Oral administration of coenzyme Q10 prevents cytochrome c release from mitochondria induced by 1-methyl-4-phenylpyridinium ion in mouse brain synaptosomes

Coenzyme Q₁₀ (CoQ₁₀) exerts neuroprotective effects in several in vivo and in vitro models of neurodegenerative disorders. However, the mechanisms of action are not fully understood. The aim in this study was to investigate whether oral administration of CoQ₁₀ could inhibit cytochrome c (cyt c) release from mitochondria induced by 1-methyl-4-phenylpyridinium ion (MPP⁺), which causes dopaminergic cell death by selective inhibition of complex I of the electron transport chain, in mouse brain synaptosomes. An increase of cyt c was detected in the cytosolic fraction from mouse brain synaptosomes treated with MPP⁺. Oral administration of CoQ₁₀ prevented the mitochondrial cyt c release in the MPP⁺-treated synaptosomes. In addition, CoQ₁₀ did not affect the MPP⁺-induced decrease in mitochondrial oxidation–reduction activity and membrane potential in brain synaptosomes. Our findings demonstrate that MPP⁺-induced mitochondrial cyt c release in brain synaptosomes is prevented by oral administration of CoQ₁₀ independently of mitochondrial dysfunction prior to the cyt c release.     

Severe encephalopathy associated to pyruvate dehydrogenase mutations and unbalanced coenzyme Q10 content

Coenzyme Q₁₀ (CoQ₁₀) deficiency is associated to a variety of clinical phenotypes including neuromuscular and nephrotic disorders. We report two unrelated boys presenting encephalopathy, ataxia, and lactic acidosis, who died with necrotic lesions in different areas of brain. Levels of CoQ₁₀ and complex II+III activity were increased in both skeletal muscle and fibroblasts, but it was a consequence of higher mitochondria mass measured as citrate synthase. In fibroblasts, oxygen consumption was also increased, whereas steady state ATP levels were decreased. Antioxidant enzymes such as NQO1 and MnSOD and mitochondrial marker VDAC were overexpressed. Mitochondria recycling markers Fis1 and mitofusin, and mtDNA regulatory Tfam were reduced. Exome sequencing showed mutations in PDHA1 in the first patient and in PDHB in the second. These genes encode subunits of pyruvate dehydrogenase complex (PDH) that could explain the compensatory increase of CoQ₁₀ and a defect of mitochondrial homeostasis. These two cases describe, for the first time, a mitochondrial disease caused by PDH defects associated with unbalanced of both CoQ₁₀ content and mitochondria homeostasis, which severely affects the brain. Both CoQ₁₀ and mitochondria homeostasis appears as new markers for PDH associated mitochondrial disorders.     

A selective defect of cytochrome c oxidase is present in brain of Alzheimer disease patients

To assess mitochondrial function and test the hypothesis of an underlying oxidative phosphorylation defect in Alzheimer disease (AD), we evaluated the activities of mitochondrial respiratory chain enzyme complexes I+III, complexes II+III, complex IV (cytochrome c oxidase, COX), succinate dehydrogenase, and citrate synthase in the frontal cortex, temporal cortex, hippocampus, and cerebellum of 23 AD patients and 13 normal human brains. The major finding was a significant decrease in COX activity in AD temporal cortex and hippocampus, both whether activities were expressed per noncollagen protein content (49 +/-4.6 versus 78+/-10.8 nmol/min/mg NCP, P = 0.006; 23+/-1.9 versus 48.6+/-8.1 nmol/min/mg NCP, p = 0.003) or corrected for citrate synthase activity (1.6+/-0.2 versus 3+/-0.4, P = 0.001; 0.76+/-0.1 versus 1.76+/-0.26, P = 0.0009). There were no significant differences in the activities of complexes I+III, II+III, and of succinate dehydrogenase in any of the brain regions examined. Our results suggest a specific defect of COX in the AD brain versus the normal human brain, which may contribute to impaired energy generation. Biochemically, the defect is confined to selected brain regions, suggesting anatomic specificity.     

