bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2021‒09‒26
fifty-three papers selected by
Catalina Vasilescu
University of Helsinki
  1. Controlling the topology of mammalian mitochondrial DNA.
  2. Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome.
  3. Assessment of Mitochondrial Protein Topology and Membrane Insertion.
  4. Redox-Mediated Regulation of Mitochondrial Biogenesis, Dynamics, and Respiratory Chain Assembly in Yeast and Human Cells.
  5. Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit.
  6. Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin.
  7. Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples.
  8. Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.
  9. Assessing the Kinetics of Metabolite Uptake and Utilization by Isolated Mitochondria Using Selective Reaction Monitoring Mass Spectrometry (SRM-MS).
  10. Mitochondrial tRNA-Derived Fragments and Their Contribution to Gene Expression Regulation.
  11. Mitochondrial-derived compartments: A "fusel alcohol generator" in yeast?
  12. Heritable shifts in redox metabolites during mitochondrial quiescence reprogramme progeny metabolism.
  13. Macroscopic characteristics of the native liver in children with MPV17-related mitochondrial DNA depletion syndrome: an indication for performing liver transplantation?
  14. A novel compound heterozygous mutation of the MTO1 gene associated with complex oxidative phosphorylation deficiency type 10.
  15. Enhanced glycolysis and GSK3 inactivation promote brain metabolic adaptations following neuronal mitochondrial stress.
  16. Neuronal mitochondrial dysfunction in sporadic amyotrophic lateral sclerosis is developmentally regulated.
  17. Assessment of Mitochondrial Protein Composition and Purity by Mass Spectroscopy.
  18. Inositol serves as a natural inhibitor of mitochondrial fission by directly targeting AMPK.
  19. Mutational Analysis of Mitochondrial tRNA Genes in 200 Patients with Type 2 Diabetes Mellitus.
  20. Skeletal muscle type-specific mitochondrial adaptation to high-fat diet relies on differential autophagy modulation.
  21. High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes.
  22. Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage.
  23. Blood cell respiration rates and mtDNA copy number: a promising tool for the diagnosis of mitochondrial disease.
  24. Physiological cyanide concentrations do not stimulate mitochondrial cytochrome c oxidase activity.
  25. Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma.
  26. Brown adipocyte ATF4 activation improves thermoregulation and systemic metabolism.
  27. A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance.
  28. Whole-Blood Mitochondrial DNA Copies Are Associated With the Prognosis of Acute Respiratory Distress Syndrome After Sepsis.
  29. Development and characterization of a mouse model for Acad9 deficiency.
  30. Measuring the Activity of DNA Repair Enzymes in Isolated Mitochondria.
  31. Leveraging the Cell Ontology to classify unseen cell types.
  32. UQCRC1 engages cytochrome c for neuronal apoptotic cell death.
  33. We are more than what we eat.
  34. Markers for Mitochondrial ROS Status.
  35. NAD+ flux is maintained in aged mice despite lower tissue concentrations.
  36. Coding and Noncoding Variation in LRRK2 and Parkinson's Disease Risk.
  37. Loss of mpv17 affected early embryonic development via mitochondria dysfunction in zebrafish.
  38. iPSC culture expansion selects against putatively actionable mutations in the mitochondrial genome.
  39. Fine-tuned repression of Drp1 driven mitochondrial fission primes a 'stem/progenitor-like state' to support neoplastic transformation.
  40. Clinico-Genetic Spectrum of POLG1 Mutation Carriers from India.
  41. MEGDEL Syndrome and Its Anesthetic Implications.
  42. RBC mitochondria trigger interferon release.
  43. Fission accomplished: Uncovering the role of Drp1 in regulating mitochondrial dysfunction and age-related muscle atrophy.
  44. Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase.
  45. In situ observation of mitochondrial biogenesis as the early event of apoptosis.
  46. Implication of folate deficiency in CYP2U1 loss of function.
  47. Inhibition of ATG3 ameliorates liver steatosis by increasing mitochondrial function.
  48. LIN28A enhances regenerative capacity of human somatic tissue stem cells via metabolic and mitochondrial reprogramming.
  49. Mutations at a split codon in the GTPase-encoding domain of OPA1 cause dominant optic atrophy through different molecular mechanisms.
  50. From variant to function in human disease genetics.
  51. FTO suppresses STAT3 activation and modulates proinflammatory interferon-stimulated gene expression.
  52. The 3D organisation of mitochondria in primate photoreceptors.
  53. Optimising the health-care experiences of babies, children, and young people.
  1. Open Biol. 2021 Sep;11(9): 210168
    Controlling the topology of mammalian mitochondrial DNA.
    Katja E Menger, Alejandro Rodríguez-Luis, James Chapman, Thomas J Nicholls.
      The genome of mitochondria, called mtDNA, is a small circular DNA molecule present at thousands of copies per human cell. MtDNA is packaged into nucleoprotein complexes called nucleoids, and the density of mtDNA packaging affects mitochondrial gene expression. Genetic processes such as transcription, DNA replication and DNA packaging alter DNA topology, and these topological problems are solved by a family of enzymes called topoisomerases. Within mitochondria, topoisomerases are involved firstly in the regulation of mtDNA supercoiling and secondly in disentangling interlinked mtDNA molecules following mtDNA replication. The loss of mitochondrial topoisomerase activity leads to defects in mitochondrial function, and variants in the dual-localized type IA topoisomerase TOP3A have also been reported to cause human mitochondrial disease. We review the current knowledge on processes that alter mtDNA topology, how mtDNA topology is modulated by the action of topoisomerases, and the consequences of altered mtDNA topology for mitochondrial function and human health.
    Keywords:  DNA topology; mitochondria; mitochondrial DNA; mitochondrial disease; topoisomerases
    DOI:  https://doi.org/10.1098/rsob.210168
  2. EMBO J. 2021 Sep 20. e108648
    Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome.
    Sergio Guerrero-Castillo, Joeri van Strien, Ulrich Brandt, Susanne Arnold.
      So-called ρ0 cells lack mitochondrial DNA and are therefore incapable of aerobic ATP synthesis. How cells adapt to survive ablation of oxidative phosphorylation remains poorly understood. Complexome profiling analysis of ρ0 cells covered 1,002 mitochondrial proteins and revealed changes in abundance and organization of numerous multiprotein complexes including previously not described assemblies. Beyond multiple subassemblies of complexes that would normally contain components encoded by mitochondrial DNA, we observed widespread reorganization of the complexome. This included distinct changes in the expression pattern of adenine nucleotide carrier isoforms, other mitochondrial transporters, and components of the protein import machinery. Remarkably, ablation of mitochondrial DNA hardly affected the complexes organizing cristae junctions indicating that the altered cristae morphology in ρ0 mitochondria predominantly resulted from the loss of complex V dimers required to impose narrow curvatures to the inner membrane. Our data provide a comprehensive resource for in-depth analysis of remodeling of the mitochondrial complexome in response to respiratory deficiency.
    Keywords:  OXPHOS; complexome profiling; mitochondria; mtDNA; rho0 cells
    DOI:  https://doi.org/10.15252/embj.2021108648
  3. Methods Mol Biol. 2022 ;2363 165-181
    Assessment of Mitochondrial Protein Topology and Membrane Insertion.
    Kerstin Schäfer, Carina Engstler, Korbinian Dischinger, Chris Carrie.
      Analyzing the membrane integrity and topology of a mitochondrial protein is essential for truly understanding its function. In this chapter, we demonstrate how to analyze mitochondrial membrane proteins using both an immunological-based assay and an in vivo self-assembling GFP approach. First, immunological approaches to investigate the solubility or membrane association of a protein within mitochondria are described. With this method, we demonstrate how the topology of soluble domains of a membrane-integrated protein can be determined. Using protein-specific antibodies, the localization of the soluble domains of a protein are analyzed by a proteolytic-cleavage approach using proteinase K in mitochondria, outer membrane-ruptured mitochondria, and solubilized mitochondrial membranes. In a second approach, we determine the topology of plant mitochondrial proteins using an in vivo self-assembling GFP localization approach.
    Keywords:  Carbonate extraction; In vivo GFP localization; Membrane integration; Membrane protein topology; Membrane solubilization; Mitochondrial membrane proteins; Mitoplasts; Osmotic swelling; Proteinase K digestion; Self-assembling GFP
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_13
  4. Front Cell Dev Biol. 2021 ;9 720656
    Redox-Mediated Regulation of Mitochondrial Biogenesis, Dynamics, and Respiratory Chain Assembly in Yeast and Human Cells.
    Stefan Geldon, Erika Fernández-Vizarra, Kostas Tokatlidis.
      Mitochondria are double-membrane organelles that contain their own genome, the mitochondrial DNA (mtDNA), and reminiscent of its endosymbiotic origin. Mitochondria are responsible for cellular respiration via the function of the electron oxidative phosphorylation system (OXPHOS), located in the mitochondrial inner membrane and composed of the four electron transport chain (ETC) enzymes (complexes I-IV), and the ATP synthase (complex V). Even though the mtDNA encodes essential OXPHOS components, the large majority of the structural subunits and additional biogenetical factors (more than seventy proteins) are encoded in the nucleus and translated in the cytoplasm. To incorporate these proteins and the rest of the mitochondrial proteome, mitochondria have evolved varied, and sophisticated import machineries that specifically target proteins to the different compartments defined by the two membranes. The intermembrane space (IMS) contains a high number of cysteine-rich proteins, which are mostly imported via the MIA40 oxidative folding system, dependent on the reduction, and oxidation of key Cys residues. Several of these proteins are structural components or assembly factors necessary for the correct maturation and function of the ETC complexes. Interestingly, many of these proteins are involved in the metalation of the active redox centers of complex IV, the terminal oxidase of the mitochondrial ETC. Due to their function in oxygen reduction, mitochondria are the main generators of reactive oxygen species (ROS), on both sides of the inner membrane, i.e., in the matrix and the IMS. ROS generation is important due to their role as signaling molecules, but an excessive production is detrimental due to unwanted oxidation reactions that impact on the function of different types of biomolecules contained in mitochondria. Therefore, the maintenance of the redox balance in the IMS is essential for mitochondrial function. In this review, we will discuss the role that redox regulation plays in the maintenance of IMS homeostasis as well as how mitochondrial ROS generation may be a key regulatory factor for ETC biogenesis, especially for complex IV.
