Oxidative Phosphorylation Defects Research Articles

Mitochondrial Mutations in Cardiovascular Diseases: Preliminary Findings

Background/Objectives: Mitochondria are the main organelles for ATP synthesis able to produce energy for several different cellular activities. Cardiac cells require high amounts of energy and, thus, they contain a high number of mitochondria. Consequently, mitochondrial dysfunction in these cells is a crucial factor for the development of cardiovascular diseases. Mitochondria constitute central regulators of cellular metabolism and energy production, producing approximately 90% of the cells’ energy needs in the form of ATP via oxidative phosphorylation. The mitochondria have their own circular, double-stranded DNA encoding 37 genes. Any mitochondrial DNA sequence anomaly may result in defective oxidative phosphorylation and lead to cardiac dysfunction. Methods: In this study, we investigated the potential association between mitochondrial DNA mutation and cardiovascular disease. Cardiac tissue and serum samples were collected from seven patients undergoing coronary artery bypass grafting. Total DNA was extracted from cardiac muscle tissue specimens and serum and each sample was subjected to polymerase chain reaction (PCR) to amplify the NADH dehydrogenase 1 (ND1) gene, which is part of the mitochondrial complex I enzyme complex and was screened for mutations. Results: We identified one patient with a homoplasmic A to G substitution mutation in cardiac tissue DNA and two patients with heteroplasmic A3397G mutation in serum DNA. Specifically, amplicon sequence analysis revealed a homoplasmic A3397G substitution in the ND1 gene in a tissue sample of the patient with ID number 1 and a heteroplasmic mutation in A3397G in serum samples of patients with ID numbers 3 and 6, respectively. The A to G substitution changes the amino acid from methionine (ATA) to valine (GTA) at position 31 of the ND1 gene. Conclusions: The detection of this novel mutation in patients with coronary artery disease may contribute to our understanding of the association between mitochondrial dysfunction and the disease, implying that mitochondria may be key players in the pathogenesis of cardiovascular diseases.

HADHA Regulates Respiratory Complex Assembly and Couples FAO and OXPHOS.

Oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) are key bioenergetics pathways. The machineries for both processes are localized in mitochondria. Secondary OXPHOS defects have been documented in patients with primary FAO deficiencies, and vice versa. However, the underlying mechanisms remain unclear. Intrigued by the observations that regulation of supercomplexes (SCs) assembly in a mouse OXPHOS deficient cell line and its derivatives is associated with the changes in lipid metabolism, a proteomics analysis is carried out and identified mitochondrial trifunctional protein (MTP) subunit alpha (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, HADHA) as a potential regulatory factor for SCs assembly. HADHA-Knockdown cells and mouse embryonic fibroblasts (MEFs) derived from HADHA-Knockout mice displayed both reduced SCs assembly and defective OXPHOS. Stimulation of OXPHOS induced in cell culture by replacing glucose with galactose and of lipid metabolism in mice with a high-fat diet (HFD) both exhibited increased HADHA expression. HADHA Heterozygous mice fed with HFD showed enhanced steatosis associated with a reduction of SCs assembly and OXPHOS function. The results indicate that HADHA participates in SCs assembly and couples FAO and OXPHOS.

Signaling Pathways Concerning Mitochondrial Dysfunction: Implications in Neurodegeneration and Possible Molecular Targets.

Mitochondrion is an important organelle present in our cells responsible for meeting energy requirements. All higher organisms rely on efficient mitochondrial bioenergetic machinery to sustain life. No other respiratory process can produce as much power as generated by mitochondria in the form of ATPs. This review is written in order to get an insight into the magnificent working of mitochondrion and its implications in cellular homeostasis, bioenergetics, redox, calcium signaling, and cell death. However, if this machinery gets faulty, it may lead to several disease states. Mitochondrial dysfunctioning is of growing concern today as it is seen in the pathogenesis of several diseases which includes neurodegenerative disorders, cardiovascular disorders, diabetes mellitus, skeletal muscle defects, liver diseases, and so on. To cover all these aspects is beyond the scope of this article; hence, our study is restricted to neurodegenerative disorders only. Moreover, faulty functioning of this organelle can be one of the causes of early ageing in individuals. This review emphasizes mutations in the mitochondrial DNA, defects in oxidative phosphorylation, generation of ROS, and apoptosis. Researchers have looked into new approaches that might be able to control mitochondrial failure and show a lot of promise as treatments.

