The pathophysiology of mitochondrial disease as modeled in the mouse

It is now clear that mitochondrial defects are associated with a plethora of clinical phenotypes in man and mouse. This is the result of the mitochondria''s central role in energy production, reactive oxygen species (ROS) biology, and apoptosis, and because the mitochondrial genome consists of roughly 1500 genes distributed across the maternal mitochondrial DNA (mtDNA) and the Mendelian nuclear DNA (nDNA). While numerous pathogenic mutations in both mtDNA and nDNA mitochondrial genes have been identified in the past 21 years, the causal role of mitochondrial dysfunction in the common metabolic and degenerative diseases, cancer, and aging is still debated. However, the development of mice harboring mitochondrial gene mutations is permitting demonstration of the direct cause-and-effect relationship between mitochondrial dysfunction and disease. Mutations in nDNA-encoded mitochondrial genes involved in energy metabolism, antioxidant defenses, apoptosis via the mitochondrial permeability transition pore (mtPTP), mitochondrial fusion, and mtDNA biogenesis have already demonstrated the phenotypic importance of mitochondrial defects. These studies are being expanded by the recent development of procedures for introducing mtDNA mutations into the mouse. These studies are providing direct proof that mtDNA mutations are sufficient by themselves to generate major clinical phenotypes. As more different mtDNA types and mtDNA gene mutations are introduced into various mouse nDNA backgrounds, the potential functional role of mtDNA variation in permitting humans and mammals to adapt to different environments and in determining their predisposition to a wide array of diseases should be definitively demonstrated.     

Increased mitochondrial biogenesis in muscle improves aging phenotypes in the mtDNA mutator mouse

Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations.     

Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis,aging and age-related diseases

As regulators of bioenergetics in the cell and the primary source of endogenous reactive oxygen species (ROS), dysfunctional mitochondria have been implicated for decades in the process of aging and age-related diseases. Mitochondrial DNA (mtDNA) is replicated and repaired by nuclear-encoded mtDNA polymerase γ (Pol γ) and several other associated proteins, which compose the mtDNA replication machinery. Here, we review evidence that errors caused by this replication machinery and failure to repair these mtDNA errors results in mtDNA mutations. Clonal expansion of mtDNA mutations results in mitochondrial dysfunction, such as decreased electron transport chain (ETC) enzyme activity and impaired cellular respiration. We address the literature that mitochondrial dysfunction, in conjunction with altered mitochondrial dynamics, is a major driving force behind aging and age-related diseases. Additionally, interventions to improve mitochondrial function and attenuate the symptoms of aging are examined.     

Gene expression profile in dilated cardiomyopathy caused by elevated frequencies of mitochondrial DNA mutations in the mouse heart.

BACKGROUND: Elevated mitochondrial DNA (mtDNA) mutations are associated with aging and age-related diseases, but their pathogenic potential is unclear. METHODS: We performed expression profiling using an Incyte cDNA array of a mouse model of elevated mtDNA mutations wherein random mutations accumulate specifically in the heart. At frequencies of about 1 mutation/10,000 base pairs, these mice show apoptosis of cardiomyocytes and development of four-chamber dilated cardiomyopathy. RESULTS: Significant Analysis of Microarrays (SAM) revealed that 117 genes were altered in their expression in the transgenic (Tg) heart at a threshold of less than one false positive, of which 34 were up-regulated and 83 were down-regulated. Some of the changes were confirmed by Northern and Western blots. By classification of these genes into functional categories, we identified changes that reflected cardiac pathology. The results indicated that cardiomyopathy caused by mtDNA mutations was largely characterized by gene expression changes indicative of increased fibrosis and cardiac remodeling of the extracellular matrix. Few changes were observed, suggesting an alteration in either mitochondrial energy production or generation of increased oxidative stress. CONCLUSIONS: Elevated frequencies of mtDNA mutations in the mouse heart lead to gene expression changes that are associated with remodeling of the extracellular matrix. Because cardiomyocytic death by apoptosis is also a feature of the dilated cardiomyopathy evident in these mice, extracellular remodeling may be a response to apoptotic signaling originating from the mitochondria with mtDNA mutations.     

