bims-mitdis Biomed News
on Mitochondrial Disorders
Issue of 2021‒06‒13
sixty-nine papers selected by
Catalina Vasilescu
University of Helsinki
  1. Mitochondrial mistranslation modulated by metabolic stress causes cardiovascular disease and reduced lifespan.
  2. Mitochondrial Functional Assessment in Mammalian Gametes Using Fluorescent Probes.
  3. Clinical heterogeneity in patients with m.4412G>A MT-TM mutation and different heteroplasmy levels.
  4. Imaging Mitochondrial Hydrogen Peroxide in Living Cells.
  5. Monitoring Mitochondrial Function in Mouse Embryonic Stem Cells (mESCs).
  6. Lipids activate skeletal muscle mitochondrial fission and quality control networks to induce insulin resistance in humans.
  7. Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.
  8. Severe multisystem pathology, metabolic acidosis, mitochondrial dysfunction, and early death associated with an X-linked AIFM1 variant.
  9. Determination of Oxidative Phosphorylation Complexes Activities.
  10. Analysis of Proapoptotic Protein Trafficking to and from Mitochondria.
  11. Update on the Histoenzymatic Methods for Visualization of the Activity of Individual Mitochondrial Respiratory Chain Complexes in the Human Frozen Sections.
  12. Acyl-CoA synthetase 6 is required for brain docosahexaenoic acid retention and neuroprotection during aging.
  13. Mito-SinCe2 Approach to Analyze Mitochondrial Structure-Function Relationship in Single Cells.
  14. Following the Dynamism of the Mitochondrial Network in T Cells.
  15. Sequence-Specific Control of Mitochondrial Gene Transcription Using Programmable Synthetic Gene Switches Called MITO-PIPs.
  16. Mitochondrial Coenzyme Q10 Determination Via Isotope Dilution Liquid Chromatography -Tandem Mass Spectrometry.
  17. Accurate genomic variant detection in single cells with primary template-directed amplification.
  18. PCR-Based Determination of Mitochondrial DNA Copy Number in Multiple Species.
  19. Live Imaging of Mitochondria in Kidney Tissue.
  20. Perinatal Manifestations of DARS2-Associated Leukoencephalopathy With Brainstem and Spinal Cord Involvement and Lactate Elevation (LBSL).
  21. An Update on Isolation of Functional Mitochondria from Cells for Bioenergetics Studies.
  22. ALS- and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy.
  23. Split Green Fluorescent Protein-Based Contact Site Sensor (SPLICS ) for Heterotypic Organelle Juxtaposition as Applied to ER -Mitochondria Proximities.
  24. Perspectives on formaldehyde dysregulation: Mitochondrial DNA damage and repair in mammalian cells.
  25. Mitochondria: Their relevance during oocyte ageing.
  26. Protocols for Mitochondria as the Target of Pharmacological Therapy in the Context of Nonalcoholic Fatty Liver Disease (NAFLD).
  27. Methods to Monitor Mitophagy and Mitochondrial Quality: Implications in Cancer, Neurodegeneration, and Cardiovascular Diseases.
  28. Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes.
  29. Qualitative Characterization of the Rat Liver Mitochondrial Lipidome Using All Ion Fragmentation on an Exactive Benchtop Orbitrap MS.
  30. mTRIP, an Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Resolution.
  31. An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial membrane.
  32. Mitochondrial Regulation Assessment by 13C-NMR Isotopomer Analysis.
  33. Live-Cell Assessment of Reactive Oxygen Species Levels Using Dihydroethidine.
  34. An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of α-synuclein.
  35. Exploring Liver Mitochondrial Function by 13C-Stable Isotope Breath Tests: Implications in Clinical Biochemistry.
  36. Computer-Aided Prediction of Protein Mitochondrial Localization.
  37. Novel Mitochondria-Targeted Triphenylphosphonium Conjugates of Linear β-Phosphorylated Nitrones : Preparation, 31P NMR Mitochondrial Distribution, EPR Spin Trapping Reporting, and Site-Directed Antiapoptotic Properties.
  38. Protein Supercomplex Recording in Living Cells Via Position-Specific Fluorescence Lifetime Sensors.
  39. Targeting the Mitochondrial Genome Via a MITO-Porter : Evaluation of mtDNA and mtRNA Levels and Mitochondrial Function.
  40. Time-Resolved Imaging of Mitochondrial Flavin Fluorescence and Its Applications for Evaluating the Oxidative State in Living Cardiac Cells.
  41. Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes.
  42. Optimizing mitochondrial maintenance in extended neuronal projections.
  43. 2-kupl: mapping-free variant detection from DNA-seq data of matched samples.
  44. iDRiP for the systematic discovery of proteins bound directly to noncoding RNA.
  45. Resurrection and characterization of ancestral CYP11A1 enzymes.
  46. Maintenance of complex I and its super-complexes by NDUF-11 is essential for mitochondrial structure, function and health.
  47. Myocardial hypoxic stress mediates functional cardiac extracellular vesicle release.
  48. The role of Mfn2 in the structure and function of ER-mitochondrial tethering in vivo.
  49. Integrating protein networks and machine learning for disease stratification in the Hereditary Spastic Paraplegias.
  50. Structural analysis of the full-length human LRRK2.
  51. BN-PAGE-Based Approach to Study Thyroid Hormones and Mitochondrial Function.
  52. Regulation of the one carbon folate cycle as a shared metabolic signature of longevity.
  53. DHODH inhibition modulates glucose metabolism and circulating GDF15, and improves metabolic balance.
  54. Spatially Selective Imaging of Mitochondrial MicroRNAs via Optically Programmable Strand Displacement Reactions.
  55. Genomic sequencing for the diagnosis of childhood mitochondrial disorders: a health economic evaluation.
  56. The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.
  57. Damaged mitochondria are discarded via migrasomes.
  58. Pharmacological or genetic inhibition of iNOS prevents cachexia-mediated muscle wasting and its associated metabolism defects.
  59. Norbormide-Based Probes and Their Application for Mitochondrial Imaging in Drosophila Melanogaster.
  60. Towards precision medicine in heart failure.
  61. MOGONET integrates multi-omics data using graph convolutional networks allowing patient classification and biomarker identification.
  62. Adult-onset mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome with progressive sensorineural hearing loss: A case report.
  63. [A review of consensus statements, practice resources, standards and guidelines for clinical applications of next-generation sequencing technologies in the United States].
  64. Aryl urea substituted fatty acids: a new class of protonophoric mitochondrial uncoupler that utilises a synthetic anion transporter.
  65. Self-replicative mRNA-mediated generation of induced pluripotent stem cell line from a 1-year-old Leigh syndrome patient with mitochondrial DNA cytochrome b mutation.
  66. Measuring PGC-1α and Its Acetylation Status in Mouse Primary Myotubes.
  67. A hitchhiker's guide to mitochondrial quantification.
  68. Identification of Peroxynitrite by Profiling Oxidation and Nitration Products from Mitochondria-Targeted Arylboronic Acid.
  69. Energy Metabolism of Osteocytes.
  1. Aging Cell. 2021 Jun 07. e13408
    Mitochondrial mistranslation modulated by metabolic stress causes cardiovascular disease and reduced lifespan.
    Tara R Richman, Judith A Ermer, Stefan J Siira, Irina Kuznetsova, Christopher A Brosnan, Giulia Rossetti, Jessica Baker, Kara L Perks, Henrietta Cserne Szappanos, Helena M Viola, Nicola Gray, Mark Larance, Livia C Hool, Steven Zuryn, Oliver Rackham, Aleksandra Filipovska.
      Changes in the rate and fidelity of mitochondrial protein synthesis impact the metabolic and physiological roles of mitochondria. Here we explored how environmental stress in the form of a high-fat diet modulates mitochondrial translation and affects lifespan in mutant mice with error-prone (Mrps12ep / ep ) or hyper-accurate (Mrps12ha / ha ) mitochondrial ribosomes. Intriguingly, although both mutations are metabolically beneficial in reducing body weight, decreasing circulating insulin and increasing glucose tolerance during a high-fat diet, they manifest divergent (either deleterious or beneficial) outcomes in a tissue-specific manner. In two distinct organs that are commonly affected by the metabolic disease, the heart and the liver, Mrps12ep / ep mice were protected against heart defects but sensitive towards lipid accumulation in the liver, activating genes involved in steroid and amino acid metabolism. In contrast, enhanced translational accuracy in Mrps12ha / ha mice protected the liver from a high-fat diet through activation of liver proliferation programs, but enhanced the development of severe hypertrophic cardiomyopathy and led to reduced lifespan. These findings reflect the complex transcriptional and cell signalling responses that differ between post-mitotic (heart) and highly proliferative (liver) tissues. We show trade-offs between the rate and fidelity of mitochondrial protein synthesis dictate tissue-specific outcomes due to commonly encountered stressful environmental conditions or aging.
    Keywords:  ageing; metabolism; mitochondria; protein synthesis
    DOI:  https://doi.org/10.1111/acel.13408
  2. Methods Mol Biol. 2021 ;2310 57-68
    Mitochondrial Functional Assessment in Mammalian Gametes Using Fluorescent Probes.
    Renata S Tavares, Sara Escada-Rebelo, Maria M Soares, Andreia F Silva, Teresa Almeida-Santos, Sandra Amaral, Ana Paula Sousa, João Ramalho-Santos.
      A positive relationship between mitochondrial functionality and gamete quality, ultimately contributing to fertilization success and normal embryo development has been established for some years now. Both mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production are major indicators of mitochondrial function, and the need for accurate biomarkers mirroring gamete quality highlights the importance of a precise assessment of mitochondrial quality and function. In this chapter, we discuss the use of some mitochondrial fluorescent probes coupled to flow cytometry and/or fluorescence microscopy to specifically assess mitochondrial ROS production and MMP in both sperm and oocytes. Furthermore, as the distribution/aggregation of mitochondria in the oocyte is of interest to determine its quality, a detailed protocol is also given. These methodologies are easy, accurate and can be safely applied in research- and/or clinical-based contexts.
    Keywords:  Fluorescent probes; Mitochondrial aggregation; Mitochondrial distribution; Mitochondrial membrane potential; Oocytes; Reactive oxygen species; Sperm
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_5
  3. Mitochondrion. 2021 Jun 02. pii: S1567-7249(21)00074-X. [Epub ahead of print]
    Clinical heterogeneity in patients with m.4412G>A MT-TM mutation and different heteroplasmy levels.
    Atsuko Imai-Okazaki, Nobuyasu Yagi, Kazuhiro R Nitta, Kei Murayama, Akira Ohtake, Yasushi Okazaki.
      The identification of the m.4412G>A MT-TM (mt-tRNAMet) mutation was first reported in 2019. The affected individual presented with childhood-onset seizures and myopathy and bilateral basal ganglia changes, with heteroplasmy levels in muscle as high as 90%. Here, we describe another adult-onset patient with the same mutation and additional phenotypes, including hearing impairment, cerebellar ataxia, progressive dementia, and myopathy. The 10% heteroplasmy level observed in skin fibroblasts from this patient are lower than those in the previously reported patient. Our report suggests possible clinical heterogeneity in patients with mitochondrial tRNA mutations based on heteroplasmy levels.
    Keywords:  clinical heterogeneity; mitochondrial DNA; mitochondrial disease; mitochondrial respiratory chain complex deficiencies; mitochondrial tRNA
    DOI:  https://doi.org/10.1016/j.mito.2021.06.001
  4. Methods Mol Biol. 2021 ;2275 127-140
    Imaging Mitochondrial Hydrogen Peroxide in Living Cells.
    Alexander R Lippert, Bryan C Dickinson, Elizabeth J New.