Cardiolipin deficiency disrupts CoQ redox state and induces steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive disorder marked by lipid accumulation, leading to steatohepatitis (MASH). A key feature of the transition to MASH involves oxidative stress resulting from defects in mitochondrial oxidative phosphorylation (OXPHOS). Here, we show that pathological alterations in the lipid composition of the inner mitochondrial membrane (IMM) directly instigate electron transfer inefficiency to promote oxidative stress. Specifically, cardiolipin (CL) was downregulated across four mouse models of MASLD. Hepatocyte-specific CL synthase knockout (CLS-LKO) led to spontaneous MASH with elevated mitochondrial electron leak. Loss of CL interfered with the ability of coenzyme Q (CoQ) to transfer electrons, promoting leak primarily at sites II F and III Q0 . Data from human liver biopsies revealed a highly robust correlation between mitochondrial CL and CoQ, co-downregulated with MASH. Thus, reduction in mitochondrial CL promotes oxidative stress and contributes to pathogenesis of MASH.

Mitochondrial bioenergetics stimulates autophagy for pathological MAPT/Tau clearance in tauopathy neurons

ABSTRACT Hyperphosphorylation and aggregation of MAPT (microtubule-associated protein tau) is a pathogenic hallmark of tauopathies and a defining feature of Alzheimer disease (AD). Pathological MAPT/tau is targeted by macroautophagy/autophagy for clearance after being sequestered within autophagosomes, but autophagy dysfunction is indicated in tauopathy. While mitochondrial bioenergetic deficits have been shown to precede MAPT/tau pathology in tauopathy brains, it is unclear whether energy metabolism deficiency is involved in the pathogenesis of autophagy defects. Here, we reveal that stimulation of anaplerotic metabolism restores defective oxidative phosphorylation (OXPHOS) in tauopathy neurons which, strikingly, leads to pronounced MAPT/tau clearance by boosting autophagy functionality through enhancements of mitochondrial biosynthesis and supply of phosphatidylethanolamine for autophagosome biogenesis. Furthermore, early anaplerotic stimulation of OXPHOS elevates autophagy activity and attenuates MAPT/tau pathology, thereby counteracting memory impairment in tauopathy mice. Taken together, our study sheds light on a pivotal role of mitochondrial bioenergetic deficiency in tauopathy-related autophagy defects and suggests a new therapeutic strategy to prevent the buildup of pathological MAPT/tau in AD and other tauopathy diseases. Abbreviation: AA: antimycin A; AD, Alzheimer disease; ATP, adenosine triphosphate; AV, autophagosome/autophagic vacuole; AZ, active zone; Baf-A1: bafilomycin A1; CHX, cycloheximide; COX, cytochrome c oxidase; DIV, days in vitro; DRG, dorsal root ganglion; ETN, ethanolamine; FRET, Förster/fluorescence resonance energy transfer; FTD, frontotemporal dementia; Gln, glutamine; HA: hydroxylamine; HsMAPT/Tau, human MAPT; IMM, inner mitochondrial membrane; LAMP1, lysosomal-associated membrane protein 1; LIs, lysosomal inhibitors; MDAV, mitochondria-derived autophagic vacuole; MmMAPT/Tau, murine MAPT; NFT, neurofibrillary tangle; OCR, oxygen consumption rate; Omy: oligomycin; OXPHOS, oxidative phosphorylation; PPARGC1A/PGC-1alpha: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PE, phosphatidylethanolamine; phospho-MAPT/tau, hyperphosphorylated MAPT; PS, phosphatidylserine; PISD, phosphatidylserine decarboxylase;SQSTM1/p62, sequestosome 1; STX1, syntaxin 1; SYP, synaptophysin; Tg, transgenic; TCA, tricarboxylic acid; TEM, transmission electron microscopy.

