Manual Mitochondrial Pathogenesis: From Genes and Apoptosis to Aging and Disease

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SOD1 is located in the mitochondrial intermembrane space and cytosol, SOD2 is located in the mitochondrial matrix, and SOD3 is tethered to the extracellular matrix. Catalase is found in peroxisomes. Therefore, it is possible that PRXs are critical for turning ROS signaling off, while GPXs are critical for buffering high levels of ROS to bring them to a level at which the cell evades damage and can initiate signaling stress responses.

Catalase has an even lower affinity for hydrogen peroxide. Regulation of activity and expression levels of these antioxidants occurs by a variety of mechanisms and functions in part to manage ROS levels. The mitochondrial free radical theory of aging postulates that the damage caused by accumulating ROS produced by mitochondria is the driving force behind aging This theory is corroborated to some extent by the inverse correlation between mitochondrial ROS production and lifespan in mammals Current evidence points to mitochondrial dysfunction as an overarching mechanism of aging and age-related disease.

It is implicated in an extensive list of aging pathologies such as cancer, intestinal barrier dysfunction, depression, chronic obstructive pulmonary disease COPD , diabetes, and others ROS are not the only aspect of flawed mitochondria that contributes to degenerating health, the emerging picture is that mitochondrial dysfunction in human aging and aging-associated diseases are not limited to accumulation of mtDNA mutations, but extend to abnormalities in mitochondria biogenesis, turnover, dynamics, and proteostasis. Mitochondria undergo constant fusion and fission to maintain a balance between mitochondrial biogenesis and mitochondrial autophagy mitophagy or apoptosis Several of the proteins involved in mitochondrial dynamics have been well characterized.

Mitofusin 1 Mfn1 , mitofusin 2 Mfn2 and optic atrophy-1 Opa1 are the major proteins involved in the mitochondrial fusion process. Dynamin related protein-1 Drp1 and fission protein 1 Fis1 are involved in the fission process. The fusion process is activated during conditions of increased mitochondrial bioenergetics. The fission process is activated during mitochondrial degradation through the autophagosome autophagy Thus, a deleterious feedback loop between ROS and dynamics leads to mitochondrial ROS dysfunction and subsequent apoptosis. Mitochondrial Sirtuins are modulators of energy metabolism, DNA repair and oxidative stress.

Sirt-1 is both a nuclear and cytoplasmic protein and has been observed in mitochon- dria, while Sirt-3, 4, and 5 are mitochondrial proteins Sirt-3 can also directly activate SOD2 activity by deacetylating it Interestingly, brain-specific overexpression of SIRT1 results in long-lived animals which, when they age, seem to have preserved mitochondrial morphology along with increased oxygen consumption and more robust physical activity Similarly, whole-body overexpression of SIRT6 can result in lifespan extension for male mice A vast body of data has accumulated linking mitochondrial redox metabolism to the aging process.

Similarly, a growing number of dietary interventions have been demonstrated to modulate mitochondrial ROS production, detoxification and oxidative damage repair. Many but not all of these dietary interventions are associated with lifespan extension, or protection against age-related disease, in mammals. Studies both in vitro and in vivo reveal that consumption of nutraceuticals, especially the ones with high antioxidant capacity, has an inverse relationship with cardiovascular diseases, various cancers, and diabetes.

On the basis of free radical theory of ageing it is postulated that any substance with a great antioxidant capacity can be a potential candidate for delaying the aging, but more evidences, show that these substances could improve mitochondrial functions also through activation of different signaling pathways 41, There has been a recent upsurge of interest in complementary and alternative medicine especially dietary supplements and functional foods in delaying the onset of age associated neurodegenerative disease. Different dietary interventions and nutraceuticals have been demonstrate to have beneficial effects on mitochondrial functionality in different physiopathological conditions Epigallocatechingallate EGCG - a natural polyphenol component of green tea - counteracts the mitochondrial energy deficit and oxidative stress found in Down's syndrome DS cells by promotion PKA activity Interestingly, all these compounds would exert anticancer effect activating the mitochondrial apoptotic pathway The therapeutic potential of nutritional mushrooms against human leukemia is demonstrate in U human monocytic leukemia cells in which promote apoptosis by increase of pro-apoptotic protein Pharmacological manipulation of cellular stress pathways is emerging as a viable approach to treating certain neurologic diseases, such as Alzheimer disease and psychiatric disorders, such as schizophrenia Conceivably, such therapeutic tactics may be of value in mitigating or perhaps preventing signs and symptoms of neurodegeneration.

