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To report the retinal phenotype and the associated genetic and systemic findings in patients with mitochondrial disease.

Aim:Pharmacoresistance is a major burden in epilepsy treatment. We aimed to identify genetic biomarkers in response to specific antiepileptic drugs (AEDs) in genetic generalized epilepsies (GGE). Materials & methods:We conducted a genome-wide association study (GWAS) of 3.3 million autosomal SNPs in 893 European subjects with GGE – responsive or nonresponsive to lamotrigine, levetiracetam and valproic acid. Results:Our GWAS of AED response revealed suggestive evidence for association at 29 genomic loci (p <10-5) but no significant association reflecting its limited power. The suggestive associations highlight candidate genes that are implicated in epileptogenesis and neurodevelopment. Conclusion:This first GWAS of AED response in GGE provides a comprehensive reference of SNP associations for hypothesis-driven candidate gene analyses in upcoming pharmacogenetic studies.

The metabolic disorder, pyridoxine-dependent epilepsy (PDE), exemplifies the principle that expeditious diagnosis and implementation of a targeted therapy, in this case, pyridoxine or vitamin B6, may prevent catastrophic neurodevelopmental outcomes. Scurvy in sailors was the first disease identified as curable by ingestion of a vitamin over 300 years ago. The association between pyridoxine and early onset neonatal epileptic encephalopathy was established by a single-case report nearly 70 years ago.1A mother's third pregnancy led to a child successfully treated for neonatal seizures once it became apparent that vitamins ingested during gestation had relieved hyperemesis gravidarum and then neonatal seizures. This followed a prior pregnancy with a devastating neurologic outcome in an older sibling, without the benefit of vitamin trials and a diagnosis.

Oxyphil cell transformation of epithelial cells due to the accumulation of mitochondria occurs often during cellular aging. To understand the pathogenic mechanisms, we studied mitochondrial DNA (mtDNA) alterations in the three cell types of the parathyroids using multiplex real-time PCR and next-generation sequencing. mtDNA was analyzed from cytochrome coxidase (COX)–positive and COX-negative areas of 19 parathyroids. Mitochondria-rich pre-oxyphil/oxyphil cells were more prone to develop COX defects than the mitochondria-poor clear chief cells (P < 0.001). mtDNA increased approximately 2.5-fold from clear chief to oxyphil cells. In COX deficiency, the increase was even more pronounced, and COX-negative oxyphil cells had approximately two times more mtDNA than COX-positive oxyphil cells (P < 0.001), illustrating the influence of COX deficiency on mtDNA biosynthesis, probably as a consequence of insufficient ATP synthesis. Next-generation sequencing revealed a broad spectrum of putative pathogenic mtDNA point mutations affecting NADH dehydrogenase and COX genes as well as regulatory elements of mtDNA. NADH dehydrogenase gene mutations preferentially accumulated in COX-positive pre-oxyphil/oxyphil cells and, therefore, could be essential for inducing oxyphil cell transformation by increasing mtDNA/mitochondrial biogenesis. In contrast, COX-negative cells predominantly harbored mutations in the MT-CO1and MT-CO3genes and in regulatory mtDNA elements, but only rarely NADH dehydrogenase mutations. Thus, multiple hits in NADH dehydrogenase and COX activity–impairing genes represent the molecular basis of oxyphil cell transformation in the parathyroids.

In search of tumor-specific mitochondrial DNA (mtDNA) mutations in head and neck squamous cell cancer, we found heteroplasmy in the blood of two individuals, i.e., these individuals carried two alleles of mtDNA. In both cases, the tumor was found to be homoplasmic, i.e., it contained only one of the two mtDNA alleles present in blood. More interestingly, in one case the tumor had acquired the wild-type allele, while in the other case it contained the mutant allele only. Sequencing of the whole 16.5kb mtDNA showed that the observed heteroplasmic positions in the D-loop region, nucleotides 152 and 16187, respectively, were the only differences between tumor and blood mtDNA genotypes in these individuals. Our findings thus strongly support the hypothesis that accumulation of mtDNA mutations in solid tumors occurs by clonal and random expansion of pre-existing alleles and is not necessary for the metabolic changes generally associated with tumor formation, the Warburg effect.

