bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023–02–26
93 papers selected by
Catalina Vasilescu, Helmholz Munich
  1. Genetics of mitochondrial diseases: Current approaches for the molecular diagnosis.
  2. Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing.
  3. Cardiac Involvement in Mitochondrial Disorders.
  4. Genomic Strategies in Mitochondrial Diagnostics.
  5. Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.
  6. TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.
  7. Investigation of oxidative phosphorylation activity and complex composition in mitochondrial disease.
  8. Mitochondrial optic neuropathies.
  9. Mitochondrial disease in neurology-Past, present, and future.
  10. Mitochondrial cristae in health and disease.
  11. Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies.
  12. Cell lineage-specific mitochondrial resilience during mammalian organogenesis.
  13. Studying Mitochondrial Nucleic Acid Synthesis Utilizing Intact Isolated Mitochondria.
  14. Experimental therapy for mitochondrial diseases.
  15. Peripheral neuropathy in mitochondrial disease.
  16. Blood biomarkers of mitochondrial disease-One for all or all for one?
  17. Mitochondrial Neurodegeneration: Lessons from Drosophila melanogaster Models.
  18. Creation of Mitochondrial Disease Models Using Mitochondrial DNA Editing.
  19. Concomitant Sequencing of Accessible Chromatin and Mitochondrial Genomes in Single Cells Using mtscATAC-Seq.
  20. Complex neurological and multisystem presentations in mitochondrial disease.
  21. Isolating Mitochondria, Mitoplasts, and mtDNA from Cultured Mammalian Cells.
  22. Single Cell Analysis of Mitochondrial DNA Deletions.
  23. Reproductive options in mitochondrial disease.
  24. Manipulation of Murine Mitochondrial DNA Heteroplasmy with mtZFNs.
  25. Visualization of mtDNA Using FISH.
  26. Ataxia and spastic paraplegia in mitochondrial disease.
  27. Role of human HSPE1 for OPA1 processing independent of HSPD1.
  28. Neuroimaging in mitochondrial disease.
  29. Currently available therapies in mitochondrial disease.
  30. 5'-End Mapping in Human Mitochondrial DNA.
  31. Assessing TFAM Binding to Human Mitochondrial DNA.
  32. Functional Assessment of Mitochondrial DNA Maintenance by Depletion and Repopulation Using 2',3'-Dideoxycytidine in Cultured Cells.
  33. The role of programmed mitophagy in germline mitochondrial DNA quality control.
  34. Mitochondrial dynamics in macrophages: divide to conquer or unite to survive?
  35. Isolation of Functional Mitochondria and Pure mtDNA from Murine Tissues.
  36. Analysis of Mitochondrial DNA Replication by Two-Dimensional Agarose Gel Electrophoresis.
  37. Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle.
  38. Progressive external ophthalmoplegia.
  39. Detection of UV-Induced Deletions in Mitochondrial DNA.
  40. High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS.
  41. Mitochondrial membrane biogenesis: A new pathway for lipid transport mediated by PERK/E-Syt1 complex.
  42. Rolling Circle Replication and Bypass of Damaged Nucleotides.
  43. Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels.
  44. Clinical trials in mitochondrial diseases.
  45. Laboratory and metabolic investigations.
  46. Stroke-like episodes in adult mitochondrial disease.
  47. Localization of Mitochondrial Nucleoids by Transmission Electron Microscopy Using the Transgenic Expression of the Mitochondrial Helicase Twinkle and APEX2.
  48. A Case Report on Pearson Syndrome With Emphasis on Genetic Screening in Patients Presenting With Sideroblastic Anemia and Lactic Acidosis.
  49. Measurement of Nucleoid Size Using STED Microscopy.
  50. Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Purification of mtDNA from Mammalian Cells.
  51. Mitochondrial phosphatidylethanolamine modulates UCP1 to promote brown adipose thermogenesis.
  52. Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ0 cells).
  53. Mitochondria-Targeted Gene Silencing Facilitated by Mito-CPDs.
  54. Delivery of mitochondria confers cardioprotection through mitochondria replenishment and metabolic compliance.
  55. Identification of Proximity Interactors of Mammalian Nucleoid Proteins by BioID.
  56. Mitochondrial DNA variants in a cohort from Argentina with suspected Leber's hereditary optic neuropathy (LHON).
  57. Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction.
  58. Involvement of Mitochondrial Dysfunction in FOXG1 Syndrome.
  59. The SLC25A47 locus controls gluconeogenesis and energy expenditure.
  60. A Patient with Mitochondrial Disease on Dialysis with Long-term Follow-up of Cardiomyopathy: An Autopsy Case Report.
  61. In Vitro Assays of TWINKLE Function.
  62. The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction.
  63. Mitochondrial Complex I, a Possible Sensible Site of cAMP Pathway in Aging.
  64. Mitochondrial-Encoded Peptide MOTS-c, Diabetes, and Aging-Related Diseases.
  65. svaRetro and svaNUMT: modular packages for annotating retrotransposed transcripts and nuclear integration of mitochondrial DNA in genome sequencing data.
  66. Nicotinamide riboside kinase-2 regulates metabolic adaptation in the ischemic heart.
  67. Leigh syndrome.
  68. Carnitine derivatives beyond fatigue: an update.
  69. Exploiting Multi-Omics Profiling and Systems Biology to Investigate Functions of TOMM34.
  70. NAD+-Consuming Enzymes in Stem Cell Homeostasis.
  71. FixItFelix: improving genomic analysis by fixing reference errors.
  72. Stress-dependent macromolecular crowding in the mitochondrial matrix.
  73. Quantitative Analysis of Nucleoside Triphosphate Pools in Mouse Muscle Using Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection.
  74. Neuropathy, Ataxia, and Retinitis Pigmentosa Syndrome.
  75. Mitochondrial Open Reading Frame of the 12S rRNA Type-c: Potential Therapeutic Candidate in Retinal Diseases.
  76. Rab8a as a mitochondrial receptor for lipid droplets in skeletal muscle.
  77. How to Quantify DNA Compaction by TFAM with Acoustic Force Spectroscopy and Total Internal Reflection Fluorescence Microscopy.
  78. NAD+ Homeostasis and NAD+-Consuming Enzymes: Implications for Vascular Health.
  79. Segregation of pathways leading to pexophagy.
  80. In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy.
  81. Remote visualization of large-scale genomic alignments for collaborative clinical research and diagnosis of rare diseases.
  82. From mitochondria to cells to humans: Targeting bioenergetics in aging and disease.
  83. Disruption of mitochondrial bioenergetics and calcium homeostasis by phytanic acid in the heart: Potential relevance for the cardiomyopathy in Refsum disease.
  84. Hepatic mitochondrial NAD+ transporter SLC25A47 activates AMPKα mediating lipid metabolism and tumorigenesis.
  85. Enhanced mitochondrial biogenesis promotes neuroprotection in human pluripotent stem cell derived retinal ganglion cells.
  86. Parkin and mitochondrial signalling.
  87. Disease mechanisms as Subtypes: Mitochondrial and bioenergetic dysfunction.
  88. PERK recruits E-Syt1 at ER-mitochondria contacts for mitochondrial lipid transport and respiration.
  89. Network expansion of genetic associations defines a pleiotropy map of human cell biology.
  90. Lipogenesis promotes mitochondrial fusion and maintains cancer stemness in human NSCLC.
  91. Skeletal Muscle Mitochondrial Dysfunction in Chronic Obstructive Pulmonary Disease: Underlying Mechanisms and Physical Therapy Perspectives.
  92. Intermittent fasting promotes adipocyte mitochondrial fusion through Sirt3-mediated deacetylation of Mdh2.
  93. Innovative technologies crowd the short-read sequencing market.
  1. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00011-7. [Epub ahead of print]194 141-165
    Genetics of mitochondrial diseases: Current approaches for the molecular diagnosis.
    Lea D Schlieben, Holger Prokisch.
      Mitochondrial diseases are a genetically and phenotypically variable set of monogenic disorders. The main characteristic of mitochondrial diseases is a defective oxidative phosphorylation. Both nuclear and mitochondrial DNA encode the approximately 1500 mitochondrial proteins. Since identification of the first mitochondrial disease gene in 1988 a total of 425 genes have been associated with mitochondrial diseases. Mitochondrial dysfunctions can be caused both by pathogenic variants in the mitochondrial DNA or the nuclear DNA. Hence, besides maternal inheritance, mitochondrial diseases can follow all modes of Mendelian inheritance. The maternal inheritance and tissue specificity distinguish molecular diagnostics of mitochondrial disorders from other rare disorders. With the advances made in the next-generation sequencing technology, whole exome sequencing and even whole-genome sequencing are now the established methods of choice for molecular diagnostics of mitochondrial diseases. They reach a diagnostic rate of more than 50% in clinically suspected mitochondrial disease patients. Moreover, next-generation sequencing is delivering a constantly growing number of novel mitochondrial disease genes. This chapter reviews mitochondrial and nuclear causes of mitochondrial diseases, molecular diagnostic methodologies, and their current challenges and perspectives.
    Keywords:  Diagnostic; Genetic; Heteroplasmy; Mitochondrial DNA; Mitochondrial disease; Multi-omic; Mutation
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00011-7
  2. Methods Mol Biol. 2023 ;2615 381-395
    Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing.
    Andrea Legati, Daniele Ghezzi, Carlo Viscomi.
      Over the last 10 years, next generation sequencing (NGS) became the gold standard for both diagnosis and discovery of new disease genes responsible for heterogeneous disorders, such as mitochondrial encephalomyopathies. The application of this technology to mtDNA mutations poses extra challenges compared to other genetic conditions because of the peculiarities of mitochondrial genetics and the requirement for proper NGS data management and analysis. Here, we describe a detailed, clinically relevant protocol to sequence the whole mtDNA and quantify heteroplasmy levels of mtDNA variants, starting from total DNA through the generation of a single PCR amplicon.
    Keywords:  Heteroplasmy; Mitochondrial DNA; Mitochondrial disease; Mitochondrial haplogroups; Next generation sequencing; Single amplicon
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_26
  3. Curr Heart Fail Rep. 2023 Feb 18.
    Cardiac Involvement in Mitochondrial Disorders.
    Tudor-Alexandru Popoiu, Jan Dudek, Christoph Maack, Edoardo Bertero.
       PURPOSE OF REVIEW: We review pathophysiology and clinical features of mitochondrial disorders manifesting with cardiomyopathy.
    RECENT FINDINGS: Mechanistic studies have shed light into the underpinnings of mitochondrial disorders, providing novel insights into mitochondrial physiology and identifying new therapeutic targets. Mitochondrial disorders are a group of rare genetic diseases that are caused by mutations in mitochondrial DNA (mtDNA) or in nuclear genes that are essential to mitochondrial function. The clinical picture is extremely heterogeneous, the onset can occur at any age, and virtually, any organ or tissue can be involved. Since the heart relies primarily on mitochondrial oxidative metabolism to fuel contraction and relaxation, cardiac involvement is common in mitochondrial disorders and often represents a major determinant of their prognosis.
    Keywords:  Cardiolipin; Cardiomyopathy; Electron transport chain; Mitochondrial DNA; Mitochondrial disease
    DOI:  https://doi.org/10.1007/s11897-023-00592-3
  4. Methods Mol Biol. 2023 ;2615 397-425
    Genomic Strategies in Mitochondrial Diagnostics.
    Dasha Deen, Charlotte L Alston, Gavin Hudson, Robert W Taylor, Angela Pyle.
      Pathogenic variants in both mitochondrial and nuclear genes contribute to the clinical and genetic heterogeneity of mitochondrial diseases. There are now pathogenic variants in over 300 nuclear genes linked to human mitochondrial diseases. Nonetheless, diagnosing mitochondrial disease with a genetic outcome remains challenging. However, there are now many strategies that help us to pinpoint causative variants in patients with mitochondrial disease. This chapter describes some of the approaches and recent advancements in gene/variant prioritization using whole-exome sequencing (WES).
    Keywords:  Clinical reporting; Genetic diagnosis; Genomics; Mitochondrial disease; Variant annotation; Variant detection; Whole-exome sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_27
  5. Methods Mol Biol. 2023 ;2615 427-441
    Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.
    Shilan Wu, Matthew J Longley, Scott A Lujan, Thomas A Kunkel, William C Copeland.
      Mitochondrial DNA (mtDNA) encodes components essential for cellular respiration. Low levels of point mutations and deletions accumulate in mtDNA during normal aging. However, improper maintenance of mtDNA results in mitochondrial diseases, stemming from progressive loss of mitochondrial function through the accelerated formation of deletions and mutations in mtDNA. To better understand the molecular mechanisms underlying the creation and propagation of mtDNA deletions, we developed the LostArc next-generation DNA sequencing pipeline to detect and quantify rare mtDNA species in small tissue samples. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA.
    Keywords:  DNA deletions; Mitochondrial DNA; Mitochondrial DNA Replication; Mitochondrial disease; Next-Generation Sequencing; POLG
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_28
  6. Nat Commun. 2023 Feb 23. 14(1): 1009
    TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.
    Lindsey Van Haute, Emily O'Connor, Héctor Díaz-Maldonado, Benjamin Munro, Kiran Polavarapu, Daniella H Hock, Gautham Arunachal, Alkyoni Athanasiou-Fragkouli, Mainak Bardhan, Magalie Barth, Dominique Bonneau, Nicola Brunetti-Pierri, Gerarda Cappuccio, Nikeisha J Caruana, Natalia Dominik, Himanshu Goel, Guy Helman, Henry Houlden, Guy Lenaers, Karine Mention, David Murphy, Bevinahalli Nandeesh, Catarina Olimpio, Christopher A Powell, Veeramani Preethish-Kumar, Vincent Procaccio, Rocio Rius, Pedro Rebelo-Guiomar, Cas Simons, Seena Vengalil, Maha S Zaki, Alban Ziegler, David R Thorburn, David A Stroud, Reza Maroofian, John Christodoulou, Claes Gustafsson, Atchayaram Nalini, Hanns Lochmüller, Michal Minczuk, Rita Horvath.
      Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongation factor responsible for enhancing the processivity of mitochondrial RNA polymerase, POLRMT. We report for the first time that TEFM variants are associated with mitochondrial respiratory chain deficiency and a wide range of clinical presentations including mitochondrial myopathy with a treatable neuromuscular transmission defect. Mechanistically, we show muscle and primary fibroblasts from the affected individuals have reduced levels of promoter distal mitochondrial RNA transcripts. Finally, tefm knockdown in zebrafish embryos resulted in neuromuscular junction abnormalities and abnormal mitochondrial function, strengthening the genotype-phenotype correlation. Our study highlights that TEFM regulates mitochondrial transcription elongation and its defect results in variable, tissue-specific neurological and neuromuscular symptoms.
