bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2021–06–06
74 papers selected by
Catalina Vasilescu, University of Helsinki
  1. Identification of a Novel Variant in MT-CO3 Causing MELAS.
  2. Drug Discovery Assay to Identify Modulators of the Mitochondrial Ca2+ Uniporter.
  3. Mitochondrial Dysfunction in Mitochondrial Medicine: Current Limitations, Pitfalls, and Tomorrow.
  4. Single-Cell Approaches for Studying the Role of Mitochondrial DNA in Neurodegenerative Disease.
  5. A Biochemical and Structural Understanding of TOM Complex Interactions and Implications for Human Health and Disease.
  6. Genetic and Clinical Predictors of Ataxia in Pediatric Primary Mitochondrial Disorders.
  7. How rotating ATP synthases can modulate membrane structure.
  8. Assessment of Mitochondrial Ca2+ Uptake.
  9. Studying Mitochondrial Network Formation by In Vivo and In Vitro Reconstitution Assay.
  10. Extensive pleiotropism and allelic heterogeneity mediate metabolic effects of IRX3 and IRX5.
  11. Mutational analysis of mitochondrial tRNA genes in 138 patients with Leber's hereditary optic neuropathy.
  12. Heterologous Inferential Analysis (HIA) and Other Emerging Concepts: In Understanding Mitochondrial Variation In Pathogenesis: There is no More Low-Hanging Fruit.
  13. Dominant ACO2 mutations are a frequent cause of isolated optic atrophy.
  14. Monitoring the Mitochondrial Presequence Import Pathway In Living Mammalian Cells with a New Molecular Biosensor.
  15. Loss of Fis1 impairs proteostasis during skeletal muscle aging in Drosophila.
  16. A New Method for Sequencing the Mitochondrial Genome by Using Long Read Technology.
  17. Movement Disorders in Children with a Mitochondrial Disease: A Cross-Sectional Survey from the Nationwide Italian Collaborative Network of Mitochondrial Diseases.
  18. Bioenergetic Profiling of Human Pluripotent Stem Cells.
  19. Mitochondrial disease patients with novel ND4 12058A > C and ND1 m.3911A > G variations: implications for a role in the phenotype following a bioinformatic investigation.
  20. 3D Optical Cryo-Imaging Method: A Novel Approach to Quantify Renal Mitochondrial Bioenergetics Dysfunction.
  21. Simultaneous Quantification of Mitochondrial ATP and ROS Production Using ATP Energy Clamp Methodology.
  22. Investigation of Mitochondrial ADP-Ribosylation Via Immunofluorescence.
  23. Mitochondria in neurogenesis: Implications for mitochondrial diseases.
  24. Quantitatively Controlled Intercellular Mitochondrial Transfer by Cell Fusion-Based Method Using a Microfluidic Device.
  25. The NDUFS4 Knockout Mouse: A Dual Threat Model of Childhood Mitochondrial Disease and Normative Aging.
  26. Whole-Cell and Mitochondrial dNTP Pool Quantification from Cells and Tissues.
  27. Preparation of "Functional" Mitochondria: A Challenging Business.
  28. Patch-Clamp Recording of the Activity of Ion Channels in the Inner Mitochondrial Membrane.
  29. The Isolation and Deep Sequencing of Mitochondrial DNA.
  30. Prohibitin 1 is essential to preserve mitochondria and myelin integrity in Schwann cells.
  31. Purification of Functional Platelet Mitochondria Using a Discontinuous Percoll Gradient.
  32. Structural basis of complex formation between mitochondrial anion channel VDAC1 and Hexokinase-II.
  33. Assessment of Mitochondrial Membrane Potential and NADH Redox State in Acute Brain Slices.
  34. Small Molecule Regulators Targeting NAD+ Biosynthetic Enzymes.
  35. Perilipin 5 links mitochondrial uncoupled respiration in brown fat to healthy white fat remodeling and systemic glucose tolerance.
  36. The dysregulated Pink1- drosophila mitochondrial proteome is partially corrected with exercise.
  37. Single-Particle Tracking Method in Fluorescence Microscopy to Monitor Bioenergetic Responses of Individual Mitochondria.
  38. Shedding light on the mitochondrial matrix through a functional membrane transporter.
  39. Molecular Epidemiology of Mitochondrial Cardiomyopathy: A Search Among Mitochondrial and Nuclear Genes.
  40. Mitochondrial Dysfunction in Podocytes Caused by CRIF1 Deficiency Leads to Progressive Albuminuria and Glomerular Sclerosis in Mice.
  41. A MT-TL1 variant identified by whole exome sequencing in an individual with intellectual disability, epilepsy, and spastic tetraparesis.
  42. Reevaluating the role of human mitochondrial uL18m in the cytosolic stress response.
  43. Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration.
  44. Childhood amyotrophic lateral sclerosis caused by excess sphingolipid synthesis.
  45. Isolation of Mitochondria from Retinal Pigment Epithelial Cell Cultures and an Application of High-Resolution Respirometric Assay (XFe96 Seahorse Assay).
  46. Mitochondrial quality control: Just walk away.
  47. High-Throughput Image Analysis of Lipid-Droplet-Bound Mitochondria.
  48. Suborganellar Localization of Mitochondrial Proteins and Transcripts in Human Cells.
  49. Isolation of Mitochondria-Associated ER Membranes (MAMs), Synaptic MAMs, and Glycosphingolipid Enriched Microdomains (GEMs) from Brain Tissues and Neuronal Cells.
  50. The parkinsonian LRRK2 R1441G mutation shows macroautophagy-mitophagy dysregulation concomitant with endoplasmic reticulum stress.
  51. HIGD‑1B inhibits hypoxia‑induced mitochondrial fragmentation by regulating OPA1 cleavage in cardiomyocytes.
  52. Efficient extra-mitochondrial aerobic ATP synthesis in neuronal membrane systems.
  53. Fluorescence-Based Assay For Measuring OMA1 Activity.
  54. GRSF1 deficiency in skeletal muscle reduces endurance in aged mice.
  55. PINK1 and parkin shape the organism-wide distribution of a deleterious mitochondrial genome.
  56. Assessment of Mitochondrial Reactive Oxygen Species and Redox Regulation in Stem Cells.
  57. Brains and brawn: Stress-induced myokine abates nervous system aging.
  58. Evaluation of Mitochondria Content and Function in Live Cells by Multicolor Flow Cytometric Analysis.
  59. Sprinkling salt on mitochondria: The metabolic and pathophysiological roles of mitochondrial Na+ signaling mediated by NCLX.
  60. Assessment of Mitochondrial Protein Glutathionylation as Signaling for CO Pathway.
  61. Mitochondrial Diagnostics: A Discovery-Based Biochemical Platform for Phenotyping Human Peripheral Blood Cell Mitochondria.
  62. Engineering Genetic Systems for Treating Mitochondrial Diseases.
  63. Mitochondrial defence tactics.
  64. Mitochondrial Bioenergetics and Dynamism in the Failing Heart.
  65. In Vivo Assessment of Mitochondrial Oxygen Consumption.
  66. Modified Blue Native Gel Approach for Analysis of Respiratory Supercomplexes.
  67. Assessment of Mitochondrial Cell Metabolism by Respiratory Chain Electron Flow Assays.
  68. Oxidative phosphorylation system and cell culture media.
  69. Accurate Measurement of Cellular and Cell-Free Circulating Mitochondrial DNA Content from Human Blood Samples Using Real-Time Quantitative PCR.
  70. Diagnosing Mitochondrial Disorders Remains Challenging in the Omics Era.
  71. Reply to: Reevaluating the role of human mitochondrial uL18m in the cytosolic stress response.
  72. The Mitochondrial Response to DNA Damage.
  73. Native Gel Electrophoresis and Immunoblotting to Analyze Electron Transport Chain Complexes.
  74. Extraction of Functional Mitochondria Based on Membrane Stiffness.
  1. Front Genet. 2021 ;12 638749
    Identification of a Novel Variant in MT-CO3 Causing MELAS.
    Manting Xu, Robert Kopajtich, Matthias Elstner, Zhaoxia Wang, Zhimei Liu, Junling Wang, Holger Prokisch, Fang Fang.
      Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a maternally inherited mitochondrial disease. Most cases of MELAS are caused by the m.3243A > G variant in the MT-TL1 gene encoding tRNALeu(UUR). However, the genetic cause in 10% of patients with MELAS is unknown. We investigated the pathogenicity of the novel mtDNA variant m.9396G > A/MT-CO3 (p.E64K), which affects an extremely conserved amino acid in the CO3 subunit of mitochondrial respiratory chain (MRC) complex IV (CIV) in a patient with MELAS. Biochemical assays of a muscle biopsy confirmed remarkable CIV deficiency, and pathological examination showed ragged red fibers and generalized COX non-reactive muscle fibers. Transfer of the mutant mtDNA into cybrids impaired CIV assembly, followed by remarkable mitochondrial dysfunction and ROS production. Our findings highlight the pathogenicity of a novel m.9396G > A variant and extend the spectrum of pathogenic mtDNA variants.
    Keywords:  MELAS; MT-CO3 gene; complex IV of respiratory chain; mitochondrial diseases; novel mitochondrial DNA variant
    DOI:  https://doi.org/10.3389/fgene.2021.638749
  2. Methods Mol Biol. 2021 ;2277 69-89
    Drug Discovery Assay to Identify Modulators of the Mitochondrial Ca2+ Uniporter.
    Daniela M Arduino, Valerie Goh, Dejana Mokranjac, Fabiana Perocchi.
      The mitochondrial calcium uniporter (MCU ) is an essential protein of the inner mitochondrial membrane that mediates the uptake of calcium into mitochondria of virtually all mammalian tissues, regulating cell metabolism, signaling, and death. MCU-mediated calcium uptake has been shown to play a pathophysiological role in diverse human disease contexts, which qualifies this channel as a druggable target for therapeutic intervention.Here, we present a protocol to perform drug screens to identify effective and specific MCU-targeting inhibitors. The methodology is based on the use of cryopreserved mitochondria that are isolated from a yeast strain engineered to express the human MCU and its essential regulator EMRE together with the luminescence calcium sensor aequorin. Yeast mitochondria with a functionally reconstituted MCU-mediated calcium uptake are then employed as a ready-to-use screening reagent. False discovery rate is further minimized by energizing mitochondria with D-lactate in a mannitol/sucrose-based medium, which provides a mean to discriminate between direct and secondary effects of drugs on mitochondrial calcium uptake. This screening assay is sensitive and robust and can be easily implemented in any laboratory.
    Keywords:  Aequorin; Calcium; Drug screening; Luminescence assay; Mitochondria; Mitochondrial calcium uniporter; Yeast
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_5
  3. Methods Mol Biol. 2021 ;2276 1-29
    Mitochondrial Dysfunction in Mitochondrial Medicine: Current Limitations, Pitfalls, and Tomorrow.
    Naig Gueguen, Guy Lenaers, Pascal Reynier, Volkmar Weissig, Marvin Edeas.
      Until recently restricted to hereditary mitochondrial diseases, mitochondrial dysfunction is now recognized as a key player and strategic factor in the pathophysiological of many human diseases, ranging from the metabolism, vascular, cardiac, and neurodegenerative diseases to cancer. Because of their participation in a myriad of cellular functions and signaling pathways, precisely identifying the cause of mitochondrial "dysfunctions" can be challenging and requires robust and controlled techniques. Initially limited to the analysis of the respiratory chain functioning, these analytical techniques now enlarge to the analyses of mitochondrial and cellular metabolism, based on metabolomic approaches.Here, we address the methods used to assay mitochondrial dysfunction, with a highlight on the techniques used in diagnosis on tissues and cells derived from patients, the information they provide, and their strength and weakness.Targeting mitochondrial dysfunction by various strategies is a huge challenge, requires robust methods of evaluation, and should be able to take into consideration the mitochondria dynamics and localization. The future of mitochondrial medicine is strongly related to a perfect comprehension of its dysfunction.
    Keywords:  Bioenergetics; Devices; Metabolomics; Mitochondria evaluation; Mitochondrial dysfunctions
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_1
  4. Methods Mol Biol. 2021 ;2277 299-329
    Single-Cell Approaches for Studying the Role of Mitochondrial DNA in Neurodegenerative Disease.
    Laura J Bailey, Joanna L Elson, Ilse S Pienaar.
