Mitochondria: Transducers or Time Bombs?

Transcription

1 Australian Biochemist Mitochondria: Transducers or Time Bombs? Hans-Henrik Dahl and David R. Thorburn The Murdoch Children s Research Institute, Royal Children s Hospital, Parvkille, Vic 3052 Mitochondria have many important roles in the cell but their main function is aerobic synthesis of ATP (oxidative phosphorylation). Electrons are donated from intermediates of the TCA cycle, via NADH and succinate, and flow through electron carriers in the respiratory chain (RC) until they eventually reduce molecular oxygen. As a consequence, an electrochemical ph gradient is created across the mitochondrial membranes and this drives the formation of ATP from ADP and phosphate. However, an inevitable consequence of electron transport via highly reactive intermediates is the generation of potentially damaging reactive oxygen species (ROS). Mitochondrial involvement in ROS metabolism and cell death has received much attention recently in relation to the pathogenesis of common neurodegenerative disorders. These processes are perhaps even more relevant and more amenable to study in primary disorders of the mitochondrial RC, and in this article we review our current understanding of the genetic basis and pathogenesis of primary RC disorders, which have a cumulative incidence of ~1/5000 births. Fig. 1. The mitochondrial respiratory chain and reactive oxygen species. Electrons are transferred from NADH to the lipid soluble carrier coenzyme Q by complex I, then to the aqueous carrier cytochrome c by complex III, and finally to oxygen by complex IV. The proton electrochemical gradient established by complexes I, III and IV is linked to ATP synthesis by complex V. Reactive oxygen species are generated by leakage of electrons, primarily from complex I, complex III, or coenzyme Q on to oxygen, resulting in a univalent reduction to generate superoxide (O 2 - ). Not shown for clarity is complex II, which consists of four nuclearencoded subunits, that catalyse transfer of electrons from succinate to coenzyme Q. Genetic basis of mitochondrial respiratory chain disorders Mitochondrial DNA Mutations. The respiratory chain consists of five multisubunit enzyme complexes (Fig. 1). Thirteen of the over 80 subunits are coded for by the mitochondrial DNA (mtdna) whereas others are coded for in the cell nucleus. When we discuss mitochondrial function and dysfunction it is therefore important to understand the interplay between and unique features of these two genomes. A mammalian cell usually contains hundreds of mitochondria (each with 5-15 mtdnas). In humans, the 16,569-base pair maternally inherited mtdna codes for 13 polypeptides (all part of the RC complexes), 22 trnas and 2 rrnas. Other gene products essential for mitochondrial function are encoded in the nuclear genome. The mtdna has a high mutation rate, assumed to be the result of ROS damage and a lack of protective histones. The mtdna is highly compact and nearly all nucleotide changes will affect a coding or control region. Each cell can have a homogenous mtdna population (homoplasmy) or a mixture of mtdnas (heteroplasmy). More than 110 nucleotide changes in the mtdna have been shown to be associated with disease (for recent reviews, see refs 1 and 2, or / These pathogenic mutations are usually heteroplasmic, i.e. each cell contains a mixture of wildtype and mutant mtdnas. The diseases usually affect multiple organs, and it is perhaps not surprising that the more energy demanding tissues, such as the central nervous system and muscles, are most often affected. The proportion of mutant mtdna (the level of heteroplasmy) can vary between cells and tissues. As there is a threshold mutant load above which the physiological effects of a mutation become increasingly severe, the mutant loads in various tissues are an important factor in determining the phenotype. Added to this are tissuespecific factors that affect the clinical presentation. For example it is not understood why some mtdna mutations cause Leber s Hereditary Optic Neuropathy, while others cause MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes) or Leigh syndrome (see later). Many mitochondrial diseases are progressive. This might reflect changes in tissue-specific energy demand or a gradual reduction in the ability of mitochondria to 11