      Hydrogen peroxide (H2O2) produced from mitochondria is intimately involved in human health and disease, but is challenging to selectively monitor inside living systems. The fluorescent probe MitoPY1 provides a practical tool for imaging mitochondrial H2O2 and has been demonstrated to function in a variety of diverse cell types. In this chapter, we describe the synthetic preparation of the small molecule probe MitoPY1 , methods for validating this probe in vitro and in live cells, and an example procedure for measuring mitochondrial H2O2 in a cell culture model of Parkinson's disease.
    Keywords:  Boronate; Fluorescence microscopy; Fluorescent probes; Hydrogen peroxide; Mitochondria; Triphenylphosphonium cations
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_8
  5. Methods Mol Biol. 2021 ;2310 47-56
    Monitoring Mitochondrial Function in Mouse Embryonic Stem Cells (mESCs).
    Bibiana Correia, Maria Inês Sousa, Ana F Branco, João Ramalho-Santos.
      Mouse embryonic stem cells (mESCs) can be grown in culture, recapitulating the different states of pluripotency of their in vivo counterparts, with notably different metabolic profiles. mESCs in a naïve pluripotent state present an ambivalent metabolism, using both glycolysis and oxidative phosphorylation as energy sources. Here, we describe a method to evaluate the oxidative function of naïve mESCs using the Seahorse Extracellular Flux Analyzer coupled to flow cytometry analysis of mitochondrial transmembrane potential using the TMRM fluorescence probe, thus assessing both oxygen consumption and mitochondrial membrane potential. This may be a useful protocol for understanding how mitochondrial oxidative function and potential of mESCs change in certain circumstances, and how is it related with various pluripotency/differentiation phenotypes.
    Keywords:  Metabolism; Mitochondrial Transmembrane potential; Pluripotency; Seahorse; Stem cells
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_4
  6. Metabolism. 2021 Jun 03. pii: S0026-0495(21)00103-7. [Epub ahead of print] 154803
    Lipids activate skeletal muscle mitochondrial fission and quality control networks to induce insulin resistance in humans.
    Christopher L Axelrod, Ciaran E Fealy, Melissa L Erickson, Gangarao Davuluri, Hisashi Fujioka, Wagner S Dantas, Emily Huang, Kathryn Pergola, Jacob T Mey, William T King, Anny Mulya, Daniel Hsia, Bartolome Burguera, Bernard Tandler, Charles L Hoppel, John P Kirwan.
      BACKGROUND AND AIMS: A diminution in skeletal muscle mitochondrial function due to ectopic lipid accumulation and excess nutrient intake is thought to contribute to insulin resistance and the development of type 2 diabetes. However, the functional integrity of mitochondria in insulin-resistant skeletal muscle remains highly controversial.METHODS: 19 healthy adults (age:28.4 ± 1.7 years; BMI:22.7 ± 0.3 kg/m2) received an overnight intravenous infusion of lipid (20% Intralipid) or saline followed by a hyperinsulinemic-euglycemic clamp to assess insulin sensitivity using a randomized crossover design. Skeletal muscle biopsies were obtained after the overnight lipid infusion to evaluate activation of mitochondrial dynamics proteins, ex-vivo mitochondrial membrane potential, ex-vivo oxidative phosphorylation and electron transfer capacity, and mitochondrial ultrastructure.
    RESULTS: Overnight lipid infusion increased dynamin related protein 1 (DRP1) phosphorylation at serine 616 and PTEN-induced kinase 1 (PINK1) expression (P = 0.003 and P = 0.008, respectively) in skeletal muscle while reducing mitochondrial membrane potential (P = 0.042). The lipid infusion also increased mitochondrial-associated lipid droplet formation (P = 0.011), the number of dilated cristae, and the presence of autophagic vesicles without altering mitochondrial number or respiratory capacity. Additionally, lipid infusion suppressed peripheral glucose disposal (P = 0.004) and hepatic insulin sensitivity (P = 0.014).
    CONCLUSIONS: These findings indicate that activation of mitochondrial fission and quality control occur early in the onset of insulin resistance in human skeletal muscle. Targeting mitochondrial dynamics and quality control represents a promising new pharmacological approach for treating insulin resistance and type 2 diabetes.
    CLINICAL TRIAL REGISTRATION: NCT02697201, ClinicalTrials.gov.
    DOI:  https://doi.org/10.1016/j.metabol.2021.154803
  7. Methods Mol Biol. 2021 ;2310 271-285
    Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.
    Yue Yang, Anthony A Sauve.
      NAD+ is a redox cofactor essential to the proper functioning of a variety of important metabolic pathways, including key steps in mitochondrial energy metabolism. In addition, it serves as a signaling substrate for enzymes such as sirtuins and the poly-ADP ribosyl-polymerase family of enzymes. Sirtuins, which are NAD+-dependent protein deacylases, harness changes in cellular NAD+ concentrations to produce changes in protein acylation status, thereby affecting downstream functions including energy metabolism, stress resistance, and cell survival. Thus, the availability of NAD+ in cells, or in specific organelles such as the mitochondrion, regulates downstream signaling and key biological processes. This concept has driven a need for researchers to easily and precisely measure NAD+ concentrations in biological samples. We herein describe several protocols for the measurement of NAD+ and NADH concentrations in tissues, cells, or subcellular compartments such as mitochondria. These protocols include a cycling assay that can quickly measure NAD+ or NADH levels using a plate reader equipped with fluorescence measurement capabilities. This plate assay relies only upon commercially available materials in addition to the biological samples of interest. In addition, we describe a protocol employing stable isotope-labeled NAD+ as an internal standard to determine biological NAD+ content by isotope-dilution methods. This method requires mass spectrometry to ratio endogenous NAD+ with exogenous isotope-labeled NAD+ to obtain quantification using HPLC and mass spectrometry.
    Keywords:  18O-NAD+; Diaphorase; HPLC; Isotopes; LC-MS; Lactate; Lactate dehydrogenase (LDH); Mitochondria isolation; NAD+; NAD+/NADH cycling assay; Resazurin; Resorufin
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_15
  8. Cold Spring Harb Mol Case Stud. 2021 Jun;pii: a006081. [Epub ahead of print]7(3):
    Severe multisystem pathology, metabolic acidosis, mitochondrial dysfunction, and early death associated with an X-linked AIFM1 variant.
    Tonya Moss, Melanie May, Heather Flanagan-Steet, Raymond Caylor, Yong-Hui Jiang, Marie McDonald, Michael Friez, Allyn McConkie-Rosell, Richard Steet.
      Variants in the X-linked gene AIFM1 (apoptosis-inducing factor mitochondria-associated 1) are associated with a highly variable clinical presentation that encompasses motor neuropathy, ataxia, encephalopathies, deafness, and cognitive impairment. AIFM1 encodes a mitochondrial flavin adenine dinucleotide (FAD)-dependent nicotinamide adenine dinucleotide (NADH) oxidoreductase, with roles in the regulation of respiratory complex assembly and function, production of reactive oxygen species, and the coordination of a caspase-independent type of apoptosis known as parthanatos. In this report, we describe a missense AIFM1 variant (absent in reference population databases; c.506C > T, p.Pro169Leu) identified in the proband and sibling of a family with three affected males. The proband, his brother, and their maternal uncle all exhibited severe multisystem pathology, metabolic acidosis, and early demise. Metabolic testing on the proband revealed normal activity of the pyruvate dehydrogenase complex in skin fibroblasts. Absent or partial deficiency of cytochrome c oxidase was found in muscle fibers, however, supporting a Complex IV mitochondrial deficiency. Functional studies carried out on fibroblasts from the proband demonstrated reduced steady state levels of the AIFM1 protein, decreased Complex I subunit abundance, elevated sensitivity to the apoptosis inducer staurosporine, and increased nuclear condensation when grown in galactose-containing media. The reduced abundance of AIFM1 in the patient cells could not be stabilized with riboflavin or protease inhibitor treatment. Together, these findings suggest that the normal function of the AIFM1 gene product within mitochondria, and its response to apoptotic stimuli, are impaired by this variant, likely accounting for the severity of the phenotype seen in these patients. These findings also imply tissue-specific effects of this variant on different mitochondrial complexes. This study expands the genetic and phenotypic spectrum associated with AIFM1 variants, with the combination of exome sequencing and functional studies allowing a diagnosis to finally be confirmed for this family.
    Keywords:  congenital lactic acidosis; infantile encephalopathy; lethal infantile mitochondrial myopathy; mitochondrial encephalopathy
    DOI:  https://doi.org/10.1101/mcs.a006081
  9. Methods Mol Biol. 2021 ;2310 17-31
    Determination of Oxidative Phosphorylation Complexes Activities.
    João S Teodoro, Ivo F Machado, Carlos M Palmeira, Anabela P Rolo.
      Mitochondria possess a genome that codes for proteins, in the same fashion as the nuclear genome. However, the small, circular mitochondrial DNA (mtDNA) molecule has a reduced base pair content, for it can only code for 2 rRNA, 22 tRNA molecules, and 13 proteins, all of them part of the mitochondrial respiratory chain. As such, all of the other mitochondrial components derive from nuclear genome. This separation leads to a requirement for a well-tuned coordination between both genomes, in order to produce fully functional mitochondria. A vast number of pathologies have been demonstrated to involve, to some extent, alterations in mitochondrial function that, no doubt, can be caused by alterations to the respiratory chain activity. As such, several methods and techniques have been developed to assess both content and function of mitochondrial proteins, in order to help understand mitochondrial involvement on the pathogenesis of disease. In this chapter, we will address some of these methods, with the main focus being on isolated mitochondria.
    Keywords:  Mitochondria; Mitochondrial protein complexes; Polarography; Respiratory chain; Spectrophotometry
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_2
  10. Methods Mol Biol. 2021 ;2310 161-178
    Analysis of Proapoptotic Protein Trafficking to and from Mitochondria.
    Ignacio Vega-Naredo, Gabriela Oliveira, Teresa Cunha-Oliveira, Teresa Serafim, Vilma A Sardão, Paulo J Oliveira.
      Mitochondria play a key role in cell death and its regulation. The permeabilization of the outer mitochondrial membrane, which is mainly controlled by proteins of the BCL-2 family, is a key event that can be directly induced by different signaling pathways, including p53-mediated, and results in the release of proapoptotic factors to the cytosol, such as cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor-of-apoptosis (IAP) binding protein with low pI (SMAC/Diablo), Omi serine protease (Omi/HtrA2), apoptosis-inducing factor (AIF), or endonuclease G (Endo-G). Hence, the determination of subcellular localization of these proteins is extremely important to predict cell fate and elucidate the specific mechanism of apoptosis. Here we describe experimental protocols that can be used to study the subcellular location of different proapoptotic proteins to be used in basic cell biology and toxicology studies.
    Keywords:  Cell fractions; Immunoblot; Immunocytochemistry; Immunoprecipitation; Mitochondria; Proapoptotic proteins
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_10
  11. Methods Mol Biol. 2021 ;2310 69-77
    Update on the Histoenzymatic Methods for Visualization of the Activity of Individual Mitochondrial Respiratory Chain Complexes in the Human Frozen Sections.
    Mariusz R Wieckowski, Maciej Pronicki, Agnieszka Karkucinska-Wieckowska.
      Investigation of mitochondrial metabolism perturbations and successful diagnosis of patients with mitochondrial abnormalities often requires assessment of human samples like muscle or liver biopsy as well as autopsy material. Immunohistochemical and histochemical examination is an important technique to investigate mitochondrial dysfunction that combined with spectrophotometric and Blue Native electrophoresis techniques can be an important tool to provide diagnosis of mitochondrial disorders. In this chapter, we focus on technical description of the methods that are suitable to detect the activity of complex I, II, and IV of mitochondrial respiratory chain in frozen sections of brain, heart, muscle, and liver biopsies/autopsy. The protocols provided can be useful not only for general assessment of mitochondrial activity in studied material, but they are also successfully used in the diagnostic procedures in case of suspicion of mitochondrial disorders. In the age of high-performance NGS sequencing, these methods can be used to confirm whether mutations are pathogenic by proving their impact on the activity of individual respiratory chain complexes.
