David R. Thorburn

A mitochondrial protein compendium elucidates complex I disease biology.

Mitochondria are complex organelles whose dysfunction underlies a broad spectrum of human diseases. Identifying all of the proteins resident in this organelle and understanding how they integrate into pathways represent major challenges in cell biology. Toward this goal, we performed mass spectrometry, GFP tagging, and machine learning to create a mitochondrial compendium of 1098 genes and their protein expression across 14 mouse tissues. We link poorly characterized proteins in this inventory to known mitochondrial pathways by virtue of shared evolutionary history. Using this approach, we predict 19 proteins to be important for the function of complex I (CI) of the electron transport chain. We validate a subset of these predictions using RNAi, including C8orf38, which we further show harbors an inherited mutation in a lethal, infantile CI deficiency. Our results have important implications for understanding CI function and pathogenesis and, more generally, illustrate how our compendium can serve as a foundation for systematic investigations of mitochondria.

Diagnostic criteria for respiratory chain disorders in adults and children

Background: Respiratory chain (RC) disorders are clinically, biochemically, and molecularly heterogeneous. The lack of standardized diagnostic criteria poses difficulties in evaluating diagnostic methodologies. Objective: To assess proposed adult RC diagnostic criteria that classify patients into “definite,” “probable,” or “possible” categories. Methods: The authors applied the adult RC diagnostic criteria retrospectively to 146 consecutive children referred for investigation of a suspected RC disorder. Data were collected from hospital, genetics, and laboratory records, and the diagnoses predicted by the adult criteria were compared with the previously assigned assessments. Results: The authors identified three major difficulties in applying the adult criteria:lack of pediatric-specific criteria; difficulty in segregating continuous data into circumscribed major and minor criteria; and lack of additivity of clinical features or enzyme tests. They therefore modified the adult criteria to allow for pediatric clinical and histologic features and for more sensitive coding of RC enzyme and functional studies. Reanalysis of the patients’ data resulted in congruence between the diagnostic certainty previously assigned by the authors’ center and that defined by the new general RC diagnostic criteria in 99% of patients. Conclusions: These general diagnostic criteria appear to improve the sensitivity of the adult criteria. They need further assessment in prospective clinical and epidemiologic studies.

Molecular Diagnosis of Infantile Mitochondrial Disease with Targeted Next-Generation Sequencing

Applying next-generation sequencing to 42 infants with mitochondrial disease highlights both the potential and the challenge of using this technology in clinical diagnosis. Getting to the Genetic Root of Mitochondrial Disease Next-generation DNA sequencing is being applied with great success in research settings to uncover new disease genes. Despite these successes, it is unclear how useful the technology will be for routine clinical diagnosis given the challenge of interpreting DNA variations in individual patients. In a new study, Calvo and colleagues apply next-generation sequencing to infants with mitochondrial disorders, a large collection of inherited diseases that are notoriously difficult to diagnose because of the multitude of candidate genes and the highly variable nature of the clinical presentation. First, the authors selected 42 unrelated infants with mitochondrial diseases that were refractory to standard clinical genetic testing. Then, for each child, they sequenced the DNA of the mitochondrial genome, the 100 genes previously linked to mitochondrial disease, and the ~1000 additional genes that are known to participate in mitochondrial biology. Of all the DNA differences present in the patients, the researchers prioritized those that were rare in the general population, predicted to disrupt protein function, and inherited in a recessive fashion. Such variants showed fivefold enrichment in the patients compared to that in healthy control individuals. In 10 patients (24%), firm molecular diagnoses were made in genes previously linked to mitochondrial diseases; 13 patients (31%) had prioritized recessive mutations in genes not previously linked to disease. For two of these genes, the authors were able to show that the mutations caused the mitochondrial disorder. These results suggest that next-generation sequencing may be able to provide a molecular diagnosis for ~25% of currently unsolved cases of infantile mitochondrial disease. An additional 25% of cases could be solved in the coming few years as more genes are formally proven to be linked to mitochondrial disease. The remaining 50% of patients in whom diagnosis was not possible underscores the challenge of interpreting DNA sequence data for clinical diagnosis. Nevertheless, the study by Calvo and colleagues will help to calibrate clinicians’ expectations regarding the diagnostic use of next-generation sequencing. Advances in next-generation sequencing (NGS) promise to facilitate diagnosis of inherited disorders. Although in research settings NGS has pinpointed causal alleles using segregation in large families, the key challenge for clinical diagnosis is application to single individuals. To explore its diagnostic use, we performed targeted NGS in 42 unrelated infants with clinical and biochemical evidence of mitochondrial oxidative phosphorylation disease. These devastating mitochondrial disorders are characterized by phenotypic and genetic heterogeneity, with more than 100 causal genes identified to date. We performed “MitoExome” sequencing of the mitochondrial DNA (mtDNA) and exons of ~1000 nuclear genes encoding mitochondrial proteins and prioritized rare mutations predicted to disrupt function. Because patients and healthy control individuals harbored a comparable number of such heterozygous alleles, we could not prioritize dominant-acting genes. However, patients showed a fivefold enrichment of genes with two such mutations that could underlie recessive disease. In total, 23 of 42 (55%) patients harbored such recessive genes or pathogenic mtDNA variants. Firm diagnoses were enabled in 10 patients (24%) who had mutations in genes previously linked to disease. Thirteen patients (31%) had mutations in nuclear genes not previously linked to disease. The pathogenicity of two such genes, NDUFB3 and AGK, was supported by complementation studies and evidence from multiple patients, respectively. The results underscore the potential and challenges of deploying NGS in clinical settings.