Exogenous administration of coenzyme Q10 restores mitochondrial oxygen consumption in the aged mouse brain

The level of coenzyme Q (CoQ) has been shown to decrease in an age-dependent manner in several types of animals. However, whether CoQ-dependent mitochondrial function decreases with aging remains unclear. In this study, we found that mitochondrial complexes I and II exhibited significantly reduced oxygen consumption in the brains of aged male mice relative to young male mice, although this decrease in oxygen consumption was not accompanied by a change in the CoQ₉ or CoQ₁₀ content. Nevertheless, the administration of exogenous CoQ₁₀ significantly increased the content of CoQ₁₀ and CoQ₉ in the brain mitochondria of aged male mice and restored complex I- and II-mediated oxygen consumption to levels comparable to those observed in young mice. These results indicate that mitochondrial oxygen consumption in the brain decreases in aged male mice. Furthermore, these results suggest that exogenous CoQ₁₀ restores mitochondrial oxygen use to levels equivalent to those observed in young mice.     

Enzyme activities of fatty acid oxidation and the respiratory chain in chronically stimulated fast-twitch muscle of the rabbit

Fast-twitch tibialis anterior muscle of the rabbit was subjected to chronic low-frequency (10 Hz, 10 h/day) stimulation for different time periods up to 28 days. Total cellular activities of carnitine: palmitoyl-CoA transferase, crotonase, 3-hydroxyacyl-CoA dehydrogenase, 3-keto-acyl-CoA thiolase, citrate synthase, NADH:cytochrome c oxidoreductase, succinate: cytochrome c oxidoreductase, and cytochrome c oxidase were measured in contralateral and stimulated muscles at various times. With the exception of crotonase, which increased only 1.6-fold after 28 days of stimulation, the other enzymes increased in parallel displaying 3-fold elevated absolute activities. These results, by supporting and extending our previous findings, indicate that the expression of the enzymes of the main metabolic systems of aerobic substrate oxidation, i.e. the citric acid cycle, the fatty acid oxidation and the respiratory chain, is regulated in a coordinate manner.     

Bypassing human CoQ10 deficiency

Primary disorders of the human coenzyme Q₁₀ (CoQ₁₀) biosynthesis pathway are a known cause of severe pediatric diseases. So far, oral administration of CoQ₁₀ is the only treatment strategy for affected individuals. However, the real benefit of CoQ₁₀ supplementation remains questionable and clinical studies regarding efficiency are lacking. Here we provide an outlook on novel treatment approaches using CoQ precursor compounds. These metabolic bypass strategies might be a promising alternative for oral CoQ₁₀ supplementation regimens.     

A novel inborn error of the coenzyme Q10 biosynthesis pathway: cerebellar ataxia and static encephalomyopathy due to COQ5 C‐methyltransferase deficiency

Primary coenzyme Q10 (CoQ₁₀; MIM# 607426) deficiencies are an emerging group of inherited mitochondrial disorders with heterogonous clinical phenotypes. Over a dozen genes are involved in the biosynthesis of CoQ₁₀, and mutations in several of these are associated with human disease. However, mutations in COQ5 (MIM# 616359), catalyzing the only C‐methylation in the CoQ₁₀ synthetic pathway, have not been implicated in human disease. Here, we report three female siblings of Iraqi‐Jewish descent, who had varying degrees of cerebellar ataxia, encephalopathy, generalized tonic‐clonic seizures, and cognitive disability. Whole‐exome and subsequent whole‐genome sequencing identified biallelic duplications in the COQ5 gene, leading to reduced levels of CoQ₁₀ in peripheral white blood cells of all affected individuals and reduced CoQ₁₀ levels in the only muscle tissue available from one affected proband. CoQ₁₀ supplementation led to clinical improvement and increased the concentrations of CoQ₁₀ in blood. This is the first report of primary CoQ₁₀ deficiency caused by loss of function of COQ5, with delineation of the clinical, laboratory, histological, and molecular features, and insights regarding targeted treatment with CoQ₁₀ supplementation.     

