Subunit composition of respiratory chain complex 1 and its responses to oxygen in mitochondria from human donor livers.

OBJECTIVE: Donor liver function in transplantation is defined by mitochondrial function and the ability of mitochondria to recover from the sequence of warm and/or cold ischemia. Mitochondrial resilience maybe related to assembly and- subunit composition of Complex 1. The aim of this study was to determine if Complex 1 subunit composition was different in donor livers of varying quality and whether oxygen exposure had any effect. RESULTS: Five human livers not suitable for transplant were split. One half placed in cold static storage and the other half exposed to 40% oxygen for 2 h. Protein was extracted for western blot. Membranes were probed with antibodies against ß-actin and the following subunits of Complex 1: MTND1, NDUFA10, NDUFB6 and NDUFV2. No difference in steady state Complex 1 subunit composition was demonstrated between donor livers of varying quality, in terms of steatosis or mode of donation. Neither did exposure to oxygen influence Complex 1 subunit composition. This small observational study on subunit levels suggest that Complex 1 is fully assembled as no degradation of subunits associated with the different parts of the enzyme was seen.

Influence of zinc and zinc chelator on HT-29 colorectal cell line.

Trace elements are involved in many key pathways involving cell cycle control. The influence of zinc and zinc chelator (TPEN) on transcription levels of the main zinc transporters (ZnT1 and ZIP1) in the HT-29 colorectal cell line has not been reported. Proliferation of HT-29 cells was measured using the methylene blue assay after exposure to zinc (two concentrations), TPEN (two concentrations), or a combination of zinc and TPEN (simultaneously and sequentially) for 4 h, 8 h, and 24 h. The transcription levels of ZnT1, ZIP1, vascular endothelial growth factor (VEGF), and caspase-3 were determined using reverse transcriptase real-time polymerase chain reaction (RT-PCR) after exposure of cells to zinc and TPEN. The zinc content in the substrate (medium used for culture) was determined using atomic absorption spectrometry. TPEN decreased cellular proliferation causing complete cell death by 8 h. Zinc had a protective effect against short periods of exposure to TPEN. There was no correlation between the transcripts of main zinc transporters and the zinc content in the substrate. The zinc content in the substrate remained constant after varying periods of cell culture. TPEN decreased the transcript levels of caspase-3 and VEGF, which are surrogate markers for apoptosis and angiogenesis. Zinc chelation of HT-29 cells causes cell death. Zinc appears to be protective for short periods of exposure to TPEN but has no protective effect on prolonged exposure. HT-29 cells are not able to counteract the effect of intracellular chelation of zinc by altering zinc transport. Further research into the mechanisms of these findings is necessary and may lead to novel therapeutic options.

Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase, POLG1).

These tables list both published and a number of unpublished mutations in genes associated with early onset defects in mitochondrial DNA (mtDNA) maintenance including C10orf2, SUCLG1, SUCLA2, TYMP, RRM2B, MPV17, DGUOK and TK2. The list should not be taken as evidence that any particular mutation is pathogenic. We have included genes known to cause mtDNA depletion, excluding POLG1, because of the existing database (http://tools.niehs.nih.gov/polg/). We have also excluded mutations in C10orf2 associated with dominant adult onset disorders.

Differential effects of PINK1 nonsense and missense mutations on mitochondrial function and morphology.

Mutations of the PINK1 gene are a cause of autosomal recessive Parkinson's disease (PD). PINK1 encodes a mitochondrial kinase of unknown function which is widely expressed in both neuronal and non-neuronal cells. We have studied fibroblast cultures from four family members harbouring the homozygous p.Q456X mutation in PINK1, three of their wild-type relatives, one individual with the homozygous p.V170G mutation and five independent controls. Results showed bioenergetic abnormalities involving decreased activities of complexes I and IV along with increased activities of complexes II and III in the missense p.V170G mutant. There were increased basal levels of mitochondrial superoxide dismutase in these cells and an exaggerated increase of reduced glutathione in response to paraquat-induced free radical formation. Furthermore, swollen and enlarged mitochondria were observed in this sample. In the p.Q456X nonsense mutants, the respiratory chain enzymes were unaffected, but ATP levels were significantly decreased. These results confirm that mutations of PINK1 cause abnormal mitochondrial morphology, bioenergetic function and oxidative metabolism in human tissues but suggest that the biochemical consequences may vary between mutations.