    Keywords:  MIA; ROS; biogenesis; mitochondria; protein import; redox signaling; respiratory chain assembly
    DOI:  https://doi.org/10.3389/fcell.2021.720656
  5. NPJ Regen Med. 2021 Sep 24. 6(1): 58
    Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit.
    Elad Jacoby, Moriya Ben Yakir-Blumkin, Shiri Blumenfeld-Kan, Yehuda Brody, Amilia Meir, Naomi Melamed-Book, Tina Napso, Gat Pozner, Esraa Saadi, Ayelet Shabtay-Orbach, Natalie Yivgi-Ohana, Noa Sher, Amos Toren.
      Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.
    DOI:  https://doi.org/10.1038/s41536-021-00167-7
  6. Methods Mol Biol. 2022 ;2363 77-84
    Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin.
    Allan G Rasmusson, Ian Max Møller, Susanne Widell.
      We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD+-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.
    Keywords:  Alamethicin; BY-2 cells; Electron transport chain; Mitochondria; Tricarboxylic acid cycle
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_7
  7. J Biol Chem. 2021 Sep 17. pii: S0021-9258(21)01006-1. [Epub ahead of print] 101204
    Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples.
    Fariha Ansari, Belem Yoval, Zoya Niatsetskaya, Sergey Sosunov, Anna Stepanova, Christian Garcia, Edward Owusu-Ansah, Vadim Ten, Ilka Wittig, Alexander Galkin.
      Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex (KGDHC), in any preparation. We show that the complex I content is 19±1 pmol/mg of protein in the brain mitochondria, while varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover which was calculated as 104 min-1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. KGDHC content was determined to be 65±5 and 123±9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single HEK293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.
    Keywords:  enzyme turnover; flavin adenine dinucleotide; flavin mononucleotide; fluorescence; ketoglutarate dehydrogenase complex; mitochondrial respiratory chain complex I; stoichiometry
    DOI:  https://doi.org/10.1016/j.jbc.2021.101204
  8. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00688-2. [Epub ahead of print]81(18): 3786-3802.e13
    Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.
    Max-Hinderk Schuler, Alyssa M English, Tianyao Xiao, Thane J Campbell, Janet M Shaw, Adam L Hughes.
      Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.
    Keywords:  MDC; Tom70; amino acid; lysosome; mitochondria; nutrient carrier; vacuole
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.021
  9. Methods Mol Biol. 2022 ;2363 85-100
    Assessing the Kinetics of Metabolite Uptake and Utilization by Isolated Mitochondria Using Selective Reaction Monitoring Mass Spectrometry (SRM-MS).
    Xuyen Le, A Harvey Millar, Chun Pong Lee.
      Transport of tricarboxylic acid (TCA) cycle substrates across mitochondrial membranes and their subsequent oxidative decarboxylation in the matrix provide reductants for respiration-coupled ATP synthesis. These processes are typically assessed together through the ability of mitochondria to consume oxygen or release carbon dioxide, however, this approach fails to assess or separate the complexity of transport and the subsequent metabolism of substrates and products. In this chapter, we provide a strategy for simultaneously measuring substrate transport and utilization by isolated mitochondria using a mass spectrometry-based technique. The results of cofeeding of isolated mitochondria with unlabeled malate and uniformly labeled pyruvate is used as an example. Mitochondria fed with substrates are separated from the extramitochondrial space by centrifugation through a single layer of silicone oil. Analysis of mitochondrial pellet and reaction supernatant enable quantitation of substrate import and product export. This method also allows an estimation of the contribution of different enzymatic pathways to the formation of a specific product. This assay opens opportunities to verify carrier functions in organello and to identify the substrate preferences of mitochondrial transporters of unknown function using targeted and/or untargeted metabolomics approaches.
    Keywords:  Metabolism; Mitochondria; Selective reaction monitoring (SRM) mass spectrometry; Silicone oil centrifugation; Transport
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_8
  10. Front Physiol. 2021 ;12 729452
    Mitochondrial tRNA-Derived Fragments and Their Contribution to Gene Expression Regulation.
    Athanasios-Nasir Shaukat, Eleni G Kaliatsi, Vassiliki Stamatopoulou, Constantinos Stathopoulos.
      Mutations in human mitochondrial tRNAs (mt-tRNAs) are responsible for several and sometimes severe clinical phenotypes, classified among mitochondrial diseases. In addition, post-transcriptional modifications of mt-tRNAs in correlation with several stress signals can affect their stability similarly to what has been described for their nuclear-encoded counterparts. Many of the perturbations related to either point mutations or aberrant modifications of mt-tRNAs can lead to specific cleavage and the production of mitochondrial tRNA-derived fragments (mt-tRFs). Although mt-tRFs have been detected in several studies, the exact biogenesis steps and biological role remain, to a great extent, unexplored. Several mt-tRFs are produced because of the excessive oxidative stress which predominantly affects mitochondrial DNA integrity. In addition, mt-tRFs have been detected in various diseases with possible detrimental consequences, but also their production may represent a response mechanism to external stimuli, including infections from pathogens. Finally, specific point mutations on mt-tRNAs have been reported to impact the pool of the produced mt-tRFs and there is growing evidence suggesting that mt-tRFs can be exported and act in the cytoplasm. In this review, we summarize current knowledge on mitochondrial tRNA-deriving fragments and their possible contribution to gene expression regulation.
    Keywords:  mitochondria; mitochondrial tRNA-derived fragments; mitochondrial tRNAs; ncRNAs; tRNA-derived fragments
    DOI:  https://doi.org/10.3389/fphys.2021.729452
  11. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00712-7. [Epub ahead of print]81(18): 3670-3671
    Mitochondrial-derived compartments: A "fusel alcohol generator" in yeast?
    Tim König, Heidi M McBride.
      Schuler et al. (2021) demonstrate that mitochondrial-derived compartments protect cells from amino acid toxicity by activation of amino acid catabolism through the Ehrlich pathway, thus highlighting the incredible plasticity of mitochondria in rewiring cellular metabolism.
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.032
  12. Nat Metab. 2021 Sep;3(9): 1259-1274
    Heritable shifts in redox metabolites during mitochondrial quiescence reprogramme progeny metabolism.
    Helin Hocaoglu, Lei Wang, Mengye Yang, Sibiao Yue, Matthew Sieber.
      Changes in maternal diet and metabolic defects in mothers can profoundly affect health and disease in their progeny. However, the biochemical mechanisms that induce the initial reprogramming events at the cellular level have remained largely unknown owing to limitations in obtaining pure populations of quiescent oocytes. Here, we show that the precocious onset of mitochondrial respiratory quiescence causes a reprogramming of progeny metabolic state. The premature onset of mitochondrial respiratory quiescence drives the lowering of Drosophila oocyte NAD+ levels. NAD+ depletion in the oocyte leads to reduced methionine cycle production of the methyl donor S-adenosylmethionine in embryos and lower levels of histone H3 lysine 27 trimethylation, resulting in enhanced intestinal lipid metabolism in progeny. In addition, we show that triggering cellular quiescence in mammalian cells and chemotherapy-resistant human cancer cell models induces cellular reprogramming events identical to those seen in Drosophila, suggesting a conserved metabolic mechanism in systems reliant on quiescent cells.
    DOI:  https://doi.org/10.1038/s42255-021-00450-3
  13. Liver Transpl. 2021 Sep 18.
    Macroscopic characteristics of the native liver in children with MPV17-related mitochondrial DNA depletion syndrome: an indication for performing liver transplantation?
    Mureo Kasahara, Seisuke Sakamoto, Akinari Fukuda, Reiko Horikawa, Reiko Ito, Hajime Uchida, Yusuke Yanagi, Seiichi Shimizu, Toshimasa Nakao, Kotaro Mimori, Chizuko Haga, Andrea Schlegel, Akira Ohtake, Masaru Shimura, Takuya Fushimi, Keiko Ichimoto, Ayako Matsunaga, Kei Murayama.
      Mitochondrial respiratory chain disorders (MRCDs) are rare conditions with various clinical manifestations that depend on the genetic type, with subsequent severity of the liver disease. Clinical presentations include acute liver failure or chronic liver failure with liver cirrhosis, which are cruelly progressive and fatal. We report six cases of LT for acute liver failure diagnosed pathogenic MPV17 variants. Three out of six cases were siblings with the MPV17-mutation (c.148C>T, p.R50W / c.149G>A; p.R50Q), also known as the childhood form of Navajo neurohepatopathy. Those three patients underwent LT for liver cirrhosis at the ages of 5, 7, and 7 years, respectively. All three recipients are currently doing well without any neurological sequelae. In contrast, the three additional patients that presented with an acute liver failure and subsequent diagnosis of mutation in MPV17 (homozygous c.451dupC: p.L151PfsX39; homozygous c.451dupC : p.L151PfsX39; c.451dupC : p.L151PfsX39 / c.71-2_79del11ins4), underwent LT at the ages of 4 months, 11 months, and 1 year, respectively. All three recipients died from pulmonary hypertension following successful LT. Interestingly, the intraoperative macroscopic findings of the native liver at the time of LT revealed distinctive, microgranular nodules and liver fibrosis with severe steatosis ("avocado-like" liver). LT might be contraindicated in MPV17 patients with c.451dupC mutation due to the extremely poor prognosis caused by fatal progressive pulmonary hypertension. The identification of the typical macroscopic finding ("avocado-like"), seen in such pediatric transplant candidates with acute liver failure, might be helpful to provide an early diagnosis of MPV17-related c.451dupC mutation.
