Kazuto Nakada

ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis.

Mutations in mitochondrial DNA (mtDNA) occur at high frequency in human tumors, but whether these mutations alter tumor cell behavior has been unclear. We used cytoplasmic hybrid (cybrid) technology to replace the endogenous mtDNA in a mouse tumor cell line that was poorly metastatic with mtDNA from a cell line that was highly metastatic, and vice versa. Using assays of metastasis in mice, we found that the recipient tumor cells acquired the metastatic potential of the transferred mtDNA. The mtDNA conferring high metastatic potential contained G13997A and 13885insC mutations in the gene encoding NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase subunit 6 (ND6). These mutations produced a deficiency in respiratory complex I activity and were associated with overproduction of reactive oxygen species (ROS). Pretreatment of the highly metastatic tumor cells with ROS scavengers suppressed their metastatic potential in mice. These results indicate that mtDNA mutations can contribute to tumor progression by enhancing the metastatic potential of tumor cells.

Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes.

Mice carrying mitochondrial DNA (mtDNA) with pathogenic mutations would provide a system in which to study how mutant mtDNAs are transmitted and distributed in tissues, resulting in expression of mitochondrial diseases. However, no effective procedures are available for the generation of these mice. Isolation of mouse cells without mtDNA (ρ0) enabled us to trap mutant mtDNA that had accumulated in somatic tissues into ρ0 cells repopulated with mtDNA (cybrids). We isolated respiration-deficient cybrids with mtDNA carrying a deletion and introduced this mtDNA into fertilized eggs. The mutant mtDNA was transmitted maternally, and its accumulation induced mitochondrial dysfunction in various tissues. Moreover, most of these mice died because of renal failure, suggesting the involvement of mtDNA mutations in the pathogeneses of new diseases.

Inter-mitochondrial complementation : mitochondria-specific system preventing mice from expression of disease phenotypes by mutant mtDNA

Here we investigated the pathogenesis of deletion mutant mitochondrial (mt)DNA by generating mice with mutant mtDNA carrying a 4696-basepair deletion (ΔmtDNA4696), and by using cytochrome c oxidase (COX) electron micrographs to identify COX activity at the individual mitochondrial level. All mitochondria in tissues with ΔmtDNA4696 showed normal COX activity until ΔmtDNA4696 accumulated predominantly; this prevented mice from expressing disease phenotypes. Moreover, we did not observe coexistence of COX-positive and -negative mitochondria within single cells. These results indicate the occurrence of inter-mitochondrial complementation through exchange of genetic contents between exogenously introduced mitochondria with ΔmtDNA4696 and host mitochondria with normal mtDNA. This complementation shows a mitochondria-specific mechanism for avoiding expression of deletion-mutant mtDNA, and opens the possibility of a gene therapy in which mitochondria possessing full-length DNA are introduced.

p62/SQSTM1 cooperates with Parkin for perinuclear clustering of depolarized mitochondria

PINK1 and Parkin were first identified as the causal genes responsible for familial forms of early‐onset Parkinson’s disease (PD), a prevalent neurodegenerative disorder. PINK1 encodes a mitochondrial serine/threonine protein kinase, whereas Parkin encodes an ubiquitin‐protein ligase. PINK1 and Parkin cooperate to maintain mitochondrial integrity; however, the detailed molecular mechanism of how Parkin‐catalyzed ubiquitylation results in mitochondrial integrity remains an enigma. In this study, we show that Parkin‐catalyzed K63‐linked polyubiquitylation of depolarized mitochondria resulted in ubiquitylated mitochondria being transported along microtubules to cluster in the perinuclear region, which was interfered by pathogenic mutations of Parkin. In addition, p62/SQSTM1 (hereafter referred to as p62) was recruited to depolarized mitochondria after Parkin‐directed ubiquitylation. Intriguingly, deletion of p62 in mouse embryonic fibroblasts resulted in a gross loss of mitochondrial perinuclear clustering but did not hinder mitochondrial degradation. Thus, p62 is required for ubiquitylation‐dependent clustering of damaged mitochondria, which resembles p62‐mediated ‘aggresome’ formation of misfolded/unfolded proteins after ubiquitylation.

Mitochondria-related male infertility

Approximately 15% of human couples are affected by infertility, and about half of these cases of infertility can be attributed to men, through low sperm motility (asthenozoospermia) or/and numbers (oligospermia). Because mitochondrial genome (mtDNA) mutations are identified in patients with fertility problems, there is a possibility that mitochondrial respiration defects contribute to male infertility. To address this possibility, we used a transmitochondrial mouse model (mito-mice) carrying wild-type mtDNA and mutant mtDNA with a pathogenic 4,696-bp deletion (ΔmtDNA). Here we show that mitochondrial respiration defects caused by the accumulation of ΔmtDNA induced oligospermia and asthenozoospermia in the mito-mice. Most sperm from the infertile mito-mice had abnormalities in the middle piece and nucleus. Testes of the infertile mito-mice showed meiotic arrest at the zygotene stage as well as enhanced apoptosis. Thus, our in vivo study using mito-mice directly demonstrates that normal mitochondrial respiration is required for mammalian spermatogenesis, and its defects resulting from accumulated mutant mtDNAs cause male infertility.