      In light of accumulating evidence suggestive of cell type-specific vulnerabilities as a result of normal aging processes that adversely affect the brain, as well as age-related neurodegenerative disorders such as Parkinson's disease (PD), the current chapter highlights how we study mitochondrial DNA (mtDNA) changes at a single-cell level. In particular, we comment on increasing questioning of the narrow neurocentric view of such pathologies, where microglia and astrocytes have traditionally been considered bystanders rather than players in related pathological processes. Here we review the contribution made by single-cell mtDNA alterations towards neuronal vulnerability seen in neurodegenerative disorders, focusing on PD as a prominent example. In addition, we give an overview of methodologies that support such experimental investigations. In considering the significant advances that have been made in recent times for developing mitochondria-specific therapies, investigations to account for cell type-specific mitochondrial patterns and how these are altered by disease hold promise for delivering more effective disease-modifying therapeutics.
    Keywords:  Age-related disorders; Cell specificity; Mitochondria; Mitochondrial DNA; Neurodegeneration; Parkinson’s disease; Single-cell analyses
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_19
  5. Cells. 2021 May 11. pii: 1164. [Epub ahead of print]10(5):
    A Biochemical and Structural Understanding of TOM Complex Interactions and Implications for Human Health and Disease.
    Ashley S Pitt, Susan K Buchanan.
      The central role mitochondria play in cellular homeostasis has made its study critical to our understanding of various aspects of human health and disease. Mitochondria rely on the translocase of the outer membrane (TOM) complex for the bulk of mitochondrial protein import. In addition to its role as the major entry point for mitochondrial proteins, the TOM complex serves as an entry pathway for viral proteins. TOM complex subunits also participate in a host of interactions that have been studied extensively for their function in neurodegenerative diseases, cardiovascular diseases, innate immunity, cancer, metabolism, mitophagy and autophagy. Recent advances in our structural understanding of the TOM complex and the protein import machinery of the outer mitochondrial membrane have made structure-based therapeutics targeting outer mitochondrial membrane proteins during mitochondrial dysfunction an exciting prospect. Here, we describe advances in understanding the TOM complex, the interactome of the TOM complex subunits, the implications for the development of therapeutics, and our understanding of the structure/function relationship between components of the TOM complex and mitochondrial homeostasis.
    Keywords:  TOM complex; TOM complex interactions; TOM subunits; mitochondrial cell signaling; mitochondrial quality control
    DOI:  https://doi.org/10.3390/cells10051164
  6. Cerebellum. 2021 May 30.
    Genetic and Clinical Predictors of Ataxia in Pediatric Primary Mitochondrial Disorders.
    Juan Se Bastian Martin-Saavedra, Sara Reis Teixeira, Cesar Augusto Pinheiro Ferreira Alves, Fabrício Guimarães Gonçalves, Luis Octavio Tierradentro-García, Martin Kidd, Colleen Muraresku, Amy Goldstein, Arastoo Vossough.
      Evaluation of ataxia in children is challenging in clinical practice. This is particularly true for highly heterogeneous conditions such as primary mitochondrial disorders (PMD). This study aims to explore cerebellar and brain abnormalities identified on MRI as potential predictors of ataxia in patients with PMD and, likewise, to determine the effect of the patient's genetic profile on these predictors as well as determination of the temporal relationship of clinical ataxia with MRI findings. We evaluated clinical, radiological, and genetic characteristics of 111 PMD patients younger than 21 years of age at The Children's Hospital of Philadelphia. Data was extracted from charts. Blinded radiological evaluations were carried out by experienced neuroradiologists. Multivariate logistic regression and generalized equation estimates were used for analysis. Ataxia was identified in 41% of patients. Cerebellar atrophy or putaminal involvement with mitochondrial DNA (mtDNA) mutations (OR 1.18, 95% CI 1.1-1.3, p < 0.001) and nuclear DNA mutation with no atrophy of the cerebellum (OR 1.14, 95% CI 1.0-1.3, p = 0.007) predicted an increased likelihood of having ataxia per year of age. Central tegmental tract predicted the presence of ataxia independent of age and pathogenic variant origin (OR 9.8, 95% CI 2-74, p = 0.009). Ataxia tended to precede the imaging finding of cerebellar atrophy. Cerebellar atrophy and putaminal involvement on MRI of pediatric-onset PMD may predict the presence of ataxia with age in patients with mtDNA mutations. This study provides predicted probabilities of having ataxia per year of age that may help in family counseling and future research of the population.
    Keywords:  Ataxia; Cerebellum; Child neurology; Primary mitochondrial disorders
    DOI:  https://doi.org/10.1007/s12311-021-01276-1
  7. Arch Biochem Biophys. 2021 May 27. pii: S0003-9861(21)00188-0. [Epub ahead of print] 108939
    How rotating ATP synthases can modulate membrane structure.
    Víctor Almendro-Vedia, Paolo Natale, David Valdivieso González, M Pilar Lillo, Juan L Aragones, Iván López-Montero.
      F1Fo-ATP synthase (ATP Synthase) is a central membrane protein that synthetizes most of the ATP in the cell through a rotational movement driven by a proton gradient across the hosting membrane. In mitochondria, ATP synthases can form dimers through specific interactions between some subunits of the protein. The dimeric form of ATP synthase provides the protein with a spontaneous curvature that sustain their arrangement at the rim of the high-curvature edges of mitochondrial membrane (cristae). Also, a direct interaction with cardiolipin, a lipid present in the inner mitochondrial membrane, induces the dimerization of ATP synthase molecules along cristae. The deletion of those biochemical interactions abolishes the protein dimerization producing an altered mitochondrial function and morphology. Mechanically, membrane bending is one of the key deformation modes by which mitochondrial membranes can be shaped. In particular, bending rigidity and spontaneous curvature are important physical factors for membrane remodelling. Here, we discuss a complementary mechanism whereby the rotatory movement of the ATP synthase might modify the mechanical properties of lipid bilayers and contribute to the formation and regulation of the membrane invaginations.
    Keywords:  Cristae; F(1)-F(o) ATP synthase; Membrane mechanics; Mitochondria; Rotation
    DOI:  https://doi.org/10.1016/j.abb.2021.108939
  8. Methods Mol Biol. 2021 ;2276 173-191
    Assessment of Mitochondrial Ca2+ Uptake.
    András T Deak, Claire Jean-Quartier, Alexander I Bondarenko, Lukas N Groschner, Roland Malli, Wolfgang F Graier, Markus Waldeck-Weiermair.
      Mitochondrial Ca2+ uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca2+ signals and elaborating the mechanisms that accomplish mitochondrial Ca2+ uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca2+ concentration ([Ca2+]mito) in a wide range of applications.
    Keywords:  Ca2+ Imaging; Calcium Green; FRET; Fura-2; Mitochondrial Ca2+ uptake; Mitochondrial membrane potential; Mitoplast; Oxidative phosphorylation; Patch-clamp recording; Rhod-2
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_13
  9. Methods Mol Biol. 2021 ;2276 333-341
    Studying Mitochondrial Network Formation by In Vivo and In Vitro Reconstitution Assay.
    Wanqing Du, Xiangjun Di, Qian Peter Su.
      Mitochondria change their morphologies from small isolated vesicles to large continuous networks across the cell cycles. The mitochondrial network formation (MNF) plays an important role in maintaining mitochondrial DNA integrity and interchanging mitochondrial materials. The disruption of the mitochondrial network affects mitochondrial functions, such as ATP production, integration of metabolism, calcium homeostasis, and regulation of apoptosis, leading to the abnormal development and several human diseases including neurodegenerative disease. In this unit, we describe the method of studying MNF, which is driven by microtubule-dependent motor protein, by in vivo imaging and single-molecule in vitro reconstitution assays.
    Keywords:  In vitro reconstitution system; KIF5B; Mitochondrial network formation (MNF); Single-molecule
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_25
  10. Science. 2021 Jun 04. 372(6546): 1085-1091
    Extensive pleiotropism and allelic heterogeneity mediate metabolic effects of IRX3 and IRX5.
    Débora R Sobreira, Amelia C Joslin, Qi Zhang, Iain Williamson, Grace T Hansen, Kathryn M Farris, Noboru J Sakabe, Nasa Sinnott-Armstrong, Grazyna Bozek, Sharon O Jensen-Cody, Kyle H Flippo, Carole Ober, Wendy A Bickmore, Matthew Potthoff, Mengjie Chen, Melina Claussnitzer, Ivy Aneas, Marcelo A Nóbrega.
      Whereas coding variants often have pleiotropic effects across multiple tissues, noncoding variants are thought to mediate their phenotypic effects by specific tissue and temporal regulation of gene expression. Here, we investigated the genetic and functional architecture of a genomic region within the FTO gene that is strongly associated with obesity risk. We show that multiple variants on a common haplotype modify the regulatory properties of several enhancers targeting IRX3 and IRX5 from megabase distances. We demonstrate that these enhancers affect gene expression in multiple tissues, including adipose and brain, and impart regulatory effects during a restricted temporal window. Our data indicate that the genetic architecture of disease-associated loci may involve extensive pleiotropy, allelic heterogeneity, shared allelic effects across tissues, and temporally restricted effects.
    DOI:  https://doi.org/10.1126/science.abf1008
  11. Ir J Med Sci. 2021 May 29.
    Mutational analysis of mitochondrial tRNA genes in 138 patients with Leber's hereditary optic neuropathy.
    Jie Shuai, Jian Shi, Ya Liang, Fangfang Ji, Luo Gu, Zhilan Yuan.
       INTRODUCTION: Mutations in mitochondrial DNA (mtDNA) are the most important causes for Leber's hereditary optic neuropathy (LHON). Of these, three primary mtDNA mutations account for more than 90% cases of this disease. However, to date, little is known regarding the relationship between mitochondrial tRNA (mt-tRNA) variants and LHON.
    AIM: In this study, we aimed to investigate the association between mt-tRNA variants and LHON.
    METHODOLOGY: One hundred thirty-eight LHON patients lacking three primary mutations (ND1 3460G > A, ND4 11778Gxs > A, and ND6 14484 T > C), as well as 266 controls were enrolled in this study. PCR-Sanger sequencing was performed to screen the mt-tRNA variants. Moreover, the phylogenetic analysis, pathogenicity scoring system, as well as mitochondrial functions were performed.
    RESULTS: We identified 8 possible pathogenic variants: tRNAPhe 593 T > C, tRNALeu(UUR) 3275C > T, tRNAGln 4363 T > C, tRNAMet 4435A > G, tRNAAla 5587 T > C, tRNAGlu 14693A > G, tRNAThr 15927G > A, and 15951A > G, which may change the structural and functional impact on the corresponding tRNAs, and subsequently lead to a failure in tRNA metabolism. Furthermore, significant reductions in mitochondrial ATP and MMP levels and an overproduction of ROS were observed in cybrid cells containing these mt-tRNA variants, suggesting that these variants may lead to mitochondrial dysfunction which was responsible for LHON.
    CONCLUSION: Our study indicated that mt-tRNA variants were associated with LHON, and screening for mt-tRNA variants were recommended for early detection, diagnosis, and prevention of maternally inherited LHON.
    Keywords:  LHON; Mitochondrial dysfunction; Mt-tRNA; Pathogenic; Variants
    DOI:  https://doi.org/10.1007/s11845-021-02656-6
  12. Methods Mol Biol. 2021 ;2277 203-245
    Heterologous Inferential Analysis (HIA) and Other Emerging Concepts: In Understanding Mitochondrial Variation In Pathogenesis: There is no More Low-Hanging Fruit.
    Antón Vila-Sanjurjo, Paul M Smith, Joanna L Elson.
      Here we summarize our latest efforts to elucidate the role of mtDNA variants affecting the mitochondrial translation machinery, namely variants mapping to the mt-rRNA and mt-tRNA genes. Evidence is accumulating to suggest that the cellular response to interference with mitochondrial translation is different from that occurring as a result of mutations in genes encoding OXPHOS proteins. As a result, it appears safe to state that a complete view of mitochondrial disease will not be obtained until we understand the effect of mt-rRNA and mt-tRNA variants on mitochondrial protein synthesis. Despite the identification of a large number of potentially pathogenic variants in the mitochondrially encoded rRNA (mt-rRNA) genes, we lack direct methods to firmly establish their pathogenicity. In the absence of such methods, we have devised an indirect approach named heterologous inferential analysis (HIA ) that can be used to make predictions concerning the disruptive potential of a large subset of mt-rRNA variants. We have used HIA to explore the mutational landscape of 12S and 16S mt-rRNA genes. Our HIA studies include a thorough classification of all rare variants reported in the literature as well as others obtained from studies performed in collaboration with physicians. HIA has also been used with non-mammalian mt-rRNA genes to elucidate how mitotypes influence the interaction of the individual and the environment. Regarding mt-tRNA variations, rapidly growing evidence shows that the spectrum of mutations causing mitochondrial disease might differ between the different mitochondrial haplogroups seen in human populations.
