Accumulation Of mtDNA Mutations Research Articles

Age-induced changes in skeletal muscle mitochondrial DNA synthesis, quantity, and quality in genetically unique rats

AbstractMitochondrial genomic integrity is a key element of physiological processes and health. Changes in the half-life of the mitochondrial genome are implicated in the generation and accumulation of age-induced mitochondrial DNA (mtDNA) mutations, which are implicated in skeletal muscle aging and sarcopenia. There are conflicting data on the half-life of mtDNA, and there is limited information on how aging affects half-life in skeletal muscle. We hypothesized that skeletal muscle mtDNA synthesis rates would decrease with age in both female and male rats concomitant with changes in mtDNA integrity reflected in mtDNA copy number and mutation frequency. We measured mitochondrial genome half-life using stable isotope labeling over a period of 14 days and assessed mtDNA copy number and deletion mutation frequency using digital PCR in the quadriceps muscle of 9-month-old and 26-month-old male and female OKC-HET rats. We found a significant age-related increase in mtDNA half-life, from 132 days at 9 months to 216 days at 26 months of age in OKC-HET quadriceps. Concomitant with the increase in mtDNA half-life, we found an age-related increase in mtDNA deletion mutation frequency in both male and female rats. Notably, 26-month-old female rats had a lower mutation frequency than male rats, and there were no changes in mtDNA copy number with sex, age, or mitochondrial genotype. These data reveal several key findings: (1) mtDNA turnover in rat skeletal muscle decreases with age, (2) mtDNA half-lives in skeletal muscle are approximately an order of magnitude longer than what is reported for other tissues, and (3) muscle mtDNA turnover differs significantly from the turnover of other mitochondrial macromolecules including components of the mitochondrial nucleoid. These findings provide insight into the factors driving age-induced mtDNA mutation accumulation, which contribute to losses of mitochondrial genomic integrity and may play a role in skeletal muscle dysfunction.

Modeling aging and retinal degeneration with mitochondrial DNA mutation burden.

Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a polymerase gamma (POLG) exonuclease-deficient model, the PolgD257A mutator mice (D257A), was used. POLG is an enzyme responsible for regulating mtDNA replication and repair. Retinas of young and older mice with this mutation were analyzed invivo and exvivo to provide new insights into the contribution of age-related mitochondrial (mt) dysfunction due to mtDNA damage. Optical coherence tomography (OCT) image analysis revealed a decrease in retinal and photoreceptor thickness starting at 6 months of age in mice with the D257A mutation compared to wild-type (WT) mice. Electroretinography (ERG) testing showed a significant decrease in all recorded responses at 6 months of age. Sections labeled with markers of different types of retinal cells, including cones, rods, and bipolar cells, exhibited decreased labeling starting at 6 months. However, electron microscopy analysis revealed differences in retinal pigment epithelium (RPE) mt morphology beginning at 3 months. Interestingly, there was no increase in oxidative stress and parkin-mediated mitophagy in the ages analyzed in the retina or RPE of D257A mice. Additionally, D257A RPE exhibited an accelerated rate of autofluorescence cytoplasmic granule formation and accumulation. Mt markers displayed different abundance in protein lysates obtained from retina and RPE samples. These findings suggest that the accumulation of mtDNA mutations leads to impaired mt function and accelerated aging, resulting in retinal degeneration.

Lower Mitochondrial Genome Turnover and Greater Mutation Frequency in Skeletal Muscle of Aged Compared to Adult OKC-HET Rats

