Ronald G. Haller

Identification and Characterization of a Common Set of Complex I Assembly Intermediates in Mitochondria from Patients with Complex I Deficiency

Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.

Splice Mutation in the Iron-Sulfur Cluster Scaffold Protein ISCU Causes Myopathy with Exercise Intolerance

A myopathy with severe exercise intolerance and myoglobinuria has been described in patients from northern Sweden, with associated deficiencies of succinate dehydrogenase and aconitase in skeletal muscle. We identified the gene for the iron-sulfur cluster scaffold protein ISCU as a candidate within a region of shared homozygosity among patients with this disease. We found a single mutation in ISCU that likely strengthens a weak splice acceptor site, with consequent exon retention. A marked reduction of ISCU mRNA and mitochondrial ISCU protein in patient muscle was associated with a decrease in the iron regulatory protein IRP1 and intracellular iron overload in skeletal muscle, consistent with a muscle-specific alteration of iron homeostasis in this disease. ISCU interacts with the Friedreich ataxia gene product frataxin in iron-sulfur cluster biosynthesis. Our results therefore extend the range of known human diseases that are caused by defects in iron-sulfur cluster biogenesis.

Aerobic conditioning: An effective therapy in McArdle's disease

Susceptibility to exertional cramps and rhabdomyolysis in myophosphorylase deficiency (McArdles disease [MD]) may lead patients to shun exercise. However, physical inactivity may worsen exercise intolerance by further reducing the limited oxidative capacity caused by blocked glycogenolysis. We investigated whether aerobic conditioning can safely improve exercise capacity in MD.

Energy deficit in Huntington disease: why it matters

Huntington disease (HD) is an autosomal dominant neurodegenerative disease with complete penetrance. Although the understanding of the cellular mechanisms that drive neurodegeneration in HD and account for the characteristic pattern of neuronal vulnerability is incomplete, defects in energy metabolism, particularly mitochondrial function, represent a common thread in studies of HD pathogenesis in humans and animal models. Here we review the clinical, biochemical, and molecular evidence of an energy deficit in HD and discuss the mechanisms underlying mitochondrial and related alterations.

Glycogen Storage Disease Type III diagnosis and management guidelines

Purpose: Glycogen storage disease type III is a rare disease of variable clinical severity affecting primarily the liver, heart, and skeletal muscle. It is caused by deficient activity of glycogen debranching enzyme, which is a key enzyme in glycogen degradation. Glycogen storage disease type III manifests a wide clinical spectrum. Individuals with glycogen storage disease type III present with hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation. Those with type IIIa have symptoms related to liver disease and progressive muscle (cardiac and skeletal) involvement that varies in age of onset, rate of disease progression, and severity. Those with type IIIb primarily have symptoms related to liver disease. This guideline for the management of glycogen storage disease type III was developed as an educational resource for health care providers to facilitate prompt and accurate diagnosis and appropriate management of patients.Methods: An international group of experts in various aspects of glycogen storage disease type III met to review the evidence base from the scientific literature and provided their expert opinions. Consensus was developed in each area of diagnosis, treatment, and management.Results: This management guideline specifically addresses evaluation and diagnosis across multiple organ systems (cardiovascular, gastrointestinal/nutrition, hepatic, musculoskeletal, and neuromuscular) involved in glycogen storage disease type III. Conditions to consider in a differential diagnosis stemming from presenting features and diagnostic algorithms are discussed. Aspects of diagnostic evaluation and nutritional and medical management, including care coordination, genetic counseling, hepatic transplantation, and prenatal diagnosis, are addressed.Conclusions: A guideline that will facilitate the accurate diagnosis and appropriate management of individuals with glycogen storage disease type III was developed. This guideline will help health care providers recognize patients with all forms of glycogen storage disease type III, expedite diagnosis, and minimize stress and negative sequelae from delayed diagnosis and inappropriate management. It will also help identify gaps in scientific knowledge that exist today and suggest future studies.

Deficiency of skeletal muscle succinate dehydrogenase and aconitase. Pathophysiology of exercise in a novel human muscle oxidative defect.

