Eric A. Schon

Deletions of mitochondrial DNA in Kearns-Sayre syndrome

We have identified large-scale deletions in muscle mitochondrial DNA (mtDN A) in seven of seven patients with Kearns-Sayre syndrome (KSS). We found no detectable deletions in the mtDNA of ten non-KSS patients with other mitochondrial myopathies or encephalomyopathies, or three normal controls. The deletions ranged in size from 2.0 to 7.0 kb, and did not localize to any single region of the mitochondrial genome. The proportion of mutated genomes in each KSS patient ranged from 45% to 75% of total mtDNA. There was no correlation between the size or site of the deletion, biochemical abnormality of mitochondrial enzymes, or clinical severity. The data bolster arguments that KSS is a unique disorder and genetic in origin.

Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy

Long-term zidovudine therapy in patients with human immunodeficiency virus (HIV) infection can cause a destructive mitochondrial myopathy with histological features of ragged-red fibres (RRF) and proliferation of abnormal mitochondria. In 9 zidovudine-treated patients with this myopathy we found severely reduced amounts (up to 78% reduction vs normal adult controls) of mitochondrial DNA (mtDNA) in muscle biopsy specimens by means of Southern blotting. In 2 HIV-positive patients who had not received zidovudine, muscle mtDNA content did not differ from that in the 4 controls. Depletion of mtDNA seems to be reversible, since 1 patient showed a substantial reduction in RRF and a concomitant pronounced increase in muscle mtDNA content after zidovudine therapy was discontinued. Depletion of muscle mtDNA is probably due to zidovudine-induced inhibition of mtDNA replication by DNA polymerase gamma and is not a secondary effect of HIV infection.

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 Disorders in the Nervous System

Mitochondrial diseases (encephalomyopathies) have traditionally been ascribed to defects of the respiratory chain, which has helped researchers explain their genetic and clinical complexity. However, other mitochondrial functions are greatly important for the nervous system, including protein importation, organellar dynamics, and programmed cell death. Defects in genes controlling these functions are attracting increasing attention as causes not only of neurological (and psychiatric) diseases but also of age-related neurodegenerative disorders. After discussing some pathogenic conundrums regarding the neurological manifestations of the respiratory chain defects, we review altered mitochondrial dynamics in the etiology of specific neurological diseases and in the physiopathology of more common neurodegenerative disorders.

Mitochondrial DNA Mutations and Pathogenesis

Approximately three years ago, this journal published a review on the clinical and molecular analysis of mitochondrial encephalomyopathies, with emphasis on defects in mitochondrial DNA (mtDNA). At that time, approximately 30 point mutations associated with a variety of maternally-inherited (or rarely, sporadic) disorders had been described. Since that time, almost twenty new pathogenic mtDNA point mutations have been described, and the pace of discovery of such mutations shows no signs of abating. This accumulating body of data has begun to reveal some patterns that may be relevant to pathogenesis.

Defects in mitochondrial protein synthesis and respiratory chain activity segregate with the tRNA(Leu(UUR)) mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes.

Cytoplasts from two unrelated patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) harboring an A----G transition at nucleotide position 3243 in the tRNA(Leu(UUR)) gene of the mitochondrial genome were fused with human cells lacking endogenous mitochondrial DNA (mtDNA) (rho 0 cells). Selected cybrid lines, containing less than 15 or greater than or equal to 95% mutated genomes, were examined for differences in genetic, biochemical, and morphological characteristics. Cybrids containing greater than or equal to 95% mutant mtDNA, but not those containing normal mtDNA, exhibited decreases in the rates of synthesis and in the steady-state levels of the mitochondrial translation products. In addition, NADH dehydrogenase subunit 1 (ND 1) exhibited a slightly altered mobility on polyacrylamide gel electrophoresis. The mutation also correlated with a severe respiratory chain deficiency. A small but consistent increase in the steady-state levels of an RNA transcript corresponding to 16S rRNA + tRNA(Leu(UUR)) + ND 1 genes was detected. However, there was no evidence of major errors in processing of the heavy-strand-encoded transcripts or of altered steady-state levels or ratios of mitochondrial rRNAs or mRNAs. These results provide evidence for a direct relationship between the tRNALeu(UUR) mutation and the pathogenesis of this mitochondrial disease.

Mitochondria: The Next (Neurode)Generation

Adult-onset neurodegenerative disorders are disabling and often fatal diseases of the nervous system whose underlying mechanisms of cell death remain unknown. Defects in mitochondrial respiration had previously been proposed to contribute to the occurrence of many, if not all, of the most common neurodegenerative disorders. However, the discovery of genes mutated in hereditary forms of these enigmatic diseases has additionally suggested defects in mitochondrial dynamics. Such disturbances can lead to changes in mitochondrial trafficking, in interorganellar communication, and in mitochondrial quality control. These new mechanisms by which mitochondria may also be linked to neurodegeneration will likely have far-reaching implications for our understanding of the pathophysiology and treatment of adult-onset neurodegenerative disorders.

Human mitochondrial DNA: roles of inherited and somatic mutations

Mutations in the human mitochondrial genome are known to cause an array of diverse disorders, most of which are maternally inherited, and all of which are associated with defects in oxidative energy metabolism. It is now emerging that somatic mutations in mitochondrial DNA (mtDNA) are also linked to other complex traits, including neurodegenerative diseases, ageing and cancer. Here we discuss insights into the roles of mtDNA mutations in a wide variety of diseases, highlighting the interesting genetic characteristics of the mitochondrial genome and challenges in studying its contribution to pathogenesis.