Mitochondrial genomic integrity, which is supported by processes such as replication, repair, and turnover, is critical in supporting metabolism and organismal health. Accumulation of mitochondrial DNA (mtDNA) mutations contributes to mitochondrial and whole-tissue dysfunction, which lead to chronic diseases including sarcopenia. The underlying mechanisms that promote generation and age-related accumulation of mtDNA mutations are unclear. mtDNA mutants may have a replicative advantage over wild-type mtDNA that propagates the accumulation of mtDNA mutations with age. However, there is limited in vivo data regarding effects of age on mtDNA replication, particularly in skeletal muscle. We hypothesized mtDNA synthesis rates would increase with age as mutation burden increased in skeletal muscle in both male and female rats. Methods: We measured mtDNA synthesis over 14 days using the stable isotope tracer deuterium oxide (D2O) and assessed mtDNA copy number and mutation deletion frequency using digital PCR in quadriceps muscle of 9- and 26 month-old (mo) male and female OKC-HET rats, which have heterogenous mtDNA backgrounds. Results: There were no differences in mtDNA copy number between sexes or ages. However, 26 month-od rats had lower rates of mtDNA synthesis compared to 9 month-old rats (9 mo: 0.509 ± 0.009 %/day, 26 month: 0.371 ± 0.048 %/day; p=0.0024) and greater mtDNA half-lives (9 mo: 132 ± 1.79 days, 216 ± 25.63 days; p=0.0009). Concomitant with lower mtDNA synthesis, 26 month-old rats had greater (p=0.003) mtDNA deletion mutation frequency (2.023e-004 ± 7.18e-005) than 9 month-old rats (9.310e-005 ± 2.404e-005). 26 month-old female rats (1.309e-004 ± 2.386e-005) had a lower (p=0.008) mutation burden than male rats (2.74e-004 ± 4.484e-005). Conclusion: Contrary to our hypothesis, mtDNA synthesis declined with age as mutation burden increased in the quadriceps. Because mtDNA copy number was not different between ages, while synthesis decreased, mtDNA turnover declined with age. Altogether, these results suggest lower mtDNA turnover rates may contribute to age-related mtDNA mutation burden. Future studies should test how modulating mtDNA turnover affects mutation frequency and mitochondrial function. This study was supported by funding from NIH grants R21AG072137 and 5T32AG052363-04. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

NAD+ dependent UPRmt activation underlies intestinal aging caused by mitochondrial DNA mutations

Aging in mammals is accompanied by an imbalance of intestinal homeostasis and accumulation of mitochondrial DNA (mtDNA) mutations. However, little is known about how accumulated mtDNA mutations modulate intestinal homeostasis. We observe the accumulation of mtDNA mutations in the small intestine of aged male mice, suggesting an association with physiological intestinal aging. Using polymerase gamma (POLG) mutator mice and wild-type mice, we generate male mice with progressive mtDNA mutation burdens. Investigation utilizing organoid technology and in vivo intestinal stem cell labeling reveals decreased colony formation efficiency of intestinal crypts and LGR5-expressing intestinal stem cells in response to a threshold mtDNA mutation burden. Mechanistically, increased mtDNA mutation burden exacerbates the aging phenotype of the small intestine through ATF5 dependent mitochondrial unfolded protein response (UPRmt) activation. This aging phenotype is reversed by supplementation with the NAD+ precursor, NMN. Thus, we uncover a NAD+ dependent UPRmt triggered by mtDNA mutations that regulates the intestinal aging.

Huntington's disease affects mitochondrial network dynamics predisposing to pathogenic mitochondrial DNA mutations.

Huntington's disease (HD) predominantly affects the brain, causing a mixed movement disorder, cognitive decline and behavioural abnormalities. It also causes a peripheral phenotype involving skeletal muscle. Mitochondrial dysfunction has been reported in tissues of HD models, including skeletal muscle, and lymphoblast and fibroblast cultures from patients with HD. Mutant huntingtin protein (mutHTT) expression can impair mitochondrial quality control and accelerate mitochondrial ageing. Here, we obtained fresh human skeletal muscle, a post-mitotic tissue expressing the mutated HTT allele at physiological levels since birth, and primary cell lines from HTT CAG repeat expansion mutation carriers and matched healthy volunteers to examine whether such a mitochondrial phenotype exists in human HD. Using ultra-deep mitochondrial DNA (mtDNA) sequencing, we showed an accumulation of mtDNA mutations affecting oxidative phosphorylation. Tissue proteomics indicated impairments in mtDNA maintenance with increased mitochondrial biogenesis of less efficient oxidative phosphorylation (lower complex I and IV activity). In full-length mutHTT expressing primary human cell lines, fission-inducing mitochondrial stress resulted in normal mitophagy. In contrast, expression of high levels of N-terminal mutHTT fragments promoted mitochondrial fission and resulted in slower, less dynamic mitophagy. Expression of high levels of mutHTT fragments due to somatic nuclear HTT CAG instability can thus affect mitochondrial network dynamics and mitophagy, leading to pathogenic mtDNA mutations. We show that life-long expression of mutant HTT causes a mitochondrial phenotype indicative of mtDNA instability in fresh post-mitotic human skeletal muscle. Thus, genomic instability may not be limited to nuclear DNA, where it results in somatic expansion of the HTT CAG repeat length in particularly vulnerable cells such as striatal neurons. In addition to efforts targeting the causative mutation, promoting mitochondrial health may be a complementary strategy in treating diseases with DNA instability such as HD.