Increased oxidative stress can damage mitochondrial proteins. Of particular relevance to the pathophysiology of major neurodegenerative disorders are findings of decreased levels of mRNA and protein subunits that are involved in the transfer of electrons in complex I of the ETC, in AD patients. Decreased efficiency of the electron transfer process within complex I and complex IV, results in increased leakage and mono-electronic reduction of molecular oxygen to form the superoxide anion 58 , with ensuing damage to proteins, lipids and DNA.

Apoptosis - Wikipedia

As well, an increasing body of evidence supports a role of immune activation as a prominent causative factor in the pathogenesis of a number of major neurologic and neuropsychiatric disorders Recent studies have demonstrated that the inflammasome modulates neuroinflammatory processes at the initial stage, with a secondary cascade of events inclusive of oxidative stress, having been shown to possess the ability to activate the inflammasome The inflammasome is a macromolecular complex that contains multiple copies of a receptor for pathogen- or damage-derived molecular patterns PAMPs, pro-caspase-1, and an adaptor, apoptotic speck-containing protein with a CARD [ASC], which induces caspase-1 maturation Active caspase-1 is responsible for rapid, lytic cell death pyroptosis.

The AIM2 inflammasome is activated by cytosolic DNA, and in addition, it has recently been demonstrated that mitochondria represent major sources of DAMPs capable of triggering neuroinflammatory responses, with resulting apoptosis, pyroptosis and autophagy Recognition of DNA by immunocompetent cells is an important immunological signature that marks the initiation of an innate immune response. For instance, inappropriate recognition of cytoplasmic self-DNA by AIM2 contributes to the development of a number of autoimmune and inflammatory diseases, as well as in neurodegenerative disorders 63, In this scenario the mushrooms offer great potential as a polypharmaceutic drug because of the complexity of their chemical contents and different varieties of bioactivities.

Available evidence suggests that mushrooms exhibit antioxidant, antitumor, antivirus, anticancer, antiinflammatory, immune modulating, anti-microbial, and antidiabetic activities In rat model diabetes mellitus the administration of medicinal mushrooms increases activity of antioxidant enzymes and reduces the amount of thiobarbituric acid reactive substances TBARS thus indicating pronounced antioxidant properties of studied mushrooms Also recently, it has also been shown that some mushroom extracts can produce direct cytotoxic effect on cancer cells through the action on the mitochondrial apoptotic pathway The accumulation of mutations ultimately leads to permanent age-related mitochondrial dysfunction, which contributes to the aging phenotype.

Because cells may have hundreds of mitochondria, and each carries multiple copies of mtDNA, the contribution of mtDNA mutations and deletions to normal aging remains a controversial issue. Because the most obvious consequence of mtDNA mutations is an impairment of energy metabolism, most studies addressing aging effects have focused on tissues that are postmitotic and display high energetic demands, such as the heart, skeletal muscle, and the brain.

Indeed, several studies have unambiguously demonstrated that mtDNA base-substitution mutations accumulate as a result of aging in a variety of tissues and species, including rodents, rhesus monkeys, and humans. In humans, initial studies focused on quantification of individual base-substitution mutations in mtDNA that were shown previously to be pathological in human inherited mitochondrial diseases. For example, the AG mtDNA mutation, which results in maternally inherited mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome MELAS , increases with age in the skeletal muscle of normal humans [ 1 ], but only a small fraction of mtDNA molecules in phenotypically normal humans is likely to carry these disease-associated mutations.