    DOI:  https://doi.org/10.1038/s41467-023-36277-7
  7. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00008-7. [Epub ahead of print]194 127-139
    Investigation of oxidative phosphorylation activity and complex composition in mitochondrial disease.
    Kyle Thompson, David A Stroud, David R Thorburn, Robert W Taylor.
      A multidisciplinary approach to the laboratory diagnosis of mitochondrial disease has long been applied, with crucial information provided by deep clinical phenotyping, blood investigations, and biomarker screening as well as histopathological and biochemical testing of biopsy material to support molecular genetic screening. In an era of second and third generation sequencing technologies, traditional diagnostic algorithms for mitochondrial disease have been replaced by gene agnostic, genomic strategies including whole-exome sequencing (WES) and whole-genome sequencing (WGS), increasingly supported by other 'omics technologies (Alston et al., 2021). Whether a primary testing strategy, or one used to validate and interpret candidate genetic variants, the availability of a range of tests aimed at determining mitochondrial function (i.e., the assessment of individual respiratory chain enzyme activities in a tissue biopsy or cellular respiration in a patient cell line) remains an important part of the diagnostic armory. In this chapter, we summarize several disciplines used in the laboratory investigation of suspected mitochondrial disease, including the histopathological and biochemical assessment of mitochondrial function, as well as protein-based techniques to assess the steady-state levels of oxidative phosphorylation (OXPHOS) subunits and assembly of OXPHOS complexes via traditional (immunoblotting) and cutting-edge (quantitative proteomic) approaches.
    Keywords:  Mitochondrial disease; Muscle pathology; Next-generation sequencing; Oxidative phosphorylation; Proteomics
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00008-7
  8. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00010-5. [Epub ahead of print]194 23-42
    Mitochondrial optic neuropathies.
    Valerio Carelli, Chiara La Morgia, Patrick Yu-Wai-Man.
      Mitochondrial optic neuropathies have a leading role in the field of mitochondrial medicine ever since 1988, when the first mutation in mitochondrial DNA was associated with Leber's hereditary optic neuropathy (LHON). Autosomal dominant optic atrophy (DOA) was subsequently associated in 2000 with mutations in the nuclear DNA affecting the OPA1 gene. LHON and DOA are both characterized by selective neurodegeneration of retinal ganglion cells (RGCs) triggered by mitochondrial dysfunction. This is centered on respiratory complex I impairment in LHON and defective mitochondrial dynamics in OPA1-related DOA, leading to distinct clinical phenotypes. LHON is a subacute, rapid, severe loss of central vision involving both eyes within weeks or months, with age of onset between 15 and 35 years old. DOA is a more slowly progressive optic neuropathy, usually apparent in early childhood. LHON is characterized by marked incomplete penetrance and a clear male predilection. The introduction of next-generation sequencing has greatly expanded the genetic causes for other rare forms of mitochondrial optic neuropathies, including recessive and X-linked, further emphasizing the exquisite sensitivity of RGCs to compromised mitochondrial function. All forms of mitochondrial optic neuropathies, including LHON and DOA, can manifest either as pure optic atrophy or as a more severe multisystemic syndrome. Mitochondrial optic neuropathies are currently at the forefront of a number of therapeutic programs, including gene therapy, with idebenone being the only approved drug for a mitochondrial disorder.
    Keywords:  Complex I; DOA; Gene therapy; Idebenone; LHON; Mitochondria; Mitochondrial DNA; Mitochondrial dynamics; Mitochondrial fission; Mitochondrial fusion; Mitochondrial optic neuropathies; OPA1; Optic atrophy; Optic nerve; Retinal ganglion cells; mtDNA
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00010-5
  9. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00001-4. [Epub ahead of print]194 3-6
    Mitochondrial disease in neurology-Past, present, and future.
    Patrick F Chinnery.
      This chapter provides a overview of this volume of the Handbook of Clinical Neurology, placing recent advances in our understanding of mitochondrial disorders in a historical context, and speculates about the future.
    Keywords:  Clinical medicine; Diagnosis; Genomics; Mitochondria; Mitochondrial diseases; Neurology; Treatments; mtDNA
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00001-4
  10. Int J Biol Macromol. 2023 Feb 20. pii: S0141-8130(23)00649-9. [Epub ahead of print] 123755
    Mitochondrial cristae in health and disease.
    Cheng Huang, Kun Deng, Minghua Wu.
      Mitochondria are centers of energy metabolism. The mitochondrial network is shaped by mitochondrial dynamics, including the processes of mitochondrial fission and fusion and cristae remodeling. The cristae folded by the inner mitochondrial membrane are sites of the mitochondrial oxidative phosphorylation (OXPHOS) system. However, the factors and their coordinated interplay in cristae remodeling and linked human diseases have not been fully demonstrated. In this review, we focus on key regulators of cristae structure, including the mitochondrial contact site and cristae organizing system, optic atrophy-1, mitochondrial calcium uniporter, and ATP synthase, which function in the dynamic remodeling of cristae. We summarized their contribution to sustaining functional cristae structure and abnormal cristae morphology, including a decreased number of cristae, enlarged cristae junctions, and cristae as concentric ring structures. These abnormalities directly impact cellular respiration and are caused by dysfunction or deletion of these regulators in diseases such as Parkinson's disease, Leigh syndrome, and dominant optic atrophy. Identifying the important regulators of cristae morphology and understanding their role in sustaining mitochondrial morphology could be applied to explore the pathologies of diseases and to develop relevant therapeutic tools.
    Keywords:  ATP synthase; Cristae ultrastructure; MICOS; MICU1; Mitochondria; OPA1
    DOI:  https://doi.org/10.1016/j.ijbiomac.2023.123755
  11. Methods Mol Biol. 2023 ;2615 79-88
    Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies.
    Xiangjun Di, Jinshan Qin, Yujie Sun, Qian Peter Su.
      Mitochondrial DNA (mtDNA) encodes a variety of rRNAs, tRNAs, and respiratory chain complex proteins. The integrity of mtDNA supports the mitochondrial functions and plays an essential role in numerous physiological and pathological processes. Mutations in mtDNA cause metabolic diseases and aging. The mtDNA within the human cells are packaged into hundreds of nucleoids within the mitochondrial matrix. Knowledge of how the nucleoids are dynamically distributed and organized within mitochondria is key to understanding mtDNA structure and functions. Therefore, visualizing the distribution and dynamics of mtDNA within mitochondria is a powerful approach to gain insights into the regulation of mtDNA replication and transcription. In this chapter, we describe the methods of observing mtDNA and its replication with fluorescence microscopy in both fixed and live cells using different labeling strategies.
    Keywords:  BrdU; EdU; Mitochondrial DNA (mtDNA); POLG2; PdG; TFAM
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_6
  12. Cell. 2023 Feb 17. pii: S0092-8674(23)00093-4. [Epub ahead of print]
    Cell lineage-specific mitochondrial resilience during mammalian organogenesis.
    Stephen P Burr, Florian Klimm, Angelos Glynos, Malwina Prater, Pamella Sendon, Pavel Nash, Christopher A Powell, Marie-Lune Simard, Nina A Bonekamp, Julia Charl, Hector Diaz, Lyuba V Bozhilova, Yu Nie, Haixin Zhang, Michele Frison, Maria Falkenberg, Nick Jones, Michal Minczuk, James B Stewart, Patrick F Chinnery.
      Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments.
    Keywords:  OXPHOS; RNA-seq; SCENIC; mitochondria; mt-Ta; mtDNA; organogenesis; single-cell
    DOI:  https://doi.org/10.1016/j.cell.2023.01.034
  13. Methods Mol Biol. 2023 ;2615 219-228
    Studying Mitochondrial Nucleic Acid Synthesis Utilizing Intact Isolated Mitochondria.
    Jelena Misic, Dusanka Milenkovic.
      Mitochondria are eukaryotic organelles of endosymbiotic origin that contain their own genetic material, mitochondrial DNA (mtDNA), and dedicated systems for mtDNA maintenance and expression. MtDNA molecules encode a limited number of proteins that are nevertheless all essential subunits of the mitochondrial oxidative phosphorylation system. Here, we describe protocols to monitor DNA and RNA synthesis in intact, isolated mitochondria. These in organello synthesis protocols are valuable techniques for studying the mechanisms and regulation of mtDNA maintenance and expression.
    Keywords:  Mitochondria; Radioactive labeling of nucleic acids; in organello replication and transcription; mtDNA; mtDNA maintenance and expression
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_16
  14. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00013-0. [Epub ahead of print]194 259-277
    Experimental therapy for mitochondrial diseases.
    Carlo Viscomi, Massimo Zeviani.
      Mitochondrial diseases are extremely heterogeneous genetic disorders due to faulty oxidative phosphorylation (OxPhos). No cure is currently available for these conditions, beside supportive interventions aimed at relieving complications. Mitochondria are under a double genetic control carried out by the mitochondrial DNA (mtDNA) and by nuclear DNA. Thus, not surprisingly, mutations in either genome can cause mitochondrial disease. Although mitochondria are usually associated with respiration and ATP synthesis, they play fundamental roles in a large number of other biochemical, signaling, and execution pathways, each being a potential target for therapeutic interventions. These can be classified as general therapies, i.e., potentially applicable to a number of different mitochondrial conditions, or therapies tailored to a single disease, i.e., personalized approaches, such as gene therapy, cell therapy, and organ replacement. Mitochondrial medicine is a particularly lively research field, and the last few years witnessed a steady increase in the number of clinical applications. This chapter will present the most recent therapeutic attempts emerged from preclinical work and an update of the currently ongoing clinical applications. We think that we are starting a new era in which the etiologic treatment of these conditions is becoming a realistic option.
    Keywords:  AAV; Gene therapy; Mitochondrial biogenesis; Mitochondrial disease; Mitophagy; OxPhos; Rapamycin
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00013-0
  15. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00014-2. [Epub ahead of print]194 99-116
    Peripheral neuropathy in mitochondrial disease.
    Rita Horvath, Jessica Medina, Mary M Reilly, Michael E Shy, Stephan Zuchner.
      Mitochondria are essential for the health and viability of both motor and sensory neurons and their axons. Processes that disrupt their normal distribution and transport along axons will likely cause peripheral neuropathies. Similarly, mutations in mtDNA or nuclear encoded genes result in neuropathies that either stand alone or are part of multisystem disorders. This chapter focuses on the more common genetic forms and characteristic clinical phenotypes of "mitochondrial" peripheral neuropathies. We also explain how these various mitochondrial abnormalities cause peripheral neuropathy. In a patient with a neuropathy either due to a mutation in a nuclear or an mtDNA gene, clinical investigations aim to characterize the neuropathy and make an accurate diagnosis. In some patients, this may be relatively straightforward, where a clinical assessment and nerve conduction studies followed by genetic testing is all that is needed. In others, multiple investigations including a muscle biopsy, CNS imaging, CSF analysis, and a wide range of metabolic and genetic tests in blood and muscle may be needed to establish diagnosis.
    Keywords:  Axonal transport; Charcot–Marie–Tooth disease; Mitochondrial DNA; Mitochondrial fusion/fission; Nuclear mitochondrial genes; Respiratory chain enzymes
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00014-2
  16. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00006-3. [Epub ahead of print]194 251-257
    Blood biomarkers of mitochondrial disease-One for all or all for one?
    Anu Suomalainen.
      The mitochondrial disease group consists of different disorders with unprecedented variability of clinical manifestations and tissue-specific symptoms. Their tissue-specific stress responses vary depending on the patients' age and type of dysfunction. These responses include secretion of metabolically active signal molecules to systemic circulation. Such signals-metabolites or metabokines-can be also utilized as biomarkers. During the past 10 years, metabolite and metabokine biomarkers have been described for mitochondrial disease diagnosis and follow-up, to complement the conventional blood biomarkers lactate, pyruvate and alanine. These new tools include metabokines FGF21 and GDF15; cofactors (NAD-forms); sets of metabolites (multibiomarkers) and the full metabolome. FGF21 and GDF15 are messengers of mitochondrial integrated stress response that together outperform the conventional biomarkers in specificity and sensitivity for muscle-manifesting mitochondrial diseases. Metabolite or metabolomic imbalance (e.g., NAD+ deficiency) is a secondary consequence to the primary cause in some diseases, but relevant as a biomarker and a potential indicator of therapy targets. For therapy trials, the optimal biomarker set needs to be tailored to match the disease of interest. The new biomarkers have increased the value of blood samples in mitochondrial disease diagnosis and follow-up, enabling prioritization of patients to different diagnostic paths and having crucial roles in follow-up of therapy effect.
    Keywords:  Bbiomarker; Diagnosis; Disease progression; FGF21; GDF15; Metabolomics; Mitochondrial disease; Multibiomarker; NAD; Treatment
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00006-3
  17. Biomolecules. 2023 Feb 16. pii: 378. [Epub ahead of print]13(2):
    Mitochondrial Neurodegeneration: Lessons from Drosophila melanogaster Models.
    Michele Brischigliaro, Erika Fernandez-Vizarra, Carlo Viscomi.
      The fruit fly-i.e., Drosophila melanogaster-has proven to be a very useful model for the understanding of basic physiological processes, such as development or ageing. The availability of straightforward genetic tools that can be used to produce engineered individuals makes this model extremely interesting for the understanding of the mechanisms underlying genetic diseases in physiological models. Mitochondrial diseases are a group of yet-incurable genetic disorders characterized by the malfunction of the oxidative phosphorylation system (OXPHOS), which is the highly conserved energy transformation system present in mitochondria. The generation of D. melanogaster models of mitochondrial disease started relatively recently but has already provided relevant information about the molecular mechanisms and pathological consequences of mitochondrial dysfunction. Here, we provide an overview of such models and highlight the relevance of D. melanogaster as a model to study mitochondrial disorders.
    Keywords:  Drosophila melanogaster; OXPHOS; mitochondrial disease; neurodegeneration
    DOI:  https://doi.org/10.3390/biom13020378
  18. Biomedicines. 2023 Feb 12. pii: 532. [Epub ahead of print]11(2):
    Creation of Mitochondrial Disease Models Using Mitochondrial DNA Editing.
    Victoria A Khotina, Andrey Y Vinokurov, Mariam Bagheri Ekta, Vasily N Sukhorukov, Alexander N Orekhov.
      Mitochondrial diseases are a large class of human hereditary diseases, accompanied by the dysfunction of mitochondria and the disruption of cellular energy synthesis, that affect various tissues and organ systems. Mitochondrial DNA mutation-caused disorders are difficult to study because of the insufficient number of clinical cases and the challenges of creating appropriate models. There are many cellular models of mitochondrial diseases, but their application has a number of limitations. The most proper and promising models of mitochondrial diseases are animal models, which, unfortunately, are quite rare and more difficult to develop. The challenges mainly arise from the structural features of mitochondria, which complicate the genetic editing of mitochondrial DNA. This review is devoted to discussing animal models of human mitochondrial diseases and recently developed approaches used to create them. Furthermore, this review discusses mitochondrial diseases and studies of metabolic disorders caused by the mitochondrial DNA mutations underlying these diseases.