Analysis of mitochondrial function is central to the study of intracellular energy metabolism, mechanisms of cell death and pathophysiology of a variety of human diseases, including myopathies, neurodegenerative diseases and cancer. However, important properties of mitochondria differ in vivo and in vitro. Here, we describe a protocol for the analysis of functional mitochondria in situ, without the isolation of organelles, in selectively permeabilized cells or muscle fibers using digitonin or saponin. A specially designed substrate/inhibitor titration approach allows the step-by-step analysis of several mitochondrial complexes. This protocol allows the detailed characterization of functional mitochondria in their normal intracellular position and assembly, preserving essential interactions with other organelles. As only a small amount of tissue is required for analysis, the protocol can be used in diagnostic settings in clinical studies. The permeabilization procedure and specific titration analysis can be completed in 2 h.

Mitochondrial dysfunction has gained considerable interest as a potential cause of epileptic seizures and therapy-resistant forms of severe epilepsy. Impairment of mitochondrial function has recently been observed in the seizure focus of human and experimental epilepsy. Additionally, a broad variety of mutation of mitochondrial DNA leading to the inhibition of mitochondrial respiratory chain or directly of mitochondrial adenosine triphosphate synthesis in epileptogenic areas of the human brain has been associated with epileptic phenotypes. Since mitochondrial oxidative phosphorylation provides the major source of adenosine triphosphate in neurons and mitochondria participate in cellular Ca2homeostasis they can modulate neuronal excitability and synaptic transmission. Furthermore, mitochondria are intimately involved in pathways leading to the neuronal cell death characteristic for the areas of epileptogenesis.

Mitochondrial pathology is an early observation in motor neurons and skeletal muscle of patients with amyotrophic lateral sclerosis (ALS). To clarify the relevance of this finding, we determined the effects of a 1-month oral administration of creatine on 1H NMR-visible metabolites in the motor cortices of 15 controls and 15 patients with sporadic ALS, most of whom had mitochondrial pathology in skeletal muscle. In the motor cortex of the ALS group the N-acetylaspartate (NAA)/creatine (Crt) metabolite ratio was lower than in our control group, indicating NAA loss. Upon creatine supplementation we observed in the controls a decline in the NAA/Crt, NAA/choline (Cho), glutamate + glutamine (Glx)/Crt, and Glx/Cho metabolite ratios. In contrast, in the ALS patient group the NAA/Crt and the NAA/Cho metabolite ratios remained unchanged, while the Glx/Crt and Glx/Cho metabolite ratios decreased. These data are compatible with the interpretation that creatine supplementation causes an increase in the diminished NAA levels in ALS motor cortex as well as an increase of choline levels in both ALS and control motor cortices. Because NAA is synthesized by mitochondria in an energy-dependent manner and the NAA/Cho metabolite ratios in the ALS motor cortices were found to be correlated to the degree of mitochondrial pathology in ALS skeletal muscle, our results can be explained by a deficiency of enzymes of mitochondrial respiratory chain in the ALS motor cortex which might affect motor neuron survival.

Mitochondria are cellular organelles crucial for energy supply and calcium homeostasis in neuronal cells, and their dysfunction causes seizure activity in some rare human epilepsies. To directly test whether mitochondrial respiratory chain enzymes are abnormal in the most common form of chronic epilepsy, temporal lobe epilepsy (TLE), living human brain specimens from 57 epileptic patients and 2 nonepileptic controls were investigated. In TLE patients with a hippocampal epileptic focus, we demonstrated a specific deficiency of complex I of the mitochondrial respiratory chain in the hippocampal CA3 region. In contrast, TLE patients with a parahippocampal epileptic focus showed reduced complex I activity only in parahippocampal tissue. Inhibitor titrations of the maximal respiration rate of intact human brain slices revealed that the observed reduction in complex I activity is sufficient to affect the adenosine triphosphate production rate. The abnormal complex I activity in the hippocampal CA3 region was paralleled by increased succinate dehydrogenase staining of neurons and marked ultrastructural abnormalities of mitochondria. Therefore, mitochondrial dysfunction is suggested to be specific for the epileptic focus and may constitute a pathomechanism contributing to altered excitability and selective neuronal vulnerability in TLE. Ann Neurol 2000;48:766–773