    Keywords:  Heterologous inferential analysis; Mitochondrial RNA; Mitochondrial haplogroups; Mitochondrial pathogenesis; Mitochondrial translation; Mitotypes
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_14
  13. Brain Commun. 2021 ;3(2): fcab063
    Dominant ACO2 mutations are a frequent cause of isolated optic atrophy.
    European ION Group
      Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells.
    Keywords:  ACO2; Krebs cycle; aconitase 2; mitochondria; optic neuropathy
    DOI:  https://doi.org/10.1093/braincomms/fcab063
  14. Methods Mol Biol. 2021 ;2276 441-452
    Monitoring the Mitochondrial Presequence Import Pathway In Living Mammalian Cells with a New Molecular Biosensor.
    Maxime Jacoupy, Emeline Hamon-Keromen, Olga Corti.
      Most mitochondrial proteins are encoded by the nuclear genome, synthesized in the cytosol, and imported into the organelle. Mitochondrial protein import is therefore vital for the maintenance of mitochondrial function and cell survival. Alterations in this process are suspected to contribute to various diseases, including neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Our understanding of the cytosolic signaling mechanisms and posttranslational modifications regulating the mitochondrial import process is still in its infancy and hampered by the lack of tools for its dynamic assessment in cells. We recently engineered an inducible molecular biosensor for monitoring one of the main mitochondrial import routes, the so-called presequence pathway, using a quantitative luminescence-based readout. Here, we provide basic guidelines for using this probe in common cell types of general use in the scientific community: HEK293T cells, human fibroblasts, and mouse primary neurons. These guidelines can serve as a starting point for the development of more elaborated protocols for the dynamic investigation of the presequence import pathway and its regulation in relevant physiological and pathological conditions.
    Keywords:  Bioluminescence assay; Biosensor; Mitochondrial protein import; Presequence pathway; TOM machinery
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_32
  15. Aging Cell. 2021 Jun 01. e13379
    Loss of Fis1 impairs proteostasis during skeletal muscle aging in Drosophila.
    Tai-Ting Lee, Po-Lin Chen, Matthew P Su, Jian-Chiuan Li, Yi-Wen Chang, Rei-Wen Liu, Hsueh-Fen Juan, Jinn-Moon Yang, Shih-Peng Chan, Yu-Chen Tsai, Sophia von Stockum, Elena Ziviani, Azusa Kamikouchi, Horng-Dar Wang, Chun-Hong Chen.
      Increased levels of dysfunctional mitochondria within skeletal muscle are correlated with numerous age-related physiopathological conditions. Improving our understanding of the links between mitochondrial function and muscle proteostasis, and the role played by individual genes and regulatory networks, is essential to develop treatments for these conditions. One potential player is the mitochondrial outer membrane protein Fis1, a crucial fission factor heavily involved in mitochondrial dynamics in yeast but with an unknown role in higher-order organisms. By using Drosophila melanogaster as a model, we explored the effect of Fis1 mutations generated by transposon Minos-mediated integration. Mutants exhibited a higher ratio of damaged mitochondria with age as well as elevated reactive oxygen species levels compared with controls. This caused an increase in oxidative stress, resulting in large accumulations of ubiquitinated proteins, accelerated muscle function decline, and mitochondrial myopathies in young mutant flies. Ectopic expression of Fis1 isoforms was sufficient to suppress this phenotype. Loss of Fis1 led to unbalanced mitochondrial proteostasis within fly muscle, decreasing both flight capabilities and lifespan. Fis1 thus clearly plays a role in fly mitochondrial dynamics. Further investigations into the detailed function of Fis1 are necessary for exploring how mitochondrial function correlates with muscle health during aging.
    Keywords:   Drosophila melanogaster ; Fis1; aging; mitochondria
    DOI:  https://doi.org/10.1111/acel.13379
  16. Methods Mol Biol. 2021 ;2277 331-343
    A New Method for Sequencing the Mitochondrial Genome by Using Long Read Technology.
    Sophie Dhorne-Pollet, Nicolas Pollet, Eric Barrey.
      We describe a protocol to prepare a multiplexed mtDNA library from a blood sample for performing a long read sequencing of the mitochondrial genome. All steps are carefully described to get a high enrichment of mtDNA relative to total DNA extracted from the blood sample. The obtained mutiplexed library allows the production of long sequence mtDNA reads up to 16.5 kbp with a quality enabling variant-calling by using a portable sequencer (MinION, Oxford Nanopore Technologies).
    Keywords:  Long read sequencing; MinION; Mitochondrial DNA; Multiple displacement amplification; Nuclear DNA depletion
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_20
  17. J Clin Med. 2021 May 12. pii: 2063. [Epub ahead of print]10(10):
    Movement Disorders in Children with a Mitochondrial Disease: A Cross-Sectional Survey from the Nationwide Italian Collaborative Network of Mitochondrial Diseases.
    Chiara Ticci, Daniele Orsucci, Anna Ardissone, Luca Bello, Enrico Bertini, Irene Bonato, Claudio Bruno, Valerio Carelli, Daria Diodato, Stefano Doccini, Maria Alice Donati, Claudia Dosi, Massimiliano Filosto, Chiara Fiorillo, Chiara La Morgia, Costanza Lamperti, Silvia Marchet, Diego Martinelli, Carlo Minetti, Maurizio Moggio, Tiziana Enrica Mongini, Vincenzo Montano, Isabella Moroni, Olimpia Musumeci, Elia Pancheri, Elena Pegoraro, Guido Primiano, Elena Procopio, Anna Rubegni, Roberta Scalise, Monica Sciacco, Serenella Servidei, Gabriele Siciliano, Costanza Simoncini, Deborah Tolomeo, Paola Tonin, Antonio Toscano, Flavia Tubili, Michelangelo Mancuso, Roberta Battini, Filippo Maria Santorelli.
      Movement disorders are increasingly being recognized as a manifestation of childhood-onset mitochondrial diseases (MDs). However, the spectrum and characteristics of these conditions have not been studied in detail in the context of a well-defined cohort of patients. We retrospectively explored a cohort of individuals with childhood-onset MDs querying the Nationwide Italian Collaborative Network of Mitochondrial Diseases database. Using a customized online questionnaire, we attempted to collect data from the subgroup of patients with movement disorders. Complete information was available for 102 patients. Movement disorder was the presenting feature of MD in 45 individuals, with a mean age at onset of 11 years. Ataxia was the most common movement disorder at onset, followed by dystonia, tremor, hypokinetic disorders, chorea, and myoclonus. During the disease course, most patients (67.7%) encountered a worsening of their movement disorder. Basal ganglia involvement, cerebral white matter changes, and cerebellar atrophy were the most commonly associated neuroradiological patterns. Forty-one patients harbored point mutations in the mitochondrial DNA, 10 carried mitochondrial DNA rearrangements, and 41 cases presented mutations in nuclear-DNA-encoded genes, the latter being associated with an earlier onset and a higher impairment in activities of daily living. Among our patients, 32 individuals received pharmacological treatment; clonazepam and oral baclofen were the most commonly used drugs, whereas levodopa and intrathecal baclofen administration were the most effective. A better delineation of the movement disorders phenotypes starting in childhood may improve our diagnostic workup in MDs, fine tuning management, and treatment of affected patients.
    Keywords:  childhood onset; mitochondrial disease; movement disorder; multicenter cross-sectional study
    DOI:  https://doi.org/10.3390/jcm10102063
  18. Methods Mol Biol. 2021 ;2277 391-403
    Bioenergetic Profiling of Human Pluripotent Stem Cells.
    Gizem Inak, Marie-Thérèse Henke, Alessandro Prigione.
      Cellular metabolism contributes to cell fate decisions. Bioenergetic profiling can therefore provide considerable insights into cellular identity and specification. Given the current importance of human pluripotent stem cells (hPSCs) for biomedical applications, assessing the bioenergetic properties of hPSCs and derivatives can unveil relevant mechanisms in the context of development biology and molecular disease modeling. Here, we describe a method to facilitate bioenergetic profiling of hPSCs in a reproducible and scalable manner. After simultaneous assessment of mitochondrial respiration and glycolytic capacity using Seahorse XFe96 Analyzer, we measure lactate concentration in the cellular media. Finally, we normalize the values based on DNA amount. We describe the procedures with specific requirements related to hPSCs . However, the same protocol can be easily adapted to other cell types, including differentiated progenies from hPSCs .
    Keywords:  Bioenergetic profiling; Mitochondria; Pluripotent stem cells; Seahorse; iPSCs
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_24
  19. Mol Biol Rep. 2021 Jun 05.
    Mitochondrial disease patients with novel ND4 12058A > C and ND1 m.3911A > G variations: implications for a role in the phenotype following a bioinformatic investigation.
    Emna Mkaouar-Rebai, Marwa Ammar, Lamia Sfaihi, Olfa Alila-Fersi, Marwa Maalej, Rahma Felhi, Mongia Hachicha, Faiza Fakhfakh.
      Mitochondrial diseases include a wide group of clinically heterogeneous disorders caused by a dysfunction of the mitochondrial respiratory chain and can be related to mutations in nuclear or mitochondrial DNA genes. In the present report, we performed a whole mitochondrial genome screening in two patients with clinical features of mitochondrial diseases. Mutational analysis revealed the presence of two undescribed heteroplasmic mitochondrial variations, the m.3911A > G (E202G) variant in the MT-ND1 gene found in two patients (P1 and P2) and the m.12058A > C (E433D) pathogenic variant in the MT-ND4 gene present only in patient P2 who had a more severe phenotype. These two substitutions were predicted to be damaging by several bioinformatics tools and lead to amino acid changes in two conserved residues localized in two important functional domains of the mitochondrial subunits of complex I. Furthermore, the 3D modeling suggested that the two amino acid changes could therefore alter the structure of the two subunits and may decrease the stability and the function of complex I. The two described pathogenic variants found in patient P2 could act synergically and alter the complex I function by affecting the proton pumping processes and the energy production and then could explain the severe phenotype compared to patient P1 presenting only the E202G substitution in ND1.
    Keywords:  MT-ND1; MT-ND4; Mitochondrial diseases; m.12058A > C; m.3911A > G; mtDNA
    DOI:  https://doi.org/10.1007/s11033-021-06452-4
  20. Methods Mol Biol. 2021 ;2276 259-270
    3D Optical Cryo-Imaging Method: A Novel Approach to Quantify Renal Mitochondrial Bioenergetics Dysfunction.
    Shima Mehrvar, Amadou K S Camara, Mahsa Ranji.
      Mitochondrial dysfunction contributes to various injuries and diseases. A mechanistic understanding of how dysfunctional mitochondria modulates metabolism is of paramount importance. Three-dimensional (3D) optical cryo-imager is a custom-designed device that can quantify the volumetric bioenergetics of organs in small animal models. The instrument captures the autofluorescence of bioenergetics indices (NADH and FAD) from tissues at cryogenic temperature. The quantified redox ratio (NADH/FAD) is used as an optical indicator of mitochondrial redox state.
    Keywords:  Bioenergetics; Fluorescence imaging; Mitochondria; Optical imaging; Redox state
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_20
  21. Methods Mol Biol. 2021 ;2276 271-283
    Simultaneous Quantification of Mitochondrial ATP and ROS Production Using ATP Energy Clamp Methodology.
    Liping Yu, Brian D Fink, William I Sivitz.
      Several methods are available to measure ATP production by isolated mitochondria or permeabilized cells but have several limitations, depending upon the particular assay employed. These limitations may include poor sensitivity or specificity, complexity of the method, poor throughput, changes in mitochondrial inner membrane potential as ATP is consumed, and/or inability to simultaneously assess other mitochondrial functional parameters. Here we describe a novel nuclear magnetic resonance (NMR)-based assay that can be carried out with high efficiency in a manner that alleviates the above problems.