Defective mitochondrial COX1 translation due to loss of COX14 function triggers ROS-induced inflammation in mouse liver

Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.

Decoding the muscle transcriptome of patients with late onset Pompe disease reveals markers of disease progression.

Late-onset Pompe Disease (LOPD) is a rare genetic disorder caused by the deficiency of acid alpha-glucosidase leading to progressive cellular dysfunction due to the accumulation of glycogen in the lysosome. The mechanism of relentless muscle damage - a classic manifestation of the disease - has been extensively studied by analysing the whole muscle tissue; however, little, if any, is known about transcriptional heterogeneity among nuclei within the multinucleated skeletal muscle cells. This is the first report of application of single nuclei RNA sequencing to uncover changes in the gene expression profile in muscle biopsies from eight patients with LOPD and four muscle samples from age and gender matched healthy controls. We matched these changes with histology findings using GeoMx Spatial Transcriptomics to compare the transcriptome of control myofibers from healthy individuals with non-vacuolated (histologically unaffected) and vacuolated (histologically affected) myofibers of LODP patients. We observed an increase in the proportion of slow and regenerative muscle fibers and macrophages in LOPD muscles. The expression of the genes involved in glycolysis was reduced, whereas the expression of the genes involved in the metabolism of lipids and amino acids was increased in non-vacuolated fibers, indicating early metabolic abnormalities. Additionally, we detected upregulation of autophagy genes, and downregulation of the genes involved in ribosomal and mitochondrial function leading to defective oxidative phosphorylation. The upregulation of the genes associated with inflammation, apoptosis and muscle regeneration was observed only in vacuolated fibers. Notably, enzyme replacement therapy - the only available therapy for the disease - showed a tendency to restore metabolism dysregulation, particularly within slow fibers. A combination of single nuclei RNA sequencing and spatial transcriptomics revealed the landscape of normal and the diseased muscle, and highlighted the early abnormalities associated with the disease progression. Thus, the application of these two new cutting-edge technologies provided insight into the molecular pathophysiology of muscle damage in LOPD and identified potential avenues for therapeutic intervention.

Clinical, imaging, and pathological characteristics of 35 cases of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome

Objective: To summarize the clinical, imaging, and pathological characteristics of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome (MELAS) to improve the diagnosis of this rare disease. Methods: A retrospective case series was conducted to collect the clinical data and results of genetic testing, muscle biopsy, and imaging studies including computed tomography (CT), magnetic resonance imaging (MRI), and magnetic resonance spectroscopy (MRS) of 35 patients with MELAS admitted to the Nanjing Drum Tower Hospital from 2012 to 2021. Descriptive statistical analysis including mean, standard deviation, and frequency percentage were carried out. Results: The average age of onset of the patients was 30.2±2.3 years; the prevalence of family history was 20%. The two main initial symptoms were limb weakness and convulsions. The clinical manifestations of the neuromuscular system were proximal muscle weakness and exercise intolerance. The endocrine system is the most affected outside the neuromuscular system, with diabetes being the most common condition. Among the five patients who underwent brain CT, four showed hypodense lesions and two had calcified lesions. Brain MRI in 26 patients showed that the lesions more often affected the parietal lobe, basal ganglia, temporal lobe, occipital lobe, and frontal lobe than the infratentorial areas. Twelve of these individuals exhibited different levels of brain atrophy. Among the 10 patients who underwent 1H-MRS, nine showed a decrease in N-acetylaspartate (NAA) levels, eight exhibited abnormal lactate elevation (Lac peaks), whereas six had both reduced NAA levels and the presence of Lac peaks. Thirty-one patients underwent genetic testing; among them, 25 were found to have the mt.3243A>G mutation, while the remaining six exhibited rare gene alterations. Muscle biopsies were performed in 21 patients, and 15 showed abnormal mitochondrial proliferation manifested by ragged red fibers and defective oxidative phosphorylation manifested by cytochrome C oxidase (COX) enzyme-deficient muscle fibers. Conclusion: The clinical manifestations of MELAS syndrome are variable and complex, and early atypical symptoms could be missed or misdiagnosed. A detailed clinical history, imaging MRS analysis, muscle biopsy, and genetic testing are necessary to confirm the accurate diagnosis of MELAS.

Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

A zebrafish tufm mutant model for the COXPD4 syndrome of aberrant mitochondrial function

Mitochondrial dysfunction is a critical factor leading to a wide range of clinically heterogeneous and often severe disorders due to its central role in generating cellular energy. Mutations in the TUFM gene are known to cause combined oxidative phosphorylation deficiency 4 (COXPD4), a rare mitochondrial disorder characterized by a comprehensive quantitative deficiency in mitochondrial respiratory chain (MRC) complexes. The development of a reliable animal model for COXPD4 is crucial for elucidating the roles and mechanisms of TUFM in disease pathogenesis and benefiting its medical management. In this study, we construct a zebrafish tufm−/− mutant that closely resembles the COXPD4 syndrome, exhibiting compromised mitochondrial protein translation, dysfunctional mitochondria with oxidative phosphorylation defects, and significant metabolic suppression of the tricarboxylic acid cycle. Leveraging this COXPD4 zebrafish model, we comprehensively validate the clinical relevance of TUFM mutations and identify probucol as a promising therapeutic approach for managing COXPD4. Our data offer valuable insights for understanding mitochondrial diseases and developing effective treatments.

Transcriptomic analysis reveals mitochondrial dysfunction in the pathogenesis of Nager syndrome in sf3b4-depleted zebrafish

Nager syndrome (NS) is a rare acrofacial dysostosis caused by heterozygous loss-of-function variants in the splicing factor 3B subunit 4 (SF3B4). The main clinical features of patients with NS are characterized by facial-mandibular and preaxial limb malformations. The migration and specification of neural crest cells are crucial for craniofacial development, and mitochondrial fitness appears to play a role in such processes. Here, by analyzing our previously published transcriptome dataset, we aim to investigate the potential involvement of mitochondrial components in the pathogenesis of craniofacial malformations, especially in sf3b4 mutant zebrafish. We identified that oxidative phosphorylation (OXPHOS) defects and overproduction of reactive oxygen species (ROS) due to decreased antioxidants defense activity, which leads to oxidative damage and mitochondrial dysfunction. Furthermore, our results highlight that fish lacking sf3b4 gene, primarily display defects in mitochondrial complex I. Altogether, our findings suggest that mitochondrial dysfunction may contribute to the development of the craniofacial anomalies observed in sf3b4-depleted zebrafish.

Polymerase and Proofreading Exonuclease Domains in the DNA Polymerase γ and Nuclear-encoded RNA Polymerase of the Human Mitochondria and Identification of Mitochondrial Disease Mutations in the Polymerase Active Site Region of DNA Polymerase γ

