Impaired energy production is a hallmark of mitochondrial oxidative phosphorylation (OXPHOS) defects. However, secondary metabolic disturbances also represent an important trigger for pathologies originating from OXPHOS aberrations. Here we show that cells with OXPHOS deficiencies accumulate triacylglycerols enriched in polyunsaturated fatty acids (PUFAs), which are stored in lipid droplets. Sequestration of PUFAs is a critical component of a broader stress response, which also includes downregulation of cellular desaturases and upregulation of glutathione peroxidase 4 (GPX4). We demonstrate that this mechanism represents a physiologically relevant protective strategy, manifesting in cells under hypoxia and in immortalised fibroblasts derived from patients with primary mitochondrial complex IV deficiency. As a proof of principle, we observe elevated PUFA-enriched triacylglycerols in the plasma of patients with Myoclonic Epilepsy with Ragged Red Fibres (MERRF). Our findings reveal a novel protective mechanism against ferroptosis, which preserves membrane integrity when mitochondrial respiration is compromised. OXPHOS-deficient cells activate a coordinated multilayered polyunsaturated fatty acid (PUFA) stress response, which preserves membrane integrity against lipid peroxidation-driven ferroptosis. It is conserved across distinct OXPHOS complex deficiencies, is recapitulated under hypoxia, and is detectable in patients with mitochondrial disease. OXPHOS-deficient cells activate a coordinated multilayered polyunsaturated fatty acid (PUFA) stress response, which preserves membrane integrity against lipid peroxidation-driven ferroptosis. It is conserved across distinct OXPHOS complex deficiencies, is recapitulated under hypoxia, and is detectable in patients with mitochondrial disease.

Background Succinate dehydrogenase (SDH), also known as complex II, connects the tricarboxylic acid cycle with oxidative phosphorylation, and mutations in its subunits are linked to mitochondrial disease and cancer. However, the molecular consequences of SDHA pathogenic variants remain poorly understood. Here, we generated a panel of patient-derived SDHA variants in an SDHA knockout HEK293 cell model and examined their effects on SDH assembly, function, and cellular metabolism. Results We found that SDHA mutations differentially affect SDH assembly and stability, yet most variants display severely impaired catalytic activity, despite partial or complete enzyme assembly. Loss of SDH function reduced succinate-driven respiration, altered the content of complexes I and IV, and shifted respiration toward NADH-supported pathways. Metabolomic and lipidomic analyses revealed extensive metabolic remodeling, including reorganization of the tricarboxylic acid cycle, succinate accumulation, and adaptive regulation of polyunsaturated fatty acid metabolism. Variants retaining partial SDH activity exhibited intermediate structural, functional, and metabolic phenotypes. Conclusions These findings define how different pathogenic variants of SDHA disrupt SDH structure and function, drive divergent metabolic adaptations, and provide mechanistic insight into the heterogeneous disease manifestations associated with SDHA deficiency.

Introduction:Mitochondrial DNA (mtDNA) is not routinely analyzed in inherited kidney disease. We evaluated mtDNA variation in families who remained genetically unresolved despite extensive testing. Methods:We reviewed pedigrees from the Wake Forest-Charles University Rare Inherited Kidney Disease Registry to identify genetically unresolved families with suspected maternal inheritance, performed mtDNA genotyping, clinically characterized variant carriers, and functionally evaluated disease-associated mitochondrial variants. Results:Among 33 families with evidence of maternal inheritance, 18 (55%) carried one of seven disease-associated mtDNA variant types, including homoplasmic recurrent single-nucleotide insertions in the second light-strand promoter (LSP2; 9 families), novel MT-TW and MT-TL2 variants (2 and 1 families, respectively), and previously reported MT-TF and heteroplasmic MT-ND5 variant (5 and 1 families, respectively). In 16 families, variants occurred on distinct haplotypes, consistent with independent mutational events and rapid enrichment to homoplasmy across generations. Maternal transmission was strongly supported, with below-normal kidney function observed in 54/60 (90%) offspring of affected mothers versus 1/17 (6%) offspring of affected fathers (p = 1.23 × 10 ⁻11 ). Pathogenicity was further supported by predicted deleterious structural effects and functional evidence of impaired mitochondrial transcription and translation, respiratory chain deficiency, and CoQ10 depletion. Affected individuals predominantly presented with chronic tubulointerstitial kidney disease, occasionally accompanied by gout and only sporadically with extrarenal manifestations. The rate of kidney disease progression appeared to vary both between and within families. Overall, 109/119 genetically affected individuals or obligate at-risk carriers were clinically affected; most unaffected carriers were younger than 45 years of age. Clinical status was unavailable for an additional 66 obligate at-risk carriers. Conclusions:These findings establish the physiological relevance of the LSP2 promoter, support routine assessment of the mitochondrial genome in inherited kidney disease, and highlight mtDNA variants as an important cause of familial and sporadic tubulointerstitial kidney disease of previously unexplained etiology. Lay Summary:Many inherited kidney diseases remain unexplained because routine genetic testing focuses on genes in the cell nucleus and does not examine mitochondrial DNA-the small genome in the cell's energy-providing mitochondria, inherited only from the mother. We studied 33 families with chronic kidney disease whose family histories suggested maternal inheritance and identified disease-causing mitochondrial DNA variants in 18 (55%). Nine families carried variants in LSP2, a recently discovered mitochondrial regulatory element, highlighting its importance in normal mitochondrial function and disease. Others carried pathogenic variants in mitochondrial tRNA genes required for mitochondrial protein synthesis. Laboratory studies showed that these variants impair mitochondrial energy conversion. In all families, the predominant manifestation was slowly progressive kidney disease, sometimes leading to dialysis or kidney transplantation. These findings identify pathogenic mitochondrial DNA variants as an underrecognized cause of inherited kidney disease and support the inclusion of mitochondrial DNA analysis in routine genetic testing.

