts from the patient exhibited decreased mitochondrial membrane potential (Deltapsim) and increased lactate production, consistent with impaired mitochondrial function. Transfer of patient mtDNA to a new nuclear background using transmitochondrial cybrid fusions confirmed the pathogenicity of the 13528A-->G mutation; Complex I-linked respiration and Deltapsim were both significantly reduced in patient mtDNA cybrids compared with controls. Inhibition of the adenine nucleotide translocase or the F1F0-ATPase with bongkrekic acid or oligomycin caused a loss of potential in patient mtDNA cybrid mitochondria, indicating a requirement for glycolytically generated ATP to maintain Deltapsim. This was confirmed by inhibition of glycolysis with 2-deoxy-D-glucose, which caused depletion of ATP and mitochondrial depolarization in patient mtDNA cybrids. These data suggest that in response to impaired respiration due to the mtDNA mutation, mitochondria consume ATP to maintain Deltapsim, representing a potential pathophysiological mechanism in human mitochondrial disease.     

Evidence for a role of FEN1 in maintaining mitochondrial DNA integrity

Although the nuclear processes responsible for genomic DNA replication and repair are well characterized, the pathways involved in mitochondrial DNA (mtDNA) replication and repair remain unclear. DNA repair has been identified as being particularly important within the mitochondrial compartment due to the organelle's high propensity to accumulate oxidative DNA damage. It has been postulated that continual accumulation of mtDNA damage and subsequent mutagenesis may function in cellular aging. Mitochondrial base excision repair (mtBER) plays a major role in combating mtDNA oxidative damage; however, the proteins involved in mtBER have yet to be fully characterized. It has been established that during nuclear long-patch (LP) BER, FEN1 is responsible for cleavage of 5′ flap structures generated during DNA synthesis. Furthermore, removal of 5′ flaps has been observed in mitochondrial extracts of mammalian cell lines; yet, the mitochondrial localization of FEN1 has not been clearly demonstrated. In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity.     

Aging and high concentrations of glucose potentiate injury to mitochondrial DNA

Deletions of mitochondrial DNA (mtDNA) are associated with aging and several chronic diseases. We have reported heterogeneous mutations between base pair 8468 and 13446 in mtDNA, the region known as the "common" deletion, in muscle of older humans with impaired glucose tolerance or diabetes mellitus. To further characterize potential effects of age and glycemia on mtDNA integrity, we studied corpulent JCR:LA-cp rats that are characterized by insulin resistance, hyperinsulinemia, and hyperlipidemia, factors strongly associated with both aging and cardiovascular disease. In addition to skeletal muscle, we isolated vascular smooth muscle cells (VSMC) from aortas of 6-, 12-, and 17-month-old rats and exposed them to 5-, 25-, 62-, and 100-mM glucose or a combination of hypoxanthine (100 microM) and xanthine oxidase (0.025 U/ml) to generate reactive oxygen species in separate cultures. Long- and short-fragment and nested polymerase chain reaction was used to detect mutations in the common deletion region. The data demonstrate that aging and the cp genotype confer susceptibility to mtDNA deletions in vivo and that high glucose concentrations can induce mtDNA mutations in vitro. Accordingly, aging and glucose-related oxidative stress and possibly hyperinsulinemia may contribute to alterations in mitochondrial gene integrity and the cp genotype appears to increase the susceptibility of muscle to the age-related accumulation of mtDNA mutations.     

The hexameric structure of the human mitochondrial replicative helicase Twinkle

The mitochondrial replicative helicase Twinkle is involved in strand separation at the replication fork of mitochondrial DNA (mtDNA). Twinkle malfunction is associated with rare diseases that include late onset mitochondrial myopathies, neuromuscular disorders and fatal infantile mtDNA depletion syndrome. We examined its 3D structure by electron microscopy (EM) and small angle X-ray scattering (SAXS) and built the corresponding atomic models, which gave insight into the first molecular architecture of a full-length SF4 helicase that includes an N-terminal zinc-binding domain (ZBD), an intermediate RNA polymerase domain (RPD) and a RecA-like hexamerization C-terminal domain (CTD). The EM model of Twinkle reveals a hexameric two-layered ring comprising the ZBDs and RPDs in one layer and the CTDs in another. In the hexamer, contacts in trans with adjacent subunits occur between ZBDs and RPDs, and between RPDs and CTDs. The ZBDs show important structural heterogeneity. In solution, the scattering data are compatible with a mixture of extended hexa- and heptameric models in variable conformations. Overall, our structural data show a complex network of dynamic interactions that reconciles with the structural flexibility required for helicase activity.     

A novel defense system of mitochondria in mice and human subjects for preventing expression of mitochondrial dysfunction by pathogenic mutant mtDNAs

Recently, we generated mtDNA-based disease mice (mito mice) by introduction of respiration-deficient mitochondria possessing pathogenic mutant mtDNA with a 4696 bp deletion (deltamtDNA4696) from somatic cells into mouse zygotes. Mito mice and cytochrome c oxidase (COX) electronmicrographs, that could identify the respiration enzyme activity at individual mitochondrial levels, enabled precise investigation of the pathogenesis of deltamtDNA4696. All the observations represented unambiguous evidence for the presence of extensive and continuous exchange of genetic contents between mitochondria. Thus, the inter-mitochondrial interaction could correspond to a very unique and effective defense system of the highly oxidative organelles for preventing mice and human subjects from expressing mitochondrial dysfunction caused by mtDNA lesions, which have been continuously created by oxidative stresses during aging. Here, we would like to propose a new hypothesis on mitochondrial biogenesis, 'the interaction theory of mammalian mitochondria': mitochondria exchange genetic contents, and thus lose individuality and function as a single dynamic cellular unit.