Recent reports have made it clear that mutations in the mitochondrial DNA (mtDNA) polymerase γ (POLG1) are a major cause of many human diseases. Mutations in the mtDNA polymerase leads to defective oxidative phosphorylation and ATP production, resulting in many mitochondrial diseases. For their mitochondrial genome replication, humans and animals use POLG1, whose catalytic site is essentially similar to the E. coli DNA polymerase I (DNA pol I). Multiple sequence alignment (MSA) analysis have shown that the POLG1 and E. coli DNA pol I use identical amino acids in their polymerase catalytic sites, viz. –943R-4EHAKI1FNYGRI955Y8G- (human DNA pol γ) as –R-4RSAKA1INFGLIY8G- (E. coli DNA pol I). However, the human POLG1 shows only 31.25% identity with the E. coli DNA pol I, suggesting a highly divergent evolution. Mutation(s) in the POLG1 gene is one of the most common causes of many inherited mitochondrial diseases in children and adults. Depending on their location within the enzyme, mutations either lead to mtDNA depletion or accumulation of multiple mtDNA deletions leading to various mitochondrial diseases. The most common POLG1 dominant mutation, viz. Y955→H/C, which lead to a severe, early-onset of multi-systemic mitochondrial disease with bilateral sensorineural hearing loss, cataract, myopathy, and liver failure is located in the template-binding pair of the polymerase catalytic site region by MSA. Another dominant mutation, R943→H/C is observed in patients with Progressive External Ophthalmoplegia (adPEO, an autosomal, dominant, heritable mitochondrial disorder) is located in the nucleoside triphosphate (NTP) selection amino acid in the polymerase catalytic site region. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the mitochondrial genes. The human mitochondrial NEP showed 39.30%, 40.12% and 26.98% identities to the NEPs of the mitochondria and chloroplasts of Arabidopsis thaliana, and T7 RNA polymerase, respectively, suggesting that the human and plant mitochondrial NEPs are distinctly different. Interestingly, the human NEP’s catalytic core is almost completely conserved in the plant mitochondrial and chloroplast NEPs, viz. –R-4KVVKQ1TVMTVVY8G- (Human) and –R-4KLVKQ1TVMTSVY8G- (A. Thaliana). Furthermore, the mitochondrial NEP’s catalytic core from human and different animal sources is remarkably conserved and is in close agreement with other NEPs of plant sources. Both the human DNA pol γ and NEP possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from the catalytic K. The PR exonucleases of POLG1 and NEP belong to the DEDD-superfamily of exonucleases and uses a Y or H as the proton acceptor, respectively.

A Novel Homozygous Germline Mutation in Transferrin Receptor 1 (TfR1) Leads to Combined Immunodeficiency and Provides New Insights into Iron-Immunity Axis

A homozygous missense mutation in the transferrin receptor 1 (TfR1), also known as CD71, leads to a rare inborn error of immunity (IEI) characterized by the impaired lymphocyte activation and proliferation due to defective iron uptake of cells. However, only one causative mutation (c.58T > C, p.Y20H) in the TFRC gene coding for TfR1 has been reported so far. We herein identified a new disease-causing homozygous germline mutation in the TFRC gene (c.64C > T, p.R22W) (referred to as TfR1R22W from now on) in a Turkish patient with combined immunodeficiency (CID). TfR1R22W results in impaired TfR1 internalization similar to previously defined TfR1Y20H mutation. We found that TfR1R22W is associated with severely restricted B and T lymphocyte clonal diversity and impaired T cell activation and cytokine production as well as defective mitochondrial oxidative phosphorylation in helper T cells. In addition, circulating NK, Treg, and MAIT cell populations were significantly decreased in the patient. Using whole transcriptome analysis, we found dysregulated immune homeostasis and novel biological processes associated with TfR1R22W. We also identified a considerable expansion of circulating low-density neutrophils (LDNs) in patient’s PBMCs. Overall, TfR1R22W mutation expands the current understanding of the IEI associated with TfR1 dysfunction and provides new insights underlying impaired immune function, lymphocyte diversity, and granulocyte homeostasis.