Mitochondria are cellular organelles crucial for energy supply and calcium homeostasis in neuronal cells, and their dysfunction causes seizure activity in some rare human epilepsies. To directly test whether mitochondrial respiratory chain enzymes are abnormal in the most common form of chronic epilepsy, temporal lobe epilepsy (TLE), living human brain specimens from 57 epileptic patients and 2 nonepileptic controls were investigated. In TLE patients with a hippocampal epileptic focus, we demonstrated a specific deficiency of complex I of the mitochondrial respiratory chain in the hippocampal CA3 region. In contrast, TLE patients with a parahippocampal epileptic focus showed reduced complex I activity only in parahippocampal tissue. Inhibitor titrations of the maximal respiration rate of intact human brain slices revealed that the observed reduction in complex I activity is sufficient to affect the adenosine triphosphate production rate. The abnormal complex I activity in the hippocampal CA3 region was paralleled by increased succinate dehydrogenase staining of neurons and marked ultrastructural abnormalities of mitochondria. Therefore, mitochondrial dysfunction is suggested to be specific for the epileptic focus and may constitute a pathomechanism contributing to altered excitability and selective neuronal vulnerability in TLE. Ann Neurol 2000;48:766–773

Amyotrophic lateral sclerosis is a neurodegenerative disease affecting the anterior horn cells of the spinal cord and cortical motor neurons. Previous findings have suggested a specific impairment of mitochondrial function in skeletal muscle of at least a limited number of patients. Applying flavoprotein/NAD(P)H autofluorescence imaging of mitochondrial function in saponin-permeabilized muscle fibres, we detected a heterogeneous distribution of the respiratory chain defect among individual fibres in muscle biopsies of patients (11 out of 17) with sporadic amyotrophic lateral sclerosis (SALS). These findings correlate with the presence of cytochrome c oxidase (COX)-negative muscle fibres detected histologically. We established the molecular basis for the decreased activities of NADH:CoQ oxidoreductase and COX in SALS muscle. In the skeletal muscle of the investigated patients, diminished levels (13 out of 17) or multiple deletions (one out of 17) of mitochondrial DNA (mtDNA) were observed. These alterations of mtDNA seem to be related to decreased levels of membrane-associated mitochondrial Mn-superoxide dismutase. Our results support the viewpoint that an oxygen radical-induced impairment of mtDNA is of pathophysiological significance in the aetiology of at least a subgroup of patients with SALS.

The quantification of mitochondrial enzyme activities in skeletal muscle samples of patients suspected of having mitochondrial myopathies is problematic. Therefore, we have evaluated different methods for the determination of activities cytochrome c oxidase and NADH:CoQ oxidoreductase in human skeletal muscle samples. The measurement of cytochrome c oxidase activity in the presence of 200 μM ferrocytochrome c and the detection of NADH:CoQ oxidoreductase as rotenone-sensitive NADH:CoQ₁ reductase resulted in comparable citrate synthase-normalized respiratory chain enzyme activities of both isolated mitochondria and homogenates from control human skeletal muscle samples. These methods allowed the precise detection of deficiencies of respiratory chain enzymes in skeletal muscle of two patients harboring only 20 and 27% of deleted mitochondrial DNA, respectively. Therefore, citrate synthase-normalized respiratory chain activities can serve as stable reference values for the determination of a putative mitochondrial defect in human skeletal muscle. Copyright 2000 Academic Press.

Changes are described that are brought about by antimycin, NoHOQnO, funiculosin, myxothiazol and mucidin in the α-, β- and γ-absorption bands of reduced and oxidized cytochromes bin the isolated complex bc1, form beef heart mitochondria. The inhibitors can be divided into 2 groups. Antimycin, funiculosin and NoHOQnO are likely to shift the spectrum of b-562 and compete for specific binding with complex bc1with each other but not with myxothiazol and mucidin. The spectral effects of the latter two inhibitors are more difficult to interpret and may involve contributions not only from b-562 but from b-566 as well. The existence of 2 independent inhibitor binding-sites in the complex bc1corroborates the Q-cycle hypothesis.

When succinate oxidation by submitochondrial particles is blocked by antimycin, NoHOQnO or funiculosin, addition of ferricyanide restores oxygen uptake coupled to membrane potential generation. The effect of ferricyanide is abolished by mucidin or myxothiazol, as well as by KCN. The data strongly favor a cyclic redox loop mechanism in site 2 and show that either heme of the ferrous cytochrome bor ubisemiquinone formed in the QH 2-oxidizing center of complex b− c1is accessible to ferricyanide at the outer (M) side of the submitochondrial particle membrane.

