Broad defects in the energy metabolism of leukocytes underlie immunoparalysis in sepsis.

The acute phase of sepsis is characterized by a strong inflammatory reaction. At later stages in some patients, immunoparalysis may be encountered, which is associated with a poor outcome. By transcriptional and metabolic profiling of human patients with sepsis, we found that a shift from oxidative phosphorylation to aerobic glycolysis was an important component of initial activation of host defense. Blocking metabolic pathways with metformin diminished cytokine production and increased mortality in systemic fungal infection in mice. In contrast, in leukocytes rendered tolerant by exposure to lipopolysaccharide or after isolation from patients with sepsis and immunoparalysis, a generalized metabolic defect at the level of both glycolysis and oxidative metabolism was apparent, which was restored after recovery of the patients. Finally, the immunometabolic defects in humans were partially restored by therapy with recombinant interferon-γ, which suggested that metabolic processes might represent a therapeutic target in sepsis.

Stress-dependent macromolecular crowding in the mitochondrial matrix.

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.

Homozygous damaging SOD2 variant causes lethal neonatal dilated cardiomyopathy.

BACKGROUND: Idiopathic dilated cardiomyopathy (DCM) is recognised to be a heritable disorder, yet clinical genetic testing does not produce a diagnosis in >50% of paediatric patients. Identifying a genetic cause is crucial because this knowledge can affect management options, cardiac surveillance in relatives and reproductive decision-making. In this study, we sought to identify the underlying genetic defect in a patient born to consanguineous parents with rapidly progressive DCM that led to death in early infancy. METHODS AND RESULTS: Exome sequencing revealed a potentially pathogenic, homozygous missense variant, c.542G>T, p.(Gly181Val), in SOD2. This gene encodes superoxide dismutase 2 (SOD2) or manganese-superoxide dismutase, a mitochondrial matrix protein that scavenges oxygen radicals produced by oxidation-reduction and electron transport reactions occurring in mitochondria via conversion of superoxide anion (O2-·) into H2O2. Measurement of hydroethidine oxidation showed a significant increase in O2-· levels in the patient's skin fibroblasts, as compared with controls, and this was paralleled by reduced catalytic activity of SOD2 in patient fibroblasts and muscle. Lentiviral complementation experiments demonstrated that mitochondrial SOD2 activity could be completely restored on transduction with wild type SOD2. CONCLUSION: Our results provide evidence that defective SOD2 may lead to toxic increases in the levels of damaging oxygen radicals in the neonatal heart, which can result in rapidly developing heart failure and death. We propose SOD2 as a novel nuclear-encoded mitochondrial protein involved in severe human neonatal cardiomyopathy, thus expanding the wide range of genetic factors involved in paediatric cardiomyopathies.

Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR-RAPTOR.

Mitochondria play a central role in cellular energy production, and their dysfunction can trigger a compensatory increase in glycolytic flux to sustain cellular ATP levels. Here, we studied the mechanism of this homeostatic phenomenon in C2C12 myoblasts. Acute (30 min) mitoenergetic dysfunction induced by the mitochondrial inhibitors piericidin A and antimycin A stimulated Glut1-mediated glucose uptake without altering Glut1 (also known as SLC2A1) mRNA or plasma membrane levels. The serine/threonine liver kinase B1 (LKB1; also known as STK11) and AMP-activated protein kinase (AMPK) played a central role in this stimulation. In contrast, ataxia-telangiectasia mutated (ATM; a potential AMPK kinase) and hydroethidium (HEt)-oxidizing reactive oxygen species (ROS; increased in piericidin-A- and antimycin-A-treated cells) appeared not to be involved in the stimulation of glucose uptake. Treatment with mitochondrial inhibitors increased NAD+ and NADH levels (associated with a lower NAD+:NADH ratio) but did not affect the level of Glut1 acetylation. Stimulation of glucose uptake was greatly reduced by chemical inhibition of Sirt2 or mTOR-RAPTOR. We propose that mitochondrial dysfunction triggers LKB1-mediated AMPK activation, which stimulates Sirt2 phosphorylation, leading to activation of mTOR-RAPTOR and Glut1-mediated glucose uptake.

OXPHOS mutations and neurodegeneration.