Thus, it is unlikely that these mutations have deleterious consequences in normal aging. Studies performed in the Attardi laboratory have established that some specific base-substitution mutations can reach high levels in fibroblast cells derived from aged individuals [ 2 ] and also in skeletal muscle [ 3 ]. The reason why these specific mutations accumulate in mtDNA is unclear, but they are tissue-specific and occur in mtDNA control sites for replication.

Interestingly, the same group has found a CT transition mutation that occurs in most or all mtDNA molecules i. This mutation is associated with a new replication origin position, suggesting that it may confer a survival advantage in humans [ 4 ]. With the development of high-throughput sequencing methods, an unbiased large-scale examination of either selected regions or the entire mtDNA sequence has become feasible.

Several studies in rodent and primate tissues are in agreement with this estimate of mtDNA mutational burden, but a study using direct cloning of mtDNA reported much lower levels [ 6 ]. This suggests that technical issues remain a problem in determining mtDNA mutation frequencies. Deletions, which can be readily detected by PCR but are not easily quantified, also increase with aging in multiple tissues in rodents [ 7 ] and humans [ 8 , 9 ] and can be clonal, as determined by analysis of individual cardiomyocytes from aged humans [ 10 ].

In agreement with the hypothesis that mtDNA deletions contribute to mammalian aging, it has been shown that they accumulate exponentially in several tissues, and do so much faster in short-lived mice as compared to long-lived humans [ 11 ]. An ongoing debate in the field relates to the issue of causality: are mtDNA mutations merely markers of biological age, or do they lead to a decline in physiological function that contributes to the aging process?

Two important age-related phenotypes have helped to address this issue. A common feature of aging in multiple species, including humans, is the age-related loss of muscle mass, termed sarcopenia. Studies using laser capture microdissection to study single muscle fibers in skeletal muscle from sarcopenic rats have shown that mtDNA deletions colocalize with electron transport system abnormalities, fiber atrophy, and splitting [ 12 ].

Interestingly, the mutations are largely clonal and absent from phenotypically normal regions within individual muscle fibers [ 13 ]. In a similar study of aged 69—82 years old human muscle biopsies, an association between a deficiency in the mitochondrially encoded cytochrome c oxidase COX and clonally expanded base-substitution mutations and deletions was shown [ 14 ]. Perhaps the strongest evidence that clonally expanded mtDNA mutations can be causal in both age-related dysfunction and disease comes from recent studies of neurons present in the substantia nigra region of the human brain.

These dopamine-rich, pigmented neurons contain very high levels of mtDNA deletions. Deleted mtDNA molecules are clonal in each neuron, and are associated with respiratory chain deficiency [ 15 ]. The level of mtDNA deletions increases with normal aging, and is higher in Parkinson's disease [ 16 ]. Cytochrome c oxidase—deficient cells have also been shown to increase with age in both hippocampal pyramidal neurons and choroid plexus epithelial cells [ 17 ].

Although these studies do not prove causality, they provide strong evidence in support of the hypothesis that mtDNA deletions contribute to aging in mammals. A significant gap in our knowledge concerns the mechanisms of age-related clonal expansion of mtDNA base-substitution mutations. Using single-cell sequence analysis, Nekhaeva et al. These clonally expanded mtDNA mutations are abundant in cells of aged individuals and result in very different mtDNA mutational spectra in these two cell types.

Specifically, epithelial cells display a mutational hotspot in a homopolymeric C 7—8 tract, whereas almost all cardiomyocyte mutations were observed within a bp sequence in the control region. This sequence was postulated to represent either a binding site for a mitochondrial protein or a secondary structure of functional importance to mitochondria [ 10 ]. A recently described observation, the accumulation of mtDNA mutations in human crypt stem cells, has also provided insights on the mechanisms of clonal mtDNA mutation accumulation.