    Keywords:  Frozen sections; Histoenzymatic methods; Mitochondrial respiratory chain complexes
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_6
  12. JCI Insight. 2021 Jun 08. pii: 144351. [Epub ahead of print]6(11):
    Acyl-CoA synthetase 6 is required for brain docosahexaenoic acid retention and neuroprotection during aging.
    Regina F Fernandez, Andrea S Pereyra, Victoria Diaz, Emily S Wilson, Karen A Litwa, Jonatan Martínez-Gardeazabal, Shelley N Jackson, J Thomas Brenna, Brian P Hermann, Jeffrey B Eells, Jessica M Ellis.
      The omega-3 fatty acid docosahexaenoic acid (DHA) inversely relates to neurological impairments with aging; however, limited nondietary models manipulating brain DHA have hindered a direct linkage. We discovered that loss of long-chain acyl-CoA synthetase 6 in mice (Acsl6-/-) depletes brain membrane phospholipid DHA levels, independent of diet. Here, Acsl6-/- brains contained lower DHA compared with controls across the life span. The loss of DHA- and increased arachidonate-enriched phospholipids were visualized by MALDI imaging predominantly in neuron-rich regions where single-molecule RNA in situ hybridization localized Acsl6 to neurons. ACSL6 is also astrocytic; however, we found that astrocyte-specific ACSL6 depletion did not alter membrane DHA because astrocytes express a non-DHA-preferring ACSL6 variant. Across the life span, Acsl6-/- mice exhibited hyperlocomotion, impairments in working spatial memory, and increased cholesterol biosynthesis genes. Aging caused Acsl6-/- brains to decrease the expression of membrane, bioenergetic, ribosomal, and synaptic genes and increase the expression of immune response genes. With age, the Acsl6-/- cerebellum became inflamed and gliotic. Together, our findings suggest that ACSL6 promotes membrane DHA enrichment in neurons, but not in astrocytes, and is important for neuronal DHA levels across the life span. The loss of ACSL6 impacts motor function, memory, and age-related neuroinflammation, reflecting the importance of neuronal ACSL6-mediated lipid metabolism across the life span.
    Keywords:  Behavior; Eicosanoids; Inflammation; Metabolism; Neurological disorders
    DOI:  https://doi.org/10.1172/jci.insight.144351
  13. Methods Mol Biol. 2021 ;2275 415-432
    Mito-SinCe2 Approach to Analyze Mitochondrial Structure-Function Relationship in Single Cells.
    B Spurlock, K Mitra.
      The cross talk between mitochondrial dynamic structure, determined primarily by mitochondrial fission and fusion events, and mitochondrial function of energetics, primarily ATP and ROS production, is widely appreciated. Understanding the mechanistic details of such cross talk between mitochondrial structure and function needs integrated quantitative analyses between mitochondrial dynamics and energetics. Here we describe our recently designed approach of mito-SinCe2 that involves high resolution confocal microscopy of genetically expressed ratiometric fluorescent probes targeted to mitochondria, and its quantitative analyses. Mito-SinCe2 analyses allows for quantitative analyses of mitochondrial structure-function relationship in single cells toward understanding the role of mitochondria and their heterogeneity in various physiological and pathological conditions.
    Keywords:  ATP; Confocal Microscopy; Fission; Fusion; Mitochondrial Dynamics; Mitochondrial Energetics; Quantitative analyses; Ratiometric Probes; Redox State; Single Cell; Structure–Function Relationships
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_27
  14. Methods Mol Biol. 2021 ;2310 287-299
    Following the Dynamism of the Mitochondrial Network in T Cells.
    Arianna Di Daniele, Luca Simula, Silvia Campello.
      The dynamism of mitochondria, considered as complex and motile organelles, is brought about by mitochondria ability to undergo cycles of fission and fusion events, whose fine balance determines their morphology in a specific physiological context. A huge body of evidence makes it possible to associate mitochondrial organization to regulation of an increasing number of key cellular processes, such as biosynthetic pathways, oxidative phosphorylation and ATP production, calcium buffering, mtDNA homeostasis, autophagy, and cell death. Here, we review the recently developed imaging methods for studying mitochondrial dynamics, including live-cell imaging, by using mitochondrial-targeted fluorescent proteins. In more details, we focus our attention on two different protocols in the T cell model, an example of nonadherent cells, which present some particularities and difficulties in the analysis of mitochondrial shape. Also, we discuss some examples of mouse models carrying mitochondria-targeted fluorescent proteins, which allow to investigate the mitochondrial morphology in vivo.
    Keywords:  Dynamic network; Fission; Fusion; Imaging; Mitochondria; Morphology
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_16
  15. Methods Mol Biol. 2021 ;2275 217-225
    Sequence-Specific Control of Mitochondrial Gene Transcription Using Programmable Synthetic Gene Switches Called MITO-PIPs.
    Takuya Hidaka, Hiroshi Sugiyama, Ganesh N Pandian.
      Mitochondria possess multiple copies of mitochondrial DNA (mtDNA) that encode 37 genes and their transcription and replication get controlled by unique molecular codes different from that in the nuclear DNA. The mtDNA has been gaining increased attention as one of the critical therapeutic targets as mutations in them impair the function of mitochondria and cause mitochondrial diseases like MELAS. In this chapter, we describe artificial control of mitochondrial transcription based on mtDNA sequence information with a new type of compounds termed MITO-PIPs, which encompasses two domains: pyrrole-imidazole polyamide as DNA recognition domain and mitochondrial penetrating peptide as the mitochondria-targeting domain. Because MITO-PIPs are amenable to tunability, they can be expanded as a synthetic strategy to modulate mitochondrial gene(s) on demand.
    Keywords:  DNA binding ligand; Fmoc solid-phase synthesis; MITO-PIP; Mitochondrial DNA; Pyrrole–imidazole polyamide; Transcription
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_13
  16. Methods Mol Biol. 2021 ;2275 329-339
    Mitochondrial Coenzyme Q10 Determination Via Isotope Dilution Liquid Chromatography -Tandem Mass Spectrometry.
    Erik Hedman, Outi Itkonen.
      Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain . Here, we describe an accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of mitochondrial CoQ10 in isolated mitochondria . In the assay, mitochondrial suspensions are spiked with CoQ10-[2H9] internal standard (IS), extracted with organic solvents and CoQ10 quantified by LC-MS/MS using multiple reaction monitoring (MRM).
    Keywords:  Coenzyme Q10; Isotope dilution; LC-MS/MS; Mitochondrial disease; Ubiquinone
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_21
  17. Proc Natl Acad Sci U S A. 2021 Jun 15. pii: e2024176118. [Epub ahead of print]118(24):
    Accurate genomic variant detection in single cells with primary template-directed amplification.
    Veronica Gonzalez-Pena, Sivaraman Natarajan, Yuntao Xia, David Klein, Robert Carter, Yakun Pang, Bridget Shaner, Kavya Annu, Daniel Putnam, Wenan Chen, Jon Connelly, Shondra Pruett-Miller, Xiang Chen, John Easton, Charles Gawad.
      Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.
    Keywords:  genome editing off-target; mutagenesis; single-cell sequencing; whole genome amplification
    DOI:  https://doi.org/10.1073/pnas.2024176118
  18. Methods Mol Biol. 2021 ;2310 91-111
    PCR-Based Determination of Mitochondrial DNA Copy Number in Multiple Species.
    Tess C Leuthner, Jessica H Hartman, Ian T Ryde, Joel N Meyer.
      Mitochondrial DNA (mtDNA) copy number is a critical component of overall mitochondrial health. In this chapter, we describe methods for simultaneous isolation of mtDNA and nuclear DNA (nucDNA), and measurement of their respective copy numbers using quantitative PCR. Methods differ depending on the species and cell type of the starting material, and availability of specific PCR reagents. We also briefly describe factors that affect mtDNA copy number and discuss caveats to its use as a biomarker.
    Keywords:  Copy number; Mitochondrial DNA; Mitochondrial disease; Mitochondrial toxicity; QPCR; mtDNA; mtDNA depletion
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_8
  19. Methods Mol Biol. 2021 ;2275 393-402
    Live Imaging of Mitochondria in Kidney Tissue.
    Milica Bugarski, Susan Ghazi, Andrew M Hall.
      Kidneys are highly aerobic organs and their function is tightly coupled to mitochondrial energy production. Renal tubular cells, particularly the proximal tubule (PT), require an abundance of mitochondria to provide sufficient energy for regulating fluid and electrolyte balance. Meanwhile, mitochondrial defects are implicated in a range of different kidney diseases. Multiphoton microscopy (MP) is a powerful tool that allows detailed study of mitochondrial morphology, dynamics, and function in kidney tissue. Here, we describe how MP can be used to image mitochondria in kidney tubular cells, either ex vivo in tissue slices or in vivo in living rodents, using both endogenous and exogenous fluorescent molecules. Moreover, changes in mitochondrial signals can be followed in real time in response to different insults or stimuli, in parallel with other important readouts of kidney tubular function, such as solute uptake and trafficking.
    Keywords:  Fluorescence imaging; Kidney; Mitochondria; Multiphoton microscopy
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_25
  20. Child Neurol Open. 2021 Jan-Dec;8:8 2329048X211019173
    Perinatal Manifestations of DARS2-Associated Leukoencephalopathy With Brainstem and Spinal Cord Involvement and Lactate Elevation (LBSL).
    Julie Ngo, Jeremy W Prokop, Jason Umfleet, Laurie H Seaver.
      Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a progressive disorder associated with deficiency of mitochondrial aspartyl-tRNA synthetase, a homodimer encoded by the gene DARS2. There is a wide range in age of onset of symptoms, typically from childhood to adulthood, with very few cases of infantile onset disease reported. We report a child at age 10 years with perinatal onset of symptoms evidenced by congenital microcephaly with progression to severe but non-lethal epileptic encephalopathy and spastic quadriplegia. A comprehensive epilepsy focused gene panel performed as a trio with parents detected a novel homozygous DARS2 variant. This variant is located at the dimer interface in a critical catalytic domain and is expected to result in markedly reduced enzyme activity which likely explains the severe and early onset symptoms in this case.
    Keywords:  DARS2; LBSL; epileptic encephalopathy; leukoencephalopathy; mitochondrial disease; neuroimaging; spasticity
    DOI:  https://doi.org/10.1177/2329048X211019173
  21. Methods Mol Biol. 2021 ;2310 79-89
    An Update on Isolation of Functional Mitochondria from Cells for Bioenergetics Studies.
    Magdalena Lebiedzinska-Arciszewska, Lech Wojtczak, Mariusz R Wieckowski.
      Mitochondria are the organelles where the most fundamental processes of energy transformation within the cell are located. They are also involved in several processes like apoptosis and autophagy, reactive oxygen species formation, and calcium signaling, which are crucial for proper cell functioning. In addition, mitochondrial genome hosts genes encoding important proteins incorporated in respiratory chain complexes and indispensable for the oxidative phosphorylation. Studying isolated mitochondria is, therefore, crucial for better understanding of cell physiology. The presented protocol describes a relatively simple and handy method for crude mitochondrial fraction isolation from different mammalian cell lines. It includes mechanical cells disruption (homogenization) and differential centrifugation. In addition, this chapter presents two basic ways to assess mitochondrial functionality: by measuring mitochondrial inner membrane potential and coupled respiration.
    Keywords:  Cell cultures; Mitochondria isolation; Mitochondrial membrane potential; Oxidative phosphorylation; Oxygen consumption
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_7
  22. Proc Natl Acad Sci U S A. 2021 Jun 15. pii: e2025053118. [Epub ahead of print]118(24):
    ALS- and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy.
    Olivia Harding, Chantell S Evans, Junqiang Ye, Jonah Cheung, Tom Maniatis, Erika L F Holzbaur.
      TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia, including missense mutations that disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type-specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals.
    Keywords:  OPTN; Parkin; TBK1; mitophagy; neurodegeneration
    DOI:  https://doi.org/10.1073/pnas.2025053118
  23. Methods Mol Biol. 2021 ;2275 363-378
    Split Green Fluorescent Protein-Based Contact Site Sensor (SPLICS ) for Heterotypic Organelle Juxtaposition as Applied to ER -Mitochondria Proximities.
    Tito Calì, Marisa Brini.
      In the last decades, membrane contact sites (MCSs) have been the object of intense investigation in different fields of cell physiology and pathology and their importance for the correct functioning of the cell is now widely recognized. MCS between any known intercellular organelles, including endoplasmic reticulum (ER), mitochondria, Golgi, endosomes, peroxisomes, lysosomes, lipid droplets, and the plasma membrane (PM), have been largely documented and in some cases the molecules responsible for the tethering also identified. They represent specific membrane hubs where a tightly coordinated exchange of ions, lipids, nutrients, and factors required to maintain proper cellular homeostasis takes place. Their delicate, dynamic, and sometimes elusive nature prevented and/or delayed the development of tools to easily image interorganelle proximity under physiological conditions and in living organisms. Nowadays, this aspect received great momentum due to the finding that MCSs' dysregulation is involved in several pathological conditions. We have recently developed modular, split-GFP-based contact site sensors (SPLICS) engineered to fluoresce when homo- and heterotypic juxtapositions between ER and mitochondria occur over a range of specific distances. Here we describe in detail, by highlighting strengths and weaknesses, the use and the application of these novel genetically encoded SPLICS sensors and how to properly quantify short- and long-range ER-mitochondria interactions.
    Keywords:  ER–Mitochondria tethering; Endoplasmic reticulum; Mitochondria; Organelle contact sites; SPLICS; Split GFP
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_23
  24. DNA Repair (Amst). 2021 May 11. pii: S1568-7864(21)00090-2. [Epub ahead of print]105 103134
    Perspectives on formaldehyde dysregulation: Mitochondrial DNA damage and repair in mammalian cells.
    Cristina A Nadalutti, Rajendra Prasad, Samuel H Wilson.
      Maintaining genome stability involves coordination between different subcellular compartments providing cells with DNA repair systems that safeguard against environmental and endogenous stresses. Organisms produce the chemically reactive molecule formaldehyde as a component of one-carbon metabolism, and cells maintain systems to regulate endogenous levels of formaldehyde under physiological conditions, preventing genotoxicity, among other adverse effects. Dysregulation of formaldehyde is associated with several diseases, including cancer and neurodegenerative disorders. In the present review, we discuss the complex topic of endogenous formaldehyde metabolism and summarize advances in research on fo dysregulation, along with future research perspectives.
    Keywords:  DNA damage; Formaldehyde; Mitochondrial DNA; One carbon metabolism
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103134
  25. Ageing Res Rev. 2021 Jun 04. pii: S1568-1637(21)00125-2. [Epub ahead of print]70 101378
    Mitochondria: Their relevance during oocyte ageing.
    Jiska van der Reest, Gustavo Nardini Cecchino, Marcia C Haigis, Paweł Kordowitzki.
      The oocyte is recognised as the largest cell in mammalian species and other multicellular organisms. Mitochondria represent a high proportion of the cytoplasm in oocytes and mitochondrial architecture is different in oocytes than in somatic cells, characterised by a rounder appearance and fragmented network. Although the number of mitochondria per oocyte is higher than in any other mammalian cell, their number and activity decrease with advancing age. Mitochondria integrate numerous processes essential for cellular function, such as metabolic processes related to energy production, biosynthesis, and waste removal, as well as Ca2+ signalling and reactive oxygen species (ROS) homeostasis. Further, mitochondria are responsible for the cellular adaptation to different types of stressors such as oxidative stress or DNA damage. When these stressors outstrip the adaptive capacity of mitochondria to restore homeostasis, it leads to mitochondrial dysfunction. Decades of studies indicate that mitochondrial function is multifaceted, which is reflected in the oocyte, where mitochondria support numerous processes during oocyte maturation, fertilization, and early embryonic development. Dysregulation of mitochondrial processes has been consistently reported in ageing and age-related diseases. In this review, we describe the functions of mitochondria as bioenergetic powerhouses and signal transducers in oocytes, how dysfunction of mitochondrial processes contributes to reproductive ageing, and whether mitochondria could be targeted to promote oocyte rejuvenation.
    Keywords:  Metabolism; Mitochondria; Oocytes; Oxidative stress; Reproductive ageing
    DOI:  https://doi.org/10.1016/j.arr.2021.101378
  26. Methods Mol Biol. 2021 ;2310 201-246
    Protocols for Mitochondria as the Target of Pharmacological Therapy in the Context of Nonalcoholic Fatty Liver Disease (NAFLD).
    Ignazio Grattagliano, Agostino Di Ciaula, Jacek Baj, Emilio Molina-Molina, Harshitha Shanmugam, Gabriella Garruti, David Q-H Wang, Piero Portincasa.
      Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent metabolic chronic liver diseases in developed countries and puts the populations at risk of progression to liver necro-inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Mitochondrial dysfunction is involved in the onset of NAFLD and contributes to the progression from NAFLD to nonalcoholic steatohepatitis (NASH). Thus, liver mitochondria could become the target for treatments for improving liver function in NAFLD patients. This chapter describes the most important steps used for potential therapeutic interventions in NAFLD patients, discusses current options gathered from both experimental and clinical evidence, and presents some novel options for potentially improving mitochondrial function in NAFLD.
    Keywords:  Fatty liver; Mitochondria; Nitrosative stress; Nonalcoholic fatty liver disease; Oxidative stress
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_12
  27. Methods Mol Biol. 2021 ;2310 113-159
    Methods to Monitor Mitophagy and Mitochondrial Quality: Implications in Cancer, Neurodegeneration, and Cardiovascular Diseases.
    Simone Patergnani, Massimo Bonora, Esmaa Bouhamida, Alberto Danese, Saverio Marchi, Giampaolo Morciano, Maurizio Previati, Gaia Pedriali, Alessandro Rimessi, Gabriele Anania, Carlotta Giorgi, Paolo Pinton.
      Mitochondria are dynamic organelles that participate in a broad array of molecular functions within the cell. They are responsible for maintaining the appropriate energetic levels and control the cellular homeostasis throughout the generation of intermediary metabolites. Preserving a healthy and functional mitochondrial population is of fundamental importance throughout the life of the cells under pathophysiological conditions. Hence, cells have evolved fine-tuned mechanisms of quality control that help to preserve the right amount of functional mitochondria to meet the demand of the cell. The specific recycling of mitochondria by autophagy, termed mitophagy, represents the primary contributor to mitochondrial quality control. During this process, damaged or unnecessary mitochondria are recognized and selectively degraded. In the past few years, the knowledge in mitophagy has seen rapid progress, and a growing body of evidence confirms that mitophagy holds a central role in controlling cellular functions and the progression of various human diseases.In this chapter, we will discuss the pathophysiological roles of mitophagy and provide a general overview of the current methods used to monitor and quantify mitophagy. We will also outline the main established approaches to investigate the mitochondrial function, metabolism, morphology, and protein damage.
    Keywords:  Cardiovascular diseases (CVD); Homeostasis; Metabolism; Mitochondrial morphology; Mitochondrial quality control; Pathology
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_9
  28. EMBO Rep. 2021 Jun 04. 22(6): e51323
    Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes.
    Hyun Ku Yeo, Tae Hyun Park, Hee Yeon Kim, Hyonchol Jang, Jueun Lee, Geum-Sook Hwang, Seong Eon Ryu, Si Hoon Park, Hyun Kyu Song, Hyun Seung Ban, Hye-Jin Yoon, Byung Il Lee.
      In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51-VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function.
    Keywords:  MAM; PTPIP51; endoplasmic reticulum; mitochondria; phospholipid
    DOI:  https://doi.org/10.15252/embr.202051323
  29. Methods Mol Biol. 2021 ;2275 379-391
    Qualitative Characterization of the Rat Liver Mitochondrial Lipidome Using All Ion Fragmentation on an Exactive Benchtop Orbitrap MS.
    Irina G Stavrovskaya, Rose M Gathungu, Susan S Bird, Bruce S Kristal.
      Untargeted lipidomics profiling by liquid chromatography -mass spectrometry (LC-MS) allows researchers to observe the occurrences of lipids in a biological sample without showing intentional bias to any specific class of lipids and allows retrospective reanalysis of data collected. Typically, and in the specific method described, a general extraction method followed by LC separation is used to achieve nonspecific class coverage of the lipidome prior to high resolution accurate mass (HRAM) MS detection . Here we describe a workflow including the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition. We also highlight how, in this method, all ion fragmentation can be used to identify species of lower abundances, often missed by data dependent fragmentation techniques. Here we describe the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition.
    Keywords:  Cardiolipins; HCD; LC-MS; Lipidomics; Lysophospholipids; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_24
  30. Methods Mol Biol. 2021 ;2275 247-263
    mTRIP, an Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Resolution.
    Laurent Chatre, Miria Ricchetti.
      Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling of mtDNA, mtRNA, and proteins or mitochondria, respectively. Altogether, qualitative and quantitative alterations of the dynamics of mtDNA processing are detected by mTRIP in human cells undergoing physiological changes, as well as stress and dysfunction. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.
    Keywords:  DNA replication; FISH; Imaging; Mitochondrial DNA; Mitochondrial disease; Single-cell; Transcription; mTRIP
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_15
  31. EMBO J. 2021 Jun 08. e106438
    An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial membrane.
    Fujiao Lv, Fei Qi, Zhi Zhang, Maorong Wen, Justin Kale, Alessandro Piai, Lingyu Du, Shuqing Wang, Liujuan Zhou, Yaqing Yang, Bin Wu, Zhijun Liu, Juan Del Rosario, Justin Pogmore, James J Chou, David W Andrews, Jialing Lin, Bo OuYang.
      Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.
    Keywords:  NMR structure; bax core dimer; functional mutagenesis; membrane lipid bilayer; pore formation
    DOI:  https://doi.org/10.15252/embj.2020106438
  32. Methods Mol Biol. 2021 ;2310 259-270
    Mitochondrial Regulation Assessment by 13C-NMR Isotopomer Analysis.
    Francisco X Carvalho, Bárbara Guerra-Carvalho, Ivana Jarak, Rui A Carvalho.
      Mitochondria play a central role in metabolic reprograming that occurs in numerous disease conditions. A precise evaluation of the extent of mitochondrial involvement in the metabolic alterations is essential for a better definition of metabolically based therapeutic strategies. In this chapter, some simple protocols are presented, using carbon 13 tracers and nuclear magnetic resonance isotopomer analysis, for the evaluation of mitochondrial contributions to intermediary metabolism and the metabolic effects of the implementation of some mitochondrial regulatory mechanisms.
    Keywords:  13C isotope tracers; Intermediary metabolism; Metabolic reprograming; Mitochondria; Mitochondrial regulation; NMR isotopomer analysis; TCA cycle
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_14
  33. Methods Mol Biol. 2021 ;2275 291-299
    Live-Cell Assessment of Reactive Oxygen Species Levels Using Dihydroethidine.
    Sander Grefte, Werner J H Koopman.
      Reactive oxygen species (ROS) play an important role in cellular (patho)physiology. Empirical evidence suggests that mitochondria are an important source of ROS, especially under pathological conditions. Here, we describe a method for ROS measurement using dihydroethidium (HEt) and live-cell microscopy.
    Keywords:  Fluorescence imaging; MitoSOX Red®; Mitochondrial membrane potential
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_18
  34. Cell Rep. 2021 Jun 08. pii: S2211-1247(21)00535-0. [Epub ahead of print]35(10): 109189
    An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of α-synuclein.