    Keywords:  acute liver failure; living donor liver transplantation; metabolic liver disease; mitochondrial DNA depletion syndrome; mitochondrial hepatopathy; mitochondrial respiratory chain disorder; pediatric liver transplantation
    DOI:  https://doi.org/10.1002/lt.26296
  14. Clin Chim Acta. 2021 Sep 18. pii: S0009-8981(21)00325-9. [Epub ahead of print]
    A novel compound heterozygous mutation of the MTO1 gene associated with complex oxidative phosphorylation deficiency type 10.
    Qing Luo, Xia Wen, Jiahong Zhou, Yang Chen, Zhiyu Lv, Xing Shen, Jinbo Liu.
      BACKGROUND: The mitochondrial tRNA translation optimization 1 (MTO1) gene, which is closely related to defective mitochondrial oxidative phosphorylation, is an evolutionarily conserved protein expressed in high energy-demanding tissues and is associated with complex oxidative phosphorylation deficiency type 10 (COXPD10) in humans. Related cases and studies are still scarce and have not been reported in the Chinese region.MATERIALS AND METHODS: Detailed clinical assessment was applied to the patient. Based on next-generation sequencing technology, we performed whole-exome sequencing of the patient and the parents. Sanger sequencing was used for validation. Bioinformatics software and protein simulations were used to predict the pathogenicity of the variants.
    RESULTS: The patient was diagnosed with a possible association with mitochondrial disease according to the clinical manifestations and physical examination. A novel frameshift mutation c.344delA (p. Asn115Thrfs*11) and a novel point mutation c.1055C>T (p. Thr352Met) in the MTO1 gene were identified. They were found to cause abnormal changes in amino acids and the protein by biochemical tools, indicating it may be pathogenic.
    CONCLUSION: We present two novel and possibly pathogenic variants in the MTO1 gene in a Chinese Han family.
    Keywords:  Autosomal recessive inheritance; COXPD10; MOT1 gene; Mitochondrial disorders
    DOI:  https://doi.org/10.1016/j.cca.2021.09.014
  15. Hum Mol Genet. 2021 Sep 24. pii: ddab282. [Epub ahead of print]
    Enhanced glycolysis and GSK3 inactivation promote brain metabolic adaptations following neuronal mitochondrial stress.
    Sofia Garcia, Amy Saldana-Caboverde, Mir Anwar, Ami Pravinkant Raval, Nadee Nissanka, Milena Pinto, Carlos Torres Moraes, Francisca Diaz.
      We analyzed early brain metabolic adaptations in response to mitochondrial dysfunction in a mouse model of mitochondrial encephalopathy with complex IV deficiency (neuron specific COX10 KO). In this mouse model the onset of the mitochondrial defect did not coincide with immediate cell death suggesting early adaptive metabolic responses to compensate for the energetic deficit. Metabolomic analysis in the knockout mice revealed increased levels of glycolytic and pentose phosphate pathway intermediates, amino acids and lysolipids. Glycolysis was modulated by enhanced activity of glycolytic enzymes, and not by their overexpression, suggesting the importance of post-translational modifications in the adaptive response. GSK3 inactivation was the most upstream regulation identified, implying that it is a key event in this adaptive mechanism. Because neurons are thought not to rely on glycolysis for ATP production in normal conditions, our results indicate that neurons still maintain their ability to upregulate this pathway when under mitochondrial respiration stress.
    Keywords:  mitochondrial diseasescomplex IV deficiencyneuron specific COX10 KOmetabolic adaptationposttranslational modificationsglycolysisGSK3
    DOI:  https://doi.org/10.1093/hmg/ddab282
  16. Sci Rep. 2021 Sep 23. 11(1): 18916
    Neuronal mitochondrial dysfunction in sporadic amyotrophic lateral sclerosis is developmentally regulated.
    Tanisha Singh, Yuanyuan Jiao, Lisa M Ferrando, Svitlana Yablonska, Fang Li, Emily C Horoszko, David Lacomis, Robert M Friedlander, Diane L Carlisle.
      Amyotrophic lateral sclerosis is an adult-onset neurodegenerative disorder characterized by loss of motor neurons. Mitochondria are essential for neuronal survival but the developmental timing and mechanistic importance of mitochondrial dysfunction in sporadic ALS (sALS) neurons is not fully understood. We used human induced pluripotent stem cells and generated a developmental timeline by differentiating sALS iPSCs to neural progenitors and to motor neurons and comparing mitochondrial parameters with familial ALS (fALS) and control cells at each developmental stage. We report that sALS and fALS motor neurons have elevated reactive oxygen species levels, depolarized mitochondria, impaired oxidative phosphorylation, ATP loss and defective mitochondrial protein import compared with control motor neurons. This phenotype develops with differentiation into motor neurons, the affected cell type in ALS, and does not occur in the parental undifferentiated sALS cells or sALS neural progenitors. Our work demonstrates a developmentally regulated unifying mitochondrial phenotype between patient derived sALS and fALS motor neurons. The occurrence of a unifying mitochondrial phenotype suggests that mitochondrial etiology known to SOD1-fALS may applicable to sALS. Furthermore, our findings suggest that disease-modifying treatments focused on rescue of mitochondrial function may benefit both sALS and fALS patients.
    DOI:  https://doi.org/10.1038/s41598-021-97928-7
  17. Methods Mol Biol. 2022 ;2363 121-152
    Assessment of Mitochondrial Protein Composition and Purity by Mass Spectroscopy.
    Anqiang Tang, Yimin Wang, Nicolas L Taylor.
      While the routine mitochondrial extraction and isolation protocols have not fundamentally changed since the introduction of density gradients, the approaches we use to examine the proteome have. The initial characterisation of mitochondrial proteomes was carried out using two-dimensional gel electrophoresis in 2001 and gel spot mass spectrometry have now largely been superseded as the throughput and sensitivity of commercial mass spectrometers increases. Whist many of these early studies established the components of the mitochondrial proteome, as gels were replaced by gel free approaches the numbers of confirmed components rapidly increased. In this chapter we present gel-based approaches for the separation and concentration of mitochondrial proteins for their characterization by mass spectrometry. We also describe two gel-free approaches which can be used to quantity the degree of contamination arising during the isolation of mitochondria. These approaches are equally suitable for studies comparing one treatment to another.
    Keywords:  Gel fractionation; Gel purification; Mass spectrometry; Mitochondria; Proteomics; Selected reaction monitoring (SRM) mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_11
  18. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00692-4. [Epub ahead of print]81(18): 3803-3819.e7
    Inositol serves as a natural inhibitor of mitochondrial fission by directly targeting AMPK.
    Che-Chia Hsu, Xian Zhang, Guihua Wang, Weina Zhang, Zhen Cai, Bo-Syong Pan, Haiwei Gu, Chuan Xu, Guoxiang Jin, Xiangshang Xu, Rajesh Kumar Manne, Yan Jin, Wei Yan, Jingwei Shao, Tingjin Chen, Emily Lin, Amit Ketkar, Robert Eoff, Zhi-Gang Xu, Zhong-Zhu Chen, Hong-Yu Li, Hui-Kuan Lin.
      Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.
    Keywords:  AMP; AMPK; IMPA1; energy stress; glucose deprivation; inosiotl sensor; inositol; inositol/AMP ratio; mitochondrial fission; mitocondrial dynamics
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.025
  19. Int J Gen Med. 2021 ;14 5719-5735
    Mutational Analysis of Mitochondrial tRNA Genes in 200 Patients with Type 2 Diabetes Mellitus.
    Liangyan Lin, Dongdong Zhang, Qingsong Jin, Yaqin Teng, Xiaoyan Yao, Tiantian Zhao, Xinmiao Xu, Yongjun Jin.
      Objective: Previous studies showed that variants in mitochondrial DNA (mtDNA) are associated with type 2 diabetes mellitus (T2DM). However, the relationships between mitochondrial tRNA (mt-tRNA) variants and T2DM remain poorly understood.Methods: In this study, we performed a mutational screening of 22 mt-tRNA genes in a cohort of 200 Han Chinese subjects with T2DM and 200 control subjects through PCR-Sanger sequencing. The identified mt-tRNA variants were assessed for their pathogenicity via the phylogenetic approach, structural and functional analysis. Furthermore, two Han Chinese pedigrees with maternally inherited diabetes and deafness (MIDD) were reported by clinical and genetic assessments.
    Results: A total of 49 genetic variants in mt-tRNA genes were identified; among them, 31 variants (17 pathogenic/likely pathogenic) were absent in controls, located at extremely conserved nucleotides, may have potential structural and functional significance, thereby considered to be T2DM-associated variants. In addition, sequence analysis of entire mitochondrial genomes of the matrilineal relatives from two MIDD pedigrees revealed the occurrence of tRNALeu(UUR) A3243G and T3290C mutations, as well as sets of polymorphisms belonging to mitochondrial haplogroups F2 and D4. However, the lack of any functional variants in connexin 26 gene (GJB2) and tRNA 5-methylaminomethyl-2-thiouridylate (TRMU) suggested that nuclear genes may not play active roles in clinical expression of MIDD in these pedigrees.
    Conclusion: Our data indicated that mt-tRNA variants were associated with T2DM, screening for mt-tRNA pathogenic mutations was recommended for early detection and prevention of mitochondrial diabetes.
