NDUFS4 deletion triggers loss of NDUFA12 in Ndufs4-/- mice and Leigh syndrome patients: A stabilizing role for NDUFAF2.

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4-/- mouse tissues. Ndufs4-/- animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4-/- mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4-/- MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4-/- mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids.

TMEM70 functions in the assembly of complexes I and V.

Protein complexes from the oxidative phosphorylation (OXPHOS) system are assembled with the help of proteins called assembly factors. We here delineate the function of the inner mitochondrial membrane protein TMEM70, in which mutations have been linked to OXPHOS deficiencies, using a combination of BioID, complexome profiling and coevolution analyses. TMEM70 interacts with complex I and V and for both complexes the loss of TMEM70 results in the accumulation of an assembly intermediate followed by a reduction of the next assembly intermediate in the pathway. This indicates that TMEM70 has a role in the stability of membrane-bound subassemblies or in the membrane recruitment of subunits into the forming complex. Independent evidence for a role of TMEM70 in OXPHOS assembly comes from evolutionary analyses. The TMEM70/TMEM186/TMEM223 protein family, of which we show that TMEM186 and TMEM223 are mitochondrial in human as well, only occurs in species with OXPHOS complexes. Our results validate the use of combining complexome profiling with BioID and evolutionary analyses in elucidating congenital defects in protein complex assembly.

Bi-allelic Mutations in the Mitochondrial Ribosomal Protein MRPS2 Cause Sensorineural Hearing Loss, Hypoglycemia, and Multiple OXPHOS Complex Deficiencies.

Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation.

A Heterozygous NDUFV1 Variant Aggravates Mitochondrial Complex I Deficiency in a Family with a Homoplasmic ND1 Variant.

We demonstrate that a heterozygous nuclear variant in the gene encoding mitochondrial complex I subunit NDUFV1 aggravates the cellular phenotype in the presence of a mitochondrial DNA variant in complex I subunit ND1. Our findings suggest that heterozygous variants could be more significant in inherited mitochondrial diseases than hitherto assumed.

NDUFAF4 variants are associated with Leigh syndrome and cause a specific mitochondrial complex I assembly defect.

Mitochondrial respiratory chain complex I consists of 44 different subunits and can be subgrouped into three functional modules: the Q-, the P- and the N-module. NDUFAF4 (C6ORF66) is an assembly factor of complex I that associates with assembly intermediates of the Q-module. Via exome sequencing, we identified a homozygous missense variant in a complex I-deficient patient with Leigh syndrome. Supercomplex analysis in patient fibroblasts revealed specifically altered stoichiometry. Detailed assembly analysis of complex I, indicative of all of its assembly routes, showed an accumulation of parts of the P- and the N-module but not the Q-module. Lentiviral complementation of patient fibroblasts with wild-type NDUFAF4 rescued complex I deficiency and the assembly defect, confirming the causal role of the variant. Our report on the second family affected by an NDUFAF4 variant further characterizes the phenotypic spectrum and sheds light into the role of NDUFAF4 in mitochondrial complex I biogenesis.

Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive.

COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts.

Mutations in ATP6V1E1 or ATP6V1A Cause Autosomal-Recessive Cutis Laxa.

Defects of the V-type proton (H+) ATPase (V-ATPase) impair acidification and intracellular trafficking of membrane-enclosed compartments, including secretory granules, endosomes, and lysosomes. Whole-exome sequencing in five families affected by mild to severe cutis laxa, dysmorphic facial features, and cardiopulmonary involvement identified biallelic missense mutations in ATP6V1E1 and ATP6V1A, which encode the E1 and A subunits, respectively, of the V1 domain of the heteromultimeric V-ATPase complex. Structural modeling indicated that all substitutions affect critical residues and inter- or intrasubunit interactions. Furthermore, complexome profiling, a method combining blue-native gel electrophoresis and liquid chromatography tandem mass spectrometry, showed that they disturb either the assembly or the stability of the V-ATPase complex. Protein glycosylation was variably affected. Abnormal vesicular trafficking was evidenced by delayed retrograde transport after brefeldin A treatment and abnormal swelling and fragmentation of the Golgi apparatus. In addition to showing reduced and fragmented elastic fibers, the histopathological hallmark of cutis laxa, transmission electron microscopy of the dermis also showed pronounced changes in the structure and organization of the collagen fibers. Our findings expand the clinical and molecular spectrum of metabolic cutis laxa syndromes and further link defective extracellular matrix assembly to faulty protein processing and cellular trafficking caused by genetic defects in the V-ATPase complex.

Mutations in mitochondrial complex I assembly factor NDUFAF3 cause Leigh syndrome.

NDUFAF3 is an assembly factor of mitochondrial respiratory chain complex I. Variants in NDUFAF3 have been identified as a cause of severe multisystem mitochondrial disease. In a patient presenting with Leigh syndrome, which has hitherto not been described as a clinical feature of NDUFAF3 deficiency, we identified a novel homozygous variant and confirmed its pathogenicity in patient fibroblasts studies. Furthermore, we present an analysis of complex I assembly routes representative of each functional module and, thereby, link NDUFAF3 to a specific step in complex I assembly. Therefore, our report expands the phenotype of NDUFAF3 deficiency and further characterizes the role of NDUFAF3 in complex I biogenesis.

