Mito-mice: animal models for mitochondrial DNA-based diseases

We have successfully produced "Mito-mice" harbouring a pathogenic mtDNA mutation. We generated the mice by introducing mitochondria with a 4696 base-pair mtDNA deletion (Delta mtDNA4696) into mouse embryos. This deletion encompasses nucleotides 7759-12 454 and includes six tRNA genes and seven structural genes. In Mito-mice, the Delta mtDNA4696 is transmitted maternally, and induces mitochondrial dysfunction in various tissues. Most of the Mito-mice with high proportions of the Delta mtDNA4696 died at about age 6 months due to renal failure. Mito-mice are the first animal model for mtDNA-based diseases and will be valuable for studying pathogenesis and for identifying effective drug and gene therapies.     

Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes.

Many human mitochondrial diseases are associated with defects in the mitochondrial DNA (mtDNA). Mutated and wild-type forms of mtDNA often coexist in the same cell in a state called heteroplasmy. Here, we report the isolation of a Caenorhabditis elegans strain bearing the 3.1-kb uaDf5 deletion that removes 11 genes from the mtDNA. The uaDf5 deletion is maternally transmitted and has been maintained for at least 100 generations in a stable heteroplasmic state in which it accounts for approximately 60% of the mtDNA content of each developmental stage. Heteroplasmy levels vary between individual animals (from approximately 20 to 80%), but no observable phenotype is detected. The total mtDNA copy number in the uaDf5 mutant is approximately twice that of the wild type. The maternal transmission of the uaDf5 mtDNA is controlled by at least two competing processes: one process promotes the increase in the average proportion of uaDf5 mtDNA in the offspring, while the second promotes a decrease. These two forces prevent the segregation of the mtDNAs to homoplasmy.     

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 (DeltamtDNA4696), and by using cytochrome c oxidase (COX) electron micrographs to identify COX activity at the individual mitochondrial level. All mitochondria in tissues with DeltamtDNA4696 showed normal COX activity until DeltamtDNA4696 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 DeltamtDNA4696 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.     

Interaction theory of mammalian mitochondria.

We generated mice with deletion mutant mtDNA by its introduction from somatic cells into mouse zygotes. Expressions of disease phenotypes are limited to tissues expressing mitochondrial dysfunction. Considering that all these mice share the same nuclear background, these observations suggest that accumulation of the mutant mtDNA and resultant expressions of mitochondrial dysfunction are responsible for expression of disease phenotypes. On the other hand, mitochondrial dysfunction and expression of clinical abnormalities were not observed until the mutant mtDNA accumulated predominantly. This protection is due to the presence of extensive and continuous interaction between exogenous mitochondria from cybrids and recipient mitochondria from embryos. Thus, we would like to propose a new hypothesis on mitochondrial biogenesis, interaction theory of mitochondria: mammalian mitochondria exchange genetic contents, and thus lost the individuality and function as a single dynamic cellular unit.     

Molecular Characterization of the Mitochondrial DNA of a New Stopper Mutant Er-3 of Neurospora Crassa

An ethidium bromide-induced stopper mutant of Neurospora crassa is characterized at the molecular level. The mutant has two populations of mitochondrial DNA: a defective predominant mutant molecule and a basal level of the wild-type molecule. The aberrant DNA resulted after a 25-kbp deletion from the wild-type mitochondrial chromosome, which included major genes such as cytb, co1 and oli2. The deletion endpoints are located in the second intron of the ND5 gene, and in a sequence 250 nucleotides upstream of the co2 gene. The recombination has taken place between two nine nucleotide repeats CCCCGCCCC, one of which is close to a PstI palindrome at its 5' end. Thus the mutant ER-3 differs from all the other stopper mutants described previously in the extent and location of the deletions in the mtDNA.     

