Valentina Strecker

Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency

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.

Molecular diagnosis in mitochondrial complex I deficiency using exome sequencing

Background Next generation sequencing has become the core technology for gene discovery in rare inherited disorders. However, the interpretation of the numerous sequence variants identified remains challenging. We assessed the application of exome sequencing for diagnostics in complex I deficiency, a disease with vast genetic heterogeneity. Methods Ten unrelated individuals with complex I deficiency were selected for exome sequencing and sequential bioinformatic filtering. Cellular rescue experiments were performed to verify pathogenicity of novel disease alleles. Results The first filter criterion was ‘Presence of known pathogenic complex I deficiency variants’. This revealed homozygous mutations in NDUFS3 and ACAD9 in two individuals. A second criterion was ‘Presence of two novel potentially pathogenic variants in a structural gene of complex I’, which discovered rare variants in NDUFS8 in two unrelated individuals and in NDUFB3 in a third. Expression of wild-type cDNA in mutant cell lines rescued complex I activity and assembly, thus providing a functional validation of their pathogenicity. Using the third criterion ‘Presence of two potentially pathogenic variants in a gene encoding a mitochondrial protein’, loss-of-function mutations in MTFMT were discovered in two patients. In three patients the molecular genetic correlate remained unclear and follow-up analysis is ongoing. Conclusion Appropriate in silico filtering of exome sequencing data, coupled with functional validation of new disease alleles, is effective in rapidly identifying disease-causative variants in known and new complex I associated disease genes.

Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells.

Background Mitochondria, 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 Findings The 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/Significance Our 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.

Homozygous missense mutation in BOLA3 causes multiple mitochondrial dysfunctions syndrome in two siblings

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.

Large pore gels to separate mega protein complexes larger than 10 MDa by blue native electrophoresis: Isolation of putative respiratory strings or patches

Here, we expand the application of blue native electrophoresis to the separation of mega protein complexes larger than 10 MDa by introducing novel large pore acrylamide gels. We tailored the bis‐acrylamide cross‐linker amounts relative to the acrylamide monomer to enlarge the pore size of acrylamide gels and to obtain elastic and sufficiently stable gels. The novel gel types were then used to search for suprastructures of mitochondrial respiratory supercomplexes, the hypothetical respiratory strings, or patches. We identified 4–8 MDa assemblies that contain respiratory complexes I, III, and IV and most likely represent dimers, trimers, and tetramers of respiratory supercomplexes. We also isolated multimeric respiratory supercomplexes with apparent masses of 35–45 MDa, the presumed core pieces of respiratory strings or patches. Electron microscopic investigations will be required to clarify whether the isolated assemblies of complexes are ordered and specific, as predicted for respiratory strings and patches in the mitochondrial membrane.

Determination of protein mobility in mitochondrial membranes of living cells.

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F(1)F(0) ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D=0.7μm(2)/s and 0.6μm(2)/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F(1)F(0) ATPase: 0.4μm(2)/s and 0.0005μm(2)/s; COX: 0.3μm(2)/s and 0.007μm(2)/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.

Aging of different avian cultured cells: Lack of ROS-induced damage and quality control mechanisms

Elevated reactive oxygen species (ROS) levels have been observed in mammals during aging, implying an important role of ROS in the aging process. Most bird species are known to live longer and to contain lower ROS levels than mammals of the same body weight. The influence of ROS on the aging process of birds has been investigated using pigeon embryonic fibroblasts (PEF) and chicken embryonic fibroblasts (CEF). ROS levels in young avian cells were much lower than in human cells. When cultivated till replicative senescence, PEF proliferated about one-third longer compared to CEF. However, both senescent avian cell populations showed no increased ROS levels or accumulation of ROS-induced damage on the mtDNA or protein level. The investigation for quality control (QC) mechanisms revealed that the autophagosomal/lysosomal pathway was not downregulated in old avian cells and stable overexpression of the autophagy protein ATG5 improved mitochondrial fitness, enhanced the resistance against oxidative stress and prolonged the life span of CEF. Oxidative stress-mediated apoptosis induced a dose-dependent cell proliferation in CEF as well as in PEF. Taken together, our data indicate that autophagy and compensatory proliferation act as QC mechanisms, while ROS did not influence the aging process in avian cells.

Biallelic Mutations in TMEM126B Cause Severe Complex I Deficiency with a Variable Clinical Phenotype

Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs∗2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.

Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays

Abstract Measuring NADPH oxidase (Nox)‐derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH‐stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1‐Nox2‐Nox4‐/‐) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH‐stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH‐stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH‐stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH‐dependent signal in assays utilizing lucigenin, L‐012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR‐/‐) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR‐/‐ abolished the signal in NADPH‐stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH‐dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. In conclusion: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays. Graphical abstract Figure. No Caption available. HighlightsNox activity of intact cells could not be recapitulated in membranes treated with NADPH.Proteomics of membranes show P450 reductase as source of NADPH‐dependent signal.Microsomes overexpressing Cytochrome P450 system produce a NADPH‐dependent signal.Knockout of P450 reductase (CRISPR/Cas9) abolished lucigenin signal in HEK cell membranes.Knockout of POR but not p22phox abolishes the basal and Angiotensin II‐stimulated NADPH‐dependent signal in SMC membranes.

Mic13 Is Essential for Formation of Crista Junctions in Mammalian Cells

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.