Mitochondria.

Following the discovery in the early 1960s that mitochondria contain their own DNA (mtDNA), there were two major advances, both in the 1980s: the human mtDNA sequence was published in 1981, and in 1988 the first pathogenic mtDNA mutations were identified. The floodgates were opened, and the 1990s became the decade of the mitochondrial genome. There has been a change of emphasis in the first few years of the new millennium, away from the "magic circle" of mtDNA and back to the nuclear genome. Various nuclear genes have been identified that are fundamentally important for mitochondrial homeostasis, and when these genes are disrupted, they cause autosomally inherited mitochondrial disease. Moreover, mitochondrial dysfunction plays an important role in the pathophysiology of several well established nuclear genetic disorders, such as dominant optic atrophy (mutations in OPA1), Friedreich's ataxia (FRDA), hereditary spastic paraplegia (SPG7), and Wilson's disease (ATP7B). The next major challenge is to define the more subtle interactions between nuclear and mitochondrial genes in health and disease.

Pathogenesis of primary defects in mitochondrial ATP synthesis.

Maternally inherited mutations in the mtDNA-encoded ATPase 6 subunit of complex V (ATP synthase) of the respiratory chain/oxidative phosphorylation system are responsible for a subgroup of severe and often-fatal disorders characterized predominantly by lesions in the brain, particularly in the striatum. These include NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh syndrome), and FBSN (familial bilateral striatal necrosis). Of the five known pathogenic mutations causing these disorders, four are located at two codons (156 and 217), each of which can suffer mutations converting a conserved leucine to either an arginine or a proline. Based on the accumulating data on both the structure of ATP synthase and the mechanism by which rotary catalysis couples proton flow to ATP synthesis, we propose a model that may help explain why mutations at codons 156 and 217 are pathogenic.

Mitochondrial DNA mutations in human disease.

The small, maternally inherited mitochondrial DNA (mtDNA) has turned out to be a Pandora's box of pathogenic mutations: 13 years into the era of "molecular mitochondrial medicine," more than 100 pathogenic point mutations and innumerable rearrangements have been associated with a striking variety of multisystemic as well as tissue-specific human diseases. After reviewing the principles of mitochondrial genetics, we consider disorders due to mutations in genes affecting mitochondrial protein synthesis and disorders due to mutations in protein-coding genes. In contrast to the remarkable progress in our understanding of etiology, pathogenesis is only partially explained by the rules of mitochondrial genetics and remains largely unclear. We review recent progress in prenatal diagnosis, epidemiology, and in the development of animal models harboring mtDNA mutations.

Both heavy strand replication origins are active in partially duplicated human mitochondrial DNAs.

The replication of human mitochondrial DNA (mtDNA) is initiated from a pair of displaced origins, one priming continuous synthesis of daughter-strand DNA from the heavy strand (OH) and the other priming continuous synthesis from the light strand (OL). In patients with sporadic large-scale rearrangements of mitochondrial DNA (i.e., partially-deleted [Delta-mtDNA] and partially-duplicated [dup-mtDNA] molecules), the dup-mtDNAs typically contain extra origins of replication, but it is unknown at present whether they are competent for initiation of replication. Using cybrids harboring each of two types of dup-mtDNAs-one containing two OHs and two OLs, and one containing two OHs and one OL-we used ligation-mediated polymerase chain reaction (LMPCR) to measure the presence and relative amounts of nascent heavy strands originating from each OH. We found that the nascent heavy strands originated almost equally from the two OHs in each cell line, indicating that the extra OH present on a partially duplicated mtDNA is competent for heavy strand synthesis. This extra OH could potentially confer a replicative advantage to dup-mtDNAs, as these molecules may have twice as many opportunities to initiate replication compared to wild-type (or partially deleted) molecules.

Mitochondrial genetics and disease.

Mitochondrial respiratory chain diseases are a highly diverse group of disorders whose main unifying characteristic is the impairment of mitochondrial function. As befits an organelle containing gene products encoded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), these diseases can be caused by inherited errors in either genome, but a surprising number are sporadic, and a few are even caused by environmental factors.

Kearns-sayre syndrome: oncocytic transformation of choroid plexus epithelium.