    Keywords:  Chinese population; mitochondrial tRNA; type 2 diabetes mellitus; variants
    DOI:  https://doi.org/10.2147/IJGM.S330973
  20. FASEB J. 2021 Oct;35(10): e21933
    Skeletal muscle type-specific mitochondrial adaptation to high-fat diet relies on differential autophagy modulation.
    Pablo E Morales, Matías Monsalves-Álvarez, Satya Murthy Tadinada, Matthew P Harris, Andrea Ramírez-Sagredo, Jafet Ortiz-Quintero, Mayarling Francisca Troncoso, Nicole De Gregorio, Ximena Calle, Renata O Pereira, Vitor A Lira, Alejandra Espinosa, E Dale Abel, Sergio Lavandero.
      In obesity, skeletal muscle mitochondrial activity changes to cope with increased nutrient availability. Autophagy has been proposed as an essential mechanism involved in the regulation of mitochondrial metabolism. Still, the contribution of autophagy to mitochondrial adaptations in skeletal muscle during obesity is unknown. Here, we show that in response to high-fat diet (HFD) feeding, distinct skeletal muscles in mice exhibit differentially regulated autophagy that may modulate mitochondrial activity. We observed that after 4 and 40 weeks of high-fat diet feeding, OXPHOS subunits and mitochondrial DNA content increased in the oxidative soleus muscle. However, in gastrocnemius muscle, which has a mixed fiber-type composition, the mitochondrial mass increased only after 40 weeks of HFD feeding. Interestingly, fatty acid-supported mitochondrial respiration was enhanced in gastrocnemius, but not in soleus muscle after a 4-week HFD feeding. This increased metabolic profile in gastrocnemius was paralleled by preserving autophagy flux, while autophagy flux in soleus was reduced. To determine the role of autophagy in this differential response, we used an autophagy-deficient mouse model with partial deletion of Atg7 specifically in skeletal muscle (SkM-Atg7+/- mice). We observed that Atg7 reduction resulted in diminished autophagic flux in skeletal muscle, alongside blunting the HFD-induced increase in fatty acid-supported mitochondrial respiration observed in gastrocnemius. Remarkably, SkM-Atg7+/- mice did not present increased mitochondria accumulation. Altogether, our results show that HFD triggers specific mitochondrial adaptations in skeletal muscles with different fiber type compositions, and that Atg7-mediated autophagy modulates mitochondrial respiratory capacity but not its content in response to an obesogenic diet.
    Keywords:  Atg7; fatty acids; obesity; skeletal muscle fiber
    DOI:  https://doi.org/10.1096/fj.202001593RR
  21. Methods Mol Biol. 2022 ;2363 111-119
    High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes.
    Lilian Vincis Pereira Sanglard, Catherine Colas des Francs-Small.
      Blue native electrophoresis (BN-PAGE) is a highly resolutive method suited to the study of high molecular weight protein complexes between 100 and >3000 kDa. One of the drawbacks of this method is that it is very time-consuming and requires high quantities of purified organelles. Here we describe a high throughput BN-PAGE method allowing to screen libraries of plants potentially altered in respiratory metabolism.
    Keywords:  Blue Native PAGE; Immunoblots; Mitochondria; Respiratory complexes
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_10
  22. J Biol Chem. 2021 Sep 21. pii: S0021-9258(21)01027-9. [Epub ahead of print] 101224
    Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage.
    Kristina Bubb, Tatjana Holzer, Janica L Nolte, Marcus Krüger, Richard Wilson, Ursula Schlötzer-Schrehardt, Jürgen Brinckmann, Janine Altmüller, Attila Aszodi, Lutz Fleischhauer, Hauke Clausen-Schaumann, Kristina Probst, Bent Brachvogel.
      Energy metabolism and extracellular matrix function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single cell RNA-seq (scRNA-seq) analysis to uncover the importance of the mitochondrial respiratory chain for extracellular matrix homeostasis in mature cartilage. This tissue produces large amounts of a specialized extracellular matrix to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocytes. These changes were associated with alterations in extracellular matrix composition, a shift in collagen/non-collagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.
    Keywords:  Extracellular matrix; MMP10; THBS1; atomic force microscopy; matrisome; matrix metalloproteinase (MMP); mitochondria; mitochondrial respiratory chain; single cell RNA sequencing; transcriptomics
    DOI:  https://doi.org/10.1016/j.jbc.2021.101224
  23. Mitochondrion. 2021 Sep 15. pii: S1567-7249(21)00122-7. [Epub ahead of print]
    Blood cell respiration rates and mtDNA copy number: a promising tool for the diagnosis of mitochondrial disease.
    Martina Alonso, Cristina Zabala, Santiago Mansilla, Laureana De Brun, Jennyfer Martínez, Mariela Garau, Gabriela Rivas, Cecilia Acosta, Daniela Lens, Alfredo Cerisola, Martín Graña, Hugo Naya, Rodrigo Puentes, Lucía Spangenberg, Víctor Raggio, Aída Lemes, Laura Castro, Celia Quijano.
      Human mitochondrial diseases are a group of heterogeneous diseases caused by defects in oxidative phosphorylation, due to mutations in mitochondrial (mtDNA) or nuclear DNA. The diagnosis of mitochondrial disease is challenging since mutations in multiple genes can affect mitochondrial function, there is considerable clinical variability and a poor correlation between genotype and phenotype. Herein we assessed mitochondrial function in peripheral blood mononuclear cells (PBMCs) and platelets from volunteers without known metabolic pathology and patients with mitochondrial disease. Oxygen consumption rates were evaluated and respiratory parameters indicative of mitochondrial function were obtained. A negative correlation between age and respiratory parameters of PBMCs from control individuals was observed. Surprisingly, respiratory parameters of PBMCs normalized by cell number were similar in patients and young controls. Considering possible compensatory mechanisms, mtDNA copy number in PBMCs was quantified and an increase was found in patients with respect to controls. Hence, respiratory parameters normalized by mtDNA copy number were determined, and in these conditions a decrease in maximum respiration rate and spare respiratory capacity was observed in patients relative to control individuals. In platelets no decay was seen in mitochondrial function with age, while a reduction in basal, ATP-independent and ATP-dependent respiration normalized by cell number was detected in patients compared to control subjects. In summary, our results offer promising perspectives regarding the assessment of mitochondrial function in blood cells for the diagnosis mitochondrial disease, minimizing the need for invasive procedures such as muscle biopsies, and for following disease progression and response to treatments.
    Keywords:  PBMC; aging; bioenergetics; mitochondrial disease; mtDNA; platelets
    DOI:  https://doi.org/10.1016/j.mito.2021.09.004
  24. Proc Natl Acad Sci U S A. 2021 09 28. pii: e2112373118. [Epub ahead of print]118(39):
    Physiological cyanide concentrations do not stimulate mitochondrial cytochrome c oxidase activity.
    Flavia Giamogante, Tito Calì, Francesco Malatesta.
      
    DOI:  https://doi.org/10.1073/pnas.2112373118
  25. Front Genet. 2021 ;12 721864
    Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma.
    Grzegorz L Ciesielski, Shalom Kim, Carolina de Bovi Pontes, Laurie S Kaguni.
      The maintenance of the mitochondrial genome depends on a suite of nucleus-encoded proteins, among which the catalytic subunit of the mitochondrial replicative DNA polymerase, Pol γα, plays a pivotal role. Mutations in the Pol γα-encoding gene, POLG, are a major cause of human mitochondrial disorders. Here we present a study of direct and functional interactions of Pol γα with the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB coordinates the activity of the enzymes at the DNA replication fork. However, the mechanism of this functional relationship is elusive, and no direct interactions between the replicative factors have been identified to date. This contrasts strikingly with the extensive interactomes of SSB proteins identified in other homologous replication systems. Here we show for the first time that mtSSB binds Pol γα directly, in a DNA-independent manner. This interaction is strengthened in the absence of the loop 2.3 structure in mtSSB, and is abolished upon preincubation with Pol γβ. Together, our findings suggest that the interaction between mtSSB and polymerase gamma holoenzyme (Pol γ) involves a balance between attractive and repulsive affinities, which have distinct effects on DNA synthesis and exonucleolysis.
    Keywords:  DNA polymerase gamma; intermolecular interactions; mitochondrial DNA replication; mitochondrial biogenesis; mitochondrial single-stranded DNA-binding protein
    DOI:  https://doi.org/10.3389/fgene.2021.721864
  26. Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01195-5. [Epub ahead of print]36(12): 109742
    Brown adipocyte ATF4 activation improves thermoregulation and systemic metabolism.
    Esther Paulo, Yun Zhang, Ruchi Masand, Tony L Huynh, Youngho Seo, Danielle L Swaney, Margaret Soucheray, Erica Stevenson, David Jimenez-Morales, Nevan J Krogan, Biao Wang.
      Cold-induced thermogenesis in endotherms demands adaptive thermogenesis fueled by mitochondrial respiration and Ucp1-mediated uncoupling in multilocular brown adipocytes (BAs). However, dietary regulation of thermogenesis in BAs isn't fully understood. Here, we describe that the deficiency of Leucine-rich pentatricopeptide repeat containing-protein (Lrpprc) in BAs reduces mtDNA-encoded ETC gene expression, causes ETC proteome imbalance, and abolishes the mitochondria-fueled thermogenesis. BA-specific Lrpprc knockout mice are cold resistant in a 4°C cold-tolerance test in the presence of food, which is accompanied by the activation of transcription factor 4 (ATF4) and proteome turnover in BAs. ATF4 activation genetically by BA-specific ATF4 overexpression or physiologically by a low-protein diet feeding can improve cold tolerance in wild-type and Ucp1 knockout mice. Furthermore, ATF4 activation in BAs improves systemic metabolism in obesogenic environment regardless of Ucp1's action. Therefore, our study reveals a diet-dependent but Ucp1-independent thermogenic mechanism in BAs that is relevant to systemic thermoregulation and energy homeostasis.