2 Volume 31, No. 2, August produce energy. Somatic mtdna mutations accumulate with time, although the extent to which these random somatic mtdna mutations affect disease progression and late onset mitochondrial diseases remains unclear. Nuclear DNA mtdna interaction. A number of nuclear genes are involved in maintenance and expression, and mutations in such genes could affect mtdna integrity. Examples of resulting disorders are the syndromes of multiple mtdna deletions and mtdna depletion (3). The multiple deletion syndromes are clinically and genetically heterogeneous. A number of autosomal recessive phenotypes have been described and in one of these, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), the genetic basis was recently shown to be mutations in the thymidine phosphorylase gene (4). As yet, we do not understand the mechanism by which abnormal function of this gene appears to cause sloppy mtdna replication and accumulation of multiple deleted forms of mtdna. The autosomal dominant form (adpeo) usually presents with adult-onset progressive external ophthalmoplegia and myopathy. The multiple mtdna deletions are most abundant in muscle. So far 3 chromosome loci have been identified in large families with adpeo: on chromosome 10q, 3p and 4q, indicating genetic heterogeneity of this disorder (1). MtDNA depletion syndrome is characterised by grossly reduced levels of mtdna. The syndrome is normally inherited in an autosomal recessive pattern; but the underlying genetic defects have still to be identified (3). Nuclear genes causing respiratory chain dysfunction. Nuclear genes encode more than 80% of RC subunits. These have to be imported into the correct location within the mitochondrion, processed and assembled into functional complexes. It is not surprising that studies in yeast, mouse models and human diseases have identified genetic defects affecting nuclearencoded subunit genes, mitochondrial protein import, assembly or processing, together with related functions such as export of iron, exchange of ATP and ADP or ROS defence systems (Table 1). RC complex IV defects illustrate this point, since studies of yeast mutants have Table 1. Genetic mechanisms of mitochondrial respiratory chain defects Gene category Examples OMIM Gene or reference locus mtdna Genes RC subunit Leigh disease MTATP6 trna MELAS MTTL1 rrna non-syndromic deafness MTRNA1 Nuclear Genes RC subunit Leigh disease NDUFS8 COX assembly Leigh disease SURF1 Haem synthesis leukodystrophy COX10 Mitochondrial copper delivery infantile cardiomyopathy SCO2 Protein import deafness dystonia syndrome DDP1 Mitochondrial iron export(?) sideroblastic anaemia/ataxia ABC7 Regulation of iron transport(?) Friedreich ataxia FRDA Metalloprotease/chaperonin Hereditary spastic paraplegia SPG7 MtDNA/nuclear DNA interaction adpeo/multiple deletions q23-q24 Only one example is given for each category but multiple examples are known for several categories, including mtdna trna and RC subunit genes and for nuclear RC subunit genes. OMIM references refer to the Online Mendelian Inheritance in Man catalogue ( shown that complex IV defects could be caused by mutations in over 30 different genes, encoding proteins involved in copper trafficking, haem synthesis, COX assembly and mtdna processing (Fig. 2). Occasional patients have been found to have mutations in the three mtdna genes encoding the catalytic subunits of complex IV (COI, COII, COIII), but no mutations in the nuclear-encoded structural subunits have been identified. Recently it was shown that most cases of complex IV deficiency are caused by mutations in the human homologues (SURF1, SCO2, COX10) of three yeast complex IV assembly genes (5-7). Pathogenesis of mitochondrial disorders Our understanding of the pathogenesis of mitochondrial RC disorders is currently incomplete. The most obvious pathogenic mechanisms relate to the effects of inadequate ATP production, increased ROS, and induction of the mitochondrial permeability transition or other mechanisms by which mitochondria regulate cell death. ATP deficit. ATP is required for a vast number of homeostatic cellular processes so an ATP deficit could impact on ion channels, protein synthesis, and various specific reactions to result in cellular Fig. 2. A speculative model for COX assembly in human cells. S1 to S4 represent assembly intermediates (COX subcomplexes) that can be detected when cells are incubated with protein synthesis inhibitors (21). COX subunits and prosthetic groups are shown on the right, and the known or putative homologues of yeast genes involved in COX assembly are shown on the left (6,7,21). COX10 and COX 11 are involved in synthesis of haem A. COX17, SCO1 and SCO2 are required for transport of copper from the plasma membrane transporter to the mitochondrial inner membrane. The individual roles of the other proteins involved in COX assembly are less clear.

