C. van den Bogert

Deficiency of the adenine nucleotide translocator in muscle of a patient with myopathy and lactic acidosis: a new mitochondrial defect

ABSTRACT: In a patient with a mitochondrial myopathy, presenting with lactic acidosis, 31P-nuclear magnetic resonance spectroscopy in resting muscle showed half the creatine phosphate level of controls. The creatine phosphate resynthesis rate after aerobic exercise was only 18% of that in controls. However, the activities of complexes I to V catalyzing oxidative phosphorylation and the pyruvate and the 2-oxoglutarate dehydrogenase complexes showed a 2- to 20-fold increase. In line with this, the uncoupled mitochondrial respiration rate was significantly higher than in controls. In contrast, the respiration of the mitochondria from the patient was less stimulated by ADP than that of control mitochondria. This finding could point to a defect in complex V, the enzyme directly involved in ATP synthesis. The activity of complex V, measured as the mitochondrial ATPase activity, and its concentration, as judged from Western blots using antisera against the F1 part of complex V, were, however, also greatly increased in the patient. Alternatively, the transport system, importing ADP into and exporting ATP out of the mitochondrial matrix, the ADP/ATP or adenine nucleotide translocator, could be affected. Immunostaining of Western blots revealed a 4-fold decrease in the concentration of the adenine nucleotide translocator in the patient. Because oxidative phosphorylation was not disturbed in fibroblasts and lymphocytes, we conclude that this patient suffers from a muscle-specific deficiency of his mitochondrial adenine nucleotide translocator, a defect unknown so far.

Isoforms of cytochrome c oxidase in tissues and cell lines of the mouse

The subunit pattern of immunopurified cytochrome c oxidase from cultured mouse cells and mature tissues of the mouse was investigated by electrophoretic analysis. In mature tissues two forms of cytochrome c oxidase could clearly be identified on the basis of differences in morbidity or staining intensity of subunits VIa and VIII. One form was present in muscle and heart, and the other in liver, kidney and spleen. In lung both forms were found. In the thymus, subunit VIII showed the characteristics of subunit VIII found in muscle and heart, whereas subunit VIa resembled subunit VIa found in liver. This suggest the existence of a third cytochrome c oxidase isoform. The subunits of cytochrome c oxidase from cultured cell lines showed no differences between the various cell lines and resembled those of mature mouse liver tissue. The cytochrome c oxidase isoform from cultured proliferating cells might therefore be the same as the one found in liver. Alternatively, it might represent either a normally occurring fetal isoform, or a form specific for poorly differentiated cultured cells.

Preferential amplification and phenotypic selection in a population of deleted and wild-type mitochondrial DNA in cultured cells

Abstract In order to study the still poorly understood dynamics of mitochondrial gene segregation, we attempted to alter the percentage of deleted mtDNA (del-mtDNA) over wild-type mtDNA in cell-culture by manipulating respiratory chain capacity. For this purpose, we used a cell-line harbouring a 6-kb mtDNA-deletion which normally was present in 70% of the molecules. The results show that in the presence of low concentrations of doxycycline (DC), an inhibitor of mitochondrial protein synthesis, the average percentage of del-mtDNA in culture steadily declined. After short-term DC treatment most cells still contained del-mtDNA and removal of DC led to a rapid increase in the proportion of del-mtDNA. In contrast, long-term DC treatment rendered del-mtDNA undetectable by Southern analysis, reflecting the complete absence of del-mtDNA in most cells. In this case, del-mtDNA in culture remained at a constant low level after removal of the drug. The results clearly show the importance of phenotypic selection in the segregation of a deleterious mtDNA mutation.

