Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage.

Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer's disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent °( mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the °cells, demonstrating that these sequences are also present in the human nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then tested for each of the five ''AD'' missense mutations by restriction endonuclease site variant assays. All five mutations were found in the nuclear CO1 and CO2 PCR products from °cells, but none were found in the PCR products obtained from cells with normal mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products were cloned and sequenced, all five missense mutations were found, as well as a linked synonymous mutation. Unlike the findings in the original report, an additional 32 base substitutions were found, including two in adjacent tRNAs and a two base pair deletion in the CO2 gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences revealed that they diverged from modern human mtDNAs early in hominid evolution about 770,000 years before present. These data would be consistent with the interpretation that the missense mutations proposed to cause AD may be the product of ancient mtDNA variants preserved as nuclear pseudogenes.

We report that oligonucleotides can be introduced into the mitochondria of living mammalian cells by annealing them to peptide nucleic acids coupled to mitochondrial targeting peptides. These complexes are imported into the mitochondrial matrix through the outer and inner membrane import channels of isolated mitochondria. They are also imported into the mitochondria of cultured cells, provided that the cytosolic uptake of the complexes is facilitated by using synthetic polycations or membrane permeabilizing toxins. Our method now promises to provide a viable strategy for the genetic modification of the mitochondria in cultured cells, animals and patients.

Changes in the gut microbiota and the mitochondrial genome are both linked with the development of disease. To investigate why, we examined the gut microbiota of mice harboring various mutations in genes that alter mitochondrial function. These studies revealed that mitochondrial genetic variations altered the composition of the gut microbiota community. In cross-fostering studies, we found that although the initial microbiota community of newborn mice was that obtained from the nursing mother, the microbiota community progressed toward that characteristic of the microbiome of unfostered pups of the same genotype within 2 months. Analysis of the mitochondrial DNA variants associated with altered gut microbiota suggested that microbiome species diversity correlated with host reactive oxygen species (ROS) production. To determine whether the abundance of ROS could alter the gut microbiota, mice were aged, treated with N-acetylcysteine, or engineered to express the ROS scavenger catalase specifically within the mitochondria. All three conditions altered the microbiota from that initially established. Thus, these data suggest that the mitochondrial genotype modulates both ROS production and the species diversity of the gut microbiome, implying that the connection between the gut microbiome and common disease phenotypes might be due to underlying changes in mitochondrial function.

Diabetes is associated with impaired glucose metabolism in the presence of excess insulin. Glucose and fatty acids provide reducing equivalents to mitochondria to generate energy, and studies have reported mitochondrial dysfunction in type II diabetes patients. If mitochondrial dysfunction can cause diabetes, then we hypothesized that increased mitochondrial metabolism should render animals resistant to diabetes. This was confirmed in mice in which the heart-muscle-brain adenine nucleotide translocator isoform 1 (ANT1) was inactivated. ANT1-deficient animals are insulin-hypersensitive, glucose-tolerant, and resistant to high fat diet (HFD)-induced toxicity. In ANT1-deficient skeletal muscle, mitochondrial gene expression is induced in association with the hyperproliferation of mitochondria. The ANT1-deficient muscle mitochondria produce excess reactive oxygen species (ROS) and are partially uncoupled. Hence, the muscle respiration under nonphosphorylating conditions is increased. Muscle transcriptome analysis revealed the induction of mitochondrial biogenesis, down-regulation of diabetes-related genes, and increased expression of the genes encoding the myokines FGF21 and GDF15. However, FGF21 was not elevated in serum, and FGF21 and UCP1 mRNAs were not induced in liver or brown adipose tissue (BAT). Hence, increased oxidation of dietary-reducing equivalents by elevated muscle mitochondrial respiration appears to be the mechanism by which ANT1-deficient mice prevent diabetes, demonstrating that the rate of mitochondrial oxidation of calories is important in the etiology of metabolic disease. mitochondria | skeletal muscle | ANT1 | insulin sensitivity