A mathematical model was developed to estimate flux control coefficients (C o) from titration studies with specific non-competitive inhibitors. In contrast to the normally used graphical determination the model pays regard to the dissociation equilibrium ( kD) that exists between inhibitor and its binding sites ( Eo) as well as to an objective estimation of the initial slope. The model was used for the analysis of titration experiments where the respiration of rat liver mitochondria was inhibited with carboxyatractyloside and antimycin A. It is shown that the graphical estimation of Eoand Colead to significant overestimation if the ratio Kd/ Eois larger than 10 −4which can be avoided by using our model.

The different flavoproteins contributing to flavin fluorescence of isolated rat liver mitochondria have distinct excitation and emission spectra. The NAD-linked flavin component was identified as α-lipoamide dehydrogenase, while the non-NAD-linked component was found to be electron transfer flavoprotein. The differences in excitation and emission properties of the mitochondrial flavoproteins permit selective recording of their redox state changes in isolated mitochondria.

Anaerobic potentiometric titrations of bcytochromes have been carried out in beef heart submitochondrial particles in the presence of several specific inhibitors of electron transfer through the b-c1-site of the respiratory chain. Whereas antimycin shows no significant effect on the titration curve of cytochrome b-562, NoHOQnO is found to shift the Emof b-562 by 20–30 mV to the positive. Funiculosin raises the Emof b-562 by > 100 mV and also appears to bring about a minor shift of b-566 midpoint potential. In the presence of myxothiazol, both bcytochromes with Emvalues 15–30 mV more positive than in the control.

The different flavoproteins contributing to flavin fluorescence of isolated rat liver mitochondria have distinct excitation and emission spectra. The NAD‐linked flavin component was identified as α‐lipoamide dehydrogenase, while the non‐NAD‐linked component was found to be electron transfer flavoprotein. The differences in excitation and emission properties of the mitochondrial flavoproteins permit selective recording of their redox state changes in isolated mitochondria.

At 488 nm argon-ion laser excitation human mononuclear cells emit flavoprotein-related autofluorescence signals. Approximately 60% of these are caused by the mitochondrial flavoproteins α-lipoamide dehydrogenase and electron transfer flavoprotein, having differences in their fluorescence emission spectra. At the emission wavelength of 530 nm the redox changes of α-lipoamide dehydrogenase fluorescence in human mononuclear cells can be monitored by flow cytometry. This allows the estimation of the steady-state reduction level of this flavoprotein being in redox equilibrium with the mitochondrial NAD-system. We applied this method to elucidate the possible impairment of mitochondrial function in subpopulations of mononuclear cells of patients harboring deletions of the mitochondrial DNA in skeletal muscle. In the monocyte fraction of three patients and in the lymphocyte fraction of one patient we observed in the presence of the mitochondrial substrate octanoate elevated steady-state reduction levels of α-lipoamide dehydrogenase. This is an indication for the presence of respiratory chain-inhibited mitochondria in mononuclear cell subpopulations of the described patients. These data were confirmed by conventional determinations of maximal oxygen consumption rates of digitonin-permeabilized cells. Therefore, the flow cytometric determination of flavoprotein-caused autofluorescence changes is a useful and sensitive method for the detection of an impairment of mitochondrial respiratory chain in subpopulations of heterogeneous cell suspensions.

Three cecal-fistulated horses were used in a 3 × 3 latin square experiment to determine the influence of diet and of cecal infusions of Na2C03on cecal fermentation and feeding behavior. The three treatments were hay, concentrate and concentrate plus hourly infusions of Na2CO3. Cecal fluid samples and cecal pH readings were taken at zero through 11 hr following feeding at the end of each experimental period, and animal activity was measured by the use of a movie camera set to take 5 sec of film every 5 minutes. Cecal pH was significantly lower at 4, 5 and 6 hr following feeding for the horses receiving the concentrate diet than for those fed hay. The concentrate-fed horses had a significantly lower percentage of cecal acetate and higher cecal propionate than those fed hay, while cecal butyrate was variable for horses receiving both diets. Cecal lactic acid was lower for the horses fed the hay diet than for those fed concentrate but the data were variable.Infusions of Na2CO3significantly increased cecal pH at 3, 4 and 5 hr post-feeding, compared with that of horses fed only the concentrate diet. Horses receiving the Na2C03infusion had higher cecal acetate and lower propionate at 1, 3 and 4 hr following feeding than those fed only the all-concentrate diet.The horses fed the concentrate diet spent significantly more time chewing wood and in coprophagy than did those fed hay. Infusions appeared to reduce the time spent in these activities by the concentrate-fed horses, however the differences were not significant. The amount of time spent chewing wood was found to be significantly correlated with cecal propionate.

