Mitochondria: Biogenesis and mitophagy balance in segregation and clonal expansion of mitochondrial DNA mutations

Mitochondria are cytoplasmic organelles containing their own multi-copy genome. They are organized in a highly dynamic network, resulting from balance between fission and fusion, which maintains homeostasis of mitochondrial mass through mitochondrial biogenesis and mitophagy. Mitochondrial DNA (mtDNA) mutates much faster than nuclear DNA. In particular, mtDNA point mutations and deletions may occur somatically and accumulate with aging, coexisting with the wild type, a condition known as heteroplasmy. Under specific circumstances, clonal expansion of mutant mtDNA may occur within single cells, causing a wide range of severe human diseases when mutant overcomes wild type. Furthermore, mtDNA deletions accumulate and clonally expand as a consequence of deleterious mutations in nuclear genes involved in mtDNA replication and maintenance, as well as in mitochondrial fusion genes (mitofusin-2 and OPA1), possibly implicating mtDNA nucleoids segregation. We here discuss how the intricacies of mitochondrial homeostasis impinge on the intracellular propagation of mutant mtDNA.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Mitochondrial DNA replication and repair defects: Clinical phenotypes and therapeutic interventions

Mitochondria is a unique cellular organelle involved in multiple cellular processes and is critical for maintaining cellular homeostasis. This semi-autonomous organelle contains its circular genome – mtDNA (mitochondrial DNA), that undergoes continuous cycles of replication and repair to maintain the mitochondrial genome integrity. The majority of the mitochondrial genes, including mitochondrial replisome and repair genes, are nuclear-encoded. Although the repair machinery of mitochondria is quite efficient, the mitochondrial genome is highly susceptible to oxidative damage and other types of exogenous and endogenous agent-induced DNA damage, due to the absence of protective histones and their proximity to the main ROS production sites. Mutations in replication and repair genes of mitochondria can result in mtDNA depletion and deletions subsequently leading to mitochondrial genome instability. The combined action of mutations and deletions can result in compromised mitochondrial genome maintenance and lead to various mitochondrial disorders. Here, we review the mechanism of mitochondrial DNA replication and repair process, key proteins involved, and their altered function in mitochondrial disorders. The focus of this review will be on the key genes of mitochondrial DNA replication and repair machinery and the clinical phenotypes associated with mutations in these genes.     

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.     

Mitochondrial genome maintenance: roles for nuclear nonhomologous end-joining proteins in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.     

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.     

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.     

Specific mutations in alpha- and gamma-subunits of F1-ATPase affect mitochondrial genome integrity in the petite-negative yeast Kluyveromyces lactis.

We have shown previously that mutations in nuclear genes, termed MGI, for mitochondrial genome integrity, can convert the petite-negative yeast Kluyveromyces lactis into a petite-positive form with the ability to produce mitochondrial genome deletion mutants (Chen and Clark-Walker, Genetics, 133, 517-525, 1993). Here we describe that two genes, MGI2 and MGI5, encode the alpha- and gamma-subunits of mitochondrial F1-ATPase. Specific mutations, Phe443-->Ser and Ala333-->Val in MGI2, and Thr275-->Ala in MGI5, confer on cells the ability to produce petite mutants spontaneously with deletions in mitochondrial (mt) DNA and the capacity to lose their mitochondrial genomes upon treatment with ethidium bromide. Structural integrity of the F1 complex seems to be needed for expression of the Mgi- phenotype as null mutations in MGI2 and MGI5 remove the ability to form mtDNA deletions. It is suggested that mgi mutations allow petites to survive because an aberrant F1 complex prevents collapse of the mitochondrial inner membrane potential that normally occurs on loss of mtDNA-encoded F0 subunits.     

Cause or casualty: The role of mitochondrial DNA in aging and age-associated disease

The mitochondrial genome (mtDNA) represents a tiny fraction of the whole genome, comprising just 16.6?kilobases encoding 37 genes involved in oxidative phosphorylation and the mitochondrial translation machinery. Despite its small size, much interest has developed in recent years regarding the role of mtDNA as a determinant of both aging and age-associated diseases. A number of studies have presented compelling evidence for key roles of mtDNA in age-related pathology, although many are correlative rather than demonstrating cause. In this review we will evaluate the evidence supporting and opposing a role for mtDNA in age-associated functional declines and diseases. We provide an overview of mtDNA biology, damage and repair as well as the influence of mitochondrial haplogroups, epigenetics and maternal inheritance in aging and longevity.     