Abstract IA020: Increased mitochondrial DNA mutation burden with age limits lung cancer

Abstract Mitochondrial DNA (mtDNA) mutations accumulate with age and are found in some cancers. However, the relationship between cancer and mtDNA mutations, and the pathogenicity of those mutations, is not known. To address this question, we created a new mouse model of non-small cell lung cancer (NSCLC) with an increased mtDNA mutation burden. This model was generated by breeding the NSCLC mice model carrying floxed alleles of p53 and KrasG12D, with the mtDNA mutator mouse bearing a germline polymerase gamma mutation that disables proofreading function (POLGD257A) that is responsible for the accumulation of mtDNA mutations over time. The mutator mice with NSCLC had reduced tumor burden and prolonged survival after tumor induction in comparison to POLG wild type mice with NSCLC. Both tumor and normal tissue from mutator mice had elevated mtDNA mutation burden in comparison to wild type mice. The prolonged survival of POLGD257A mice with NSCLC was even more pronounced in older animals where mtDNA mutation burden was further increased. The same was true for a mouse model with a single pathogenic mtDNA mutation found in mitochondrial disease, indicating that it is not the number of mutations, but rather the disabling of mitochondrial function by mutation that causes reduced tumor growth. This suggested that elevated mtDNA mutation with age may suppress NSCLC. To address the mechanism by which accumulation of mtDNA mutations suppress tumor growth we examined metabolism and immune functions. We found that mtDNA mutations caused tumor cell metabolic dysfunction including increased glycolysis and decreased respiration with dependency of glycose and serine and glycine as carbon sources in vitro. Moreover, mtDNA mutations caused tumor dependence on serine and glycine in vivo. Thus, elevated mtDNA mutation burden with age may increase probability of suppression of mitochondrial function, resulting in specific metabolic dependencies in NSCLC. Thus, tumors with mtDNA mutation accumulation may be susceptible to therapeutic dietary restriction of serine and glycine. Citation Format: Eileen White. Increased mitochondrial DNA mutation burden with age limits lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr IA020.

REC drives recombination to repair double-strand breaks in animal mtDNA.

Mechanisms that safeguard mitochondrial DNA (mtDNA) limit the accumulation of mutations linked to mitochondrial and age-related diseases. Yet, pathways that repair double-strand breaks (DSBs) in animal mitochondria are poorly understood. By performing a candidate screen for mtDNA repair proteins, we identify that REC-an MCM helicase that drives meiotic recombination in the nucleus-also localizes to mitochondria in Drosophila. We show that REC repairs mtDNA DSBs by homologous recombination in somatic and germline tissues. Moreover, REC prevents age-associated mtDNA mutations. We further show that MCM8, the human ortholog of REC, also localizes to mitochondria and limits the accumulation of mtDNA mutations. This study provides mechanistic insight into animal mtDNA recombination and demonstrates its importance in safeguarding mtDNA during ageing and evolution.

Targeting Mitochondrial Function with Chemoptogenetics.