The brindled mottled mouse (Mobr), an animal model of the Menkes' copper deficiency syndrome, was used for the investigation of changes in respiratory flux control associated with cytochrome coxidase deficiency in muscle. Enzymatic analysis of cardiac and skeletal muscles showed an approximately 2-fold decrease in cytochrome coxidase activity of brindled mutants in both types of muscles as compared with controls. The activities of NADH-cytochrome coxidoreductase (respiratory chain segment I-III) and succinate-cytochrome coxidoreductase (segment II-III) were normal. Assessment of mitochondrial respiratory function was performed using chemically skinned musculus quadriceps or heart muscle fibers isolated from control and brindled mottled mice. In skeletal muscle, there was no difference found in maximal rates of respiration. In the Mobrhearts, this parameter was slightly lower than control. Alternately, the determination of flux control coefficients of cytochrome coxidase performed by a step by step inhibition of respiration with increasing concentrations of azide or cyanide revealed significantly sharper inhibition curves for brindled mice than for control, indicating more than 2-fold elevated flux control coefficients of cytochrome coxidase. This investigation proved essential in characterizing the metabolic effect of a cytochrome coxidase deficiency. We conclude, therefore, that application of metabolic control analysis can be a valuable approach to study defects of mitochondrial oxidative phosphorylation.

In the presence of ascorbate, hexaamineruthenium mediates rapid reduction of cytochrome b-562 in submitochondrial particles but not in mitochondria. The reaction is obsreved in the combined presence of antimycin (or funiculosin) and myxothiazol, which implies direct interaction of Ru(NH 3) 2+6with bcytochrome(s). We assume that contrary to previous conclusions (Case and Leigh (1976) Biochem. J., 160, 769-783) redox centre of at least one of the oxidized cytochromes b, most probably of b-562, is exposed to the M-aqueous phase.

Changes are described that are brought about by antimycin, NoHOQnO, funiculosin, myxothiazol and mucidin in the α‐, β‐ and γ‐absorption bands of reduced and oxidized cytochromes bin the isolated complex bc1, form beef heart mitochondria. The inhibitors can be divided into 2 groups. Antimycin, funiculosin and NoHOQnO are likely to shift the spectrum of b‐562 and compete for specific binding with complex bc1with each other but not with myxothiazol and mucidin. The spectral effects of the latter two inhibitors are more difficult to interpret and may involve contributions not only from b‐562 but from b‐566 as well. The existence of 2 independent inhibitor binding‐sites in the complex bc1corroborates the Q‐cycle hypothesis.

Anaerobic potentiometric titrations of bcytochromes have been carried out in beef heart submitochondrial particles in the presence of several specific inhibitors of electron transfer through the b‐c1‐site of the respiratory chain. Whereas antimycin shows no significant effect on the titration curve of cytochrome b‐562, NoHOQnO is found to shift the Emof b‐562 by 20–30 mV to the positive. Funiculosin raises the Emof b‐562 by > 100 mV and also appears to bring about a minor shift of b‐566 midpoint potential. In the presence of myxothiazol, both bcytochromes with Emvalues 15–30 mV more positive than in the control.

When succinate oxidation by submitochondrial particles is blocked by antimycin, NoHOQnO or funiculosin, addition of ferricyanide restores oxygen uptake coupled to membrane potential generation. The effect of ferricyanide is abolished by mucidin or myxothiazol, as well as by KCN. The data strongly favor a cyclic redox loop mechanism in site 2 and show that either heme of the ferrous cytochrome bor ubisemiquinone formed in the QH2‐oxidizing center of complex b−c1is accessible to ferricyanide at the outer (M) side of the submitochondrial particle membrane.