Cerebellar ataxia and severe muscle CoQ10 deficiency in a patient with a novel mutation in ADCK3

Inherited ataxias are a group of heterogeneous disorders in children or adults but their genetic definition remains still undetermined in almost half of the patients. However, CoQ₁₀ deficiency is a rare cause of cerebellar ataxia and ADCK3 is the most frequent gene associated with this defect. We herein report a 48 year old man, who presented with dysarthria and walking difficulties. Brain magnetic resonance imaging showed a marked cerebellar atrophy. Serum lactate was elevated. Tissues obtained by muscle and skin biopsies were studied for biochemical and genetic characterization. Skeletal muscle biochemistry revealed decreased activities of complexes I+III and II+III and a severe reduction of CoQ₁₀, while skin fibroblasts showed normal CoQ₁₀ levels. A mild loss of maximal respiration capacity was also found by high‐resolution respirometry. Molecular studies identified a novel homozygous deletion (c.504del_CT) in ADCK3, causing a premature stop codon. Western blot analysis revealed marked reduction of ADCK3 protein levels. Treatment with CoQ₁₀ was started and, after 1 year follow‐up, patient neurological condition slightly improved. This report suggests the importance of investigating mitochondrial function and, in particular, muscle CoQ₁₀ levels, in patients with adult‐onset cerebellar ataxia. Moreover, clinical stabilization by CoQ₁₀ supplementation emphasizes the importance of an early diagnosis.     

Fatal neonatal encephalopathy and lactic acidosis caused by a homozygous loss-of-function variant in COQ9

Coenzyme Q₁₀ (CoQ₁₀) has an important role in mitochondrial energy metabolism by way of its functioning as an electron carrier in the respiratory chain. Genetic defects disrupting the endogenous biosynthesis pathway of CoQ₁₀ may lead to severe metabolic disorders with onset in early childhood. Using exome sequencing in a child with fatal neonatal lactic acidosis and encephalopathy, we identified a homozygous loss-of-function variant in COQ9. Functional studies in patient fibroblasts showed that the absence of the COQ9 protein was concomitant with a strong reduction of COQ7, leading to a significant accumulation of the substrate of COQ7, 6-demethoxy ubiquinone₁₀. At the same time, the total amount of CoQ₁₀ was severely reduced, which was reflected in a significant decrease of mitochondrial respiratory chain succinate-cytochrome c oxidoreductase (complex II/III) activity. Lentiviral expression of COQ9 restored all these parameters, confirming the causal role of the variant. Our report on the second COQ9 patient expands the clinical spectrum associated with COQ9 variants, indicating the importance of COQ9 already during prenatal development. Moreover, the rescue of cellular CoQ₁₀ levels and respiratory chain complex activities by CoQ₁₀ supplementation points to the importance of an early diagnosis and immediate treatment.     

Mitochondrial energetic impairment in a patient with late‐onset glutaric acidemia Type 2

Glutaric acidemia type 2 (GA2), also called multiple acyl‐CoA dehydrogenase deficiency, is an autosomal recessive disorder of fatty acid, amino acid, and choline metabolism resulting in excretion of multiple organic acids and glycine conjugates as well as elevation of various plasma acylcarnitine species (C4–C18). It is caused by mutations in the ETFA, ETFB, or ETFDH genes which are involved in the transfer of electrons from 11 flavin‐containing dehydrogenases to Coenzyme Q₁₀ (CoQ₁₀) of the mitochondrial electron transport chain (ETC). We report a patient who was originally reported as the first case with primary myopathic CoQ₁₀ deficiency when he presented at 11.5 years with exercise intolerance and myopathy that improved after treatment with ubiquinone and carnitine. At age 23, his symptoms relapsed despite increasing doses of ubiquinone and he was shown to have biallelic mutations in the ETFDH gene. Treatment with riboflavin was started and ubiquinone was changed to ubiquinol. After 4 months, the patient recovered his muscle strength with normalization of laboratory exams and exercise tolerance. Functional studies on fibroblasts revealed decreased levels of ETFDH as well as of very long‐chain acyl‐CoA dehydrogenase and trifunctional protein α. In addition, the mitochondrial mass was decreased, with increased formation of reactive oxygen species and oxygen consumption rate, but with a decreased spared respiratory capacity, and decreased adenosine triphosphate level. These findings of widespread dysfunction of fatty acid oxidation and ETC enzymes support the impairment of a larger mitochondrial ETC supercomplex in our patient.     

Rat liver mitochondrial enzyme activities in hypoxia.