Variation of the mitochondrial genome in the evolution of Drosophila

The evidence on mitochondrial genome variation and its role in evolution of the genus Drosophila are reviewed. The mitochondrial genome is represented by a circular double-stranded DNA molecule 16 to 19 kb in length. The genome contains no introns involved in recombination. The entire mitochondrial genome can be arbitrarily divided into three parts: (1) protein-coding genes; (2) genes encoding rRNA and tRNA; and (3) the noncoding regulatory region (A + T region). The selective importance of mutations within different mtDNA regions is therefore unequal. In Drosophila, the content of the A + T pairs in mtDNA is extremely low and a pattern of nucleotide substitution is characterized by a low transition/transversion ratio (and a low threshold of mutation saturation). The deletions and duplications are of common occurrence in the mitochondrial genome. However, this genome lacks such characteristic for the nuclear genome aberrations as the inversions and transpositions. The phenomena of introgression and heteroplasmy provide an opportunity to study the adaptive role of the mitochondrial genome and its role in speciation. Analysis of evidence concerning mtDNA variation in different species of the genus Drosophila made it possible to ascertain data on phylogenetic relationships among species obtained by studying nuclear genome variation. In some species, mtDNA variation may serve as a reliable marker for population differentiation within a species, although evidence on the population dynamics of the mtDNA variation is very scarce.     

Changes in the human mitochondrial genome after treatment of malignant disease

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in human cells. The mitochondrial genome encodes essential information for the synthesis of the mitochondrial respiratory chain. Inherited defects of this genome are an important cause of human disease. In addition, the mitochondrial genome seems to be particularly prone to DNA damage and acquired mutations may have a role in ageing, cancer and neurodegeneration. We wished to determine if radiotherapy and chemotherapy used in the treatment of cancer could induce changes in the mitochondrial genome. Such changes would be an important genetic marker of DNA damage and may explain some of the adverse effects of treatment. We studied samples from patients who had received radiotherapy and chemotherapy for point mutations within the mtDNA control region, and for large-scale deletions. In blood samples from patients, we found a significantly increased number of point mutations compared to the control subjects. In muscle biopsies from 7 of 8 patients whom had received whole body irradiation as well as chemotherapy, the level of a specific mtDNA deletion was significantly greater than in control subjects. Our studies have shown that in patients who have been treated for cancer there is an increased level of mtDNA damage.     

Age-associated mitochondrial DNA deletions in mouse skeletal muscle: Comparison of different regions of the mitochondrial genome

The abundance of mitochondrial DNA (mtDNA) deletions has been shown to increase with age in a number of species and may contribute to the aging process. Estimating the total mtDNA deletion load of an individual is essential in evaluating the potential physiological impact. In this study, we compared three 5-kb regions of the mitochondrial genome: one in the major arc, one in the minor arc, and a third containing the light strand origin of replication. Through PCR analysis of mouse skeletal muscle, we have determined that not all regions produce equal numbers of age-associated deletions. There are, on average, twofold more detectable deletions in the major arc region than in the minor arc region. Deletions that result in the loss of the light strand origin of replication are rarely detected. Furthermore, the mechanism of deletion formation seems to be similar in both the major and minor arcs, with direct repeats playing an important, although not essential, role. © 1996 Wiley-Liss, Inc.     

Variation of the Mitochondral Genome in the Evolution of Drosophila

The evidence on mitochondrial genome variation and its role in evolution of the genus Drosophila are reviewed. The mitochondrial genome is represented by a circular double-stranded DNA molecule 16 to 19 kb in length. Mitochondrial genes lack introns and recombination. The entire mitochondrial genome can be arbitrarily divided into three parts: (1) protein-coding genes; (2) genes encoding rRNA and tRNA; and (3) the noncoding regulatory region (A + T region). The selective importance of mutations within different mtDNA regions is therefore unequal. In Drosophila, the content of the A + T pairs in mtDNA is extremely high and a pattern of nucleotide substitution is characterized by a low transition/transversion ratio (and a low threshold of mutation saturation). The deletions and duplications are of common occurrence in the mitochondrial genome. However, this genome lacks such characteristic for the nuclear genome aberrations as inversions and transpositions. The phenomena of introgression and heteroplasmy provide an opportunity to study the adaptive role of the mitochondrial genome and its role in speciation. Analysis of evidence concerning mtDNA variation in different species of the genus Drosophilamade it possible to ascertain data on phylogenetic relationships among species obtained by studying nuclear genome variation. In some species, mtDNA variation may serve as a reliable marker for population differentiation within a species, although evidence on the population dynamics of the mtDNA variation is very scarce.     

Next generation molecular diagnosis of mitochondrial disorders

Mitochondrial disorders are by far the most genetically heterogeneous group of diseases, involving two genomes, the 16.6 kb mitochondrial genome and ~ 1500 genes encoded in the nuclear genome. For maternally inherited mitochondrial DNA disorders, a complete molecular diagnosis requires several different methods for the detection and quantification of mtDNA point mutations and large deletions. For mitochondrial disorders caused by autosomal recessive, dominant, and X-linked nuclear genes, the diagnosis has relied on clinical, biochemical, and molecular studies to point to a group of candidate genes followed by stepwise Sanger sequencing of the candidate genes one-by-one. The development of Next Generation Sequencing (NGS) has revolutionized the diagnostic approach. Using massively parallel sequencing (MPS) analysis of the entire mitochondrial genome, mtDNA point mutations and deletions can be detected and quantified in one single step. The NGS approach also allows simultaneous analyses of a group of genes or the whole exome, thus, the mutations in causative gene(s) can be identified in one-step. New approaches make genetic analyses much faster and more efficient. Huge amounts of sequencing data produced by the new technologies brought new challenges to bioinformatics, analytical pipelines, and interpretation of numerous novel variants. This article reviews the clinical utility of next generation sequencing for the molecular diagnoses of complex dual genome mitochondrial disorders.     

