Identification of mitochondrial DNA polymorphisms that alter mitochondrial matrix pH and intracellular calcium dynamics

Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.     

Mitochondrial function in the human oocyte and embryo and their role in developmental competence

The role of mitochondria as a nexus of developmental regulation in mammalian oogenesis and early embryogenesis is emerging from basic research in model species and from clinical studies in infertility treatments that require in vitro fertilization and embryo culture. Here, mitochondrial bioenergetic activities and roles in calcium homeostasis, regulation of cytoplasmic redox state, and signal transduction are discussed with respect to outcome in general, and as possible etiologies of chromosomal defects, maturation and fertilization failure in human oocytes, and as causative factors in early human embryo demise. At present, the ability of mitochondria to balance ATP supply and demand is considered the most critical factor with respect to fertilization competence for the oocyte and developmental competence for the embryo. mtDNA copy number, the timing of mtDNA replication during oocyte maturation, and the numerical size of the mitochondrial complement in the oocyte are evaluated with respect to their relative contribution to the establishment of developmental competence. Rather than net cytoplasmic bioenergetic capacity, the notion of functional compartmentalization of mitochondria is presented as a means by which ATP may be differentially supplied and localized within the cytoplasm by virtue of stage-specific changes in mitochondrial density and potential (ΔΨm). Abnormal patterns of calcium release and sequestration detected at fertilization in the human appear to have coincident effects on levels of mitochondrial ATP generation. These aberrations are not uncommon in oocytes obtained after ovarian hyperstimulation for in vitro fertilization. The possibility that defects in mitochondrial calcium regulation or bioenergetic homeostasis could have negative downstream development consequences, including imprinting disorders, is discussed in the context of signaling pathways and cytoplasmic redox state.     

An Association Analysis between Mitochondrial DNA A10398G Polymorphism and Temperament in Japanese Young Adults

The mitochondrial (mt) DNA C5178A and A10398G polymorphisms have been reported to be associated with mental disorders such as bipolar disorder. However, the effects of these polymorphisms on temperament in healthy people are poorly understood. Evaluating healthy subjects can have the advantage of providing new strategies for maintaining psychological health and preventing mental illness. We examined the association between mtDNA polymorphisms and temperament in Japanese students. There was no significant difference in examined temperament when analysed by genotypes, 5178–10398 haplotypes, or sex. The subgroup analysis based on sex indicated that there was an interactive effect of the mtDNA A10398G polymorphism and sex on anxiety and obsession. This finding is preliminary and cannot exclude the possibility of false-positive due to small sample size (144 subjects) and multiple statistical testing. Further studies involving a larger sample size or other ethnic groups are necessary to confirm that mtDNA A10398G polymorphism can be a genetic factor for temperament.     

Sequence and functional analyses of mtDNA in a maternally inherited family with bipolar disorder and depression

Recent studies suggest that mutations/polymorphisms of mitochondrial DNA (mtDNA) are associated with neuropsychiatric diseases. We identified a patient with major depression and epilepsy. Some family members in the pedigree of the proband had bipolar disorder, depression, suicide, or psychotic disorder not otherwise specified. The mode of inheritance was compatible with maternal inheritance with low penetration. We assumed that the mental disorder in this family might be associated with maternally inherited mitochondrial DNA (mtDNA) mutation. We sequenced the entire mtDNA of the proband. Among the 34 base substitutions detected in the proband, two homoplasmic, nonsynonymous single substitutions of mtDNA, T3394C in MT-ND1 and A9115G in MT-ATP6, were suspected to cause functional impairment, because the former was reported to be disease-related and the latter is vary rare. To study the functional outcome of these substitutions, we examined mitochondrial membrane potential and the activity of mitochondrial ATP synthesis in the transmitochondrial cybrids, but no significant impairment was detected. The data did not support our hypothesis that these disorders in this family are caused by mtDNA mutation(s).     

Does premature aging of the mtDNA mutator mouse prove that mtDNA mutations are involved in natural aging?