Mitochondria are ATP-generating organelles in eukaryotic cells that produce reactive oxygen species (ROS) during oxidative phosphorylation (OXPHOS). Mitochondrial DNA (mtDNA) is packaged within nucleoids and, due to its close proximity to ROS production, endures oxidative base damage. This damage can be repaired by base excision repair (BER) within the mitochondria, or it can be degraded via exonucleases or mitophagy. Persistent mtDNA damage may drive the production of dysfunctional OXPHOS components that generate increased ROS, or OXPHOS components may be directly damaged by ROS, which then can cause more mtDNA damage and create a vicious cycle of ROS production and mitochondrial dysfunction. If mtDNA damage is left unrepaired, mtDNA mutations including deletions can result. The accumulation of mtDNA mutations has been associated with conditions ranging from the aging process to cancer and neurodegenerative conditions, but the sequence of events leading to mtDNA mutations and deletions is yet unknown. Researchers have utilized many systems and agents for generating ROS in mitochondria to observe the downstream effects on mtDNA, ROS, and mitochondrial function; yet, there are various drawbacks to these methodologies that limit their precision. Here, we describe a novel chemoptogenetic approach to target oxidative damage to mitochondria and mtDNA with a high spatial and temporal resolution so that the downstream effects of ROS-induced damage can be measured with a high precision in order to better understand the mechanism of mitochondrial dysfunction in aging, cancer, and neurodegenerative diseases.

Premature aging is associated with higher levels of 8-oxoguanine and increased DNA damage in the Polg mutator mouse.

Mitochondrial dysfunction plays an important role in the aging process. However, the mechanism by which this dysfunction causes aging is not fully understood. The accumulation of mutations in the mitochondrial genome (or “mtDNA”) has been proposed as a contributor. One compelling piece of evidence in support of this hypothesis comes from the Polg D257A/D257A mutator mouse (Polg mut/mut ). These mice express an error‐prone mitochondrial DNA polymerase that results in the accumulation of mtDNA mutations, accelerated aging, and premature death. In this paper, we have used the Polg mut/mut model to investigate whether the age‐related biological effects observed in these mice are triggered by oxidative damage to the DNA that compromises the integrity of the genome. Our results show that mutator mouse has significantly higher levels of 8‐oxoguanine (8‐oxoGua) that are correlated with increased nuclear DNA (nDNA) strand breakage and oxidative nDNA damage, shorter average telomere length, and reduced mtDNA integrity. Based on these results, we propose a model whereby the increased level of reactive oxygen species (ROS) associated with the accumulation of mtDNA mutations in Polg mut/mut mice results in higher levels of 8‐oxoGua, which in turn lead to compromised DNA integrity and accelerated aging via increased DNA fragmentation and telomere shortening. These results suggest that mitochondrial play a central role in aging and may guide future research to develop potential therapeutics for mitigating aging process.

Mitochondrial DNA Is a Vital Driving Force in Ischemia-Reperfusion Injury in Cardiovascular Diseases.

According to the latest Global Burden of Disease Study, cardiovascular disease (CVD) is the leading cause of death, and ischemic heart disease and stroke are the cause of death in approximately half of CVD patients. In CVD, mitochondrial dysfunction following ischemia-reperfusion (I/R) injury results in heart failure. The proper functioning of oxidative phosphorylation (OXPHOS) and the mitochondrial life cycle in cardiac mitochondria are closely related to mitochondrial DNA (mtDNA). Following myocardial I/R injury, mitochondria activate multiple repair and clearance mechanisms to repair damaged mtDNA. When these repair mechanisms are insufficient to restore the structure and function of mtDNA, irreversible mtDNA damage occurs, leading to mtDNA mutations. Since mtDNA mutations aggravate OXPHOS dysfunction and affect mitophagy, mtDNA mutation accumulation leads to leakage of mtDNA and proteins outside the mitochondria, inducing an innate immune response, aggravating cardiovascular injury, and leading to the need for external interventions to stop or slow the disease course. On the other hand, mtDNA released into the circulation after cardiac injury can serve as a biomarker for CVD diagnosis and prognosis. This article reviews the pathogenic basis and related research findings of mtDNA oxidative damage and mtDNA leak-triggered innate immune response associated with I/R injury in CVD and summarizes therapeutic options that target mtDNA.