Taylor et al. Interestingly, the pattern of distribution of these cells in individual crypts is not random, suggesting that mutations arising in adult stem cells result in the accumulation of such mutations in the tissue. But how do mtDNA mutations become clonal within a cell in the first place? Because the spectrum of expanded mutations is very different between cardiomyocytes and epithelial cells, different mechanisms of expansion, namely random segregation or positive selection, have been proposed for these cell types [ 10 ].

Interestingly, modeling of mtDNA replication in human cells suggests that genetic drift and expansion of mutations that occur in early adult life may account for the abundance of specific mtDNA mutations within individual cells [ 20 ]. The finding that the clonal expansion of mtDNA base-substitution mutations is a widespread process in human somatic cells may have profound implications for both aging and age-related diseases.

Normal human aging is a gradual, cumulative process that spans decades and most likely involves multiple mechanisms. Information on the specific contribution of mtDNA instability to human aging can be inferred through the analysis of disorders associated with increased mtDNA mutation or deletion frequency. Tissues most affected by disorders associated with inherited mtDNA mutations are the same tissues markedly affected by normal aging; these include the brain, heart, skeletal muscle, kidney and the endocrine system [ 21 ].

Disorders associated with increased levels of mtDNA mutations generally fall into two classes: those associated with specific, maternally-inherited mtDNA mutations; and, those associated with mutations in nucleus-encoded genes important for maintaining the fidelity of mtDNA replication and mtDNA stability. Because disorders in the latter category result in random accumulation of many different mtDNA mutations and deletions, they may better represent the potential consequences of age-related mtDNA mutation accumulation in humans. Disease onset typically occurs after the mid-twenties and can be associated with a variety of symptoms, including ophthalmoplegia, cataracts, progressive muscle weakness, parkinsonism, premature ovarian failure, male infertility, hearing loss presbycusis , and cardiac dysfunction [ 24 — 30 ].

At the molecular level, these mutations are often associated with the accumulation of mtDNA deletions in multiple tissues. The few dominant POLG mutations reported in PEO occur within the polymerase domain and tend to disrupt the interaction between the polymerase and the incoming nucleotide; this can cause misincorporation of nucleotides and may also lead to large deletions between direct repeats [ 31 , 32 ].

Interestingly, sequencing of mtDNA deletions from patients suggests that replication stalling may be the major mechanism of deletion formation [ 33 ]. Mutations in black are associated with PEO, those in blue are associated with Alpers syndrome, red indicates mutations present in both PEO and Alpers, and green indicates mutations associated with other disorders. Italics indicate changes in DNA sequence. A POLG. The light green and light blue segments represent the exonuclease and polymerase domains, respectively.

Highly conserved motifs within each are shown as red segments. The pink domain is the primase-helicase linker region, as identified by homology to T7 phage protein [ 34 ]. C ANT1. In addition to the pathogenic mutations shown within the protein, a 3. These mutations seem to enhance dNTPase activity and thus may lower the pool of nucleotides available for mtDNA replication. In humans, the classical progeroid diseases Hutchinson-Gilford syndrome and Werner's syndrome are associated with defects in nucleus-encoded genes involved in nuclear architecture [ 42 — 44 ] and DNA damage repair [ 45 , 46 ], respectively.

The absence of a more general progeroid syndrome in humans carrying mutations that lead to mtDNA instability suggests that mtDNA mutations do not contribute to general aspects of normal human aging. However, the association of genetic disorders of mtDNA instability with cataracts, presbycusis, Parkinson's disease, early menopause, and decreased cardiac and skeletal muscle function suggests that these aging phenotypes are most likely to be influenced by the age-related accumulation of mtDNA base-substitution mutations and deletions.

A number of transgenic and knock-in mouse models have been developed to test the in vivo effects of increased mtDNA mutation accumulation. The lack of proofreading activity in Polg DA mice results in mitochondrial mutation frequencies that are increased by at least 3- to fold in multiple tissues, with accumulation of mtDNA base-substitution mutations beginning in development. Deletions of mtDNA can also be detected in these mice [ 48 ]. The two models have very similar phenotypes resembling aspects of premature aging; these include hair graying and loss, reduced bone density and increased incidence of kyphosis, reduced muscle mass, severe reduction in body fat, early loss of fertility, dilated cardiac hypertrophy, accelerated thymic atrophy, presbycusis, and reduced survival.