    Keywords:  animal model; cellular model; gene editing; mitochondrial diseases; mitochondrial mutations
    DOI:  https://doi.org/10.3390/biomedicines11020532
  19. Methods Mol Biol. 2023 ;2611 269-282
    Concomitant Sequencing of Accessible Chromatin and Mitochondrial Genomes in Single Cells Using mtscATAC-Seq.
    Leif S Ludwig, Caleb A Lareau.
      Mitochondria are unique organelles of eukaryotic cells that carry their own multicopy number and circular genome. In most mammals, including humans and mice, the size of the chromosome is ~16,000 base pairs and unlike nuclear DNA, mitochondrial DNA (mtDNA) is not densely compacted. This results in mtDNA to be highly accessible for enzymes such as the Tn5 transposase, commonly used for accessible chromatin profiling of nuclear chromatinized DNA. Here, we describe a method for the concomitant sequencing of mtDNA and accessible chromatin in thousands of individual cells via the mitochondrial single-cell assay for transposase accessible chromatin by sequencing (mtscATAC-seq). Our approach extends the utility of existing scATAC-seq products and protocols as we (Nam et al, Nat Rev Genet 22:3-18, 2021) fix cells using formaldehyde to retain mitochondria and its mtDNA within its originating cell, (Buenrostro et al, Nat Methods 10:1213-1218, 2013) modify lysis conditions to permeabilize cells and mitochondria, and (Corces et al, Nat Methods 14:959-962, 2017) optimize bioinformatic processing protocols to collectively increase mitochondrial genome coverage for downstream analysis. Here, we discuss the essentials for the experimental and computational methodologies to generate and analyze thousands of multiomic profiles of single cells over the course of a few days, enabling the profiling of accessible chromatin and mtDNA genotypes to reconstruct clonal relationships and studies of mitochondrial genetics and disease.
    Keywords:  Accessible chromatin profiling; Lineage tracing; Mitochondrial DNA; Mitochondrial disease; Pathogenic mutation; Single cell multiomics; Somatic mutation
    DOI:  https://doi.org/10.1007/978-1-0716-2899-7_14
  20. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00003-8. [Epub ahead of print]194 117-124
    Complex neurological and multisystem presentations in mitochondrial disease.
    Michelangelo Mancuso.
      Mitochondrial diseases typically involve organs highly dependent on aerobic metabolism and are often progressive with high morbidity and mortality. In the previous chapters of this book, classical mitochondrial phenotypes and syndromes are extensively described. However, these well-known clinical pictures are more the exception rather than the rule in mitochondrial medicine. In fact, more complex, unspecified, incomplete, and/or overlap clinical entities may be even more frequent, with multisystem appearance or progression. In this chapter, we describe some complex neurological presentations, as well as the multisystem manifestations of mitochondrial diseases, ranging from the brain to the other organs.
    Keywords:  Cardiomyopathies; Hearing loss; Leukoencephalopathies; Mitochondrial diseases; Multisystem involvement; Parkinsonism; mtDNA
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00003-8
  21. Methods Mol Biol. 2023 ;2615 17-30
    Isolating Mitochondria, Mitoplasts, and mtDNA from Cultured Mammalian Cells.
    Katja E Menger, Thomas J Nicholls.
      Mitochondria are double membrane-bound eukaryotic organelles with roles in a range of cellular activities including energy conversion, apoptosis, cell signalling, and the biosynthesis of enzyme cofactors. Mitochondria contain their own genome, called mtDNA, which encodes subunits of the oxidative phosphorylation machinery as well as the rRNA and tRNA molecules required for their translation within mitochondria. The ability to isolate highly purified mitochondria from cells has been instrumental in a number of studies of mitochondrial function. Differential centrifugation is a long-established method for the isolation of mitochondria. Cells are subjected to osmotic swelling and disruption, followed by centrifugation in isotonic sucrose solutions to separate mitochondria from other cellular components. We present a method using this principle for the isolation of mitochondria from cultured mammalian cell lines. Mitochondria purified by this method can be further fractionated to investigate protein localization, or act as a starting point to purify mtDNA.
    Keywords:  DNA extraction; Mitochondria; Mitochondrial DNA; Mitochondrial isolation; Protein localization
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_2
  22. Methods Mol Biol. 2023 ;2615 443-463
    Single Cell Analysis of Mitochondrial DNA Deletions.
    Helen A L Tuppen, Amy K Reeve, Amy E Vincent.
      Mitochondrial DNA (mtDNA) deletions underpin mitochondrial dysfunction in human tissues in aging and disease. The multicopy nature of the mitochondrial genome means these mtDNA deletions can occur in varying mutation loads. At low levels, these deletions have no impact, but once the proportion of molecules harbouring a deletion exceeds a threshold level, then dysfunction occurs. The location of the breakpoints and the size of the deletion impact upon the mutation threshold required to cause deficiency of an oxidative phosphorylation complex, and this varies for each of the different complexes. Furthermore, mutation load and deletion species can vary between adjacent cells in a tissue, with a mosaic pattern of mitochondrial dysfunction observed. As such, it is often important for understanding human aging and disease to be able to characterise the mutation load, breakpoints and size of deletion(s) from a single human cell. Here, we detail protocols for laser micro-dissection and single cell lysis from tissues, and the subsequent analysis of deletion size, breakpoints and mutation load using long-range PCR, mtDNA sequencing and real-time PCR, respectively.
    Keywords:  Breakpoint; Heteroplasmy; Mutation load; mtDNA deletion
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_29
  23. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00004-X. [Epub ahead of print]194 207-228
    Reproductive options in mitochondrial disease.
    Hubert J M Smeets, Suzanne C E H Sallevelt, Mary Herbert.
      Mitochondrial diseases require customized approaches for reproductive counseling, addressing differences in recurrence risks and reproductive options. The majority of mitochondrial diseases is caused by mutations in nuclear genes and segregate in a Mendelian way. Prenatal diagnosis (PND) or preimplantation genetic testing (PGT) are available to prevent the birth of another severely affected child. In at least 15%-25% of cases, mitochondrial diseases are caused by mitochondrial DNA (mtDNA) mutations, which can occur de novo (25%) or be maternally inherited. For de novo mtDNA mutations, the recurrence risk is low and PND can be offered for reassurance. For maternally inherited, heteroplasmic mtDNA mutations, the recurrence risk is often unpredictable, due to the mitochondrial bottleneck. PND for mtDNA mutations is technically possible, but often not applicable given limitations in predicting the phenotype. Another option for preventing the transmission of mtDNA diseases is PGT. Embryos with mutant load below the expression threshold are being transferred. Oocyte donation is another safe option to prevent the transmission of mtDNA disease to a future child for couples who reject PGT. Recently, mitochondrial replacement therapy (MRT) became available for clinical application as an alternative to prevent the transmission of heteroplasmic and homoplasmic mtDNA mutations.
    Keywords:  Mitochondrial bottleneck; Mitochondrial disease; Mitochondrial replacement therapy; Preimplantation genetic diagnosis; Prenatal diagnosis; Reproductive options; mtDNA disease
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00004-X
  24. Methods Mol Biol. 2023 ;2615 329-344
    Manipulation of Murine Mitochondrial DNA Heteroplasmy with mtZFNs.
    Pavel A Nash, Michal Minczuk.
      Mouse models of mitochondrial DNA mutations hold promise in the development and optimization of mitochondrial gene therapy technology and for gathering pre-clinical data prior to human trials. Their suitability for this purpose stems from the high similarity of human and murine mitochondrial genomes and the increasing availability of rationally designed AAV vectors capable of selectively transducing murine tissues. Our laboratory routinely optimizes mitochondrially targeted zinc finger nucleases (mtZFNs), the compactness of which makes them highly suitable for downstream AAV-based in vivo mitochondrial gene therapy. This chapter discusses the necessary precautions for the robust and precise genotyping of the murine mitochondrial genome as well as the optimization of mtZFNs intended for subsequent use in vivo.
    Keywords:  Gene therapy; Heteroplasmy; MEF; Mitochondria; Mouse; Zinc Finger nuclease; mtDNA; mtZFN
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_23
  25. Methods Mol Biol. 2023 ;2615 89-98
    Visualization of mtDNA Using FISH.
    Xie Xie, Xuefeng Zhu.
      Proper mitochondrial DNA (mtDNA) levels are critical for many cellular biological functions and are associated with aging and many mitochondria disorders. Defects in core subunits of the mtDNA replication machinery lead to decreased mtDNA levels. Other indirect mitochondrial contexts including ATP concentration, lipid composition, and nucleotide composition also contribute to mtDNA maintenance. Furthermore, mtDNA molecules are distributed evenly throughout the mitochondrial network. This uniform distribution pattern is required for oxidative phosphorylation and ATP production and has been linked to many diseases when perturbed. Thus, it is important to visualize mtDNA in the cellular context. Here we provide detailed protocols for cellular visualization of mtDNA using fluorescence in situ hybridization (FISH). The fluorescent signals are targeted to the mtDNA sequence directly, ensuring both sensitivity and specificity. This mtDNA FISH method can be combined with immunostaining and used for visualizing mtDNA-protein interactions and dynamics.
    Keywords:  FISH; Imaging; Microscopy; Mitochondria; Visualization; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_7
  26. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00009-9. [Epub ahead of print]194 79-98
    Ataxia and spastic paraplegia in mitochondrial disease.
    Matthis Synofzik, Elena Rugarli, Evan Reid, Rebecca Schüle.
      Degenerative ataxias and hereditary spastic paraplegias (HSPs) form a continuous, often overlapping disease spectrum sharing not only phenotypic features and underlying genes, but also cellular pathways and disease mechanisms. Mitochondrial metabolism presents a major molecular theme underlying both multiple ataxias and HSPs, thus indicating a heightened vulnerability of Purkinje cells, spinocerebellar tracts, and motor neurons to mitochondrial dysfunction, which is of particular interest for translational approaches. Mitochondrial dysfunction might be the primary (upstream) or secondary (downstream) result of a genetic defect, with underlying genetic defects in nuclear-encoded genes being much more frequent than in mtDNA genes in both, ataxias and HSPs. Here, we outline the substantial number of ataxias, spastic ataxias and HSPs caused by mutated genes implicated in (primary or secondary) mitochondrial dysfunction, highlighting several key "mitochondrial" ataxias and HSPs which are of particular interest for their frequency, pathogenesis and translational opportunities. We then showcase prototypic mitochondrial mechanisms by which disruption of these ataxia and HSP genes contributes to Purkinje cells or corticospinal neuron dysfunction, thus elucidating hypotheses on Purkinje cells and corticospinal neuron vulnerability to mitochondrial dysfunction.
    Keywords:  Ataxia; Axon; Cerebellum; Genetic; Genetics; Hereditary spastic paraplegia; Mitochondrion; Motor neuron; Spastic ataxia; Translation
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00009-9
  27. iScience. 2023 Feb 17. 26(2): 106067
    Role of human HSPE1 for OPA1 processing independent of HSPD1.
    Nelson Yeung, Daisuke Murata, Miho Iijima, Hiromi Sesaki.
      The human mtHSP60/HSPD1-mtHSP10/HSPE1 system prevents protein misfolding and maintains proteostasis in the mitochondrial matrix. Altered activities of this chaperonin system have been implicated in human diseases, such as cancer and neurodegeneration. However, how defects in HSPD1 and HSPE1 affect mitochondrial structure and dynamics remains elusive. In the current study, we address this fundamental question in a human cell line, HEK293T. We found that the depletion of HSPD1 or HSPE1 results in fragmentation of mitochondria, suggesting a decrease in mitochondrial fusion. Supporting this notion, HSPE1 depletion led to proteolytic inactivation of OPA1, a dynamin-related GTPase that fuses the mitochondrial membrane. This OPA1 inactivation was mediated by a stress-activated metalloprotease, OMA1. In contrast, HSPD1 depletion did not induce OMA1 activation or OPA1 cleavage. These data suggest that HSPE1 controls mitochondrial morphology through a mechanism separate from its chaperonin activity.
    Keywords:  Biological sciences; Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2023.106067
  28. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00016-6. [Epub ahead of print]194 173-185
    Neuroimaging in mitochondrial disease.
    Felix Distelmaier, Thomas Klopstock.
      The anatomic complexity of the brain in combination with its high energy demands makes this organ specifically vulnerable to defects of mitochondrial oxidative phosphorylation. Therefore, neurodegeneration is a hallmark of mitochondrial diseases. The nervous system of affected individuals typically shows selective regional vulnerability leading to distinct patterns of tissue damage. A classic example is Leigh syndrome, which causes symmetric alterations of basal ganglia and brain stem. Leigh syndrome can be caused by different genetic defects (>75 known disease genes) with variable disease onset ranging from infancy to adulthood. Other mitochondrial diseases are characterized by focal brain lesions, which is a core feature of MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes). Apart from gray matter, also white matter can be affected by mitochondrial dysfunction. White matter lesions vary depending on the underlying genetic defect and may progress into cystic cavities. In view of the recognizable patterns of brain damage in mitochondrial diseases, neuroimaging techniques play a key role in diagnostic work-up. In the clinical setting, magnetic resonance imaging (MRI) and MR spectroscopy (MRS) are the mainstay of diagnostic work-up. Apart from visualization of brain anatomy, MRS allows the detection of metabolites such as lactate, which is of specific interest in the context of mitochondrial dysfunction. However, it is important to note that findings like symmetric basal ganglia lesions on MRI or a lactate peak on MRS are not specific, and that there is a broad range of disorders that can mimic mitochondrial diseases on neuroimaging. In this chapter, we will review the spectrum of neuroimaging findings in mitochondrial diseases and discuss important differential diagnoses. Moreover, we will give an outlook on novel biomedical imaging tools that may provide interesting insights into mitochondrial disease pathophysiology.
    Keywords:  Brain; Central nervous system; Leigh disease; Magnetic resonance imaging; Neurodegeneration; OXPHOS
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00016-6
  29. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00007-5. [Epub ahead of print]194 189-206
    Currently available therapies in mitochondrial disease.
    Cornelia Kornblum, Costanza Lamperti, Sumit Parikh.