Detecting respiratory chain defects in osteoblasts from osteoarthritic patients using imaging mass cytometry.

Osteoporosis is a skeletal disease which is characterised by reduced bone mass and microarchitecture, with a subsequent loss of strength that predisposes to fragility and risk of fractures. The pathogenesis of falling bone mineral density, ultimately leading to a diagnosis of osteoporosis is incompletely understood but the disease is currently thought to be multifactorial. Humans are known to accumulate mitochondrial mutations and respiratory chain deficiency with age and mounting evidence suggests that this may indeed be the overarching cause intrinsic to the changing phenotype in advancing age and age-related disease. Mitochondrial mutations are detectable from the age of about 30years onwards. Mitochondria contain their own genome which encodes 13 essential mitochondrial proteins and accumulates somatic variants at up to 10 times the rate of the nuclear genome. Once the concentration of any pathogenic mitochondrial genome variant exceeds a threshold, respiratory chain deficiency and cellular dysfunction occur. The PolgD257A/D257A mouse model is a knock-in mutant that expresses a proof-reading-deficient version of PolgA, a nuclear encoded subunit of mtDNA polymerase. These mice are a useful model of age-related accumulation of mtDNA mutations in humans since their defective proof-reading mechanism leads to a mitochondrial DNA mutation rate 3-5 times higher than in wild-type mice. These mice showed enhanced levels of age-related osteoporosis along with respiratory chain deficiency in osteoblasts. To explore whether respiratory chain deficiency is also seen in human osteoblasts, we developed a protocol and analysis framework for imaging mass cytometry in bone tissue sections to analyse osteoblasts in situ. By comparing bone tissue sampled at one timepoint from femoral neck of 10 older healthy volunteers aged 40-85 with samples from young patients aged 1-19, we have identified complex I defect in osteoblasts from 6 out of 10 older volunteers, complex II defect in 2 out of 10 older volunteers, complex IV defect in 1 out of 10 older volunteers and complex V defect in 4 out of 10 older volunteers. These observations are consistent with findings from the PolgD257A/D257A mouse model and suggest that respiratory chain deficiency, as a consequence of the accumulation of age-related pathogenic mitochondrial DNA mutations, may play a significant role in the pathogenesis of human age-related osteoporosis.

Mitochondrial genome and aging of cardiomyocytes

The review presents data on the importance of mitochondrial DNA in aging of cardiomocytes. The mechanisms of accumulation of mutations in mtDNA and reduction of its content, as well as the consequences of these phenomena in cardiomyocytes are described. The similarity of the aging processes of cardiomyocytes and skeletal muscle cells and comparison with the aging processes occurring in mononuclear cells of peripheral blood is indicated. The death of cardiomyocytes and skeletal muscle cells leads to the destruction of mutant forms of mtDNA, as a result of which the content of mutant forms of mtDNA, constantly increasing with age, does not exceed 1-2% of the total number of mtDNA molecules. In addition, the death of cardiomyocytes and myocytes is accompanied by the release of CpG-motive cells mtDNA, which can cause local and general inflammation in old age. It is concluded, that in the treatment of elderly patients it is desirable to take into account the degree of aging ("biological age”) of their myocardial and their presence of chronic myocarditis, for which appropriate diagnostic methods should be developed.

Mitochondrial DNA Mutagenesis: Feature of and Biomarker for Environmental Exposures and Aging.