Kearns-Sayre syndrome (KSS) is a sporadic multisystem disorder due to a defect of oxidative phosphorylation and associated with clonally-expanded rearrangements of mitochondrial DNA (mtDNA) deletions (Delta-mtDNAs) and/or duplications (dup-mtDNAs). To gain further insight into the pathogenesis of CNS dysfunction in KSS, we studied the choroid plexus from two autoptic cases using in situ hybridization (ISH) of mtDNA, and immunohistochemistry to detect mtDNA and nuclear DNA-encoded subunits of the respiratory chain. Neuropathological examination of both cases showed oncocytic transformation of choroid plexus epithelial cells. In the same cells, ISH demonstrated that the predominant species of mtDNA were Delta-mtDNAs, and immunohistochemistry showed a decreased expression of mtDNA-encoded proteins. We suggest that mitochondrial abnormalities due to the presence of abundant Delta-mtDNAs in the choroid plexus play an important role in causing the increased cerebrospinal fluid (CSF) protein and reduced folic-acid levels that are characteristic of KSS.

Chromosomal non-disjunction in human oocytes: is there a mitochondrial connection?

The frequency of chromosome abnormalities due to non-disjunction of maternal chromosomes during meiosis is a function of age, with a sharp increase in the slope of the trisomy-age curve between the ages of 30 and 40 years. The basis of this increase, which is a major cause of birth defects, is unknown at present. In recent years, mutations in mitochondrial (mt) DNA have been associated with a growing number of disorders, including those associated with spontaneous deletions of mtDNA (deltamt DNAs). Intriguingly, these pathogenic deltamtDNAs, which are present at extremely high levels in certain patients, are also present at extremely low levels (detectable only by polymerase chain reaction) in normal individuals. The proportion of such deltamtDNAs in normal muscle is a function of age; the shape of this curve is exponential, with the accelerating part of the curve beginning at approximately 30-40 years. We postulate that, as well as muscle and brain, a similar time-dependent accumulation of deltamtDNAs also occurs in normal oocytes. Since deltamtDNAs are functionally inactive, an accumulation of such aberrant genomes could eventually compromise ATP-dependent energy-utilization in these cells. Furthermore, these deficiencies would also affect the function of the somatic follicular cells that surround, and secrete important paracrine factors to, the oocyte. If there is indeed an age-associated relationship between deltamtDNAs and oocyte age, perhaps errors in meiosis (which is almost certainly an energy, and ATP, dependent process) are related to mutations in mtDNA (primarily deletions, but perhaps point mutations as well) in oocytes and/or the surrounding somatic cells, which result in deficiencies in both mitochondrial function in general and oxidative energy metabolism in particular. This hypothesis would explain many of the non-Mendelian features associated with maternal age-related trisomies, e.g. Down's syndrome.

Maintenance of human rearranged mitochondrial DNAs in long-term cultured transmitochondrial cell lines.

Large-scale rearrangements of mitochondrial DNA (mtDNA; i.e., partial duplications [dup-mtDNAs] and deletions [Delta-mtDNAs]) coexist in tissues in a subset of patients with sporadic mitochondrial disorders. In order to study the dynamic relationship among rearranged and wild-type mtDNA (wt-mtDNA) species, we created transmitochondrial cell lines harboring various proportions of wt-, Delta-, and dup-mtDNAs from two patients. After prolonged culture in nonselective media, cells that contained initially 100% dup-mtDNAs became heteroplasmic, containing both wild-type and rearranged mtDNAs, likely generated via intramolecular recombination events. However, in cells that contained initially a mixture of both wt- and Delta-mtDNAs, we did not observe any dup-mtDNAs or other new forms of rearranged mtDNAs, perhaps because the two species were physically separated and were therefore unable to recombine. The ratio of wt-mtDNA to Delta-mtDNAs remained stable in all cells examined, suggesting that there was no replicative advantage for the smaller deleted molecules. Finally, in cells containing a mixture of monomeric and dimeric forms of a specific Delta-mtDNA, we found that the mtDNA population shifted towards homoplasmic dimers, suggesting that there may be circumstances under which the cells favor molecules with multiple replication origins, independent of the size of the molecule.

A human SCO2 mutation helps define the role of Sco1p in the cytochrome oxidase assembly pathway.