Mitochondrial oxidative phosphorylation (OXPHOS) sustains organelle function and plays a central role in cellular energy metabolism. The OXPHOS system consists of 5 multisubunit complexes (CI-CV) that are built up of 92 different structural proteins encoded by the nuclear (nDNA) and mitochondrial DNA (mtDNA). Biogenesis of a functional OXPHOS system further requires the assistance of nDNA-encoded OXPHOS assembly factors, of which 35 are currently identified. In humans, mutations in both structural and assembly genes and in genes involved in mtDNA maintenance, replication, transcription, and translation induce 'primary' OXPHOS disorders that are associated with neurodegenerative diseases including Leigh syndrome (LS), which is probably the most classical OXPHOS disease during early childhood. Here, we present the current insights regarding function, biogenesis, regulation, and supramolecular architecture of the OXPHOS system, as well as its genetic origin. Next, we provide an inventory of OXPHOS structural and assembly genes which, when mutated, induce human neurodegenerative disorders. Finally, we discuss the consequences of mutations in OXPHOS structural and assembly genes at the single cell level and how this information has advanced our understanding of the role of OXPHOS dysfunction in neurodegeneration.

Rotenone inhibits primary murine myotube formation via Raf-1 and ROCK2.

Rotenone (ROT) is a widely used inhibitor of complex I (CI), the first complex of the mitochondrial oxidative phosphorylation (OXPHOS) system. However, particularly at high concentrations ROT was also described to display off-target effects. Here we studied how ROT affected in vitro primary murine myotube formation. We demonstrate that myotube formation is specifically inhibited by ROT (10-100nM), but not by piericidin A (PA; 100nM), another CI inhibitor. At 100nM, both ROT and PA fully blocked myoblast oxygen consumption. Knock-down of Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) and, to a lesser extent ROCK1, prevented the ROT-induced inhibition of myotube formation. Moreover, the latter was reversed by inhibiting Raf-1 activity. In contrast, ROT-induced inhibition of myotube formation was not prevented by knock-down of RhoA. Taken together, our results support a model in which ROT reduces primary myotube formation independent of its inhibitory effect on CI-driven mitochondrial ATP production, but via a mechanism primarily involving the Raf-1/ROCK2 pathway.

Skeletal muscle mitochondria of NDUFS4-/- mice display normal maximal pyruvate oxidation and ATP production.

Mitochondrial ATP production is mediated by the oxidative phosphorylation (OXPHOS) system, which consists of four multi-subunit complexes (CI-CIV) and the FoF1-ATP synthase (CV). Mitochondrial disorders including Leigh Syndrome often involve CI dysfunction, the pathophysiological consequences of which still remain incompletely understood. Here we combined experimental and computational strategies to gain mechanistic insight into the energy metabolism of isolated skeletal muscle mitochondria from 5-week-old wild-type (WT) and CI-deficient NDUFS4-/- (KO) mice. Enzyme activity measurements in KO mitochondria revealed a reduction of 79% in maximal CI activity (Vmax), which was paralleled by 45-72% increase in Vmax of CII, CIII, CIV and citrate synthase. Mathematical modeling of mitochondrial metabolism predicted that these Vmax changes do not affect the maximal rates of pyruvate (PYR) oxidation and ATP production in KO mitochondria. This prediction was empirically confirmed by flux measurements. In silico analysis further predicted that CI deficiency altered the concentration of intermediate metabolites, modestly increased mitochondrial NADH/NAD+ ratio and stimulated the lower half of the TCA cycle, including CII. Several of the predicted changes were previously observed in experimental models of CI-deficiency. Interestingly, model predictions further suggested that CI deficiency only has major metabolic consequences when its activity decreases below 90% of normal levels, compatible with a biochemical threshold effect. Taken together, our results suggest that mouse skeletal muscle mitochondria possess a substantial CI overcapacity, which minimizes the effects of CI dysfunction on mitochondrial metabolism in this otherwise early fatal mouse model.

Mitochondrial dysfunction in primary human fibroblasts triggers an adaptive cell survival program that requires AMPK-α.