The growth of Escherichia coli strain TG 1 was monitored, measuring simultaneously the culture fluorescence and the 360° reflection at 578 nm with a two-channel optical sensor. It was observed that the culture fluorescence at 366 nm excitation was approximately three times higher than the NADH fluorescence of washed E. coli cells whereas the 360° reflection at 578 nm was comparable. The reason for this effect was found to be the accumulation of riboflavin in the cultivation liquid of the E. coli cells being equal to approximately 0.05 mg/g biomass. In shaken batch cultivations of the same strain the amount of riboflavin in the cell-free cultivation liquid correlated with the biomass being a very sensitive indicator of E. coli growth.

In the presence of ascorbate, hexaamineruthenium mediates rapid reduction of cytochrome b‐562 in submitochondrial particles but not in mitochondria. The reaction is obsreved in the combined presence of antimycin (or funiculosin) and myxothiazol, which implies direct interaction of Ru(NH3)2+6with bcytochrome(s). We assume that contrary to previous conclusions (Case and Leigh (1976) Biochem. J., 160, 769‐783) redox centre of at least one of the oxidized cytochromes b, most probably of b‐562, is exposed to the M‐aqueous phase.

Abstract

The oxygen dependence of cytochrome coxidase control on succinate oxidation was investigated in saponin‐permeabilized muscle fibers and isolated mitochondria from mouse quadriceps muscle applying metabolic control analysis. For this cyanide titrations of the oxygen consumption in the presence of succinate+rotenone were performed at different oxygen concentrations in the medium. While with isolated mitochondria high flux control coefficients were detected only at oxygen concentrations close to the KMvalue of cytochrome coxidase, with saponin‐permeabilized fibers a significant increase of cytochrome coxidase flux control was already observed below 130 μM oxygen. The result is in line with the high oxygen sensitivity of maximal respiration of saponin‐permeabilized muscle fibers (P50=33 μM) caused most probably by oxygen diffusion gradients through the fiber lattice. The oxygen dependence of cytochrome coxidase flux control in muscle fibers can explain the pathological phenotype of mild cytochrome coxidase deficiencies in mitochondrial myopathies.

The addition of 15 mM caffeine to saponin-skinned human muscle fibers from M. vastus lateralis caused in the presence of 2 mM ATP an approx. 2-fold stimulation of respiration with glutamate + malate. This effect can be abolished by either the addition of the Ca 2+chelator EGTA, the inhibitor of Ca 2+transport Ruthenium red and the inhibitor of the myosin ATPase vanadate. The caffeine concentration dependency of respiration of fibers coincided with the caffeine-caused stimulation of myosin ATPase activity. The activation of oxidative phosphorylation in saponin-skinned human muscle fibers by caffeine can be explained by a stimulation of myosin ATPase caused by Ca 2+release from sarcoplasmic reticulum.

The addition of 15 mM caffeine to saponin‐skinned human muscle fibers from M. vastus lateralis caused in the presence of 2 mM ATP an approx. 2‐fold stimulation of respiration with glutamate + malate. This effect can be abolished by either the addition of the Ca2+chelator EGTA, the inhibitor of Ca2+transport Ruthenium red and the inhibitor of the myosin ATPase vanadate. The caffeine concentration dependency of respiration of fibers coincided with the caffeine‐caused stimulation of myosin ATPase activity. The activation of oxidative phosphorylation in saponin‐skinned human muscle fibers by caffeine can be explained by a stimulation of myosin ATPase caused by Ca2+release from sarcoplasmic reticulum.

The oxygen dependence of cytochrome coxidase control on succinate oxidation was investigated in saponin-permeabilized muscle fibers and isolated mitochondria from mouse quadriceps muscle applying metabolic control analysis. For this cyanide titrations of the oxygen consumption in the presence of succinate+rotenone were performed at different oxygen concentrations in the medium. While with isolated mitochondria high flux control coefficients were detected only at oxygen concentrations close to the KMvalue of cytochrome coxidase, with saponin-permeabilized fibers a significant increase of cytochrome coxidase flux control was already observed below 130 μM oxygen. The result is in line with the high oxygen sensitivity of maximal respiration of saponin-permeabilized muscle fibers (P 50=33 μM) caused most probably by oxygen diffusion gradients through the fiber lattice. The oxygen dependence of cytochrome coxidase flux control in muscle fibers can explain the pathological phenotype of mild cytochrome coxidase deficiencies in mitochondrial myopathies.

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Targeting the cGMP effector VASP may enhance metabolism by stimulating the activity and abundance of brown adipocytes.