    Keywords:  ATP; Bioenergetics; H2O2; Mitochondria; NMR; Reactive oxygen species; Superoxide
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_21
  22. Methods Mol Biol. 2021 ;2276 165-171
    Investigation of Mitochondrial ADP-Ribosylation Via Immunofluorescence.
    Ann-Katrin Hopp, Michael O Hottiger.
      ADP-ribosylation is a posttranslational protein modification, involved in various cellular processes, ranging from DNA-damage repair to apoptosis. While its function has been studied amply with respect to genotoxic stress-associated nuclear ADP-ribosylation, the functional relevance of mitochondrial ADP-ribosylation remains so far poorly studied. This is mainly attributed to the absence of powerful techniques able to detect the modification. However, the usage of recently developed anti-ADP-ribose-specific antibodies allows now to investigate mitochondrial ADP-ribosylation under physiological and pathophysiological conditions. In the below method, we describe in detail how to efficiently detect and quantify mitochondrial ADP-ribosylation via immunofluorescence.
    Keywords:  ADP-ribosylation; Antibodies; Immunofluorescence; MacroD1; Mitochondria; NAD+; Posttranslational modifications
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_12
  23. Stem Cells. 2021 Jun 05.
    Mitochondria in neurogenesis: Implications for mitochondrial diseases.
    Dario Brunetti, Werner Dykstra, Stephanie Le, Annika Zink, Alessandro Prigione.
      Mitochondria are organelles with recognized key roles in cellular homeostasis, including bioenergetics, redox, calcium signaling, and cell death. Mitochondria are essential for neuronal function, given the high energy demands of the human brain. Consequently, mitochondrial diseases affecting oxidative phosphorylation (OXPHOS) commonly exhibit neurological impairment. Emerging evidence suggests that mitochondria are important not only for mature postmitotic neurons but also for the regulation of neural progenitor cells (NPCs) during the process of neurogenesis. These recent findings put mitochondria as central regulator of cell fate decisions during brain development. OXPHOS mutations may disrupt the function of NPCs and thereby impair the metabolic programming required for neural fate commitment. Promoting the mitochondrial function of NPCs could therefore represent a novel interventional approach against incurable mitochondrial diseases.
    Keywords:  NPCs; iPSCs; mitochondria; mitochondrial diseases; neurogenesis
    DOI:  https://doi.org/10.1002/stem.3425
  24. Methods Mol Biol. 2021 ;2277 39-47
    Quantitatively Controlled Intercellular Mitochondrial Transfer by Cell Fusion-Based Method Using a Microfluidic Device.
    Ken-Ichi Wada, Kazuo Hosokawa, Yoshihiro Ito, Mizuo Maeda.
      Quantitative control of mitochondrial transfer is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA) because it enables precise modulation of heteroplasmy. Furthermore, single mitochondrion transfer from a mtDNA mutation-accumulated cell to a mtDNA-less (ρ0) cell potentially achieves homoplasmy of mutated mtDNA. Here we describe the method for quantitative control of mitochondrial transfer including achieving single mitochondrion transfer between live single cells using a microfluidic device.
    Keywords:  Cell fusion; Heteroplasmy; Homoplasmy; Microfluidics; Microtunnel; Mitochondrial DNA; Mitochondrial transfer
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_3
  25. Methods Mol Biol. 2021 ;2277 143-155
    The NDUFS4 Knockout Mouse: A Dual Threat Model of Childhood Mitochondrial Disease and Normative Aging.
    Anthony S Grillo, Alessandro Bitto, Matt Kaeberlein.
      Mice missing the Complex I subunit NADH:Ubiquinone Oxidoreductase Fe-S Protein 4 (NDUFS4) of the electron transport chain are a leading model of the severe mitochondrial disease Leigh syndrome. These mice have enabled a better understanding of mitochondrial dysfunction in human disease, as well as in the discovery of interventions that can potentially suppress mitochondrial disease manifestations. In addition, increasing evidence suggests significant overlap between interventions that increase survival in NDUFS4 knockout mice and that extend life span during normative aging. This chapter discusses the practical aspects of handling and studying these mice, which can be challenging due to their severe disease phenotype. Common procedures such as breeding, genotyping, weaning, or treating these transgenic mice are also discussed.
    Keywords:  Aging; Complex I; Electron transport chain; Hypoxia; Leigh syndrome; Mitochondrial disease; Mitochondrial dysfunction; NAD; NDUFS4; Rapamycin; mTOR
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_10
  26. Methods Mol Biol. 2021 ;2276 143-151
    Whole-Cell and Mitochondrial dNTP Pool Quantification from Cells and Tissues.
    Juan C Landoni, Liya Wang, Anu Suomalainen.
      Deoxynucleoside 5'-triphosphates (dNTPs) are the molecular building blocks for DNA synthesis, and their balanced concentration in the cell is fundamental for health. dNTP imbalance can lead to genomic instability and other metabolic disturbances, resulting in devastating mitochondrial diseases.The accurate and efficient measurement of dNTPs from different biological samples and cellular compartments is vital to understand the mechanisms behind these diseases and develop and scrutinize their possible treatments. This chapter describes an update on the most recent development of the traditional radiolabeled polymerase extension method and its adaptation for the measurement of whole-cell and mitochondrial dNTP pools from cultured cells and tissue samples. The solid-phase reaction setting enables an increase in efficiency, accuracy, and measurement scale.
    Keywords:  Mitochondrial DNA depletion syndrome; Nucleotide pools; Solid-phase detection; dNTP; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_10
  27. Methods Mol Biol. 2021 ;2276 31-39
    Preparation of "Functional" Mitochondria: A Challenging Business.
    Stefan Lehr, Sonja Hartwig, Jorg Kotzka.
      As the powerhouse of the cell, mitochondria, plays a crucial role in many aspects of life, whereby mitochondrial dysfunctions are associated with pathogenesis of many diseases, like neurodegenerative diseases, obesity, cancer, and metabolic as well as cardiovascular disorders. Mitochondria analysis frequently starts with isolation and enrichment procedures, which have become increasingly important in biomedical research. Unfortunately, isolation procedures can easily cause changes in the structural integrity of mitochondria during in vitro handling having impact on their function. This carries the risk that conclusions about isolated mitochondria may be drawn on the basis of experimental artifacts. Here we critically review a commonly used isolation procedure for mitochondria utilizing differential (gradient) centrifugation and depict major challenges to achieve "functional" mitochondria as basis for comprehensive physiological studies.
    Keywords:  Differential gradient centrifugation; Isolation of mitochondria; Mitochondrial integrity
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_2
  28. Methods Mol Biol. 2021 ;2276 235-248
    Patch-Clamp Recording of the Activity of Ion Channels in the Inner Mitochondrial Membrane.
    Piotr Bednarczyk, Rafał P Kampa, Shur Gałecka, Aleksandra Sęk, Agnieszka Walewska, Piotr Koprowski.
      Mitochondria are intracellular organelles, which play a crucial role in the generation of ATP. Mitochondria are surrounded by a double membrane, consisting of a smooth outer membrane (OMM) and a markedly folded inner mitochondrial membrane (IMM). Mitochondrion that has been stripped of its outer membrane, leaving the inner membrane intact is called mitoplast. There is a number of different transport proteins located in the inner mitochondrial membrane including ion channels that mediate fluxes of potassium, calcium, and chloride ions. These channels regulate the mitochondrial membrane potential, respiration, and production of reactive oxygen species. The stability of mitoplasts offers the possibility of measuring the activity of ion channels from IMM using the patch-clamp technique. Electrophysiological measurements of currents through ion channels in the IMM permit discovery of unique properties of these channels with the aim of new specific pharmacological therapies. In this chapter, we describe the isolation of mitochondria, preparation of mitoplast for patch-clamp recordings and single-mitoplast PCR experiments, which can be helpful in mastering the technique of recording the activity of mitochondrial ion channels.
    Keywords:  Inner mitochondrial membrane; Ion channel; Mitochondria; Mitoplast; PCR; Patch-clamp technique
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_18
  29. Methods Mol Biol. 2021 ;2277 433-447
    The Isolation and Deep Sequencing of Mitochondrial DNA.
    Alexander G Bury, Fiona M Robertson, Angela Pyle, Brendan A I Payne, Gavin Hudson.
      In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .
    Keywords:  Massively parallel sequencing; Mitochondrial DNA; Mitochondrial isolation deep sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_27
  30. Nat Commun. 2021 06 02. 12(1): 3285
    Prohibitin 1 is essential to preserve mitochondria and myelin integrity in Schwann cells.
    Gustavo Della-Flora Nunes, Emma R Wilson, Leandro N Marziali, Edward Hurley, Nicholas Silvestri, Bin He, Bert W O'Malley, Bogdan Beirowski, Yannick Poitelon, Lawrence Wrabetz, M Laura Feltri.
      In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.
    DOI:  https://doi.org/10.1038/s41467-021-23552-8
  31. Methods Mol Biol. 2021 ;2276 57-66
    Purification of Functional Platelet Mitochondria Using a Discontinuous Percoll Gradient.
    Jacob L Léger, Nicolas Pichaud, Luc H Boudreau.
      The isolation of mitochondria is gaining importance in experimental and clinical laboratory settings. Of interest, mitochondria and mitochondrial components (i.e., circular mitochondrial DNA, N-formylated peptides, cardiolipin) have been involved in several human inflammatory pathologies, such as cancer, Alzheimer's disease, Parkinson's disease, and rheumatoid arthritis. While several mitochondrial isolation methods have been previously published, these techniques are aimed at yielding mitochondria from cell types other than platelets. In addition, little information is known on the number of platelet-derived microvesicles that can contaminate the mitochondrial preparation or even the overall quality as well as functional and structural integrity of mitochondria. Here we describe a purification method, using a discontinuous Percoll gradient, yielding mitochondria of high purity and integrity from human platelets.
    Keywords:  Mitochondria isolation; Mitochondria membrane integrity; Percoll extraction method; Platelet-derived microvesicles; Platelet-derived mitochondria
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_4
  32. Commun Biol. 2021 Jun 03. 4(1): 667
    Structural basis of complex formation between mitochondrial anion channel VDAC1 and Hexokinase-II.
    Nandan Haloi, Po-Chao Wen, Qunli Cheng, Meiying Yang, Gayathri Natarajan, Amadou K S Camara, Wai-Meng Kwok, Emad Tajkhorshid.
      Complex formation between hexokinase-II (HKII) and the mitochondrial VDAC1 is crucial to cell growth and survival. We hypothesize that HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we devised a hybrid approach. First, we describe membrane binding of HKII with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion capturing membrane insertion of its H-anchor. The insertion depth of the H-anchor was then used to derive positional restraints in subsequent millisecond-scale Brownian dynamics (BD) simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models for the complex were further refined and their structural stability probed with additional MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1's permeation pathway, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. We also show how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and has implications in mitochondria-mediated cell death.
    DOI:  https://doi.org/10.1038/s42003-021-02205-y
  33. Methods Mol Biol. 2021 ;2276 193-202
    Assessment of Mitochondrial Membrane Potential and NADH Redox State in Acute Brain Slices.
    Andrey Y Vinokurov, Viktor V Dremin, Gennadii A Piavchenko, Olga A Stelmashchuk, Plamena R Angelova, Andrey Y Abramov.
      Brain is one of the most energy-demanding organs. Energy in the form of ATP is produced in brain cells predominantly in oxidative phosphorylation coupled to mitochondrial respiration. Any alteration of the mitochondrial metabolism or prolonged ischemic or anoxic conditions can lead to serious neurological conditions, including neurodegenerative disorders. Assessment of mitochondrial metabolism is important for understanding physiological and pathological processes in the brain. Bioenergetics in central nervous system is dependent on multiple parameters including neuron-glia interactions and considering this, in vivo or ex vivo, the measurements of mitochondrial metabolism should also be complimenting the experiments on isolated mitochondria or cell cultures. To assess the mitochondrial function, there are several key bioenergetic parameters which indicate mitochondrial health. One of the major characteristics of mitochondria is the mitochondrial membrane potential (ΔΨm) which is used as a proton motive force for ATP production and generated by activity of the electron transport chain. Major donor of electrons for the mitochondrial respiratory chain is NADH. Here we demonstrate how to measure mitochondrial NADH/NAD(P)H autofluorescence and ΔΨm in acute brain slices in a time-dependent manner and provide information for the identification of NADH redox index, mitochondrial NADH pool, and the rate of NADH production in the Krebs cycle. Additionally, non-mitochondrial NADH/NADPH autofluorescence can signify the level of activity of the pentose phosphate pathway.