Recent studies have demonstrated that transgenic mice with an increased rate of somatic point mutations in mitochondrial DNA (mtDNA mutator mice) display a premature aging phenotype reminiscent of human aging. These results are widely interpreted as implying that mtDNA mutations may be a central mechanism in mammalian aging. However, the levels of mutations in the mutator mice typically are more than an order of magnitude higher than typical levels in aged humans. Furthermore, most of the aging-like features are not specific to the mtDNA mutator mice, but are shared with several other premature aging mouse models, where no mtDNA mutations are involved. We conclude that, although mtDNA mutator mouse is a very useful model for studies of phenotypes associated with mtDNA mutations, the aging-like phenotypes of the mouse do not imply that mtDNA mutations are necessarily involved in natural mammalian aging. On the other hand, the fact that point mutations in aged human tissues are much less abundant than those causing premature aging in mutator mice does not mean that mtDNA mutations are not involved in human aging. Thus, mtDNA mutations may indeed be relevant to human aging, but they probably differ by origin, type, distribution, and spectra of affected tissues from those observed in mutator mice.     

Do mtDNA deletions drive premature aging in mtDNA mutator mice?

Deletions in mitochondrial DNA (mtDNA) have long been suspected to be involved in mammalian aging, but their role remains controversial. Recent research has demonstrated that relatively higher levels of mtDNA deletions correlate with premature aging in mtDNA mutator mice, which led to the conclusion that premature aging in these mice is driven by mtDNA deletions. However, it is reported here that the absolute level of deletions in mutator mice is quite low, especially when compared with the level of point mutations in these mice. It is thus argued that the available data are insufficient to conclude that mtDNA mutations drive premature aging in mtDNA mutator mice. It remains possible that clonal expansion of mtDNA deletions may result in sufficiently high levels to play a role in age-related dysfunction in some cells, but assessing this possibility will require studies of the distribution of these deletions among different cell types and in individual cells.     

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice

In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria‐targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro‐apoptotic and pro‐inflammatory redox signaling pathways.     

Mitochondrial DNA mutations activate programmed cell survival in the mouse heart

Increased frequencies of mitochondrial DNA (mtDNA) mutations characterize the aging heart and are also found in idiopathic dilated cardiomyopathy and end-stage heart failure. The pathogenic potential of such mutations is unclear. Transgenic mice showing accelerated accumulation of mtDNA mutations and dilated cardiomyopathy due to expression of an error-prone mtDNA polymerase specifically in the heart were characterized by Western blot analysis and immunohistochemistry for the levels of pro- and antiapoptotic proteins. By 8 wk of age, when frequencies of mtDNA mutations were approximately 0.01% and all transgenic mice showed four-chamber cardiac dilation, a vigorous prosurvival response was evident. Upregulated were Bcl-2, Bcl-xl, Bfl1, heat shock protein 27, and X-linked inhibitor of apoptosis protein, all of which function to inhibit apoptosis. Although translocation of Bax to mitochondria was also seen, it was not integrated into the mitochondrial membrane. Treatment of transgenic mice with doxorubicin failed to induce apoptosis, in contrast to controls, showing that the prosurvival response protected cardiomyocytes from a death stimulus. Increased apoptosis and release of cytochrome c appeared to precede the establishment of the prosurvival state suggesting that it may reflect a response to activation of programmed cell death pathways. It has been proposed that a programmed cell survival response is activated in the failing and aging heart. We show that elevated frequencies of mtDNA mutations may serve as one trigger for the activation of such a response.     

The complete nucleotide sequence of Chinese hamster (Cricetulus griseus) mitochondrial DNA.

Mammalian mitochondria contain their own approximately 16.5 kb circular genome. Mitochondrial DNA (mtDNA) encodes for a subset of the proteins involved in the electron transport chain and depletion or mutation of the sequence is implicated in a number of human disease processes. The recent finding is that mitochondrial damage mediates genotoxicity after exposure to chemical carcinogens has focused attention on the role of mtDNA mutations in the development of cancer. Although the entire genome has been sequenced for a number of mammals, only a small fraction of the mtDNA sequence is available for hamsters. We have obtained here the entire 16,284 bp sequence of the Chinese hamster mitochondrial genome, which will enable detailed analysis of mtDNA mutations caused by exposure to mutagens in hamster-derived cell lines.     

