Impaired ubiquitin-proteasome-mediated PGC-1α protein turnover and induced mitochondrial biogenesis secondary to complex-I deficiency.

Most eukaryotic cells depend on mitochondrial OXidative PHOSphorylation (OXPHOS) in their ATP supply. The cellular consequences of OXPHOS defects and the pathophysiological mechanisms in related disorders are incompletely understood. Using a quantitative proteomics approach we provide evidence that a genetic defect of complex-I of the OXPHOS system may associate with transcriptional derangements of mitochondrial biogenesis through stabilization of the master transcriptional regulator PPARγ co-activator 1α (PGC-1α) protein. Chronic oxidative stress suppresses the gene expression of PGC-1α but concomitant inhibition of the ubiquitin-proteasome system (UPS) can stabilize this co-activator protein, thereby inducing its downstream metabolic gene expression programs. Thus, mitochondrial biogenesis, which lays at the heart of the homeostatic control of energy metabolism, can be deregulated by secondary impairments of the protein turnover machinery.

Mitochondrial enzymes discriminate between mitochondrial disorders and chronic fatigue syndrome.

We studied the extent of mitochondrial involvement in chronic fatigue syndrome (CFS) and investigated whether measurement of mitochondrial respiratory chain complex (RCC) activities discriminates between CFS and mitochondrial disorders. Mitochondrial content was decreased in CFS compared to healthy controls, whereas RCC activities corrected for mitochondrial content were not. Conversely, mitochondrial content did not discriminate between CFS and two groups of mitochondrial disorders, whereas ATP production rate and complex I, III and IV activity did, all with higher activities in CFS. We conclude that the ATP production rate and RCC activities can reliably discriminate between mitochondrial disorders and CFS.

Mitochondrial dysfunction in muscle tissue of complex regional pain syndrome type I patients.

Reactive oxygen species (ROS) are known to be involved in the pathophysiology of complex regional pain syndrome type I (CRPS I). Since the mitochondrial respiratory chain is a major source of ROS, we hypothesized that mitochondria play a role in the pathophysiology of CRPS I. The hypothesis was tested by studying mitochondrial energy metabolism in muscle tissue from amputated limbs of CRPS I patients. We observed that mitochondria obtained from CRPS I muscle tissue displayed reduced mitochondrial ATP production and substrate oxidation rates in comparison to control muscle tissue. Moreover, we observed reactive oxygen species evoked damage to mitochondrial proteins and reduced MnSOD levels. It remains to be established if the mitochondrial dysfunction that is apparent at the end-stage of CRPS I is also present in earlier stages of the disease, or are secondary to CRPS I. The observation of a reduced mitochondrial energy production combined with reactive oxygen species induced damage in muscle tissue from CRPS I patients warrants further studies into the involvement of mitochondrial dysfunctioning in the pathophysiology of CRPS I.

High-throughput assay to measure oxygen consumption in digitonin-permeabilized cells of patients with mitochondrial disorders.

BACKGROUND: Muscle biopsy analysis is regarded as the gold standard in diagnostic workups of patients with suspected mitochondrial disorders. Analysis of cultured fibroblasts can provide important additional diagnostic information. The measurement of individual OXPHOS complexes does not always provide sufficient information about the functional state of the complete mitochondrial energy-generating system. Thus, we optimized a high-throughput fluorescence-based methodology for oxygen consumption analysis in patient-derived cells. METHODS: We analyzed mitochondrial respiration in digitonin-permeabilized cells in the presence of a substrate mix containing pyruvate and malate, using a phosphorescent probe, 96-well plates, and a fluorescence plate reader. RESULTS: In control fibroblasts, we observed clear stimulation by ADP of the pyruvate + malate-driven respiration. Known inhibitors of the OXPHOS system and the Krebs cycle significantly reduced respiration. In patient fibroblasts with different OXPHOS deficiencies, ADP-stimulated respiratory activity was decreased in comparison to control cells. In several patients with reduced ATP production rate in muscle tissue but with normal OXPHOS enzyme activities, the fibroblasts displayed reduced respiratory activity. Finally, we observed a clear difference between control and complex I-deficient transmitochondrial cybrid cells. CONCLUSIONS: These results confirm the validity of the assay as a high-throughput screening method for mitochondrial function in digitonin-permeabilized cells. The assay allows primary and secondary mitochondrial abnormalities in muscle to be differentiated, which is of great importance with respect to counseling, and also will facilitate the search for new genetic defects that lead to mitochondrial disease.