In patients with testicular germ cell tumours (TGCT), sperm cryopreservation prior to anti-cancer treatment represents the main fertility preservation approach. However, it is associated with a low sperm recovery rate after thawing. Since sperm is a high-energy demanding cell, which is supplied by glycolysis and oxidative phosphorylation (OXPHOS), mitochondrial dysfunctionality can directly result in sperm anomalies. In this study, we investigated the bioenergetic pattern of cryopreserved sperm of TGCT patients in comparison with normozoospermic samples using two state-of-the-art methods: the Extracellular Flux Analyzer (XF Analyzer) and two-photon fluorescence lifetime imaging microscopy (2P-FLIM), in order to assess the contributions of OXPHOS and glycolysis to energy provision. A novel protocol for the combined measurement of OXPHOS (oxygen consumption rate: OCR) and glycolysis (extracellular acidification rate: ECAR) using the XF Analyzer was developed together with a unique customized AI-based approach for semiautomated processing of 2P-FLIM images. Our study delivers optimized low-HEPES modified human tubal fluid media (mHTF) for sperm handling during pre-analytical and analytical phases, to maintain sperm physiological parameters and optimal OCR, equivalent to OXPHOS. The negative effect of cryopreservation was signified by the deterioration of both bioenergetic pathways represented by modified OCR and ECAR curves and the derived parameters. This was true for normozoospermic as well as samples from TGCT patients, which showed even stronger damage within the respiratory chain compared to the level of glycolytic activity impairment. The impact of cryopreservation and pathology are supported by 2P-FLIM analysis, showing a significant decrease in bound NADH in contrast to unbound NAD(P)H, which reflects decreased metabolic activity in samples from TGCT patients. Our study provides novel insights into the impact of TGCT on sperm bioenergetics and delivers a verified protocol to be used for the assessment of human sperm metabolic activity, which can be a valuable tool for further research and clinical andrology.

Mitochondrial diseases are severe, inherited metabolic disorders that affect the paediatric population. They affect the functioning of mitochondrial oxidative phosphorylation (OXPHOS) apparatus either directly or indirectly. Since mutations in mtDNA are responsible for only 25% of paediatric cases and next-generation sequencing does not always provide a conclusive diagnosis, the biochemical approach still represents a valuable tool in diagnostics. Mitochondrial defects can be identified in tissue biopsies (muscle or skin). However, they also often manifest in peripheral blood cells. We developed a protocol for isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs) from 5 ml of children's blood using Ficoll centrifugation which can be utilised for subsequent functional measurements on thawed samples. Furthermore, we evaluated the diagnostic utility of the optimised high-resolution oxygraphy protocol using digitonin-permeabilized cryopreserved PBMCs on 47 samples from patients with confirmed or suspected mitochondrial disease. Overall, the diagnosis was confirmed in 72% of cases, while the analysis of cryopreserved PBMCs provided a false negative outcome in 13% of cases. Our study demonstrates a sensitive, fast, and non-invasive approach for the diagnostics of various types of mitochondrial disorders, especially those of nuclear genetic origin manifesting in paediatric patients.

