Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency.

Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity of RCS in vitro and in vivo, independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization. We demonstrate that, accordingly, the efficiency of mitochondria-dependent cell growth depends on cristae shape. Thus, RCS assembly emerges as a link between membrane morphology and function.

A new D-galactose treatment monitoring index for PGM1-CDG.

Phosphoglucomutase 1 (PGM1) catalyzes the interconversion of glucose-6-phosphate to glucose-1-phosphate and is a key enzyme of glycolysis, glycogenesis, and glycogenolysis. PGM1 deficiency (OMIM: 614921) was initially defined as a glycogen storage disorder (type XIV), and later re-classified as a PGM1-congenital disorder of glycosylation (PGM1-CDG). Serum transferrin (Tf) glycan isoform analysis by liquid chromatography-mass spectrometry (LC-MS) is used as a primary diagnostic screen tool, and reveals a very unique CDG profile described as a mixture of CDG-type I and CDG-type II patterns. Oral d-galactose supplementation shows significant clinical and metabolic improvements, which are indicated by the Tf glycan isoform normalization over time in patients with PGM1-CDG. Thus, there is a need for biomarkers to guide d-galactose dosage in patients in order to maintain effective and safe drug levels. Here, we present a simplified algorithm called PGM1-CDG Treatment Monitoring Index (PGM1-TMI) for assessing the response of PGM1-CDG patients to d-galactose supplementation. For our single-center cohort of 16 PGM1-CDG patients, the Tf glycan profile analysis provided the biochemical diagnosis in all of them. In addition, the PGM1-TMI was reduced in PGM1-CDG patients under d-galactose supplementation as compared with their corresponding values before treatment, indicating that glycosylation proceeds towards normalization. PGM1-TMI allows tracking Tf glycan isoform normalization over time when the patients are on d-galactose supplementation.

Natural underlying mtDNA heteroplasmy as a potential source of intra-person hiPSC variability.

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability, the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.

Spot14/Mig12 heterocomplex sequesters polymerization and restrains catalytic function of human acetyl-CoA carboxylase 2.

Acetyl-CoA carboxylase 2 (ACC2) is an isoform of ACC functioning as a negative regulator of fatty acid ß-oxidation. Spot14, a thyroid hormone responsive protein, and Mig12, a Spot14 paralog, have recently been identified as regulators of fatty acid synthesis targeting ACC1, a distinctive subtype of ACC. Here, we examined whether Spot14/Mig12 modulates ACC2. Nanoscale protein topography mapped putative protein-protein interactions between purified human Spot14/Mig12 and ACC2, validated by functional assays. Human ACC2 displayed consistent enzymatic activity, and homogeneous particle distribution was probed by atomic force microscopy. Citrate-induced polymerization and enzymatic activity of ACC2 were restrained by the addition of the recombinant Spot14/Mig12 heterocomplex but only partially by the oligo-heterocomplex, demonstrating that the heterocomplex is a designated metabolic inhibitor of human ACC2. Moreover, Spot14/Mig12 demonstrated a sequestering role preventing an initial ACC2 nucleation step during filamentous polymer formation. Thus, the Spot14/Mig12 heterocomplex controls human ACC2 polymerization and catalytic function, emerging as a previously unrecognized molecular regulator in catalytic lipid metabolism.

Allotopic expression of mitochondrial-encoded genes in mammals: achieved goal, undemonstrated mechanism or impossible task?

Mitochondrial-DNA diseases have no effective treatments. Allotopic expression-synthesis of a wild-type version of the mutated protein in the nuclear-cytosolic compartment and its importation into mitochondria-has been proposed as a gene-therapy approach. Allotopic expression has been successfully demonstrated in yeast, but in mammalian mitochondria results are contradictory. The evidence available is based on partial phenotype rescue, not on the incorporation of a functional protein into mitochondria. Here, we show that reliance on partial rescue alone can lead to a false conclusion of successful allotopic expression. We recoded mitochondrial mt-Nd6 to the universal genetic code, and added the N-terminal mitochondrial-targeting sequence of cytochrome c oxidase VIII (C8) and the HA epitope (C8Nd6HA). The protein apparently co-localized with mitochondria, but a significant part of it seemed to be located outside mitochondria. Complex I activity and assembly was restored, suggesting successful allotopic expression. However, careful examination of transfected cells showed that the allotopically-expressed protein was not internalized in mitochondria and that the selected clones were in fact revertants for the mt-Nd6 mutation. These findings demonstrate the need for extreme caution in the interpretation of functional rescue experiments and for clear-cut controls to demonstrate true rescue of mitochondrial function by allotopic expression.