Diagnostic value of succinate ubiquinone reductase activity in the identification of patients with mitochondrial DNA depletion.

Mitochondrial DNA (mtDNA) depletion syndrome (McKusick 251880) is characterized by a progressive quantitative loss of mtDNA resulting in severe mitochondrial dysfunction. A diagnosis of mtDNA depletion can only be confirmed after Southern blot analysis of affected tissue. Only a limited number of centres have the facilities to offer this service, and this is frequently on an irregular basis. There is therefore a need for a test that can refine sample selection as well as complementing the molecular analysis. In this study we compared the activities of the nuclear-encoded succinate ubiquinone reductase (complex II) to the activities of the combined mitochondrial and nuclear-encoded mitochondrial electron transport chain (ETC) complexes; NADH:ubiquinone reductase (complex I), ubiquinol-cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV), in skeletal muscle biopsies from 7 patients with confirmed mtDNA depletion. In one patient there was no evidence of an ETC defect. However, the remaining 6 patients exhibited reduced complex I and IV activities. Five of these patients also displayed reduced complex II-III (succinate:cytochrome-c reductase) activity. Individual measurement of complex II and complex III activities demonstrated normal levels of complex II activity compared to complex III, which was reduced in the 5 biopsies assayed. These findings suggest a possible diagnostic value for the detection of normal levels of complex II activity in conjunction with reduced complex I, III and IV activity in the identification of likely candidates for mtDNA depletion syndrome

Immunological phenotyping of fibroblast cultures from patients with a mitochondrial respiratory chain deficit.

Conventional approaches to the diagnosis of mitochondrial respiratory chain diseases, using enzyme assays and histochemistry, are laborious and give limited information concerning the genetic basis of a deficiency. We have evaluated the diagnostic value of 12 monoclonal antibodies to subunits of the four respiratory chain enzyme complexes and F(1)F(0)-ATP synthase. Antibodies were used in immunological studies with skin fibroblast cultures derived from patients with diverse mitochondrial diseases, including patients in which the disease was caused by a nuclear genetic defect and patients known to harbor a heteroplasmic mutation in a mitochondrial tRNA gene. Immunoblotting experiments permitted the identification of specific enzyme assembly deficits and immunocytochemical studies provided clues regarding the genetic origin of the disease. The immunological findings were in agreement with the biochemical and genetic data of the patients. Our study demonstrates that characterization of the fibroblast cultures with the monoclonal antibodies provides a convenient technique to complement biochemical assays and histochemistry in the diagnosis of mitochondrial respiratory chain disorders.

A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure.

Complex III (CIII; ubiquinol cytochrome c reductase of the mitochondrial respiratory chain) catalyzes electron transfer from succinate and nicotinamide adenine dinucleotide-linked dehydrogenases to cytochrome c. CIII is made up of 11 subunits, of which all but one (cytochrome b) are encoded by nuclear DNA. CIII deficiencies are rare and manifest heterogeneous clinical presentations. Although pathogenic mutations in the gene encoding mitochondrial cytochrome b have been described, mutations in the nuclear-DNA-encoded subunits have not been reported. Involvement of various genes has been indicated in assembly of yeast CIII (refs. 8-11). So far only one such gene, BCS1L, has been identified in human. BCS1L represents, therefore, an obvious candidate gene in CIII deficiency. Here, we report BCS1L mutations in six patients, from four unrelated families and presenting neonatal proximal tubulopathy, hepatic involvement and encephalopathy. Complementation study in yeast confirmed the deleterious effect of these mutations. Mutation of BCS1L would seem to be a frequent cause of CIII deficiency, as one-third of our patients have BCS1L mutations.

A novel mutation in SURF1 causes skipping of exon 8 in a patient with cytochrome c oxidase-deficient leigh syndrome and hypertrichosis.