      Mitochondrial diseases are a heterogeneous group of multisystem disorders caused by impaired mitochondrial function. These disorders occur at any age and involve any tissue, typically affecting organs highly dependent on aerobic metabolism. Diagnosis and management are extremely difficult due to various underlying genetic defects and a wide range of clinical symptoms. Preventive care and active surveillance are strategies to try to reduce morbidity and mortality by timely treatment of organ-specific complications. More specific interventional therapies are in early phases of development and no effective treatment or cure currently exists. A variety of dietary supplements have been utilized based on biological logic. For several reasons, few randomized controlled trials have been completed to assess the efficacy of these supplements. The majority of the literature on supplement efficacy represents case reports, retrospective analyses and open-label studies. We briefly review selected supplements that have some degree of clinical research support. In mitochondrial diseases, potential triggers of metabolic decompensation or medications that are potentially toxic to mitochondrial function should be avoided. We shortly summarize current recommendations on safe medication in mitochondrial diseases. Finally, we focus on the frequent and debilitating symptoms of exercise intolerance and fatigue and their management including physical training strategies.
    Keywords:  Dietary supplements; Drugs; Exercise intolerance; Exercise training; Fatigue; Management; Medication; Mitochondrial disease; Surveillance; Therapy; Treatment
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00007-5
  30. Methods Mol Biol. 2023 ;2615 315-325
    5'-End Mapping in Human Mitochondrial DNA.
    Andranik Durgaryan, Anders R Clausen.
      Here, we describe an assay that enables mapping of 5'-ends across the genome using next-generation sequencing on an Illumina platform, 5'-End-sequencing (5'-End-seq). We use this method to map free 5'-ends in mtDNA isolated from fibroblasts. This method can be used to answer key questions regarding DNA integrity, DNA replication mechanisms and to identify priming events, primer processing, nick processing, and double strand break processing on the entire genome.
    Keywords:  5′-End-seq; Bioinformatics; DNA polymerase; DNA replication; Genomics; Next-generation sequencing; RNase H1; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_22
  31. Methods Mol Biol. 2023 ;2615 139-151
    Assessing TFAM Binding to Human Mitochondrial DNA.
    Takehiro Yasukawa, Dongchon Kang.
      Mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA)-binding protein that plays a crucial dual role in the initiation of mitochondrial transcription initiation and mtDNA maintenance. Because TFAM directly interacts with mtDNA, assessing its DNA-binding property can provide useful information. This chapter describes two in vitro assay methods, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay with recombinant TFAM proteins, which both require simple agarose gel electrophoresis. These are used to investigate the effects of mutations, truncation, and posttranslational modifications on this key mtDNA regulatory protein.
    Keywords:  DNA-unwinding assay; EMSA; Electrophoretic mobility shift assay; Mitochondrial DNA; Mitochondrial nucleoids; Mitochondrial transcription factor A; TFAM; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_11
  32. Methods Mol Biol. 2023 ;2615 229-240
    Functional Assessment of Mitochondrial DNA Maintenance by Depletion and Repopulation Using 2',3'-Dideoxycytidine in Cultured Cells.
    Gábor Zsurka, Genevieve Trombly, Susanne Schöler, Daniel Blei, Wolfram S Kunz.
      The manipulation of mitochondrial DNA (mtDNA) copy number in cultured cells, using substances that interfere with DNA replication, is a useful tool to investigate various aspects of mtDNA maintenance. Here we describe the use of 2',3'-dideoxycytidine (ddC) to induce a reversible reduction of mtDNA copy number in human primary fibroblasts and human embryonic kidney (HEK293) cells. Once the application of ddC is stopped, cells depleted for mtDNA attempt to recover normal mtDNA copy numbers. The dynamics of repopulation of mtDNA provide a valuable measure for the enzymatic activity of the mtDNA replication machinery.
    Keywords:  DNA polymerase γ (POLG); Nucleoside reverse transcriptase inhibitor (NRTI); Quantitative PCR; Replication of mtDNA; Zalcitabine; mtDNA copy number
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_17
  33. Autophagy. 2023 Feb 20.
    The role of programmed mitophagy in germline mitochondrial DNA quality control.
    Jonathan M Palozzi, Thomas R Hurd.
      Mitochondrial DNA (mtDNA) is prone to the accumulation of mutations. To prevent harmful mtDNA mutations from being passed on to the next generation, the female germline, through which mtDNA is exclusively inherited, has evolved extensive mtDNA quality control. To dissect the molecular underpinnings of this process, we recently performed a large RNAi screen in Drosophila and uncovered a programmed germline mitophagy (PGM) that is essential for mtDNA quality control. We found that PGM begins as germ cells enter meiosis, induced, at least in part, by the inhibition of the mTor (mechanistic Target of rapamycin) complex 1 (mTorC1). Interestingly, PGM requires the general macroautophagy/autophagy machinery and the mitophagy adaptor BNIP3, but not the canonical mitophagy genes Pink1 and park (parkin), even though they are critical for germline mtDNA quality control. We also identified the RNA-binding protein Atx2 as a major regulator of PGM. This work is the first to identify and implicate a programmed mitophagy event in germline mtDNA quality control, and it highlights the utility of the Drosophila ovary for studying developmentally regulated mitophagy and autophagy in vivo.
    Keywords:  Drosophila; autophagy; germline; mitochondria; mitochondrial DNA; mitophagy; mtDNA; purifying selection
    DOI:  https://doi.org/10.1080/15548627.2023.2182595
  34. Biochem Soc Trans. 2023 Feb 23. pii: BST20220014. [Epub ahead of print]
    Mitochondrial dynamics in macrophages: divide to conquer or unite to survive?
    Syeda Farhana Afroz, Karoline D Raven, Grace M E P Lawrence, Ronan Kapetanovic, Kate Schroder, Matthew J Sweet.
      Mitochondria have long been appreciated as the metabolic hub of cells. Emerging evidence also posits these organelles as hubs for innate immune signalling and activation, particularly in macrophages. Macrophages are front-line cellular defenders against endogenous and exogenous threats in mammals. These cells use an array of receptors and downstream signalling molecules to respond to a diverse range of stimuli, with mitochondrial biology implicated in many of these responses. Mitochondria have the capacity to both divide through mitochondrial fission and coalesce through mitochondrial fusion. Mitochondrial dynamics, the balance between fission and fusion, regulate many cellular functions, including innate immune pathways in macrophages. In these cells, mitochondrial fission has primarily been associated with pro-inflammatory responses and metabolic adaptation, so can be considered as a combative strategy utilised by immune cells. In contrast, mitochondrial fusion has a more protective role in limiting cell death under conditions of nutrient starvation. Hence, fusion can be viewed as a cellular survival strategy. Here we broadly review the role of mitochondria in macrophage functions, with a focus on how regulated mitochondrial dynamics control different functional responses in these cells.
    Keywords:  inflammation; macrophages; mitochondrial dynamics; mitochondrial fission; mitochondrial fusion; neuroinflammation
    DOI:  https://doi.org/10.1042/BST20220014
  35. Methods Mol Biol. 2023 ;2615 3-16
    Isolation of Functional Mitochondria and Pure mtDNA from Murine Tissues.
    Dieu Hien Rozsivalova, Milica Popovic, Harshita Kaul, Aleksandra Trifunovic.
      Detailed analysis of mitochondrial function cannot be achieved without good quality preparations of isolated mitochondria. Ideally, the isolation protocol should be quick, while producing a reasonably pure pool of mitochondria that are still intact and coupled. Here, we describe a fast and simple method for the purification of mammalian mitochondria relying on isopycnic density gradient centrifugation. We describe specific steps that should be taken into consideration when functional mitochondria from different tissues should be isolated. This protocol is suitable for the analysis of many aspects of the organelle's structure and function.
    Keywords:  Blue native PAGE; Electron transport chain; Isopycnic density gradient centrifugation; Mitochondria; Organelle isolation; Oxidative phosphorylation; Respirometry
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_1
  36. Methods Mol Biol. 2023 ;2615 241-266
    Analysis of Mitochondrial DNA Replication by Two-Dimensional Agarose Gel Electrophoresis.
    Steffi Goffart, Jaakko Pohjoismäki.
      Two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been employed for nearly two decades in the analysis of replication and maintenance processes of animal mitochondrial DNA, but the method's potential has not been fully exploited. Here, we describe the various steps involved in this technique, from DNA isolation, to two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE), Southern hybridization and interpretation. We also provide examples of the applicability of 2D-AGE to investigate the different features of mtDNA maintenance and regulation.
    Keywords:  2D-AGE; Gel electrophoresis; Mitochondrial DNA; Recombination; Replication intermediates; Replication mechanism; Replication pausing
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_18
  37. bioRxiv. 2023 Feb 15. pii: 2023.02.09.527880. [Epub ahead of print]
    Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle.
    Erik McShane, Mary Couvillion, Robert Ietswaart, Gyan Prakash, Brendan M Smalec, Iliana Soto, Autum R Baxter-Koenigs, Karine Choquet, L Stirling Churchman.
      Mitochondria play critical roles in cellular metabolism, primarily by serving as the site of assembly and function of the oxidative phosphorylation (OXPHOS) machinery. The OXPHOS proteins are encoded by mitochondrial DNA (mtDNA) and nuclear DNA, which reside and are regulated within separate compartments. To unravel how the two gene expression systems collaborate to produce the OXPHOS complexes, the regulatory principles controlling the production of mtDNA-encoded proteins need to be elucidated. In this study, we performed a quantitative analysis of the mitochondrial messenger RNA (mt-mRNA) life cycle to determine which steps of gene expression experience strong regulatory control. Our analysis revealed that the high accumulation of mt-mRNA despite their rapid turnover was made possible by a 700-fold higher transcriptional output than nuclear-encoded OXPHOS genes. In addition, we observed that mt-mRNA processing and its association with the mitochondrial ribosome occur rapidly and that these processes are linked mechanistically. Based on these data, we developed a model of mtDNA expression that is predictive across human cell lines, revealing that differences in turnover and translation efficiency are the major contributors to mitochondrial-encoded protein synthesis. Applying this framework to a disease model of Leigh syndrome, French-Canadian type, we found that the disease-associated nuclear-encoded gene, LRPPRC , acts predominantly by stabilizing mt-mRNA. Our findings provide a comprehensive view of the intricate regulatory mechanisms governing mtDNA-encoded protein synthesis, highlighting the importance of quantitatively analyzing the mitochondrial RNA life cycle in order to decode the regulatory principles of mtDNA expression.
    DOI:  https://doi.org/10.1101/2023.02.09.527880
  38. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00018-X. [Epub ahead of print]194 9-21
    Progressive external ophthalmoplegia.
    Michio Hirano, Robert D S Pitceathly.
      Progressive external ophthalmoplegia (PEO), characterized by ptosis and impaired eye movements, is a clinical syndrome with an expanding number of etiologically distinct subtypes. Advances in molecular genetics have revealed numerous pathogenic causes of PEO, originally heralded in 1988 by the detection of single large-scale deletions of mitochondrial DNA (mtDNA) in skeletal muscle of people with PEO and Kearns-Sayre syndrome. Since then, multiple point variants of mtDNA and nuclear genes have been identified to cause mitochondrial PEO and PEO-plus syndromes, including mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and sensory ataxic neuropathy dysarthria ophthalmoplegia (SANDO). Intriguingly, many of those nuclear DNA pathogenic variants impair maintenance of the mitochondrial genome causing downstream mtDNA multiple deletions and depletion. In addition, numerous genetic causes of nonmitochondrial PEO have been identified.
    Keywords:  Kearns–Sayre syndrome; MNGIE; Mitochondria; Mitochondrial DNA; Ophthalmoplegia; SANDO
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00018-X
  39. Methods Mol Biol. 2023 ;2615 281-292
    Detection of UV-Induced Deletions in Mitochondrial DNA.
    Gabriele A Fontana, Hailey L Gahlon.
      Mitochondrial DNA (mtDNA) mutations are found in several human pathologies and are associated with aging. Deletion mutations in mtDNA result in the loss of essential genes for mitochondrial function. Over 250 deletion mutations have been reported and the common deletion is the most frequent mtDNA deletion linked to disease. This deletion removes 4977 base pairs of mtDNA. It has previously been shown that exposure to UVA radiation can promote the formation of the common deletion. Furthermore, aberrations in mtDNA replication and repair are associated with formation of the common deletion. However, molecular mechanisms describing the formation of this deletion are poorly characterized. This chapter describes a method to irradiate human skin fibroblasts with physiological doses of UVA and the subsequent detection of the common deletion by quantitative PCR analysis.
    Keywords:  Common Deletion; Deletion Mutations; Mitochondrial DNA; Replication and Repair; UVA radiation
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_20
  40. Bio Protoc. 2023 Feb 05. pii: e4602. [Epub ahead of print]13(3):
    High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS.
    Hironori Saito, Tatsuya Osaki, Yoshiho Ikeuchi, Shintaro Iwasaki.
      In addition to cytosolic protein synthesis, mitochondria also utilize another translation system that is tailored for mRNAs encoded in the mitochondrial genome. The importance of mitochondrial protein synthesis has been exemplified by the diverse diseases associated with in organello translation deficiencies. Various methods have been developed to monitor mitochondrial translation, such as the classic method of labeling newly synthesized proteins with radioisotopes and the more recent ribosome profiling. However, since these methods always assess the average cell population, measuring the mitochondrial translation capacity in individual cells has been challenging. To overcome this issue, we recently developed mito-fluorescent noncanonical amino acid tagging (FUNCAT) fluorescence-activated cell sorting (FACS), which labels nascent peptides generated by mitochondrial ribosomes with a methionine analog, L-homopropargylglycine (HPG), conjugates the peptides with fluorophores by an in situ click reaction, and detects the signal in individual cells by FACS equipment. With this methodology, the hidden heterogeneity of mitochondrial translation in cell populations can be addressed.
    Keywords:   FACS ; FUNCAT ; Mitochondria ; Mitoribosome ; Translation
    DOI:  https://doi.org/10.21769/BioProtoc.4602
  41. J Cell Biol. 2023 Mar 06. pii: e202301132. [Epub ahead of print]222(3):
    Mitochondrial membrane biogenesis: A new pathway for lipid transport mediated by PERK/E-Syt1 complex.
    Sébastien Leterme, Morgane Michaud.
      Despite decades of extensive research, mitochondrial lipid transport is a process far from fully understood. In this issue, Sassano et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202206008) identified a new complex, composed of E-Syt1 and PERK, which mediates lipid transport at ER-mitochondria contact sites and regulates mitochondrial functions in human cells.
    DOI:  https://doi.org/10.1083/jcb.202301132
  42. Methods Mol Biol. 2023 ;2615 203-217
    Rolling Circle Replication and Bypass of Damaged Nucleotides.
    Josefin M E Forslund, Gorazd Stojkovič, Sjoerd Wanrooij.