Dominant membrane uncoupling by mutant adenine nucleotide translocase in mitochondrial diseases

Adenine nucleotide translocase (Ant) is the most abundant protein on the mitochondrial inner membrane (MIM) primarily involved in ADP/ATP exchange. Ant also possesses a discrete membrane uncoupling activity. Specific mis-sense mutations in the human Ant1 cause autosomal dominant Progressive External Ophthalmoplegia (adPEO), mitochondrial myopathy and cardiomyopathy, which are commonly manifested by fractional mitochondrial DNA (mtDNA) deletions. It is currently thought that the pathogenic mutations alter substrate preference (e.g. ATP versus ADP) thereby dominantly disturbing adenine nucleotide homeostasis in mitochondria. This may interfere with mtDNA replication, consequently affecting mtDNA stability and oxidative phosphorylation. Here, we showed that the adPEO-type A128P, A106D and M114P mutations in the yeast Aac2p share the following common dominant phenotypes: electron transport chain damage, intolerance to moderate over-expression, synthetic lethality with low Deltapsi(m) conditions, hypersensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and mtDNA instability. More interestingly, the aac2(A137D) allele mimicking ant1(A123D) in mitochondrial myopathy and cardiomyopathy exhibits similar dominant phenotypes. Because Aac2(A137D) is known to completely lack transport activity, it is strongly argued that the dominant mitochondrial damages are not caused by aberrant nucleotide transport. The four pathogenic mutations occur in a structurally dynamic gating region on the cytosolic side. We provided direct evidence that the mutant alleles uncouple mitochondrial respiration. The pathogenic mutations likely enhance the intrinsic proton-conducting activity of Ant, which excessively uncouples the MIM thereby affecting energy transduction and mitochondrial biogenesis. mtDNA disintegration is a phenotype co-lateral to mitochondrial damages. These findings provide mechanistic insights into the pathogenesis of the Ant1-induced diseases.     

Age-dependent accumulation of mtDNA mutations in murine hematopoietic stem cells is modulated by the nuclear genetic background

Alterations in mitochondrial DNA (mtDNA) and consequent loss of mitochondrial function underlie the mitochondrial theory of aging. In this study, we systematically analyzed the mtDNA control region somatic mutation pattern in 2864 single hematopoietic stem cells (HSCs) and progenitors, isolated by flow cytometry sorting on Lin(-)Kit(+)CD34(-) parameters from young and old C57BL/6 (B6) and BALB/cBy (BALB) mice, to test the hypothesis that the accumulated mtDNA mutations in HSCs were strain-correlated and associated with HSC functional senescence during aging. An increased level of mtDNA mutations in single HSCs was observed in old B6 when compared with young B6 mice (P=0.003); in contrast, no significant age-dependent accumulation of mutations was observed in BALB mice (old versus young, P=0.202) and the level of mutations in both young and old BALB mice was close to that of old B6 mice (P>0.280). Cellular reactive oxygen species (ROS) in mouse HSCs could not be correlated with the level of mtDNA mutations in these cells, although B6 mice had a higher proportion of ROS(-) cells when compared with the BALB mice. Propagation assays of single HSCs showed B6 cells form larger colonies compared with cells from BALB mice, irrespective of age and mtDNA mutation load. We infer from our data that age-related mtDNA somatic mutation accumulation in mouse HSCs is influenced by the nuclear genetic background and that these mutations may not obviously correlate to either cellular ROS content or HSC senescence.     

A national perspective on prenatal testing for mitochondrial disease

Mitochondrial diseases affect >1 in 7500 live births and may be due to mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). Genetic counselling for families with mitochondrial diseases, especially those due to mtDNA mutations, provides unique and difficult challenges particularly in relation to disease transmission and prevention. We have experienced an increasing demand for prenatal diagnostic testing from families affected by mitochondrial disease since we first offered this service in 2007. We review the diagnostic records of the 62 prenatal samples (17 mtDNA and 45 nDNA) analysed since 2007, the reasons for testing, mutation investigated and the clinical outcome. Our findings indicate that prenatal testing for mitochondrial disease is reliable and informative for the nuclear and selected mtDNA mutations we have tested. Where available, the results of mtDNA heteroplasmy analyses from other family members are helpful in interpreting the prenatal mtDNA test result. This is particularly important when the mutation is rare or the mtDNA heteroplasmy is observed at intermediate levels. At least 11 cases of mitochondrial disease were prevented following prenatal testing, 3 of which were mtDNA disease. On the basis of our results, we believe that prenatal testing for mitochondrial disease is an important option for couples where appropriate genetic analyses and pre/post-test counselling can be provided.     