    Eleanna Kara, Alessandro Crimi, Anne Wiedmer, Marc Emmenegger, Claudia Manzoni, Sara Bandres-Ciga, Karishma D'Sa, Regina H Reynolds, Juan A Botía, Marco Losa, Veronika Lysenko, Manfredi Carta, Daniel Heinzer, Merve Avar, Andra Chincisan, Cornelis Blauwendraat, Sonia García-Ruiz, Daniel Pease, Lorene Mottier, Alessandra Carrella, Dezirae Beck-Schneider, Andreia D Magalhães, Caroline Aemisegger, Alexandre P A Theocharides, Zhanyun Fan, Jordan D Marks, Sarah C Hopp, Andrey Y Abramov, Patrick A Lewis, Mina Ryten, John Hardy, Bradley T Hyman, Adriano Aguzzi.
      Neuropathological and experimental evidence suggests that the cell-to-cell transfer of α-synuclein has an important role in the pathogenesis of Parkinson's disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook a small interfering RNA (siRNA), genome-wide screen to identify genes regulating the cell-to-cell transfer of α-synuclein. A genetically encoded reporter, GFP-2A-αSynuclein-RFP, suitable for separating donor and recipient cells, was transiently transfected into HEK cells stably overexpressing α-synuclein. We find that 38 genes regulate the transfer of α-synuclein-RFP, one of which is ITGA8, a candidate gene identified through a recent PD genome-wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) show that those hits cluster in networks that include known PD genes more frequently than expected by random chance. The findings expand our understanding of the mechanism of α-synuclein spread.
    Keywords:  Braak hypothesis; GWAS; ITGA8; high-throughput screen; siRNA; weighted gene co-expression network analysis; weighted protein-protein network interaction analysis; α-synuclein
    DOI:  https://doi.org/10.1016/j.celrep.2021.109189
  35. Methods Mol Biol. 2021 ;2310 179-199
    Exploring Liver Mitochondrial Function by 13C-Stable Isotope Breath Tests: Implications in Clinical Biochemistry.
    Emilio Molina-Molina, Harshitha Shanmugam, Domenica Di Palo, Ignazio Grattagliano, Piero Portincasa.
      The liver is at the crossroad of key metabolic processes, which include detoxification, glycolipidic storage and export, and protein synthesis. The gut-liver axis, moreover, provides hepatocytes with a series of bacterial products and metabolites, which contribute to maintain liver function in health and disease. Breath tests (BTs) are developed as diagnostic tools for indirect, rapid, noninvasive assessment of several metabolic processes in the liver. BTs monitor the appearance of CO2 in breath as a marker of a specific substrate metabolized in the liver, typically within microsomes, cytosol, or mitochondria. The noninvasiveness of BTs originates from the use of the, nonradioactive, naturally occurring stable isotope 13C marking a specific substrate which is metabolized in the liver, leading to the appearance of 13CO2 in expired air. Some substrates (ketoisocaproic acid, methionine, and octanoic acid) provide information about dynamic liver mitochondrial function in health and disease. In humans, the application of 13C-breath tests ranges from nonalcoholic and alcoholic liver diseases to liver cirrhosis, hepatocarcinoma, preoperative and postoperative assessment of liver function, and drug-induced liver damage. 13C-BTs are an indirect, cost-effective, and easy method to evaluate dynamic liver function and gastric kinetics in health and disease, with ongoing studies focusing on further applications in clinical medicine.
    Keywords:  Breath test; Hepatic mitochondrial function; Hepatocellular carcinoma; Ketoisocaproic acid; Liver diseases; Liver steatosis; Methionine; Octanoic acid; β-oxidation
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_11
  36. Methods Mol Biol. 2021 ;2275 433-452
    Computer-Aided Prediction of Protein Mitochondrial Localization.
    Pier Luigi Martelli, Castrense Savojardo, Piero Fariselli, Giacomo Tartari, Rita Casadio.
      Protein sequences, directly translated from genomic data, need functional and structural annotation. Together with molecular function and biological process, subcellular localization is an important feature necessary for understanding the protein role and the compartment where the mature protein is active. In the case of mitochondrial proteins, their precursor sequences translated by the ribosome machinery include specific patterns from which it is possible not only to recognize their final destination within the organelle but also which of the mitochondrial subcompartments the protein is intended for. Four compartments are routinely discriminated, including the inner and the outer membranes, the intermembrane space, and the matrix. Here we discuss to which extent it is feasible to develop computational methods for detecting mitochondrial targeting peptides in the precursor sequence and to discriminate their final destination in the organelle. We benchmark two of our methods on the general task of recognizing human mitochondrial proteins endowed with an experimentally characterized targeting peptide (TPpred3) and predicting which submitochondrial compartment is the final destination (DeepMito). We describe how to adopt our web servers in order to discriminate which human proteins are endowed with mitochondrial targeting peptides, the position of cleavage sites, and which submitochondrial compartment are intended for. By this, we add some other 1788 human proteins to the 450 ones already manually annotated in UniProt with a mitochondrial targeting peptide, providing for each of them also the characterization of the suborganellar localization.
    Keywords:  Arginine motifs; Cleavage site; Machine and deep learning; Prediction of subcellular localization; Targeting peptide
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_28
  37. Methods Mol Biol. 2021 ;2275 65-85
    Novel Mitochondria-Targeted Triphenylphosphonium Conjugates of Linear β-Phosphorylated Nitrones : Preparation, 31P NMR Mitochondrial Distribution, EPR Spin Trapping Reporting, and Site-Directed Antiapoptotic Properties.
    Consuelo Petrocchi, Sophie Thétiot-Laurent, Marcel Culcasi, Sylvia Pietri.
      The mitochondrion can be considered as the metabolic powerhouse of the cell, having a key impact on energy production, cell respiration, and intrinsic cell death. Mitochondria are also the main source of endogenous reactive oxygen species , including free radicals (FR), which are physiologically involved in signaling pathways but may promote cell damage when unregulated or excessively formed in inappropriate locations. A variety of chronic pathologies have been associated with FR-induced mitochondrial dysfunctions , such as cancer, age-related neurodegenerative diseases, and metabolic syndrome.In recent years drug design based on specific mitochondria-targeted antioxidants has become a very attractive therapeutic strategy and, among target compounds, nitrones have received growing attention because of their specific affinity toward FR. Here, we describe protocols dealing with the preparation, mitochondria permeation assessment, electron paramagnetic resonance (EPR) spin trapping setting, and antiapoptotic properties evaluation of a series of new linear nitrones vectorized by a triphenylphosphonium cation and labeled with a diethoxyphosphoryl moiety as 31P nuclear magnetic resonance (NMR) reporter with antioxidant property.
    Keywords:  31P NMR; EPR spin trapping; H2O2-induced apoptosis; Mitochondria-targeted nitrones; Mitochondrial permeability
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_5
  38. Methods Mol Biol. 2021 ;2275 301-314
    Protein Supercomplex Recording in Living Cells Via Position-Specific Fluorescence Lifetime Sensors.
    Bettina Rieger, Karin B Busch.
      Our group has previously established a strategy utilizing fluorescence lifetime probes to image membrane protein supercomplex (SC) formation in situ. We showed that a probe at the interface between individual mitochondrial respiratory complexes exhibits a decreased fluorescence lifetime when a supercomplex is formed. This is caused by electrostatic interactions with the adjacent proteins. Fluorescence lifetime imaging microscopy (FLIM) records the resulting decrease of the lifetime of the SC-probe. Here we present the details of our method for performing SC-FLIM, including the evaluation of fluorescence lifetimes from the FLIM images. To validate the feasibility of the technique for monitoring adaptive SC formation, we compare data obtained under different metabolic conditions. The results confirm that SC formation is dynamic.
    Keywords:  FLIM; Fluorescence sensor; Live cell imaging; Mitochondria; Respiratory supercomplexes
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_19
  39. Methods Mol Biol. 2021 ;2275 227-245
    Targeting the Mitochondrial Genome Via a MITO-Porter : Evaluation of mtDNA and mtRNA Levels and Mitochondrial Function.
    Yuma Yamada, Hideyoshi Harashima.
      Genetic mutations and defects in mitochondrial DNA (mtDNA) are associated with certain types of mitochondrial dysfunctions, ultimately resulting in the emergence of a variety of human diseases. To achieve an effective mitochondrial gene therapy, it will be necessary to deliver therapeutic agents to the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool. We recently developed a MITO-Porter, a liposome-based nanocarrier that delivers cargo to mitochondria via a membrane-fusion mechanism. In this chapter, we discuss the methodology used to deliver bioactive molecules to the mitochondrial matrix using a Dual Function (DF)-MITO-Porter, a liposome-based nanocarrier that delivers it cargo by means of a stepwise process, and an evaluation of mtDNA levels and mitochondrial activities in living cells. We also discuss mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using the MITO-Porter system.
    Keywords:  MITO-Porter; Mitochondria; Mitochondrial DNA; Mitochondrial RNA knockdown; Mitochondrial drug delivery; Mitochondrial gene therapy; Mitochondrial matrix; Nucleic acid delivery
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_14
  40. Methods Mol Biol. 2021 ;2275 403-414
    Time-Resolved Imaging of Mitochondrial Flavin Fluorescence and Its Applications for Evaluating the Oxidative State in Living Cardiac Cells.
    A Marcek Chorvatova, M Cagalinec, D Chorvat.
      Time-resolved fluorescence spectrometry is a highly valuable technological tool to detect and characterize mitochondrial metabolic oxidative changes by means of endogenous fluorescence. Here, we describe detection and measurement of endogenous mitochondrial flavin fluorescence directly in living cardiac cells using fluorescence lifetime imaging microscopy (FLIM) after excitation with 473 nm picoseconds (ps) laser. Time-correlated single photon counting (TCSPC) method is employed.
    Keywords:  Endogenous flavin fluorescence; Energy metabolism; FLIM; Mitochondrial oxidative state; TCSPC
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_26
  41. Redox Biol. 2021 Jun 01. pii: S2213-2317(21)00179-8. [Epub ahead of print]45 102021
    Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes.
    Sehwan Jang, Xavier R Chapa-Dubocq, Yulia Y Tyurina, Claudette M St Croix, Alexandr A Kapralov, Vladimir A Tyurin, Hülya Bayır, Valerian E Kagan, Sabzali Javadov.
      Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10) and the oxoglutarate carrier (OGC, SLC25A11) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro. In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.
    Keywords:  Ferroptosis; Glutathione; Heart; Ischemia-reperfusion; Mitochondria; Oxidized phosphatidylethanolamine
    DOI:  https://doi.org/10.1016/j.redox.2021.102021
  42. PLoS Comput Biol. 2021 Jun 09. 17(6): e1009073
    Optimizing mitochondrial maintenance in extended neuronal projections.
    Anamika Agrawal, Elena F Koslover.
      Neurons rely on localized mitochondria to fulfill spatially heterogeneous metabolic demands. Mitochondrial aging occurs on timescales shorter than the neuronal lifespan, necessitating transport of fresh material from the soma. Maintaining an optimal distribution of healthy mitochondria requires an interplay between a stationary pool localized to sites of high metabolic demand and a motile pool capable of delivering new material. Interchange between these pools can occur via transient fusion / fission events or by halting and restarting entire mitochondria. Our quantitative model of neuronal mitostasis identifies key parameters that govern steady-state mitochondrial health at discrete locations. Very infrequent exchange between stationary and motile pools optimizes this system. Exchange via transient fusion allows for robust maintenance, which can be further improved by selective recycling through mitophagy. These results provide a framework for quantifying how perturbations in organelle transport and interactions affect mitochondrial homeostasis in neurons, a key aspect underlying many neurodegenerative disorders.
    DOI:  https://doi.org/10.1371/journal.pcbi.1009073
  43. BMC Bioinformatics. 2021 Jun 05. 22(1): 304
    2-kupl: mapping-free variant detection from DNA-seq data of matched samples.