Leigh syndrome is a rare pediatric neurodegenerative disorder attributed to impaired mitochondrial energy metabolism. Mutations in SURF1 have been described in several patients with Leigh syndrome associated with cytochrome c oxidase deficiency. We report a new 18-bp deletion (821del18), spanning the splice donor junction of exon 8 of SURF1, in an infant presenting with cytochrome c oxidase-deficient Leigh syndrome and hypertrichosis. cDNA sequencing demonstrated that this deletion results in a messenger lacking exon 8. RT-PCR experiments suggested a rapid degradation of the aberrant mRNA species from the 5'-end.

Assembly of cytochrome c oxidase: what can we learn from patients with cytochrome c oxidase deficiency?

Cytochrome c oxidase is an intricate metalloprotein that transfers electrons from cytochrome c to oxygen in the last step of the mitochondrial respiratory chain. It uses the free energy of this reaction to sustain a transmembrane electrochemical gradient of protons. Site-directed mutagenesis studies of bacterial terminal oxidases and the recent availability of refined crystal structures of the enzyme are rapidly expanding the understanding of the coupling mechanism between electron transfer and proton translocation. In contrast, relatively little is known about the assembly pathway of cytochrome c oxidase. Studies in yeast have indicated that assembly is dependent on numerous proteins in addition to the structural subunits and prosthetic groups. Human homologues of a number of these assembly factors have been identified and some are now known to be involved in disease. To dissect the assembly pathway of cytochrome c oxidase, we are characterizing tissues and cell cultures derived from patients with genetically defined cytochrome c oxidase deficiency, using biochemical, biophysical and immunological techniques. These studies have allowed us to identify some of the steps of the assembly process.

Expression of mutant alpha-synuclein causes increased susceptibility to dopamine toxicity.

Mutations of the alpha-synuclein gene have been identified in autosomal dominant Parkinson's disease (PD). Transgenic mice overexpressing wild-type human alpha-synuclein develop motor impairments, intraneuronal inclusions and loss of dopaminergic terminals in the striatum. To study the mechanism of action through which mutant alpha-synuclein toxicity is mediated, we have generated stable, inducible cell models expressing wild-type or PD-associated mutant (G209A) alpha-synuclein in human-derived HEK293 cells. Increased expression of either wild-type or mutant alpha-synuclein resulted in the formation of cytoplasmic aggregates which were associated with the vesicular (including monoaminergic) compartment. Expression of mutant alpha-synuclein induced a significant increase in sensitivity to dopamine toxicity compared with the wild-type protein expression. These results provide an explanation for the preferential dopaminergic neuronal degeneration seen in both the PD G209A mutant alpha-synuclein families and suggest that similar mechanisms may underlie or contribute to cell death in sporadic PD.

A novel frameshift mutation of the mtDNA COIII gene leads to impaired assembly of cytochrome c oxidase in a patient affected by Leigh-like syndrome.

We report on a novel frameshift mutation in the mtDNA gene encoding cytochrome c oxidase (COX) subunit III. The proband is an 11-year-old girl with a negative family history and an apparently healthy younger brother. Since 4 years of age, she has developed a progressive spastic paraparesis associated with ophthalmoparesis and moderate mental retardation. The presence of severe lactic acidosis and Leigh-like lesions of putamina prompted us to perform muscle and skin biopsies. In both, a profound, isolated defect of COX was found by histochemical and biochemical assays. Sequence analysis of muscle mtDNA resulted in the identification of a virtually homoplasmic frameshift mutation in the COIII gene, due to the insertion of an extra C at nucleotide position 9537 of mtDNA. Although the 9537C(ins) does not impair transcription of COIII, no full-length COX III protein was detected in mtDNA translation assays in vivo. Western blot analysis of two-dimensional blue-native electrophoresis showed a reduction of specific crossreacting material and the accumulation of early-assembly intermediates of COX, whereas the fully assembled complex was absent. One of these intermediates had an electrophoretic mobility different from those seen in controls, suggesting the presence of a qualitative abnormality of COX assembly. Immunostaining with specific antibodies failed to detect the presence of several smaller subunits in the complex lacking COX III, in spite of the demonstration that these subunits were present in the crude mitochondrial fraction of patient's cultured fibroblasts. Taken together, the data indicate a role for COX III in the incorporation and maintenance of smaller COX subunits within the complex.