Preneoplastic cells switch to Warburg metabolism from their inception exposing multiple vulnerabilities for targeted elimination

Otto Warburg described tumour cells as displaying enhanced aerobic glycolysis whilst maintaining defective oxidative phosphorylation (OXPHOS) for energy production almost 100 years ago [1, 2]. Since then, the ‘Warburg effect’ has been widely accepted as a key feature of rapidly proliferating cancer cells [3–5]. What is not clear is how early “Warburg metabolism” initiates in cancer and whether changes in energy metabolism might influence tumour progression ab initio. We set out to investigate energy metabolism in the HRASG12V driven preneoplastic cell (PNC) at inception, in a zebrafish skin PNC model. We find that, within 24 h of HRASG12V induction, PNCs upregulate glycolysis and blocking glycolysis reduces PNC proliferation, whilst increasing available glucose enhances PNC proliferation and reduces apoptosis. Impaired OXPHOS accompanies enhanced glycolysis in PNCs, and a mild complex I inhibitor, metformin, selectively suppresses expansion of PNCs. Enhanced mitochondrial fragmentation might be underlining impaired OXPHOS and blocking mitochondrial fragmentation triggers PNC apoptosis. Our data indicate that altered energy metabolism is one of the earliest events upon oncogene activation in somatic cells, which allows a targeted and effective PNC elimination.

IL-15 reprogramming compensates for NK cell mitochondrial dysfunction in HIV-1 infection.

Dynamic regulation of cellular metabolism is important for maintaining homeostasis and can directly influence immune cell function and differentiation, including NK cell responses. Persistent HIV-1 infection leads to a state of chronic immune activation, NK cell subset redistribution, and progressive NK cell dysregulation. In this study, we examined the metabolic processes that characterize NK cell subsets in HIV-1 infection, including adaptive NK cell subpopulations expressing the activating receptor NKG2C, which expand during chronic infection. These adaptive NK cells exhibit an enhanced metabolic profile in HIV-1- individuals infected with human cytomegalovirus (HCMV). However, the bioenergetic advantage of adaptive CD57+NKG2C+ NK cells is diminished during chronic HIV-1 infection, where NK cells uniformly display reduced oxidative phosphorylation (OXPHOS). Defective OXPHOS was accompanied by increased mitochondrial depolarization, structural alterations, and increased DRP-1 levels promoting fission, suggesting that mitochondrial defects are restricting the metabolic plasticity of NK cell subsets in HIV-1 infection. The metabolic requirement for the NK cell response to receptor stimulation was alleviated upon IL-15 pretreatment, which enhanced mammalian target of rapamycin complex 1 (mTORC1) activity. IL-15 priming enhanced NK cell functionality to anti-CD16 stimulation in HIV-1 infection, representing an effective strategy for pharmacologically boosting NK cell responses.

Expanding the genetic spectrum of mitochondrial diseases in Tunisia: novel variants revealed by whole-exome sequencing.

Introduction: Inherited mitochondrial diseases are the most common group of metabolic disorders caused by a defect in oxidative phosphorylation. They are characterized by a wide clinical and genetic spectrum and can manifest at any age. In this study, we established novel phenotype-genotype correlations between the clinical and molecular features of a cohort of Tunisian patients with mitochondrial diseases. Materials and methods: Whole-exome sequencing was performed on five Tunisian patients with suspected mitochondrial diseases. Then, a combination of filtering and bioinformatics prediction tools was utilized to assess the pathogenicity of genetic variations. Sanger sequencing was subsequently performed to confirm the presence of potential deleterious variants in the patients and verify their segregation within families. Structural modeling was conducted to study the effect of novel variants on the protein structure. Results: We identified two novel homozygous variants in NDUFAF5 (c.827G>C; p.Arg276Pro) and FASTKD2 (c.496_497del; p.Leu166GlufsTer2) associated with a severe clinical form of Leigh and Leigh-like syndromes, respectively. Our results further disclosed two variants unreported in North Africa, in GFM2 (c.569G>A; p.Arg190Gln) and FOXRED1 (c.1261G>A; p.Val421Met) genes, and we described the first case of fumaric aciduria in a Tunisian patient harboring the c.1358T>C; p.Leu453Pro FH variant. Conclusion: Our study expands the mutational and phenotypic spectrum of mitochondrial diseases in Tunisia and highlights the importance of next-generation sequencing to decipher the pathomolecular mechanisms responsible for these disorders in an admixed population.