    Yunfeng Wang, Haoliang Xue, Christine Pourcel, Yang Du, Daniel Gautheret.
      BACKGROUND: The detection of genome variants, including point mutations, indels and structural variants, is a fundamental and challenging computational problem. We address here the problem of variant detection between two deep-sequencing (DNA-seq) samples, such as two human samples from an individual patient, or two samples from distinct bacterial strains. The preferred strategy in such a case is to align each sample to a common reference genome, collect all variants and compare these variants between samples. Such mapping-based protocols have several limitations. DNA sequences with large indels, aggregated mutations and structural variants are hard to map to the reference. Furthermore, DNA sequences cannot be mapped reliably to genomic low complexity regions and repeats.RESULTS: We introduce 2-kupl, a k-mer based, mapping-free protocol to detect variants between two DNA-seq samples. On simulated and actual data, 2-kupl achieves higher accuracy than other mapping-free protocols. Applying 2-kupl to prostate cancer whole exome sequencing data, we identify a number of candidate variants in hard-to-map regions and propose potential novel recurrent variants in this disease.
    CONCLUSIONS: We developed a mapping-free protocol for variant calling between matched DNA-seq samples. Our protocol is suitable for variant detection in unmappable genome regions or in the absence of a reference genome.
    Keywords:  Contigs; DNAseq; Mapping-free; PRAD; Recurrent variants; WES; WGS; k-mers
    DOI:  https://doi.org/10.1186/s12859-021-04185-6
  44. Nat Protoc. 2021 Jun 09.
    iDRiP for the systematic discovery of proteins bound directly to noncoding RNA.
    Hsueh-Ping Chu, Anand Minajigi, Yunfei Chen, Robert Morris, Chia-Yu Guh, Yu-Hung Hsieh, Myriam Boukhali, Wilhelm Haas, Jeannie T Lee.
      More than 90% of the human genome is transcribed into noncoding RNAs, but their functional characterization has lagged behind. A major bottleneck in the understanding of their functions and mechanisms has been a dearth of systematic methods for identifying interacting protein partners. There now exist several methods, including identification of direct RNA interacting proteins (iDRiP), chromatin isolation by RNA purification (ChIRP), and RNA antisense purification, each previously applied towards identifying a proteome for the prototype noncoding RNA, Xist. iDRiP has recently been modified to successfully identify proteomes for two additional noncoding RNAs of interest, TERRA and U1 RNA. Here we describe the modified protocol in detail, highlighting technical differences that facilitate capture of various noncoding RNAs. The protocol can be applied to short and long RNAs in both cultured cells and tissues, and requires ~1 week from start to finish. Here we also perform a comparative analysis between iDRiP and ChIRP. We obtain partially overlapping profiles, but find that iDRiP yields a greater number of specific proteins and fewer mitochondrial contaminants. With an increasing number of essential long noncoding RNAs being described, robust RNA-centric protein capture methods are critical for the probing of noncoding RNA function and mechanism.
    DOI:  https://doi.org/10.1038/s41596-021-00555-9
  45. FEBS J. 2021 Jun 06.
    Resurrection and characterization of ancestral CYP11A1 enzymes.
    Philip Hartz, Silja J Strohmaier, Basma M El-Gayar, Ammar Abdulmughni, Michael C Hutter, Frank Hannemann, Elizabeth M J Gillam, Rita Bernhardt.
      Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (~25 °C increase in T50 ) compared to the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated by CYP11A_N1, may have allowed them to stay active when presented with incompatible electron transfer systems and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.
    Keywords:  Ancestral sequence reconstruction; CYP11A1; cholesterol; cytochromes P450; pregnenolone formation; protein engineering; side-chain cleavage; steroid hormone biosynthesis; thermostable enzymes
    DOI:  https://doi.org/10.1111/febs.16054
  46. J Cell Sci. 2021 Jun 09. pii: jcs.258399. [Epub ahead of print]
    Maintenance of complex I and its super-complexes by NDUF-11 is essential for mitochondrial structure, function and health.
    Amber Knapp-Wilson, Gonçalo C Pereira, Emma Buzzard, Holly C Ford, Andrew Richardson, Robin A Corey, Chris Neal, Paul Verkade, Andrew P Halestrap, Vicki A M Gold, Patricia Kuwabara, Ian Collinson.
      Mitochondrial super-complexes form around a conserved core of monomeric complex I and dimeric complex III; wherein subunit NDUFA11, of the former, is conspicuously situated at the interface. We identified B0491.5 (NDUF-11) as the C. elegans homologue, of which animals homozygous for a CRISPR-Cas9 generated knockout allele arrested at the L2 development stage. Reducing (but not eliminating) expression by RNAi allowed development to adulthood, enabling characterisation of the consequences: destabilisation of complex I and its super-complexes, and perturbation of respiratory function. The loss of NADH-dehydrogenase activity is compensated by enhanced complex II activity, with the potential for detrimental ROS-production. Electron cryo-tomography highlight aberrant cristae morphology and inter-membrane-space widening and cristae-junctions. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development presumably arises due to its involvement in complex I/ super-complex maintenance. This highlights the importance of respiratory complex integrity for health and the potential of its perturbation for mitochondrial disease.
    Keywords:  Caenorhabditis elegans; Electron-transfer chain; Mitochondria; Mitochondrial ultrastructure; electron cryo-tomography; NDUF-11; Respirasome; Respiration; Super-complexes; Worm
    DOI:  https://doi.org/10.1242/jcs.258399
  47. Eur Heart J. 2021 Jun 09. pii: ehab247. [Epub ahead of print]
    Myocardial hypoxic stress mediates functional cardiac extracellular vesicle release.
    Achille Anselmo, Derk Frank, Laura Papa, Chiara Viviani Anselmi, Elisa Di Pasquale, Marta Mazzola, Cristina Panico, Francesca Clemente, Cristiana Soldani, Christina Pagiatakis, Rabea Hinkel, Ruth Thalmann, Reiner Kozlik-Feldmann, Michele Miragoli, Pierluigi Carullo, Marco Vacchiano, Antonio Chaves-Sanjuan, Nadia Santo, Maria Angela Losi, Matteo Carlo Ferrari, Annibale Alessandro Puca, Vincent Christiansen, Hatim Seoudy, Sandra Freitag-Wolf, Norbert Frey, Astrid Dempfle, Mark Mercola, Giovanni Esposito, Carlo Briguori, Christian Kupatt, Gianluigi Condorelli.
      AIMS : Increased shedding of extracellular vesicles (EVs)-small, lipid bilayer-delimited particles with a role in paracrine signalling-has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown.METHODS AND RESULTS : Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts.
    CONCLUSION : We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.
    Keywords:  Aortic stenosis; CD172a; Cardiomyocytes; Extracellular vesicles; Myocardium
    DOI:  https://doi.org/10.1093/eurheartj/ehab247
  48. J Cell Sci. 2021 Jun 10. pii: jcs.253443. [Epub ahead of print]
    The role of Mfn2 in the structure and function of ER-mitochondrial tethering in vivo.
    Song Han, Fanpeng Zhao, Jeffrey Hsia, Xiaopin Ma, Yi Liu, Sandy Torres, Hisashi Fujioka, Xiongwei Zhu.
      The mitochondria-ER contacts (MERCs) plays an essential role in multiple cell physiological process. While Mfn2 was the first protein implicated in the formation of MERCs, it is debated whether it acts as a tether or antagonizer, largely based on in vitro studies. To understand the role of Mfn2 in MERCs in vivo, we characterized ultrastructural and biochemical changes of MERCs in pyramidal neurons of hippocampus in Mfn2 conditional knockout (KO) mice and in Mfn2 overexpression (OE) mice and found Mfn2 ablation caused reduced close contacts while Mfn2 OE caused increased close contacts between ER and mitochondria in vivo. Functional studies on SH-SY5Y cells with Mfn2 KO or overexpression demonstrating similar biochemical changes found that mitochondrial calcium uptake along with IP3R3-Grp75 interaction was decreased in Mfn2 KO cells but increased in the Mfn2 OE cells. Lastly, we found Mfn2 KO decreased and Mfn2 OE increased the interaction between the ER-mitochondria tethering pair of VAPB-PTPIP51. In conclusion, our study supports the notion that Mfn2 plays a critical role in ER-mitochondrial tethering and the formation of close contacts in neuronal cells in vivo.
    Keywords:  ER-mitochondria tethering; Mfn2; Mitochondria-ER contact; Mitochondria-associated membrane; Mitochondrial calcium uptake; VAPB
    DOI:  https://doi.org/10.1242/jcs.253443
  49. iScience. 2021 May 21. 24(5): 102484
    Integrating protein networks and machine learning for disease stratification in the Hereditary Spastic Paraplegias.
    Nikoleta Vavouraki, James E Tomkins, Eleanna Kara, Henry Houlden, John Hardy, Marcus J Tindall, Patrick A Lewis, Claudia Manzoni.
      The Hereditary Spastic Paraplegias are a group of neurodegenerative diseases characterized by spasticity and weakness in the lower body. Owing to the combination of genetic diversity and variable clinical presentation, the Hereditary Spastic Paraplegias are a strong candidate for protein-protein interaction network analysis as a tool to understand disease mechanism(s) and to aid functional stratification of phenotypes. In this study, experimentally validated human data were used to create a protein-protein interaction network based on the causative genes. Network evaluation as a combination of topological analysis and functional annotation led to the identification of core proteins in putative shared biological processes, such as intracellular transport and vesicle trafficking. The application of machine learning techniques suggested a functional dichotomy linked with distinct sets of clinical presentations, indicating that there is scope to further classify conditions currently described under the same umbrella-term of Hereditary Spastic Paraplegias based on specific molecular mechanisms of disease.
    Keywords:  Bioinformatics; Biological sciences; Molecular network; Network
    DOI:  https://doi.org/10.1016/j.isci.2021.102484
  50. Cell. 2021 Jun 03. pii: S0092-8674(21)00601-2. [Epub ahead of print]
    Structural analysis of the full-length human LRRK2.
    Alexander Myasnikov, Hanwen Zhu, Patricia Hixson, Boer Xie, Kaiwen Yu, Aaron Pitre, Junmin Peng, Ji Sun.
      Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2G2019S. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.
    Keywords:  LRRK2; LRRK2 dimer; LRRK2 mutations; Parkinson's disease; kinase
    DOI:  https://doi.org/10.1016/j.cell.2021.05.004
  51. Methods Mol Biol. 2021 ;2310 33-45
    BN-PAGE-Based Approach to Study Thyroid Hormones and Mitochondrial Function.
    Elena Silvestri, Assunta Lombardi, Federica Cioffi, Fernando Goglia.
      In recent years, a number of advancements have been made in the study of entire mitochondrial proteomes in both physiological and pathological conditions. Naturally occurring iodothyronines (i.e., T3 and T2) greatly influence mitochondrial oxidative capacity, directly or indirectly affecting the structure and function of the respiratory chain components. Blue native PAGE (BN-PAGE) can be used to isolate enzymatically active oxidative phosphorylation (OXPHOS) complexes in one step, allowing the clinical diagnosis of mitochondrial metabolism by monitoring OXPHOS catalytic and/or structural features. Protocols for isolating mammalian liver mitochondria and subsequent one-dimensional (1D) BN-PAGE will be described in relation to the impact of thyroid hormones on mitochondrial bioenergetics.
    Keywords:  BN-PAGE; Iodothyronine; Mitochondrion; Respiratory chain; Thyroid hormone
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_3
  52. Nat Commun. 2021 06 09. 12(1): 3486
    Regulation of the one carbon folate cycle as a shared metabolic signature of longevity.
    Andrea Annibal, Rebecca George Tharyan, Maribel Fides Schonewolff, Hannah Tam, Christian Latza, Markus Max Karl Auler, Adam Antebi.