Mitochondria: Participation to infertility as source of energy and cause of senescence

Mitochondria is a powerhouse organelle involved in ATP synthesis, calcium signaling, reactive oxygen species (ROS) by oxidative stress production, cell cycle arrest via apoptosis and sex steroid hormones biosynthesis. Improvement of sperm parameters such as motility, capacitation, acrosome reaction, and oocyte interaction, involve regulation of ROS levels by the mitochondria. In human, the relation between the quantitative level of mitochondrial DNA (mtDNA), oocyte cytoplasm maturation and fertilization potential, is not clear. It has been hypothesized that oocytes without sufficient wild type mtDNA and therefore able to generate ATP, would not normally be ovulated. This is reflected in the low numbers of mtDNA observed in degenerate oocytes obtained through super ovulation protocols during assisted reproductive technology programs. Different theories place mitochondria in a central role of oxidative damage to cells and tissues related to infertility declining and aging. Mitochondria-dependent apoptosis seems to be responsible for the pre and post-natal decline in germ cells, embryo development, implantation failure, and miscarriages.     

Mitochondrial Mutations in Subjects with Psychiatric Disorders

A considerable body of evidence supports the role of mitochondrial dysfunction in psychiatric disorders and mitochondrial DNA (mtDNA) mutations are known to alter brain energy metabolism, neurotransmission, and cause neurodegenerative disorders. Genetic studies focusing on common nuclear genome variants associated with these disorders have produced genome wide significant results but those studies have not directly studied mtDNA variants. The purpose of this study is to investigate, using next generation sequencing, the involvement of mtDNA variation in bipolar disorder, schizophrenia, major depressive disorder, and methamphetamine use. MtDNA extracted from multiple brain regions and blood were sequenced (121 mtDNA samples with an average of 8,800x coverage) and compared to an electronic database containing 26,850 mtDNA genomes. We confirmed novel and rare variants, and confirmed next generation sequencing error hotspots by traditional sequencing and genotyping methods. We observed a significant increase of non-synonymous mutations found in individuals with schizophrenia. Novel and rare non-synonymous mutations were found in psychiatric cases in mtDNA genes: ND6, ATP6, CYTB, and ND2. We also observed mtDNA heteroplasmy in brain at a locus previously associated with schizophrenia (T16519C). Large differences in heteroplasmy levels across brain regions within subjects suggest that somatic mutations accumulate differentially in brain regions. Finally, multiplasmy, a heteroplasmic measure of repeat length, was observed in brain from selective cases at a higher frequency than controls. These results offer support for increased rates of mtDNA substitutions in schizophrenia shown in our prior results. The variable levels of heteroplasmic/multiplasmic somatic mutations that occur in brain may be indicators of genetic instability in mtDNA.     

Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψ_m) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψ_m in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.     

Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice

A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical damage to the mitochondria could be causally involved in motor neuron death in amyotrophic lateral sclerosis (ALS) and in familial ALS associated with mutations of Cu,Zn superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of patients with sporadic ALS. To investigate further the role of mitochondrial alterations in the pathogenesis of ALS, we studied mitochondria from transgenic mice expressing wild type and G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer chain, and ATP synthesis were severely defective in G93A mice at the time of onset of the disease. We also found evidence of oxidative damage to mitochondrial proteins and lipids. On the other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of hSOD1 did not show significant mitochondrial abnormalities. Our findings suggest that G93A-mutated hSOD1 in mitochondria may cause mitochondrial defects, which contribute to precipitating the neurodegenerative process in motor neurons.     