Deficiencies in cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, are most often caused by an inability to complete assembly of the enzyme. Pathogenic mutations in SCO2, which encodes a cytochrome oxidase assembly factor, were recently described in several cases of fatal infantile cardioencephalomyopathy. To determine the molecular etiology of these disorders, we describe the generation and characterization of the parallel mutations in the homologous yeast SCO1 gene. We show that the E155K yeast sco1 mutant is respiration-competent, whereas the S240F mutant is not. Interestingly, the S240F mutation allows partial but incorrect assembly of cytochrome oxidase, as judged by an altered cytochrome aa(3) peak. Immunoblot analysis reveals a specific absence of subunit 2 from the cytochrome oxidase in this mutant. Taken together, our data suggest that Sco1p provides copper to the Cu(A) site on subunit 2 at a step occurring late in the assembly pathway. This is the first instance of a yeast cytochrome oxidase assembly mutant that is partially assembled. The S240F mutant also represents a powerful new tool with which to elucidate further steps in the cytochrome oxidase assembly pathway.

Intragenic inversion of mtDNA: a new type of pathogenic mutation in a patient with mitochondrial myopathy.

We report an unusual molecular defect in the mitochondrially encoded ND1 subunit of NADH ubiquinone oxidoreductase (complex I) in a patient with mitochondrial myopathy and isolated complex I deficiency. The mutation is an inversion of seven nucleotides within the ND1 gene, which maintains the reading frame. The inversion, which alters three highly conserved amino acids in the polypeptide, was heteroplasmic in the patient's muscle but was not detectable in blood. This is the first report of a pathogenic inversion mutation in human mtDNA.

Rearrangements of human mitochondrial DNA (mtDNA): new insights into the regulation of mtDNA copy number and gene expression.

Mitochondria from patients with Kearns-Sayre syndrome harboring large-scale rearrangements of human mitochondrial DNA (mtDNA; both partial deletions and a partial duplication) were introduced into human cells lacking endogenous mtDNA. Cytoplasmic hybrids containing 100% wild-type mtDNA, 100% mtDNA with partial duplications, and 100% mtDNA with partial deletions were isolated and characterized. The cell lines with 100% deleted mtDNAs exhibited a complete impairment of respiratory chain function and oxidative phosphorylation. In contrast, there were no detectable respiratory chain or protein synthesis defects in the cell lines with 100% duplicated mtDNAs. Unexpectedly, the mass of mtDNA was identical in all cell lines, despite the fact that different lines contained mtDNAs of vastly different sizes and with different numbers of replication origins, suggesting that mtDNA copy number may be regulated by tightly controlled mitochondrial dNTP pools. In addition, quantitation of mtDNA-encoded RNAs and polypeptides in these lines provided evidence that mtDNA gene copy number affects gene expression, which, in turn, is regulated at both the post-transcriptional and translational levels.

Mitochondrial encephalomyopathies: 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. We review available and experimental therapeutic approaches, which fall into seven categories: (1) palliative therapy; (2) removal of noxious metabolites; (3) administration of artificial electron acceptors; (4) administration of metabolites and cofactors; (5) administration of oxygen radical scavengers; (6) gene therapy; and (7) genetic counseling. Progress in each of these approaches provides some glimmer of hope for the future, although much work remains to be done.

Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene.

Mammalian cytochrome c oxidase (COX) catalyses the transfer of reducing equivalents from cytochrome c to molecular oxygen and pumps protons across the inner mitochondrial membrane. Mitochondrial DNA (mtDNA) encodes three COX subunits (I-III) and nuclear DNA (nDNA) encodes ten. In addition, ancillary proteins are required for the correct assembly and function of COX (refs 2, 3, 4, 5, 6). Although pathogenic mutations in mtDNA-encoded COX subunits have been described, no mutations in the nDNA-encoded subunits have been uncovered in any mendelian-inherited COX deficiency disorder. In yeast, two related COX assembly genes, SCO1 and SCO2 (for synthesis of cytochrome c oxidase), enable subunits I and II to be incorporated into the holoprotein. Here we have identified mutations in the human homologue, SCO2, in three unrelated infants with a newly recognized fatal cardioencephalomyopathy and COX deficiency. Immunohistochemical studies implied that the enzymatic deficiency, which was most severe in cardiac and skeletal muscle, was due to the loss of mtDNA-encoded COX subunits. The clinical phenotype caused by mutations in human SCO2 differs from that caused by mutations in SURF1, the only other known COX assembly gene associated with a human disease, Leigh syndrome.

Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep.