    Keywords:  Acute brain slices; Mitochondria; Mitochondrial membrane potential; NADH
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_14
  34. Curr Med Chem. 2021 May 31.
    Small Molecule Regulators Targeting NAD+ Biosynthetic Enzymes.
    Alyson Curry, Dawanna White, Yana Cen.
      Nicotinamide adenine dinucleotide (NAD+) is a key player in many metabolic pathways as an activated carrier of electrons. In addition to being the cofactor for redox reactions, NAD+ also serves as the substrate for various enzymatic transformations such as adenylation and ADP-ribosylation. Maintaining cellular NAD+ homeostasis has been suggested as an effective anti-aging strategy. Given the importance of NAD+ in regulating a broad spectrum of cellular events, small molecules targeting NAD+ metabolism have been pursued as therapeutic interventions for the treatment of mitochondrial disorders and age-related diseases. In this article, small molecule regulators of NAD+ biosynthetic enzymes will be reviewed. The focus will be given to the discovery and development of these molecules, the mechanism of action as well as their therapeutic potentials.
    Keywords:  NAD+; aging; biosynthesis; de novo; metabolism; salvage; small molecule regulator
    DOI:  https://doi.org/10.2174/0929867328666210531144629
  35. Nat Commun. 2021 06 03. 12(1): 3320
    Perilipin 5 links mitochondrial uncoupled respiration in brown fat to healthy white fat remodeling and systemic glucose tolerance.
    Violeta I Gallardo-Montejano, Chaofeng Yang, Lisa Hahner, John L McAfee, Joshua A Johnson, William L Holland, Rodrigo Fernandez-Valdivia, Perry E Bickel.
      Exposure of mice or humans to cold promotes significant changes in brown adipose tissue (BAT) with respect to histology, lipid content, gene expression, and mitochondrial mass and function. Herein we report that the lipid droplet coat protein Perilipin 5 (PLIN5) increases markedly in BAT during exposure of mice to cold. To understand the functional significance of cold-induced PLIN5, we created and characterized gain- and loss-of-function mouse models. Enforcing PLIN5 expression in mouse BAT mimics the effects of cold with respect to mitochondrial cristae packing and uncoupled substrate-driven respiration. PLIN5 is necessary for the maintenance of mitochondrial cristae structure and respiratory function during cold stress. We further show that promoting PLIN5 function in BAT is associated with healthy remodeling of subcutaneous white adipose tissue and improvements in systemic glucose tolerance and diet-induced hepatic steatosis. These observations will inform future strategies that seek to exploit thermogenic adipose tissue as a therapeutic target for type 2 diabetes, obesity, and nonalcoholic fatty liver disease.
    DOI:  https://doi.org/10.1038/s41467-021-23601-2
  36. Aging (Albany NY). 2021 Jun 01. 13
    The dysregulated Pink1- drosophila mitochondrial proteome is partially corrected with exercise.
    Brad Ebanks, Thomas L Ingram, Gunjan Katyal, John R Ingram, Nicoleta Moisoi, Lisa Chakrabarti.
      One of the genes which has been linked to the onset of juvenile/early onset Parkinson's disease (PD) is PINK1. There is evidence that supports the therapeutic potential of exercise in the alleviation of PD symptoms. It is possible that exercise may enhance synaptic plasticity, protect against neuro-inflammation and modulate L-Dopa regulated signalling pathways. We explored the effects of exercise on Pink1 deficient Drosophila melanogaster which undergo neurodegeneration and muscle degeneration. We used a 'power-tower' type exercise platform to deliver exercise activity to Pink1- and age matched wild-type Drosophila. Mitochondrial proteomic profiles responding to exercise were obtained. Of the 516 proteins identified, 105 proteins had different levels between Pink1- and wild-type non-exercised Drosophila. Gene ontology enrichment analysis and STRING network analysis highlighted proteins and pathways with altered expression within the mitochondrial proteome. Comparison of the Pink1- exercised proteome to wild-type proteomes showed that exercising the Pink1- Drosophila caused their proteomic profile to return towards wild-type levels.
    Keywords:  PINK1; drosophila; exercise; mitochondria; proteomics
    DOI:  https://doi.org/10.18632/aging.203128
  37. Methods Mol Biol. 2021 ;2276 153-163
    Single-Particle Tracking Method in Fluorescence Microscopy to Monitor Bioenergetic Responses of Individual Mitochondria.
    Camille Colin, Emmanuel Suraniti, Emma Abell, Audrey Sémont, Neso Sojic, Philippe Diolez, Stéphane Arbault.
      The spectroscopic methods commonly used to study mitochondria bioenergetics do not show the diversity of responses within a population of mitochondria (isolated or in a cell), and/or cannot measure individual dynamics. New methodological developments are necessary in order to improve quantitative and kinetic resolutions and eventually gain further insights on individual mitochondrial responses, such as studying activities of the mitochondrial permeability transition pore (mPTP ). The work reported herein is devoted to study responses of single mitochondria within a large population after isolation from cardiomyocytes. Mitochondria were preloaded with a commonly used membrane potential sensitive dye (TMRM), they are then deposited on a plasma-treated glass coverslip and subsequently energized or inhibited by additions of usual bioenergetics effectors. Responses were analyzed by fluorescence microscopy over few thousands of mitochondria simultaneously with a single organelle resolution. We report an automatic method to analyze each image of time-lapse stacks based on the TrackMate-ImageJ plug-in and specially made Python scripts. Images are processed to eliminate defects of illumination inhomogeneity, improving by at least two orders of magnitude the signal/noise ratio. This method enables us to follow the track of each mitochondrion within the observed field and monitor its fluorescence changes, with a time resolution of 400 ms, uninterrupted over the course of the experiment. Such methodological improvement is a prerequisite to further study the role of mPTP in single mitochondria during calcium transient loading.
    Keywords:  Bioenergetics; Fiji software; Fluorescence microscopy; Membrane potential; Mitochondria; Single organelle; Single particle tracking; TrackMate
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_11
  38. Chem Sci. 2019 Dec 09. 11(4): 1052-1065
    Shedding light on the mitochondrial matrix through a functional membrane transporter.
    Alberto Blázquez-Moraleja, Ines Sáenz-de-Santa María, María D Chiara, Delia Álvarez-Fernández, Inmaculada García-Moreno, Ruth Prieto-Montero, Virginia Martínez-Martínez, Iñigo López Arbeloa, Jose Luis Chiara.
      The first fluorescent probes that are actively channeled into the mitochondrial matrix by a specific mitochondrial membrane transporter in living cells have been developed. The new functional probes (BCT) have a minimalist structural design based on the highly efficient and photostable BODIPY chromophore and carnitine as a biotargeting element. Both units are orthogonally bonded through the common boron atom, thus avoiding the use of complex polyatomic connectors. In contrast to known mitochondria-specific dyes, BCTs selectively label these organelles regardless of their transmembrane potential and in an enantioselective way. The obtained experimental evidence supports carnitine-acylcarnitine translocase (CACT) as the key transporter protein for BCTs, which behave therefore as acylcarnitine biomimetics. This simple structural design can be readily extended to other structurally diverse starting F-BODIPYs to obtain BCTs with varied emission wavelengths along the visible and NIR spectral regions and with multifunctional capabilities. BCTs are the first fluorescent derivatives of carnitine to be used in cell microscopy and stand as promising research tools to explore the role of the carnitine shuttle system in cancer and metabolic diseases. Extension of this approach to other small-molecule mitochondrial transporters is envisaged.
    DOI:  https://doi.org/10.1039/c9sc04852a
  39. Int J Mol Sci. 2021 May 27. pii: 5742. [Epub ahead of print]22(11):
    Molecular Epidemiology of Mitochondrial Cardiomyopathy: A Search Among Mitochondrial and Nuclear Genes.
    Cristina Mazzaccara, Bruno Mirra, Ferdinando Barretta, Martina Caiazza, Barbara Lombardo, Olga Scudiero, Nadia Tinto, Giuseppe Limongelli, Giulia Frisso.
      Mitochondrial Cardiomyopathy (MCM) is a common manifestation of multi-organ Mitochondrial Diseases (MDs), occasionally present in non-syndromic cases. Diagnosis of MCM is complex because of wide clinical and genetic heterogeneity and requires medical, laboratory, and neuroimaging investigations. Currently, the molecular screening for MCM is fundamental part of MDs management and allows achieving the definitive diagnosis. In this article, we review the current genetic knowledge associated with MDs, focusing on diagnosis of MCM and MDs showing cardiac involvement. We searched for publications on mitochondrial and nuclear genes involved in MCM, mainly focusing on genetic screening based on targeted gene panels for the molecular diagnosis of the MCM, by using Next Generation Sequencing. Here we report twelve case reports, four case-control studies, eleven retrospective studies, and two prospective studies, for a total of twenty-nine papers concerning the evaluation of cardiac manifestations in mitochondrial diseases. From the analysis of published causal mutations, we identified 130 genes to be associated with mitochondrial heart diseases. A large proportion of these genes (34.3%) encode for key proteins involved in the oxidative phosphorylation system (OXPHOS), either as directly OXPHOS subunits (22.8%), and as OXPHOS assembly factors (11.5%). Mutations in several mitochondrial tRNA genes have been also reported in multi-organ or isolated MCM (15.3%). This review highlights the main disease-genes, identified by extensive genetic analysis, which could be included as target genes in next generation panels for the molecular diagnosis of patients with clinical suspect of mitochondrial cardiomyopathies.
    Keywords:  diagnosis; genetic testing; mitochondrial DNA; mitochondrial cardiomyopathy; mitochondrial disease; mutation; next generation sequencing
    DOI:  https://doi.org/10.3390/ijms22115742
  40. Int J Mol Sci. 2021 May 02. pii: 4827. [Epub ahead of print]22(9):
    Mitochondrial Dysfunction in Podocytes Caused by CRIF1 Deficiency Leads to Progressive Albuminuria and Glomerular Sclerosis in Mice.
    Ki Ryang Na, Jin Young Jeong, Jin Ah Shin, Yoon-Kyung Chang, Kwang-Sun Suh, Kang Wook Lee, Dae Eun Choi.
      Recent studies have implicated mitochondrial disruption in podocyte dysfunction, which is a characteristic feature of primary and diabetic glomerular diseases. However, the mechanisms by which primary mitochondrial dysfunction in podocytes affects glomerular renal diseases are currently unknown. To investigate the role of mitochondrial oxidative phosphorylation (OxPhos) in podocyte dysfunction, glomerular function was examined in mice carrying a loss of function mutation of the gene encoding CR6-interacting factor-1 (CRIF1), which is essential for intramitochondrial production and the subsequent insertion of OxPhos polypeptides into the inner mitochondrial membrane. Homozygotic deficiency of CRIF1 in podocytes resulted in profound and progressive albuminuria from 3 weeks of age; the CRIF1-deficient mice also developed glomerular and tubulointerstitial lesions by 10 weeks of age. Furthermore, marked glomerular sclerosis and interstitial fibrosis were observed in homozygous CRIF1-deficient mice at 20 weeks of age. In cultured mouse podocytes, loss of CRIF1 resulted in OxPhos dysfunction and marked loss or abnormal aggregation of F-actin. These findings indicate that the OxPhos status determines the integrity of podocytes and their ability to maintain a tight barrier and control albuminuria. Analyses of the glomerular function of the podocyte-specific primary OxPhos dysfunction model mice demonstrate a link between podocyte mitochondrial dysfunction, progressive glomerular sclerosis, and tubulointerstitial diseases.
    Keywords:  CRIF1; albuminuria; glomerular sclerosis; mitochondrial oxidative phosphorylation; podocyte
    DOI:  https://doi.org/10.3390/ijms22094827
  41. Eur J Hum Genet. 2021 Jun 01.
    A MT-TL1 variant identified by whole exome sequencing in an individual with intellectual disability, epilepsy, and spastic tetraparesis.
    Solve-RD SNV-indel working group
      The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC_012920.1:m.3291T > C; NC_012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line.