H+-slip correlated to rotor free-wheeling as cause of F1FO-ATPase dysfunction in primary mitochondrial disorders.

Inborn errors of metabolism are related to mitochondrial disorders caused by dysfunction of the oxidative phosphorylation (OXPHOS) system. Congenital hypermetabolism in the infant is a rare disease belonging to Luft syndrome, nonthyroidal hypermetabolism, arising from a singular example of a defect in OXPHOS. The mitochondria lose coupling of mitochondrial substrates oxidation from the ADP phosphorylation. Since Luft syndrome is due to uncoupled cell respiration responsible for deficient in ATP production that originates in the respiratory complexes, a de novo heterozygous variant in the catalytic subunit of mitochondrial F1FO-ATPase arises as the main cause of an autosomal dominant syndrome of hypermetabolism associated with dysfunction in ATP production, which does not involve the respiratory complexes. The F1FO-ATPase works as an embedded molecular machine with a rotary action using two different motor engines. The FO, which is an integral domain in the membrane, dissipates the chemical potential difference for H+, a proton motive force (Δp), across the inner membrane to generate a torsion. The F1 domain-the hydrophilic portion responsible for ATP turnover-is powered by the molecular rotary action to synthesize ATP. The structural and functional coupling of F1 and FO domains support the energy transduction for ATP synthesis. The dissipation of Δp by means of an H+ slip correlated to rotor free-wheeling of the F1FO-ATPase has been discovered to cause enzyme dysfunction in primary mitochondrial disorders. In this insight, we try to offer commentary and analysis of the molecular mechanism in these impaired mitochondria.

TARS2 Variants Cause Combination Oxidative Phosphorylation Deficiency-21: A Case Report and Literature Review.

The aim of the study is to explore the clinical and genetic characteristics of the combined oxidative phosphorylation defect type 21 (COXPD21) caused by the TARS2 compound heterozygous pathogenic variants, and to improve clinicians' awareness of the disease. The proband was a girl of first birth, with repeated refractory hypokalemia, hearing impairment, developmental delay, intellectual disability, developmental retardation after infection, high limb muscle tension, and increased serum lactate as the clinical phenotype. The clinical performance, diagnosis, treatment process, and gene characteristics of COXPD21 caused by TARS2 of the case were analyzed, reviewed, and compared with the literature from the CNKI, Wanfang Data, and biomedical literature database (PubMed) until November 2021. The child was diagnosed with COXPD21 after two heterozygous variants in the TARS2 gene were found via whole exome sequencing. One of the variants was c.1679(exon14) A > C (p.Asp560Ala) missense, derived from the mother, and the other was c.1036(exon10)C > T (p.Arg346Cys) missense, derived from the father. The literature was searched and reviewed with the keywords "mitochondrial encephalomyopathy," "TARS2," and "combination oxidative phosphorylation deficiency type 21." A total of four complete domestic and foreign cases were collected from the literature search. COXPD21 onset by a complex heterozygous variant of TARS2 causes refractory hypokalemia, which is rarely reported in China and abroad.

The energetics of cellular life transitions.

Major life transitions are always difficult because change costs energy. Recent findings have demonstrated how mitochondrial oxidative phosphorylation (OxPhos) defects increase the energetic cost of living, and that excessive integrated stress response (ISR) signaling may prevent cellular identity transitions during development. In this perspective, we discuss general bioenergetic principles of life transitions and the costly molecular processes involved in reprograming the cellular hardware/software as cells shift identity. The energetic cost of cellular differentiation has not been directly quantified, representing a gap in knowledge. We propose that the ISR is an energetic checkpoint evolved to i) prevent OxPhos-deficient cells from engaging in excessively costly transitions, and ii) allow ISR-positive cells to recruit systemic energetic resources by signaling via the brain.