      Faithful mitochondrial DNA (mtDNA) replication is critical for the proper function of the oxidative phosphorylation system. Problems with mtDNA maintenance, such as replication stalling upon encountering DNA damage, impair this vital function and can potentially lead to disease. An in vitro reconstituted mtDNA replication system can be used to investigate how the mtDNA replisome deals with, for example, oxidatively or UV-damaged DNA. In this chapter, we provide a detailed protocol on how to study the bypass of different types of DNA damage using a rolling circle replication assay. The assay takes advantage of purified recombinant proteins and can be adapted to the examination of various aspects of mtDNA maintenance.
    Keywords:  DNA damage; DNA polymerase; DNA replication; Mitochondrial DNA; Rolling circle
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_15
  43. Methods Mol Biol. 2023 ;2615 293-314
    Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels.
    Choco Michael Gorospe, Bruno Marçal Repolês, Paulina H Wanrooij.
      Impaired mitochondrial DNA (mtDNA) maintenance, due to, e.g., defects in the replication machinery or an insufficient dNTP supply, underlies a number of mitochondrial disorders. The normal process of mtDNA replication leads to the incorporation of multiple single ribonucleotides (rNMPs) per mtDNA molecule. Given that embedded rNMPs alter the stability and properties of the DNA, they may have consequences for mtDNA maintenance and thereby for mitochondrial disease. They also serve as a readout of the intramitochondrial NTP/dNTP ratios. In this chapter, we describe a method for the determination of mtDNA rNMP content using alkaline gel electrophoresis and Southern blotting. This procedure is suited for the analysis of mtDNA in total genomic DNA preparations as well as in purified form. Moreover, it can be performed using equipment found in most biomedical laboratories, allows the simultaneous analysis of 10-20 samples depending on the gel system employed, and can be modified for the analysis of other mtDNA modifications.
    Keywords:  Alkaline gels; Alkaline hydrolysis; Denaturing gels; Ribonucleotides; Southern blot; rNMPs
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_21
  44. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00002-6. [Epub ahead of print]194 229-250
    Clinical trials in mitochondrial diseases.
    Amel Karaa, Thomas Klopstock.
      Primary mitochondrial diseases are some of the most common and complex inherited inborn errors of metabolism. Their molecular and phenotypic diversity has led to difficulties in finding disease-modifying therapies and clinical trial efforts have been slow due to multiple significant challenges. Lack of robust natural history data, difficulties in finding specific biomarkers, absence of well-validated outcome measures, and small patient numbers have made clinical trial design and conduct difficult. Encouragingly, new interest in treating mitochondrial dysfunction in common diseases and regulatory incentives to develop therapies for rare conditions have led to significant interest and efforts to develop drugs for primary mitochondrial diseases. Here, we review past and present clinical trials and future strategies of drug development in primary mitochondrial diseases.
    Keywords:  Antioxidants; Clinical trials; Gene therapy; Mitochondria; Mitochondrial biogenesis; Mitophagy; Nucleosides; Primary mitochondrial disease; Treatment
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00002-6
  45. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00012-9. [Epub ahead of print]194 167-172
    Laboratory and metabolic investigations.
    Eva Morava, Devin Oglesbee.
      Clinical variability and substantial overlap between mitochondrial disorders and other genetic disorders and inborn errors make the clinical and metabolic diagnosis of mitochondrial disorders quite challenging. Evaluating specific laboratory markers is essential in the diagnostic process, but mitochondrial disease can be present in the absence of any abnormal metabolic markers. In this chapter, we share the current consensus guidelines for metabolic investigations, including investigations in blood, urine, and the cerebral spinal fluid and discuss different diagnostic approaches. As personal experience might significantly vary and there are different recommendations published as diagnostic guidelines, the Mitochondrial Medicine Society developed a consensus approach based on literature review for metabolic diagnostics in a suspected mitochondrial disease. According to the guidelines, the work-up should include the assessment of complete blood count, creatine phosphokinase, transaminases, albumin, postprandial lactate and pyruvate (lactate/pyruvate ratio when the lactate level is elevated), uric acid, thymidine, amino acids, acylcarnitines in blood, and urinary organic acids (especially screening for 3-methylglutaconic acid). Urine amino acid analysis is recommended in mitochondrial tubulopathies. CSF metabolite analysis (lactate, pyruvate, amino acids, and 5-methyltetrahydrofolate) should be included in the presence of central nervous system disease. We also suggest a diagnostic strategy based on the mitochondrial disease criteria (MDC) scoring system in mitochondrial disease diagnostics; evaluating muscle-, neurologic-, and multisystem involvement, and the presence of metabolic markers and abnormal imaging. The consensus guideline encourages a primary genetic approach in diagnostics and only suggests a more invasive diagnostic approach with tissue biopsies (histology, OXPHOS measurements, etc.) after nonconclusive genetic testing.
    Keywords:  3MGA; Alanine; Ethylmalonic acid; Lactic acid; Metabolomics; Methylmalonic acid; Mitochondrial disease criteria; Pyruvate; Thymidine
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00012-9
  46. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00005-1. [Epub ahead of print]194 65-78
    Stroke-like episodes in adult mitochondrial disease.
    Yi Shiau Ng, Gráinne S Gorman.
      Stroke-like episode is a paroxysmal neurological manifestation which affects a specific group of patients with mitochondrial disease. Focal-onset seizures, encephalopathy, and visual disturbances are prominent findings associated with stroke-like episodes, with a predilection for the posterior cerebral cortex. The most common cause of stroke-like episodes is the m.3243A>G variant in MT-TL1 gene followed by recessive POLG variants. This chapter aims to review the definition of stroke-like episode and delineate the clinical phenomenology, neuroimaging and EEG findings typically seen in patients. In addition, several lines of evidence supporting neuronal hyper-excitability as the key mechanism of stroke-like episodes are discussed. The management of stroke-like episodes should focus on aggressive seizure management and treatment for concomitant complications such as intestinal pseudo-obstruction. There is no robust evidence to prove the efficacy of l-arginine for both acute and prophylactic settings. Progressive brain atrophy and dementia are the sequalae of recurrent stroke-like episode, and the underlying genotype in part predicts prognosis.
    Keywords:  MELAS; Neuronal hyper-excitability; POLG; Seizures; Status epilepticus; m.3243A>G
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00005-1
  47. Methods Mol Biol. 2023 ;2615 173-188
    Localization of Mitochondrial Nucleoids by Transmission Electron Microscopy Using the Transgenic Expression of the Mitochondrial Helicase Twinkle and APEX2.
    David Pla-Martín, Felix Babatz, Astrid C Schauss.
      Reminiscent of their evolutionary origin, mitochondria contain their own genome (mtDNA) compacted into the mitochondrial chromosome or nucleoid (mt-nucleoid). Many mitochondrial disorders are characterized by disruption of mt-nucleoids, either by direct mutation of genes involved in mtDNA organization or by interfering with other vital proteins for mitochondrial function. Thus, changes in mt-nucleoid morphology, distribution, and structure are a common feature in many human diseases and can be exploited as an indicator of cellular fitness. Electron microscopy provides the highest possible resolution that can be achieved, delivering spatial and structural information about all cellular structures. Recently, the ascorbate peroxidase APEX2 has been used to increase transmission electron microscopy (TEM) contrast by inducing diaminobenzidine (DAB) precipitation. DAB has the ability to accumulate osmium during classical EM sample preparation and, due to its high electron density, provides strong contrast for TEM. Among the nucleoid proteins, the mitochondrial helicase Twinkle fused with APEX2 has been successfully used to target mt-nucleoids, providing a tool to visualize these subcellular structures with high contrast and with the resolution of an electron microscope. In the presence of H2O2, APEX2 catalyzes the polymerization of DAB, generating a brown precipitate that can be visualized in specific regions of the mitochondrial matrix. Here, we provide a detailed protocol to generate murine cell lines expressing a transgenic variant of Twinkle, suitable to target and visualize mt-nucleoids. We also describe all the necessary steps to validate the cell lines prior to electron microscopy imaging and offer examples of anticipated results.
    Keywords:  APEX2; Mitochondria; Nucleoid; TEM
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_13
  48. Cureus. 2023 Jan;15(1): e33963
    A Case Report on Pearson Syndrome With Emphasis on Genetic Screening in Patients Presenting With Sideroblastic Anemia and Lactic Acidosis.
    Celeste Shoeleh, Umberto M Donato, Andrew Galligan, Julie Vitko.
      Pearson marrow-pancreas syndrome is a rare multisystem mitochondrial disease that is a result of defective oxidative phosphorylation caused by mitochondrial DNA mutations. The average prognosis of infants diagnosed with this disease is death within four years of age. The disease often carries an atypical presentation during the neonatal period causing this rare syndrome to be frequently misdiagnosed. The current report details the diagnosis of Pearson syndrome in a three-month-old male with a history of pancytopenia.
    Keywords:  anemia; genetic screening; mitochondrial disorders; sideroblastic anemia; “pancytopenia”
    DOI:  https://doi.org/10.7759/cureus.33963
  49. Methods Mol Biol. 2023 ;2615 107-117
    Measurement of Nucleoid Size Using STED Microscopy.
    Elisa Motori.
      Mitochondria are equipped with their own DNA (mtDNA), which is packed into structures termed nucleoids . While nucleoids can be visualized in situ by fluorescence microscopy , the advent of super-resolution microscopy , and in particular of stimulated emission depletion (STED), has recently enabled the visualization of nucleoids at sub-diffraction resolution. Super-resolution microscopy has proved an invaluable tool for addressing fundamental questions in mitochondrial biology. In this chapter I describe how to achieve efficient labeling of mtDNA and how to quantify nucleoid diameter using an automated approach in fixed cultured cells by STED microscopy .
    Keywords:  Fluorescence microscopy; ImageJ; Immunocytochemistry; Mitochondrial DNA; Nucleoid; STED; Stimulated emission depletion microscopy
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_9
  50. Methods Mol Biol. 2023 ;2615 31-40
    Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Purification of mtDNA from Mammalian Cells.
    Andrew M Shaw, Payam A Gammage.
      Direct analysis of mtDNA using PCR-free methods is limited by the presence of persistent, contaminating nucleic acids originating from the nuclear genome, even following stringent mitochondrial isolations. Here we describe a method developed in our laboratory that couples existing, commercially available mtDNA isolation protocols with exonuclease treatment and size exclusion chromatography (DIFSEC). This protocol produces highly enriched mtDNA extracts from small-scale cell culture, with near-undetectable nuclear DNA contamination.
    Keywords:  DNA; Gel filtration; Purification; Size exclusion chromatography; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_3
  51. Sci Adv. 2023 Feb 24. 9(8): eade7864
    Mitochondrial phosphatidylethanolamine modulates UCP1 to promote brown adipose thermogenesis.
    Jordan M Johnson, Alek D Peterlin, Enrique Balderas, Elahu G Sustarsic, J Alan Maschek, Marisa J Lang, Alejandro Jara-Ramos, Vanja Panic, Jeffrey T Morgan, Claudio J Villanueva, Alejandro Sanchez, Jared Rutter, Irfan J Lodhi, James E Cox, Kelsey H Fisher-Wellman, Dipayan Chaudhuri, Zachary Gerhart-Hines, Katsuhiko Funai.
      Thermogenesis by uncoupling protein 1 (UCP1) is one of the primary mechanisms by which brown adipose tissue (BAT) increases energy expenditure. UCP1 resides in the inner mitochondrial membrane (IMM), where it dissipates membrane potential independent of adenosine triphosphate (ATP) synthase. Here, we provide evidence that phosphatidylethanolamine (PE) modulates UCP1-dependent proton conductance across the IMM to modulate thermogenesis. Mitochondrial lipidomic analyses revealed PE as a signature molecule whose abundance bidirectionally responds to changes in thermogenic burden. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) made mice cold intolerant and insensitive to β3 adrenergic receptor agonist-induced increase in whole-body oxygen consumption. High-resolution respirometry and fluorometry of BAT mitochondria showed that loss of mitochondrial PE specifically lowers UCP1-dependent respiration without compromising electron transfer efficiency or ATP synthesis. These findings were confirmed by a reduction in UCP1 proton current in PE-deficient mitoplasts. Thus, PE performs a previously unknown role as a temperature-responsive rheostat that regulates UCP1-dependent thermogenesis.
    DOI:  https://doi.org/10.1126/sciadv.ade7864
  52. Curr Protoc. 2023 Feb;3(2): e679
    Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ0 cells).
    Natalya Khozhukhar, Domenico Spadafora, Yelitza A Rodriguez Rodriguez, Rafik Fayzulin, Mikhail Alexeyev.
      To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR).
    Keywords:  cybrids; mtDNA; mtDNA copy number; mtDNA damage; ρ0 cells
    DOI:  https://doi.org/10.1002/cpz1.679
  53. Chemistry. 2023 Feb 20. e202204021
    Mitochondria-Targeted Gene Silencing Facilitated by Mito-CPDs.
    Wenjie Lang, Wei Tan, Bizhong Zhou, Yuli Zhuang, Bei Zhang, Linye Jiang, Shao Q Yao, Jingyan Ge.
      Mitochondrial DNA (mtDNA) plays an essential role in maintaining normal cellular activities. Its heteroplasmic mutations are known to cause various genetic diseases. Current genetic engineering strategies, such as those based on RNA interference (RNAi) and antisense technology, are difficult to genetically alter mtDNA, however, due to the inability of highly negatively charged oligonucleotides to translocate across the double-membrane mitochondria. We report herein a universal mitochondria-targeted gene-delivery approach by using cell-penetrating poly(disulfide)s (CPDs). Novel CPD-based mitochondrial transporters, named Mito-CPDs, were synthesized by using triphenylphosphonium (TPP)-fused propagating monomers containing either disulfide or diselenide backbones. Upon spontaneous complex formation with an oligonucleotide (single- or double-stranded), the resulting nanoscale Mito-CPD@Oligo exhibited excellent properties in common biological media. While the intracellular gene-delivery efficiency of these Mito-CPDs was comparable to that of commercial transfection agents, their unique mitochondria-localized properties enabled effective release of the loaded cargo inside these organelles. Subsequent mitochondrial delivery of siRNA and antisense oligonucleotides against suitable mtDNA-encoded proteins showed successful down-regulation of target protein expression, leading to profound effects on mitochondrial functions. Mito-CPDs thus provide a useful tool for future investigations of mitochondrial biology and treatment of mitochondria-related diseases.
    Keywords:  Antisense oligonucleotides; Cell-penetrating poly(disulfide)s; Mitochondrial functions; Mitochondrial gene; RNA interference
    DOI:  https://doi.org/10.1002/chem.202204021
  54. Mol Ther. 2023 Feb 18. pii: S1525-0016(23)00079-5. [Epub ahead of print]
    Delivery of mitochondria confers cardioprotection through mitochondria replenishment and metabolic compliance.