    DOI:  https://doi.org/10.1038/s41431-021-00900-2
  42. Nat Struct Mol Biol. 2021 May 31.
    Reevaluating the role of human mitochondrial uL18m in the cytosolic stress response.
    Austin Choi, Antoni Barrientos.
      
    DOI:  https://doi.org/10.1038/s41594-021-00599-1
  43. Life (Basel). 2021 May 11. pii: 432. [Epub ahead of print]11(5):
    Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration.
    Hope I Needs, Margherita Protasoni, Jeremy M Henley, Julien Prudent, Ian Collinson, Gonçalo C Pereira.
      The fact that >99% of mitochondrial proteins are encoded by the nuclear genome and synthesised in the cytosol renders the process of mitochondrial protein import fundamental for normal organelle physiology. In addition to this, the nuclear genome comprises most of the proteins required for respiratory complex assembly and function. This means that without fully functional protein import, mitochondrial respiration will be defective, and the major cellular ATP source depleted. When mitochondrial protein import is impaired, a number of stress response pathways are activated in order to overcome the dysfunction and restore mitochondrial and cellular proteostasis. However, prolonged impaired mitochondrial protein import and subsequent defective respiratory chain function contributes to a number of diseases including primary mitochondrial diseases and neurodegeneration. This review focuses on how the processes of mitochondrial protein translocation and respiratory complex assembly and function are interlinked, how they are regulated, and their importance in health and disease.
    Keywords:  mitochondrial dysfunction; mitochondrial proteostasis; neurodegeneration; protein import; respiratory complex assembly; supercomplexes
    DOI:  https://doi.org/10.3390/life11050432
  44. Nat Med. 2021 May 31.
    Childhood amyotrophic lateral sclerosis caused by excess sphingolipid synthesis.
    Payam Mohassel, Sandra Donkervoort, Museer A Lone, Matthew Nalls, Kenneth Gable, Sita D Gupta, A Reghan Foley, Ying Hu, Jonas Alex Morales Saute, Ana Lucila Moreira, Fernando Kok, Alessandro Introna, Giancarlo Logroscino, Christopher Grunseich, Alec R Nickolls, Naemeh Pourshafie, Sarah B Neuhaus, Dimah Saade, Andrea Gangfuß, Heike Kölbel, Zoe Piccus, Claire E Le Pichon, Chiara Fiorillo, Cindy V Ly, Ana Töpf, Lauren Brady, Sabine Specht, Aliza Zidell, Helio Pedro, Eric Mittelmann, Florian P Thomas, Katherine R Chao, Chamindra G Konersman, Megan T Cho, Tracy Brandt, Volker Straub, Anne M Connolly, Ulrike Schara, Andreas Roos, Mark Tarnopolsky, Ahmet Höke, Robert H Brown, Chia-Hsueh Lee, Thorsten Hornemann, Teresa M Dunn, Carsten G Bönnemann.
      Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.
    DOI:  https://doi.org/10.1038/s41591-021-01346-1
  45. Methods Mol Biol. 2021 ;2277 423-431
    Isolation of Mitochondria from Retinal Pigment Epithelial Cell Cultures and an Application of High-Resolution Respirometric Assay (XFe96 Seahorse Assay).
    Masaaki Ishii, Gyda Beeson, Craig Beeson, Bärbel Rohrer.
      Intracellular Ca2+ is strictly regulated to maintain optimal levels for function of cellular organelles as well as mitochondrial respiratory signaling at the tricarboxylic acid cycle and electron transport chain level. Optimal Ca2+ concentration for these processes vary between cell types. Furthermore, exposure of mitochondria to sustained, elevated levels of Ca2+ induces mitochondrial Ca2+ overload and damage to mitochondrial oxidative phosphorylation and ATP production. Isolated mitochondria are widely used to study mitochondrial physiology and drug effects on mitochondrial metabolism and respiratory function. However, isolated mitochondria are easily damaged during the mitochondrial isolation process. The present article describes a mitochondrial isolation method using Ca2+-chelation to minimize mitochondrial damage. We follow up the isolation process with an application that requires an optimized buffer Ca2+ concentration: the characterization of their respiratory function using a high-resolution respirometric assay.
    Keywords:  Calcium chelator; Mitochondrial isolation; Organelle isolation
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_26
  46. Cell Metab. 2021 Jun 01. pii: S1550-4131(21)00228-X. [Epub ahead of print]33(6): 1069-1071
    Mitochondrial quality control: Just walk away.
    Douglas R Green.
      The repair and removal of damaged mitochondria is essential for sustaining cellular and tissue homeostasis. Now in Cell, Jiao et al. (2021) describe a novel mechanism of such quality control in which damaged mitochondria move to the plasma membrane where they are "packaged" and left behind the trailing edge of migrating cells.
    DOI:  https://doi.org/10.1016/j.cmet.2021.05.011
  47. Methods Mol Biol. 2021 ;2276 285-303
    High-Throughput Image Analysis of Lipid-Droplet-Bound Mitochondria.
    Nathanael Miller, Dane Wolf, Nour Alsabeeh, Kiana Mahdaviani, Mayuko Segawa, Marc Liesa, Orian S Shirihai.
      Changes to mitochondrial architecture are associated with various adaptive and pathogenic processes. However, quantification of changes to mitochondrial structures is limited by the yet unmet challenge of defining the borders of each individual mitochondrion within an image. Here, we describe a novel method for segmenting primary brown adipocyte (BA) mitochondria images. We describe a granular approach to quantifying subcellular structures, particularly mitochondria in close proximity to lipid droplets: peridroplet mitochondria. In addition, we lay out a novel machine-learning-based mitochondrial segmentation method that eliminates the bias of manual mitochondrial segmentation and improves object recognition compared to conventional thresholding analyses. By applying these methods, we discovered a significant difference between cytosolic and peridroplet BA mitochondrial H2O2 production and validated the machine-learning algorithm in BA via norepinephrine-induced mitochondrial fragmentation and comparing manual analyses to the automated analysis. This approach provides a high-throughput analysis protocol to quantify ratiometric probes in subpopulations of mitochondria in adipocytes.
    Keywords:  Brown adipocyte morphology; Image analysis; Machine learning; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_22
  48. Methods Mol Biol. 2021 ;2277 157-173
    Suborganellar Localization of Mitochondrial Proteins and Transcripts in Human Cells.
    Smirnova Anna, Richert Ludovic, Smirnov Alexandre, Mély Yves, Tarassov Ivan.
      Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.
    Keywords:  3D STORM; Colocalization analysis; Confocal microscopy; Immunolabeling; RNA in situ hybridization; Submitochondrial ultrastructure
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_11
  49. Methods Mol Biol. 2021 ;2277 357-370
    Isolation of Mitochondria-Associated ER Membranes (MAMs), Synaptic MAMs, and Glycosphingolipid Enriched Microdomains (GEMs) from Brain Tissues and Neuronal Cells.
    Ida Annunziata, Jason Andrew Weesner, Alessandra d'Azzo.
      Subcellular fractionation is a valuable procedure in cell biology to separate and purify various subcellular constituents from one another, i.e., nucleus, cytosol, membranes/organelles, and cytoskeleton. The procedure relies on the use of differential centrifugation of cell and tissue homogenates. Fractionated subcellular organelles may be subjected to additional purification steps that enable the isolation of specific cellular sub-compartments, including interorganellar membrane contact sites. Here we outline a protocol tailored to the isolation of mitochondria, mitochondria-associated ER membranes (MAMs), and glycosphingolipid enriched microdomains (GEMs) from the adult mouse brain, primary neurospheres, and murine embryonic fibroblasts (MEFs). We also provide a detailed protocol for the purification of synaptosomes and their corresponding MAMs .
    Keywords:  Brain; Centrifugation; ER; GEMs; MAMs; Mitochondria; Neuronal cells; Synaptosomes
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_22
  50. Cell Biol Toxicol. 2021 May 31.
    The parkinsonian LRRK2 R1441G mutation shows macroautophagy-mitophagy dysregulation concomitant with endoplasmic reticulum stress.
    Sokhna M S Yakhine-Diop, Mario Rodríguez-Arribas, Saray Canales-Cortés, Guadalupe Martínez-Chacón, Elisabet Uribe-Carretero, Mercedes Blanco-Benítez, Gema Duque-González, Marta Paredes-Barquero, Eva Alegre-Cortés, Vicente Climent, Ana Aiastui, Adolfo López de Munain, José M Bravo-San Pedro, Mireia Niso-Santano, José M Fuentes, Rosa A González-Polo.
      Autophagy is a mechanism responsible for the degradation of cellular components to maintain their homeostasis. However, autophagy is commonly altered and compromised in several diseases, including neurodegenerative disorders. Parkinson's disease (PD) can be considered a multifactorial disease because environmental factors, genetic factors, and aging are involved. Several genes are involved in PD pathology, among which the LRRK2 gene and its mutations, inherited in an autosomal dominant manner, are responsible for most genetic PD cases. The R1441G LRRK2 mutation is, after G2019S, the most important in PD pathogenesis. Our results demonstrate a relationship between the R1441G LRRK2 mutation and a mechanistic dysregulation of autophagy that compromises cell viability. This altered autophagy mechanism is associated with organellar stress including mitochondrial (which induces mitophagy) and endoplasmic reticulum (ER) stress, consistent with the fact that patients with this mutation are more vulnerable to toxins related to PD, such as MPP+.
    Keywords:  Autophagy; MAMs; Mitochondrial dysfunction; Neurodegeneration; Parkinson disease
    DOI:  https://doi.org/10.1007/s10565-021-09617-w
  51. Mol Med Rep. 2021 Aug;pii: 549. [Epub ahead of print]24(2):
    HIGD‑1B inhibits hypoxia‑induced mitochondrial fragmentation by regulating OPA1 cleavage in cardiomyocytes.
    Yan Pang, Zhide Zhu, Zhihao Wen, Junshen Lu, Hao Lin, Meiling Tang, Zhiliang Xu, Jianqi Lu.
      The dynamic regulation of mitochondrial morphology is key for eukaryotic cells to manage physiological challenges. Therefore, it is important to understand the molecular basis of mitochondrial dynamic regulation. The aim of the present study was to explore the role of HIG1 hypoxia inducible domain family member 1B (HIGD‑1B) in hypoxia‑induced mitochondrial fragmentation. Protein expression was determined via western blotting. Immunofluorescence assays were performed to detect the subcellular location of HIGD‑1B. Cell Counting Kit‑8 assays and flow cytometry were carried out to measure cell viability and apoptosis, respectively. Protein interactions were evaluated by co‑immunoprecipitation. In the present study, it was found that HIGD‑1B serves a role in cell survival by maintaining the integrity of the mitochondria under hypoxic conditions. Knockdown of HIGD‑1B promoted mitochondrial fragmentation, while overexpression of HIGD‑1B increased survival by preventing activation of caspase‑3 and ‑9. HIGD‑1B expression was associated with cell viability and apoptosis in cardiomyocytes. Furthermore, HIGD‑1B delayed the cleavage process of optic atrophy 1 (OPA1) and stabilized mitochondrial morphology by interacting with OPA1. Collectively, the results from the present study identified a role for HIGD‑1B as an inhibitor of the mitochondrial fission in cardiomyocytes.
    Keywords:  HIG1 hypoxia inducible domain family member 1B; cardiomyocyte; hypoxia; mitochondria; optic atrophy 1
    DOI:  https://doi.org/10.3892/mmr.2021.12188
  52. J Neurosci Res. 2021 Jun 03.
    Efficient extra-mitochondrial aerobic ATP synthesis in neuronal membrane systems.
    Silvia Ravera, Martina Bartolucci, Daniela Calzia, Alessandro M Morelli, Isabella Panfoli.