Mammalian Copper Chaperone Cox17p Has an Essential Role in Activation of Cytochrome c Oxidase and Embryonic Development

ABSTRACT Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-. Although mouse embryos homozygous for COX17 disruption die between embryonic days E8.5 and E10, they develop normally until E6.5. This phenotype is strikingly similar to embryos of Ctr1(−/−), a cell surface copper transporter, in its lethality around the time of gastrulation. COX17-deficient embryos exhibit severe reductions in CCO activity at E6.5. Succinate dehydrogenase activity and immunoreactivities for anti-COX subunit antibodies were normal in the COX17(−/−) embryos, indicating that this defect was not caused by the deficiency of other complexes and/or subunits but was caused by impaired CCO activation by Cox17p. Since other copper chaperone (Atox1 and CCS)-deficient mice show a more moderate defect, the disruption of the COX17 locus causes the expression of only the phenotype of Ctr1(−/−). We found that the activity of lactate dehydrogenase was also normal in E6.5 embryos, implying that the activation of CCO by Cox17p may not be essential to the progress of embryogenesis before gastrulation.

Mitochondrial DNA Mutations Regulate Metastasis of Human Breast Cancer Cells

Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously expressed a highly metastatic potential, mitochondrial respiration defects, and ROS overproduction. Since mitochondrial respiratory function is controlled by both mtDNA and nuclear DNA, it is possible that nuclear DNA mutations contribute to the mitochondrial respiration defects and the highly metastatic potential found in MDA-MB-231 cells. To examine this possibility, we carried out mtDNA replacement of MDA-MB-231 cells by normal human mtDNA. For the complete mtDNA replacement, first we isolated mtDNA-less (ρ0) MDA-MB-231 cells, and then introduced normal human mtDNA into the ρ0 MDA-MB-231 cells, and isolated trans-mitochondrial cells (cybrids) carrying nuclear DNA from MDA-MB-231 cells and mtDNA from a normal subject. The normal mtDNA transfer simultaneously induced restoration of mitochondrial respiratory function and suppression of the highly metastatic potential expressed in MDA-MB-231 cells, but did not suppress ROS overproduction. These observations suggest that mitochondrial respiration defects observed in MDA-MB-231 cells are caused by mutations in mtDNA but not in nuclear DNA, and are responsible for expression of the high metastatic potential without using ROS-mediated pathways. Thus, human tumor cells possess an mtDNA-mediated metastatic pathway that is required for expression of the highly metastatic potential in the absence of ROS production.

Dynamics of mitochondrial DNA nucleoids regulated by mitochondrial fission is essential for maintenance of homogeneously active mitochondria during neonatal heart development

ABSTRACT 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.

Rare creation of recombinant mtDNA haplotypes in mammalian tissues

The problem of whether recombinant mtDNAs are created in mammalian cells has been controversial for many years. We show convincing evidence for the very rare creation of recombinant mtDNA haplotypes by isolating human somatic hybrid cells and by generating mice carrying two different mtDNA haplotypes. To avoid misinterpretation of PCR-jumping products as recombinants, we used purified mtDNAs for cloning and sequencing. The results showed that only three of 318 clones of mtDNA purified from mouse tissues corresponded to recombinant mtDNA haplotypes, whereas no recombinants were found in human somatic hybrid cells. Such an extremely low frequency of mtDNA recombination does not require any revision of important concepts on human evolution that are based on its absence. Considering the high concentration of reactive oxygen species around the mtDNA and its frequent strand breakage, recombinant clones would correspond to gene conversion products created by repair of nucleotide mismatches.

Mitochondrial respiration defects modulate differentiation but not proliferation of hematopoietic stem and progenitor cells

Mitochondrial energy production is involved in various cellular processes. Here we show that ATP content is significantly increased in lineage‐restricted progenitor cells compared with hematopoietic stem and progenitor cells (HSPCs) or more differentiated cells. Transplantation analysis using a mouse model of mitochondrial disease revealed that mitochondrial respiration defects resulted in a significant decrease in the total number and repopulating activity of bone marrow cells, although the number of HSPCs increased. The proliferative activity of HSPCs and lineage‐restricted progenitor cells was not impaired by reduction of ATP content and there seems to be no associated increase in reactive oxygen species levels and apoptosis. Our findings indicate that mitochondrial respiration defects modulate HSPC commitment/differentiation into lineage‐restricted progenitor cells.