    Alian Zhang, Yangyang Liu, Jianan Pan, Francesca Pontanari, Andrew Chia-Hao Chang, Honghui Wang, Shuang Gao, Changqian Wang, Alex Cy Chang.
      Mitochondrial dysfunction is a hallmark of heart failure. Mitochondrial transplantation has been demonstrated to be able to restore heart function but its mechanism of action remains unresolved. Using an in-house optimized mitochondrial isolation method, we tested efficacy of mitochondria transplantation in two different heart failure models. First using the doxorubicin-induced heart failure model, we demonstrate that mitochondrial transplantation prior to doxorubicin challenge protects cardiac function in vivo, prevents myocardial apoptosis, but contraction improvement relies on the metabolic compatibility between transplanted mitochondria and treated cardiomyocytes. Second, using mutation driven dilated cardiomyopathic human induced pluripotent stem cell-derived cardiomyocyte model, we demonstrate that mitochondrial transplantation preferentially boosts contraction in ventricular myocytes. Last, using single cell RNASeq, we show that mitochondria transplantation boosts contractility in dystrophic cardiomyocytes with little transcriptomic alterations. Together, we provide evidence that mitochondria transplantation confers myocardial protection and may serve as a potential therapeutic option for heart failure.
    Keywords:  dilated cardiomyopathy; doxorubicin; iPSC; mitochondria delivery
    DOI:  https://doi.org/10.1016/j.ymthe.2023.02.016
  55. Methods Mol Biol. 2023 ;2615 153-172
    Identification of Proximity Interactors of Mammalian Nucleoid Proteins by BioID.
    Mari J Aaltonen, Hana Antonicka.
      Mitochondrial nucleoids are compact nucleoprotein complexes, in which mtDNA is located, replicated, and transcribed. Several proteomic approaches have been previously employed to identify nucleoid proteins; however, a consensus list of nucleoid-associated proteins has not been generated. Here we describe a proximity-biotinylation assay, BioID, which allows identification of proximity interactors of mitochondrial nucleoid proteins. It uses a promiscuous biotin ligase fused to a protein of interest which covalently attaches biotin to lysine residues of its proximal neighbors. Biotinylated proteins can be further enriched by a biotin-affinity purification and identified by mass-spectrometry. BioID can identify transient and weak interactions and can be used to identify changes in the interactions upon different cellular treatments, for different protein isoforms or for pathogenic variants.
    Keywords:  BioID; Nucleoid; Proximity interactors; Proximity-dependent biotinylation; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_12
  56. PLoS One. 2023 ;18(2): e0275703
    Mitochondrial DNA variants in a cohort from Argentina with suspected Leber's hereditary optic neuropathy (LHON).
    Paula I Buonfiglio, Sebastián Menazzi, Liliana Francipane, Vanesa Lotersztein, Verónica Ferreiro, Ana Belén Elgoyhen, Viviana Dalamón.
      The present study investigates the spectrum and analysis of mitochondrial DNA (mtDNA) variants associated with Leber hereditary optic neuropathy (LHON) in an Argentinean cohort, analyzing 3 LHON-associated mitochondrial genes. In 32% of the cases, molecular confirmation of the diagnosis could be established, due to the identification of disease-causing variants. A total of 54 variants were observed in a cohort of 100 patients tested with direct sequencing analysis. The frequent causative mutations m.11778G>A in MT-ND4, m.3460G>A in MT-ND1, and m.14484T>C in MT-ND6 were identified in 28% of the cases of our cohort. Secondary mutations in this Argentinean LHON cohort were m.11253T>C p.Ile165Thr in MT-ND4, identified in three patients (3/100, 3%) and m.3395A>G p.Tyr30Cys in MT-ND1, in one of the patients studied (1%). This study shows, for the first time, the analysis of mtDNA variants in patients with a probable diagnosis of LHON in Argentina. Standard molecular methods are an effective first approach in order to achieve genetic diagnosis of the disease, leaving NGS tests for those patients with negative results.
    DOI:  https://doi.org/10.1371/journal.pone.0275703
  57. Cell Chem Biol. 2023 Feb 17. pii: S2451-9456(23)00033-8. [Epub ahead of print]
    Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction.
    Jun Yang, Peihao Chen, Yu Cao, Shanshan Liu, Wei Wang, Lin Li, Jiaojiao Li, Zhaodi Jiang, Yan Ma, She Chen, Sanduo Zheng, Xiangbing Qi, Hui Jiang.
      Mitochondrial fission is critical for mitochondrial dynamics and homeostasis. The dynamin superfamily GTPase DRP1 is recruited by three functionally redundant receptors, MFF, MiD49, and MiD51, to mitochondria to drive fission. Here, we exploit high-content live-cell imaging to screen for mitochondrial fission inhibitors and have developed a covalent compound, mitochondrial division inhibitor (MIDI). MIDI treatment potently blocks mitochondrial fragmentation induced by mitochondrial toxins and restores mitochondrial morphology in fusion-defective cells carrying pathogenic mitofusin and OPA1 mutations. Mechanistically, MIDI does not affect DRP1 tetramerization nor DRP1 GTPase activity but does block DRP1 recruitment to mitochondria. Subsequent biochemical and cellular characterizations reveal an unexpected mechanism that MIDI targets DRP1 interaction with multiple receptors via covalent interaction with DRP1-C367. Taken together, beyond developing a potent mitochondrial fission inhibitor that profoundly impacts mitochondrial morphogenesis, our study establishes proof of concept for developing protein-protein interaction inhibitors targeting DRP1.
    Keywords:  DRP1 inhibitor; MFF; MIDI; MiD49/51; OPA1; mitochondrial dynamics; mitochondrial fission; mitofusin
    DOI:  https://doi.org/10.1016/j.chembiol.2023.02.002
  58. Genes (Basel). 2023 Jan 17. pii: 246. [Epub ahead of print]14(2):
    Involvement of Mitochondrial Dysfunction in FOXG1 Syndrome.
    Victoria A Bjerregaard, Amanda M Levy, Mille S Batz, Ravina Salehi, Mathis Hildonen, Trine B Hammer, Rikke S Møller, Claus Desler, Zeynep Tümer.
      FOXG1 (Forkhead box g1) syndrome is a neurodevelopmental disorder caused by a defective transcription factor, FOXG1, important for normal brain development and function. As FOXG1 syndrome and mitochondrial disorders have shared symptoms and FOXG1 regulates mitochondrial function, we investigated whether defective FOXG1 leads to mitochondrial dysfunction in five individuals with FOXG1 variants compared to controls (n = 6). We observed a significant decrease in mitochondrial content and adenosine triphosphate (ATP) levels and morphological changes in mitochondrial network in the fibroblasts of affected individuals, indicating involvement of mitochondrial dysfunction in FOXG1 syndrome pathogenesis. Further investigations are warranted to elucidate how FOXG1 deficiency impairs mitochondrial homeostasis.
    Keywords:  FOXG1 syndrome; mitochondrial dysfunction; mitochondrial homeostasis; mitochondrial morphology; mitochondrial respiratory capacity; neurodevelopmental disorders
    DOI:  https://doi.org/10.3390/genes14020246
  59. Proc Natl Acad Sci U S A. 2023 Feb 28. 120(9): e2216810120
    The SLC25A47 locus controls gluconeogenesis and energy expenditure.
    Jin-Seon Yook, Zachary H Taxin, Bo Yuan, Satoshi Oikawa, Christopher Auger, Beste Mutlu, Pere Puigserver, Sheng Hui, Shingo Kajimura.
      Mitochondria provide essential metabolites and adenosine triphosphate (ATP) for the regulation of energy homeostasis. For instance, liver mitochondria are a vital source of gluconeogenic precursors under a fasted state. However, the regulatory mechanisms at the level of mitochondrial membrane transport are not fully understood. Here, we report that a liver-specific mitochondrial inner-membrane carrier SLC25A47 is required for hepatic gluconeogenesis and energy homeostasis. Genome-wide association studies found significant associations between SLC25A47 and fasting glucose, HbA1c, and cholesterol levels in humans. In mice, we demonstrated that liver-specific depletion of SLC25A47 impaired hepatic gluconeogenesis selectively from lactate, while significantly enhancing whole-body energy expenditure and the hepatic expression of FGF21. These metabolic changes were not a consequence of general liver dysfunction because acute SLC25A47 depletion in adult mice was sufficient to enhance hepatic FGF21 production, pyruvate tolerance, and insulin tolerance independent of liver damage and mitochondrial dysfunction. Mechanistically, SLC25A47 depletion leads to impaired hepatic pyruvate flux and malate accumulation in the mitochondria, thereby restricting hepatic gluconeogenesis. Together, the present study identified a crucial node in the liver mitochondria that regulates fasting-induced gluconeogenesis and energy homeostasis.
    Keywords:  bioenergetics; metabolism; mitochondria; obesity; type 2 diabetes
    DOI:  https://doi.org/10.1073/pnas.2216810120
  60. Intern Med. 2023 Feb 22.
    A Patient with Mitochondrial Disease on Dialysis with Long-term Follow-up of Cardiomyopathy: An Autopsy Case Report.
    Shoko Ochiai, Hiroko Inagaki, Shuichi Hisanaga, Hiroyuki Tanaka, Masao Kikuchi, Shouichi Fujimoto.
      A 59-year-old man developed diabetes at 24 years old and underwent hemodialysis at 42 years old. At 54 years old, cardiac dysfunction with left ventricular hypertrophy was detected, followed by complete atrioventricular block at 57 years old. The patient was diagnosed with mitochondrial disease based on a myocardial biopsy and the presence of a mitochondrial DNA mutation (3243A>G). He died of septic shock at 59 years old, and an autopsy confirmed mitochondrial cardiomyopathy. If progressive cardiac hypertrophy and conduction disturbances are observed in patients with diabetes mellitus on long-term hemodialysis, mitochondrial disease needs to be considered.
    Keywords:  hemodialysis; maternally inherited diabetes with deafness; mitochondrial cardiomyopathy; mitochondrial disease
    DOI:  https://doi.org/10.2169/internalmedicine.0656-22
  61. Methods Mol Biol. 2023 ;2615 191-201
    In Vitro Assays of TWINKLE Function.
    Jay P Uhler, Ulrika Alexandersson, Maria Falkenberg.
      TWINKLE is an essential helicase that unwinds the duplex mitochondrial genome during DNA replication. In vitro assays using purified recombinant forms of the protein have been an instrumental tool for gaining mechanistic insights about TWINKLE and its function at the replication fork. Here we present methods to probe the helicase and ATPase activities of TWINKLE. For the helicase assay, TWINKLE is incubated with a radiolabeled oligonucleotide annealed to an M13mp18 single-stranded DNA template. TWINKLE will displace the oligonucleotide, which is then visualized by gel electrophoresis and autoradiography. To measure the ATPase activity of TWINKLE, a colorimetric assay is used, which quantifies the release of phosphate upon ATP hydrolysis by TWINKLE.
    Keywords:  DNA helicase; In vitro; Mitochondria; Replication; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_14
  62. bioRxiv. 2023 Feb 13. pii: 2023.02.11.528148. [Epub ahead of print]
    The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction.
    Wenjing Liang, Rachel Y Diao, Justin M Quiles, Rita H Najor, Liguo Chi, Benjamin P Woodall, Leonardo J Leon, Jason Duran, David M Cauvi, Antonio De Maio, Eric D Adler, Ã Sa B Gustafsson.
      Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.
    DOI:  https://doi.org/10.1101/2023.02.11.528148
  63. Antioxidants (Basel). 2023 Jan 18. pii: 221. [Epub ahead of print]12(2):
    Mitochondrial Complex I, a Possible Sensible Site of cAMP Pathway in Aging.
    Anna Signorile, Domenico De Rasmo.
      In mammals during aging, reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, cause oxidative damage of macromolecules leading to respiratory chain dysfunction, which in turn increases ROS mitochondrial production. Many efforts have been made to understand the role of oxidative stress in aging and age-related diseases. The complex I of the mitochondrial respiratory chain is the major source of ROS production and its dysfunctions have been associated with several forms of neurodegeneration, other common human diseases and aging. Complex I-ROS production and complex I content have been proposed as the major determinants for longevity. The cAMP signal has a role in the regulation of complex I activity and the decrease of ROS production. In the last years, an increasing number of studies have attempted to activate cAMP signaling to treat age-related diseases associated with mitochondrial dysfunctions and ROS production. This idea comes from a long-line of studies showing a main role of cAMP signal in the memory consolidation mechanism and in the regulation of mitochondrial functions. Here, we discuss several evidences on the possible connection between complex I and cAMP pathway in the aging process.
    Keywords:  aging; cAMP; complex I; mitochondria; signaling
    DOI:  https://doi.org/10.3390/antiox12020221
  64. Diabetes Metab J. 2023 Feb 24.
    Mitochondrial-Encoded Peptide MOTS-c, Diabetes, and Aging-Related Diseases.
    Byung Soo Kong, Changhan Lee, Young Min Cho.
      Mitochondria are complex metabolic organelles with manifold pathophysiological implications in diabetes. Currently published mitochondrial-encoded peptides, which are expressed from the mitochondrial open reading frame of the 12S ribosomal RNA type-c (MOTS-c), 16S rRNA (humanin and short humanin like peptide 1-6 [SHLP1-6]), or small human mitochondrial open reading frame over serine tRNA (SHMOOSE) are associated with regulation of cellular metabolism and insulin action in age-related diseases, such as type 2 diabetes mellitus. This review focuses mainly on recent advances in MOTS-c research with regards to diabetes, including both type 1 and type 2. The emerging understanding of MOTS-c in diabetes may provide insight into the development of new therapies for diabetes and other age or senescence-related diseases.
    Keywords:  Aging; Diabetes mellitus, type 2; Intracellular signaling peptides and proteins; Mitochondria
    DOI:  https://doi.org/10.4093/dmj.2022.0333
  65. GigaByte. 2022 ;2022 gigabyte70
    svaRetro and svaNUMT: modular packages for annotating retrotransposed transcripts and nuclear integration of mitochondrial DNA in genome sequencing data.
    Ruining Dong, Daniel Cameron, Justin Bedo, Anthony T Papenfuss.
      Nuclear integration of mitochondrial genomes and retrocopied transcript insertion are biologically important but often-overlooked aspects of structural variant (SV) annotation. While tools for their detection exist, these typically rely on reanalysis of primary data using specialised detectors rather than leveraging calls from general purpose structural variant callers. Such reanalysis potentially leads to additional computational expense and does not take advantage of advances in general purpose structural variant calling. Here, we present svaRetro and svaNUMT; R packages that provide functions for annotating novel genomic events, such as nonreference retrocopied transcripts and nuclear integration of mitochondrial DNA. The packages were developed to work within the Bioconductor framework. We evaluate the performance of these packages to detect events using simulations and public benchmarking datasets, and annotate processed transcripts in a public structural variant database. svaRetro and svaNUMT provide modular, SV-caller agnostic tools for downstream annotation of structural variant calls.
