Wild-type Mitochondrial Genome Research Articles

Mitochondrial DNA Mutation Load

Mitochondrial DNA Mutation Load

Mitochondrial Diseases: Therapeutic Approaches

Therapy of mitochondrial encephalomyopathies (defined restrictively as defects of the mitochondrial respiratory chain) is woefully inadequate, despite great progress in our understanding of the molecular bases of these disorders. In this review, we consider sequentially several different therapeutic approaches. Palliative therapy is dictated by good medical practice and includes anticonvulsant medication, control of endocrine dysfunction, and surgical procedures. Removal of noxious metabolites is centered on combating lactic acidosis, but extends to other metabolites. Attempts to bypass blocks in the respiratory chain by administration of electron acceptors have not been successful, but this may be amenable to genetic engineering. Administration of metabolites and cofactors is the mainstay of real-life therapy and is especially important in disorders due to primary deficiencies of specific compounds, such as carnitine or coenzyme Q10. There is increasing interest in the administration of reactive oxygen species scavengers both in primary mitochondrial diseases and in neurodegenerative diseases directly or indirectly related to mitochondrial dysfunction. Aerobic exercise and physical therapy prevent or correct deconditioning and improve exercise tolerance in patients with mitochondrial myopathies due to mitochondrial DNA (mtDNA) mutations. Gene therapy is a challenge because of polyplasmy and heteroplasmy, but interesting experimental approaches are being pursued and include, for example, decreasing the ratio of mutant to wild-type mitochondrial genomes (gene shifting), converting mutated mtDNA genes into normal nuclear DNA genes (allotopic expression), importing cognate genes from other species, or correcting mtDNA mutations with specific restriction endonucleases. Germline therapy raises ethical problems but is being considered for prevention of maternal transmission of mtDNA mutations. Preventive therapy through genetic counseling and prenatal diagnosis is becoming increasingly important for nuclear DNA-related disorders. Progress in each of these approaches provides some glimmer of hope for the future, although much work remains to be done.

Accumulation of Mitochondrial DNA Deletion Mutations in Aged Muscle Fibers: Evidence for a Causal Role in Muscle Fiber Loss

Although mitochondrial mutation abundance has been recognized to increase in an age-dependent manner, the impact of mutation has been more difficult to establish. Using quantitative polymerase chain reaction, we measured the intracellular abundance of mutant and wild-type mitochondrial genomes along the length of individual laser-captured microdissected muscle fibers from aged rat quadriceps. Aged muscle fibers possessed segmental, clonal intracellular expansions of unique somatically derived mitochondrial DNA (mtDNA) deletion mutations. When the mutation abundance surpassed 90% of the total mitochondrial genomes, the fiber lost cytochrome c oxidase activity and exhibited an increase in succinate dehydrogenase activity. In addition to the mitochondrial enzymatic abnormalities, some fibers displayed abnormal morphology such as fiber splitting, atrophy, and breakage. Deletion mutation accumulation was linked to these aberrant morphologies with more severe cellular pathologies resulting from higher deletion mutation abundance. In summary, our measurements indicate that age-induced mtDNA deletion mutations expand within individual muscle fibers, eliciting fiber dysfunction and breakage.

Progressive depletion of mtDNA in mitochondrial myopathy

The authors studied seven patients with mitochondrial DNA (mtDNA) myopathy. Over time, there was a progressive depletion of mtDNA, which preferentially affected wild-type mitochondrial genomes. This suggests that loss of wild-type mtDNA is a major feature of mtDNA myopathy, and preventing wild-type mtDNA depletion has treatment implications.