A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy.

To identify the biochemical and molecular genetic defect in a 16-year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy suspected for a mitochondrial disorder.Measurement of the mitochondrial energy-generating system (MEGS) capacity in muscle and enzyme analysis in muscle and fibroblasts were performed. Relevant parts of the mitochondrial DNA were analysed by sequencing.A homoplasmic nonsense mutation m.8529G→A (p.Trp55X) was found in the mitochondrial ATP8 gene in the patient's fibroblasts and muscle tissue. Reduced complex V activity was measured in the patient's fibroblasts and muscle tissue, and was confirmed in cybrid clones containing patient-derived mitochondrial DNAWe describe the first pathogenic mutation in the mitochondrial ATP8 gene, resulting in an improper assembly and reduced activity of the complex V holoenzyme.

Muscle 3243A-->G mutation load and capacity of the mitochondrial energy-generating system.

OBJECTIVE: The mitochondrial energy-generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate production rates, which we refer to as the "MEGS capacity." Currently, little is known about MEGS pathology in patients with mutations in the mitochondrial DNA. Because MEGS capacity is an indicator for the overall mitochondrial function related to energy production, we searched for a correlation between MEGS capacity and 3243A-->G mutation load in muscle of patients with the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) syndrome. METHODS: In muscle tissue of 24 patients with the 3243A-->G mutation, we investigated the MEGS capacity, the respiratory chain enzymatic activities, and the 3243A-->G mutation load. To exclude coinciding mutations, we sequenced all 22 mitochondrial transfer RNA genes in the patients, if possible. RESULTS: We found highly significant differences between patients and control subjects with respect to the MEGS capacity and complex I, III, and IV activities. MEGS-related measurements correlated considerably better with the mutation load than respiratory chain enzyme activities. We found no additional mutations in the mitochondrial transfer RNA genes of the patients. INTERPRETATION: The results show that MEGS capacity has a greater sensitivity than respiratory chain enzymatic activities for detection of subtle mitochondrial dysfunction. This is important in the workup of patients with rare or new mitochondrial DNA mutations, and with low mutation loads. In these cases we suggest to determine the MEGS capacity.

Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts.

BACKGROUND: A reliable and sensitive complex I assay is an essential tool for the diagnosis of mitochondrial disorders, but current spectrophotometric assays suffer from low sensitivity, low specificity, or both. This deficiency is mainly due to the poor solubility of coenzyme-Q analogs and reaction mixture turbidity caused by the relatively high concentrations of tissue extract that are often required to measure complex I. METHODS: We developed a new spectrophotometric assay to measure complex I in mitochondrial fractions and applied it to muscle and cultured fibroblasts. The method is based on measuring 2,6-dichloroindophenol reduction by electrons accepted from decylubiquinol, reduced after oxidation of NADH by complex I. The assay thus is designed to avoid nonspecific NADH oxidation because electrons produced in these reactions are not accepted by decylubiquinone, resulting in high rotenone sensitivity. RESULTS: The assay was linear with time and amount of mitochondria. The K(m) values for NADH and 2,6-dichloroindophenol in muscle mitochondria were 0.04 and 0.017 mmol/L, respectively. The highest complex I activities were measured with 0.07 mmol/L decylubiquinone and 3.5 g/L bovine serum albumin. The latter was an essential component of the reaction mixture, increasing the solubility of decylubiquinone and rotenone. In patients with previously diagnosed complex I deficiencies, the new assay detected the complex I deficiencies in both muscle and fibroblasts. CONCLUSIONS: This spectrophotometric assay is reproducible, sensitive, and specific for complex I activity because of its high rotenone sensitivity, and it can be applied successfully to the diagnosis of complex I deficiencies.

