CHCHD10P80L knock-in zebrafish display a mild ALS-like phenotype.

Mutations in the nuclear-encoded mitochondrial gene CHCHD10 have been observed in patients with a spectrum of diseases that include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathogenic nature of disease-associated variants of CHCHD10 we generated a zebrafish knock-in (KI) model expressing the orthologous ALS-associated CHCHD10P80L variant (zebrafish: Chchd10P83L). Larval chchd10P83L/P83L fish displayed reduced Chchd10 protein expression levels, motor impairment, reduced survival and abnormal neuromuscular junctions (NMJ). These deficits were not accompanied by changes in transcripts involved in the integrated stress response (ISR), phenocopying previous findings in our knockout (chchd10-/-). Adult, 11-month old chchd10P83L/P83L zebrafish, displayed smaller slow- and fast-twitch muscle cell cross-sectional areas compared to wild type zebrafish muscle cells. Motoneurons in the spinal cord of chchd10P83L/P83L zebrafish displayed similar cross-sectional areas to that of wild type motor neurons and significantly fewer motor neurons were observed when compared to chchd2-/- adult spinal cords. Bulk RNA sequencing using whole spinal cords of 7-month old fish revealed transcriptional changes associated with neuroinflammation, apoptosis, amino acid metabolism and mt-DNA inflammatory response in our chchd10P83L/P83L model. The findings presented here, suggest that the CHCHD10P80L variant confers an ALS-like phenotype when expressed in zebrafish.

Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.

Mammalian mitochondrial DNA (mtDNA) is a highly polymorphic, high-copy-number genome that is maternally inherited. New mutations in mtDNA segregate rapidly in the female germline due to a genetic bottleneck in early oogenesis and as a result most individuals are homoplasmic for a single species of mtDNA. Sequence variants thus accumulate along maternal lineages without genetic recombination. Most of the extant variation in mtDNA in mammalian populations has been assumed to be neutral with respect to selection; however, comparisons of the ratio of replacement to silent nucleotide substitutions between species suggest that the evolution of mammalian mtDNA is not strictly neutral. To test directly whether polymorphic mtDNAs behave as neutral variants, we examined the segregation of two different mtDNA genotypes in the tissues of heteroplasmic mice. We find evidence for random genetic drift in some tissues, but in others strong, tissue-specific and age-related, directional selection for different mtDNA genotypes in the same animal. These surprising data suggest that the coding sequence of mtDNA may represent a compromise between the competing demands of different tissues and point to the existence of unknown, tissue-specific nuclear genes important in the interaction between the nuclear and mitochondrial genomes.

Random genetic drift in the female germline explains the rapid segregation of mammalian mitochondrial DNA.

Mitochondrial DNA (mtDNA) is maternally inherited in mammals. Despite the high genome copy number in mature oocytes (10(5)) and the relatively small number of cell divisions in the female germline, mtDNA sequence variants segregate rapidly between generations. To investigate the molecular basis for this apparent paradox we created lines of heteroplasmic mice carrying two mtDNA genotypes. We show that the pattern of segregation can be explained by random genetic drift occurring in early oogenesis, and that the effective number of segregating units for mtDNA is approximately 200 in mice. These results provide the basis for estimating recurrence risks for mitochondrial disease due to pathogenic mtDNA mutations and for predicting the rate of fixation of neutral mtDNA mutations in maternal lineages.

SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome.

Leigh Syndrome (LS) is a severe neurological disorder characterized by bilaterally symmetrical necrotic lesions in subcortical brain regions that is commonly associated with systemic cytochrome c oxidase (COX) deficiency. COX deficiency is an autosomal recessive trait and most patients belong to a single genetic complementation group. DNA sequence analysis of the genes encoding the structural subunits of the COX complex has failed to identify a pathogenic mutation. Using microcell-mediated chromosome transfer, we mapped the gene defect in this disorder to chromosome 9q34 by complementation of the respiratory chain deficiency in patient fibroblasts. Analysis of a candidate gene (SURF1) of unknown function revealed several mutations, all of which predict a truncated protein. These data suggest a role for SURF1 in the biogenesis of the COX complex and define a new class of gene defects causing human neurodegenerative disease.