      The nervous system displays high energy consumption, apparently not fulfilled by mitochondria, which are underrepresented therein. The oxidative phosphorylation (OxPhos) activity, a mitochondrial process that aerobically provides ATP, has also been reported also in the myelin sheath and the rod outer segment (OS) disks. Thus, commonalities and differences between the extra-mitochondrial and mitochondrial aerobic metabolism were evaluated in bovine isolated myelin (IM), rod OS, and mitochondria-enriched fractions (MIT). The subcellular fraction quality and the absence of contamination fractions have been estimated by western blot analysis. Oxygen consumption and ATP synthesis were stimulated by conventional (pyruvate + malate or succinate) and unconventional (NADH) substrates, observing that oxygen consumption and ATP synthesis by IM and rod OS are more efficient than by MIT, in the presence of both kinds of respiratory substrates. Mitochondria did not utilize NADH as a respiring substrate. When ATP synthesis by either sample was assayed in the presence of 10-100 µM ATP in the assay medium, only in IM and OS it was not inhibited, suggesting that the ATP exportation by the mitochondria is limited by extravesicular ATP concentration. Interestingly, IM and OS but not mitochondria appear able to synthesize ATP at a later time with respect to exposure to respiratory substrates, supporting the hypothesis that the proton gradient produced by the electron transport chain is buffered by membrane phospholipids. The putative transfer mode of the OxPhos molecular machinery from mitochondria to the extra-mitochondrial structures is also discussed, opening new perspectives in the field of neurophysiology.
    Keywords:  RRID:AB_10696805; RRID:AB_11183050; RRID:AB_1845182; RRID:AB_2818988; RRID:AB_2851910; RRID:SCR_002798; RRID:SCR_014210; aerobic metabolism; bioenergetics; myelin sheath; oxidative phosphorylation; phototransduction; rod outer segments disks
    DOI:  https://doi.org/10.1002/jnr.24865
  53. Methods Mol Biol. 2021 ;2276 325-332
    Fluorescence-Based Assay For Measuring OMA1 Activity.
    Julia Tobacyk, Lee Ann MacMillan-Crow.
      Mitochondrial fusion depends on proteolytic processing of the dynamin-related GTPase protein, OPA1, which is regulated by the mitochondrial zinc metalloproteinase, OMA1. Last year we published a report describing a novel approach to directly measure the enzymatic activity of OMA1 in whole cell lysates. This fluorescence-based reporter assay utilizes an eight amino acid peptide sequence referred to as the S1 cleavage site where OMA1 cleaves within OPA1 and is flanked by a fluorophore and quencher. In this chapter, we provide additional insight into the OMA1 activity assay.
    Keywords:  Fluorescence-based reporter assay; Fusion; Mitochondria; OMA1; Protease
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_24
  54. Aging (Albany NY). 2021 Jun 02. 13
    GRSF1 deficiency in skeletal muscle reduces endurance in aged mice.
    Riley K Driscoll, Linda K Krasniewski, Samuel G Cockey, Jen-Hao Yang, Yulan Piao, Elin Lehrmann, Yongqing Zhang, Marc Michel, Ji Heon Noh, Chang-Yi Cui, Myriam Gorospe.
      GRSF1 is a mitochondrial RNA-binding protein important for maintaining mitochondrial function. We found that GRSF1 is highly expressed in cultured skeletal myoblasts differentiating into myotubes. To understand the physiological function of GRSF1 in vivo, we generated mice in which GRSF1 was specifically ablated in skeletal muscle. The conditional knockout mice (Grsf1cKO) appeared normal until 7-9 months of age. Importantly, however, a reduction of muscle endurance compared to wild-type controls was observed in 16- to 18-month old Grsf1cKO mice. Transcriptomic analysis revealed more than 200 mRNAs differentially expressed in Grsf1cKO muscle at this age. Notably, mRNAs encoding proteins involved in mitochondrial function, inflammation, and ion transport, including Mgarp, Cxcl10, Nfkb2, and Sln mRNAs, were significantly elevated in aged Grsf1cKO muscle. Our findings suggest that GRSF1 deficiency exacerbates the functional decline of aged skeletal muscle, likely through multiple downstream effector proteins.
    Keywords:  GRSF1; RNA-binding protein; mouse aging; skeletal muscle aging
    DOI:  https://doi.org/10.18632/aging.203151
  55. Cell Rep. 2021 Jun 01. pii: S2211-1247(21)00552-0. [Epub ahead of print]35(9): 109203
    PINK1 and parkin shape the organism-wide distribution of a deleterious mitochondrial genome.
    Arnaud Ahier, Chuan-Yang Dai, Ina Kirmes, Nadia Cummins, Grace Ching Ching Hung, Jürgen Götz, Steven Zuryn.
      In multiple species, certain tissue types are prone to acquiring greater loads of mitochondrial genome (mtDNA) mutations relative to others, but the mechanisms that drive these heteroplasmy differences are unknown. We find that the conserved PTEN-induced putative kinase (PINK1/PINK-1) and the E3 ubiquitin-protein ligase parkin (PDR-1), which are required for mitochondrial autophagy (mitophagy), underlie stereotyped differences in heteroplasmy of a deleterious mitochondrial genome mutation (ΔmtDNA) between major somatic tissues types in Caenorhabditis elegans. We demonstrate that tissues prone to accumulating ΔmtDNA have lower mitophagy responses than those with low mutation levels. Moreover, we show that ΔmtDNA heteroplasmy increases when proteotoxic species that are associated with neurodegenerative disease and mitophagy inhibition are overexpressed in the nervous system. These results suggest that PINK1 and parkin drive organism-wide patterns of heteroplasmy and provide evidence of a causal link between proteotoxicity, mitophagy, and mtDNA mutation levels in neurons.
    Keywords:  Alzheimer's disease; PINK1; heteroplasmy; mitochondria; mitophagy; mtDNA; parkin; polyglutamate; proteotoxicity; tau
    DOI:  https://doi.org/10.1016/j.celrep.2021.109203
  56. Methods Mol Biol. 2021 ;2277 289-297
    Assessment of Mitochondrial Reactive Oxygen Species and Redox Regulation in Stem Cells.
    Madhavee Thumiah-Mootoo, Tina Podinic, Mireille Khacho.
      Mitochondrial reactive oxygen species (mtROS) and redox regulation play an important role in stem cell maintenance and cell fate decisions. Although changes in mtROS and redox homeostasis represent a physiological mechanism to drive stem cell commitment and differentiation, dysregulation of this system can lead to defects in stem cell maintenance and regenerative capacity. This chapter explains the methods used to assess mitochondrial superoxide levels and redox regulation in stem cell populations.
    Keywords:  Antioxidant; Electron transport chain; Metabolism; Mitochondria; Oxidative stress; Reactive oxygen species (ROS); Redox; Stem cell fate; Stem cells
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_18
  57. Cell Metab. 2021 Jun 01. pii: S1550-4131(21)00224-2. [Epub ahead of print]33(6): 1067-1069
    Brains and brawn: Stress-induced myokine abates nervous system aging.
    C Kimberly Tsui, Julian G Dishart, Andrew Dillin.
      Skeletal muscle secretes numerous systemic factors, termed myokines, which can regulate homeostasis of distal tissues. In this issue, Rai et al. (2021) identify and characterize a novel myokine, Amyrel, which is secreted under muscle proteasome stress and protects central nervous system health and function by enhancing protein quality control during aging.
    DOI:  https://doi.org/10.1016/j.cmet.2021.05.007
  58. Methods Mol Biol. 2021 ;2276 203-213
    Evaluation of Mitochondria Content and Function in Live Cells by Multicolor Flow Cytometric Analysis.
    Hsiu-Han Fan, Tsung-Lin Tsai, Ivan L Dzhagalov, Chia-Lin Hsu.
      To evaluate how a cell responds to the external stimuli, treatment, or alteration of the microenvironment, the quantity and quality of mitochondria are commonly used as readouts. However, it is challenging to apply mitochondrial analysis to the samples that are composed of mixed cell populations originating from tissues or when multiple cell populations are of interest, using methods such as Western blot, electron microscopy, or extracellular flux analysis.Flow cytometry is a technique allowing the detection of individual cell status and its identity simultaneously when used in combination with surface markers. Here we describe how to combine mitochondria-specific dyes or the dyes targeting the superoxide produced by mitochondria with surface marker staining to measure the mitochondrial content and activity in live cells by flow cytometry. This method can be applied to all types of cells in suspension and is particularly useful for analysis of samples composed of heterogeneous cell populations.
    Keywords:  FACS; Flow cytometry; Mitochondria; Quantification; Reactive oxygen species
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_15
  59. Cell Calcium. 2021 May 19. pii: S0143-4160(21)00070-1. [Epub ahead of print]97 102416
    Sprinkling salt on mitochondria: The metabolic and pathophysiological roles of mitochondrial Na+ signaling mediated by NCLX.
    Essam A Assali, Israel Sekler.
      NCLX, the mitochondrial Na+/Ca2+ transporter is a key player in Ca2+ signaling. However, its role in Na+ signaling is poorly understood. In this review we focus on Na+ signaling by NCLX, and discuss recent physiological and pathophysiological roles attributed to the Na+ influx into mitochondria.
    Keywords:  Hypoxia; Ischemia; Mitochondrial Ca(2+) efflux; NCLX; Na(+) signaling
    DOI:  https://doi.org/10.1016/j.ceca.2021.102416
  60. Methods Mol Biol. 2021 ;2276 249-257
    Assessment of Mitochondrial Protein Glutathionylation as Signaling for CO Pathway.
    Ana S Almeida, Cláudia Figueiredo-Pereira, Helena L A Vieira.
      Protein glutathionylation is a posttranslational process that regulates protein function in response to redox cellular changes. Furthermore, carbon monoxide-induced cellular pathways involve reactive oxygen species (ROS) signaling and mitochondrial protein glutathionylation. Herein, it is described as a technique to assess mitochondrial glutathionylation due to low concentrations of CO exposure. Mitochondria are isolated from cell culture or tissue, followed by an immunoprecipitation assay, which allows the capture of any glutathionylated mitochondrial protein using a specific antibody coupled to a solid matrix that binds to glutathione antigen. The precipitated protein is further identified and quantified by immunoblotting analysis.
    Keywords:  Carbon monoxide; Glutathione; Glutathionylation; Immunoprecipitation; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_19
  61. Methods Mol Biol. 2021 ;2277 371-389
    Mitochondrial Diagnostics: A Discovery-Based Biochemical Platform for Phenotyping Human Peripheral Blood Cell Mitochondria.
    Margaret A M Nelson, Kelsey H Fisher-Wellman.
      In vitro experiments using permeabilized cells and/or isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across multiple scientific fields to shed valuable insight on mitochondrial-linked pathologies. The present chapter is intended to serve as a methodological blueprint for comprehensively phenotyping peripheral blood cell mitochondria. While primarily adapted for peripheral blood cells, the protocols outlined herein could easily be made amenable to most all cell types with minimal modifications.
    Keywords:  ATP synthesis; Creatine kinase clamp; Matrix dehydrogenase activity; Mitochondrial bioenergetics; Peripheral blood cells; Respiratory flux
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_23
  62. Pharmaceutics. 2021 May 28. pii: 810. [Epub ahead of print]13(6):
    Engineering Genetic Systems for Treating Mitochondrial Diseases.
    Yoon-Ha Jang, Sae Ryun Ahn, Ji-Yeon Shim, Kwang-Il Lim.
      Mitochondria are intracellular energy generators involved in various cellular processes. Therefore, mitochondrial dysfunction often leads to multiple serious diseases, including neurodegenerative and cardiovascular diseases. A better understanding of the underlying mitochondrial dysfunctions of the molecular mechanism will provide important hints on how to mitigate the symptoms of mitochondrial diseases and eventually cure them. In this review, we first summarize the key parts of the genetic processes that control the physiology and functions of mitochondria and discuss how alterations of the processes cause mitochondrial diseases. We then list up the relevant core genetic components involved in these processes and explore the mutations of the components that link to the diseases. Lastly, we discuss recent attempts to apply multiple genetic methods to alleviate and further reverse the adverse effects of the core component mutations on the physiology and functions of mitochondria.
    Keywords:  gene therapy; heteroplasmy; mitochondrial DNA; mitochondrial disease; mitochondrial gene delivery
    DOI:  https://doi.org/10.3390/pharmaceutics13060810
  63. Nat Rev Neurosci. 2021 Jun 04.
    Mitochondrial defence tactics.
    Sian Lewis.
      
    DOI:  https://doi.org/10.1038/s41583-021-00482-4
  64. Life (Basel). 2021 May 12. pii: 436. [Epub ahead of print]11(5):
    Mitochondrial Bioenergetics and Dynamism in the Failing Heart.
    Giampaolo Morciano, Veronica Angela Maria Vitto, Esmaa Bouhamida, Carlotta Giorgi, Paolo Pinton.