      The metabolome represents a complex network of biological events that reflects the physiologic state of the organism in health and disease. Additionally, specific metabolites and metabolic signaling pathways have been shown to modulate animal ageing, but whether there are convergent mechanisms uniting these processes remains elusive. Here, we used high resolution mass spectrometry to obtain the metabolomic profiles of canonical longevity pathways in C. elegans to identify metabolites regulating life span. By leveraging the metabolomic profiles across pathways, we found that one carbon metabolism and the folate cycle are pervasively regulated in common. We observed similar changes in long-lived mouse models of reduced insulin/IGF signaling. Genetic manipulation of pathway enzymes and supplementation with one carbon metabolites in C. elegans reveal that regulation of the folate cycle represents a shared causal mechanism of longevity and proteoprotection. Such interventions impact the methionine cycle, and reveal methionine restriction as an underlying mechanism. This comparative approach reveals key metabolic nodes to enhance healthy ageing.
    DOI:  https://doi.org/10.1038/s41467-021-23856-9
  53. iScience. 2021 May 21. 24(5): 102494
    DHODH inhibition modulates glucose metabolism and circulating GDF15, and improves metabolic balance.
    Juan Zhang, Graciela Terán, Mihaela Popa, Harsha Madapura, Marcus James Graeme Watson Ladds, Danai Lianoudaki, Jacob Grünler, Marie Arsenian-Henriksson, Emmet McCormack, Martin Enrique Rottenberg, Sergiu-Bogdan Catrina, Sonia Laín, Suhas Darekar.
      Dihydroorotate dehydrogenase (DHODH) is essential for the de novo synthesis of pyrimidine ribonucleotides, and as such, its inhibitors have been long used to treat autoimmune diseases and are in clinical trials for cancer and viral infections. Interestingly, DHODH is located in the inner mitochondrial membrane and contributes to provide ubiquinol to the respiratory chain. Thus, DHODH provides the link between nucleotide metabolism and mitochondrial function. Here we show that pharmacological inhibition of DHODH reduces mitochondrial respiration, promotes glycolysis, and enhances GLUT4 translocation to the cytoplasmic membrane and that by activating tumor suppressor p53, increases the expression of GDF15, a cytokine that reduces appetite and prolongs lifespan. In addition, similar to the antidiabetic drug metformin, we observed that in db/db mice, DHODH inhibitors elevate levels of circulating GDF15 and reduce food intake. Further analysis using this model for obesity-induced diabetes revealed that DHODH inhibitors delay pancreatic β cell death and improve metabolic balance.
    Keywords:  Biological sciences; Cellular physiology; Diabetology; Endocrinology; Physiology
    DOI:  https://doi.org/10.1016/j.isci.2021.102494
  54. Angew Chem Int Ed Engl. 2021 Jun 12.
    Spatially Selective Imaging of Mitochondrial MicroRNAs via Optically Programmable Strand Displacement Reactions.
    Jian Zhao, Zhixiang Li, Yulei Shao, Wenping Hu, Lele Li.
      MicroRNA (miRNA) functions are tightly regulated by their sub-compartmental location in living cells, and the ability to imaging of mitochondrial miRNAs (mitomiRs) is essential for understanding of the related pathological processes. However, most existing DNA-based methods could not be used for this purpose. Here, we report the development of a DNA nanoreporter technology for imaging of mitomiRs in living cells through near-infrared (NIR) light-controlled DNA strand displacement reactions. The sensing function of the DNA nanoreporters are silent (OFF) during the delivery process, but can be photoactivated (ON) with NIR light after targeted mitochondrial localization, enabling spatially-restricted imaging of two types of cancer-related mitomiRs with improved detection accuracy. Furthermore, we demonstrate imaging of mitomiRs in vivo through spatiotemporally-controlled delivery and activation. Therefore, this study illustrates a simple methodology that may be broadly applicable for investigating the mitomiRs-associated physiological events.
    Keywords:  DNA-based probes; Mitochondria; microRNA imaging; optical control
    DOI:  https://doi.org/10.1002/anie.202105696
  55. Eur J Hum Genet. 2021 Jun 08.
    Genomic sequencing for the diagnosis of childhood mitochondrial disorders: a health economic evaluation.
    You Wu, Shanti Balasubramaniam, Rocio Rius, David R Thorburn, John Christodoulou, Ilias Goranitis.
      The diagnostic and clinical benefits of genomic sequencing are being increasingly demonstrated across multiple rare genetic conditions. Despite the expanding clinical literature, there is a significant paucity of health economics evidence to inform the prioritization and implementation of genomic sequencing. This study aims to evaluate whether genomic sequencing for pediatric-onset mitochondrial disorders (MDs) is cost-effective and cost-beneficial relative to conventional care from an Australian healthcare system perspective. Two independent and complementary health economic modeling approaches were used. Approach 1 used a decision tree to model the costs and outcomes associated with genomic sequencing and conventional care. Approach 2 used a discrete-event simulation to incorporate heterogeneity in the condition and clinical practice. Deterministic and probabilistic sensitivity analyses were performed. Genomic sequencing was less costly and more effective compared with conventional care, saving AU$1997 (Approach 1) to AU$8823 (Approach 2) per child tested, while leading to an additional 11 (Approach 1) to 14 (Approach 2) definitive diagnoses per 100 children tested. The mean monetary value of the incremental benefits of genomic sequencing was estimated at AU$5890 (95% CI: AU$5730-$6046). Implementation of genomic sequencing for MDs in Australia could translate to an annual cost-saving of up to AU$0.7 million. Genomic sequencing is cost-saving relative to traditional investigative approaches, while enabling more diagnoses to be made in a timely manner, offering substantial personal benefits to children and their families. Our findings support the prioritization of genomic sequencing for children with MDs.
    DOI:  https://doi.org/10.1038/s41431-021-00916-8
  56. J Biol Chem. 2021 Apr 29. pii: S0021-9258(21)00525-1. [Epub ahead of print] 100736
    The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.
    Victor Vitvitsky, Roshan Kumar, Marouane Libiad, Allison Maebius, Aaron Landry, Ruma Banerjee.
      Hydrogen sulfide is synthesized by enzymes involved in sulfur metabolism and oxidized via a dedicated mitochondrial pathway that intersects with the electron transport chain (ETC) at the level of complex III. Studies with H2S are challenging since it is volatile and also reacts with oxidized thiols in the culture medium, forming sulfane sulfur species. The half-life of exogenously added H2S to cultured cells is unknown. In this study, we first examined the half-life of exogenously added H2S to human colonic epithelial cells. In plate cultures, H2S disappeared with a t1/2 of 3-4 min at 37°C with a small fraction being trapped as sulfane sulfur species. In suspension cultures, the rate of abiotic loss of H2S was slower, and we demonstrated that sulfide stimulated aerobic glycolysis, which was sensitive to the mitochondrial but not the cytoplasmic NADH pool. Oxidation of mitochondrial NADH using the genetically encoded mito-LbNOX tool, blunted the cellular sensitivity to sulfide-stimulated aerobic glycolysis and enhanced its oxidation to thiosulfate. In contrast, sulfide did not affect flux through the oxidative pentose phosphate pathway or the TCA cycle. Knockdown of sulfide quinone oxidoreductase, which commits H2S to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of H2S to activate oxidative phosphorylation at low concentrations, but inhibit it at high concentrations, suggests that it might play a role in tuning electron flux and therefore, cellular energy metabolism, particularly during cell proliferation.
    Keywords:  Hydrogen sulfide; aerobic glycolysis; electron transport chain; sulfide quinone oxidoreductase
    DOI:  https://doi.org/10.1016/j.jbc.2021.100736
  57. Nat Rev Mol Cell Biol. 2021 Jun 09.
    Damaged mitochondria are discarded via migrasomes.
    Kim Baumann.
      
    DOI:  https://doi.org/10.1038/s41580-021-00388-0
  58. EMBO Mol Med. 2021 Jun 07. e13591
    Pharmacological or genetic inhibition of iNOS prevents cachexia-mediated muscle wasting and its associated metabolism defects.
    Jason Sadek, Derek T Hall, Bianca Colalillo, Amr Omer, Anne-Marie K Tremblay, Virginie Sanguin-Gendreau, William Muller, Sergio Di Marco, Marco Emilio Bianchi, Imed-Eddine Gallouzi.
      Cachexia syndrome develops in patients with diseases such as cancer and sepsis and is characterized by progressive muscle wasting. While iNOS is one of the main effectors of cachexia, its mechanism of action and whether it could be targeted for therapy remains unexplored. Here, we show that iNOS knockout mice and mice treated with the clinically tested iNOS inhibitor GW274150 are protected against muscle wasting in models of both septic and cancer cachexia. We demonstrate that iNOS triggers muscle wasting by disrupting mitochondrial content, morphology, and energy production processes such as the TCA cycle and acylcarnitine transport. Notably, iNOS inhibits oxidative phosphorylation through impairment of complexes II and IV of the electron transport chain and reduces ATP production, leading to energetic stress, activation of AMPK, suppression of mTOR, and, ultimately, muscle atrophy. Importantly, all these effects were reversed by GW274150. Therefore, our data establish how iNOS induces muscle wasting under cachectic conditions and provide a proof of principle for the repurposing of iNOS inhibitors, such as GW274150 for the treatment of cachexia.
    Keywords:  cachexia; cancer; iNOS; inflammation; metabolism
    DOI:  https://doi.org/10.15252/emmm.202013591
  59. Methods Mol Biol. 2021 ;2275 279-289
    Norbormide-Based Probes and Their Application for Mitochondrial Imaging in Drosophila Melanogaster.
    Alessia Forgiarini, Zifei Wang, Sergio Bova, Margaret Anne Brimble, Brian Hopkins, David Rennison, Genny Orso.
      Fluorescent live imaging on Drosophila melanogaster is a microscopy technique in rapid expansion. The growing number of probes available to detect cellular components and the relatively easy genetic manipulation of fruit fly make this model one of the most used for in vivo analysis of several physiological and/or pathological processes. Here we describe the chemical synthesis of two norbormide-derived BODIPY-conjugated fluorescent probes (NRBMC009 and NRBZLW0047). Moreover, we describe the larval dissection method, and subsequent live imaging acquisition. Both probes are able to label mitochondria in different Drosophila larval tissues, which allows for the characterization of mitochondrial morphological alterations by using a simple and quick method that avoids the fixation artefacts that often occur in immunofluorescence studies.
    Keywords:  Confocal microscopy; Drosophila melanogaster; Fluorescent probes; Live imaging; Norbormide
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_17
  60. Nat Rev Cardiol. 2021 Jun 09.
    Towards precision medicine in heart failure.
    Chad S Weldy, Euan A Ashley.
      The number of therapies for heart failure (HF) with reduced ejection fraction has nearly doubled in the past decade. In addition, new therapies for HF caused by hypertrophic and infiltrative disease are emerging rapidly. Indeed, we are on the verge of a new era in HF in which insights into the biology of myocardial disease can be matched to an understanding of the genetic predisposition in an individual patient to inform precision approaches to therapy. In this Review, we summarize the biology of HF, emphasizing the causal relationships between genetic contributors and traditional structure-based remodelling outcomes, and highlight the mechanisms of action of traditional and novel therapeutics. We discuss the latest advances in our understanding of both the Mendelian genetics of cardiomyopathy and the complex genetics of the clinical syndrome presenting as HF. In the phenotypic domain, we discuss applications of machine learning for the subcategorization of HF in ways that might inform rational prescribing of medications. We aim to bridge the gap between the biology of the failing heart, its diverse clinical presentations and the range of medications that we can now use to treat it. We present a roadmap for the future of precision medicine in HF.
    DOI:  https://doi.org/10.1038/s41569-021-00566-9
  61. Nat Commun. 2021 06 08. 12(1): 3445
    MOGONET integrates multi-omics data using graph convolutional networks allowing patient classification and biomarker identification.