Ethanol: novel end product of vertebrate anaerobic metabolism.

During periods of short-term experimental anoxia, the goldfish produces metabolic carbon dioxide and does not accumulate lactate to the extent expected. The goldfish has evolved a novel pathway of vertebrate anaerobic metabolism in which glucose carbon is metabolized to ethanol and excreted.

Response of the 23Na-NMR double-quantum filtered signal to changes in Na+ ion concentration in model biological solutions and human erythrocytes.

Double quantum filtered (DQF) 23Na-NMR signals were evaluated as a function of [Na+] at constant temperature in two model systems (bovine serum albumin (BSA) and Ficoll 400) and in human red blood cells (RBCs). In model systems, the ratio of double quantum filtered to single quantum (SQ) signal intensities was independent of [Na+], even over a wide range of Na+/K+ ratios. Varying the DQF preparation time affected only the DQF signal intensity. In contrast, in human red blood cells (RBCs) the shape and phase of the DQF intracellular Na+ signal (Na+in) varied as a function of preparation time. Similar observations in cartilage [Eliav, U., Shinar, H. and Navon, G. (1992) J. Magn. Reson. 28, 223-229] have been attributed to the generation of a second- and a third-rank tensor by the DQF pulse sequence, resulting from Na+ ion ordering. By using a DQF sequence which isolates the second-rank tensor only, this component was found to originate from the intracellular Na+ ion pool in human RBCs, as well as from interactions of Na+ ions with the extracellular face of the plasma membrane. The residual quadrupolar splitting for the signal originating from the former environment was shown to be less than the SQ linewidth, explaining its absence in SQ spectra, and this was confirmed by two-dimensional DQF 23Na-NMR experiments. By isolating the contribution from the third-rank tensor exclusively, the ratio of DQF:SQ signal intensities for Na+in in human RBCs was shown to be constant over a 4-fold change in [Na+in] produced by addition of an ionophore (nystatin). This indicates that such changes in physiological state do not alter the efficiency of DQF signal generation in human RBCs.

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue beta-guanidinopropionic acid.

Rats were fed a diet containing 1% beta-guanidinopropionic acid (GPA) for 6-10 weeks to deplete their skeletal muscle of creatine. 31P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 mumol X g-1) and the [ATP] to 50% (3.3 mumol X g-1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 mumol X g-1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM X s-1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM X s-1). The rate of PGPA resynthesis during recovery (0.018 mM X s-1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM X s-1). The creatine kinase flux (creatine phosphate----ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM X s-1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.

Regulation of creatine kinase during steady-state isometric twitch contraction in rat skeletal muscle.

In vivo 31P-NMR saturation transfer measurements of the creatine kinase exchange flux in the direction creatine phosphate----ATP were made in the gastrocnemius muscle of rats at rest and during steady-state isometric twitch contraction at frequencies from 0.25 to 2 Hz. There was no correlation between creatine kinase exchange flux and either free [ADP] or oxygen consumption, both of which increase with stimulation frequency. The flux was found to be nearly constant over all conditions at about 16 mM X s-1, 10-times greater than the highest estimated ATP turnover in this study. The kinetic properties of skeletal muscle creatine kinase in vivo are similar to, but not completely predictable from, the equilibrium exchange fluxes measured on the isolated enzyme. These results are not consistent with strong functional coupling between ATP synthesis and mitochondrial creatine kinase.

Creatine kinase kinetics, ATP turnover, and cardiac performance in hearts depleted of creatine with the substrate analogue beta-guanidinopropionic acid.

Rats were fed a diet containing 1% of the creatine substrate analogue beta-guanidinopropionic acid for 6-10 weeks. 31P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 mumol/g dry wt in guanidinopropionic acid-fed animals but no change in [Pi], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine----ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 mumol/g dry wt per s in control hearts and decreased 4-fold to 2.5-2.8 mumol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2-7.8 mumol/g dry per s. These rates are 1.5-3-times greater than the rate of the phosphocreatine----ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.