RAGE-Induced Cytosolic ROS Promote Mitochondrial Superoxide Generation in Diabetes

Damaged mitochondria generate an excess of superoxide, which may mediate tissue injury in diabetes. We hypothesized that in diabetic nephropathy, advanced glycation end-products (AGEs) lead to increases in cytosolic reactive oxygen species (ROS), which facilitate the production of mitochondrial superoxide. In normoglycemic conditions, exposure of primary renal cells to AGEs, transient overexpression of the receptor for AGEs (RAGE) with an adenoviral vector, and infusion of AGEs to healthy rodents each induced renal cytosolic oxidative stress, which led to mitochondrial permeability transition and deficiency of mitochondrial complex I. Because of a lack of glucose-derived NADH, which is the substrate for complex I, these changes did not lead to excess production of mitochondrial superoxide; however, when we performed these experiments in hyperglycemic conditions in vitro or in diabetic rats, we observed significant generation of mitochondrial superoxide at the level of complex I, fueled by a sustained supply of NADH. Pharmacologic inhibition of AGE-RAGE-induced mitochondrial permeability transition in vitro abrogated production of mitochondrial superoxide; we observed a similar effect in vivo after inhibiting cytosolic ROS production with apocynin or lowering AGEs with alagebrium. Furthermore, RAGE deficiency prevented diabetes-induced increases in renal mitochondrial superoxide and renal cortical apoptosis in mice. Taken together, these studies suggest that AGE-RAGE-induced cytosolic ROS production facilitates mitochondrial superoxide production in hyperglycemic environments, providing further evidence of a role for the advanced glycation pathway in the development and progression of diabetic nephropathy.

High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function. We established genetic diagnoses in 13 of 60 previously unsolved cases using confirmatory experiments, including cDNA complementation to show that mutations in NUBPL and FOXRED1 can cause complex I deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can be used to identify causal mutations in individual cases.

Biochemical assays of respiratory chain complex activity.

Publisher Summary This chapter focuses on the biochemical assays of the respiratory chain (RC) complex activity. It presents the preparation of mitochondrial fractions from tissues and cultured cells for RC enzymology; the measurement of activity of the individual complexes I, II, III, IV, V, the mitochondrial matrix marker enzyme citrate synthase; and the combined activity of complexes II + III. RC enzyme activities are frequently expressed relative to its activity of citrate synthase. Such ratios are more robust than absolute activities because of the variability inherent in cell culture conditions, such as passage number and the degree of confluence, and the proliferation of mitochondria seen in tissues of many patients with mitochondrial disease. The effects of postmortem delay on RC enzymes from liver samples frozen at varying times after death were analyzed to assess the stability of RC enzyme activities postmortem. There can be considerable loss of RC enzyme activity postmortem, particularly in liver, but the observations suggest that muscle collected and frozen at -70°C within 6 h of death and liver within 2 h remain suitable for RC enzyme analysis. The chapter presents the effects of tissue pathology by comparing RC enzymes in tissues from patients without RC complexes I–IV defects with normal controls. The possibility of secondary decreases in enzyme activity and the broadening of reference ranges in the presence of tissue pathology should be considered in interpreting RC enzyme profiles.

Respiratory chain complex I deficiency: an underdiagnosed energy generation disorder.