Mitochondrial DNA (mtDNA) haplogroups are valuable for investigations in forensic science, molecular anthropology, and human genetics. In this study, we developed a custom panel of 61 mtDNA markers for high-throughput classification of European, African, and Native American/Asian mitochondrial haplogroup lineages. Using these mtDNA markers we constructed a mitochondrial haplogroup classification tree and classified 18,832 participants from the National Health and Nutrition Examination Surveys (NHANES). To our knowledge, this is the largest study to date characterizing mitochondrial haplogroups in a population-based sample from the United States, and the first study characterizing mitochondrial haplogroup distributions in self-identified Mexican Americans separately from Hispanic Americans of other descent. We observed clear differences in the distribution of maternal genetic ancestry consistent with proposed admixture models for these subpopulations, underscoring the genetic heterogeneity of the United States Hispanic population. The mitochondrial haplogroup distributions in the other self-identified racial/ethnic groups within NHANES were largely comparable to previous studies. Mitochondrial haplogroup classification was highly concordant with self-identified race/ethnicity (SIRE) in non-Hispanic whites (94.8%), but was considerably lower in admixed populations including non-Hispanic blacks (88.3%), Mexican Americans (81.8%), and other Hispanics (61.6%), suggesting SIRE does not accurately reflect maternal genetic ancestry, particularly in populations with greater proportions of admixture. Thus, it is important to consider inconsistencies between SIRE and genetic ancestry when performing genetic association studies. The mitochondrial haplogroup data that we have generated, coupled with the epidemiologic variables in NHANES, is a valuable resource for future studies investigating the contribution of mtDNA variation to human health and disease.

The peptide nucleic acid (PNA)-directed PCR clamping technique was modified and applied to the detection of mitochondrial DNA mutations with low heteroplasmy. This method is extremely specific, eliminating false positives in the absence of mutant molecules, and highly sensitive, being capable of detecting mutations at the level of 0.1% of total molecules. Moreover, the reaction can be multiplexed to identify more than one mutation per reaction. Using this technique, the levels of three point mutations, the tRNA(Leu(UUA)) 3243 mutation causing mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS); the tRNA(Lys) 8344 mutation causing myoclonic epilepsy and ragged red fibers (MERRF); and the nucleotide position 414 mutation adjacent to the control region promoters, were evaluated in human brain and muscle from individuals of various ages. While none of the mutations were detected in brain samples from individuals ranging in age from 23 to 93, the 414 mutation could be detected in muscle from individuals 30 years and older. These data demonstrate that the 3243 and 8344 mutations do not accumulate with age to levels greater than 0.1% in brain and muscle. By contrast, the 414 mutation accumulates with age in normal human muscle, though not in brain. The reason for the striking absence of the 414 mutation in aging brain is unknown.

Mice deficient in the heart/muscle specific isoform of the adenine nucleotide translocator (ANT1) exhibit many of the hallmarks of human oxidative phosphorylation (OXPHOS) disease, including a dramatic proliferation of skeletal muscle mitochondria. Because many of the genes necessary for mitochondrial biosynthesis, OXPHOS function, and response to OXPHOS disease might be expected to be up-regulated in the Ant1 ؊/؊ mouse, we used differential display reverse transcription-polymerase chain reaction techniques in an effort to identify these genes. 17 genes were identified as upregulated in Ant1-deficient mice, and they fall into four categories: 1) nuclear and mitochondrial genes encoding OXPHOS components, 2) mitochondrial tRNA and rRNA genes, 3) genes involved in intermediary metabolism, and 4) an eclectic group of other genes. Among the latter genes, we identified the gene encoding anti-apoptotic Mcl-1, the Skd3 gene, and the WS-3 gene, which were previously unknown to be related to mitochondrial function. These results indicate that identification of genes up-regulated in the skeletal muscle of the Ant1-deficient mouse provides a novel method for identifying mammalian genes required for mitochondrial biogenesis.The mitochondria generate most of the cells' energy via the process oxidative phosphorylation (OXPHOS).1 OXPHOS is catalyzed by five multi-subunit enzyme complexes (complexes I-V) located within the inner mitochondrial membrane. The protein subunits necessary for oxidative phosphorylation are encoded by both the nuclear and mitochondrial genome. The mitochondrial genome encodes 13 polypeptides involved in oxidative phosphorylation, including seven subunits of complex I (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6), one subunit (cytochrome b) of complex III, three subunits of complex IV (COI, COII, and COIII), and two subunits of complex V (ATPases 6 and 8) along with 22 tRNAs and 2 rRNA subunits necessary for translation of these polypeptides. Transcription of the mtDNA initiates at two sites, yielding polycistronic messages in which regions coding for protein are interspersed by regions coding for tRNA (1). The mature mRNAs, rRNAs, and tRNAs are released by cleavage. The nuclear genome encodes all of the remaining genes for mitochondrial biogenesis. These include the remaining subunits for OXPHOS; all of the proteins for mitochondrial regulation, transcription, and translation; and all proteins for mitochondrial intermediary metabolism (2).Mutations in both the mitochondrial DNA and the nuclear DNA have been shown to cause OXPHOS disease. OXPHOS diseases have a highly variable clinical spectrum, showing the progressive defects in tissues most reliant on cellular energy, with central nervous system, skeletal muscle, and heart frequently being affected. Mitochondrial myopathies are characterized pathologically by degeneration of the contractile elements and the proliferation of subsarcolemmal mitochondria, resulting in ragged red muscle fibers as revealed by the modified Gomori-trichrome stain. The proliferati...