De novo double mutation in PAX6 and mtDNA tRNA(Lys) associated with atypical aniridia and mitochondrial disease.

We report on the clinical, molecular and biochemical findings of a patient with the rare event (<4.02 x 10(-9) per generation) of coinciding de novo mutations in the nuclear PAX6 (c.1252-1267del16) and the mitochondrial mt.RNA (Lys) (8347A-->G) genes. The boy suffers from exercise intolerance, ptosis, nystagmus, macular hypoplasia and anterior segment abnormalities evocative of Axenfeld-Rieger anomaly. The PAX6 mutation is predicted to cause haploinsufficiency. The novel mt.RNA (Lys) mutation is located close to the classic myoclonic epilepsy with ragged-red-fibers mutation, but the patient exhibits neither myoclonic epilepsy nor ragged-red-fibers. The degree of mutant mtDNA heteroplasmy, as determined by a very accurate pyrosequencing assay, varies between 31% (muscle) and 38% (fibroblasts). We discuss a potential effect of the PAX6 mutation on the mtDNA mutation rate.

Clinical and biochemical characteristics in patients with a high mutant load of the mitochondrial T8993G/C mutations.

We retrospectively analyzed the clinical, histological, and biochemical data of 11 children, five of which carried the maternally-inherited mitochondrial T8993C and six carrying the T8993G point mutations in the ATP synthase 6 gene. The percentage of heteroplasmy was 95% or higher in muscle and in blood. All patients had an early clinical presentation with muscle hypotonia, severe extrapyramidal dysfunction and Leigh disease demonstrated by the cranial MRI. A slower clinical progression and more frequent sensory-neuronal involvement were noted in the patients carrying the T8993C mutation in a high mutation load in muscle and blood. No histological abnormality was found. In 9 out of 11 patients a decreased ATP production was detected, and complex V activity was deficient in all children. The activities of the respiratory enzyme complexes II and IV were normal, whereas an associated combined complex I and III deficiency were present in two patients. No obvious difference was found between the biochemical parameters of the two patient groups harboring different mutations in the same gene. No correlation was found between the degree of complex V enzyme deficiency and the severity of the phenotype. We confirmed an impaired assembly/stability of complex V in our patients. This is the first report of decreased activity and impaired assembly/stability of complex V in patients with T8993C mutations measured in muscle tissue.

Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology.

BACKGROUND: Diagnosis of mitochondrial disorders usually requires a muscle biopsy to examine mitochondrial function. We describe our diagnostic procedure and results for 29 patients with mitochondrial disorders. METHODS: Muscle biopsies were from 43 healthy individuals and 29 patients with defects in one of the oxidative phosphorylation (OXPHOS) complexes, the pyruvate dehydrogenase complex (PDHc), or the adenine nucleotide translocator (ANT). Homogenized muscle samples were used to determine the oxidation rates of radiolabeled pyruvate, malate, and succinate in the absence or presence of various acetyl Co-A donors and acceptors, as well as specific inhibitors of tricarboxylic acid cycle or OXPHOS enzymes. We determined the rate of ATP production from oxidation of pyruvate. RESULTS: Each defect in the energy-generating system produced a specific combination of substrate oxidation impairments. PDHc deficiencies decreased substrate oxidation reactions containing pyruvate. Defects in complexes I, III, and IV decreased oxidation of pyruvate plus malate, with normal to mildly diminished oxidation of pyruvate plus carnitine. In complex V defects, pyruvate oxidation improved by addition of carbonyl cyanide 3-chlorophenyl hydrazone, whereas other oxidation rates were decreased. In most patients, ATP production was decreased. CONCLUSION: The proposed method can be successfully applied to the diagnosis of defects in PDHc, OXPHOS complexes, and ANT.

Decreased intracellular ATP content and intact mitochondrial energy generating capacity in human cystinotic fibroblasts.