A novel NDUFA1 mutation leads to a progressive mitochondrial complex I-specific neurodegenerative disease

Mitochondrial diseases have been shown to result from mutations in mitochondrial genes located in either the nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Mitochondrial OXPHOS complex I has 45 subunits encoded by 38 nuclear and 7 mitochondrial genes. Two male patients in a putative X-linked pedigree exhibiting a progressive neurodegenerative disorder and a severe muscle complex I enzyme defect were analyzed for mutations in the 38 nDNA and seven mtDNA encoded complex I subunits. The nDNA X-linked NDUFA1 gene (MWFE polypeptide) was discovered to harbor a novel missense mutation which changed a highly conserved glycine at position 32 to an arginine, shown to segregate with the disease. When this mutation was introduced into a NDUFA1 null hamster cell line, a substantial decrease in the complex I assembly and activity was observed. When the mtDNA of the patient was analyzed, potentially relevant missense mutations were observed in the complex I genes. Transmitochondrial cybrids containing the patient’s mtDNA resulted in a mild complex I deficiency. Interestingly enough, the nDNA encoded MWFE polypeptide has been shown to interact with various mtDNA encoded complex I subunits. Therefore, we hypothesize that the novel G32R mutation in NDUFA1 is causing complex I deficiency either by itself or in synergy with additional mtDNA variants.     

Neuropathologic aspects of cytochrome C oxidase deficiency

Cytochrome c oxidase (COX) deficiency is an important cause of myopathy or encephalomyopathy. Considering the structural complexity of COX, its dual genetic control, and the several nuclear genes needed for its proper assembly, the phenotypic heterogeneity is not surprising. From a morphologic view point, the application of histochemistry and immunohistochemistry to the study of COX deficiency in muscle has revealed specific patterns that -we believe- are helpful both for diagnosis and for directing sequencing studies of either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) genes. Similar studies in brain have shown that patients with mutations in mtDNA appear to have different patterns of COX deficiency from patients with mutations in nDNA genes. The recent discovery of mutations in COX assembly genes coupled with the potential to generate knock-out mice with these mutations holds the promise of providing the neuropathologist with the animal models needed to study the pathogenesis of COX deficiency in brain and muscle.     

Mitochondrial Encephalomyopathies: Defects of Nuclear DNA

The term "mitochondrial diseases" encompasses a heterogeneous group of disorders in which a primary mitochondrial dysfunction is suspected or proven by morphologic, genetic, or biochemical criteria. Clinically, these progressive disorders usually affect muscle, either alone (mitochondrial myopathies) or in combination with other systems, most often brain (encephalomyopathies). Mitochondria are unique among intracellular organelles in that mitochondrial proteins are encoded by two genomes, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). The vast majority of mitochondrial proteins are encoded by the nuclear genome, whereas mtDNA (a circular, double stranded 16.5 kb molecule) encodes only 13 polypeptides, all of them subunits of respiratory chain complexes. In addition to structural genes, mtDNA also codes for 22 transfer RNAs and two ribosomal RNAs. Our understanding of mitochondrial diseases has grown at an impressive rate in the past few years, and most of the progress has been in the area of mtDNA genetics, where several mtDNA mutations have been associated with specific diseases (reviewed in this issue by Zeviani et al.). In comparison, our understanding of mitochondrial disorders due to nDNA lesions has lagged behind and, to date, molecular defects of nuclear genes have been documented in only a few patients. We will review which alterations in the nuclear genome can cause mitochondrial disorders and which criteria are useful in identifying such mutations. While several examples will be provided, this is not intended as a complete review of the subject.     

Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells

Inheritance of one of three primary mutations at positions 11778, 3460 or 14484 of the mitochondrial genome in subunits of Complex I causes Leber's Hereditary Optic Neuropathy (LHON), a specific degeneration of the optic nerve, resulting in bilateral blindness. It has been unclear why inheritance of a systemic mitochondrial mutation would result in a specific neurodegeneration. To address the neuron-specific degenerative phenotype of the LHON genotype, we have created cybrids using a neuronal precursor cell line, Ntera 2/D1 (NT2), containing mitochondria from patient lymphoblasts bearing the most common LHON mutation (11778) and the most severe LHON mutation (3460). The undifferentiated LHON-NT2 mutant cells were not significantly different from the parental cell control in terms of mtDNA/nDNA ratio, mitochondrial membrane potential, reactive oxygen species (ROS) production or the ability to reduce Alamar Blue. Differentiation of NT2s resulted in a neuronal morphology and neuron-specific pattern of gene expression, and a 3-fold reduction in mtDNA/nDNA ratio in both mutant and control cells; however, the differentiation protocol yielded significantly less LHON cells than controls, by 30%, indicating either a decreased proliferative potential or increased cell death of the LHON-NT2 cells. Differentiation of the cells to the neuronal form also resulted in significant increases in ROS production in the LHON-NT2 neurons versus controls, which is abolished by rotenone, a specific inhibitor of Complex I. We infer that the LHON genotype requires a differentiated neuronal environment in order to induce increased mitochondrial ROS, which may be the cause of the reduced NT2 yield; and suggest that the LHON degenerative phenotype may be the result of an increase in mitochondrial superoxide which is caused by the LHON mutations, possibly mediated through neuron-specific alterations in Complex I structure.     

Mitochondrial genotype associated with longevity and its inhibitory effect on mutagenesis

Mitochondria are not only the major site of ATP production in cells but also an important source of reactive oxygen species (ROS) under certain pathological conditions. Because mitochondrial DNA (mtDNA) in the mitochondrial matrix is exposed to ROS that leak from the respiratory chain, this extranuclear genome is prone to mutations. Therefore, the mitochondrial genome is a rich source of single nucleotide polymorphisms (SNPs) and the functional significance of SNPs in the mitochondrial genome is comparable to that of SNPs in the entire nuclear genome. To demonstrate the contribution of mitochondrial SNPs to the susceptibility to adult-onset diseases, we analyzed the mtDNA from Japanese centenarians and identified a longevity-associated mitochondrial genotype, Mt5178A. Because this genotype was demonstrated to suppress the occurrence of mtDNA mutations in the oocytes, it also would seem to decelerate the accumulation of mtDNA mutations in the somatic cells with increasing age. This genotype is likely to confer resistance to adult-onset diseases by suppressing obesity and atherosclerosis.     

Mitochondrial medicine--molecular pathology of defective oxidative phosphorylation

Different tissues display distinct sensitivities to defective mitochondrial oxidative phosphorylation (OXPHOS). Tissues highly dependent on oxygen such as the cardiac muscle, skeletal and smooth muscle, the central and peripheral nervous system, the kidney, and the insulin-producing pancreatic beta-cell are especially susceptible to defective OXPHOS. There is evidence that defective OXPHOS plays an important role in atherogenesis, in the pathogenesis of Alzheimer's disease, Parkinson's disease, diabetes, and aging. Defective OXPHOS may be caused by abnormal mitochondrial biosynthesis due to inherited or acquired mutations in the nuclear (n) or mitochondrial (mt) deoxyribonucleic acid (DNA). For instance, the presence of a mutation of the mtDNA in the pancreatic beta-cell impairs adenosine triphosphate (ATP) generation and insulin synthesis. The nuclear genome controls mitochondrial biosynthesis, but mtDNA has a much higher mutation rate than nDNA because it lacks histones and is exposed to the radical oxygen species (ROS) generated by the electron transport chain, and the mtDNA repair system is limited. Defective OXPHOS may be caused by insufficient fuel supply, by defective electron transport chain enzymes (Complexes I - IV), lack of the electron carrier coenzyme Q10, lack of oxygen due to ischemia or anemia, or excessive membrane leakage, resulting in insufficient mitochondrial inner membrane potential for ATP synthesis by the F0F1-ATPase. Human tissues can counteract OXPHOS defects by stimulating mitochondrial biosynthesis; however, above a certain threshold the lack of ATP causes cell death. Many agents affect OXPHOS. Several nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit or uncouple OXPHOS and induce the 'topical' phase of gastrointestinal ulcer formation. Uncoupled mitochondria reduce cell viability. The Helicobacter pylori induces uncoupling. The uncoupling that opens the membrane pores can activate apoptosis. Cholic acid in experimental atherogenic diets inhibits Complex IV, cocaine inhibits Complex I, the poliovirus inhibits Complex II, ceramide inhibits Complex III, azide, cyanide, chloroform, and methamphetamine inhibit Complex IV. Ethanol abuse and antiviral nucleoside analogue therapy inhibit mtDNA replication. By contrast, melatonin stimulates Complexes I and IV and Gingko biloba stimulates Complexes I and III. Oral Q10 supplementation is effective in treating cardiomyopathies and in restoring plasma levels reduced by the statin type of cholesterol-lowering drugs.     