A comparison of in vivo catalysis by creatine kinase in avian skeletal muscles with different fibre composition.

The maximum activity of creatine kinase in vitro is similar in the pectoralis major muscle of the chicken and the duck. However, the flux (phosphocreatine to ATP) as measured by 31P saturation transfer NMR in vivo is almost 2-fold higher in the duck. This apparent discrepancy can be accounted for by the differences in the cytosolic free ADP concentrations in resting muscle.

Characterization of the mitochondrial DNA abnormalities in the skeletal muscle of patients with inclusion body myositis.

Inclusion body myositis (IBM) is a late-onset inflammatory myopathy with distinctive clinical and histopathological features. The molecular basis for the disease remains unknown, but abnormal nuclear morphology and the accumulation of a protein that binds single-stranded DNA in a sequence-independent fashion suggest a nuclear defect. Evidence of mitochondrial respiratory chain dysfunction (ragged-red fibers, multiple mtDNA deletions) has been reported in IBM muscle. Here we have investigated the relationship of the mtDNA abnormalities in sporadic and familial IBM patients to the pathogenesis of the disease. In situ hybridization analysis with mtDNA probes revealed several different mtDNA abnormalities in cytochrome c oxidase-negative muscle fibers including large-scale mtDNA deletions and mtDNA depletion, but no evidence for nonspecific DNA binding. Contrary to previous reports, we did not observe mtDNA deletions on Southern blot analysis, consistent with the presence of multiple different deleted mtDNA species demonstrated by single fiber PCR. There was no consistent correlation between the mitochondrial abnormalities and markers of muscle regeneration, inflammation, or microscopically detectable pathological alterations of myonuclei in the same fibers. Thus, early molecular abnormalities in IBM may simply accelerate the accumulation of mtDNA abnormalities that occurs with natural aging.

Variable distribution of mutant mitochondrial DNAs (tRNA(Leu[3243])) in tissues of symptomatic relatives with MELAS: the role of mitotic segregation.

We studied multiple different postmortem tissue samples from a woman and two of her daughters with the MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) tRNA(Leu(UUR)) mutation at nucleotide 3243 in mitochondrial DNA (mtDNA). All tissues examined were heteroplasmic for the mutation. The mean proportion of mutant mtDNAs in the mother's tissues (0.30 +/- 0.10) was significantly lower than that of each of her daughters' (0.76 +/- 0.11, p < 0.03, and 0.72 +/- 0.13, p < 0.001); there was no difference in the fraction of mutant mtDNAs between the daughters (p < 0.71). This difference in the mean proportion of mtDNA mutants between family members correlates with their clinical profiles; the mother had the latest onset of disease and lived longest, while the two daughters had a strikingly similar clinical course. In individual patients, the mean proportion of mutant mtDNAs was not different in tissues deriving from ectodermal, mesodermal, and endodermal germ layers. Variance in the mutant:wild-type mtDNA ratio was normally distributed about the mean, both when all tissues were considered together and when different regions of the CNS were considered separately. Thus, the proportion of mtDNAs carrying the tRNA(Leu(3243)) mutation was not uniform in members of this pedigree and did not undergo rapid mitotic segregation along germ-layer divisions. These findings are consistent with the hypothesis that the overall proportion of mtDNAs carrying this mutation is primarily determined by segregation during oogenesis or early embryologic development and that random replicative (mitotic) segregation, subsequent to the establishment of primary germ layers, is responsible for the variation between tissues.

In vivo muscle magnetic resonance spectroscopy in the clinical investigation of mitochondrial disease.