    Keywords:  ATF4; brown adipocyte; thermogenesis
    DOI:  https://doi.org/10.1016/j.celrep.2021.109742
  27. Sci Adv. 2021 Sep 24. 7(39): eabi7514
    A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance.
    Giulia Rossetti, Judith A Ermer, Maike Stentenbach, Stefan J Siira, Tara R Richman, Dusanka Milenkovic, Kara L Perks, Laetitia A Hughes, Emma Jamieson, Gulibaikelamu Xiafukaiti, Natalie C Ward, Satoru Takahashi, Nicola Gray, Helena M Viola, Livia C Hool, Oliver Rackham, Aleksandra Filipovska.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/sciadv.abi7514
  28. Front Immunol. 2021 ;12 737369
    Whole-Blood Mitochondrial DNA Copies Are Associated With the Prognosis of Acute Respiratory Distress Syndrome After Sepsis.
    Tamara Hernández-Beeftink, Beatriz Guillen-Guio, Héctor Rodríguez-Pérez, Itahisa Marcelino-Rodríguez, Jose M Lorenzo-Salazar, Almudena Corrales, Miryam Prieto-González, Aurelio Rodríguez-Pérez, Demetrio Carriedo, Jesús Blanco, Alfonso Ambrós, Elena González-Higueras, Nancy G Casanova, Manuel González-Garay, Elena Espinosa, Arturo Muriel, David Domínguez, Abelardo García de Lorenzo, José M Añón, Marina Soro, Javier Belda, Joe G N Garcia, Jesús Villar, Carlos Flores.
      Acute respiratory distress syndrome (ARDS) is an inflammatory process of the lungs that develops primarily in response to pulmonary or systemic sepsis, resulting in a disproportionate death toll in intensive care units (ICUs). Given its role as a critical activator of the inflammatory and innate immune responses, previous studies have reported that an increase of circulating cell-free mitochondrial DNA (mtDNA) is a biomarker for fatal outcome in the ICU. Here we analyzed the association of whole-blood mtDNA (wb-mtDNA) copies with 28-day survival from sepsis and sepsis-associated ARDS. We analyzed mtDNA data from 687 peripheral whole-blood samples within 24 h of sepsis diagnosis from unrelated Spanish patients with sepsis (264 with ARDS) included in the GEN-SEP study. The wb-mtDNA copies were obtained from the array intensities of selected probes, with 100% identity with mtDNA and with the largest number of mismatches with the nuclear sequences, and normalized across the individual-probe intensities. We used Cox regression models for testing the association with 28-day survival. We observed that wb-mtDNA copies were significantly associated with 28-day survival in ARDS patients (hazard ratio = 3.65, 95% confidence interval = 1.39-9.59, p = 0.009) but not in non-ARDS patients. Our findings support that wb-mtDNA copies at sepsis diagnosis could be considered an early prognostic biomarker in sepsis-associated ARDS patients. Future studies will be needed to evaluate the mechanistic links of this observation with the pathogenesis of ARDS.
    Keywords:  ARDS; DAMPs; mitochondria; mtDNA; survival; whole blood
    DOI:  https://doi.org/10.3389/fimmu.2021.737369
  29. Mol Genet Metab. 2021 Sep 14. pii: S1096-7192(21)00778-2. [Epub ahead of print]
    Development and characterization of a mouse model for Acad9 deficiency.
    Andrew Sinsheimer, Al-Walid Mohsen, Kailyn Bloom, Anuradha Karunanidhi, Sivakama Bharathi, Yijen L Wu, Manuel Schiff, Yudong Wang, Eric S Goetzman, Lina Ghaloul-Gonzalez, Jerry Vockley.
      Acyl CoA Dehydrogenase 9 (ACAD9) is a member of the family of flavoenzymes that catalyze the dehydrogenation of acyl-CoAs to 2,3 enoyl-CoAs in mitochondrial fatty acid oxidation (FAO). Inborn errors of metabolism of all family members, including ACAD9, have been described in humans, and represent significant causes of morbidity and mortality particularly in children. ACAD9 deficiency leads to a combined defect in fatty acid oxidation and oxidative phosphorylation (OXPHOS) due to a dual role in the pathways. In addition to its function in mitochondrial FAO, ACAD9 has a second function as one of 14 factors responsible for assembly of complex I of the electron transport chain (ETC). Considerable controversy remains over the relative role of these two functions in normal physiology and the disparate clinical findings described in patients with ACAD9 deficiency. To better understand the normal function of ACAD9 and the pathophysiology of its deficiency, several knock out mouse models were developed. Homozygous total body knock out appeared to be lethal as no ACAD9 animals were obtained. Cre-lox technology was then used to generate tissue-specific deletion of the gene. Cardiac-specific ACAD9 deficient animals had severe neonatal cardiomyopathy and died by 17 days of age. They had severe mitochondrial dysfunction in vitro. Muscle-specific mutants were viable but exhibited muscle weakness. Additional studies of heart muscle from the cardiac specific deficient animals were used to examine the evolutionarily conserved signaling Intermediate in toll pathway (ECSIT) protein, a known binding partner of ACAD9 in the electron chain complex I assembly pathway. As expected, ECSIT levels were significantly reduced in the absence of ACAD9 protein, consistent with the demonstrated impairment of the complex I assembly. The various ACAD9 deficient animals should serve as useful models for development of novel therapeutics for this disorder.
    Keywords:  Acyl-CoA dehydrogenase 9; Cardiomyopathy; Fatty acid oxidation; Mouse models; Myopathy; Respiratory chain; Supercomplexes
    DOI:  https://doi.org/10.1016/j.ymgme.2021.09.002
  30. Methods Mol Biol. 2022 ;2363 321-334
    Measuring the Activity of DNA Repair Enzymes in Isolated Mitochondria.
    Beatriz Ferrando, Ian Max Møller, Tinna Stevnsner.
      Nuclear, mitochondrial and plastidic DNA is constantly exposed to conditions, such as ultraviolet radiation or reactive oxygen species, which will induce chemical modifications to the nucleotides. Unless repaired, these modifications can lead to mutations, so the nucleus, mitochondria and plastids each contains a number of DNA repair systems. We here describe assays for measuring the enzyme activities associated with the base-excision repair pathway in potato tuber mitochondria. As the name implies, this pathway involves removing a modified base and replacing it with an undamaged base. Activity of each of the enzymes involved, DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase and DNA ligase can be measured by incubating a mitochondrial extract with a specifically designed oligonucleotide. After incubation, the reaction mixture is separated on a polyacrylamide gel, and the amounts of specific products formed is estimated by autoradiography, which makes it possible to calculate the enzymatic activity.
    Keywords:  Base excision repair; DNA repair; Plant mitochondria; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_21
  31. Nat Commun. 2021 Sep 21. 12(1): 5556
    Leveraging the Cell Ontology to classify unseen cell types.
    Sheng Wang, Angela Oliveira Pisco, Aaron McGeever, Maria Brbic, Marinka Zitnik, Spyros Darmanis, Jure Leskovec, Jim Karkanias, Russ B Altman.
      Single cell technologies are rapidly generating large amounts of data that enables us to understand biological systems at single-cell resolution. However, joint analysis of datasets generated by independent labs remains challenging due to a lack of consistent terminology to describe cell types. Here, we present OnClass, an algorithm and accompanying software for automatically classifying cells into cell types that are part of the controlled vocabulary that forms the Cell Ontology. A key advantage of OnClass is its capability to classify cells into cell types not present in the training data because it uses the Cell Ontology graph to infer cell type relationships. Furthermore, OnClass can be used to identify marker genes for all the cell ontology categories, regardless of whether the cell types are present or absent in the training data, suggesting that OnClass goes beyond a simple annotation tool for single cell datasets, being the first algorithm capable to identify marker genes specific to all terms of the Cell Ontology and offering the possibility of refining the Cell Ontology using a data-centric approach.
    DOI:  https://doi.org/10.1038/s41467-021-25725-x
  32. Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01178-5. [Epub ahead of print]36(12): 109729
    UQCRC1 engages cytochrome c for neuronal apoptotic cell death.
    Yu-Chien Hung, Kuan-Lin Huang, Po-Lin Chen, Jeng-Lin Li, Serena Huei-An Lu, Jui-Chih Chang, Han-Yi Lin, Wen-Chun Lo, Shu-Yi Huang, Tai-Ting Lee, Tai-Yi Lin, Yuzuru Imai, Nobutaka Hattori, Chin-San Liu, Su-Yi Tsai, Chun-Hong Chen, Chin-Hsien Lin, Chih-Chiang Chan.
      Human ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an evolutionarily conserved core subunit of mitochondrial respiratory chain complex III. We recently identified the disease-associated variants of UQCRC1 from patients with familial parkinsonism, but its function remains unclear. Here we investigate the endogenous function of UQCRC1 in the human neuronal cell line and the Drosophila nervous system. Flies with neuronal knockdown of uqcrc1 exhibit age-dependent parkinsonism-resembling defects, including dopaminergic neuron reduction and locomotor decline, and are ameliorated by UQCRC1 expression. Lethality of uqcrc1-KO is also rescued by neuronally expressing UQCRC1, but not the disease-causing variant, providing a platform to discern the pathogenicity of this mutation. Furthermore, UQCRC1 associates with the apoptosis trigger cytochrome c (cyt-c), and uqcrc1 deficiency increases cyt-c in the cytoplasmic fraction and activates the caspase cascade. Depleting cyt-c or expression of the anti-apoptotic p35 ameliorates uqcrc1-mediated neurodegeneration. Our findings identify a role for UQCRC1 in regulating cyt-c-induced apoptosis.