Assaying the probabilities of obtaining maternally inherited heteroplasmy as the basis for modeling OXPHOS diseases in animals

Gross alterations in cell energy metabolism underlie manifestations of hereditary OXPHOS (oxidative phosphorylation) diseases, many of which depend on proportion of mutant mitochondrial DNA (mtDNA) in tissues. An animal model of OXPHOS disease with maternal inheritance of mitochondrial heteroplasmy might help understanding the peculiarities of abnormal mtDNA distribution and its effect on pre- and postnatal development. Previously we obtained mice that carry human mtDNA in some tissues. It co-existed with murine mtDNA (heteroplasmy) and was transmitted maternally to the progeny of animals developed from zygotes injected with human mitochondria. To analyze the probability of obtaining heteroplasmic mice we increased the number of experiments with early embryos and obtained more specimens from F1. About 33% of zygotes injected with human mtDNA developed into post-implantation embryos (7th-13th days). Lower amount of such developed into neonate mice (ca. 21%). Among post-implantation embryos and in generations F0 and F1 percentages of human mtDNA-carriers were ca. 14-16%. Such percentages are sufficient for modeling maternally inherited heteroplasmy in small animal groups. More data are needed to understand the regularities of anomalous mtDNA distribution among cells and tissues and whether heart and muscles frequently carrying human mtDNA in our experiments are particularly susceptible to heteroplasmy.     

Construction of transgenic mice with tissue-specific acceleration of mitochondrial DNA mutagenesis

Transgenic mice having rapid accumulation of mitochondrial DNA (mtDNA) mutations specifically in the heart were created. These mice contained a transgene encoding a proofreading-deficient, mouse mitochondrial DNA polymerase (pol gamma) driven by the promoter for the cardiac-specific alpha-myosin heavy chain. Starting shortly after birth greater than 95% of all pol gamma mRNA in the heart was transgene derived; expression in other tissues was low or absent. Mutations in cardiac mtDNA began to accumulate by 7 days after birth. At 1 month of age the frequency of point mutations was 0.014% as determined by DNA sequencing of cloned mtDNA. By long-extension PCR multiple different deletion mutations that had removed several thousand basepairs of genomic sequence were also detected. Sequencing of two deletion molecules showed that one was flanked at the breakpoint by direct repeat sequences. The expression of proofreading-deficient pol gamma had no apparent deleterious effect on mitochondrial DNA and protein content, gene expression, or respiratory function. However, associated with the rise in mtDNA mutation levels was the development of cardiomyopathy as evidenced by enlarged hearts in the transgenic mice. These mice may prove to be useful models to study the pathogenic effects of elevated levels of mitochondrial DNA mutations in specific tissues.     

Maternal inheritance of deleted mitochondrial DNA in a family with mitochondrial myopathy

Skeletal muscles from a mother and her daughter both with chronic progressive ophthalmoplegia were analyzed. Histological and biochemical analyses of their muscle samples showed typical features of this type of mitochondrial myopathy. Southern blot analysis revealed that, in both patients, there were two species of mitochondrial DNA (mtDNA): normal one and partially deleted one. The sizes of the deletion were different; the mutant mtDNAs from the mother and the daughter had about 2.5- and 5-kilobase deletions, respectively. The two mutant mtDNAs shared a common deleted region of 1.2-kilobase. However, both the start and the end of deletion were different between them, implying a novel mode of inheritance. This is the first report that the mutant mtDNA is responsible for the maternal inheritance of a human disease.     

Transcomplementation between different types of respiration-deficient mitochondria with different pathogenic mutant mitochondrial DNAs

Two cell lines were used for determination of whether interaction occurred between different types of respiration-deficient mitochondria. One was a respiration-deficient rho- cell line having mutant mitochondrial DNA (mtDNA) with a 5,196-base pair deletion including five tRNA genes (tRNAGly, Arg, Ser(AGY), Leu(CUN), His), DeltamtDNA5196, causing Kearns-Sayre syndrome. The other was a respiration-deficient syn- cell line having mutant mtDNA with an A to G substitution at 4,269 in the tRNAIle gene, mtDNA4269, causing fatal cardiomyopathy. The occurrence of mitochondrial interaction was examined by determining whether cybrids constructed by fusion of enucleated rho- cells with syn- cells became respiration competent by exchanging their tRNAs. No cybrids were isolated in selection medium, where only respiration-competent cells could survive, suggesting that no interaction occurred, or that it occurred so slowly that sufficient recovery of mitochondrial respiratory function was not attained by the time of selection. The latter possibility was confirmed by the observations that heteroplasmic cybrids with both mutant mtDNA4269 and DeltamtDNA5196 isolated without selection showed restored mitochondrial respiration activity. This demonstration of transcomplementation between different respiration-deficient mitochondria will help in understanding the relationship between somatic mutant mtDNAs and the roles of such mutations in aging processes.     