We have investigated the sensitivity and specificity of a rapid phosphorus magnetic resonance spectroscopy (MRS) protocol for detecting metabolic abnormalities in vivo in skeletal muscle of patients with mitochondrial disease. We examined 17 patients with mitochondrial myopathies. Sixteen had only mild or minimal myopathic signs and symptoms. Phosphorus magnetic resonance spectra from the resting gastrocnemius muscles showed an abnormal intracellular energy state (marked by an increased intracellular inorganic phosphate concentration) in 14/17. In 3/17, this was associated with a decreased phosphocreatine concentration. We also studied 20 patients with other diseases of muscle (inflammatory myopathies, metabolic myopathies, muscular dystrophies, and myasthenia gravis) that can present with similar clinical features. Spectra showed increased intracellular inorganic phosphate concentrations in 6/20. All of these muscle diseases were associated with evidence of muscle fiber necrosis. Abnormalities in the muscle energy state in these cases may be due to secondary mitochondrial dysfunction. Except for cases of polymyositis and dermatomyositis, these 6 other myopathies could be readily distinguished from the mitochondrial myopathies on the basis of the clinical examination and blood tests. We conclude that phosphorus MRS of resting muscle is practical in a clinical setting and has a useful sensitivity and specificity for mitochondrial myopathies when used in conjunction with standard noninvasive tests.

Pedigree analysis of French Canadian families with T14484C Leber's hereditary optic neuropathy.

We analyzed the clinical phenotype and determined the recurrence risks to relatives of patients with T14484C Leber's hereditary optic neuropathy (LHON). LHON is a maternally inherited optic neuropathy that primarily affects adolescent males. It is usually associated with one of three mtDNA mutations: G3460A, G11778A, or T14484C. Definition of recurrence risks for the T14484C mutation previously has not been possible due to the relative scarcity of families with this mutation. We obtained blood samples from index patients and their consenting family members, all of whom were of French Canadian ancestry and screened for LHON mutations in mtDNA. Referring ophthalmologists furnished clinical summaries and patients provided pedigree data. T14484C was the most common mutation in the pedigrees analyzed and was always homoplasmic. In these pedigrees, the ratio of affected males to females was 8:1. Median age at onset for males was 19 years (95th percentile, 40.8 years; range, 6 to 48 years). Some improvement of vision was observed in 58% of patients. Recurrence risks to brothers were 28%, sisters 5%, nephews 30%, nieces 3%, male matrilineal first cousins 19%, and female matrilineal first cousins 4%. Recurrence risks to brothers and nephews were not different; however, recurrence risks to brothers and male cousins and to nephews and male cousins were significantly different. There were no differences in recurrence risks to sisters and nieces or to either group compared with their female cousins. Affected females did not have more affected children than unaffected females. The clinical characteristics of French Canadian patients with T14484C LHON were strikingly similar to those in previous reports, suggesting that recurrence risks are generalizable to other T14484C LHON populations for genetic counseling of T14484C LHON families.

Homozygosity (E140K) in SCO2 causes delayed infantile onset of cardiomyopathy and neuropathy.

OBJECTIVE: To report three unrelated infants with a distinctive phenotype of Leigh-like syndrome, neurogenic muscular atrophy, and hypertrophic obstructive cardiomyopathy. The patients all had a homozygous missense mutation in SCO2. BACKGROUND: SCO2 encodes a mitochondrial inner membrane protein, thought to function as a copper transporter to cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. Mutations in SCO2 have been described in patients with severe COX deficiency and early onset fatal infantile hypertrophic cardioencephalomyopathy. All patients so far reported are compound heterozygotes for a missense mutation (E140K) near the predicted CxxxC metal binding motif; however, recent functional studies of the homologous mutation in yeast failed to demonstrate an effect on respiration. METHODS: Here we present clinical, biochemical, morphologic, functional, MRI, and MRS data in two infants, and a short report in an additional patient, all carrying a homozygous G1541A transition (E140K). RESULTS: The disease onset and symptoms differed significantly from those in compound heterozygotes. MRI and muscle morphology demonstrated an age-dependent progression of disease with predominant involvement of white matter, late appearance of basal ganglia lesions, and neurogenic muscular atrophy in addition to the relatively late onset of hypertrophic cardiomyopathy. The copper uptake of cultured fibroblasts was significantly increased. CONCLUSIONS: The clinical spectrum of SCO2 deficiency includes the delayed development of hypertrophic obstructive cardiomyopathy and severe neurogenic muscular atrophy. There is increased copper uptake in patients' fibroblasts indicating that the G1541A mutation effects cellular copper metabolism.

Familial myopathy with conspicuous depletion of mitochondria in muscle fibers: a morphologically distinct disease.