The helicase CaHmi1p is required for wild-type mitochondrial DNA organization in Candida albicans

The mechanistic details of mtDNA maintenance in petite-negative yeasts have remained largely unexplored. We report here that the DNA helicase Hmi1p plays a crucial role in mtDNA stability in Candida albicans. Like its counterpart in Saccharomyces cerevisiae, Hmi1p in C. albicans (CaHmi1p) contains a C-terminal mitochondrial targeting signal that is functional in both organisms. Biochemical analysis demonstrates that CaHmi1p is a protein possessing ATP-dependent 3'-5' DNA-unwinding activity. Deletion of both HMI1 alleles does not lead to complete loss of mtDNA in C. albicans; however, substantial fragmentation of the wild-type mitochondrial genome, reduction of mtDNA mass and loss of wild-type nucleoid distribution occur. Specific regions of the mitochondrial genome give rise to mtDNA molecule populations with altered characteristics upon CaHMI1 deletion. Fragmentation of the mitochondrial genome can be reversed by reintroduction of CaHmi1p. This is the first time that a gene required for wild-type mtDNA maintenance in S. cerevisiae has been demonstrated to be nonessential in a petite-negative yeast.

Approaches to the treatment of mitochondrial diseases

Therapy for mitochondrial diseases is woefully inadequate. However, lack of a cure does not equate with lack of treatment. Palliative therapy is dictated by good medical practice and includes anticonvulsant medication, control of endocrine dysfunction, and surgical procedures. Removal of noxious metabolites is centered on combating lactic acidosis, but extends to other metabolites. Attempts to bypass blocks in the respiratory chain by administration of electron acceptors have not been successful, but this may be amenable to genetic engineering. Administration of metabolites and cofactors is the mainstay of real-life therapy and is especially important in disorders due to primary deficiencies of specific compounds, such as carnitine or coenzyme Q10 (CoQ10). There is increasing interest in the administration of reactive oxygen radicals (ROS) scavengers, both in primary mitochondrial diseases and in neurodegenerative diseases. Gene therapy is a challenge because of polyplasmy and heteroplasmy, but novel experimental approaches are being pursued. One important strategy is to decrease the ratio of mutant to wild-type mitochondrial genomes ("gene shifting") by different means: (1) converting mutated mitochondrial DNA (mtDNA) genes into normal nuclear DNA genes ("allotopic expression"); (2) importing cognate genes from other species ("xenotopic expression"); (3) correcting mtDNA mutations by importing specific restriction endonucleases; (4) selecting for respiratory function; and (5) inducing muscle regeneration. Germline therapy raises ethical problems but is being considered for prevention of maternal transmission of mtDNA mutations. Preventive therapy through genetic counseling and prenatal diagnosis is becoming increasingly important for nuclear DNA-related disorders.

Rpm2p, a Component of Yeast Mitochondrial RNase P, Acts as a Transcriptional Activator in the Nucleus

Rpm2p, a protein subunit of yeast mitochondrial RNase P, has another function that is essential in cells lacking the wild-type mitochondrial genome. This function does not require the mitochondrial leader sequence and appears to affect transcription of nuclear genes. Rpm2p expressed as a fusion protein with green fluorescent protein localizes to the nucleus and activates transcription from promoters containing lexA-binding sites when fused to a heterologous DNA binding domain, lexA. The transcriptional activation region of Rpm2p contains two leucine zippers that are required for transcriptional activation and are conserved in the distantly related yeast Candida glabrata. The presence of a mitochondrial leader sequence does not prevent a portion of Rpm2p from locating to the nucleus, and several observations suggest that the nuclear location and transcriptional activation ability of Rpm2p are physiologically significant. The ability of RPM2 alleles to suppress tom40-3, a temperature-sensitive mutant of a component of the mitochondrial import apparatus, correlates with their ability to transactivate the reporter genes with lexA-binding sites. In cells lacking mitochondrial DNA, Rpm2p influences the levels of TOM40, TOM6, TOM20, TOM22, and TOM37 mRNAs, which encode components of the mitochondrial import apparatus, but not that of TOM70 mRNA. It also affects HSP60 and HSP10 mRNAs that encode essential mitochondrial chaperones. Rpm2p also increases the level of Tom40p, as well as Hsp60p, but not Atp2p, suggesting that some, but not all, nucleus-encoded mitochondrial components are affected.

Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae

Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates rho(-) and rho(0) cells. Here we demonstrate that, in rho(-) yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric rho(-) mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1-5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1Delta yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.