Rat liver mitochondrial enzyme activities were measured after exposing the animals to the atmospheric pressure of 380 mm Hg for 5 h and 14 days. Succinate dehydrogenase and succinate oxidase activities increased significantly during the hypoxic period of 14 days. No change was observed in cytochrome oxidase activity. Malate dehydrogenase and glutamate dehydrogenase activities increased somewhat, but not significantly. The efficiency of oxidative phosphorylation (the ADP:O ratio) in the isolated mitochondria remained unchanged. The exact mitochondrial protein values showed a 15% decrease as compared with the control group. The concentrations of cytochromes did not change significantly in the hypoxic group. During the short hypoxic period succinate dehydrogenase, succinate oxidase and cytochrome oxidase activities increased as compared with those in the control group.     

Comparison between developmental and senescent changes in enzyme activities linked to energy metabolism in rat heart

The developmental and senescent patterns of a number of heart enzyme activities linked to energy metabolism have been studied in rats aged between 4 days and 21 months. A morphometric study of mitochondrial volume fractions and numbers has been also carried out. Developmental changes result in a rise of most mitochondrial enzymes (NADP⁺-isocitrate dehydrogenase, malic enzyme, succinate dehydrogenase, citrate synthase) and mitochondrial volume fractions. Exceptions are NAD⁺-isocitarte dehydrogenase, which declines from 4 days onwards, and NAD⁺-malate dehydrogenase, which declines and then rises over the same period. Senescent changes follow two different trends. While pyruvate kinase and those mitochondrial enzymes lying between citrate formation and isocitrate oxidation (citrate synthase, NADP⁺- and NAD⁺-isocitrate dehydrogenases) decline to some degree, mitochondrial succinate dehydrogenase and NAD⁺-malate dehydrogenase activities increase over the same period. This could point towards a partial impairment of Krebs cycle function, and a reduced energy-producing capacity in the aged rat heart.     

Effective and safe therapy with coenzyme Q10 for cardiomyopathy

Summary Coenzyme Q₁₀ (CoQ₁₀) is indispensable in mitochondrial bioenergetics and for human life to exist. 88/115 patients completed a trial of therapy with CoQ₁₀ for cardiomyopathy. Patients were selected on the basis of clinical criteria,X-rays, electrocardiograms, echocardiography, and coronary angiography. Responses were monitored by ejection fractions, cardiac output, and improvements in functional classifications (NYHA). Of the 88 patients 75%–85% showed statistically significant increases in two monitored cardiac parameters. Patients with the lowest ejection fractions (approx. 10%–30%) showed the highest increases (115%–210%) and those with higher ejection fractions (50%–80%) showed increases of approx. 10%–25% on therapy. By functional classification, 17/21 in class IV, 52/62 in class III, and 4/5 in class II improved to lower classes. Clinical responses appeared over variable times, and are presumably based on mechanisms of DNA-RNA-protein synthesis of apoenzymes which restore levels of CoQ₁₀ enzymes in a deficiency state. 10/21 (48%) of patients in class IV, 26/62 (42%) in class III, and 2/5 (40%) in class II had exceptionally low control blood levels of CoQ₁₀. Clinical responses on therapy with CoQ₁₀ appear maximal with blood levels of approx. 2.5 µg CoQ₁₀/ml and higher during therapy.Abbreviations CHF Congestive heart failure - CO Cardiac output - CoQ₁₀ Coenzyme Q₁₀ - EF Ejection fraction - IC Impedance cardiography - NYHA New York Heart Association - STI Systolic time interval     

Primary coenzyme Q10 deficiency presenting as fatal neonatal multiorgan failure

Coenzyme Q₁₀ deficiency is a clinically and genetically heterogeneous disorder, with manifestations that may range from fatal neonatal multisystem failure, to adult-onset encephalopathy. We report a patient who presented at birth with severe lactic acidosis, proteinuria, dicarboxylic aciduria, and hepatic insufficiency. She also had dilation of left ventricle on echocardiography. Her neurological condition rapidly worsened and despite aggressive care she died at 23 h of life. Muscle histology displayed lipid accumulation. Electron microscopy showed markedly swollen mitochondria with fragmented cristae. Respiratory-chain enzymatic assays showed a reduction of combined activities of complex I+III and II+III with normal activities of isolated complexes. The defect was confirmed in fibroblasts, where it could be rescued by supplementing the culture medium with 10 μM coenzyme Q₁₀. Coenzyme Q₁₀ levels were reduced (28% of controls) in these cells. We performed exome sequencing and focused the analysis on genes involved in coenzyme Q₁₀ biosynthesis. The patient harbored a homozygous c.545T>G, p.(Met182Arg) alteration in COQ2, which was validated by functional complementation in yeast. In this case the biochemical and morphological features were essential to direct the genetic diagnosis. The parents had another pregnancy after the biochemical diagnosis was established, but before the identification of the genetic defect. Because of the potentially high recurrence risk, and given the importance of early CoQ₁₀ supplementation, we decided to treat with CoQ₁₀ the newborn child pending the results of the biochemical assays. Clinicians should consider a similar management in siblings of patients with CoQ10 deficiency without a genetic diagnosis.     