Impaired energy production is a hallmark of mitochondrial oxidative phosphorylation (OXPHOS) defects. However, secondary metabolic disturbances also represent an important trigger for pathologies originating from OXPHOS aberrations. We have discovered that cells with OXPHOS deficiencies accumulate triacylglycerols enriched in polyunsaturated fatty acids (PUFAs), which are stored in lipid droplets. Sequestration of PUFAs is a critical component of a broader stress response, which also includes downregulation of cellular desaturases and upregulation of glutathione peroxidase 4 (GPX4). Here, we demonstrate that this mechanism represents a physiologically relevant protective strategy, manifesting in the cells under hypoxia and fibroblasts derived from patients with primary mitochondrial complex IV deficiency. As proof of principle, we observed elevated PUFA- enriched triacylglycerols in the plasma of patients with Myoclonic Epilepsy with Ragged Red Fibres (MERRF). Our findings reveal a novel protective mechanism against ferroptosis, which preserves membrane integrity when mitochondrial respiration is compromised. Highlights ### Competing Interest Statement The authors have declared no competing interest. Czech Science Foundation, https://ror.org/01pv73b02, 21-18993S Ministry of Education Youth and Sports, https://ror.org/037n8p820, LX22NPO5104 Agentura Pro Zdravotnický Výzkum České Republiky, https://ror.org/05m8t8e28, NU22-01-00499 Charles University, 283423/2023 Czech Academy of Sciences, Institute of Physiology, https://ror.org/05xw0ep96, 67985823 Czech Academy of Sciences, Institute of Molecular Genetics, 68378050

COX6B is one of the eleven nuclear-encoded subunits of the human mitochondrial cytochrome c oxidase (cIV). Within the cIV structure, COX6B is located in the intermembrane space-facing region. In mammals, there are two COX6B isoforms: the ubiquitous COX6B1, expressed in all cell types and tissues, and COX6B2 present only in testes and certain types of cancer. Rare COX6B1 missense pathogenic variants have been identified as the cause of childhood-onset mitochondrial encephalopathy associated with cIV deficiency. Despite the relevance of COX6B1 in mitochondrial physiopathology, its molecular role in cIV biogenesis and/or maintenance had not been thoroughly characterized. In spite of the assigned role for COX6B1 as a late incorporation subunit into an almost complete cIV, a COX6B1 human cell line knock-out (KO) exhibited a total loss of cIV. To get a deeper insight into the mechanisms that drive the lack of assembly or destabilization of cIV in the absence of COX6B1, we also used the COX6B1 KO cell background to express an alternative oxidase (AOX) and COX6B1 carrying the R20C and R20H pathogenic variants. These analyses have led us to conclude that the COX6B1 subunit does not only contribute to the stabilization of cIV in the late assembly stages, but is also indispensable for redox-sensitive early cIV assembly steps. In addition, this study has evidenced the incorporation of partially assembled cIV modules directly into supercomplex structures, supporting the "cooperative assembly" model for respiratory chain biogenesis. ### Competing Interest Statement The authors have declared no competing interest.

Neuronal differentiation requires extensive metabolic remodeling to support increased energetic and biosynthetic demands. Here, we present an integrated multi-omics and functional characterization of metabolic transitions during early differentiation of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons using doxycycline-inducible overexpression of neurogenin-2 (NGN2). We analyzed parental iPSCs and induced neurons (iNs) at days 7 and 14 of differentiation, integrating gene expression profiling, label-free quantitative proteomics, high-resolution respirometry, fluorescence lifetime imaging microscopy (FLIM), and 13C₆-glucose metabolic flux analysis. Our data reveal progressive metabolic remodeling associated with neuronal maturation, including enhanced oxidative phosphorylation, increased mitochondrial content, and respiratory capacity. Proteomic analyses showed upregulation of mitochondrial and antioxidant pathways, while FLIM indicated a progressive increase in enzyme-bound NAD(P)H, consistent with a shift toward oxidative metabolism. Notably, 13C₆-glucose tracing revealed delayed labeling of the intracellular pool of fully labeled glucose and tricarboxylic acid cycle metabolites, together with enhanced labeling of pentose phosphate pathway intermediates and glutathione in iNs, indicating a shift toward biosynthetic and antioxidant glucose utilization during differentiation. Despite this enhancement in mitochondrial function, differentiated neurons maintained glycolytic activity, suggesting metabolic flexibility. Our results define the first week of differentiation as a critical window of metabolic specialization and establish NGN2-iPSC-derived cortical neurons as a versatile and well-characterized model system for investigating bioenergetic remodeling during early human neurodevelopment. It provides a robust foundation for mechanistic insights and high-throughput evaluation of metabolic pathways relevant to human disease.