Mitochondrial dysfunction in congenital nephrotic syndrome.

The molecular mechanisms maintaining the kidney glomerular filtration barrier remain poorly understood. Recent evidence suggests that mitochondrial dysfunction is a characteristic feature of kidney glomeruli in congenital nephrotic syndrome of the Finnish type (CNF). Here we searched for detailed functional evidence of mitochondrial lesion in CNF kidneys. We used histochemical and immunohistochemical methods, quantitative measurement of mitochondrial DNA, and superoxide production to characterize the mitochondrial function. The results unequivocally show down-regulation of mitochondria-encoded respiratory chain components, whereas the respective nuclearly encoded subunits were close to normal. These results give detailed evidence of distinct mitochondrial dysfunction and of the resulting abnormal production of reactive oxygen species in CNF and suggest a critical role for mitochondria in maintaining the glomerular permeability barrier.

A mutation in the human heme A:farnesyltransferase gene (COX10 ) causes cytochrome c oxidase deficiency.

Cytochrome c oxidase (COX) defects are found in a clinically and genetically heterogeneous group of mitochondrial disorders. To date, mutations in only two nuclear genes causing COX deficiency have been described. We report here a genetic linkage study of a consanguineous family with an isolated COX defect and subsequent identification of a mutation in a third nuclear gene causing a deficiency of the enzyme. A genome-wide search for homozygosity allowed us to map the disease gene to chromosome 17p13.1-q11.1 (Z (max)= 2.46; theta = 0.00 at the locus D17S799). This region encompasses two genes, SCO1 and COX10, encoding proteins involved in COX assembly. Mutation analysis followed by a complementation study in yeast permitted us to ascribe the COX deficiency to a homozygous missense mutation in the COX10 gene. This gene encodes heme A:farnesyltransferase, which catalyzes the first step in the conversion of protoheme to the heme A prosthetic groups of the enzyme. All three nuclear genes now linked to isolated COX deficiency are involved in the maturation and assembly of COX, emphasizing the major role of such genes in COX pathology.

Cytochrome oxidase immunohistochemistry: clues for genetic mechanisms.

Cytochrome c oxidase (COX) is encoded by three mitochondrial and nine nuclear genes. COX deficiency is genetically heterogeneous but current diagnostic methods cannot easily distinguish between mitochondrial and nuclear defects. We hypothesized that there may be differential expression of COX subunits depending on the underlying mutation. COX subunit expression was investigated in five patients with known mtDNA mutations. Severe and selective reduction of mtDNA-encoded COX subunits I and II was consistently observed in all these patients and was restricted to COX-deficient fibres. Immunostaining of nuclear-encoded subunits COX IV and Va was normal, whilst subunit VIc, also nuclear-encoded, was decreased. Twelve of 36 additional patients with histochemically defined COX deficiency also had this pattern of staining, suggesting that they had mtDNA defects. Clinical features in this group were heterogeneous, including infantile encephalopathy, multisystem disease, cardiomyopathy and childhood-onset isolated myopathy. The remaining patients did not have the same pattern of immunostaining. Fourteen had reduced staining of all subunits, whilst 10 had normal staining of all subunits despite reduced enzyme activity. Patients with COX deficiency secondary to mtDNA mutations have a specific pattern of subunit loss, but the majority of children with COX deficiency do not have this pattern of subunit loss and are likely to have nuclear gene defects.

Biochemical, genetic and immunoblot analyses of 17 patients with an isolated cytochrome c oxidase deficiency.