3 Australian Biochemist dysfunction. As an example, when rodent insulinoma cell lines are depleted of mtdna, they are unable to increase their ATP/ADP ratio sufficiently to block K + channels and trigger depolarisation, calcium influx and insulin secretion (8). The inability of pancreatic β cells to increase their ATP/ADP ratio is thus the probable cause of insulin secretion defects in some mitochondrial patients, although as yet we do not understand why this is a common feature with some mtdna mutations, but not others. Reactive oxygen species. In nonphagocytic cells, the mitochondrial respiratory chain is the main source of ROS, with leakage of electrons resulting in the generation of superoxide (O 2 - ). Superoxide can be dismutated to hydrogen peroxide (H 2 O 2 ) by mitochondrial manganese superoxide dismutase (MnSOD) (Fig. 1). Hydrogen peroxide can then be converted to water by glutathione peroxidase or catalase. Neither superoxide nor hydrogen peroxide is very toxic, but the latter can be converted to the highly reactive hydroxyl radical (OH - ) in the presence of free iron or copper by the Fenton reaction. Hydroxyl radical can damage any biomolecules. Nitric oxide (NO ) is another weak ROS that is continually synthesised by several types of endogenous or inducible nitric oxide synthases. In the presence of superoxide, however, it generates highly reactive peroxynitrite (ONOO-), which can nitrate tyrosine residues in proteins, resulting in their inactivation. Until recently, the experimental evidence for involvement of ROS in the pathogenesis of RC defects was based largely on in vitro experiments, but recent data provide more direct support. Homozygous (-/-) MnSOD knockout mice die from dilated cardiomyopathy at about 8 days of age with inactivation of oxidantsensitive mitochondrial iron-sulfur proteins. The cardiac defect can be rescued by treatment with the SOD-mimetic MnTBAP, but since this does not cross the blood-brain barrier, the animals develop a debilitating movement disorder (1). Heterozygous (+/-) MnSOD mice are healthy but show increased rates of superoxide production, decreased activity of iron-sulfur enzymes in various tissues, and an increased rate of induction of the mitochondrial permeability transition (1) (see later). They also develop larger cerebral infarcts following cerebral artery ischaemia and reperfusion (9), suggesting that even relatively modest changes in mitochondrial ROS metabolism can have profound effects on susceptibility to cell death, not just in cell models but in live animals. The other relevant mouse model was generated by genetic inactivation of the heart and muscle isoform of the adenine nucleotide translocator (Ant1-/-), which results in a classic mitochondrial myopathy. Skeletal muscle mitochondria in these mice are unable to exchange ADP and ATP across the inner mitochondrial membrane, but they also showed approximately 8-fold increases in the rate of hydrogen peroxide generation, and in MnSOD activity. The mitochondria had a more electronegative membrane potential, suggesting that a lack of matrix ADP decreased the proton flux due to a stalled ATP synthase, resulting in a more highly reduced respiratory chain, and increased rates of superoxide generation (1). The increased ROS generation in this mouse model is particularly striking given that the primary defect is not in the RC itself. Currently, the best example of a human disease in which mitochondrial ROS play a key pathogenic role is Friedreich ataxia, which is characterised by progressive cerebellar ataxia, with onset typically in adolescence, and often accompanied by hypertrophic cardiomyopathy and diabetes. It is caused by mutations in the frataxin gene, which encodes a mitochondrial protein apparently involved in controlling mitochondrial iron efflux. Frataxin mutations result in increased accumulation of intramitochondrial iron, presumably causing increased formation of hydroxyl radical and oxidative damage (10). Support for this hypothesis comes from studies showing that cardiac muscle from Friedreich ataxia patients had decreased activities of iron sulfur enzymes (analogous to the MnSOD knockout mouse model), and that the cardiomyopathy in these patients can be dramatically improved by treatment with idebenone, a short chain analogue of coenzyme Q (11). Mitochondria and cell death. Mitochondria play a key role in many pathways of apoptotic and necrotic cell death (12,13). Anti-apoptotic members of the Bcl-2 family may regulate apoptosis by interacting with the Mitochondrial Permeability Transition Pore, which spans the mitochondrial inner and outer membrane (Fig. 3). Under normal conditions, this pore must remain closed in order to maintain tight mitochondrial coupling. Factors that promote pore opening include low mitochondrial membrane potential, Ca 2+, Pi, ROS and pro-apoptotic members of the Fig. 3. A model of the mitochondrial permeability transition pore, which appears to be created at the inner and outer mitochondrial membrane contact points by the interaction of Adenine Nucleotide Translocase (ANT), the Voltage-Dependent Anion Channel (VDAC), the Peripheral Benzodiazepine Receptor (PBR), cyclophilin D (CyD), and Bax (1,12,13). The antiapoptotic effect of Bcl-2 is mediated in part by inhibition of pore opening. Either because of pore opening or by another mechanism, pro-apoptotic proteins including cytochrome c (cyt c) and Apoptosis Initiating Factor (AIF) are released from the inter-membrane space into the cytosol. AIF translocates to the nucleus, initiating chromatin destruction. Cytochrome c binds to Apaf-1 and, in the presence of datp, activates the cytosolic caspase cascade, culminating in apoptotic cell death. 13