Altered mitochondrial gene expression in a maternal distorted leaf mutant of Arabidopsis induced by chloroplast mutator.

Chloroplast mutator (chm) of Arabidopsis is a recessive nuclear mutation that causes green and white variegation in leaves and is inherited in a non-Mendelian fashion. In this study, we have identified and characterized a mutant observed in F1 and backcrossed BC1 populations from a cross between chm1-3 and ecotype Columbia. This mutant, maternal distorted leaf (MDL), grows very poorly and is distinguished by distorted rough leaves and aborted flowering organs. Electron microscopic observation showed that in MDL plants, a significant portion of mitochondria are abnormal and appear to be nonfunctional. DNA gel blot and sequence analysis of the MDL mitochondrial DNA (mtDNA) revealed rearrangements in two mtDNA fragments associated with rps3-rpl16 genes (encoding ribosomal proteins S3 and L16, respectively). One rearrangement resulted in the insertion of the rps3-rpl16 operon downstream of atp9. An independent deletion in this region had eliminated the majority of rps3. In contrast, another rearrangement deleted part of rpl16, whereas rps3 remained intact. RNA gel blot analysis indicated that expression of these genes is also altered as a consequence of the mtDNA rearrangements. Thus, a mutation at the CHM locus affects mitochondrial gene expression, and impaired mitochondrial function may result in the distorted phenotype.     

Mutant copies of mitochondrial DNA in tissues and plasma of mice subjected to X-ray irradiation

Impairments of mitochondrial genome are associated with a wide spectrum of degenerative diseases, development of tumors, aging, and cell death. We studied the content of mitochondrial DNA (mtDNA) with mutations and the total content of mutations in the brain and the spleen of mice subjected to X-ray irradiation at a dose of 1–5 Gy at 8–28 days after treatment. In these mice, we studied the number of mutant copies of extracellular mtDNA (ec-mtDNA) and its total content in blood plasma. We estimated mutations in control and irradiated mice using cleavage of heteroduplexes prepared by hybridization of PCR amplicons of mtDNA (D-loop region) mediated by CEL-I endonuclease, an enzyme that specifically cleaves unpaired bases. Changes in the total number of mtDNA copies relative to nuclear DNA were assessed by real time PCR using the ND-4 and GAPDH genes, respectively. We found that the number of mutant mtDNA copies was significantly increased in the brain and the spleen of irradiated mice and reached the maximum level at the eighth day after treatment; it then decreased by the 28th day after treatment. In nuclear genes similar to mutagenesis, mutagenesis of mtDNA in the brain and spleen tissues linearly depended on irradiation dose. In contrast to mutant nuclear genes, most mutant mtDNA copies were eliminated in the brain and spleen tissues, whereas the total content of mtDNA did not change within 28 days after irradiation. Our data show that, during this period, a high level of ec-mtDNA with mutations was observed in DNA circulating in blood plasma with the maximum level found at the 14th day. We suppose that mutant mtDNA copies are eliminated from cells of animals subjected to irradiation during the posttreatment period. Higher content of ec-mtDNA in blood plasma can be considered as a potential marker of radiation damage to the body.     

Mitochondrial DNA deletions are associated with non-B DNA conformations

Mitochondrial DNA (mtDNA) deletions are a primary cause of mitochondrial disease and are believed to contribute to the aging process and to various neurodegenerative diseases. Despite strong observational and experimental evidence, the molecular basis of the deletion process remains obscure. In this study, we test the hypothesis that the primary cause of mtDNA vulnerability to breakage resides in the formation of non-B DNA conformations, namely hairpin, cruciform and cloverleaf-like elements. Using the largest database of human mtDNA deletions built thus far (753 different cases), we show that site-specific breakage hotspots exist in the mtDNA. Furthermore, we discover that the most frequent deletion breakpoints occur within or near predicted structures, a result that is supported by data from transgenic mice with mitochondrial disease. There is also a significant association between the folding energy of an mtDNA region and the number of breakpoints that it harbours. In particular, two clusters of hairpins (near the D-loop 3'-terminus and the L-strand origin of replication) are hotspots for mtDNA breakage. Consistent with our hypothesis, the highest number of 5'- and 3'-breakpoints per base is found in the highly structured tRNA genes. Overall, the data presented in this study suggest that non-B DNA conformations are a key element of the mtDNA deletion process.     