We evaluated a 22-yr-old Swedish man with lifelong exercise intolerance marked by premature exertional muscle fatigue, dyspnea, and cardiac palpitations with superimposed episodes lasting days to weeks of increased muscle fatigability and weakness associated with painful muscle swelling and pigmenturia. Cycle exercise testing revealed low maximal oxygen uptake (12 ml/min per kg; healthy sedentary men = 39 +/- 5) with exaggerated increases in venous lactate and pyruvate in relation to oxygen uptake (VO2) but low lactate/pyruvate ratios in maximal exercise. The severe oxidative limitation was characterized by impaired muscle oxygen extraction indicated by subnormal systemic arteriovenous oxygen difference (a-v O2 diff) in maximal exercise (patient = 4.0 ml/dl, normal men = 16.7 +/- 2.1) despite normal oxygen carrying capacity and Hgb-O2 P50. In contrast maximal oxygen delivery (cardiac output, Q) was high compared to sedentary healthy men (Qmax, patient = 303 ml/min per kg, normal men 238 +/- 36) and the slope of increase in Q relative to VO2 (i.e., delta Q/delta VO2) from rest to exercise was exaggerated (delta Q/delta VO2, patient = 29, normal men = 4.7 +/- 0.6) indicating uncoupling of the normal approximately 1:1 relationship between oxygen delivery and utilization in dynamic exercise. Studies of isolated skeletal muscle mitochondria in our patient revealed markedly impaired succinate oxidation with normal glutamate oxidation implying a metabolic defect at the level of complex II of the mitochondrial respiratory chain. A defect in Complex II in skeletal muscle was confirmed by the finding of deficiency of succinate dehydrogenase as determined histochemically and biochemically. Immunoblot analysis showed low amounts of the 30-kD (iron-sulfur) and 13.5-kD proteins with near normal levels of the 70-kD protein of complex II. Deficiency of succinate dehydrogenase was associated with decreased levels of mitochondrial aconitase assessed enzymatically and immunologically whereas activities of other tricarboxylic acid cycle enzymes were increased compared to normal subjects. The exercise findings are consistent with the hypothesis that this defect impairs muscle oxidative metabolism by limiting the rate of NADH production by the tricarboxylic acid cycle.

Resistance training in patients with single, large-scale deletions of mitochondrial DNA

Dramatic tissue variation in mitochondrial heteroplasmy has been found to exist in patients with sporadic mitochondrial DNA (mtDNA) mutations. Despite high abundance in mature skeletal muscle, levels of the causative mutation are low or undetectable in satellite cells. The activation of these typically quiescent mitotic cells and subsequent shifting of wild-type mtDNA templates to mature muscle have been proposed as a means of restoring a more normal mitochondrial genotype and function in these patients. Because resistance exercise is known to serve as a stimulus for satellite cell induction within active skeletal muscle, this study sought to assess the therapeutic potential of resistance training in eight patients with single, large-scale mtDNA deletions by assessing: physiological determinants of peak muscle strength and oxidative capacity and muscle biopsy-derived measures of damage, mtDNA mutation load, level of oxidative impairment and satellite cell numbers. Our results show that 12 weeks of progressive overload leg resistance training led to: (i) increased muscle strength; (ii) myofibre damage and regeneration; (iii) increased proportion of neural cell adhesion molecule (NCAM)-positive satellite cells; (iv) improved muscle oxidative capacity. Taken together, we believe these findings support the hypothesis of resistance exercise-induced mitochondrial gene-shifting in muscle containing satellite cells which have low or absent levels of deleted mtDNA. Further investigation is warranted to refine parameters of the exercise training protocol in order to maximize the training effect on mitochondrial genotype and treatment potential for patients with selected, sporadic mutations of mtDNA in skeletal muscle.