Restoration of electron transport without proton pumping in mammalian mitochondria.

We have restored the CoQ oxidative capacity of mouse mtDNA-less cells (rho degrees cells) by transforming them with the alternative oxidase Aox of Emericella nidulans. Cotransforming rho degrees cells with the NADH dehydrogenase of Saccharomyces cerevisiae, Ndi1 and Aox recovered the NADH DH/CoQ reductase and the CoQ oxidase activities. CoQ oxidation by AOX reduces the dependence of rho degrees cells on pyruvate and uridine. Coexpression of AOX and NDI1 further improves the recycling of NAD(+). Therefore, 2 single-protein enzymes restore the electron transport in mammalian mitochondria substituting >80 nuclear DNA-encoded and 11 mtDNA-encoded proteins. Because those enzymes do not pump protons, we were able to split electron transport and proton pumping (ATP synthesis) and inquire which of the metabolic deficiencies associated with the loss of oxidative phosphorylation should be attributed to each of the 2 processes.

Bilateral subdural hematomas and retinal hemorrhages mimicking nonaccidental trauma in a patient with D-2-hydroxyglutaric aciduria.

INTRODUCTION: Nonaccidental trauma (NAT) is considered when pediatric patients present with intracranial injuries and a negative history of an accidental injury or concomitant medical diagnosis. The evaluation of NAT should include the consideration of possible medical causes including coagulation, hematologic, metabolic and other genetic disorders, as well as witnessed and unwitnessed accidental injuries. CASE PRESENTATION: We present a 7-month-old male with spells and incidental findings of bilateral subdural hematomas, retinal hemorrhages, and secondary macrocephaly, leading to investigation for NAT. Biochemical analysis showed excretion of a large amount of D-2-hydroxyglutaric in urine consistent with a biochemical diagnosis of D-2-hydroxyglutaric aciduria, a rare neurometabolic disorder characterized by developmental delay, epilepsy, hypotonia, and psychomotor retardation. None of these symptoms were present in our patient at the time of diagnosis. Molecular genetic testing revealed a pathogenic splice site variant (c.685-2A>G) and a variant of uncertain significance (c.1256G>T) with evidence of pathogenicity in the D2HGDH gene, consistent with a molecular diagnosis of D-2-hydroxyglutaric aciduria type I (OMIM #600721). CONCLUSION: Since several metabolic disorders, including D-2-hydroxyglutaric aciduria type I, can present solely with symptoms suggestive of NAT (subdural and retinal hemorrhages), an early metabolic evaluation by urine organic acid analysis should be included in clinical protocols evaluating NAT. A methodical and nonjudgmental approach coordinated between pediatricians and metabolic specialists is also necessary to ensure that rare genetic conditions are not overlooked to prevent devastating social, legal, and financial consequences of suspected child abuse.

m.3243A > G-Induced Mitochondrial Dysfunction Impairs Human Neuronal Development and Reduces Neuronal Network Activity and Synchronicity.

Epilepsy, intellectual and cortical sensory deficits, and psychiatric manifestations are the most frequent manifestations of mitochondrial diseases. How mitochondrial dysfunction affects neural structure and function remains elusive, mostly because of a lack of proper in vitro neuronal model systems with mitochondrial dysfunction. Leveraging induced pluripotent stem cell technology, we differentiated excitatory cortical neurons (iNeurons) with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function on an isogenic nuclear DNA background from patients with the common pathogenic m.3243A > G variant of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). iNeurons with high heteroplasmy exhibited mitochondrial dysfunction, delayed neural maturation, reduced dendritic complexity, and fewer excitatory synapses. Micro-electrode array recordings of neuronal networks displayed reduced network activity and decreased synchronous network bursting. Impaired neuronal energy metabolism and compromised structural and functional integrity of neurons and neural networks could be the primary drivers of increased susceptibility to neuropsychiatric manifestations of mitochondrial disease.

Quantification of Etoposide Hypersensitivity: A Sensitive, Functional Method for Assessing Pluripotent Stem Cell Quality.

Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high-throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time-consuming, and incompatible with high-throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells' documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419-42-0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of <300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells. Stem Cells Translational Medicine 2017;6:1829-1839.

The CoQH2/CoQ Ratio Serves as a Sensor of Respiratory Chain Efficiency.