Mitochondrial dysfunction is a hallmark of aging. Mitochondrial genome (mtDNA) instability contributes to mitochondrial dysfunction, and mtDNA mutagenesis may contribute to aging. However, the origin of mtDNA mutations remains somewhat controversial. The goals of this review are to introduce and review recent literature on mtDNA mutagenesis and aging, address recent animal and epidemiological evidence for the effects of chemicals on mtDNA damage and mutagenesis, propose hypotheses regarding the contribution of environmental toxicant exposure to mtDNA mutagenesis in the context of aging, and suggest future directions and approaches for environmental health researchers. Stressors such as pollutants, pharmaceuticals, and ultraviolet radiation can damage the mitochondrial genome or disrupt mtDNA replication, repair, and organelle homeostatic processes, potentially influencing the rate of accumulation of mtDNA mutations. Accelerated mtDNA mutagenesis could contribute to aging, diseases of aging, and sensitize individuals with pathogenic mtDNA variants to stressors. We propose three potential mechanisms of toxicant-induced effects on mtDNA mutagenesis over lifespan: (1) increased de novo mtDNA mutations, (2) altered frequencies of mtDNA mutations, or (3) both. There are remarkably few studies that have investigated the impact of environmental chemical exposures on mtDNA instability and mutagenesis, and even fewer in the context of aging. More studies are warranted because people are exposed to tens of thousands of chemicals, and are living longer. Finally, we suggest that toxicant-induced mtDNA damage and mutational signatures may be a sensitive biomarker for some exposures.

The role of mitophagy during oocyte aging in human, mouse, and Drosophila: implications for oocyte quality and mitochondrial disease.

There is a worldwide trend for women to have their first pregnancy later in life. However, as oocyte quality declines with maternal aging, this trend leads to an increase in subfertility. The cellular mechanisms underlying this decline in oocyte competence are poorly understood. Oocyte mitochondria are the subcellular organelles that supply the energy that drives early embryogenesis, and thus their quality is critical for successful conception. Mitochondria contain their own DNA (mtDNA) and mutations in mtDNA cause mitochondrial diseases with severe symptoms, such as neurodegeneration and heart disease. Since mitochondrial function declines in tissues as humans age accompanied by an accumulation of mtDNA mutations, mtDNA is implicated as a cause of declining oocyte quality in older mothers. While this mutation load could be caused by declining accuracy of the mitochondrial replisome, age-related decline in mitochondrial quality control likely contributes, however knowledge is lacking. Mitophagy, a cellular process which specifically targets and recycles damaged mitochondria may be involved, but studies are scarce. And although assisted reproductive technologies can help older mothers, how these techniques affect the mechanisms that regulate mitochondrial and oocyte quality have not been studied. With the long-term goal of understanding the molecular mechanisms that control mitochondrial quality in the oocyte, model systems including Drosophila and mouse as well as human oocytes have been used. In this review, we explore the contribution of mitophagy to oocyte quality and the need for further systematic investigation in oocytes during maternal aging using different systems.Lay summaryMitochondria are small parts of cells called organelles that generate the chemical energy needed for life. Hundreds of thousands of mitochondria in the developing eggs of the mother support the initial growth and development of the fertilized egg. However, due to increasingly diminished function over time, mitochondria generate less energy as we age, posing real problems for older women considering pregnancy. It is possible that this declining energy could be responsible for declining fertility as women age. Energy may decline because mitochondria fail and the cell’s way of keeping them healthy become less efficient as we age. This review summarizes what is known about mitochondrial quality control in developing eggs as they age. In the future, understanding how the best mitochondria are selected and maintained in the egg, and hence the future baby, may enable older women with or without mitochondrial problems, to have healthy children.

Mitochondrial Dysfunction in Vascular Wall Cells and Its Role in Atherosclerosis.

Altered mitochondrial function is currently recognized as an important factor in atherosclerosis initiation and progression. Mitochondrial dysfunction can be caused by mitochondrial DNA (mtDNA) mutations, which can be inherited or spontaneously acquired in various organs and tissues, having more or less profound effects depending on the tissue energy status. Arterial wall cells are among the most vulnerable to mitochondrial dysfunction due to their barrier and metabolic functions. In atherosclerosis, mitochondria cause alteration of cellular metabolism and respiration and are known to produce excessive amounts of reactive oxygen species (ROS) resulting in oxidative stress. These processes are involved in vascular disease and chronic inflammation associated with atherosclerosis. Currently, the list of known mtDNA mutations associated with human pathologies is growing, and many of the identified mtDNA variants are being tested as disease markers. Alleviation of oxidative stress and inflammation appears to be promising for atherosclerosis treatment. In this review, we discuss the role of mitochondrial dysfunction in atherosclerosis development, focusing on the key cell types of the arterial wall involved in the pathological processes. Accumulation of mtDNA mutations in isolated arterial wall cells, such as endothelial cells, may contribute to the development of local inflammatory process that helps explaining the focal distribution of atherosclerotic plaques on the arterial wall surface. We also discuss antioxidant and anti-inflammatory approaches that can potentially reduce the impact of mitochondrial dysfunction.