    Tongxin Wang, Wei Shao, Zhi Huang, Haixu Tang, Jie Zhang, Zhengming Ding, Kun Huang.
      To fully utilize the advances in omics technologies and achieve a more comprehensive understanding of human diseases, novel computational methods are required for integrative analysis of multiple types of omics data. Here, we present a novel multi-omics integrative method named Multi-Omics Graph cOnvolutional NETworks (MOGONET) for biomedical classification. MOGONET jointly explores omics-specific learning and cross-omics correlation learning for effective multi-omics data classification. We demonstrate that MOGONET outperforms other state-of-the-art supervised multi-omics integrative analysis approaches from different biomedical classification applications using mRNA expression data, DNA methylation data, and microRNA expression data. Furthermore, MOGONET can identify important biomarkers from different omics data types related to the investigated biomedical problems.
    DOI:  https://doi.org/10.1038/s41467-021-23774-w
  62. Radiol Case Rep. 2021 Jul;16(7): 1865-1869
    Adult-onset mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome with progressive sensorineural hearing loss: A case report.
    T M Trang, P C Chien, B T Dung, N T H Thu, N T T Truc, V N C Khang.
      Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most common maternally inherited mitochondrial disorders, with no specific treatment available. We report a case of a 34-year-old female in whom symptoms of MELAS were initially misdiagnosed as herpes simplex encephalitis (HSE). Her clinical course was marked by an acute episode of consciousness disturbance with newly developed lesions on brain MRI five years after disease onset and followed by progressive sensorineural hearing loss. Brain imaging sequences throughout the seven years of her illness are presented. The final diagnosis of MELAS syndrome was confirmed by m.3243A>G mitochondrial mutation. In conclusion, understanding the overlapping imaging features between MELAS syndrome and other mimickers, such as HSE or ischemic stroke, is crucial to help establish early diagnosis and initiate appropriate treatment.
    Keywords:  Herpes Simplex Encephalopathy; MELAS; Magnetic Resonance Imaging; Mitochondrial Encephalomyopathy
    DOI:  https://doi.org/10.1016/j.radcr.2021.04.049
  63. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2021 Jun 10. 38(6): 513-520
    [A review of consensus statements, practice resources, standards and guidelines for clinical applications of next-generation sequencing technologies in the United States].
    Chen Zhao, Xiaolei Xie, Weizhen Ji, Ming Qi, Qing Zhou, Mengrong Li, Peining Li, Yonghui Jiang, Hui Zhang.
      The use of whole exome sequencing (WES) for the detection of disease-causing variants of genetic diseases and for non-invasive prenatal screening (NIPS) of fetal aneuploidies are two major clinical applications of next generation sequencing (NGS). This article has summarized the official documents developed and updated by the American College of Medical Genetics and Genomics (ACMG) on governing WES and NIPS. These include the development of expert consensus policies and position statements on an ongoing basis to guide clinical application of NGS technology and variant analysis, establish evidence-based practical resources, as well as standards and guidelines to govern diagnosis and screening. These ACMG documents are valuable references to Chinese geneticists, but direct adoption of these standards and guidelines may not be practical due to the differences in disease-associated variant frequencies in Chinese population, socioeconomic status, and medical practice between the two countries. It is hoped that this review could facilitate the development of NGS and NIPS standards and guidelines that are consistent with international standards and concordant with medical genetics practice in China to provide high-quality, efficient and safe clinical services for patients and their families with genetic diseases.
    DOI:  https://doi.org/10.3760/cma.j.cn511374-20200924-00691
  64. Chem Sci. 2020 Aug 19. 11(47): 12677-12685
    Aryl urea substituted fatty acids: a new class of protonophoric mitochondrial uncoupler that utilises a synthetic anion transporter.
    Tristan Rawling, Hugo MacDermott-Opeskin, Ariane Roseblade, Curtis Pazderka, Callum Clarke, Kirsi Bourget, Xin Wu, William Lewis, Benjamin Noble, Philip A Gale, Megan L O'Mara, Charles Cranfield, Michael Murray.
      Respiring mitochondria establish a proton gradient across the mitochondrial inner membrane (MIM) that is used to generate ATP. Protein-independent mitochondrial uncouplers collapse the proton gradient and disrupt ATP production by shuttling protons back across the MIM in a protonophoric cycle. Continued cycling relies on the formation of MIM-permeable anionic species that can return to the intermembrane space after deprotonation in the mitochondrial matrix. Previously described protonophores contain acidic groups that are part of delocalised π-systems that provide large surfaces for charge delocalisation and facilitate anion permeation across the MIM. Here we present a new class of protonophoric uncoupler based on aryl-urea substituted fatty acids in which an acidic group and a π-system are separated by a long alkyl chain. The aryl-urea group in these molecules acts as a synthetic anion receptor that forms intermolecular hydrogen bonds with the fatty acid carboxylate after deprotonation. Dispersal of the negative charge across the aryl-urea system produces lipophilic dimeric complexes that can permeate the MIM and facilitate repeated cycling. Substitution of the aryl-urea group with lipophilic electron withdrawing groups is critical to complex lipophilicity and uncoupling activity. The aryl-urea substituted fatty acids represent the first biological example of mitochondrial uncoupling mediated by the interaction of a fatty acid and an anion receptor moiety, via self-assembly.
    DOI:  https://doi.org/10.1039/d0sc02777d
  65. Stem Cell Res. 2021 May 21. pii: S1873-5061(21)00238-5. [Epub ahead of print]54 102392
    Self-replicative mRNA-mediated generation of induced pluripotent stem cell line from a 1-year-old Leigh syndrome patient with mitochondrial DNA cytochrome b mutation.
    Daryeon Son, Jie Zheng, In Yong Kim, Seungkwon You.
      Leigh syndrome is a progressive neurodegenerative disease due to defects in the mitochondrial genes, including mitochondrial DNA cytochrome b (MTCYB) mutation, that typically begins in infancy or early childhood. Exercise intolerance and fatigue are common symptoms of mitochondrial disorders. Here, we generated induced pluripotent stem cell (iPSC) line from a 1-year-old patient with Leigh syndrome with MTCYB through temporal expression of exogenes, synthetic self-replicative mRNAs which were regulated by B18R protein. The established iPSCs showed expression of various pluripotency markers, a normal karyotype and differentiation potential to three germ layers in vitro while retaining MTCYB mutation.
    DOI:  https://doi.org/10.1016/j.scr.2021.102392
  66. Methods Mol Biol. 2021 ;2310 301-309
    Measuring PGC-1α and Its Acetylation Status in Mouse Primary Myotubes.
    João A Amorim, David A Sinclair.
      Metabolic flexibility is vital for organisms to respond to and survive changes in energy availability. A critical metabolic flexibility regulator is peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), which regulates various transcription factors and nuclear receptors that, in turn, regulate mitochondrial homeostasis and fatty acid oxidation. PGC-1α is itself regulated, with one of the significant modes of regulation being acetylation. Thus, measuring the acetylation status of PGC-1α is a critical indicator of cells' metabolic flexibility. In this chapter, we describe a method of evaluating PGC-1α acetylation in primary mouse myotubes. This method can also be used with other cell types and tissues.
    Keywords:  Acetylation; Aging; Cachexia; Diabetes; GCN5; Mitochondrial biogenesis; PGC-1α; Respiration; SIRT1; Sarcopenia; Sirtuin deacetylase
    DOI:  https://doi.org/10.1007/978-1-0716-1433-4_17
  67. Mitochondrion. 2021 Jun 05. pii: S1567-7249(21)00078-7. [Epub ahead of print]
    A hitchhiker's guide to mitochondrial quantification.
    Irene Mgm Hemel, Bob Ph Engelen, Nicole Luber, Mike Gerards.
      The variety of available mitochondrial quantification tools makes it difficult to select the most reliable and accurate quantification tool. Here, we performed elaborate analyses on five open source ImageJ tools. Excessive clustering of mitochondrial structures was observed in four tools, caused by the global thresholding applied by these tools. The Mitochondrial Analyzer, which uses adaptive thresholding, outperformed the other examined tools, with accurate structural segregation and identification. Additionally, we showed that the Mitochondrial Analyzer successfully identifies mitochondrial morphology differences. Based on the observed performance, we consider the Mitochondrial Analyzer the best open source tool for mitochondrial network morphology quantification.
    Keywords:  Image analysis; ImageJ; Mitochondria; Mitochondrial Dynamics; Mitochondrial quantification
    DOI:  https://doi.org/10.1016/j.mito.2021.06.005
  68. Methods Mol Biol. 2021 ;2275 315-327
    Identification of Peroxynitrite by Profiling Oxidation and Nitration Products from Mitochondria-Targeted Arylboronic Acid.
    Jacek Zielonka, Adam Sikora, Radoslaw Podsiadly, Micael Hardy, B Kalyanaraman.
      The development of boronic probes enabled reliable detection and quantitative analysis of hydrogen peroxide , other nucleophilic hydroperoxides, hypochlorite , and peroxynitrite . The major product, in which boronate moiety of the probe is replaced by the hydroxyl group, is, however, common for all those oxidants. Here, we describe how ortho-isomer of mitochondria-targeted phenylboronic acid can be used to detect and differentiate peroxynitrite-dependent and independent probe oxidation. This method highlights detection and quantification of both the major, phenolic product and the minor, peroxynitrite-specific cyclic and nitrated products of probe oxidation.
    Keywords:  Boronic probes; HPLC-MS; Hydrogen peroxide; Mitochondria-targeted probes; Peroxynitrite; o-MitoPhB(OH)2
    DOI:  https://doi.org/10.1007/978-1-0716-1262-0_20
  69. Curr Osteoporos Rep. 2021 Jun 12.
    Energy Metabolism of Osteocytes.
    Vivin Karthik, Anyonya R Guntur.
      PURPOSE OF REVIEW: In this review, we provide a recent update on bioenergetic pathways in osteocytes and identify potential future areas of research interest. Studies have identified a role for regulation of bone formation and bone resorption through osteocyte mechanosensing and osteocyte secreted factors. Nevertheless, there is a paucity of studies on the bioenergetics and energy metabolism of osteocytes, which are required for the regulation of bone remodeling.RECENT FINDINGS: Osteocytes are cells of the osteoblast lineage embedded in bone. The osteocyte lacunocanalicular network within the skeletal matrix is exposed to a unique hypoxic environment. Therefore, the bioenergetic requirements of these cells could differ from other bone cells due to its location in the ossified matrix and its role in bone regulation transduced by mechanical signals. Recent findings highlighted in this review provide some evidence that metabolism of these cells is dependent on their location due to the substrates present in the microenvironment and metabolic cues from stress pathways. Both glycolysis (glucose metabolism) and oxidative phosphorylation (mitochondrial dynamics, ROS generation) affect osteocyte function and viability. In this review, we provide evidence that is currently available about information regarding bioenergetics pathways in osteocytes. We discuss published studies showing a role for hypoxia-driven glucose metabolism in regulating osteocyte bioenergetics. We also provide information on various substrates that osteocytes could utilize to fuel energetic needs, namely pyruvate, amino acids, and fatty acids. This is based on some preliminary experimental evidence that is available in literature. The role of parathyroid hormone PTH and parathryoid hormone-related peptide PTHrP in bone anabolism and resorption, along with regulation of metabolic pathways in the cells of the skeletal niche, needs to be explored further. Mitochondrial metabolism has a role in osteocyte bioenergetics through substrate utilization, location of the osteocyte in the bone cortex, and mitochondrial biogenesis. While there are limitations in studying metabolic flux in traditional cell lines, there are now novel cell lines and sophisticated tools available to study osteocyte bioenergetics to help harness its potential in vivo in the future.
    Keywords:  Glycolysis; Metabolism; Mitochondria; Osteocytes; Oxidative phosphorylation
    DOI:  https://doi.org/10.1007/s11914-021-00688-6
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