Aep3p Stabilizes the Mitochondrial Bicistronic mRNA Encoding Subunits 6 and 8 of the H+-translocating ATP Synthase of Saccharomyces cerevisiae

Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/6 mRNA, which differ in the length of the 5'-untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to approximately 35% that of wild type. Localization of a hemagglutinin-tagged version of Aep3p showed that the protein is an extrinsic constituent of the mitochondrial inner membrane facing the matrix.

Mitochondrial encephalomyopathies: therapeutic approach.

Therapy for mitochondrial diseases is woefully inadequate. How-ever, lack of cure does not equate with lack of treatment. In this review, we consider sequentially several different therapeutic approaches. Palliative therapy is dictated by good medical practice and includes anticonvulsant medication, control of endocrine dysfunction, and surgical procedures. Removal of noxious metabolites is centered on combating lactic acidosis, but it extends to other metabolites, such as thymidine in patients with the mitochondrial neurogastrointestinal encephalomyopathy syndrome. Attempts to bypass blocks in the respiratory chain by administration of artificial electron acceptors have not been successful, but this concept may be amenable to genetic engineering. Administration of metabolites and cofactors is the mainstay of real-life therapy and includes both components of the respiratory chain and other natural compounds. There is increasing interest in the administration of reactive oxygen species scavengers both in primary mitochondrial diseases and in neurodegenerative diseases directly or indirectly related to mitochondrial dysfunction. Aerobic exercise and physical therapy prevent or correct deconditioning and improve exercise tolerance in patients with mitochondrial myopathies due to mtDNA mutations. Gene therapy is a challenge because of polyplasmy and heteroplasmy, but interesting experimental approaches are being pursued and include, for example, decreasing the ratio of mutant to wild-type mitochondrial genomes (gene shifting), converting mutated mtDNA genes into normal nDNA genes (allotropic expression), importing cognate genes from other species, or correcting mtDNA mutations with specific restriction endonucleases. Germline therapy raises ethical problems but is being seriously considered to prevent maternal transmission of mtDNA mutations. Preventive therapy through genetic counseling and prenatal diagnosis is still limited for mtDNA-related disorders but is becoming increasingly important for nDNA-related disorders.

Rpm2p: separate domains promote tRNA and Rpm1r maturation in Saccharomyces cerevisiae mitochondria.

Rpm2p is a protein subunit of yeast mitochondrial RNase P and is also required for the maturation of Rpm1r, the mitochondrially-encoded RNA subunit of the enzyme. Previous work demonstrated that an insertional disruption of RPM2, which produces the C-terminally truncated protein Rpm2-DeltaCp, supports growth on glucose but cells lose some or all of their mitochondrial genome and become petite. These petites, even if they retain the RPM1 locus, lose their ability to process the 5'-ends of mitochondrial tRNA. We report here that if strains containing the truncated RPM2 allele are created and maintained on respiratory carbon sources they have wild-type mitochondrial genomes, and a significant portion of tRNA transcripts are processed. In contrast, precursor Rpm1r transcripts accumulate and mature Rpm1r is not made. These data show that one function of the deleted C-terminal region is in the maturation of Rpm1r, and that this region and mature Rpm1r are not absolutely required for RNase P activity. Finally, we demonstrate that full activity can be restored if the N-terminal and C-terminal domains of Rpm2p are supplied in trans.

A major deletion in the cucumber mitochondrial genome sorts with the MSC phenotype.

The cucumber mitochondrial genome is unique because of its large size, paternal transmission, and the existence of a paternally transmitted mosaic (MSC) phenotype spontaneously appearing after independent tissue culture experiments. Transmission studies eliminated paternal imprinting as the genetic basis for the MSC phenotype. We identified a 13 kb region (JLV5-DEL) in the wild-type mitochondrial genome that was absent from three MSC lines. This deleted region was paternally transmitted with the MSC phenotype through the F3 and test-cross generations. Southern hybridizations and PCR amplifications using primers within the JLV5 region revealed that rare wild-type sorters possessed the wild-type region. MSC plants possess the wild-type region at levels (approximately 0.002) below detection by standard PCR reactions and Southern hybridizations, using genomic DNA. Sequence analysis of the wild-type contig revealed no homologies to mitochondrial genes and no open reading frames within JLV5-DEL. PCR amplifications across JLV5-DEL using MSC mtDNA revealed that the loss of this region was not a simple deletion and may be associated with a rearrangement. Because no genic regions were identified within the wild-type JLV5 region, the specific lesion conditioning the MSC phenotype may be associated with the putative rearrangement or another mutation that occurred during tissue culture or exists substoichiometrically in the parental line and is transmitted together with JLV5 within the same mitochondrion.