Electrons feed into the mitochondrial electron transport chain (mETC) from NAD- or FAD-dependent enzymes. A shift from glucose to fatty acids increases electron flux through FAD, which can saturate the oxidation capacity of the dedicated coenzyme Q (CoQ) pool and result in the generation of reactive oxygen species. To prevent this, the mETC superstructure can be reconfigured through the degradation of respiratory complex I, liberating associated complex III to increase electron flux via FAD at the expense of NAD. Here, we demonstrate that this adaptation is driven by the ratio of reduced to oxidized CoQ. Saturation of CoQ oxidation capacity induces reverse electron transport from reduced CoQ to complex I, and the resulting local generation of superoxide oxidizes specific complex I proteins, triggering their degradation and the disintegration of the complex. Thus, CoQ redox status acts as a metabolic sensor that fine-tunes mETC configuration in order to match the prevailing substrate profile.

Metabolic regulation of redox status in stem cells.

SIGNIFICANCE: Metabolism-dependent generation of reactive oxygen species (ROS) and associated oxidative damage have been traditionally linked to impaired homeostasis and cellular death. Beyond the adverse effects of ROS accumulation, increasing evidence implicates redox status as a regulator of vital cellular processes. RECENT ADVANCES: Emerging studies on the molecular mechanisms guiding stem cell fate decisions indicate a role for energy metabolism in regulating the fundamental ability of maintaining stemness versus undergoing lineage-specific differentiation. Stem cells have evolved protective metabolic phenotypes to minimize reactive oxygen generation through oxidative metabolism and support antioxidant scavenging through glycolysis and the pentose phosphate pathway. CRITICAL ISSUES: While the dynamics in ROS generation has been correlated with stem cell function, the intimate mechanisms by which energy metabolism regulates ROS to impact cellular fate remain to be deciphered. FUTURE DIRECTIONS: Decoding the linkage between nutrient sensing, energy metabolism, and ROS in regulating cell fate decisions would offer a redox-dependent strategy to regulate stemness and lineage specification.

Supercomplex assembly determines electron flux in the mitochondrial electron transport chain.

The textbook description of mitochondrial respiratory complexes (RCs) views them as free-moving entities linked by the mobile carriers coenzyme Q (CoQ) and cytochrome c (cyt c). This model (known as the fluid model) is challenged by the proposal that all RCs except complex II can associate in supercomplexes (SCs). The proposed SCs are the respirasome (complexes I, III, and IV), complexes I and III, and complexes III and IV. The role of SCs is unclear, and their existence is debated. By genetic modulation of interactions between complexes I and III and III and IV, we show that these associations define dedicated CoQ and cyt c pools and that SC assembly is dynamic and organizes electron flux to optimize the use of available substrates.

NDUFA4 is a subunit of complex IV of the mammalian electron transport chain.

The oxidative phosphorylation system is one of the best-characterized metabolic pathways. In mammals, the protein components and X-ray structures are defined for all complexes except complex I. Here, we show that NDUFA4, formerly considered a constituent of NADH Dehydrogenase (CI), is instead a component of the cytochrome c oxidase (CIV). Deletion of NDUFA4 does not perturb CI. Rather, proteomic, genetic, evolutionary, and biochemical analyses reveal that NDUFA4 plays a role in CIV function and biogenesis. The change in the attribution of the NDUFA4 protein requires renaming of the gene and reconsideration of the structure of CIV. Furthermore, NDUFA4 should be considered a candidate gene for CIV rather than CI deficiencies in humans.

Induction of the mitochondrial NDUFA4L2 protein by HIF-1α decreases oxygen consumption by inhibiting Complex I activity.

The fine regulation of mitochondrial function has proved to be an essential metabolic adaptation to fluctuations in oxygen availability. During hypoxia, cells activate an anaerobic switch that favors glycolysis and attenuates the mitochondrial activity. This switch involves the hypoxia-inducible transcription factor-1 (HIF-1). We have identified a HIF-1 target gene, the mitochondrial NDUFA4L2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2). Our results, obtained employing NDUFA4L2-silenced cells and NDUFA4L2 knockout murine embryonic fibroblasts, indicate that hypoxia-induced NDUFA4L2 attenuates mitochondrial oxygen consumption involving inhibition of Complex I activity, which limits the intracellular ROS production under low-oxygen conditions. Thus, reducing mitochondrial Complex I activity via NDUFA4L2 appears to be an essential element in the mitochondrial reprogramming induced by HIF-1.

Five entry points of the mitochondrially encoded subunits in mammalian complex I assembly.

Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.