Dynamics of Mitochondrial DNA Nucleoids Regulated by Mitochondrial Fission Is Essential for Maintenance of Homogeneously Active Mitochondria during Neonatal Heart Development

Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis, including mitochondrial DNA (mtDNA) distribution. Cardiac myocytes have a specialized cytoplasmic structure where large mitochondria are aligned into tightly packed myofibril bundles; however, recent studies have revealed that mitochondrial dynamics also plays an important role in the formation and maintenance of cardiomyocytes. Here, we precisely analyzed the role of mitochondrial fission in vivo. The mitochondrial fission GTPase, Drp1, is highly expressed in the developing neonatal heart, and muscle-specific Drp1 knockout (Drp1-KO) mice showed neonatal lethality due to dilated cardiomyopathy. The Drp1 ablation in heart and primary cultured cardiomyocytes resulted in severe mtDNA nucleoid clustering and led to mosaic deficiency of mitochondrial respiration. The functional and structural alteration of mitochondria also led to immature myofibril assembly and defective cardiomyocyte hypertrophy. Thus, the dynamics of mtDNA nucleoids regulated by mitochondrial fission is required for neonatal cardiomyocyte development by promoting homogeneous distribution of active mitochondria throughout the cardiomyocytes.     

Mitochondrial Abeta: a potential cause of metabolic dysfunction in Alzheimer's disease

Deficits in mitochondrial function are a characteristic finding in Alzheimer's disease (AD), though the mechanism remains to be clarified. Recent studies revealed that amyloid beta peptide (Abeta) gains access into mitochondrial matrix, which was much more pronounced in both AD brain and transgenic mutant APP mice than in normal controls. Abeta progressively accumulates in mitochondria and mediates mitochondrial toxicity. Interaction of mitochondrial Abeta with mitochondrial enzymes such as amyloid beta binding alcohol dehydrogenase (ABAD) exaggerates mitochondrial stress by inhibiting the enzyme activity, releasing reactive oxygen species (ROS), and affecting glycolytic, Krebs cycle and/or the respiratory chain pathways through the accumulation of deleterious intermediate metabolites. The pathways proposed may play a key role in the pathogenesis of this devastating neurodegenerative disorder, Alzheimer's disease.     

Mitochondria as the decision makers for cancer cell fate: from signaling pathways to therapeutic strategies

As pivotal players in cellular metabolism, mitochondria have a double-faceted role in the final decision of cell fate. This is true for all cell types, but it is even more important and intriguing in the cancer setting.Mitochondria regulate cell fate in many diverse ways: through metabolism, by producing ATP and other metabolites deemed vital or detrimental for cancer cells; through the regulation of Ca²⁺ homeostasis, especially by the joint participation of the endoplasmic reticulum in a membranous tethering system for Ca²⁺ signaling called mitochondria-ER associated membranes (MAMs); and by regulating signaling pathways involved in the survival of cancer cells such as mitophagy. Recent studies have shown that mitochondria can also play a role in the regulation of inflammatory pathways in cancer cells, for example, through the release of mitochondrial DNA (mtDNA) involved in the activation of the cGAS-cGAMP-STING pathway.In this review, we aim to explore the role of mitochondria as decision makers in fostering cancer cell death or survival depending on the tumor cell stage and describe novel anticancer therapeutic strategies targeting mitochondria.     

Transgenic expression of the EXT2 gene in developing chondrocytes enhances the synthesis of heparan sulfate and bone formation in mice

Hereditary multiple exostoses (HME), a dominantly inherited disorder characterized by multiple cartilaginous tumors, is caused by mutations in the gene for, EXT1 or EXT2. Recent studies have revealed that EXT1 and EXT2 are required for the biosynthesis of heparan sulfate and exert maximal transferase activity as a complex. The Drosophila homologue of EXT1 (tout-velu) regulates the movement and signaling of Hedgehog protein, which plays an important role in the regulation of chondrocyte differentiation and bone development. In this study, to investigate the biological role of EXT2 in bone development in vivo and the pathological role of HME mutations in the development of exostoses, we generated transgenic mice expressing EXT2 or mutant EXT2 in developing chondrocytes. Histological analyses and micro-CT scanning showed that the biosynthesis of heparan sulfate and the formation of trabeculae were upregulated in EXT2-transgenic mice, but not in mutant EXT2-transgenic mice. The expression of EXT1 is concomitantly upregulated in EXT2-transgenic and even mutant EXT2-transgenic mice, suggesting an interactive regulation of EXT1 and EXT2 expression. These findings support that the EXT2 gene encodes an essential component of the glycosyltransferase complex required for the biosynthesis of heparan sulfate, which may eventually modulate the signaling involved in bone formation.