Reintroduction of a characterized Mit tRNA glycine mutation into yeast mitochondria provides a new tool for the study of human neurodegenerative diseases.

We report the identification and characterization of a new mutation (ts9) in the Saccharomyces cerevisiae mitochondrial genome, which was first genetically mapped in the tRNAgly region and further identified by means of sequencing as consisting of a G to A transition at position 30 in the tRNA. The mutation causes an almost complete disappearance of mature tRNAgly, while a second mitochondrial mutation with a compensatory C to T change restores it in normal quantities; this points to the importance of the strong bond between bases 30 and 40 of the anticodon stem in the stabilization of the tRNA. In addition to resulting in a clear-cut heat-sensitive phenotype, the ts9 mutation creates a new EcoRV restriction site. Both properties were used as markers to monitor the successful (re) introduction of the mutated allele into a wild-type mitochondrial genome through biolistic transformation. The mutant frequency in the progeny as well as the correct integration of the mutated allele at its proper site demonstrate the feasibility of this method for creating and investigating specific mitochondrial tRNA mutations. The method will provide important applications for the use of yeast as a model system of human mitochondrial pathologies.

The CBP2 gene from Saccharomyces douglasii is a functional homologue of the Saccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome.

In Saccharomyces cerevisiae the only known role of the CBP2 gene is the excision of the fifth intron of the mitochondrial cyt b gene (bI5). We have cloned the CBP2 gene from Saccharomyces douglasii (a close relative of S. cerevisiae). A comparison of the S. douglasii and S. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that the S. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome, but not in the presence of an intronless S. cerevisiae mitochondrial genome. Also the S. douglasii and S. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from the S. douglasii mitochondrial genome.

Phenotype-genotype correlations in skeletal muscle of patients with mtDNA deletions.

Large-scale deletions of mitochondrial DNA (mtDNA) have been associated with a subgroup of mitochondrial encephalomyopathies, usually characterized by progressive external ophthalmoplegia (PEO) and mitochondrial proliferation in muscle fibers. We and others have shown that muscle from patients with mtDNA deletions have variable cytochrome c oxidase (COX) deficiency and reduction of mitochondrially-synthesized polypeptides in affected muscle fibers. The present work summarizes the phenotype-genotype correlations observed in patients' muscle. In situ hybridization revealed that, while most COX-deficient fibers had increased levels of mutant mtDNA, they almost invariably had reduced levels of normal mtDNA. PCR quantitation of both deleted and wild-type mtDNAs in normal and respiration-deficient muscle fibers from patients with the "common deletion" showed that deleted mtDNAs were present in normal fibers (31 +/- 26%), but their percentages were much higher in affected fibers (95% +/- 2%). Absolute levels of deleted mtDNA were also increased in affected fibers, whereas absolute levels of wild-type mtDNA were significantly reduced. Taken together, our results suggest that although a specific ratio between mutant and wild-type mitochondrial genomes is probably the major determinant of the respiratory chain deficiency associated with mtDNA deletions, the reduction in the absolute amounts of wild-type mtDNA may also play a significant pathogenetic role.

Induction of respiration-deficient mutants in Saccharomyces cerevisiae by chelerythrine

Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae, chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.

Induction of respiration-deficient mutants inSaccharomyces cerevisiaeby chelerythrine

Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae, chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.

Complementation of mutant and wild-type human mitochondrial DNAs coexisting since the mutation event and lack of complementation of DNAs introduced separately into a cell within distinct organelles.