Three patients (two of them siblings) presented with easy fatiguability and prominent postexercise pain. Muscle biopsy showed that large areas of about one third of the type II fibers were completely devoid of mitochondria. The remaining mitochondria were unusually large in size, but otherwise normal ultrastructurally. In two patients, 31P in vivo MRS showed low phosphocreatine (PCr), high ADP, low phosphorylation potential at rest and slow ADP and PCr recovery after aerobic exercise. This appears to be a pathologically unique form of metabolic myopathy. The cause of the focal mitochondrial depletion is not known. It should be distinguished from the mtDNA depletion syndrome in which muscle mitochondria are not reduced, but proliferate.

Cytochrome c oxidase deficiency.

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain, catalyzing the transfer of electrons from reduced cytochrome c to molecular oxygen. It is composed of 13 structural subunits, three of which are encoded in mtDNA and form the catalytic core of the enzyme. In addition to these structural subunits, a large number of accessory factors are necessary for the assembly and maintenance of the active holoenzyme complex. Most isolated COX deficiencies are inherited as autosomal recessive disorders; mutations in the mtDNA-encoded COX subunit genes are relatively rare. These mutations are associated with a wide spectrum of clinical phenotypes ranging from isolated myopathy to multisystem disease, with onset from late childhood to adulthood. Autosomal recessive COX deficiencies generally have a very early age of onset and a fatal outcome. Several clinical presentations have been described including Leigh Syndrome, hypertrophic cardiomyopathy and myopathy, and fatal infantile lactic acidosis. Surprisingly, mutations in the nuclear-encoded structural COX subunits have not been found in association with any of these phenotypes. Mutations have, however, been identified in several COX assembly factors: SURF1 (Leigh Syndrome), SCO2 (hypertrophic cardiomyopathy), SCO1 (hepatic failure, ketoacidotic coma), and COX10 (encephalopathy, tubulopathy). As all of these assembly factors are ubiquitously expressed, the molecular basis for the different clinical presentations remains unexplained. Although the genetic defects in the majority of patients with COX deficiency are unknown, it is likely that most will be solved in the near future using functional complementation techniques.

Early metabolic changes following chemotherapy of human gliomas in vivo demonstrated by phosphorus magnetic resonance spectroscopy.

The authors have used phosphorus magnetic resonance spectroscopy to monitor pH changes in malignant gliomas following treatment with intravenous and intra-arterial 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). Image-guided, localized phosphorus spectra of human gliomas in situ were obtained using a 1.5-T whole body combined imaging and spectroscopy system. Initial intravenous BCNU treatment was followed by a transient decrease of tumor intracellular pH by 0.15 +/- 0.03 pH units (mean +/- SD). Superselective intra-arterial administration of the same drug was followed by an increase of tumor intracellular pH by 0.15 +/- 0.6 pH units (mean +/- SD). These changes occurred prior to any changes on x-ray, computed tomography (CT), or magnetic resonance imaging (MRI). In addition to enhancing our understanding of the metabolic effects of BCNU, such changes may correlate with drug efficacy or toxicity and may be useful in guiding therapy in the future.

An animal model of mitochondrial myopathy: a biochemical and physiological investigation of rats treated in vivo with the NADH-CoQ reductase inhibitor, diphenyleneiodonium.

Chronic administration of the NADH-CoQ reductase inhibitor, diphenyleneiodonium to rats at two dose levels, 1.0 and 1.5 mg/kg per day, caused a 40% and 60% reduction, respectively, in the in vitro rate of NAD-linked respiration by skeletal muscle mitochondria. At the highest dose, muscle fatigue, lactic acidosis and an over-utilization of phosphocreatine was observed in the gastrocnemius muscle during mild stimulation of 1 Hz frequency. The resynthesis of phosphocreatine following muscle stimulation was about 2 fold slower in the treated animal group. At the low dose, no significant biochemical changes were observed during muscle stimulation at 4 Hz. The results are discussed in terms of skeletal muscle "oxidative reserve", twitch tension maintenance and the relevance to the human diseased state of mitochondrial myopathy.