    Keywords:  Parkinson’s disease; UQCRC1; apoptosis; complex III; cytochrome c; mitochondrial respiratory chain
    DOI:  https://doi.org/10.1016/j.celrep.2021.109729
  33. Nat Metab. 2021 Sep;3(9): 1144-1145
    We are more than what we eat.
    Cara L Green, Dudley W Lamming.
      
    DOI:  https://doi.org/10.1038/s42255-021-00434-3
  34. Methods Mol Biol. 2022 ;2363 199-213
    Markers for Mitochondrial ROS Status.
    Monika Ostaszewska-Bugajska, Anna Podgórska, Bożena Szal.
      Mitochondria actively participate in oxygenic metabolism and are one of the major sources of reactive oxygen species (ROS) production in plant cells. However, instead of measuring ROS concentrations in organelles it is more worthwhile to observe active ROS generation or downstream oxidation products, because the steady state level of ROS is easily buffered. Here, we describe how to measure the in vitro production of superoxide anion radicals (O2·-) by mitochondria and the release of O2·- into the cytosol. A method to determine glutathione, which is the most abundant mitochondrial low-mass antioxidant, is presented since changes in the redox state of glutathione can be indicative of the oxidative action of ROS. The identification of oxidative damage to mitochondrial components is the ultimate symptom that ROS homeostasis is not under control. We present how to determine the extent of oxidation of membrane lipids and the carbonylation of mitochondrial proteins. In summary, oxidative stress symptoms have to be analyzed at different levels, including ROS production, scavenging capacity, and signs of destruction, which only together can be considered markers of mitochondrial ROS status.
    Keywords:  Carbonylated proteins; Glutathione; Lipid peroxidation; Mitochondria; Mitochondrial integrity; Oxidized proteins; Reactive oxygen species; Superoxide anion radical
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_15
  35. Cell Syst. 2021 Sep 16. pii: S2405-4712(21)00338-0. [Epub ahead of print]
    NAD+ flux is maintained in aged mice despite lower tissue concentrations.
    Melanie R McReynolds, Karthikeyani Chellappa, Eric Chiles, Connor Jankowski, Yihui Shen, Li Chen, Hélène C Descamps, Sarmistha Mukherjee, Yashaswini R Bhat, Siddharth R Lingala, Qingwei Chu, Paul Botolin, Faisal Hayat, Tomohito Doke, Katalin Susztak, Christoph A Thaiss, Wenyun Lu, Marie E Migaud, Xiaoyang Su, Joshua D Rabinowitz, Joseph A Baur.
      NAD+ is an essential coenzyme for all living cells. NAD+ concentrations decline with age, but whether this reflects impaired production or accelerated consumption remains unclear. We employed isotope tracing and mass spectrometry to probe age-related changes in NAD+ metabolism across tissues. In aged mice, we observed modest tissue NAD+ depletion (median decrease ∼30%). Circulating NAD+ precursors were not significantly changed, and isotope tracing showed the unimpaired synthesis of nicotinamide from tryptophan. In most tissues of aged mice, turnover of the smaller tissue NAD+ pool was modestly faster such that absolute NAD+ biosynthetic flux was maintained, consistent with more active NAD+-consuming enzymes. Calorie restriction partially mitigated age-associated NAD+ decline by decreasing consumption. Acute inflammatory stress induced by LPS decreased NAD+ by impairing synthesis in both young and aged mice. Thus, the decline in NAD+ with normal aging is relatively subtle and occurs despite maintained NAD+ production, likely due to increased consumption.
    Keywords:  CD38; NAD; NADH; PARP; PARP1; SIRT1; aging; flux; mononucleotide; niacin; nicotinamide; redox; riboside; sirtuins
    DOI:  https://doi.org/10.1016/j.cels.2021.09.001
  36. Mov Disord. 2021 Sep 20.
    Coding and Noncoding Variation in LRRK2 and Parkinson's Disease Risk.
    Julie Lake, Xylena Reed, Rebekah G Langston, Mike A Nalls, Ziv Gan-Or, Mark R Cookson, Andrew B Singleton, Cornelis Blauwendraat, Hampton L Leonard, .
      BACKGROUND: The leucine-rich repeat kinase 2 (LRRK2) gene harbors both rare highly damaging missense variants (eg, p.G2019S) and common noncoding variants (eg, rs76904798) with lower effect sizes that are associated with Parkinson's disease (PD) risk.OBJECTIVES: This study aimed to investigate in a large meta-analysis whether the LRRK2 Genome-Wide Association Study (GWAS) signal represented by rs76904798 is independently associated with PD risk from LRRK2 coding variation and whether complex linkage disequilibrium structures with p.G2019S and the 5' noncoding haplotype account for the association of LRRK2 coding variants.
    METHODS: We performed a meta-analysis using imputed genotypes from 17,838 patients, 13,404 proxy patients, and 173,639 healthy controls of European ancestry. We excluded carriers of p.G2019S and/or rs76904798 to clarify the role of LRRK2 coding variation in mediating disease risk and excluded carriers of relatively rare LRRK2 coding variants to assess the independence of rs76904798. We also investigated the co-inheritance of LRRK2 coding variants with p.G2019S, rs76904798, and p.N2081D.
    RESULTS: LRRK2 rs76904798 remained significantly associated with PD after excluding the carriers of relatively rare LRRK2 coding variants. LRRK2 p.R1514Q and p.N2081D were frequently co-inherited with rs76904798, and the allele distribution of p.S1647T significantly changed among patients after removing rs76904798 carriers.
    CONCLUSIONS: These data suggest that the LRRK2 coding variants previously related to PD (p.N551K, p.R1398H, p.M1646T, and p.N2081D) do not drive the 5' noncoding GWAS signal. These data, however, do not preclude the independent association of the haplotype p.N551K-p.R1398H and p.M1646T with altered disease risk. © 2021 International Parkinson and Movement Disorder Society.
    Keywords:  LRRK2; Parkinson's disease; association; genetics
    DOI:  https://doi.org/10.1002/mds.28787
  37. Cell Death Discov. 2021 Sep 18. 7(1): 250
    Loss of mpv17 affected early embryonic development via mitochondria dysfunction in zebrafish.
    Wan-Ping Bian, Shi-Ya Pu, Shao-Lin Xie, Chao Wang, Shun Deng, Phyllis R Strauss, De-Sheng Pei.
      MVP17 encodes a mitochondrial inner-membrane protein, and mutation of human MVP17 can cause mitochondria DNA depletion syndrome (MDDS). However, the underlying function of mpv17 is still elusive. Here, we developed a new mutant with mpv17 knockout by using the CRISPR/Cas9 system. The mpv17-/- zebrafish showed developmental defects in muscles, liver, and energy supply. The mpv17-/- larvae hardly survived beyond a month, and they showed abnormal growth during the development stage. Abnormal swimming ability was also found in the mpv17-/- zebrafish. The transmission electron microscope (TEM) observation indicated that the mpv17-/- zebrafish underwent severe mitochondria dysfunction and the disorder of mitochondrial cristae. As an energy producer, the defects of mitochondria significantly reduced ATP content in mpv17-/- zebrafish, compared to wild-type zebrafish. We hypothesized that the disorder of mitochondria cristae was contributed to the dysfunction of muscle and liver in the mpv17-/- zebrafish. Moreover, the content of major energy depot triglycerides (TAG) was decreased dramatically. Interestingly, after rescued with normal exogenous mitochondria by microinjection, the genes involved in the TAG metabolism pathway were recovered to a normal level. Taken together, this is the first report of developmental defects in muscles, liver, and energy supply via mitochondria dysfunction, and reveals the functional mechanism of mpv17 in zebrafish.
    DOI:  https://doi.org/10.1038/s41420-021-00630-w
  38. Stem Cell Reports. 2021 Sep 14. pii: S2213-6711(21)00434-3. [Epub ahead of print]
    iPSC culture expansion selects against putatively actionable mutations in the mitochondrial genome.
    Maike Kosanke, Colin Davenport, Monika Szepes, Lutz Wiehlmann, Tim Kohrn, Marie Dorda, Jonas Gruber, Kaja Menge, Maike Sievert, Anna Melchert, Ina Gruh, Gudrun Göhring, Ulrich Martin.
      Therapeutic application of induced pluripotent stem cell (iPSC) derivatives requires comprehensive assessment of the integrity of their nuclear and mitochondrial DNA (mtDNA) to exclude oncogenic potential and functional deficits. It is unknown, to which extent mtDNA variants originate from their parental cells or from de novo mutagenesis, and whether dynamics in heteroplasmy levels are caused by inter- and intracellular selection or genetic drift. Sequencing of mtDNA of 26 iPSC clones did not reveal evidence for de novo mutagenesis, or for any selection processes during reprogramming or differentiation. Culture expansion, however, selected against putatively actionable mtDNA mutations. Altogether, our findings point toward a scenario in which intracellular selection of mtDNA variants during culture expansion shapes the mutational landscape of the mitochondrial genome. Our results suggest that intercellular selection and genetic drift exert minor impact and that the bottleneck effect in context of the mtDNA genetic pool might have been overestimated.
    Keywords:  genomic integrity; induced pluripotent stem cells; mitochondrial genome; prolonged expansion culture; reprogramming; selection; small-scale mutations
    DOI:  https://doi.org/10.1016/j.stemcr.2021.08.016
  39. Elife. 2021 Sep 21. pii: e68394. [Epub ahead of print]10
    Fine-tuned repression of Drp1 driven mitochondrial fission primes a 'stem/progenitor-like state' to support neoplastic transformation.
    Brian Spurlock, Danitra Parker, Malay Kumar Basu, Anita Hjelmeland, Sajina Gc, Shanrun Liu, Gene P Siegal, Alan Gunter, Aida Moran, Kasturi Mitra.