EVOLUTION AND MITOCHONDRIAL DNA IN NEUROSPORA CRASSA

We studied mitochondrial DNA variability in 19 natural Neurospora crassa isolates and one wild-type isolate to examine evolution of these fungi and their mitochondrial DNA (mtDNA). We combined restriction endonuclease analysis of natural isolate mtDNA with DNA-DNA hybridization to cloned EcoR I fragments of a wild-type genome to discriminate between length mutations and site changes due to nucleotide substitution. Most variability was due to length mutations (insertions and deletions); genome size could vary 25% between pairs of isolates. Length-mutation distribution was not random, nor simply explained by the presence of coding versus noncoding regions. Restriction-site changes were few; the estimated amount of nucleotide substitution per nucleotide between the most divergent pair of isolates was 0.78%. Evolutionary relationships among isolates based on both types of mutations were compatible, and suggest that geographically distinct populations of mitochondrial DNA exist in the biological species, N. crassa. In contrast, no such correlation was shown by the previously determined distribution of nuclear heterokaryon incompatibility genes in the same isolates (Mylyk, 1975, 1976).     

Mutations in Mgi Genes Convert Kluyveromyces Lactis into a Petite-Positive Yeast

Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes.     

Mechanisms linking mtDNA damage and aging

In the past century, considerable efforts were made to understand the role of mitochondrial DNA (mtDNA) mutations and of oxidative stress in aging. The classic mitochondrial free radical theory of aging, in which mtDNA mutations cause genotoxic oxidative stress, which in turn creates more mutations, has been a central hypothesis in the field for decades. In the past few years, however, new elements have discredited this original theory. The major sources of mitochondrial DNA mutations seem to be replication errors and failure of the repair mechanisms, and the accumulation of these mutations as observed in aged organisms seems to occur by clonal expansion and not to be caused by a reactive oxygen species-dependent vicious cycle. New hypotheses of how age-associated mitochondrial dysfunction may lead to aging are based on the role of reactive oxygen species as signaling molecules and on their role in mediating stress responses to age-dependent damage. Here, we review the changes that mtDNA undergoes during aging and the past and most recent hypotheses linking these changes to the tissue failure observed in aging.     

Evidence that specific mtDNA point mutations may not accumulate in skeletal muscle during normal human aging.

It is unclear at present whether specific mtDNA point mutations accumulate during normal human aging. In order to address this question, we used quantitative PCR of total DNA isolated from skeletal muscle from normal individuals of various ages to search for the presence and amount of spontaneous mtDNA point mutations in two small regions of the human mitochondrial genome. We observed low levels of somatic mutations above background in both regions, but there was no correlation between the amount of mutation detected and the age of the subject. These results contrasted with our finding of an age-related increase in the amount of the mtDNA "common deletion" in these very samples. Thus, it appears that both somatic mtDNA point mutations and mtDNA deletions can arise at low frequency in normal individuals but that, unlike deletions, there is no preferential amplification or accumulation of specific point mutations in skeletal muscle over the course of the normal human life span.     

Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia.

The in vivo cellular impact of age-associated mitochondrial DNA mutations is unknown. We hypothesized that mitochondrial DNA deletion mutations contribute to the fiber atrophy and loss that cause sarcopenia, the age-related decline of muscle mass and function. We examined 82,713 rectus femoris muscle fibers from Fischer 344 x Brown Norway F1 hybrid rats of ages 5, 18, and 38 months through 1000 microns by serial cryosectioning and histochemical staining for cytochrome c oxidase and succinate dehydrogenase. Between 5 and 38 months of age, the rectus femoris muscle in the hybrid rat demonstrated a 33% decrease in mass concomitant with a 30% decrease in total fibers at the muscle mid-belly. We observed significant increases in the number of mitochondrial abnormalities with age from 289 +/- 8 ETS abnormal fibers in the entire 5-month-old rectus femoris to 1094 +/- 126 in the 38-month-old as calculated from the volume density of these abnormalities. Segmental mitochondrial abnormalities contained mitochondrial DNA deletion mutations as revealed by laser capture microdissection and whole mitochondrial genome amplification. Muscle fibers harboring mitochondrial deletions often displayed atrophy, splitting and increased steady-state levels of oxidative nucleic damage. These data suggest a causal role for age-associated mitochondrial DNA deletion mutations in sarcopenia.