Eukaryotic cells contain two distinct genomes. One is located in the nucleus (nDNA) and is transmitted in a mendelian fashion, whereas the other is located in mitochondria (mtDNA) and is transmitted by maternal inheritance. Cloning of mammals typically has been achieved via nuclear transfer, in which a donor somatic cell is fused by electoporation with a recipient enucleated oocyte. During this whole-cell electrofusion, nDNA as well as mtDNA ought to be transferred to the oocyte. Thus, the cloned progeny should harbour mtDNAs from both the donor and recipient cytoplasms, resulting in heteroplasmy. Although the confirmation of nuclear transfer has been established using somatic cell-specific nDNA markers, no similar analysis of the mtDNA genotype has been reported. We report here the origin of the mtDNA in Dolly, the first animal cloned from an established adult somatic cell line, and in nine other nuclear transfer-derived sheep generated from fetal cells. The mtDNA of each of the ten nuclear-transfer sheep was derived exclusively from recipient enucleated oocytes, with no detectable contribution from the respective somatic donor cells. Thus, although these ten sheep are authentic nuclear clones, they are in fact genetic chimaeras, containing somatic cell-derived nuclear DNA but oocyte-derived mtDNA.

A calcium signaling defect in the pathogenesis of a mitochondrial DNA inherited oxidative phosphorylation deficiency.

In recent years, genetic defects of the mitochondrial genome (mtDNA) were shown to be associated with a heterogeneous group of disorders, known as mitochondrial diseases, but the cellular events deriving from the molecular lesions and the mechanistic basis of the specificity of the syndromes are still incompletely understood. Mitochondrial calcium (Ca2+) homeostasis depends on close contacts with the endoplasmic reticulum and is essential in modulating organelle function. Given the strong dependence of mitochondrial Ca2+ uptake on the membrane potential and the intracellular distribution of the organelle, both of which may be altered in mitochondrial diseases, we investigated the occurrence of defects in mitochondrial Ca2+ handling in living cells with either the tRNALys mutation of MERRF (myoclonic epilepsy with ragged-red fibers) or the ATPase mutation of NARP (neurogenic muscle weakness, ataxia and retinitis pigmentosa). There was a derangement of mitochondrial Ca2+ homeostasis in MERRF, but not in NARP cells, whereas cytosolic Ca2+ responses were normal in both cell types. Treatment of MERRF cells with drugs affecting organellar Ca2+ transport mostly restored both the agonist-dependent mitochondrial Ca2+ uptake and the ensuing stimulation of ATP production. These results emphasize the differences in the cellular pathogenesis of the various mtDNA defects and indicate specific pharmacological approaches to the treatment of some mitochondrial diseases.

Kearns-Sayre syndrome: unusual pattern of expression of subunits of the respiratory chain in the cerebellar system.

Kearns-Sayre syndrome (KSS) is a sporadic multisystem disorder of oxidative phosphorylation associated with clonally expanded rearrangements of mitochondrial DNA (mtDNA). Mitochondrial dysfunction in the central nervous system of patients with KSS accounts for the neurological manifestations of the disease. To gain further insight into the pathogenesis of neuronal dysfunction in KSS, we used antibodies against mtDNA-encoded and nuclear DNA-encoded subunits of the mitochondrial respiratory chain to study the expression of these proteins in the cerebellar cortex, dentate nucleus, and inferior olivary nucleus from 2 autoptic cases of KSS. Neuropathological examination showed a moderate loss of Purkinje cells and spongiform degeneration of the cerebellar white matter. By using immunohistochemistry, we found a decreased expression of mtDNA-encoded proteins only in neurons of the dentate nucleus. We suggest that mitochondrial abnormalities in the dentate nucleus in conjunction with loss of Purkinje cells and spongiform degeneration of the cerebellar white matter may be important factors in the genesis of the cerebellar dysfunction in KSS.

Mitochondrial involvement in Alzheimer's disease.

The causes of most neurodegenerative diseases, including sporadic Alzheimer's disease (AD), remain enigmatic. There is, however, increasing evidence implicating mitochondrial dysfunction resulting from deafferentiation of disconnected neural circuits in the pathogenesis of energy deficit in AD. The patterns of reduced expression of both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) encoded genes is consistent with a physiological down-regulation of the mitochondrial respiratory chain in response to reduced neuronal activity. On the other hand, the role(s) of somatic cell or maternally inherited mtDNA mutations in the pathogenesis of mitochondrial dysfunction in AD are still controversial.

Oligomycin induces a decrease in the cellular content of a pathogenic mutation in the human mitochondrial ATPase 6 gene.

A T --> G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.