    DOI:  https://doi.org/10.46471/gigabyte.70
  66. J Mol Med (Berl). 2023 Feb 19.
    Nicotinamide riboside kinase-2 regulates metabolic adaptation in the ischemic heart.
    Hezlin Marzook, Anamika Gupta, Dhanendra Tomar, Mohamed A Saleh, Kiran Patil, Mohammad H Semreen, Rifat Hamoudi, Nelson C Soares, Rizwan Qaisar, Firdos Ahmad.
      Ischemia-induced metabolic remodeling plays a critical role in the pathogenesis of adverse cardiac remodeling and heart failure however, the underlying molecular mechanism is largely unknown. Here, we assess the potential roles of nicotinamide riboside kinase-2 (NRK-2), a muscle-specific protein, in ischemia-induced metabolic switch and heart failure through employing transcriptomic and metabolomic approaches in ischemic NRK-2 knockout mice. The investigations revealed NRK-2 as a novel regulator of several metabolic processes in the ischemic heart. Cardiac metabolism and mitochondrial function and fibrosis were identified as top dysregulated cellular processes in the KO hearts post-MI. Several genes linked to mitochondrial function, metabolism, and cardiomyocyte structural proteins were severely downregulated in the ischemic NRK-2 KO hearts. Analysis revealed significantly upregulated ECM-related pathways which was accompanied by the upregulation of several key cell signaling pathways including SMAD, MAPK, cGMP, integrin, and Akt in the KO heart post-MI. Metabolomic studies identified profound upregulation of metabolites mevalonic acid, 3,4-dihydroxyphenylglycol, 2-penylbutyric acid, and uridine. However, other metabolites stearic acid, 8,11,14-eicosatrienoic acid, and 2-pyrrolidinone were significantly downregulated in the ischemic KO hearts. Taken together, these findings suggest that NRK-2 promotes metabolic adaptation in the ischemic heart. The aberrant metabolism in the ischemic NRK-2 KO heart is largely driven by dysregulated cGMP and Akt and mitochondrial pathways. KEY MESSAGES: Post-myocardial infarction metabolic switch critically regulates the pathogenesis of adverse cardiac remodeling and heart failure. Here, we report NRK-2 as a novel regulator of several cellular processes including metabolism and mitochondrial function post-MI. NRK-2 deficiency leads to downregulation of genes important for mitochondrial pathway, metabolism, and cardiomyocyte structural proteins in the ischemic heart. It was accompanied by upregulation of several key cell signaling pathways including SMAD, MAPK, cGMP, integrin, and Akt and dysregulation of numerous metabolites essential for cardiac bioenergetics. Taken together, these findings suggest that NRK-2 is critical for metabolic adaptation of the ischemic heart.
    Keywords:  MIBP; Metabolic adaptation; Mitochondrial dysfunction; Myocardial infarction; NMRK2; Transcriptomic
    DOI:  https://doi.org/10.1007/s00109-023-02296-6
  67. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00015-4. [Epub ahead of print]194 43-63
    Leigh syndrome.
    Shamima Rahman.
      Leigh syndrome, or subacute necrotizing encephalomyelopathy, was initially recognized as a neuropathological entity in 1951. Bilateral symmetrical lesions, typically extending from the basal ganglia and thalamus through brainstem structures to the posterior columns of the spinal cord, are characterized microscopically by capillary proliferation, gliosis, severe neuronal loss, and relative preservation of astrocytes. Leigh syndrome is a pan-ethnic disorder usually with onset in infancy or early childhood, but late-onset forms occur, including in adult life. Over the last six decades it has emerged that this complex neurodegenerative disorder encompasses more than 100 separate monogenic disorders associated with enormous clinical and biochemical heterogeneity. This chapter discusses clinical, biochemical and neuropathological aspects of the disorder, and postulated pathomechanisms. Known genetic causes, including defects of 16 mitochondrial DNA (mtDNA) genes and approaching 100 nuclear genes, are categorized into disorders of subunits and assembly factors of the five oxidative phosphorylation enzymes, disorders of pyruvate metabolism and vitamin and cofactor transport and metabolism, disorders of mtDNA maintenance, and defects of mitochondrial gene expression, protein quality control, lipid remodeling, dynamics, and toxicity. An approach to diagnosis is presented, together with known treatable causes and an overview of current supportive management options and emerging therapies on the horizon.
    Keywords:  Diagnosis; Genetics; History; Leigh syndrome; Neuropathology; Pathomechanisms; Subacute necrotizing encephalomyelopathy; Treatment
    DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00015-4
  68. Curr Opin Gastroenterol. 2023 Mar 01. 39(2): 125-128
    Carnitine derivatives beyond fatigue: an update.
    Michele Malaguarnera, Vito Emanuele Catania, Mariano Malaguarnera.
       PURPOSE OF REVIEW: Carnitine is an essential micronutrient that transfer long-chain fatty acids from the cytoplasm into the mitochondrial matrix for the β-oxidation. Carnitine is also needed for the mitochondrial efflux of acyl groups in the cases wherein substrate oxidation exceeds energy demands.
    RECENT FINDINGS: Carnitine deficiency can affect the oxidation of free fatty acids in the mitochondria resulting in the aggregation of lipids in the cytoplasm instead of entering the citric acid cycle. The aggregation leads a lack of energy, acetyl coenzyme A accumulation in the mitochondria and cytotoxic production.
    SUMMARY: Carnitine and its derivatives show great clinical therapeutic effect without significant side effects.
    DOI:  https://doi.org/10.1097/MOG.0000000000000906
  69. Biology (Basel). 2023 Jan 28. pii: 198. [Epub ahead of print]12(2):
    Exploiting Multi-Omics Profiling and Systems Biology to Investigate Functions of TOMM34.
    Ekaterina V Poverennaya, Mikhail A Pyatnitskiy, Georgii V Dolgalev, Viktoria A Arzumanian, Olga I Kiseleva, Ilya Yu Kurbatov, Leonid K Kurbatov, Igor V Vakhrushev, Daniil D Romashin, Yan S Kim, Elena A Ponomarenko.
      Although modern biology is now in the post-genomic era with vastly increased access to high-quality data, the set of human genes with a known function remains far from complete. This is especially true for hundreds of mitochondria-associated genes, which are under-characterized and lack clear functional annotation. However, with the advent of multi-omics profiling methods coupled with systems biology algorithms, the cellular role of many such genes can be elucidated. Here, we report genes and pathways associated with TOMM34, Translocase of Outer Mitochondrial Membrane, which plays role in the mitochondrial protein import as a part of cytosolic complex together with Hsp70/Hsp90 and is upregulated in various cancers. We identified genes, proteins, and metabolites altered in TOMM34-/- HepG2 cells. To our knowledge, this is the first attempt to study the functional capacity of TOMM34 using a multi-omics strategy. We demonstrate that TOMM34 affects various processes including oxidative phosphorylation, citric acid cycle, metabolism of purine, and several amino acids. Besides the analysis of already known pathways, we utilized de novo network enrichment algorithm to extract novel perturbed subnetworks, thus obtaining evidence that TOMM34 potentially plays role in several other cellular processes, including NOTCH-, MAPK-, and STAT3-signaling. Collectively, our findings provide new insights into TOMM34's cellular functions.
    Keywords:  HepG2; TOMM34; de novo network enrichment; gene knockout; multi-omics profiling; pathway analysis
    DOI:  https://doi.org/10.3390/biology12020198
  70. Oxid Med Cell Longev. 2023 ;2023 4985726
    NAD+-Consuming Enzymes in Stem Cell Homeostasis.
    Xiuna Ji, Mingyue Zheng, Tao Yu, Jie Kang, Tingjun Fan, Bin Xu.
      Nicotinamide adenine dinucleotide (NAD+) is a coenzyme used in redox reactions, energy metabolism, and mitochondrial biogenesis. NAD+ is also required as a cofactor by nonredox NAD+-dependent enzymes. Hundreds of enzymes that consume NAD+ have been identified. The NAD+-consuming enzymes are involved in a variety of cellular processes such as signal transduction, DNA repair, cellular senescence, and stem cell (SC) homeostasis. In this review, we discussed how different types of NAD+-consuming enzymes regulate SC functions and summarized current research on the roles of the NAD+ consumers in SC homeostasis. We hope to provide a more global and integrative insight to the mechanism and intervention of SC homeostasis via the regulation of the NAD+-consuming enzymes.
    DOI:  https://doi.org/10.1155/2023/4985726
  71. Genome Biol. 2023 Feb 21. 24(1): 31
    FixItFelix: improving genomic analysis by fixing reference errors.
    Sairam Behera, Jonathon LeFaive, Peter Orchard, Medhat Mahmoud, Luis F Paulin, Jesse Farek, Daniela C Soto, Stephen C J Parker, Albert V Smith, Megan Y Dennis, Justin M Zook, Fritz J Sedlazeck.
      The current version of the human reference genome, GRCh38, contains a number of errors including 1.2 Mbp of falsely duplicated and 8.04 Mbp of collapsed regions. These errors impact the variant calling of 33 protein-coding genes, including 12 with medical relevance. Here, we present FixItFelix, an efficient remapping approach, together with a modified version of the GRCh38 reference genome that improves the subsequent analysis across these genes within minutes for an existing alignment file while maintaining the same coordinates. We showcase these improvements over multi-ethnic control samples, demonstrating improvements for population variant calling as well as eQTL studies.
    Keywords:  GIAB; GRCh38; INDEL; Medically relevant genes; Reference; Remapping; SNV; T2T-CHM13; Variant; eQTL
    DOI:  https://doi.org/10.1186/s13059-023-02863-7
  72. EMBO J. 2023 Feb 24. e108533
    Stress-dependent macromolecular crowding in the mitochondrial matrix.
    Elianne P Bulthuis, Cindy E J Dieteren, Jesper Bergmans, Job Berkhout, Jori A Wagenaars, Els M A van de Westerlo, Emina Podhumljak, Mark A Hink, Laura F B Hesp, Hannah S Rosa, Afshan N Malik, Mariska Kea-Te Lindert, Peter H G M Willems, Han J G E Gardeniers, Wouter K den Otter, Merel J W Adjobo-Hermans, Werner J H Koopman.
      Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.
    Keywords:  FRAP; chloramphenicol; diffusion; macromolecular crowding; mitochondria
    DOI:  https://doi.org/10.15252/embj.2021108533
  73. Methods Mol Biol. 2023 ;2615 267-280
    Quantitative Analysis of Nucleoside Triphosphate Pools in Mouse Muscle Using Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection.
    Sushma Sharma, Ziqing Kong, Shaodong Jia, Phong Tran, Anna Karin Nilsson, Andrei Chabes.
      Defects in deoxyribonucleoside triphosphate (dNTP) metabolism are associated with a number of mitochondrial DNA (mtDNA) depletion syndromes (MDS). These disorders affect the muscles, liver, and brain, and the concentrations of dNTPs in these tissues are already normally low and are, therefore, difficult to measure. Thus, information about the concentrations of dNTPs in tissues of healthy animals and animals with MDS are important for mechanistic studies of mtDNA replication, analysis of disease progression, and the development of therapeutic interventions. Here, we present a sensitive method for the simultaneous analysis of all four dNTPs as well as all four ribonucleoside triphosphates (NTPs) in mouse muscles using hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry. The simultaneous detection of NTPs allows them to be used as internal standards for the normalization of dNTP concentrations. The method can be applied for measuring dNTP and NTP pools in other tissues and organisms.
    Keywords:  Deoxyribonucleoside triphosphates; Differentiated tissues; Liquid chromatography; Triple quadrupole mass spectrometry; ZIC–HILIC
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_19
  74. J Clin Neuromuscul Dis. 2023 Mar 01. 24(3): 140-146
    Neuropathy, Ataxia, and Retinitis Pigmentosa Syndrome.
    Josef Finsterer.
       OBJECTIVES: To provide an overview about the phenotype, genotype, treatment, and outcome of neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome.
    METHODS: Systematic review by application of appropriate search terms.
    RESULTS: NARP syndrome is a syndromic mitochondrial disorder due to pathogenic variants in MT-ATP6. The canonical phenotypic features of NARP syndrome include proximal muscle weakness, axonal neuropathy, cerebellar ataxia, and retinitis pigmentosa. Noncanonical phenotypic features in NARP include epilepsy, cerebral or cerebellar atrophy, optic atrophy, cognitive impairment, dementia, sleep apnea syndrome, hearing impairment, renal insufficiency, and diabetes. So far, 10 pathogenic variants in MT-ATP6 have been associated with NARP, NARP-like syndrome, or NARP/maternally inherited Leigh overlap syndrome. Most pathogenic MT-ATP6 variants are missense, but a few truncating pathogenic variants have been reported. The most common variant responsible for NARP is the transversion m.8993T>G. Only symptomatic treatment for NARP syndrome is available. In most of the cases, patients die prematurely. Patients with late-onset NARP survive longer.
    CONCLUSIONS: NARP is a rare, syndromic, monogenic mitochondrial disorder due to pathogenic variants in MT-ATP6. The nervous system and the eyes are most commonly affected. Although only symptomatic treatment is available, the outcome is usually fair.
    DOI:  https://doi.org/10.1097/CND.0000000000000422
  75. Antioxidants (Basel). 2023 Feb 18. pii: 518. [Epub ahead of print]12(2):
    Mitochondrial Open Reading Frame of the 12S rRNA Type-c: Potential Therapeutic Candidate in Retinal Diseases.
    Zahra Mohtashami, Mithalesh Kumar Singh, Farid Thomaz Neto, Nasim Salimiaghdam, Hossein Hasanpour, M Cristina Kenney.
      Mitochondrial open reading frame of the 12S rRNA type-c (MOTS-c) is the most unearthed peptide encoded by mitochondrial DNA (mtDNA). It is an important regulator of the nuclear genome during times of stress because it promotes an adaptive stress response to maintain cellular homeostasis. Identifying MOTS-c specific binding partners may aid in deciphering the complex web of mitochondrial and nuclear-encoded signals. Mitochondrial damage and dysfunction have been linked to aging and the accelerated cell death associated with many types of retinal degenerations. Furthermore, research on MOTS-c ability to revive oxidatively stressed RPE cells has revealed a significant protective role for the molecule. Evidence suggests that senescent cells play a role in the development of age-related retinal disorders. This review examines the links between MOTS-c, mitochondria, and age-related diseases of the retina. Moreover, the untapped potential of MOTS-c as a treatment for glaucoma, diabetic retinopathy, and age-related macular degeneration is reviewed.