Isocitrate Dehydrogenase 2 Mutation Allows Myeloid Differentiation but Impairs Bone Marrow Macrophage Polarization and Function Via Metabolic Dysregulation

Isocitrate dehydrogenase 2 (IDH2) mutations, while less common than other mutations associated with myelodysplastic syndromes (MDS), are also found in clonal hematopoiesis and are associated with increased risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite derived from alpha-ketoglutarate (aKG), which disrupts hematopoiesis. However, the mechanisms that induce clonal progression and transformation remain incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutations on mature myeloid cell functions and the bone marrow microenvironment remains to be elucidated. Previous literature suggested that hypermethylation via this mutant leads to incomplete differentiation and failed mature myeloid cell generation. Our lab previously demonstrated that efferocytic defects in bone marrow macrophages can skew hematopoietic stem and progenitor cells via increased inflammatory mediators. Based on these data, we hypothesized that IDH2 mutations prevent complete polarization down pro-efferocytic phenotypes in macrophages due to blocking of metabolic pathways required for mature macrophage function. Congenic CD45.1+ mice were transplanted with retrovirally infected hematopoietic stem cells containing human WT or R172K IDH2 mutant with an mCherry reporter and showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes by 16 weeks post-transplant. In contrast to prior literature, IDH2 mutants gave rise to mature reporter positive monocytes, macrophages, and neutrophils in the bone marrow and peripheral blood. As expected, we observed myeloid skewed hematopoiesis, a trademark of MDS. When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow macrophages when fed apoptotic neutrophils. We then generated a genetic murine model using the Vav1 Cre IDH2 R140Q floxed mutant. The genetic model allowed us to study disease progression in the absence of radiation induced stress, and to observe mature myeloid populations while clinically defined MDS was absent. We performed ATACseq and bulk RNAseq analysis on bone marrow macrophages isolated from 3-month-old and 6-month-old mice, which demonstrated a significant decrease in global chromosomal accessibility; however, this had limited impact on the transcriptional program. Based on existing literature, the majority of macrophages in the bone marrow are in a poised state and have not yet activated genes associated with pro-efferocytic/pro-resolution or pro-inflammatory phenotypes. Since aKG is accumulated in macrophages during polarization toward reparative and pro-efferocytic phenotypes and is necessary for metabolic reprogramming for oxidative phosphorylation, we hypothesized that, in the presence of IDH2 mutations, which drain aKG from the system, pro-efferocytic polarized macrophages would be metabolically stunned. To test this theory, we isolated bone marrow macrophages from IDH2 mutants and cultured them to generate pro-efferocytic polarized macrophages. We then tested their efferocytic potential and metabolic capacity by seahorse assay. Continual efferocytosis, a hallmark of pro-efferocytic macrophages, was impaired in IDH2 mutant macrophages as early as 2-3 months of age. Consistent with this finding, mito stress testing by seahorse demonstrated that IDH2 mutant macrophages had severely impaired maximal respiratory capacity compared to WT macrophages. Treatment with global hypomethylating agent azacytidine or a cell permeable aKG analog were unable to restore respiratory capacity or efferocytic activity in the IDH2 mutant. These data strongly suggest that the efferocytic defect in IDH2 mutant macrophages is due to metabolic rather than epigenetic reprogramming. In summary, we identify defects in efferocytic function and oxidative phosphorylation in IDH2 mutant macrophages. This represents a novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and leukemic transformation. Furthermore, our data suggest that the metabolic dysregulation induced by IDH mutations may play a greater role than epigenetic impairment in IDH2 mutant mature myeloid cells, which could explain why approaches targeting hypermethylation have shown limited efficacy in clinical settings.