Anemia and intestinal dysplasia are also seen. A progressive decline in respiratory function of mitochondrially encoded complexes was evident as early as 12 weeks, resulting in decreased oxygen consumption and ATP production [ 48 , 49 ].


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No increase in DNA, RNA, protein, or lipid markers of oxidative stress was observed in these mice and antioxidant defense systems were likewise not upregulated [ 47 , 49 ]. Instead, mtDNA mutation accumulation was associated with the activation of apoptosis in multiple tissues as measured by TUNEL and cleaved caspase-3 assays [ 47 ]. Additional mitochondrial mutator mouse models have employed tissue-specific Polg DA exonuclease-deficient transgenes, expressed primarily in the heart [ 6 ] or in the brain [ 50 ].

Both base-substitution mutations and mtDNA deletions accumulated in these models. Respiratory function remained comparable to controls [ 51 ]. Similarly to the Polg knock-in mice, the transgenic hearts did not display increased oxidative damage to proteins including oxidation-sensitive aconitase enyzme activity or mtDNA, nor elevated antioxidant defenses [ 52 ]. Cytosolic fractions from transgenic hearts contained cytochrome c [ 51 ], mitochondrial release of which is a hallmark of apoptosis.


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  • This survival response was of functional consequence, in that it could protect the transgenic hearts from further apoptotic stress induced by doxorubicin [ 53 ]. Alternatively, those cells with the highest mutational burden could release cell-extrinsic factors that induce widespread gene expression changes throughout the heart. Cell death seems to be a key element driving the pathology of mtDNA mutations in the heart because cyclosporin A, a cell-death inhibitor that blocks the opening of the mitochondrial permeability transition pore, prevents the cardiomyopathy of the transgenic mice [ 54 ].

    As discussed earlier, mutations in human POLG are associated with chronic PEO, with some patients exhibiting mood disorders [ 29 , 30 ]. Furthermore, mitochondrial dysfunction and altered energy metabolism have been implicated in the etiology of bipolar disorder by magnetic resonance spectroscopy, mtDNA polymorphism association, and detection of mtDNA deletions in bipolar patient brains for reviews, see [ 55 — 57 ]. These behaviors were worsened by treatment with amitriptyline hydrochloride, an antidepressant that can induce mania in individuals with bipolar disorder.

    Although total wheel-running activity decreased, a 5-d pattern of peak activity coinciding with the estrus cycle was observed in female transgenic mice; treatment with lithium, commonly used as a mood stabilizer in the treatment of bipolar disorder, diminished this periodicity. No measurements of respiratory function, apoptosis, or oxidative stress were reported for this model. Mutations in these genes result in reduction or loss of mtDNA content and mice deficient for some of these genes die during development [ 58 , 62 ].

    For example, several mouse models with general [ 58 ] or tissue-specific [ 64 — 67 ] deficiencies in Tfam have been generated, all based on a loxP -flanked Tfam allele Tfam loxP , and are associated with mtDNA depletion see Table 1. All of these Tfam mouse models exhibit a delay between onset of cre expression and the occurrence of respiratory dysfunction, which can be attributed to the time needed to turn over Tfam, mtDNA, and respiratory enzyme subunits.

    Transgenic mice expressing mutant Twinkle isoforms modeled after those mutations seen in human disease display progressive localized mitochondrial respiratory deficiencies, particularly in individual muscle fibers and neuronal subpopulations transgene expression was noted in heart, muscle, and brain , and mild myopathy at about 1 y of age [ 68 ]. Premature aging does not appear to be a feature of these Twinkle transgenic mice, although it is unclear if transgene expression was achieved in most tissues or cell types.