COX6B1 is a nuclear-encoded subunit of the human mitochondrial cytochrome c oxidase (cIV) located in its intermembrane space-facing region. The relevance of COX6B1 in mitochondrial physiopathology was highlighted by the missense pathogenic variants associated with cIV deficiency. Despite the assigned COX6B1 role as a late incorporation subunit, the COX6B1 human cell line KO exhibited a total loss of cIV. To get a deeper insight into the mechanisms driving the lack of cIV assembly or destabilization in the absence of COX6B1, we used the COX6B1 KO cell background to express alternative oxidase and COX6B1 pathogenic variants. These analyses uncovered that the COX6B1 subunit is indispensable for redox-sensitive early cIV assembly steps, besides its contribution to the stabilization of cIV in the late assembly stages. In addition, we have evidenced the incorporation of partially assembled cIV modules directly into supercomplex structures, supporting the "cooperative assembly" model for respiratory chain biogenesis.

Metabolic syndrome is a growing concern in developed societies and due to its polygenic nature, the genetic component is only slowly being elucidated. Common mitochondrial DNA sequence variants have been associated with symptoms of metabolic syndrome and may, therefore, be relevant players in the genetics of metabolic syndrome. We investigate the effect of mitochondrial sequence variation on the metabolic phenotype in conplastic rat strains with identical nuclear but unique mitochondrial genomes, challenged by high-fat diet. We find that the variation in mitochondrial rRNA sequence represents risk factor in the insulin resistance development, which is associated with diacylglycerols accumulation, induced by tissue-specific reduction of the oxidative capacity. These metabolic perturbations stem from the 12S rRNA sequence variation affecting mitochondrial ribosome assembly and translation. Our work demonstrates that physiological variation in mitochondrial rRNA might represent a relevant underlying factor in the progression of metabolic syndrome. Detailed characterization of mtDNA haplotypes uncovers the impact of mitochondrial rRNA variations on mitochondrial translation and oxidative capacity and their metabolic consequences including insulin resistance development induced by high-fat diet.

Succinate dehydrogenase (SDH) connects the tricarboxylic acid (TCA) cycle and the respiratory chain. Mutations in SDH subunits have been associated with tumorigenesis and mitochondrial disease. In this project, we focused on subunit A of SDH (SDHA), primarily associated with inherited mitochondrial disease, and investigated the consequences of its loss or re-expression of mutant variants in HEK cells (SDHA KO). Lack of SDHA led to a downregulation of all SDH subunits and a secondary downregulation of the majority of mitochondrial complex I and IV subunits. Cellular respiratory capacity was severely decreased in the model, SDH-dependent respiration completely abolished and complex I-dependent respiration attenuated, reflecting the downregulation of respiratory chain complexes in general. Finally, the NAD+/NADH ratio was increased in SDHA KO, indicating complex rearrangement of the TCA. It resulted in higher glycolytic activity and lipid accumulation. Supported by Czech Science Foundation (21-18993S), Grant Agency of Charles University (283423) and Czech Health Research Council (NU22-01-00499).

Individual complexes of the mitochondrial oxidative phosphorylation system (OXPHOS) are not linked solely by their function; they also share dependencies at the maintenance/assembly level, where one complex depends on the presence of a different individual complex. Despite the relevance of this "interdependence" behavior for mitochondrial diseases, its true nature remains elusive. To understand the mechanism that can explain this phenomenon, we examined the consequences of the aberration of different OXPHOS complexes in human cells. We demonstrate here that the complete disruption of each of the OXPHOS complexes resulted in a decrease in the complex I (cI) level and that the major reason for this is linked to the downregulation of mitochondrial ribosomal proteins. We conclude that the secondary cI defect is due to mitochondrial protein synthesis attenuation, while the responsible signaling pathways could differ based on the origin of the OXPHOS defect.