Complementary Square-Wave Voltammetry and LC-MS/MS Analysis to Elucidate Induced Damaged and Mutated Mitochondrial and Nuclear DNA from in Vivo Knockdown of the BRCA1 Gene in the Mouse Skeletal Muscle.

Breast cancer 1 gene (BRCA1) DNA mutations impact skeletal muscle functions. Inducible skeletal muscle specific Brca1 homozygote knockout (Brca1KOsmi, KO) mice accumulate mitochondrial DNA (mtDNA) mutations resulting in loss of muscle quality.1 Complementary electrochemical andmass spectrometry analyses were utilized to rapidly assess mtDNA or nuclear DNA (nDNA) extracted directly from mouse skeletal muscles. Oxidative peak currents (Ip) from DNA immobilized layer by layer (LbL) were monitored using square-wave voltammetry (SWV) via Ru(bpy)32+ electrocatalysis. Ip significantly decreased (p < 0.05) for KO mtDNA compared to heterozygous KO (Het) or wild type (WT), indicative of decreases in the guanine content. nDNA Ip significantly increased in KO compared to WT (p < 0.05), suggesting an accumulation of damaged nDNA. Guanine or oxidatively damaged guanine content was monitored via appropriate m/z mass transitions using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Guanine in both KO mtDNA and nDNA was significantly lower, while oxidatively damaged guanine in KO nDNA was significantly elevated versus WT. These data demonstrate a loss of guanine content consistent with mtDNA mutation accumulation. Oxidative damage in KO nDNA suggests that repair processes associated with Brca1 are impacted. Overall, electrochemical and LC-MS/MS analysis can provide chemical-level answers to biological model phenotypic responses as a rapid and cost-effective analysis alternative to established assays.

A Slc25a46 Mouse Model Simulating Age-Associated Motor Deficit, Redox Imbalance, and Mitochondria Dysfunction.

The mitochondrial theory of aging postulates that accumulation of mtDNA mutations and mitochondrial dysfunction are responsible for producing aging phenotypes. To more comprehensively explore the complex relationship between aging and mitochondria dysfunction, we have developed a mouse model with Slc25a46 knockout, a nuclear gene described as encoding mitochondrial carriers, by CRISPR/Cas9 gene editing to mimic some typical aging phenotypes in human. Slc25a46-/- mice present segmental premature aging phenotypes characterized by shortened life span of no more than 2 months, obviously defective motor ability, gastrocnemius muscle atrophy, and imbalance of redox level in brain and liver. The underlying mechanism for multiple organ disorder may attribute to mitochondrial dysfunction, which is mainly manifested in the damaged mitochondrial structure (eg, vacuolar structure, irregular swelling, and disorganized cristae) and an age-associated decrease in respiratory chain enzyme (mainly complex I and IV) activity. In summary, our study suggests that the Slc25a46-/- mouse is a valid animal model for segmental aging-related pathologies studies based on mitochondrial theory, generating a new platform to both understand mechanisms between aging and mitochondria dysfunction as well as to design mitochondria-based therapeutic strategies to improve mitochondrial quality, and thereby the overall healthspan.