META-IODOBENZYLGUANIDINE (MIBG) INHIBITS MALATE AND SUCCINATE DRIVEN MITOCHONDRIAL ATP SYNTHESIS IN THE HUMAN NEUROBLASTOMA CELL LINE SK-N-BE(2C)

In this paper, we report on our studies of the effects of MIBG, a structural analogue of norepinephrine, on SK-N-BE(2c) cells. In micromolar concentrations, MIBG caused almost complete inhibition of the proliferation of SK-N-BE(2c) cells. In intact SK-N-BE(2c) cells, addition of MIBG led to a decrease of the ATP to ADP ratio. A progressive increase of the lactate to pyruvate ratio (due to increased lactate production) was observed after incubation of the cells with glucose and increasing concentrations of MIBG. In cells treated with digitonin, MIBG inhibited malate driven ATP synthesis. Comparable inhibition of ATP synthesis with succinate as a substrate required higher concentrations of MIBG. These results indicate that, apart from inhibition of complex I, MIBG was capable of inhibiting at least one other complex of the respiratory chain. Although maximal inhibition of ATP synthesis was observed at a concentration of 10 microM, optimal inhibition of cell proliferation occurred at a MIBG concentration > 25 microM. This suggests that MIBG also influences other cellular processes apart from mitochondrial ATP synthesis, resulting in additional inhibition of cell proliferation.

Permanent increase of immunocytochemical reactivity for γ-aminobutyric acid (GABA), glutamic acid decarboxylase, mitochondrial enzymes, and glial fibrillary acidic protein in rat cerebral cortex damaged by early postnatal hypoxia-ischemia

A former study indicated that hypoxicischemic encephalopathy in rat sustained during early postnatal life may result in permanent epileptic activity in the baseline electroencephalogram. We, therefore, investigated whether the presumed higher firing frequency and metabolic activity of neurons in such hypoxia-damaged cortical areas would be reflected by an enhanced light microscopic immunoreactivity of γ-aminobutyric acid (GABA), the two isoforms of glutamic acid decarboxylase (GAD67 and GAD65), the mitochondrial enzymes cytochrome c oxidase and ATP synthase, and/or glial fibrillary acidic, protein (GFAP). To that end rat pups, 12–13 days of age, were unilaterally exposed to hypoxic-ischemic conditions and, after a survival period of 2 and 61/2 months, respectively, killed by perfusion fixation. After dissection of the brain, coronal vibratome sections of animals showing cortical damage were immunostained for the presence of the abovementioned antigens. Subsequent qualitative analysis revealed that the surroundings of cortical infarctions were unambiguously characterized by a disordered neural network containing numerous nerve cells, fibers and/or endings showing an enhanced immunoreactivity for GABA, both isoforms of glutamic acid decarboxylase, and cytochrome c oxidase and ATP synthase, while the astrocytes showed an enhanced immunoreactivity for GFAP. The diverse patterns of enhanced immunoreactivity suggested, furthermore, a wider low-to-high range of metabolic activities in both excitatory and inhibitory neurons.

Altered kinetics of cytochrome c oxidase in a patient with severe mitochondrial encephalomyopathy

Deficiency of cytochrome c oxidase activity was established in a girl born to consanguineous parents. She showed symptoms of dysmaturity, generalized hypotonia, myoclonic seizures and progressive respiratory failure, leading to death on the seventh day of life. Structural abnormalities of the central nervous system consisted of severe cerebellar hypoplasia and optic nerve atrophy. Biochemical analysis of a muscle biopsy specimen demonstrated deficiency of cytochrome c oxidase activity. Cultured fibroblasts from this patient also showed a selective decrease in the activity of cytochrome c oxidase, excluding a muscle-specific type of deficiency. Further investigations in cultured fibroblasts revealed that synthesis, assembly and stability of both the mitochondrial and the nuclear subunits of the enzyme were entirely normal. The steady-state concentration of cytochrome c oxidase in the fibroblasts of the patient was also normal, suggesting that the kinetic properties of the enzyme were altered. Analysis of the kinetic parameters of cytochrome c oxidase demonstrated an aberrant interaction between cytochrome c oxidase and its substrate, cytochrome c, most likely because of a mutation in one of the nuclear subunits of the enzyme.