      Gene knockout of the master regulator of mitochondrial fission, Drp1, prevents neoplastic transformation. Also, mitochondrial fission and its opposing process of mitochondrial fusion are emerging as crucial regulators of stemness. Intriguingly, stem/progenitor cells maintaining repressed mitochondrial fission are primed for self-renewal and proliferation. Using our newly derived carcinogen transformed human cell model we demonstrate that fine-tuned Drp1 repression primes a slow cycling 'stem/progenitor-like state', which is characterized by small networks of fused mitochondria and a gene-expression profile with elevated functional stem/progenitor markers (Krt15, Sox2 etc) and their regulators (Cyclin E). Fine tuning Drp1 protein by reducing its activating phosphorylation sustains the neoplastic stem cell markers. Whereas, fine-tuned reduction of Drp1 protein maintains the characteristic mitochondrial shape and gene-expression of the primed 'stem/progenitor-like state' to accelerate neoplastic transformation, and more complete reduction of Drp1 protein prevents it. Therefore, our data highlights a 'goldilocks'; level of Drp1 repression supporting stem/progenitor state dependent neoplastic transformation.
    Keywords:  cancer biology; cell biology; mouse
    DOI:  https://doi.org/10.7554/eLife.68394
  40. J Mol Neurosci. 2021 Sep 24.
    Clinico-Genetic Spectrum of POLG1 Mutation Carriers from India.
    Josef Finsterer.
      
    Keywords:  Heterogeneity; Mitochondrial disorder; POLG1; Phenotype; mtDNA
    DOI:  https://doi.org/10.1007/s12031-021-01905-8
  41. Cureus. 2021 Sep;13(9): e17761
    MEGDEL Syndrome and Its Anesthetic Implications.
    Balazs Horvath, Kathleen M Pfister, Alexis Rupp, Benjamin Kloesel.
      MEGDEL syndrome gains its name for its following features: 3-methylglutaconic aciduria (MEG), deafness (D), encephalopathy (E), Leigh-like syndrome (L). This syndrome is caused by biallelic mutations in the serine active site-containing protein 1 (SERAC1 ) gene. When these patients present with hepatopathy (H) in addition to the above manifestations the syndrome is labeled as MEGD(H)EL. The pathology of the disease shares features with different types of inborn errors of metabolism. We present the anesthetic management of a neonate who was diagnosed with MEGD(H)EL syndrome and underwent diagnostic magnetic resonance imaging of the brain at 14 days of postnatal age. We describe the epidemiology and important features of this rare disease that are pertinent for the anesthesiologist.
    Keywords:  aciduria; megd(h)el; megdel; mitochondrial disease; pediatric anesthesia; phospholipid metabolism
    DOI:  https://doi.org/10.7759/cureus.17761
  42. Nat Rev Nephrol. 2021 Sep 20.
    RBC mitochondria trigger interferon release.
    Monica Wang.
      
    DOI:  https://doi.org/10.1038/s41581-021-00494-4
  43. J Physiol. 2021 Sep 23.
    Fission accomplished: Uncovering the role of Drp1 in regulating mitochondrial dysfunction and age-related muscle atrophy.
    Paula M Miotto, Giang M Dao, Henver S Brunetta.
      
    DOI:  https://doi.org/10.1113/JP282197
  44. Front Cardiovasc Med. 2021 ;8 724846
    Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase.
    Bowen Li, Kun Chen, Fangfang Liu, Juan Zhang, Xihui Chen, Tangdong Chen, Qi Chen, Yan Yao, Weihong Hu, Li Wang, Yuanming Wu.
      Background: Mitochondrial aminoacyl-tRNA synthetases (mtARSs) catalyze the binding of specific amino acids to their cognate tRNAs and play an essential role in the synthesis of proteins encoded by mitochondrial DNA. Defects in mtARSs have been linked to human diseases, but their tissue-specific pathophysiology remains elusive. Here we examined the role of mitochondrial phenylalanyl-tRNA synthetase (FARS2) in developmental angiogenesis and its potential contribution to the pathogenesis of cardiovascular disease. Methods: Morpholinos were injected into fertilized zebrafish ova to establish an in vivo fars2 knock-down model. A visualization of the vasculature was achieved by using Tg (fli1: EGFP) y1 transgenic zebrafish. In addition, small interference RNAs (siRNAs) were transferred into human umbilical vein endothelial cells (HUVECs) to establish an in vitro FARS2 knock-down model. Cell motility, proliferation, and tubulogenesis were determined using scratch-wound CCK8, transwell-based migration, and tube formation assays. In addition, mitochondria- and non-mitochondria-related respiration were evaluated using a Seahorse XF24 analyzer and flow cytometry assays. Analyses of the expression levels of transcripts and proteins were performed using qRT-PCR and western blotting, respectively. Results: The knock-down of fars2 hampered the embryonic development in zebrafish and delayed the formation of the vasculature in Tg (fli1: EGFP) y1 transgenic zebrafish. In addition, the siRNA-mediated knock-down of FARS2 impaired angiogenesis in HUVECs as indicated by decreased cell motility and tube formation capacity. The knock-down of FARS2 also produced variable decreases in mitochondrial- and non-mitochondrial respiration in HUVECs and disrupted the regulatory pathways of angiogenesis in both HUVECs and zebrafish. Conclusion: Our current work offers novel insights into angiogenesis defects and cardiovascular diseases induced by FARS2 deficiency.
    Keywords:  FARS2; HUVECs; angiogenesis; mitochondrial dysfunction; mtARSs; zebrafish
    DOI:  https://doi.org/10.3389/fcvm.2021.724846
  45. iScience. 2021 Sep 24. 24(9): 103038
    In situ observation of mitochondrial biogenesis as the early event of apoptosis.
    Chang-Sheng Shao, Xiu-Hong Zhou, Yu-Hui Miao, Peng Wang, Qian-Qian Zhang, Qing Huang.
      Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria and monitored the mitochondrial changes in the living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial mass, mitochondrial morphology, and protein expression related to mitochondrial biogenesis, as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and it was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed.
    Keywords:  Biochemistry methods; Biomolecular engineering; Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2021.103038
  46. J Exp Med. 2021 Nov 01. pii: e20210846. [Epub ahead of print]218(11):
    Implication of folate deficiency in CYP2U1 loss of function.
    Claire Pujol, Anne Legrand, Livia Parodi, Priscilla Thomas, Fanny Mochel, Dario Saracino, Giulia Coarelli, Marijana Croon, Milica Popovic, Manon Valet, Nicolas Villain, Shahira Elshafie, Mahmoud Issa, Stephane Zuily, Mathilde Renaud, Cécilia Marelli-Tosi, Marine Legendre, Aurélien Trimouille, Isabelle Kemlin, Sophie Mathieu, Joseph G Gleeson, Foudil Lamari, Daniele Galatolo, Rana Alkouri, Chantal Tse, Diana Rodriguez, Claire Ewenczyk, Florence Fellmann, Thierry Kuntzer, Emilie Blond, Khalid H El Hachimi, Frédéric Darios, Alexandre Seyer, Anastasia D Gazi, Patrick Giavalisco, Silvina Perin, Jean-Luc Boucher, Laurent Le Corre, Filippo M Santorelli, Cyril Goizet, Maha S Zaki, Serge Picaud, Arnaud Mourier, Sophie Marie Steculorum, Cyril Mignot, Alexandra Durr, Aleksandra Trifunovic, Giovanni Stevanin.
      Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders. Understanding of their pathogenic mechanisms remains sparse, and therapeutic options are lacking. We characterized a mouse model lacking the Cyp2u1 gene, loss of which is known to be involved in a complex form of these diseases in humans. We showed that this model partially recapitulated the clinical and biochemical phenotypes of patients. Using electron microscopy, lipidomic, and proteomic studies, we identified vitamin B2 as a substrate of the CYP2U1 enzyme, as well as coenzyme Q, neopterin, and IFN-α levels as putative biomarkers in mice and fluids obtained from the largest series of CYP2U1-mutated patients reported so far. We also confirmed brain calcifications as a potential biomarker in patients. Our results suggest that CYP2U1 deficiency disrupts mitochondrial function and impacts proper neurodevelopment, which could be prevented by folate supplementation in our mouse model, followed by a neurodegenerative process altering multiple neuronal and extraneuronal tissues.
    DOI:  https://doi.org/10.1084/jem.20210846
  47. J Hepatol. 2021 Sep 20. pii: S0168-8278(21)02038-9. [Epub ahead of print]
    Inhibition of ATG3 ameliorates liver steatosis by increasing mitochondrial function.
    Natália da Silva Lima, Marcos F Fondevila, Eva Nóvoa, Xabier Buqué, Maria Mercado-Gómez, Sarah Gallet, Maria J González-Rellan, Uxia Fernandez, Anne Loyens, Maria Garcia-Vence, Maria Del Pilar Chantada-Vazquez, Susana B Bravo, Patricia Marañon, Ana Senra, Adriana Escudero, Magdalena Leiva, Diana Guallar, Miguel Fidalgo, Pedro Gomes, Marc Claret, Guadalupe Sabio, Marta Varela-Rey, Teresa C Delgado, Rocio Montero-Vallejo, Javier Ampuero, Miguel López, Carlos Diéguez, Laura Herrero, Dolors Serra, Markus Schwaninger, Vincent Prevot, Rocio Gallego-Duran, Manuel Romero-Gomez, Paula Iruzubieta, Javier Crespo, Maria L Martinez-Chantar, Carmelo Garcia-Monzon, Agueda Gonzalez-Rodriguez, Patricia Aspichueta, Ruben Nogueiras.