The Assembly Pathway of Mitochondrial Respiratory Chain Complex I.

Mitochondrial complex I is the largest integral membrane enzyme of the respiratory chain and consists of 44 different subunits encoded in the mitochondrial and nuclear genome. Its biosynthesis is a highly complicated and multifaceted process involving at least 14 additional assembly factors. How these subunits assemble into a functional complex I and where the assembly factors come into play is largely unknown. Here, we applied a dynamic complexome profiling approach to elucidate the assembly of human mitochondrial complex I and its further incorporation into respiratory chain supercomplexes. We delineate the stepwise incorporation of all but one subunit into a series of distinct assembly intermediates and their association with known and putative assembly factors, which had not been implicated in this process before. The resulting detailed and comprehensive model of complex I assembly is fully consistent with recent structural data and the remarkable modular architecture of this multiprotein complex.

Mutations in Complex I Assembly Factor TMEM126B Result in Muscle Weakness and Isolated Complex I Deficiency.

Mitochondrial complex I deficiency results in a plethora of often severe clinical phenotypes manifesting in early childhood. Here, we report on three complex-I-deficient adult subjects with relatively mild clinical symptoms, including isolated, progressive exercise-induced myalgia and exercise intolerance but with normal later development. Exome sequencing and targeted exome sequencing revealed compound-heterozygous mutations in TMEM126B, encoding a complex I assembly factor. Further biochemical analysis of subject fibroblasts revealed a severe complex I deficiency caused by defective assembly. Lentiviral complementation with the wild-type cDNA restored the complex I deficiency, demonstrating the pathogenic nature of these mutations. Further complexome analysis of one subject indicated that the complex I assembly defect occurred during assembly of its membrane module. Our results show that TMEM126B defects can lead to complex I deficiencies and, interestingly, that symptoms can occur only after exercise.

Unraveling the complexity of mitochondrial complex I assembly: A dynamic process.

Mammalian complex I is composed of 44 different subunits and its assembly requires at least 13 specific assembly factors. Proper function of the mitochondrial respiratory chain enzyme is of crucial importance for cell survival due to its major participation in energy production and cell signaling. Complex I assembly depends on the coordination of several crucial processes that need to be tightly interconnected and orchestrated by a number of assembly factors. The understanding of complex I assembly evolved from simple sequential concept to the more sophisticated modular assembly model describing a convoluted process. According to this model, the different modules assemble independently and associate afterwards with each other to form the final enzyme. In this review, we aim to unravel the complexity of complex I assembly and provide the latest insights in this fundamental and fascinating process. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

Increased mitochondrial ATP production capacity in brain of healthy mice and a mouse model of isolated complex I deficiency after isoflurane anesthesia.

We reported before that the minimal alveolar concentration (MAC) of isoflurane is decreased in complex I-deficient mice lacking the NDUFS4 subunit of the respiratory chain (RC) (1.55 and 0.81% at postnatal (PN) 22-25 days and 1.68 and 0.65% at PN 31-34 days for wildtype (WT) and CI-deficient KO, respectively). A more severe respiratory depression was caused by 1.0 MAC isoflurane in KO mice (respiratory rate values of 86 and 45 at PN 22-25 days and 69 and 29 at PN 31-34 days for anesthetized WT and KO, respectively). Here, we address the idea that isoflurane anesthesia causes a much larger decrease in brain mitochondrial ATP production in KO mice thus explaining their increased sensitivity to this anesthetic. Brains from WT and KO mice of the above study were removed immediately after MAC determination at PN 31-34 days and a mitochondria-enriched fraction was prepared. Aliquots were used for measurement of maximal ATP production in the presence of pyruvate, malate, ADP and creatine and, after freeze-thawing, the maximal activity of the individual RC complexes in the presence of complex-specific substrates. CI activity was dramatically decreased in KO, whereas ATP production was decreased by only 26% (p < 0.05). The activities of CII, CIII, and CIV were the same for WT and KO. Isoflurane anesthesia decreased the activity of CI by 30% (p < 0.001) in WT. In sharp contrast, it increased the activity of CII by 37% (p < 0.001) and 50% (p < 0.001) and that of CIII by 37% (p < 0.001) and 40% (p < 0.001) in WT and KO, respectively, whereas it tended to increase that of CIV in both WT and KO. Isoflurane anesthesia increased ATP production by 52 and 69% in WT (p < 0.05) and KO (p < 0.01), respectively. Together these findings indicate that isoflurane anesthesia interferes positively rather than negatively with the ability of CI-deficient mice brain mitochondria to convert their main substrate pyruvate into ATP.

Mutations in COA6 cause cytochrome c oxidase deficiency and neonatal hypertrophic cardiomyopathy.

COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6-deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady-state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse-chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII2 /CIVn -supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6-deficient patients is provided.

Disrupting mitochondrial-nuclear coevolution affects OXPHOS complex I integrity and impacts human health.

The mutation rate of the mitochondrial DNA (mtDNA), which is higher by an order of magnitude as compared with the nuclear genome, enforces tight mitonuclear coevolution to maintain mitochondrial activities. Interruption of such coevolution plays a role in interpopulation hybrid breakdown, speciation events, and disease susceptibility. Previously, we found an elevated amino acid replacement rate and positive selection in the nuclear DNA-encoded oxidative phosphorylation (OXPHOS) complex I subunit NDUFC2, a phenomenon important for the direct interaction of NDUFC2 with the mtDNA-encoded complex I subunit ND4. This finding underlines the importance of mitonuclear coevolution to physical interactions between mtDNA and nuclear DNA-encoded factors. Nevertheless, it remains unclear whether this interaction is important for the stability and activity of complex I. Here, we show that siRNA silencing of NDUFC2 reduced growth of human D-407 retinal pigment epithelial cells, significantly diminished mitochondrial membrane potential, and interfered with complex I integrity. Moreover, site-directed mutagenesis of a positively selected amino acid in NDUFC2 significantly interfered with the interaction of NDUFC2 with its mtDNA-encoded partner ND4. Finally, we show that a genotype combination involving this amino acid (NDUFC2 residue 46) and the mtDNA haplogroup HV likely altered susceptibility to type 2 diabetes mellitus in Ashkenazi Jews. Therefore, mitonuclear coevolution is important for maintaining mitonuclear factor interactions, OXPHOS, and for human health.

A mutation in the human CBP4 ortholog UQCC3 impairs complex III assembly, activity and cytochrome b stability.

Complex III (cytochrome bc1) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c. Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83, hereafter named UQCC3, to be the ortholog of the fungal complex III assembly factor CBP4. We describe a homozygous c.59T>A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T>A mutation has a causal role in complex III deficiency.

Clinical and biochemical features guiding the diagnostics in neurometabolic cutis laxa.

Patients with cutis laxa (CL) have wrinkled, sagging skin with decreased elasticity. Skin symptoms are associated with variable systemic involvement. The most common, genetically highly heterogeneous form of autosomal recessive CL, ARCL2, is frequently associated with variable metabolic and neurological symptoms. Progeroid symptoms, dysmorphic features, hypotonia and psychomotor retardation are highly overlapping in the early phase of these disorders. This makes the genetic diagnosis often challenging. In search for discriminatory symptoms, we prospectively evaluated clinical, neurologic, metabolic and genetic features in our patient cohort referred for suspected ARCL. From a cohort of 26 children, we confirmed mutations in genes associated with ARCL in 16 children (14 probands), including 12 novel mutations. Abnormal glycosylation and gyration abnormalities were mostly, but not always associated with ATP6V0A2 mutations. Epilepsy was most common in ATP6V0A2 defects. Corpus callosum dysgenesis was associated with PYCR1 and ALDH18A1 mutations. Dystonic posturing was discriminatory for PYCR1 and ALDH18A1 defects. Metabolic markers of mitochondrial dysfunction were found in one patient with PYCR1 mutations. So far unreported white matter abnormalities were found associated with GORAB and RIN2 mutations. We describe a large cohort of CL patients with neurologic involvement. Migration defects and corpus callosum hypoplasia were not always diagnostic for a specific genetic defect in CL. All patients with ATP6V0A2 defects had abnormal glycosylation. To conclude, central nervous system and metabolic abnormalities were discriminatory in this genetically heterogeneous group, although not always diagnostic for a certain genetic defect in CL.

ACAD9, a complex I assembly factor with a moonlighting function in fatty acid oxidation deficiencies.

Oxidative phosphorylation and fatty acid oxidation are two major metabolic pathways in mitochondria. Acyl-CoA dehydrogenase 9 (ACAD9), an enzyme assumed to play a role in fatty acid oxidation, was recently identified as a factor involved in complex I biogenesis. Here we further investigated the role of ACAD9's enzymatic activity in fatty acid oxidation and complex I biogenesis. We provide evidence indicating that ACAD9 displays enzyme activity in vivo. Knockdown experiments in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts revealed that ACAD9 is responsible for the production of C14:1-carnitine from oleate and C12-carnitine from palmitate. These results explain the origin of these obscure acylcarnitines that are used to diagnose VLCAD deficiency in humans. Knockdown of ACAD9 in control fibroblasts did not reveal changes in the acylcarnitine profiles upon fatty acid loading. Next, we investigated whether catalytic activity of ACAD9 was necessary for complex I biogenesis. Catalytically inactive ACAD9 gave partial-to-complete rescue of complex I biogenesis in ACAD9-deficient cells and was incorporated in high-molecular-weight assembly intermediates. Our results underscore the importance of the ACAD9 protein in complex I assembly and suggest that the enzymatic activity is a rudiment of the duplication event.