The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions. Among mitochondrial transformants derived from an individual affected by the MERRF (myoclonus epilepsy associated with ragged red fibers) encephalomyopathy and carrying in heteroplasmic form the mitochondrial tRNA(Lys) mutation associated with that syndrome, normal protein synthesis and respiration was observed when the wild-type mitochondrial DNA exceeded 10% of the total complement. In these transformants, the protective effect of wild-type mitochondrial DNA was shown to involve interactions of the mutant and wild-type gene products. Very different results were obtained in experiments in which two mitochondrial DNAs carrying nonallelic disease-causing mutations were sequentially introduced within distinct organelles into the same human mitochondrial DNA-less (rho 0) cell. In transformants exhibiting different ratios of the two genomes, no evidence of cooperation between their products was observed, even 3 months after the introduction of the second mutation. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which mitochondria carrying a chloramphenicol resistance-inducing mitochondrial DNA mutation were introduced into chloramphenicol-sensitive cells. A plausible interpretation of the different results obtained in the latter two sets of experiments, compared with the complementation behavior observed in the heteroplasmic MERRF transformants, is that in the latter, the mutant and wild-type genomes coexisted in the same organelles from the time of the mutation. This would imply that the way in which mitochondrial DNA is sorted among different organelles plays a fundamental role in determining the oxidative-phosphorylation phenotype in mammalian cells. These results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.

Complementation of mutant and wild-type human mitochondrial DNAs coexisting since the mutation event and lack of complementation of DNAs introduced separately into a cell within distinct organelles.

The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions. Among mitochondrial transformants derived from an individual affected by the MERRF (myoclonus epilepsy associated with ragged red fibers) encephalomyopathy and carrying in heteroplasmic form the mitochondrial tRNA(Lys) mutation associated with that syndrome, normal protein synthesis and respiration was observed when the wild-type mitochondrial DNA exceeded 10% of the total complement. In these transformants, the protective effect of wild-type mitochondrial DNA was shown to involve interactions of the mutant and wild-type gene products. Very different results were obtained in experiments in which two mitochondrial DNAs carrying nonallelic disease-causing mutations were sequentially introduced within distinct organelles into the same human mitochondrial DNA-less (rho 0) cell. In transformants exhibiting different ratios of the two genomes, no evidence of cooperation between their products was observed, even 3 months after the introduction of the second mutation. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which mitochondria carrying a chloramphenicol resistance-inducing mitochondrial DNA mutation were introduced into chloramphenicol-sensitive cells. A plausible interpretation of the different results obtained in the latter two sets of experiments, compared with the complementation behavior observed in the heteroplasmic MERRF transformants, is that in the latter, the mutant and wild-type genomes coexisted in the same organelles from the time of the mutation. This would imply that the way in which mitochondrial DNA is sorted among different organelles plays a fundamental role in determining the oxidative-phosphorylation phenotype in mammalian cells. These results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.

Deletion of mitochondrial DNA in a case of early-onset diabetes mellitus, optic atrophy, and deafness (Wolfram syndrome, MIM 222300).

The Wolfram syndrome (MIM 222300) is a disease of unknown origin consisting of diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Here we report on a generalized deficiency of the mitochondrial respiratory enzyme activities in skeletal muscle and lymphocyte homogenate of a girl suffering from the Wolfram syndrome. In addition, we provide evidence for a 7.6-kilobase pair heteroplasmic deletion (spanning nucleotides 6465-14135) of the mitochondrial DNA in the two tissues and show that directly repeated sequences (11 bp) were present in the wild-type mitochondrial genome at the boundaries of the deletion. Neither of the patient's parents was found to bear rearranged molecules. This study supports the view that a respiratory chain defect can present with insulin-dependent diabetes mellitus as the onset symptom. It also suggests that a defect of oxidative phosphorylation should be considered when investigating other cases of Wolfram syndrome, especially because this syndrome fulfills the criteria for a genetic defect of the mitochondrial energy supply: (a) an unexplained association of symptoms (b) with early onset and rapidly progressive course, (c) involving seemingly unrelated organs and tissues.