The Origin of Mitochondria and their Role in the Evolution of Life and Human Health

In studies of many medical and biological problems, there is a clear underestimation of the fundamental role of mitochondria in the evolution of eukaryotic organisms on the planet, including fungi, plants and fauna. It is important to take into consideration numerous fundamental functions of mitochondria when studying the human physiology and pathology, aging mechanisms. In this lecture, we briefly discuss the origin of mitochondria and their importance in the emergence of plants, which appeared on the planet 1–1.5 billion years later than eukaryotes with mitochondria, and served as the food basis for the rapid evolution of all species of the animal world. In the course of transformation of protobacteria into mitochondria, approximately 1000–1500 genes were transferred to the nucleus of eukaryotes, and the remaining 37 mitochondrial genes (mtDNA) exist in all types of animals. The presence of mitochondria in eukaryotes led to increased production of reactive oxygen species and accelerated mutations in mtDNA. The spread of sexual reproduction may have been the way of protection against the accumulation of harmful mutations in mtDNA. At the same time, in all animal species mtDNA is inherited only maternally. In humans, the maternal inheritance of mtDNA led to uneven distribution, in small or inbred populations in particular, of a number of diseases with large prevalence in men, as well as male infertility, and in general accelerated aging and shorter lifespan in men, as compared with women. These negative consequences of the maternal inheritance of mtDNA were termed as “mother’s curse”. Recent studies have shown that mtDNA mutations are not the cause of human aging. The crisis of the mitochondrial free radical theory of aging is largely associated with the neglect of the protonated form of the superoxide radical, the perhydroxyl radical, which activates the peroxidation of polyunsaturated fatty acids in mitochondrial phospholipids. This results in production of various biologically active molecules and toxins. Therefore, initially aging may lack the manifestation of specific symptoms because of gradual accumulation of a wide variety of disorders and damages to the structure and functions of mitochondria and cells that finally lead to the development of diseases. The pathologies appear primarily in those organs that have a wide range of functional activity and a high dependence on oxygen consumption, namely the heart, brain, skeletal muscles and vascular epithelium.

Unbiased PCR-free spatio-temporal mapping of the mtDNA mutation spectrum reveals brain region-specific responses to replication instability

BackgroundThe accumulation of mtDNA mutations in different tissues from various mouse models has been widely studied especially in the context of mtDNA mutation-driven ageing but has been confounded by the inherent limitations of the most widely used approaches. By implementing a method to sequence mtDNA without PCR amplification prior to library preparation, we map the full unbiased mtDNA mutation spectrum across six distinct brain regions from mice.ResultsWe demonstrate that ageing-induced levels of mtDNA mutations (single nucleotide variants and deletions) reach stable levels at 50 weeks of age but can be further elevated specifically in the cortex, nucleus accumbens (NAc), and paraventricular thalamic nucleus (PVT) by expression of a proof-reading-deficient mitochondrial DNA polymerase, PolgD181A. The increase in single nucleotide variants increases the fraction of shared SNVs as well as their frequency, while characteristics of deletions remain largely unaffected. In addition, PolgD181A also induces an ageing-dependent accumulation of non-coding control-region multimers in NAc and PVT, a feature that appears almost non-existent in wild-type mice.ConclusionsOur data provide a novel view of the spatio-temporal accumulation of mtDNA mutations using very limited tissue input. The differential response of brain regions to a state of replication instability provides insight into a possible heterogenic mitochondrial landscape across the brain that may be involved in the ageing phenotype and mitochondria-associated disorders.

Mitochondrial DNA mutation exacerbates female reproductive aging via impairment of the NADH/NAD+ redox.

Mammals' aging is correlated with the accumulation of somatic heteroplasmic mitochondrial DNA (mtDNA) mutations. Whether and how aging accumulated mtDNA mutations modulate fertility remains unknown. Here, we analyzed oocyte quality of young (≤30 years old) and elder (≥38 years old) female patients and show the elder group had lower blastocyst formation rate and more mtDNA point mutations in oocytes. To test the causal role of mtDNA point mutations on infertility, we used polymerase gamma (POLG) mutator mice. We show that mtDNA mutation levels inversely correlate with fertility, interestingly mainly affecting not male but female fertility. mtDNA mutations decrease female mice's fertility by reducing ovarian primordial and mature follicles. Mechanistically, accumulation of mtDNA mutations decreases fertility by impairing oocyte's NADH/NAD+ redox state, which could be rescued by nicotinamide mononucleotide treatment. For the first time, we answer the fundamental question of the causal effect of age‐accumulated mtDNA mutations on fertility and its sex dependence, and show its distinct metabolic controlling mechanism.