Mitochondrial respiratory chain defects involving cytochrome c oxidase (COX) are found in a clinically heterogeneous group of diseases, yet the molecular basis of these disorders have been determined in only a limited number of cases. Here, we report the clinical, biochemical and molecular findings in 17 patients who all had isolated COX deficiency and expressed the defect in cultured skin fibroblasts. Immunoblot analysis of mitochondrial fractions with nine subunit specific monoclonal antibodies revealed that in most patients, including in a patient with a novel mutation in the SURF1 gene, steady-state levels of all investigated COX subunits were decreased. Distinct subunit expression patterns were found, however, in different patients. The severity of the enzymatic defect matched the decrease in immunoreactive material in these patients, suggesting that the remnant enzyme activity reflects the amount of remaining holo-enzyme. Four patients presented with a clear defect of COX activity but had near normal levels of COX subunits. An increased affinity for cytochrome c was observed in one of these patients. Our findings indicate a genetic heterogeneity of COX deficiencies and are suggestive of a prominent involvement of nuclear genes acting on the assembly and maintenance of cytochrome c oxidase.

A missense mutation of cytochrome oxidase subunit II causes defective assembly and myopathy.

We report the first missense mutation in the mtDNA gene for subunit II of cytochrome c oxidase (COX). The mutation was identified in a 14-year-old boy with a proximal myopathy and lactic acidosis. Muscle histochemistry and mitochondrial respiratory-chain enzymology demonstrated a marked reduction in COX activity. Immunohistochemistry and immunoblot analyses with COX subunit-specific monoclonal antibodies showed a pattern suggestive of a primary mtDNA defect, most likely involving CO II, for COX subunit II (COX II). mtDNA-sequence analysis demonstrated a novel heteroplasmic T-->A transversion at nucleotide position 7,671 in CO II. This mutation changes a methionine to a lysine residue in the middle of the first N-terminal membrane-spanning region of COX II. The immunoblot studies demonstrated a severe reduction in cross-reactivity, not only for COX II but also for the mtDNA-encoded subunit COX III and for nuclear-encoded subunits Vb, VIa, VIb, and VIc. Steady-state levels of the mtDNA-encoded subunit COX I showed a mild reduction, but spectrophotometric analysis revealed a dramatic decrease in COX I-associated heme a3 levels. These observations suggest that, in the COX protein, a structural association of COX II with COX I is necessary to stabilize the binding of heme a3 to COX I.

Mitochondrial DNA depletion syndrome is expressed in amniotic fluid cell cultures.

Mitochondrial DNA depletion syndrome is an autosomal inherited disease associated with grossly reduced cellular levels of mitochondrial DNA in infancy. Most patients are born after a full and uncomplicated pregnancy, are normal at birth, but develop symptoms in the early neonatal period. These observations have led to the suggestion that the patients have a defect affecting the control of mitochondrial DNA copy number after birth. Using immunocytochemical techniques, we demonstrated that the disease is already expressed in amniotic fluid cells. Detection of mitochondrial DNA depletion in these fetal cells indicates that the defect may already be expressed early in embryological development.

The mitochondrial genome: structure, transcription, translation and replication.

Mitochondria play a central role in cellular energy provision. The organelles contain their own genome with a modified genetic code. The mammalian mitochondrial genome is transmitted exclusively through the female germ line. The human mitochondrial DNA (mtDNA) is a double-stranded, circular molecule of 16569 bp and contains 37 genes coding for two rRNAs, 22 tRNAs and 13 polypeptides. The mtDNA-encoded polypeptides are all subunits of enzyme complexes of the oxidative phosphorylation system. Mitochondria are not self-supporting entities but rely heavily for their functions on imported nuclear gene products. The basic mechanisms of mitochondrial gene expression have been solved. Cis-acting mtDNA sequences have been characterised by sequence comparisons, mapping studies and mutation analysis both in vitro and in patients harbouring mtDNA mutations. Characterisation of trans-acting factors has proven more difficult but several key enzymes involved in mtDNA replication, transcription and protein synthesis have now been biochemically identified and some have been cloned. These studies revealed that, although some factors may have an additional function elsewhere in the cell, most are unique to mitochondria. It is expected that cell cultures of patients with mitochondrial diseases will increasingly be used to address fundamental questions about mtDNA expression.