Dysfunction of complex I (CI) of the mitochondrial electron transport chain (ETC) features prominently in human pathology. Cell models of ETC dysfunction display adaptive survival responses that still are poorly understood but of relevance for therapy development. Here we comprehensively examined how primary human skin fibroblasts adapt to chronic CI inhibition. CI inhibition triggered transient and sustained changes in metabolism, redox homeostasis and mitochondrial (ultra)structure but no cell senescence/death. CI-inhibited cells consumed no oxygen and displayed minor mitochondrial depolarization, reverse-mode action of complex V, a slower proliferation rate and futile mitochondrial biogenesis. Adaptation was neither prevented by antioxidants nor associated with increased PGC1-α/SIRT1/mTOR levels. Survival of CI-inhibited cells was strictly glucose-dependent and accompanied by increased AMPK-α phosphorylation, which occurred without changes in ATP or cytosolic calcium levels. Conversely, cells devoid of AMPK-α died upon CI inhibition. Chronic CI inhibition did not increase mitochondrial superoxide levels or cellular lipid peroxidation and was paralleled by a specific increase in SOD2/GR, whereas SOD1/CAT/Gpx1/Gpx2/Gpx5 levels remained unchanged. Upon hormone stimulation, fully adapted cells displayed aberrant cytosolic and ER calcium handling due to hampered ATP fueling of ER calcium pumps. It is concluded that CI dysfunction triggers an adaptive program that depends on extracellular glucose and AMPK-α. This response avoids cell death by suppressing energy crisis, oxidative stress induction and substantial mitochondrial depolarization.

Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction.

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-content microscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells.

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.

Mitoenergetic Dysfunction Triggers a Rapid Compensatory Increase in Steady-State Glucose Flux.

ATP can be produced in the cytosol by glycolytic conversion of glucose (GLC) into pyruvate. The latter can be metabolized into lactate, which is released by the cell, or taken up by mitochondria to fuel ATP production by the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS) system. Altering the balance between glycolytic and mitochondrial ATP generation is crucial for cell survival during mitoenergetic dysfunction, which is observed in a large variety of human disorders including cancer. To gain insight into the kinetic properties of this adaptive mechanism we determined here how acute (30 min) inhibition of OXPHOS affected cytosolic GLC homeostasis. GLC dynamics were analyzed in single living C2C12 myoblasts expressing the fluorescent biosensor FLII(12)Pglu-700µÎ´6 (FLII). Following in situ FLII calibration, the kinetic properties of GLC uptake (V1) and GLC consumption (V2) were determined independently and used to construct a minimal mathematical model of cytosolic GLC dynamics. After validating the model, it was applied to quantitatively predict V1 and V2 at steady-state (i.e., when V1 = V2 = Vsteady-state) in the absence and presence of OXPHOS inhibitors. Integrating model predictions with experimental data on lactate production, cell volume, and O2 consumption revealed that glycolysis and mitochondria equally contribute to cellular ATP production in control myoblasts. Inhibition of OXPHOS induced a twofold increase in Vsteady-state and glycolytic ATP production flux. Both in the absence and presence of OXPHOS inhibitors, GLC was consumed at near maximal rates, meaning that GLC consumption is rate-limiting under steady-state conditions. Taken together, we demonstrate here that OXPHOS inhibition increases steady-state GLC uptake and consumption in C2C12 myoblasts. This activation fully compensates for the reduction in mitochondrial ATP production, thereby maintaining the balance between cellular ATP supply and demand.

Function and regulation of the Na+-Ca2+ exchanger NCX3 splice variants in brain and skeletal muscle.

Isoform 3 of the Na(+)-Ca(2+) exchanger (NCX3) is crucial for maintaining intracellular calcium ([Ca(2+)]i) homeostasis in excitable tissues. In this sense NCX3 plays a key role in neuronal excitotoxicity and Ca(2+) extrusion during skeletal muscle relaxation. Alternative splicing generates two variants (NCX3-AC and NCX3-B). Here, we demonstrated that NCX3 variants display a tissue-specific distribution in mice, with NCX3-B as mostly expressed in brain and NCX-AC as predominant in skeletal muscle. Using Fura-2-based Ca(2+) imaging, we measured the capacity and regulation of the two variants during Ca(2+) extrusion and uptake in different conditions. Functional studies revealed that, although both variants are activated by intracellular sodium ([Na(+)]i), NCX3-AC has a higher [Na(+)]i sensitivity, as Ca(2+) influx is observed in the presence of extracellular Na(+). This effect could be partially mimicked for NCX3-B by mutating several glutamate residues in its cytoplasmic loop. In addition, NCX3-AC displayed a higher capacity of both Ca(2+) extrusion and uptake compared with NCX3-B, together with an increased sensitivity to intracellular Ca(2+). Strikingly, substitution of Glu(580) in NCX3-B with its NCX3-AC equivalent Lys(580) recapitulated the functional properties of NCX3-AC regarding Ca(2+) sensitivity, Lys(580) presumably acting through a structure stabilization of the Ca(2+) binding site. The higher Ca(2+) uptake capacity of NCX3-AC compared with NCX3-B is in line with the necessity to restore Ca(2+) levels in the sarcoplasmic reticulum during prolonged exercise. The latter result, consistent with the high expression in the slow-twitch muscle, suggests that this variant may contribute to the Ca(2+) handling beyond that of extruding Ca(2+).

Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II, III, IV and V.

The mitochondrial oxidative phosphorylation (OXPHOS) system consists of four electron transport chain (ETC) complexes (CI-CIV) and the FoF1-ATP synthase (CV), which sustain ATP generation via chemiosmotic coupling. The latter requires an inward-directed proton-motive force (PMF) across the mitochondrial inner membrane (MIM) consisting of a proton (ΔpH) and electrical charge (Δψ) gradient. CI actively participates in sustaining these gradients via trans-MIM proton pumping. Enigmatically, at the cellular level genetic or inhibitor-induced CI dysfunction has been associated with Δψ depolarization or hyperpolarization. The cellular mechanism of the latter is still incompletely understood. Here we demonstrate that chronic (24h) CI inhibition in HEK293 cells induces a proton-based Δψ hyperpolarization in HEK293 cells without triggering reverse-mode action of CV or the adenine nucleotide translocase (ANT). Hyperpolarization was associated with low levels of CII-driven O2 consumption and prevented by co-inhibition of CII, CIII or CIV activity. In contrast, chronic CIII inhibition triggered CV reverse-mode action and induced Δψ depolarization. CI- and CIII-inhibition similarly reduced free matrix ATP levels and increased the cell's dependence on extracellular glucose to maintain cytosolic free ATP. Our findings support a model in which Δψ hyperpolarization in CI-inhibited cells results from low activity of CII, CIII and CIV, combined with reduced forward action of CV and ANT.

A mutation in the FAM36A gene, the human ortholog of COX20, impairs cytochrome c oxidase assembly and is associated with ataxia and muscle hypotonia.

The mitochondrial respiratory chain complex IV (cytochrome c oxidase) is a multi-subunit enzyme that transfers electrons from cytochrome c to molecular oxygen, yielding water. Its biogenesis requires concerted expression of mitochondria- and nuclear-encoded subunits and assembly factors. In this report, we describe a homozygous missense mutation in FAM36A from a patient who displays ataxia and muscle hypotonia. The FAM36A gene is a remote, putative ortholog of the fungal complex IV assembly factor COX20. Messenger RNA (mRNA) and protein co-expression analyses support the involvement of FAM36A in complex IV function in mammals. The c.154A>C mutation in the FAM36A gene, a mutation that is absent in sequenced exomes, leads to a reduced activity and lower levels of complex IV and its protein subunits. The FAM36A protein is nearly absent in patient's fibroblasts. Cells affected by the mutation accumulate subassemblies of complex IV that contain COX1 but are almost devoid of COX2 protein. We observe co-purification of FAM36A and COX2 proteins, supporting that the FAM36A defect hampers the early step of complex IV assembly at the incorporation of the COX2 subunit. Lentiviral complementation of patient's fibroblasts with wild-type FAM36A increases the complex IV activity as well as the amount of holocomplex IV and of individual subunits. These results establish the function of the human gene FAM36A/COX20 in complex IV assembly and support a causal role of the gene in complex IV deficiency.

Interactions between mitochondrial reactive oxygen species and cellular glucose metabolism.

Mitochondrial reactive oxygen species (ROS) production and detoxification are tightly balanced. Shifting this balance enables ROS to activate intracellular signaling and/or induce cellular damage and cell death. Increased mitochondrial ROS production is observed in a number of pathological conditions characterized by mitochondrial dysfunction. One important hallmark of these diseases is enhanced glycolytic activity and low or impaired oxidative phosphorylation. This suggests that ROS is involved in glycolysis (dys)regulation and vice versa. Here we focus on the bidirectional link between ROS and the regulation of glucose metabolism. To this end, we provide a basic introduction into mitochondrial energy metabolism, ROS generation and redox homeostasis. Next, we discuss the interactions between cellular glucose metabolism and ROS. ROS-stimulated cellular glucose uptake can stimulate both ROS production and scavenging. When glucose-stimulated ROS production, leading to further glucose uptake, is not adequately counterbalanced by (glucose-stimulated) ROS scavenging systems, a toxic cycle is triggered, ultimately leading to cell death. Here we inventoried the various cellular regulatory mechanisms and negative feedback loops that prevent this cycle from occurring. It is concluded that more insight in these processes is required to understand why they are (un)able to prevent excessive ROS production during various pathological conditions in humans.

Solute diffusion is hindered in the mitochondrial matrix.