    Mitochondrial Medicine – Molecular Pathology of Defective Oxidative Phosphorylation

    Similarly, mutations in the heart- and muscle-specific isoform of the adenine nucleotide transporter 1 ANT1 gene are present in human PEO families [ 41 ]. Accumulation of mtDNA deletions or rearrangements was observed, with levels in line with the extent of induced antioxidant defenses. In the heart, which showed maximal H 2 O 2 production i.

    An additional mouse model carrying mtDNA deletions has been generated via a methodology distinct from gene targeting in mouse embryonic stem cells. The resulting cytoplasmic hybrid cells cybrids were screened by PCR to identify those containing a high proportion of mtDNA deletions.

    Such cybrid clones were enucleated and fused to donor embryos to create heteroplasmic founder females that could transmit the mtDNA deletion—containing mitochondria through their germline [ 72 ]. Germline transmission of mtDNA deletions in humans is rare [ 73 ], and partially duplicated mtDNA intermediates were postulated to allow for such transmission in the mice [ 72 ]. Similar mosaic respiratory deficits were noted in heart and kidney. An atrioventricular conduction block was reported, but in the absence of cardiac dilation [ 74 ].

    Mito-mice were anemic and died from renal failure by days of age.

    The pathophysiology of mitochondrial disease as modeled in the mouse

    No phenotypes traditionally present in mitochondrial disease or aging were reported. An increase in TUNEL staining was seen in the kidneys of mito-mice, implicating apoptosis as an important mechanism of pathology [ 75 ]. The molecular nature of the mtDNA deletions was not characterized. Generation of transmitochondrial mice has also been extended to include mtDNA base-substitution mutations. No further characterization of aging phenotypes is available yet. Transmitochondrial mice bearing TC 16S rRNA—mutated mtDNAs denoted CAP R mice, because the mutation confers resistance to chloramphenicol displayed growth retardation, myopathy, dilated cardiomyopathy, and embryonic or perinatal lethality [ 78 ].

    These models of homoplasmic mtDNA base-substitution mutations are more reflective of the inherited mitochondrial disease situation in humans, as opposed to the more random accumulation of mutations and deletions that occurs in normal aging. Although direct comparisons of mouse models derived through gene targeting, insertional transgenesis, and cybrid approaches is complicated by differences in gene dosage and tissue-specific expression patterns, it is curious to note that multi-system aging-like phenotypes are much more obvious in models bearing increased base-substitution mutations and deletions such as Polg DA mice, as opposed to those with only increased deletions see Table 1.

    Whether this is biologically meaningful or reflects technical differences in the methodology of mouse generation remains to be determined. POLG, the catalytic subunit, contains the polymerase domain, an editing exonuclease domain, as well as a deoxyribose phosphate lyase activity necessary for DNA repair.

    POLG2, the accessory subunit, increases the affinity of the complex for DNA, elevating polymerase processivity [ 79 ] and repair [ 80 ]. The human holoenzyme consists of a heterotrimer of two accessory subunits attached to one catalytic subunit [ 81 ]. Detailed kinetics experiments with and without the accessory subunit and exonuclease domain have yielded important insights into the mechanism of polymerase fidelity [ 23 , 82 , 83 ]. It is important to note that polymerase infidelity has been hypothesized to be the major cause of mutation in human mtDNA and may be responsible for many of the mutational hotspots that appear across individuals [ 84 ].

    Although in vitro the POLG exonuclease domain plays only a small role in the overall fidelity of the enzyme as compared to the discrimination between incoming dNTPs by the catalytic domain [ 82 ], this proofreading activity has been demonstrated to be essential in preventing the accumulation of mutations with age in mice [ 6 , 47 , 48 , 50 ] and human cells in culture [ 85 ].

    Overexpression of the exonuclease-deficient protein in human cells had a dominant negative effect, resulting in the accumulation of mtDNA base-substitution mutations over time. After 3 mo in culture, one mutation was found for every 1, bp of mtDNA [ 85 ]. These results demonstrate the importance of proper proofreading to prevent mtDNA base-substitution mutations that cause cell and tissue dysfunction with age.