鼠毛及脑线粒体DNA片段缺失与增龄的关系

以聚合酶链反应（ＰＣＲ）技术检测不同年龄Ｂａｌｂ／ｃ小鼠脑细胞线粒体ＤＮＡ片段缺失与增龄的关系．发现老年鼠脑细胞线粒体３８６７ｂｐ片段缺失率为５０％;而断奶鼠与青年鼠均无此缺失片段出现;用鼠毛为材料进行无损伤检测亦获类似的结果．有人认为线粒体ＤＮＡ片段缺失率可作为生物衰老的一种生物学标志     

Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys

Mitochondrial DNA (mtDNA) deletion mutations co-localize with electron transport system (ETS) abnormalities in rhesus monkey skeletal muscle fibers. Using laser capture microdissection in conjunction with PCR and DNA sequence analysis, mitochondrial genomes from single sections of ETS abnormal fibers were characterized. All ETS abnormal fibers contained mtDNA deletion mutations. Deletions were large, removing 20-78% of the genome, with some to nearly all of the functional genes lost. In one-third of the deleted genomes, the light strand origin was deleted, whereas the heavy strand origin of replication was conserved in all fibers. A majority (27/39) of the deletion mutations had direct repeat sequences at their breakpoints and most (36/39) had one breakpoint within or in close proximity to the cytochrome b gene. Several pieces of evidence support the clonality of the mtDNA deletion mutation within an ETS abnormal region of a fiber: (a) only single, smaller than wild-type, PCR products were obtained from each ETS abnormal region; (b) the amplification of mtDNA from two regions of the same ETS abnormal fiber identified identical deletion mutations, and (c) a polymorphism was observed at nucleotide position 16103 (A and G) in the wild-type mtDNA of one animal (sequence analysis of an ETS abnormal region revealed that mtDNA deletion mutations contained only A or G at this position). Species-specific differences in the regions of the genomes lost as well as the presence of direct repeat sequences at the breakpoints suggest mechanistic differences in deletion mutation formation between rodents and primates.     

Organization and expression of the mitochondrial genome in the Nicotiana sylvestris CMSII mutant.

Previous analyses suggested that the Nicotiana sylvestris CMSII mutant carried a large deletion in its mitochondrial genome. Here, we show by cosmid mapping that the deletion is 60 kb in length and contains several mitochondrial genes or ORFs, including the complex I nad7 gene. However, due to the presence of large duplications in the progenitor mitochondrial genome, the only unique gene that appears to be deleted is nad7. RNA gel blot data confirm the absence of nad7 expression, strongly suggesting that the molecular basis for the CMSII abnormal phenotype, poor growth and male sterility, is the altered complex I structure. The CMSII mitochondrial genome appears to consist essentially of one of two subgenomes resulting from recombination between direct short repeats. In the progenitor mitochondrial genome both recombination products are detected by PCR and, reciprocally, the parental fragments are detected at the substoichiometric level in the mutant. The CMSII mtDNA organization has been maintained through six sexual generations.     

Production of mitochondrial DNA transgenic mice using zygotes

Several animal models of human disease, which have been developed by random or targeted modifications of genomic DNA sequences, have furthered our understanding of pathogenesis and the development of therapeutics. However, these models have not facilitated studies on mitochondrial diseases, since modifications to mitochondrial DNA (mtDNA) sequences are not possible using current recombination techniques. Consequently, information on human mitochondrial diseases is relatively sparse, and issues related to mitochondrial pathogenesis and inheritance remain unresolved. Recently, we reported the development of a new technique to generate mice carrying mutant mtDNA from a mouse cell line. In this report, we describe our techniques in detail, with emphasis on the preparation of donor cytoplasts and the micromanipulative procedures for electrofusion of cytoplasts and recipient zygotes. These steps are critically important for the successful introduction of exogenous mtDNA into embryos, and thereby into animals, so that the mutant mtDNA is efficiently propagated in subsequent generations.