Tirandamycin: inhibition of oxidative phosphorylation in rat liver mitochondria

Tirandamycin inhibits respiration and phosphorylation in rat liver mitochondria. An investigation of individual reaction sequences occurring within the respiratory chain showed that the antibiotic stimulates reduced nicotinamide adenine dinucleotide (NADH)- and succinate-linked coenzyme Q reductase. NADH-linked reduction of tetrazolium salts remains unaffected by tirandamycin. Succinotetrazolium salt reductase is inhibited significantly. Reduction of cytochrome c by succinate is blocked by the antibiotic; NADH-cytochrome c reductase is inhibited but not completely blocked. Cytochrome c oxidase remains unaffected. Mitochondrial difference spectra prepared in the presence of tirandamycin indicate that the reduction of cytochrome b is not impaired but no reduction of cytochromes c or a is apparent. These results indicate that tirandamycin interferes with the respiratory chain at a point beyond the cytochrome b and prior to the cytochrome c reduction site. Tirandamycin acts also as a potent inhibitor of ribonucleic acid polymerase as discussed in the foregoing paper.     

Prolonged coenzyme Q10 treatment in Down syndrome patients: effect on DNA oxidation

Oxidative stress is known to play a relevant role in Down syndrome (DS) and its effects are documented from embryonic life. Oxidative DNA damage has been shown to be significantly elevated in Down syndrome patients, and this has been indicated as an early event promoting neurodegeneration and Alzheimer type dementia. The aim of this study was to investigate the efficacy of coenzyme Q₁₀ (CoQ₁₀) in delaying the effect of oxidative damage in these patients. In our previous study we demonstrated a mild protective effect of CoQ₁₀ on DNA, although the treatment was unable to modify the overall extent of oxidative damage at the patient level. Possible limitations of the previous study were: time of treatment (6 months) or spectrum of DNA lesions detected. In order to overcome these limitations we planned a continuation of the trial aimed at evaluating the effects of CoQ₁₀ following a prolonged treatment. Our results highlight an age-specific reduction in the percentage of cells showing the highest amount of oxidized bases, indicating a potential role of CoQ₁₀ in modulating DNA repair mechanisms.     

The effect of high-intensity exhaustive exercise studied in isolated mitochondria from human skeletal muscle

Six young men performed five 1-min bicycle exercise bouts to exhaustion. Muscle lactate increased to congruent with 114 mmol x kg(-1) dwt and pH decreased to congruent with 6.6. Mitochondria were prepared from a needle biopsy sample taken from m. vastus lateralis immediately after the last exercise bout. No significant effect of exhaustion on the proton permeability and amount of cytochromes c and aa3 in isolated mitochondria was detected. The activities of the following enzymes and systems were not altered either: citrate synthase, succinate dehydrogenase, cytochrome oxidase, succinate + glutamate respiration, malate + glutamate respiration, the respiratory chain, and the reactions involved in ATP synthesis. Thus, the mitochondria did not appear globally altered upon exhaustion. However, the following NAD-linked activities were significantly lowered: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, glutamate dehydrogenase and fatty acid beta-oxidation. The activities of alpha-glycerophosphate dehydrogenase and exo-NADH oxidase, enzymes that might catalyze the oxidation of sarcoplasmic NADH, were increased. These changes may be due to the action of reactive oxygen species, protons and Ca2+. Transient opening of the permeability transition pore may also be involved. Some effects may have been reversed during isolation of the mitochondria and the changes in mitochondrial function in situ upon exhaustion may have been more extensive than observed.     

