An isolated defect of respiratory chain complex I activity is a frequent biochemical abnormality in mitochondrial disorders. Despite intensive investigation in recent years, in most instances, the molecular basis underpinning complex I defects remains unknown. We report whole-exome sequencing of a single individual with severe, isolated complex I deficiency. This analysis, followed by filtering with a prioritization of mitochondrial proteins, led us to identify compound heterozygous mutations in ACAD9, which encodes a poorly understood member of the mitochondrial acyl-CoA dehydrogenase protein family. We demonstrated the pathogenic role of the ACAD9 variants by the correction of the complex I defect on expression of the wildtype ACAD9 protein in fibroblasts derived from affected individuals. ACAD9 screening of 120 additional complex I-defective index cases led us to identify two additional unrelated cases and a total of five pathogenic ACAD9 alleles.

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Secondary mitochondrial dysfunction is a feature in a wide variety of human protein aggregate diseases caused by mutations in different proteins, both in the central nervous system and in striated muscle. The functional relationship between the expression of a mutated protein and mitochondrial dysfunction is largely unknown. In particular, the mechanism how this dysfunction drives the disease process is still elusive. To address this issue for protein aggregate myopathies, we performed a comprehensive, multi-level analysis of mitochondrial pathology in skeletal muscles of human patients with mutations in the intermediate filament protein desmin and in muscles of hetero- and homozygous knock-in mice carrying the R349P desmin mutation. We demonstrate that the expression of mutant desmin causes disruption of the extrasarcomeric desmin cytoskeleton and extensive mitochondrial abnormalities regarding subcellular distribution, number and shape. At the molecular level, we uncovered changes in the abundancy and assembly of the respiratory chain complexes and supercomplexes. In addition, we revealed a marked reduction of mtDNA- and nuclear DNA-encoded mitochondrial proteins in parallel with large-scale deletions in mtDNA and reduced mtDNA copy numbers. Hence, our data demonstrate that the expression of mutant desmin causes multi-level damage of mitochondria already in early stages of desminopathies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1592-7) contains supplementary material, which is available to authorized users.

Mitochondrial cristae are connected to the inner boundary membrane via crista junctions which are implicated in the regulation of oxidative phosphorylation, apoptosis, and import of lipids and proteins. The MICOS complex determines formation of crista junctions. We performed complexome profiling and identified Mic13, also termed Qil1, as a subunit of the MICOS complex. We show that MIC13 is an inner membrane protein physically interacting with MIC60, a central subunit of the MICOS complex. Using the CRISPR/Cas method we generated the first cell line deleted for MIC13. These knockout cells show a complete loss of crista junctions demonstrating that MIC13 is strictly required for the formation of crista junctions. MIC13 is required for the assembly of MIC10, MIC26, and MIC27 into the MICOS complex. However, it is not needed for the formation of the MIC60/MIC19/MIC25 subcomplex suggesting that the latter is not sufficient for crista junction formation. MIC13 is also dispensable for assembly of respiratory chain complexes and for maintaining mitochondrial network morphology. Still, lack of MIC13 resulted in a moderate reduction of mitochondrial respiration. In summary, we show that MIC13 has a fundamental role in crista junction formation and that assembly of respiratory chain supercomplexes is independent of mitochondrial cristae shape.

Defects of mitochondrial oxidative phosphorylation constitute a clinical and genetic heterogeneous group of disorders affecting multiple organ systems at varying age. Biochemical analysis of biopsy material demonstrates isolated or combined deficiency of mitochondrial respiratory chain enzyme complexes. Co-occurrence of impaired activity of the pyruvate dehydrogenase complex has been rarely reported so far and is not yet fully understood. We investigated two siblings presenting with severe neonatal lactic acidosis, hypotonia, and intractable cardiomyopathy; both died within the first months of life. Muscle biopsy revealed a peculiar biochemical defect consisting of a combined deficiency of respiratory chain complexes I, II, and II+III accompanied by a defect of the pyruvate dehydrogenase complex. Joint exome analysis of both affected siblings uncovered a homozygous missense mutation in BOLA3. The causal role of the mutation was validated by lentiviral-mediated expression of the mitochondrial isoform of wildtype BOLA3 in patient fibroblasts, which lead to an increase of both residual enzyme activities and lipoic acid levels. Our results suggest that BOLA3 plays a crucial role in the biogenesis of iron-sulfur clusters necessary for proper function of respiratory chain and 2-oxoacid dehydrogenase complexes. We conclude that broad sequencing approaches combined with appropriate prioritization filters and experimental validation enable efficient molecular diagnosis and have the potential to discover new disease loci.

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BackgroundMitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy.ResultsWe describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation.Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers.ConclusionsOur data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.Electronic supplementary materialThe online version of this article (10.1186/s13023-018-0784-8) contains supplementary material, which is available to authorized users.

BackgroundMitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism.Methodology/Principal FindingsThe question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes.Conclusion/SignificanceOur results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.