Cystinosis is an autosomal recessive lysosomal storage disorder caused by a defect in the lysosomal cystine carrier cystinosin. Cystinosis is the most common cause of inherited Fanconi syndrome leading to renal failure, in which the pathogenesis is still enigmatic. Based on studies of proximal tubules loaded with cystine dimethyl ester (CDME), altered mitochondrial adenosine triphosphate (ATP) production was proposed to be an underlying pathologic mechanism. Thus far, however, experimental evidence supporting this hypothesis in humans is lacking. In this study, energy metabolism was extensively investigated in primary fibroblasts derived from eight healthy subjects and eight patients with cystinosis. Patient's fibroblasts accumulated marked amounts of cystine and displayed a significant decrease in intracellular ATP content. Remarkably, overall energy-generating capacity, activity of respiratory chain complexes, ouabain-dependent rubidium uptake reflecting Na,K-ATPase activity, and bradykinin-stimulated mitochondrial ATP production were all normal in these cells. In conclusion, the data presented demonstrate that mitochondrial energy-generating capacity and Na,K-ATPase activity are intact in cultured cystinotic fibroblasts, thus questioning the idea of altered mitochondrial ATP synthesis as a keystone for the pathogenesis of cystinosis.

Congenital hypertrophic cardiomyopathy, cataract, mitochondrial myopathy and defective oxidative phosphorylation in two siblings with Sengers-like syndrome.

UNLABELLED: We describe two siblings with a Sengers-like syndrome, who presented with congenital hypertrophic cardiomyopathy, infantile cataract, mitochondrial myopathy, lactic acidosis and normal mental development. A mitochondrial adenine nucleotide translocator 1 (ANT1) defect was detected since the ANT1 protein was not detectable by immmunoblotting in muscle samples of the patients. Additionally to these features of classical Sengers syndrome (OMIM 212350), we found that the mitochondrial oxidative phosphorylation, measured by biochemical analysis, was severely compromised in skeletal muscle in both children. Biochemical and morphological analysis of the fibroblasts revealed normal results. The association of significantly decreased pyruvate oxidation rates, deficient energy production and decreased multiple mitochondrial enzyme-complex activities in the muscle samples of our patients is a new finding which differs from previous results in patients with Sengers syndrome. CONCLUSION: we recommend a muscle biopsy and the biochemical analysis of the oxidative phosphorylation system in patients with muscle hypotonia, cardiomyopathy and congenital or infantile cataract.

Coenzyme Q-responsive Leigh's encephalopathy in two sisters.

A 31-year-old woman had encephalopathy, growth retardation, infantilism, ataxia, deafness, lactic acidosis, and increased signals of caudate and putamen on brain magnetic resonance imaging. Muscle biochemistry showed succinate:cytochrome c oxidoreductase (complex II-III) deficiency. Both clinical and biochemical abnormalities improved remarkably with coenzyme Q10 supplementation. Clinically, when taking 300mg coenzyme Q10 per day, she resumed walking, gained weight, underwent puberty, and grew 20cm between 24 and 29 years of age. Coenzyme Q10 was markedly decreased in cerebrospinal fluid, muscle, lymphoblasts, and fibroblasts, suggesting the diagnosis of primary coenzyme Q10 deficiency. An older sister has similar clinical course and biochemical abnormalities. These findings suggest that coenzyme Q10 deficiency can present as adult Leigh's syndrome.

Septo-optic dysplasia associated with a new mitochondrial cytochrome b mutation.

We report on a 25-year-old patient with isolated mitochondrial complex III deficiency and a new heteroplasmic mutation (T14849C) in the cytochrome b gene. He suffered from septo-optic dysplasia, retinitis pigmentosa, exercise intolerance, hypertrophic cardiomyopathy, and rhabdomyolysis. A HESX1 mutation was excluded as a cause of his septo-optic dysplasia. Low alpha-tocopherol concentrations in his muscles and an elevated urinary leukotriene E(4) excretion indicate increased production of reactive oxygen species.