Fatal neonatal liver failure and depletion of mitochondrial DNA in three children of one family

Mitochondria contain enzymes for important pathways in intermediary metabolism and are the main suppliers of cellular energy since they produce ATP as the result of oxidative phosphorylation. The oxidative phosphorylation system comprises approximately 100 polypeptides of which the majority function as subunits of five large enzyme complexes. Normally, each mitochondrion contains a number of copies of circular DNA molecules (mtDNA) which encode 13 different subunits of four of the enzyme complexes (complexes I, III, IV and V) that are involved in the oxidative phosphorylation process. Dysfunction of these enzyme complexes is a frequent observation in mitochondrial diseases and point mutations and deletions of mtDNA are regular findings in patients with mitochondrial encephalomyopathies (DiMauro and Moraes 1993). Depletion of mtDNA, characterized by very low levels of normal mtDNA in the affected tissues, can also be associated with mitochondrial encephalomyopathies (Moraes 1993). Severe infantile liver disorders with oxidative phosphorylation defects are relatively rare. However, in three cases these diseases were associated with depletion of mtDNA (Moraes et al 1991 ; Mazziotta et al 1992 ; Maaswinkel-Mooy et al 1995). In addition to these single cases, we identified a family in which three of the four children of healthy consanguineous parents died in the neonatal period because of progressive liver failure that was associated with severe depletion of mtDNA.

Adenine nucleotide translocator deficiency in muscle: Potential therapeutic value of vitamin E

The major function of mitochondria is to provide energy in the form of ATP and creatine phosphate to enable cells and tissues to drive cellular energy-requiring functions. The biochemical defects in mitochondrial energy metabolism can be classified into four groups: (1) defects in oxidative phosphorylation, (2) defects in mitochondrial dehydrogenases, (3) defects in mitochondrial transport processes and (4) defects in mitochondrial enzymes catalysing the synthesis of oxidative substrates (non-redox reactions) (Scholte 1988). Most of the patients suffering from these defects have increased lactate levels and/or abnormal concentrations of organic acids in their body fluids

Quantification of mitochondrial proteins in cultured cells by immuno-flow cytometry.

Immuno-flow cytometry was tested as a tool to estimate the cellular concentration of mitochondrial proteins in cultured cells, using cytochrome c oxidase as a model enzyme. Cells labelled with antibodies against cytochrome c oxidase, in which the amount of the enzyme was reduced by various extents, showed a linear relationship between the size of the signal obtained by immuno-flow cytometry and the amount of the enzyme. The determination by immuno-flow cytometry resulted in data comparable to the results obtained by immunoprecipitation and activity measurements. Since immuno-flow cytometry requires only limited numbers of cells, the method could especially be of value for diagnostic purposes. This is illustrated by the results obtained by comparing activity measurements and immuno-flow cytometry in the initial screening of cell lines derived from patients with deficiencies in the activity of cytochrome c oxidase.

Progressive generalized brain atrophy and infantile spasms associated with cytochrome c oxidase deficiency

Severe neonatal acidosis has been described in association with inborn errors of the enzymes involved in oxidative phosphorylation and the pyruvate dehydrogenase complex. The patients develop irritability, feeding difficulties, hypotonia and respiratory insufficiency within hours or days after birth. The clinical course is rapidly progressive and most patients die within a few days owing to uncontrollable lactic acidosis. Possible presenting signs and symptoms are nonspecific ; differential diagnosis with more common neonatal problems such as sepsis, intracranial haemorrhage and meningitis can thus be difficult. Cytochrome c oxidase (COX ; EC 1.9.3.1) or complex IV, the last component of the respiratory chain, catalyses the transfer of electrons from cytochrome c to molecular oxygen. Fatal neonatal lactic acidosis in association with COX deficiency was first described by Trijbels et al (1983), but a benign form of complex IV deficiency in which severe neonatal onset is followed by spontaneous improvement with increasing activity of the enzyme has also been demonstrated (DiMauro et al 1983, 1994). We report a patient with COX deficiency who developed lactic acidosis (lactate in plasma 28 mmol/L, normal values <2 ; pyruvate in plasma 0.28mmol/L, normal values <0.08) at the age of 1 day. Despite appropriate medication to reduce the lactate and pyruvate concentrations, the patient developed signs of severe encephalopathy almost 6 weeks after normalization of these concentrations.