      The heart is responsible for pumping blood, nutrients, and oxygen from its cavities to the whole body through rhythmic and vigorous contractions. Heart function relies on a delicate balance between continuous energy consumption and generation that changes from birth to adulthood and depends on a very efficient oxidative metabolism and the ability to adapt to different conditions. In recent years, mitochondrial dysfunctions were recognized as the hallmark of the onset and development of manifold heart diseases (HDs), including heart failure (HF). HF is a severe condition for which there is currently no cure. In this condition, the failing heart is characterized by a disequilibrium in mitochondrial bioenergetics, which compromises the basal functions and includes the loss of oxygen and substrate availability, an altered metabolism, and inefficient energy production and utilization. This review concisely summarizes the bioenergetics and some other mitochondrial features in the heart with a focus on the features that become impaired in the failing heart.
    Keywords:  bioenergetics; heart failure; mitochondria; mitochondrial dynamics
    DOI:  https://doi.org/10.3390/life11050436
  65. Methods Mol Biol. 2021 ;2277 175-185
    In Vivo Assessment of Mitochondrial Oxygen Consumption.
    Floor A Harms, Egbert G Mik.
      The Protoporphyrin IX-Triplet State Lifetime Technique (PpIX-TSLT) has been proposed by us as a potential clinical noninvasive tool for monitoring mitochondrial function. We have been working on the development of mitochondrial respirometry for monitoring mitochondrial oxygen tension (mitoPO2) and mitochondrial oxygen consumption (mitoVO2) in skin. In this work, we describe the principles of the method in small experimental animals.
    Keywords:  Mitochondrial oxygen consumption (mitoVO2); Mitochondrial oxygen tension (mitoPO2); Oxygen-dependent quenching; Phosphorescence quenching; Tissue oxygenation
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_12
  66. Methods Mol Biol. 2021 ;2276 227-234
    Modified Blue Native Gel Approach for Analysis of Respiratory Supercomplexes.
    Sergiy M Nadtochiy, Megan Ngai, Paul S Brookes.
      In mitochondrial oxidative phosphorylation (Ox-Phos), individual electron transport chain complexes are thought to assemble into supramolecular entities termed supercomplexes (SCs). The technique of blue native (BN) gel electrophoresis has emerged as the method of choice for analyzing SCs. However, the process of sample extraction for BN gel analysis is somewhat tedious and introduces the possibility for experimental artifacts. Here we outline a streamlined method that eliminates a centrifugation step and provides a more representative sampling of a population of mitochondria on the final gel. Using this method, we show that SC composition does not appear to change dynamically with altered mitochondrial function.
    Keywords:  Blue-native; Clear-native; Mitochondria; Permeability transition pore; Respiration; Supercomplexes
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_17
  67. Methods Mol Biol. 2021 ;2276 129-141
    Assessment of Mitochondrial Cell Metabolism by Respiratory Chain Electron Flow Assays.
    Flavia Radogna, Déborah Gérard, Mario Dicato, Marc Diederich.
      Cellular energy metabolism is regulated by complex metabolic pathways. Although anaerobic glycolysis was reported as a primary source of energy in cancer leading to a high rate of lactate production, current evidence shows that the main energy source supporting cancer cell metabolism relies on mitochondrial metabolism. Mitochondria are the key organelle maintaining optimal cellular energy levels. MitoPlate™ S-1 provides a highly reproducible bioenergetics tool to analyze the electron flow rate in live cells. Measuring the rates of electron flow into and through the electron transport chain using different NADH and FADH2-producing metabolic substrates enables the assessment of mitochondrial functionality. MitoPlate™ S-1 are 96-well microplates pre-coated with different substrates used as probes to examine the activity of mitochondrial metabolic pathways based on a colorimetric assay. A comparative metabolic analysis between cell lines or primary cells allows to establish a specific metabolic profile and to detect possible alterations of the mitochondrial function of a tumor cell. Moreover, the direct measurements of electron flux triggered by metabolic pathway activation could highlight targets for potential drug candidates.
    Keywords:  Bioinformatics; Cancer metabolism; Electron transport chain; Mitochondrial respiration; Tricarboxylic acid cycle
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_9
  68. Trends Cell Biol. 2021 May 26. pii: S0962-8924(21)00095-7. [Epub ahead of print]
    Oxidative phosphorylation system and cell culture media.
    M Pilar Bayona-Bafaluy, Julio Montoya, Eduardo Ruiz-Pesini.
      Traditional culture media do not resemble the metabolic composition of human blood. The concentration of different metabolites in these media influences mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) function. This knowledge is essential for the interpretation of results obtained from cellular models used for the study of OXPHOS function.
    Keywords:  cell culture media; cell model; mitochondrial biogenesis; oxidative phosphorylation
    DOI:  https://doi.org/10.1016/j.tcb.2021.05.003
  69. Methods Mol Biol. 2021 ;2277 247-268
    Accurate Measurement of Cellular and Cell-Free Circulating Mitochondrial DNA Content from Human Blood Samples Using Real-Time Quantitative PCR.
    Hannah Rosa, Afshan N Malik.
      Changes in circulating mitochondrial DNA (mtDNA) are widely used to indicate mitochondrial dysfunction in common non-genetic diseases where mitochondrial dysfunction may play a role. However, the methodology being used is not always specific and reproducible, and most studies use whole blood rather than evaluating cellular and cell-free mtDNA separately. Cellular mtDNA is contained within the mitochondrion and encodes vital subunits of the OXPHOS machinery. Conversely, cell-free mtDNA can have harmful effects, triggering inflammatory responses and potentially contributing to pathogenic processes. In this chapter, we describe a protocol to accurately measure the amount of cellular and cell-free human mtDNA in peripheral blood. Absolute quantification is carried out using real-time quantitative PCR (qPCR) to quantify cellular mtDNA, measured as the mitochondrial genome to nuclear genome ratio (designated the Mt/N ratio) in whole blood and peripheral blood mononuclear cells (PBMCs) and the number of mtDNA copies per μL in plasma and serum. We describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions, (2) prepare DNA from each of these fractions, (3) prepare dilution standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.
    Keywords:  Absolute quantification; Circulating mtDNA; Mitochondrial DNA; Mt/N ratio; PBMCs; Plasma; Serum; mtDNA; mtDNA content; mtDNA copy number; qPCR
    DOI:  https://doi.org/10.1007/978-1-0716-1270-5_15
  70. Neurol Genet. 2021 Jun;7(3): e597
    Diagnosing Mitochondrial Disorders Remains Challenging in the Omics Era.
    Patrick Forny, Emma Footitt, James E Davison, Amanda Lam, Cathy E Woodward, Spyros Batzios, Sanjay Bhate, Anupam Chakrapani, Maureen Cleary, Paul Gissen, Stephanie Grunewald, Jane A Hurst, Richard Scott, Simon Heales, Thomas S Jacques, Thomas Cullup, Shamima Rahman.
       Objective: We hypothesized that novel investigative pathways are needed to decrease diagnostic odysseys in pediatric mitochondrial disease and sought to determine the utility of clinical exome sequencing in a large cohort with suspected mitochondrial disease and to explore whether any of the traditional indicators of mitochondrial disease predict a confirmed genetic diagnosis.
    Methods: We investigated a cohort of 85 pediatric patients using clinical exome sequencing and compared the results with the outcome of traditional diagnostic tests, including biochemical testing of routine parameters (lactate, alanine, and proline), neuroimaging, and muscle biopsy with histology and respiratory chain enzyme activity studies.
    Results: We established a genetic diagnosis in 36.5% of the cohort and report 20 novel disease-causing variants (1 mitochondrial DNA). Counterintuitively, routine biochemical markers were more predictive of mitochondrial disease than more invasive and elaborate muscle studies.
    Conclusions: We propose using biochemical markers to support the clinical suspicion of mitochondrial disease and then apply first-line clinical exome sequencing to identify a definite diagnosis. Muscle biopsy studies should only be used in clinically urgent situations or to confirm an inconclusive genetic result.
    Classification of Evidence: This is a Class II diagnostic accuracy study showing that the combination of CSF and plasma biochemical tests plus neuroimaging could predict the presence or absence of exome sequencing confirmed mitochondrial disorders.
    DOI:  https://doi.org/10.1212/NXG.0000000000000597
  71. Nat Struct Mol Biol. 2021 May 31.
    Reply to: Reevaluating the role of human mitochondrial uL18m in the cytosolic stress response.
    Xingqian Zhang, Quanquan Ji, Shu-Bing Qian.
      
    DOI:  https://doi.org/10.1038/s41594-021-00600-x
  72. Front Cell Dev Biol. 2021 ;9 669379
    The Mitochondrial Response to DNA Damage.
    Ziye Rong, Peipei Tu, Peiqi Xu, Yan Sun, Fangfang Yu, Na Tu, Lixia Guo, Yanan Yang.
      Mitochondria are double membrane organelles in eukaryotic cells that provide energy by generating adenosine triphosphate (ATP) through oxidative phosphorylation. They are crucial to many aspects of cellular metabolism. Mitochondria contain their own DNA that encodes for essential proteins involved in the execution of normal mitochondrial functions. Compared with nuclear DNA, the mitochondrial DNA (mtDNA) is more prone to be affected by DNA damaging agents, and accumulated DNA damages may cause mitochondrial dysfunction and drive the pathogenesis of a variety of human diseases, including neurodegenerative disorders and cancer. Therefore, understanding better how mtDNA damages are repaired will facilitate developing therapeutic strategies. In this review, we focus on our current understanding of the mtDNA repair system. We also discuss other mitochondrial events promoted by excessive DNA damages and inefficient DNA repair, such as mitochondrial fusion, fission, and mitophagy, which serve as quality control events for clearing damaged mtDNA.
    Keywords:  DNA repair; mitochondrial DNA; mitochondrial fission; mitochondrial fusion; mitophagy
    DOI:  https://doi.org/10.3389/fcell.2021.669379
  73. Methods Mol Biol. 2021 ;2276 103-112
    Native Gel Electrophoresis and Immunoblotting to Analyze Electron Transport Chain Complexes.
    Gisela Beutner, George A Porter.
      Native electrophoresis is a powerful tool to analyze the mitochondrial electron transport chain complexes (Cx) I-V and their assembly into supercomplexes. Valuable information regarding the composition and bioenergetic regulation in physiological and pathological conditions can be obtained. This chapter compares different types of native electrophoresis to analyze mitochondrial supercomplexes.
    Keywords:  Mitochondrial supercomplexes; Native electrophoresis (blue native, colorless native, clear native, hybrid)
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_7
  74. Methods Mol Biol. 2021 ;2276 343-355
    Extraction of Functional Mitochondria Based on Membrane Stiffness.
    Md Habibur Rahman, Qinru Xiao, Shirui Zhao, An-Chi Wei, Yi-Ping Ho.
      The abnormal functionality of mitochondria has been linked to many life-threatening diseases such as cancers, failure of cardiovascular functions, and neurodegenerative disorders. Therefore, in vitro analysis of mitochondria has garnered great interest for understanding the mechanism of mitochondrial dysfunction-related disease development and therapeutics. However, due to the intrinsic heterogeneity of cell membrane stiffness, it remains challenging to standardize the protocols for the extraction of mitochondria and adequate disruption of the cellular membrane while retaining the functionality of mitochondria. We have previously developed a microfluidics-based cell shredder capable of serving the purpose. In this protocol, we describe the step-by-step procedures to empirically identify the threshold shear stress using this microfluidics-based cell shredder for mitochondrial extraction. The optimal shear stress to disrupt human embryonic kidney cell (HEK 293) and mice muscle cell (C2C12) has been characterized at around 16.4 Pa, whereas cell lines with stiffer membrane stiffness, for example, neuroblastoma cells (SH-SY5Y), require 27.4 Pa to effectively lyse the cells. This protocol also provides detailed procedures to determine the quality of extracted mitochondria based on the membrane potential and the integrity of extracted mitochondria. A comparison with the widely employed Dounce homogenizer has shown that the proposed microscale cell shredder can yield at least 40% more functional mitochondria and retain higher integrity regarding extracted mitochondria than the counterparts extracted from Dounce homogenizer, especially for low cell concentrations (5-20 × 104 cells/mL) and small sample volume (<200 μL).
    Keywords:  Cell membrane stiffness; Membrane disruption; Mitochondrial integrity; Mitochondrial membrane potential; Shear stress
    DOI:  https://doi.org/10.1007/978-1-0716-1266-8_26
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