    The vast majority of DNA polymorphisms and disease-causing base-substitution mutations that have been detected in human mtDNA are transition mutations [ 86 ]. Transition mutations are also the predominant type of mutation in both wild-type and Polg DA mice [ 47 , 48 ]. This can be partly explained by the slight infidelity of the POLG enzyme, which allows G:T mismatches to occur as a relatively frequent event [ 83 ]. These particular misincorporation events can be exacerbated by dNTP pool imbalances. As shown in rats, dGTP is present at a much higher concentration than dATP in mitochondria from many postmitotic tissues, including heart and skeletal muscle, possibly increasing the frequency of G:T mismatches [ 87 ].

    In contrast, dTTP is present at the lowest concentration of the four deoxynucleotides in mitochondria from these tissues. These pool imbalances do not differ between young and old animals. At this time, it is unknown what role dNTP pool imbalances play in the generation of the other specific types of mtDNA mutations that occur with age, such as transversion or deletion mutations. In the traditional strand-asynchronous model, replication begins at the heavy guanine-rich strand origin and proceeds approximately two-thirds of the way around the mitochondrial genome before initiation of light cytosine-rich strand synthesis begins [ 88 ].

    There is a positive correlation between the rate of accumulation of base-substitution mutations in mammalian mitochondrial genomes and the distance from the origin of light strand replication, relating to the amount of time mtDNA is single stranded during replication [ 89 ]. This suggests that mtDNA may be particularly susceptible to oxidative damage when single stranded. Pathogenic mitochondrial base-substitution mutations are found at a disproportionately high level in mitochondrial tRNA genes and it has been hypothesized that this high frequency is due to a stem-loop structure formed when these regions are single stranded during mtDNA replication [ 90 ].

    However, further evidence is needed to support or refute this suggestion. The spectrum of base-substitution mutations that accumulate in aged individuals differs across tissues [ 3 ]. This may be due to variations in the mechanism of replication in different tissues.

    Specifically, evidence for coupled leading and lagging strand mtDNA synthesis has emerged in recent years [ 91 ]. If coupled-strand replication differs from the strand-asynchronous mechanism in its susceptibility to mutation generation, then differential reliance on the two modes of replication among tissue types or under different cellular conditions might contribute to tissue-specific mutation patterns.

    Alternately, even when replication proceeds primarily via the strand-asynchronous model, utilization of alternate origins of light strand replication may influence mutation specificity by variations in proximity to the heavy strand replication origin and, thus, differences in the time that mtDNA is present in single-stranded form [ 88 ]. Mitochondria do not have the enzymes necessary for nucleotide excision repair of DNA. They do, however, possess base excision repair enzymes that are capable of repairing oxidatively damaged bases in mtDNA, and many of these repair enzymes are alternatively spliced variants of nucleus-targeted proteins [ 92 ].

    Mitochondrial base excision repair activity declines in the aging mouse brain [ 93 ]; if applicable to tissues in general, this may contribute to the accumulation of mtDNA mutations with age. Base-substitution mutations may occur as a result of POLG replicating across these lesions. In mitochondria, 8-oxoguanine is the most abundant oxidative lesion and can cause transversion mutations if unrepaired [ 94 ].

    Mice lacking Ogg1 had fold higher levels of 8-oxoguanine in mtDNA isolated from liver [ 95 ] but this did not lead to respiratory defects [ 96 ]. The mechanism of deletion formation is unknown. However, many deletions are thought to involve base pairing by direct repeat sequences [ ] and this occurs more frequently during oxidative stress, perhaps due to polymerase stalling, slipping, and mispairing during replication Table 2.