The instability of the mitochondrial genome in individuals harboring pathogenic mutations in the catalytic subunit of mitochondrial DNA (mtDNA) polymerase γ (POLG) is well recognized, but the underlying molecular mechanisms remain to be elucidated. In 5 pediatric patients with severe myoclonic epilepsy and valproic acid-induced liver failure, we identified 1 novel and 4 previously described pathogenic mutations in the linker region of this enzyme. Although muscle biopsies in these patients showed unremarkable histologic features, postmortem liver tissue available from 1 individual exhibited large cytochrome c oxidase-negative areas. These cytochrome c oxidase-negative areas contained 4-fold less mtDNA than cytochrome c oxidase-positive areas. Decreased copy numbers of mtDNA were observed not only in the liver, skeletal muscle, and brain but also in blood samples from all patients. There were also patient-specific patterns of multiple mtDNA deletions in different tissues, and in 2 patients, there were clonally expanded mtDNA point mutations. The low amount of deleted mtDNA molecules makes it unlikely that the deletions contribute significantly to the general biochemical defect. The clonal expansion of a few individual-specific deletions and point mutations indicates an accelerated segregation of early mtDNA mutations that likely are a consequence of low mtDNA copy numbers. Moreover, these results suggest a potential diagnostic approach for identifying mtDNA depletion in patients.

To assess the detailed expression pattern of mitochondrial-encoded proteins in skeletal muscle of patients with mitochondrial diseases we performed determinations of cytochrome content and enzyme activities of respiratory chain complexes of 12 patients harboring large-scale deletions and of 10 patients harboring the A3243G mutation. For large-scale deletions we observed a mutation gene dose-dependent linear decline of cytochrome aa3 content, cytochrome c oxidase (COX) activity, and complex I activity. The content of cytochromes b and the complex III activity was either not affected or only weakly affected by the deletion mutation and did not correlate to the degree of heteroplasmy. In contrast, in skeletal muscle harboring the A3243G mutation all investigated enzymes containing mitochondrial-encoded subunits were equally affected by the mutation, but we observed milder enzyme deficiencies at a comparable mutation gene dose. The results of single fiber analysis of selected biopsies supported these findings but revealed differences in the distribution of COX deficiency. Whereas predominantly type 1 fibers were affected in A3243G and deletion CPEO biopsies, we observed in MELAS and KSS biopsies higher quantities of COX-deficient type 2 fibers. Our findings indicate different pathomechanisms of deletion and A3243G mutations.

Mutations of the human plectin gene (Plec1) cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Here, we report on molecular mechanisms leading to severe dystrophic muscle alterations in EBS-MD. Analysis of a 25-yr-old EBS-MD patient carrying a novel homozygous 16-bp insertion mutation (13803ins16/13803ins16) close to the intermediate filament (IF) binding site of plectin showed severe disorganization of the myogenic IF cytoskeleton. Intermyofibrillar and subsarcolemmal accumulations of assembled but highly unordered desmin filaments may be attributed to impaired desmin binding capability of the mutant plectin. This IF pathology was also associated with severe mitochondrial dysfunction, suggesting that the muscle pathology of EBS-MD caused by IF disorganization leads not only to defects in mechanical force transduction but also to metabolic dysfunction. Beyond EBS-MD, our data may contribute to the understanding of other myopathies characterized by sarcoplasmic IF accumulations such as desminopathies or α-B-crystallinopathies.

Abnormalities of the sarcotubular system presenting as tubular aggregates (TAs) have been described in a variety of neuromuscular disorders. Here, we report on immunohistochemical and biochemical findings in 7 patients (2 familial and 5 sporadic cases) suffering from myopathies with TAs. In muscle biopsy specimens from 5 of the 7 patients, TAs were immunopositive for the ryanodine receptor (RYR 1) of the sarcoplasmic reticulum (SR), the SR Ca2+ pump (SERCA2-ATPase), and the intraluminal SR Ca2+ binding protein calsequestrin, indicating an SR origin of these aggregates. Furthermore, these 5 cases showed decreased respiratory chain enzyme activities (NADH:CoQ oxidoreductase, complex I and cytochrome c oxidase [COX], complex IV), while the remaining 2 patients exhibited normal values. Our findings indicate a functional link between mitochondrial dysfunction and the presence of TAs originating from the sarcoplasmic reticulum.