Objective: To define the spectrum of clinical and biochemical features in 51 children with isolated complex I deficiency. Background: Mitochondrial respiratory chain defects are one of the most commonly diagnosed inborn errors of metabolism. Until recently there have been technical problems with the diagnosis of respiratory chain complex I defects, and there is a lack of information about this underreported cause of respiratory chain dysfunction. Methods: A retrospective review of clinical features and laboratory findings was undertaken in all diagnosed patients who had samples referred over a 22-year period. Results: Presentations were heterogeneous, ranging from severe multisystem disease with neonatal death to isolated myopathy. Classic indicators of respiratory chain disease were not present in 16 of 42 patients in whom blood lactate levels were normal on at least one occasion, and in 23 of 37 patients in whom muscle morphology was normal or nonspecific. Ragged red fibers were present in only five patients. Tissue specificity was observed in 19 of 41 patients in whom multiple tissues were examined, thus the diagnosis may be missed if the affected tissue is not analyzed. Nine patients had only skin fibroblasts available, the diagnosis being based on enzyme assay and functional tests. Modes of inheritance include autosomal recessive (suggested in five consanguineous families), maternal (mitochondrial DNA point mutations in eight patients), and possibly X-linked (slight male predominance of 30:21). Recurrence risk was estimated as 20 to 25%. Conclusion: Heterogeneous clinical features, tissue specificity, and absence of lactic acidosis or abnormal mitochondrial morphology in many patients have resulted in underdiagnosis of respiratory chain complex I deficiency.

The inheritance of mitochondrial DNA heteroplasmy: random drift, selection or both?

The mammalian mitochondrial genome (mtDNA) is a small double-stranded DNA molecule that is exclusively transmitted down the maternal line. Pathogenic mtDNA mutations are usually heteroplasmic, with a mixture of mutant and wild-type mtDNA within the same organism. A woman harbouring one of these mutations transmits a variable amount of mutant mtDNA to each offspring. This can result in a healthy child or an infant with a devastating and fatal neurological disorder. Understanding the biological basis of this uncertainty is one of the principal challenges facing scientists and clinicians in the field of mitochondrial genetics.

Understanding mitochondrial complex I assembly in health and disease.

Complex I (NADH:ubiquinone oxidoreductase) is the largest multimeric enzyme complex of the mitochondrial respiratory chain, which is responsible for electron transport and the generation of a proton gradient across the mitochondrial inner membrane to drive ATP production. Eukaryotic complex I consists of 14 conserved subunits, which are homologous to the bacterial subunits, and more than 26 accessory subunits. In mammals, complex I consists of 45 subunits, which must be assembled correctly to form the properly functioning mature complex. Complex I dysfunction is the most common oxidative phosphorylation (OXPHOS) disorder in humans and defects in the complex I assembly process are often observed. This assembly process has been difficult to characterize because of its large size, the lack of a high resolution structure for complex I, and its dual control by nuclear and mitochondrial DNA. However, in recent years, some of the atomic structure of the complex has been resolved and new insights into complex I assembly have been generated. Furthermore, a number of proteins have been identified as assembly factors for complex I biogenesis and many patients carrying mutations in genes associated with complex I deficiency and mitochondrial diseases have been discovered. Here, we review the current knowledge of the eukaryotic complex I assembly process and new insights from the identification of novel assembly factors. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Analysis of the Assembly Profiles for Mitochondrial- and Nuclear-DNA-Encoded Subunits into Complex I

ABSTRACT Complex I of the respiratory chain is composed of at least 45 subunits that assemble together at the mitochondrial inner membrane. Defects in human complex I result in energy generation disorders and are also implicated in Parkinsons disease and altered apoptotic signaling. The assembly of this complex is poorly understood and is complicated by its large size and its regulation by two genomes, with seven subunits encoded by mitochondrial DNA (mtDNA) and the remainder encoded by nuclear genes. Here we analyzed the assembly of a number of mtDNA- and nuclear-gene-encoded subunits into complex I. We found that mtDNA-encoded subunits first assemble into intermediate complexes and require significant chase times for their integration into the holoenzyme. In contrast, a set of newly imported nuclear-gene-encoded subunits integrate with preexisting complex I subunits to form intermediates and/or the fully assembly holoenzyme. One of the intermediate complexes represents a subassembly associated with the chaperone B17.2L. By using isolated patient mitochondria, we show that this subassembly is a productive intermediate in complex I assembly since import of the missing subunit restores complex I assembly. Our studies point to a mechanism of complex I biogenesis involving two complementary processes, (i) synthesis of mtDNA-encoded subunits to seed de novo assembly and (ii) exchange of preexisting subunits with newly imported ones to maintain complex I homeostasis. Subunit exchange may also act as an efficient mechanism to prevent the accumulation of oxidatively damaged subunits that would otherwise be detrimental to mitochondrial oxidative phosphorylation and have the potential to cause disease.