A Heterozygous Truncating Mutation in RRM2B Causes Autosomal-Dominant Progressive External Ophthalmoplegia with Multiple mtDNA Deletions

Autosomal-dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder that is characterized by accumulation of multiple mitochondrial DNA (mtDNA) deletions in postmitotic tissues. The disorder is heterogeneous, with five known nuclear disease genes that encode the proteins ANT1, Twinkle, POLG, POLG2, and OPA1. Defects in these proteins affect mtDNA maintenance, probably leading to stalled replication forks, consequent mtDNA deletion formation, and progressive respiratory chain deficiency. Here we present a large adPEO family with multiple mtDNA deletions, whose disease was not explained by mutations in any of the known adPEO loci. We mapped the disease locus in this family to chromosome 8q22.1-q23.3. The critical linkage region contained the RRM2B gene, which encodes the small subunit of the ribonucleotide reductase p53R2, which has previously been shown to be essential for the maintenance of mtDNA copy number. Mutation screening of RRM2B revealed a heterozygous nonsense mutation in exon 9 (c.979C-->T [p.R327X]) in all affected individuals that was absent in 380 control chromosomes. The same mutation was found to segregate in another adPEO family. The mutant mRNA escaped nonsense-mediated decay and resulted in a protein with truncation of 25 highly conserved C-terminal amino acids essential for the interaction with the ribonucleotide reductase subunit R1. We conclude that dominant-negative or gain-of-function mutations in RRM2B are a cause of multiple mtDNA deletions and adPEO.

Exercise intolerance, lactic acidosis, and abnormal cardiopulmonary regulation in exercise associated with adult skeletal muscle cytochrome c oxidase deficiency.

A 27-yr-old woman with lifelong severe exercise intolerance manifested by muscle fatigue, lactic acidosis, and prominent symptoms of dyspnea and tachycardia induced by trivial exercise was found to have a skeletal muscle respiratory chain defect characterized by low levels of reducible cytochromes a + a3 and b in muscle mitochondria and marked deficiency of cytochrome c oxidase (complex IV) as assessed biochemically and immunologically. Investigation of the pathophysiology of the exercise response in the patient revealed low maximal oxygen uptake (1/3 that of normal sedentary women) in cycle exercise and impaired muscle oxygen extraction as indicated by profoundly low maximal systemic arteriovenous oxygen difference (5.8 ml/dl; controls = 15.4 +/- 1.4, mean +/- SD). The increases in cardiac output and ventilation during exercise, normally closely coupled to muscle metabolic rate, were markedly exaggerated (more than two- to threefold normal) relative to oxygen uptake and carbon dioxide production accounting for prominent tachycardia and dyspnea at low workloads. Symptoms in our patient are similar to those reported in other human skeletal muscle respiratory chain defects involving complexes I and III, and the exaggerated circulatory response resembles that seen during experimental inhibition of the mitochondrial respiratory chain. These results suggest that impaired oxidative phosphorylation in working muscle disrupts the normal regulation of cardiac output and ventilation relative to muscle metabolic rate in exercise.

Mitochondrial myopathy with succinate dehydrogenase and aconitase deficiency. Abnormalities of several iron-sulfur proteins.

Recently, we described a patient with severe exercise intolerance and episodic myoglobinuria, associated with marked impairment of succinate oxidation and deficient activity of succinate dehydrogenase and aconitase in muscle mitochondria (1). We now report additional enzymatic and immunological characterization of mitochondria. In addition to severe deficiency of complex II, manifested by reduction of succinate dehydrogenase and succinate:coenzyme Q oxidoreductase activities to 12 and 22% of normal, respectively, complex III activity was reduced to 37% and rhodanese to 48% of normal. Furthermore, although complex I activity was not measured, immunoblot analysis of complex I showed deficiency of the 39-, 24-, 13-, and 9-kD peptides with lesser reductions of the 51- and 18-kD peptides. Immunoblots of complex III showed markedly reduced levels of the mature Rieske protein in mitochondria and elevated levels of its precursor in the cytosol, suggesting deficient uptake into mitochondria. Immunoreactive aconitase was also low. These data, together with the previous documentation of low amounts of the 30-kD iron-sulfur protein and the 13.5-kD subunit of complex II, compared to near normal levels of the 70-kD protein suggest a more generalized abnormality of the synthesis, import, processing, or assembly of a group of proteins containing iron-sulfur clusters.