A new mtDNA mutation in the tRNALeu(UUR) gene associated with maternally inherited cardiomyopathy

We report a new mutation, a C to T transition at nt 3303 of mtDNA, in seven members of a family with cardiomyopathy and myopathy: the proband and two siblings had fatal infantile cardiomyopathy, whereas in three maternal relatives the disease manifested later in life as sudden cardiac death or as mitochondrial myopathy with cardiomyopathy. The mutation was homoplasmic in all tissues (including blood) from the proband and her brother, but heteroplasmic in blood from five oligosymptomatic or asymptomatic maternal relatives. This mutation disrupts a conserved base pair in the aminoacyl stem of the tRNA^Leu(UUR). None of 70 controls carried the mutation. Our data indicate that this mutation is the genetic cause of the disorder in this family, and confirm that the tRNA^Leu(UUR) is a “hot spot” for mutations in mtDNA. © 1994 Wiley-Liss, Inc.     

Homozygous mutations in C1QBP as cause of progressive external ophthalmoplegia (PEO) and mitochondrial myopathy with multiple mtDNA deletions

Biallelic mutations in the C1QBP gene have been associated with mitochondrial cardiomyopathy and combined respiratory‐chain deficiencies, with variable onset (including intrauterine or neonatal forms), phenotypes, and severity. We studied two unrelated adult patients from consanguineous families, presenting with progressive external ophthalmoplegia (PEO), mitochondrial myopathy, and without any heart involvement. Muscle biopsies from both patients showed typical mitochondrial alterations and the presence of multiple mitochondrial DNA deletions, whereas biochemical defects of the respiratory chain were present only in one subject. Using next‐generation sequencing approaches, we identified homozygous mutations in C1QBP. Immunoblot analyses in patients' muscle samples revealed a strong reduction in the amount of the C1QBP protein and varied impairment of respiratory chain complexes, correlating with disease severity. Despite the original study indicated C1QBP mutations as causative for mitochondrial cardiomyopathy, our data indicate that mutations in C1QBP have to be considered in subjects with PEO phenotype or primary mitochondrial myopathy and without cardiomyopathy.     

High aggregate burden of somatic mtDNA point mutations in aging and Alzheimer's disease brain.

The mitochondrial theory of aging proposes that mitochondrial DNA (mtDNA) accumulates mutations with age, and that these mutations contribute to physiological decline in aging and degenerative diseases. Although a great deal of indirect evidence supports this hypothesis, the aggregate burden of mtDNA mutations, particularly point mutations, has not been systematically quantified in aging or neurodegenerative disorders. Therefore, we directly assessed the aggregate burden of brain mtDNA point mutations in 17 subjects with Alzheimer's disease (AD), 10 elderly control subjects and 14 younger control subjects, using a PCR-cloning-sequencing strategy. We found that brain mtDNA from elderly subjects had a higher aggregate burden of mutations than brain mtDNA from younger subjects. The average aggregate mutational burden in elderly subjects was 2 x 10(-4) mutations/bp. The bulk of these mutations were individually rare point mutations, 60% of which changed an amino acid. Control experiments ensure that these results were not due to artifacts arising from PCR error, mistaken identification of nuclear pseudogenes or ex vivo oxidation. Cytochrome oxidase activity correlated negatively with increasing mutational burden. These findings significantly bolster the mitochondrial theory of aging.     

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L-L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination.     

Direct repeats in mitochondrial DNA and mammalian lifespan

Mitochondria have long been suspected to be among the leading determinants of aging due to their functional importance and accelerated deterioration caused by accumulation of mutations in the mitochondrial DNA. Direct repeats are known to contribute to deletion formation in mtDNA and are a powerful source of reactive oxygen species (ROS)-independent mutagenesis. To evaluate the potential importance of homology-based deletion formation, we have analyzed the association between direct repeats in the mtDNA sequence and the lifespans of 65 mammalian species. Here, we report a significant negative correlation between the mutagenic potential of direct repeats and the mammalian lifespan, which is especially evident in closely related species.