      BACKGROUND & AIMS: Autophagy-related gene 3 (ATG3) is an enzyme mainly known for its actions in the LC3 lipidation process, which is essential for autophagy. Whether ATG3 plays a role in lipid metabolism or contributes to nonalcoholic fatty liver disease (NAFLD) remains unknown.METHODS: By performing a liver proteomic analysis from mice with genetic manipulation of hepatic p63, a regulator of fatty acid metabolism, we identified ATG3 as a new target downstream of p63. ATG3 was evaluated in liver samples of patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the liver of mice.
    RESULTS: ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and NASH) compared with those without liver disease. Moreover, genetic knockdown of ATG3 in mice and human hepatocytes ameliorates p63- and diet-induced steatosis, while its overexpression increases the lipid load in hepatocytes. The inhibition of hepatic ATG3 improves fatty acid metabolism by reducing c-Jun N-terminal protein kinase 1 (JNK1), which increases sirtuin 1 (SIRT1), carnitine palmitoiltransferase I (CPT1a), and mitochondrial function. Hepatic knockdown of SIRT1 and CPT1a blunts the effects of ATG3 on mitochondrial activity. Unexpectedly, these effects are independent of an autophagic action.
    CONCLUSIONS: Collectively, these findings indicate that ATG3 is a novel protein implicated in the development of steatosis.
    LAY SUMMARY: We show that autophagy-related gene 3 (ATG3) contributes to the progression of NAFLD in humans and mice. Hepatic knockdown of ATG3 ameliorates the development of NAFLD, by stimulating SIRT1, CPT1a and mitochondrial function. Thus, ATG3 is an important factor implicated in steatosis.
    Keywords:  ATG3; lipid metabolism; sirtuin 1
    DOI:  https://doi.org/10.1016/j.jhep.2021.09.008
  48. Cell Death Differ. 2021 Sep 23.
    LIN28A enhances regenerative capacity of human somatic tissue stem cells via metabolic and mitochondrial reprogramming.
    Kelvin Pieknell, Yanuar Alan Sulistio, Noviana Wulansari, Wahyu Handoko Wibowo Darsono, Mi-Yoon Chang, Ji-Yun Ko, Jong Wook Chang, Min-Jeong Kim, Man Ryul Lee, Sang A Lee, Hyunbeom Lee, Gakyung Lee, Byung Hwa Jung, Hyunbum Park, Geun-Ho Kim, Doory Kim, Gayoung Cho, Chun-Hyung Kim, Dat Da Ly, Kyu-Sang Park, Sang-Hun Lee.
      Developing methods to improve the regenerative capacity of somatic stem cells (SSCs) is a major challenge in regenerative medicine. Here, we propose the forced expression of LIN28A as a method to modulate cellular metabolism, which in turn enhances self-renewal, differentiation capacities, and engraftment after transplantation of various human SSCs. Mechanistically, in undifferentiated/proliferating SSCs, LIN28A induced metabolic reprogramming from oxidative phosphorylation (OxPhos) to glycolysis by activating PDK1-mediated glycolysis-TCA/OxPhos uncoupling. Mitochondria were also reprogrammed into healthy/fused mitochondria with improved functional capacity. The reprogramming allows SSCs to undergo cell proliferation more extensively with low levels of oxidative and mitochondrial stress. When the PDK1-mediated uncoupling was untethered upon differentiation, LIN28A-SSCs differentiated more efficiently with an increase of OxPhos by utilizing the reprogrammed mitochondria. This study provides mechanistic and practical approaches of utilizing LIN28A and metabolic reprogramming in order to improve SSCs utility in regenerative medicine.
    DOI:  https://doi.org/10.1038/s41418-021-00873-1
  49. Hum Mol Genet. 2021 Sep 24. pii: ddab286. [Epub ahead of print]
    Mutations at a split codon in the GTPase-encoding domain of OPA1 cause dominant optic atrophy through different molecular mechanisms.
    Nicole Weisschuh, Valerio Marino, Karin Schäferhoff, Paul Richter, Joohyun Park, Tobias B Haack, Daniele Dell'Orco.
      Exonic (i.e. coding) variants in genes associated with disease can exert pathogenic effects both at the protein and mRNA level, either by altering the amino acid sequence or by affecting pre-mRNA splicing. The latter is often neglected due to the lack of RNA analyses in genetic diagnostic testing. In this study we considered both pathomechanisms and performed a comprehensive analysis of nine exonic nucleotide changes in OPA1, which is the major gene underlying autosomal dominant optic atrophy (DOA) and is characterized by pronounced allelic heterogeneity. We focused on the GTPase-encoding domain of OPA1, which harbors most of the missense variants associated with DOA. Given that the consensus splice sites extend into the exons, we chose a split codon, namely codon 438, for our analyses. Variants at this codon are the second most common cause of disease in our large cohort of DOA patients harboring disease-causing variants in OPA1. In silico splice predictions, heterologous splice assays, analysis of patient's RNA when available, and protein modeling revealed different molecular outcomes for variants at codon 438. The wildtype aspartate residue at amino acid position 438 is directly involved in the dimerization of OPA1 monomers. We found that six amino acid substitutions at codon 438 (i.e. all substitutions of the first and second nucleotide of the codon) destabilized dimerization while only substitutions of the first nucleotide of the codon caused exon skipping. Our study highlights the value of combining RNA analysis and protein modeling approaches to accurately assign patients to future precision therapies.
    DOI:  https://doi.org/10.1093/hmg/ddab286
  50. Science. 2021 09 24. 373(6562): 1464-1468
    From variant to function in human disease genetics.
    Tuuli Lappalainen, Daniel G MacArthur.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/science.abi8207
  51. J Mol Biol. 2021 Sep 16. pii: S0022-2836(21)00480-0. [Epub ahead of print] 167247
    FTO suppresses STAT3 activation and modulates proinflammatory interferon-stimulated gene expression.
    Michael J McFadden, Matthew T Sacco, Kristen A Murphy, Moonhee Park, Nandan S Gokhale, Kim Y Somfleth, Stacy M Horner.
      Signaling initiated by type I interferon (IFN) results in the induction of hundreds of IFN-stimulated genes (ISGs). The type I IFN response is important for antiviral restriction, but aberrant activation of this response can lead to inflammation and autoimmunity. Regulation of this response is incompletely understood. We previously reported that the mRNA modification m6A and its deposition enzymes, METTL3 and METTL14 (METTL3/14), promote the type I IFN response by directly modifying the mRNA of a subset of ISGs to enhance their translation. Here, we determined the role of the RNA demethylase fat mass and obesity-associated protein (FTO) in the type I IFN response. FTO, which can remove either m6A or cap-adjacent m6Am RNA modifications, has previously been associated with obesity and body mass index, type 2 diabetes, cardiovascular disease, and inflammation. We found that FTO suppresses the transcription of a distinct set of ISGs, including many known pro-inflammatory genes, and that this regulation requires its catalytic activity but is not through the actions of FTO on m6Am. Interestingly, depletion of FTO led to activation of the transcription factor STAT3, whose role in the type I IFN response is not well understood. This activation of STAT3 increased the expression of a subset of ISGs. Importantly, this increased ISG induction resulting from FTO depletion was partially ablated by depletion of STAT3. Together, these results reveal that FTO negatively regulates STAT3-mediated signaling that induces proinflammatory ISGs during the IFN response, highlighting an important role for FTO in suppression of inflammatory genes.
    Keywords:  Fat mass and obesity-associated (FTO); Inflammation; Interferon (IFN); Interferon-stimulated gene (ISG); N6-methyladenosine (m(6)A)
    DOI:  https://doi.org/10.1016/j.jmb.2021.167247
  52. Sci Rep. 2021 Sep 22. 11(1): 18863
    The 3D organisation of mitochondria in primate photoreceptors.
    Matthew J Hayes, Dhani Tracey-White, Jaimie Hoh Kam, Michael B Powner, Glen Jeffery.
      Vertebrate photoreceptors contain large numbers of closely-packed mitochondria which sustain the high metabolic demands of these cells. These mitochondria populations are dynamic and undergo fusion and fission events. This activity serves to maintain the population in a healthy state. In the event of mitochondrial damage, sub-domains, or indeed whole mitochondria, can be degraded and population homeostasis achieved. If this process is overwhelmed cell death may result. Death of photoreceptors contributes to loss of vision in aging individuals and is associated with many eye diseases. In this study we used serial block face scanning electron microscopy of adult Macaca fascicularis retinae to examine the 3D structure of mitochondria in rod and cone photoreceptors. We show that healthy-looking photoreceptors contain mitochondria exhibiting a range of shapes which are associated with different regions of the cell. In some photoreceptors we observe mitochondrial swelling and other changes often associated with cellular stress. In rods and cones that appear stressed we identify elongated domains of mitochondria with densely-packed normal cristae associated with photoreceptor ciliary rootlet bundles. We observe mitochondrial fission and mitochondrion fragments localised to these domains. Swollen mitochondria with few intact cristae are located towards the periphery of the photoreceptor inner-segment in rods, whilst they are found throughout the cell in cones. Swollen mitochondria exhibit sites on the mitochondrial inner membrane which have undergone complex invagination resulting in membranous, electron-dense aggregates. Membrane contact occurs between the mitochondrion and the photoreceptor plasma membrane in the vicinity of these aggregates, and a series of subsequent membrane fusions results in expulsion of the mitochondrial aggregate from the photoreceptor. These events are primarily associated with rods. The potential fate of this purged material and consequences of its clearance by retinal pigment epithelia are discussed.
    DOI:  https://doi.org/10.1038/s41598-021-98409-7
  53. Lancet. 2021 Sep 21. pii: S0140-6736(21)02124-3. [Epub ahead of print]
    Optimising the health-care experiences of babies, children, and young people.
    Tamsin Ford, Kwabena Kusi-Mensah, Tamsin Newlove-Delgado, Paul Ramchandani, Fiona Price-Kuehne, David Rowitch.
      
    DOI:  https://doi.org/10.1016/S0140-6736(21)02124-3
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