Neuronal degeneration and mitochondrial dysfunction

Over the last decade, the underlying genetic bases of several neurodegenerative disorders, including Huntington disease (HD), Friedreich ataxia, hereditary spastic paraplegia, and rare familial forms of Parkinson disease (PD), Alzheimer disease (AD), and amyotrophic lateral sclerosis (ALS), have been identified. However, the etiologies of sporadic AD, PD, and ALS, which are among the most common neurodegenerative diseases, are still unclear, as are the pathogenic mechanisms giving rise to the various, and often highly stereotypical, clinical features of these diseases. Despite the differential clinical features of the various neurodegenerative disorders, the fact that neurons are highly dependent on oxidative energy metabolism has suggested a unified pathogenetic mechanism of neurodegeneration, based on an underlying dysfunction in mitochondrial energy metabolism. Mitochondria are the seat of a number of important cellular functions, including essential pathways of intermediate metabolism, amino acid biosynthesis, fatty acid oxidation, steroid metabolism, and apoptosis. Of key importance to our discussion here is the role of mitochondria in oxidative energy metabolism. Oxidative phosphorylation (OXPHOS) generates most of the cell’s ATP, and any impairment of the organelle’s ability to produce energy can have catastrophic consequences, not only due to the primary loss of ATP, but also due to indirect impairment of “downstream” functions, such as the maintenance of organellar and cellular calcium homeostasis. Moreover, deficient mitochondrial metabolism may generate reactive oxygen species (ROS) that can wreak havoc in the cell. It is for these reasons that mitochondrial dysfunction is such an attractive candidate for an “executioner’s” role in neuronal degeneration. The mitochondrion is the only organelle in the cell, aside from the nucleus, that contains its own genome and genetic machinery. The human mitochondrial genome (1) is a tiny 16.6-kb circle of double-stranded mitochondrial DNA (mtDNA) (Figure ​(Figure1).1). It encodes 13 polypeptides, all of which are components of the respiratory chain/OXPHOS system, plus 24 genes, specifying two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs), that are required to synthesize the 13 polypeptides. Obviously, an organelle as complex as a mitochondrion requires more than 37 gene products; in fact, about 850 polypeptides, all encoded by nuclear DNA (nDNA), are required to build and maintain a functioning organelle. These proteins are synthesized in the cytoplasm and are imported into the organelle, where they are partitioned into the mitochondrion’s four main compartments — the outer mitochondrial membrane (OMM), the inner mitochondrial membrane (IMM), the intermembrane space (IMS), and the matrix, located in the interior (the organelle’s “cytoplasm”). Of the 850 proteins, approximately 75 are structural components of the respiratory complexes (Figure ​(Figure2)2) and at least another 20 are required to assemble and maintain them in working order. The five complexes of the respiratory chain/OXPHOS system — complexes I (NADH ubiquinone oxidoreductase), II (succinate ubiquinone oxidoreductase), III (ubiquinone–cytochrome c reductase), IV (cytochrome c oxidase), and V (ATP synthase) — are all located in the IMM. There are also two electron carriers, ubiquinone (also called coenzyme Q), located in the IMM, and cytochrome c, located in the IMS. Figure 1 Map of the human mitochondrial genome (1). Polypeptide-coding genes (boldface) are outside the circle and specify seven subunits of NADH dehydrogenase–coenzyme Q oxidoreductase (ND), one subunit of coenzyme Q–cytochrome c oxidoreductase ... Figure 2 Schematic representation of the mitochondrion with its electron transport chain (ETC). The ETC is the principal source of ROS in the cell. In addition to mutations in mtDNA- and nDNA-encoded components of the ETC, a number of mutant mitochondrial proteins ... Besides the fact that it operates under dual genetic control, four other features unique to the behavior of this organelle are important in understanding mitochondrial function in neurodegeneration. First, as opposed to the nucleus, in which there are two sets of chromosomes, there are thousands of mtDNAs in each cell, with approximately five mtDNAs per organelle. Second, organellar division and mtDNA replication operate independently of the cell cycle, both in dividing cells (such as glia) and in postmitotic nondividing cells (such as neurons). Third, upon cell division, the mitochondria (and their mtDNAs) are partitioned randomly between the daughter cells (mitotic segregation). Finally, the number of organelles varies among cells, depending in large part on the metabolic requirements of that cell. Thus, skin fibroblasts contain a few hundred mitochondria, whereas neurons may contain thousands and cardiomyocytes tens of thousands of organelles. Taken together, these features highlight the fact that mitochondria obey the laws of population genetics, not mendelian genetics.

The kinase domain of mitochondrial PINK1 faces the cytoplasm

Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinsons disease (PD). Efforts in deducing the PINK1 signaling pathway have been hindered by controversy around its subcellular and submitochondrial localization and the authenticity of its reported substrates. We show here that this mitochondrial protein exhibits a topology in which the kinase domain faces the cytoplasm and the N-terminal tail is inside the mitochondria. Although deletion of the transmembrane domain disrupts this topology, common PD-linked PINK1 mutations do not. These results are critical in rectifying the location and orientation of PINK1 in mitochondria, and they should help decipher its normal physiological function and potential pathogenic role in PD.