RENAL HANDLING OF CITRATE DURING HEAT-INDUCED HYPOCITRICEMIA.

A model of steady-state hypocitricemia, characterized by hypocitraturia and reduced kidney cortex citrate, has been demonstrated in the rate chronically exposed to environmental heat. The renal citrate extraction ratio remains unchanged. The physiological mechanism that brings about the reduction in circulating citrate has not been determined. Hypocitraturia likely results from a decreased filtered citrate load. Although it is generally contended that filtered citrate load. Although it is generally contended that alkalosis increases and acidosis decreases renal excretion of citrate, observations of mild alkalosis and hypocitraturia during heat exposure suggest that factors other than pH can alter renal handling of citrate. Kidney mitochondrial function, as determined by in vitro measurements of citrate-stimulated respiratory rates and specific activities of isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, and cytochrome c oxidase, appears to be unaffected by environmental heat.     

Regulation of heme metabolism during senescence: activity of several heme-containing enzymes and heme oxygenase in the liver and kidney of aging rats

The activities of several heme-containing enzymes plus succinate dehydrogenase, the content of mitochondrial cytochromes, the amount of microsomal cytochrome P-450, and the activity of heme oxygenase, the major enzyme of heme degradation, have been compared in young, mature and senescent rats. Measurements were made in liver, a tissue previously reported to have an age-related decrease in δ-aminolevulinic acid synthetase, and in kidney, a tissue previously reported to have no age-related change in this enzyme, the rate-limiting enzyme of heme biosynthesis (Paterniti, Lin and Beattie, Arch. Biochem. Biophys., 191 (1978) 792–797). The activity of cytochrome oxidase in liver mitochondria did not decrease with age while this activity in kidney mitochondria was highest in animals one year old and decreased in the two-year-olds. By contrast, succinate dehydrogenase of both kidney and liver mitochondria decreased markedly in the aging rats. No age-related change in the content of cytochromes b, c or aa₃ was observed in liver mitochondria; however, a marked age-related decrease in cytochrome aa₃ was observed in kidney mitochondria. Similarly no change in cytochrome P-450 levels was observed in either tissue obtained from aging animals. In the liver, catalase activity increased while in the kidney it decreased in senescent as compared to mature animals. Heme degradation does not decrease with age as the activity of heme oxygenase increased in both liver and kidney of two-year-old rats as compared to one-year-olds. These results suggest that the lower activity of δ-aminolevulinic acid synthetase observed in the aging rat may not be correlated with a decrease in the activity of heme-containing proteins and that the regulation of the heme pool used for the synthesis of various intracellular hemo-proteins may be complex.     

Distribution of Capillary Transit Times in Isolated Lungs of Oxygen-Tolerant Rats

Rats pre-exposed to 85% O₂ for 5–7 days tolerate the otherwise lethal effects of 100% O₂. The objective was to evaluate the effect of rat exposure to 85% O₂ for 7 days on lung capillary mean transit time ([`(t)]<sub>\textc</sub> ) (\bar{t}_{\text{c}} ) and distribution of capillary transit times (h <sub>c</sub>(t)). This information is important for subsequent evaluation of the effect of this hyperoxia model on the redox metabolic functions of the pulmonary capillary endothelium. The venous concentration vs. time outflow curves of fluorescein isothiocyanate labeled dextran (FITC-dex), an intravascular indicator, and coenzyme Q<sub>1</sub> hydroquinone (CoQ<sub>1</sub>H<sub>2</sub>), a compound which rapidly equilibrates between blood and tissue on passage through the pulmonary circulation, were measured following their bolus injection into the pulmonary artery of isolated perfused lungs from rats exposed to room air (normoxic) or 85% O<sub>2</sub> for 7 days (hyperoxic). The moments (mean transit time and variance) of the measured FITC-dex and CoQ<sub>1</sub>H<sub>2</sub> outflow curves were determined for each lung, and were then used in a mathematical model [Audi et al. J. Appl. Physiol. 77: 332–351, 1994] to estimate [`(t)]_\textc \bar{t}_{\text{c}} and the relative dispersion (RD_c) of h _c(t). Data analysis reveals that exposure to hyperoxia decreases lung [`(t)]_\textc \bar{t}_{\text{c}} by 42% and increases RD_c, a measure h _c(t) heterogeneity, by 40%.