4 14 Bcl-2 family. Coincident with pore opening, or because of it, pro-apoptotic proteins are released into the cytosol. Both apoptosis and necrosis can be induced by the mitochondrial permeability transition, and the mechanism determining which mode of cell death occurs is not entirely clear, although apoptosis can probably occur only if cellular ATP levels remain high. Evidence supporting the role of ROS and mitochondrial cell death in the pathogenesis of RC defects is now accumulating. Fibroblast cell lines from some patients with RC complex I defects show increased rates of superoxide generation, while others have normal superoxide generation but 10-fold increased rates of generation of hydroxyl radical and toxic aldehydes (14). Some patient cell lines show increased MnSOD activity, while others induce Bcl- 2, presumably to protect against apoptotic cell death (15). Muscle biopsies from patients with some mtdna mutations show apoptotic features such as increases in TUNEL staining and p75, Fas and caspase- 3 immunoreactivity (16). Clearly, our knowledge of the genetic basis and pathophysiology of mitochondrial disorders is now considerable, but the two examples in the accompanying Boxes demonstrate that we need to find and organise several more pieces before we truly understand the pathogenic puzzle. Summary We have made substantial progress in understanding the genetic basis and some of the key pathogenic mechanisms in mitochondrial disorders. The examples in the Boxes demonstrate, however, that we are as yet unable to satisfactorily explain many features. For example, why does the A3243G mutation cause stroke in some patients and diabetes or deafness in others. Why does this mutation rarely, if ever, seem to cause symptoms such as the ataxia common with the T8993G mutation BOX 1 The A3243G mutation in the trna-leu(uur) gene is the most common cause of MELAS, a syndrome in which the distinctive feature is the occurrence of stroke-like episodes, usually commencing in the early teens. However, many patients never experience cerebral infarcts, but instead develop one or more other symptoms such as diabetes, sensorineural deafness or muscle weakness. Some of the key discoveries relevant to pathogenesis of this mutation are as follows: In cultured cells, the mutation causes a RC enzyme defect and impaired mitochondrial protein synthesis at mutant loads above a threshold of ~85%, apparently by reducing the amount of aminoacylated trna-leu (17); The mutation also disrupts a highly conserved binding site in mtdna for the mterf transcription termination factor, which may interfere with mtdna-encoded mrna synthesis (18); The proportion of mutant mtdna varies between individuals and between organs and tissues within an individual, and there is a correlation between the frequency of clinical features and the level of mutant mtdna in skeletal muscle (19); In blood and some cultured cell lines, the A3243G mutant load tends to decrease with time, while in muscle and other cell lines the mutant load tends to increase with time (19); Patients who experience stroke-like episodes tend to have a uniform distribution of mutant mtdna between skeletal muscle fibres, while those with only muscle weakness tend to have lower mutant loads overall but much greater variation between individual muscle fibres (19). BOX 2 Leigh Syndrome is one of the best-known mitochondrial disorders of childhood. Most infants with Leigh Syndrome develop normally for the first six months of life, but then (usually following a viral illness) develop a progressive neurodegenerative disease, resulting in death at two to three years of age (20). There is a characteristic neuropathology of symmetrical spongiform lesions, particularly affecting the basal ganglia and brainstem, with gliosis and capillary proliferation but relative sparing of neurons. The genetic basis of Leigh syndrome is remarkably diverse, despite this relatively distinct phenotype, and can be caused by isolated defects of RC complexes I, II, IV, or V, combined defects of RC complexes, or deficiency of the pyruvate dehydrogenase complex (20). To date, molecular defects have been identified in 4 mtdna trna genes, 3 mtdna protein subunit genes, 4 autosomal genes, and the X-chromosomal PDHA1 gene. This genetic diversity implies that the specific neuropathology is not so much a function of the primary defect, but a specific response of certain cell types to a particular degree or mechanism of energy deprivation. Volume 31, No. 2, August 2000 or the lipomas associated with the A8344G mutation? In order to solve the pathogenic puzzle, we need more information about the complex interplay between the known pathogenic mechanisms and factors such as the expression pattern of nuclear encoded mitochondrial proteins (the nuclear genetic background), the occurrence and distribution of germline and somatic mtdna mutations, the mtdna copy number and haplotype, and environmental factors. To obtain this information will require the development of a wider range of animal models (particularly for mtdna mutations) and further detailed studies of cell model systems and patients. References 1. Wallace, D. C. (1999) Science 283, Leonard, J. V., and Schapira, A. H. (2000) Lancet 355, Leonard, J. 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5 mtdna subunits: ndna subunits: Intermembrane Space H + H + H + Cyt c Fig. 1 Inner Membrane Matrix I CoQ III IV NADH + H + NAD + O - 2 e - 1/2 O 2 O 2 H 2 O MnSOD ADP + P i V H + ATP Fe 2+ H 2 O 2 GSH Px OH H 2 O S1 (COXI) COX10,COX11 Heme A, Heme A 3 Fig. 2 COX17,SCO1,SCO2 COX14,COX15 CuB COXIV COX17,SCO1,SCO2 S2 COXII CuA PET100,PET117,PET191, SURF1,OXA1 COXIII, Va, Vb, VIb, COX VIc, VIIa, VIIc, VIII LON,AFG3,RCA1 S3 COXVIa,VIIb S4 Fig. 3 Outer membrane Bcl2 PBR Bax VDAC Apaf-1 datp Caspase-3 Caspase-9 Apoptosis Inter-membrane space Inner membrane CyD ANT cyt c AIF