Intracellular chemical reactions generally constitute reaction-diffusion systems located inside nanostructured compartments like the cytosol, nucleus, endoplasmic reticulum, Golgi, and mitochondrion. Understanding the properties of such systems requires quantitative information about solute diffusion. Here we present a novel approach that allows determination of the solvent-dependent solute diffusion constant (D(solvent)) inside cell compartments with an experimentally quantifiable nanostructure. In essence, our method consists of the matching of synthetic fluorescence recovery after photobleaching (FRAP) curves, generated by a mathematical model with a realistic nanostructure, and experimental FRAP data. As a proof of principle, we assessed D(solvent) of a monomeric fluorescent protein (AcGFP1) and its tandem fusion (AcGFP1(2)) in the mitochondrial matrix of HEK293 cells. Our results demonstrate that diffusion of both proteins is substantially slowed by barriers in the mitochondrial matrix (cristae), suggesting that cells can control the dynamics of biochemical reactions in this compartment by modifying its nanostructure.

Isoflurane anesthetic hypersensitivity and progressive respiratory depression in a mouse model with isolated mitochondrial complex I deficiency.

BACKGROUND: Children with mitochondrial disorders are frequently anesthetized for a wide range of operations. These disorders may interfere with the response to surgery and anesthesia. We examined anesthetic sensitivity to and respiratory effects of isoflurane in the Ndufs4 knockout (KO) mouse model. These mice exhibit an isolated mitochondrial complex I (CI) deficiency of the respiratory chain, and they also display clinical signs and symptoms resembling those of patients with mitochondrial CI disease. METHODS: We investigated seven Ndufs4(-/-) knockout (KO), five Ndufs4(+/-) heterozygous (HZ) and five Ndufs4(+/+) wild type (WT) mice between 22 and 25 days and again between 31 and 34 days post-natal. Animals were placed inside an airtight box, breathing spontaneously while isoflurane was administered in increasing concentrations. Minimum alveolar concentration (MAC) was determined with the bracketing study design, using the response to electrical stimulation to the hind paw. RESULTS: MAC for isoflurane was significantly lower in KO mice than in HZ and WT mice: 0.81% ± 0.01 vs 1.55 ± 0.05% and 1.55 ± 0.13%, respectively, at 22-25 days, and 0.65 ± 0.05%, 1.65 ± 0.08% and 1.68 ± 0.08% at 31-34 days. The KO mice showed severe respiratory depression at lower isoflurane concentrations than the WT and HZ mice. CONCLUSION: We observed an increased isoflurane anesthetic sensitivity and severe respiratory depression in the KO mice. The respiratory depression during anesthesia was strongly progressive with age. Since the pathophysiological consequences from complex I deficiency are mainly reflected in the central nervous system and our mouse model involves progressive encephalopathy, further investigation of isoflurane effects on brain mitochondrial function is warranted.

Subunit-specific incorporation efficiency and kinetics in mitochondrial complex I homeostasis.

Studies employing native PAGE suggest that most nDNA-encoded CI subunits form subassemblies before assembling into holo-CI. In addition, in vitro evidence suggests that some subunits can directly exchange in holo-CI. Presently, data on the kinetics of these two incorporation modes for individual CI subunits during CI maintenance are sparse. Here, we used inducible HEK293 cell lines stably expressing AcGFP1-tagged CI subunits and quantified the amount of tagged subunit in mitoplasts and holo-CI by non-native and native PAGE, respectively, to determine their CI incorporation efficiency. Analysis of time courses of induction revealed three subunit-specific patterns. A first pattern, represented by NDUFS1, showed overlapping time courses, indicating that imported subunits predominantly incorporate into holo-CI. A second pattern, represented by NDUFV1, consisted of parallel time courses, which were, however, not quantitatively overlapping, suggesting that imported subunits incorporate at similar rates into holo-CI and CI assembly intermediates. The third pattern, represented by NDUFS3 and NDUFA2, revealed a delayed incorporation into holo-CI, suggesting their prior appearance in CI assembly intermediates and/or as free monomers. Our analysis showed the same maximum incorporation into holo-CI for NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS4, NDUFA2, and NDUFA12 with nearly complete loss of endogenous subunit at 24 h of induction, indicative of an equimolar stoichiometry and unexpectedly rapid turnover. In conclusion, the results presented demonstrate that newly formed nDNA-encoded CI subunits rapidly incorporate into holo-CI in a subunit-specific manner.

A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome.

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K(M) of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K(M) observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.

Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts.

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).