Reported testicular loss rates following paediatric testicular torsion often reflect the surgical decision-making process, rather than long-term survival of the testes.We aim to perform systematic analysis and meta-analysis to investigate testicular salvage rates and to assess predictors of long-term viability.Systematic review according to PRISMA guidelines was performed to investigate immediate and long-term (>12 months) testicular loss rates following torsion in the paediatric population. Literature search and study inclusion were performed by two investigators. A study quality score was derived and attributed to each study. Predictors of testicular loss were described. Proportions meta-analysis was performed with random effects modelling, and testing for heterogeneity.Twelve studies were includedm, 6 reporting early orchidectomy rates, and 6 reporting long-term outcomes. Study quality was generally low.The mean early testicular loss rate was 39%, whereas meta-analysis revealed late loss to approach 50%. Predictors of outcomes include prehospital symptom duration, location of presentation, transfer to a tertiary centre, social affluence and use of ultrasound prior to diagnosis or transfer.This study has shown a considerable late testicular loss rate, which must be relayed to families even after testicular salvage. Delay in time to presentation is consistently found to predict poor outcomes.

The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be, respectively, sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain-dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome stalling or readthrough.

The cause of relapse in childhood acute lymphoblastic leukemia (ALL) is often associated with resistance to the standard chemotherapy treatment. The primary goal of this project was to elucidate the resistance mechanism of L-asparaginase (ASNase), one of the crucial drugs used in ALL therapy. The cytotoxic effect of ASNase relies on the depletion of extracellular asparagine (Asn) and glutamine, which is disastrous for leukemic cells since they have minimal activity of de novo synthesis of these amino acids that paradoxically have essential roles in leukemic cells' metabolism. We previously showed that ASNase caused metabolic reprogramming by which leukemic cells escaped the cytostatic effect of the treatment. In the present study, we investigated the role of the main aspects of the in vivo environment on the resistance mechanism of leukemic cells (BCP-ALL cell lines: NALM-6, REH, RS4-11 and SUP-B15 and primary ALL cells). By co-culturing them with mesenchymal stem cells (MSCs) and treating them with ASNase-pretreated culture media, we mimicked the bone marrow matrix and the in vivo half-life of the drug (1.28±0.35 days). The ASNase concentrations used in the ASNase-pretreated culture media were 0.04, 0.4 and 4 IU/mL. In concordance with previous results, we showed that leukemic cell survival was increased in the co-culture model compared to the "classical" in vitro treatment after five days using flow cytometry (NALM-6 - 0.04IU/mL: 17.37±2.8% p<0.0001, 0.4IU/mL: 18±2.8% p<0.0001, 4IU/mL: 25.87±2.3% p<0.0001; REH - 0.04IU/mL: 27.51±3.3% p<0.0001, 0.4IU/mL: 22.06±3.3% p<0.0001, 4IU/mL: 23.96±3.3% p<0.0001; RS4:11 - 0.04IU/mL: 50.07±2.9% p<0.0001, 0.4UI/mL: 60.14±2.9% p<0.0001, 4IU/mL: 34.50±3% p<0.0001; SUP-B15 - 0.04IU/mL: 13.90±2% p<0.0001, 0.4UI/mL: 19.55±2.3% p<0.0001, 4IU/mL: 5.72±2% p<0.05). While ASNase-mediated metabolic rewiring of leukemic cells persisted in both mono and co-culture: reduced glycolysis and increased fatty acid oxidation, the activity of mTOR-regulated biosynthetic pathways differed. The latter pathway was assessed by western blot quantification of the downstream targets of mTOR, S6 and CAD, which are protein and nucleotide synthesis mediators, respectively. In both cultures, the phosphorylated forms of S6 and CAD were inhibited after ASNase treatment (4IU/mL). However, the effect was significantly less profound in the co-culture model (REH: p-S6 (1.826 log (2) fold change, p=0.0043); p-CAD (2.385 log (2) fold change, p=0.0152), NALM-6: p-S6 (1.380 log (2) fold change, p=0.0106); p-CAD (0.78 log (2) fold change, p=n.s)). Similar changes in phospo-S6 were observed in primary BCP-ALL cells isolated from pediatric patients treated with ASNase. As shown by stable isotope tracing, asparagine synthesized de novo and released from MSCs compensated for asparagine depletion (after ASNase administration) and induced resistance of leukemic cells. Asparagine was sufficient to restore protein and nucleotide synthesis and partially rescued the viability of leukemic cells. In conclusion, the presence of MSCs sustains biosynthetic pathways, making leukemic cells more accessible to bioenergetic rewiring, which may counteract ASNase cytotoxicity. These findings present a potential therapeutical target for resistant patients. (Supported by GAČR GA20-27132S and GAUK 1262120)