    Topoisomerase II cleavage and other DNA double strand breaks have also been proposed as possible mechanisms of deletion formation [ , ]. A study analyzing deletions in human mtDNA suggests that most deletion formation may be linked to two bp repeats in mtDNA [ ]. Mechanisms for mtDNA Mutation. Data from mitochondrial mutator mouse models support the hypothesis that mtDNA mutations can promote tissue dysfunction through the loss of critical irreplaceable cells due to activation of apoptosis. In support of this hypothesis, human cells bearing mutations causing Leber's hereditary optic neuropathy, an inherited mtDNA disease, are sensitized to Fas-induced apoptosis [ ].

    Is apoptosis required for development of mtDNA-induced phenotypes, and how might mtDNA mutations trigger the apoptotic process? Loss of respiratory function is associated with activation of apoptosis e. In the first, channels in the outer mitochondrial membrane can open in a process regulated by Bcl-2 family members without the involvement of inner mitochondrial membrane components.

    In the second, opening of a permeablility transition PT pore, involving components of the outer mitochondrial membrane VDAC, Bax, and Bcl-2 , inner mitochondrial membrane ANT , and matrix Cyp D results in osmotic mitochondrial swelling, outer mitochondrial membrane rupture, and release of apoptogenic factors. The observation that cyclosporin A—mediated inhibition of PT pore opening was successful in preventing cardiomyopathy in the heart-specific mitochondrial mutator model [ 54 ] implicates a central role for the PT pore generally, and cyclophilin D Cyp D in particular, in mtDNA mutation-mediated cell-death signaling in the heart, because Cyp D is the main mitochondrial binding target of cyclosporin A [ ].

    However, mitochondria from the Polg DA transgenic hearts are purportedly more resistant to calcium-induced PT pore opening than those from control hearts [ ], an effect attributed to the protective actions of induced Bcl-2 in the pro-survival response. Thus, other functions of Cyp D aside from its role in PT pore opening such as its chaperone activity may be important.

    Mouse models deficient for many of the genes involved with apoptotic regulation e. Examining the effects of these apoptotic modulators on the aging phenotypes of mitochondrial mutator mice should help to establish whether apoptosis is required for the downstream effects of mitochondrial mutations.

    Background

    Recently, Zassenhaus and colleagues proposed an intriguing mechanism whereby mtDNA mutations would generate a pool of misfolded mitochondrial proteins, some small proportion of which might have the conformation necessary to bind to Bax or Bak and thereby activate apoptosis or perhaps bind to Cyp D and inhibit its chaperone function [ ]. This hypothesis could explain how heteroplasmic mtDNA mutations could elicit a cell-death response in the presence of many wild-type copies of mtDNA.

    A long-standing tenet of the mitochondrial free radical theory of aging is the expectation of increased ROS production in mitochondria compromised by respiration-inactivating mtDNA mutations i. However, we [ 47 ] and others [ 49 , 52 ] have clearly demonstrated that mitochondrial mutator mice do not have increased levels of oxidative stress. Mitochondria treated with specific chemical electron transport chain ETC inhibitors can indeed produce increased ROS levels [ ]. Upstream complexes can still function, resulting in electron stalling and transfer to O 2 to generate the superoxide anion.

    By contrast, in the mitochondrial mutator mice, a variety of mutations is present and multiple upstream complexes could be nonfunctional or be lacking subunits if mitochondrial rRNA or tRNA mutations are numerous. Thus, electron flow through all the complexes except nucleus-encoded complex II may be impaired and reduced intermediates may not be accumulating. In the case where mtDNA mutation levels are much lower, the presence of many wild-type copies of mtDNA will mask the effects of specific respiratory mutations.

    If mtDNA mutations do not lead to increased ROS damage in mitochondrial mutator mice, how does this finding fit into the field of oxidative stress and aging? Certainly, oxidative stress could be playing a role in the generation of mtDNA mutations in wild-type animals. The rate of mitochondrial ROS production, extent of mtDNA but not nuclear DNA oxidative damage, and degree of membrane fatty acid unsaturation a determinant of vulnerability to lipid peroxidation are all inversely correlated with longevity across species [ — ].