    Keywords:  MOTS-c; age-related macular degeneration; diabetic retinopathy; glaucoma; mitochondrial dysfunction
    DOI:  https://doi.org/10.3390/antiox12020518
  76. Dev Cell. 2023 Feb 09. pii: S1534-5807(23)00038-2. [Epub ahead of print]
    Rab8a as a mitochondrial receptor for lipid droplets in skeletal muscle.
    Qian Ouyang, Qiaoli Chen, Shunyuan Ke, Longfei Ding, Xinyu Yang, Ping Rong, Weikuan Feng, Ye Cao, Qi Wang, Min Li, Shu Su, Wen Wei, Minjun Liu, Jin Liu, Xu Zhang, John Zhong Li, Hong-Yu Wang, Shuai Chen.
      Dynamic interaction between lipid droplets (LDs) and mitochondria controls the mobilization of long-chain fatty acids (LCFAs) from LDs for mitochondrial β-oxidation in skeletal muscle in response to energy stress. However, little is known about the composition and regulation of the tethering complex mediating LD-mitochondrion interaction. Here, we identify Rab8a as a mitochondrial receptor for LDs forming the tethering complex with the LD-associated PLIN5 in skeletal muscle. In rat L6 skeletal muscle cells, the energy sensor AMPK increases the GTP-bound active Rab8a that promotes LD-mitochondrion interaction through binding to PLIN5 upon starvation. The assembly of the Rab8a-PLIN5 tethering complex also recruits the adipose triglyceride lipase (ATGL), which couples LCFA mobilization from LDs with its transfer into mitochondria for β-oxidation. Rab8a deficiency impairs fatty acid utilization and decreases endurance during exercise in a mouse model. These findings may help to elucidate the regulatory mechanisms underlying the beneficial effects of exercise on lipid homeostasis control.
    Keywords:  AMPK; Rab8a; energy metabolism; exercise; fatty acid oxidation; lipid droplets; mitochondria; organelle interaction; skeletal muscle
    DOI:  https://doi.org/10.1016/j.devcel.2023.01.007
  77. Methods Mol Biol. 2023 ;2615 121-137
    How to Quantify DNA Compaction by TFAM with Acoustic Force Spectroscopy and Total Internal Reflection Fluorescence Microscopy.
    Martial Martucci, Louis Debar, Siet van den Wildenberg, Geraldine Farge.
      Mitochondrial transcription factor A (TFAM) plays a key role in the organization and compaction of the mitochondrial genome. However, there are only a few simple and accessible methods available to observe and quantify TFAM-dependent DNA compaction. Acoustic Force Spectroscopy (AFS) is a straightforward single-molecule force spectroscopy technique. It allows one to track many individual protein-DNA complexes in parallel and to quantify their mechanical properties. Total internal reflection fluorescence (TIRF) microscopy is a high-throughput single-molecule technique that permits the real-time visualization of the dynamics of TFAM on DNA, parameters inaccessible with classical biochemistry tools. Here we describe, in detail, how to set up, perform, and analyze AFS and TIRF measurements to study DNA compaction by TFAM.
    Keywords:  Acoustic Force Spectroscopy (AFS); Single molecule biophysics; Total Internal Reflection Microscopy (TIRF); mitochondrial DNA compaction; mitochondrial Transcription Factor A (TFAM)
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_10
  78. Antioxidants (Basel). 2023 Feb 04. pii: 376. [Epub ahead of print]12(2):
    NAD+ Homeostasis and NAD+-Consuming Enzymes: Implications for Vascular Health.
    Roberto Campagna, Arianna Vignini.
      Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous metabolite that takes part in many key redox reactions. NAD+ biosynthesis and NAD+-consuming enzymes have been attracting markedly increasing interest since they have been demonstrated to be involved in several crucial biological pathways, impacting genes transcription, cellular signaling, and cell cycle regulation. As a consequence, many pathological conditions are associated with an impairment of intracellular NAD+ levels, directly or indirectly, which include cardiovascular diseases, obesity, neurodegenerative diseases, cancer, and aging. In this review, we describe the general pathways involved in the NAD+ biosynthesis starting from the different precursors, analyzing the actual state-of-art of the administration of NAD+ precursors or blocking NAD+-dependent enzymes as strategies to increase the intracellular NAD+ levels or to counteract the decline in NAD+ levels associated with ageing. Subsequently, we focus on the disease-related and age-related alterations of NAD+ homeostasis and NAD+-dependent enzymes in endothelium and the consequent vascular dysfunction, which significantly contributes to a wide group of pathological disorders.
    Keywords:  NAD+; NNMT; PARP1; SIRT1; endothelium; nicotinamide adenine dinucleotide; vascular health
    DOI:  https://doi.org/10.3390/antiox12020376
  79. Life Sci Alliance. 2023 May;pii: e202201825. [Epub ahead of print]6(5):
    Segregation of pathways leading to pexophagy.
    Francesco G Barone, Sylvie Urbé, Michael J Clague.
      Peroxisomes are organelles with key roles in metabolism including long-chain fatty acid production. Their metabolic functions overlap and interconnect with those of mitochondria, with which they share an overlapping but distinct proteome. Both organelles are degraded by selective autophagy processes termed pexophagy and mitophagy. Although mitophagy has received intense attention, the pathways linked to pexophagy and associated tools are less well developed. We have identified the neddylation inhibitor MLN4924 as a potent activator of pexophagy and show that this is mediated by the HIF1α-dependent up-regulation of BNIP3L/NIX, a known adaptor for mitophagy. We show that this pathway is distinct from pexophagy induced by the USP30 deubiquitylase inhibitor CMPD-39, for which we identify the adaptor NBR1 as a central player. Our work suggests a level of complexity to the regulation of peroxisome turnover that includes the capacity to coordinate with mitophagy, via NIX, which acts as a rheostat for both processes.
    DOI:  https://doi.org/10.26508/lsa.202201825
  80. Methods Mol Biol. 2023 ;2615 99-106
    In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy.
    John A Smolka, Samantha C Lewis.
      Metabolic labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) enables the selective labeling of DNA synthesis in live cells. Newly synthesized EdU-containing DNA can be covalently modified after extraction or in fixed cells using copper-catalyzed azide-alkyne cycloaddition "click chemistry" reactions, enabling bioconjugation to various substrates including fluorophores for imaging studies. While often used to study nuclear DNA replication, EdU labeling can also be leveraged to detect the synthesis of organellar DNA in the cytoplasm of Eukaryotic cells. In this chapter, we outline methods for the application of EdU labeling to the study of mitochondrial genome synthesis in fixed cultured human cells, using fluorescent labeling and superresolution light microscopy.
    Keywords:  Click chemistry; EdU; Metabolic labeling; Mitochondrial DNA; Superresolution microscopy
    DOI:  https://doi.org/10.1007/978-1-0716-2922-2_8
  81. Cell Genom. 2023 Feb 08. 3(2): 100246
    Remote visualization of large-scale genomic alignments for collaborative clinical research and diagnosis of rare diseases.
    Alberto Corvò, Leslie Matalonga, Dylan Spalding, Alexander Senf, Steven Laurie, Daniel Picó-Amador, Marcos Fernandez-Callejo, Ida Paramonov, Anna Foix Romero, Emilio Garcia-Rios, Jorge Izquierdo Ciges, Anand Mohan, Coline Thomas, Andres Felipe Silva Valencia, Csaba Halmagyi, Mallory Ann Freeberg, Ana Töpf, Rita Horvath, Gary Saunders, Ivo Gut, Thomas Keane, Davide Piscia, Sergi Beltran.
      The Solve-RD project objectives include solving undiagnosed rare diseases (RD) through collaborative research on shared genome-phenome datasets. The RD-Connect Genome-Phenome Analysis Platform (GPAP), for data collation and analysis, and the European Genome-Phenome Archive (EGA), for file storage, are two key components of the Solve-RD infrastructure. Clinical researchers can identify candidate genetic variants within the RD-Connect GPAP and, thanks to the developments presented here as part of joint ELIXIR activities, are able to remotely visualize the corresponding alignments stored at the EGA. The Global Alliance for Genomics and Health (GA4GH) htsget streaming application programming interface (API) is used to retrieve alignment slices, which are rendered by an integrated genome viewer (IGV) instance embedded in the GPAP. As a result, it is no longer necessary for over 11,000 datasets to download large alignment files to visualize them locally. This work highlights the advantages, from both the user and infrastructure perspectives, of implementing interoperability standards for establishing federated genomics data networks.
    Keywords:  data sharing; data visualization; diagnosis; exome analysis; federated infrastructures; genome analysis; rare diseases; remote data access; standards
    DOI:  https://doi.org/10.1016/j.xgen.2022.100246
  82. Int J Biochem Cell Biol. 2023 Feb 17. pii: S1357-2725(23)00030-4. [Epub ahead of print]157 106391
    From mitochondria to cells to humans: Targeting bioenergetics in aging and disease.
    Brandon J Berry, Gavin A Pharaoh, David J Marcinek.
      In vivo control over metabolism is at the cutting edge of biomedical research. The particulars of mitochondrial function are especially important to understand in vivo to progress metabolic therapies that will be relevant for diseases of aging. Understanding the differences between how mitochondria function in vitro versus in vivo will be a necessary challenge to overcome to achieve mitochondrial medicine. In this article we outline how discoveries in invertebrate models will be informative for understanding the basic biology of mitochondria to streamline translation to mammals and eventually to humans. Further, we highlight examples of how what is known about mitochondria in vitro is translatable to in vivo models and, in some cases, to human diseases.
    Keywords:  Aging; Bioenergetics; Membrane potential; Metabolism; Protonmotive force; Respiration
    DOI:  https://doi.org/10.1016/j.biocel.2023.106391
  83. Biochim Biophys Acta Bioenerg. 2023 Feb 20. pii: S0005-2728(23)00007-5. [Epub ahead of print]1864(2): 148961
    Disruption of mitochondrial bioenergetics and calcium homeostasis by phytanic acid in the heart: Potential relevance for the cardiomyopathy in Refsum disease.
    Ângela Beatriz Zemniaçak, Ana Cristina Roginski, Rafael Teixeira Ribeiro, Julia Gabrieli Bender, Rafael Aguiar Marschner, Simone Magagnin Wajner, Moacir Wajner, Alexandre Umpierrez Amaral.
      Refsum disease is an inherited peroxisomal disorder caused by severe deficiency of phytanoyl-CoA hydroxylase activity. Affected patients develop severe cardiomyopathy of poorly known pathogenesis that may lead to a fatal outcome. Since phytanic acid (Phyt) concentrations are highly increased in tissues of individuals with this disease, it is conceivable that this branched-chain fatty acid is cardiotoxic. The present study investigated whether Phyt (10-30 μM) could disturb important mitochondrial functions in rat heart mitochondria. We also determined the influence of Phyt (50-100 μM) on cell viability (MTT reduction) in cardiac cells (H9C2). Phyt markedly increased mitochondrial state 4 (resting) and decreased state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respirations, besides reducing the respiratory control ratio, ATP synthesis and the activities of the respiratory chain complexes I-III, II, and II-III. This fatty acid also reduced mitochondrial membrane potential and induced swelling in mitochondria supplemented by exogenous Ca2+, which were prevented by cyclosporin A alone or combined with ADP, suggesting the involvement of the mitochondrial permeability transition (MPT) pore opening. Mitochondrial NAD(P)H content and Ca2+ retention capacity were also decreased by Phyt in the presence of Ca2+. Finally, Phyt significantly reduced cellular viability (MTT reduction) in cultured cardiomyocytes. The present data indicate that Phyt, at concentrations found in the plasma of patients with Refsum disease, disrupts by multiple mechanisms mitochondrial bioenergetics and Ca2+ homeostasis, which could presumably be involved in the cardiomyopathy of this disease.
    Keywords:  Ca(2+) homeostasis; Mitochondrial bioenergetics; Mitochondrial permeability transition; Phytanic acid; Refsum disease
    DOI:  https://doi.org/10.1016/j.bbabio.2023.148961
  84. Hepatology. 2023 Feb 21.
    Hepatic mitochondrial NAD+ transporter SLC25A47 activates AMPKα mediating lipid metabolism and tumorigenesis.
    Lili Cheng, R N V Krishna Deepak, Guoqiang Wang, Ziyi Meng, Lei Tao, Mengqing Xie, Wenna Chi, Yuming Zhang, Mingming Yang, Yilie Liao, Ruiqun Chen, Yu Liang, Junyu Zhang, Yuedong Huang, Weihua Wang, Zhiying Guo, Yunfang Wang, Jiandie D Lin, Hao Fan, Ligong Chen.
       BACKGROUND AIMS: SLC25A47 was initially identified as mitochondrial hepatocellular carcinoma (HCC)-downregulated carrier protein, but its physiological functions and transport substrates are unknown. We aimed to investigate the physiological role of SLC25A47 in hepatic metabolism.
    APPROACH RESULTS: Treatment of hepatocytes with metformin found that metformin can transcriptionally activate the expression of Slc25a47, which is required for AMPKα phosphorylation. Slc25a47-deficient mice had increased hepatic lipid content, triglycerides and cholesterol levels, and we found that Slc25a47-deficiency suppressed AMPKα phosphorylation and led to an increased accumulation of nuclear SREBPs with elevated fatty acid and cholesterol biosynthetic activities. Conversely, when Slc25a47 was overexpressed in mouse liver, AMPKα was activated and resulted in inhibition of lipogenesis. Moreover, using a diethylnitrosamine (DEN)-induced mouse HCC model, we found that the deletion of Slc25a47 promoted HCC tumorigenesis and development through the activated mTOR cascade. Employing homology modeling of SLC25A47 and virtual screening of the human metabolome database, we demonstrated that NAD+ was an endogenous substrate for SLC25A47 and the activity of NAD+-dependent SIRT3 declined in Slc25a47-deficient mice, followed by inactivation of AMPKα.
    CONCLUSIONS: Our findings reveal that SLC25A47, a hepatocyte-specific mitochondrial NAD+ transporter, is one of the pharmacological targets of metformin and regulates lipid homeostasis through AMPKα, and may serve as a potential drug target for treating non-alcoholic fatty liver disease (NAFLD) and HCC.
    DOI:  https://doi.org/10.1097/HEP.0000000000000314
  85. Commun Biol. 2023 Feb 24. 6(1): 218
    Enhanced mitochondrial biogenesis promotes neuroprotection in human pluripotent stem cell derived retinal ganglion cells.
    Michelle Surma, Kavitha Anbarasu, Sayanta Dutta, Leonardo J Olivera Perez, Kang-Chieh Huang, Jason S Meyer, Arupratan Das.
      Mitochondrial dysfunctions are widely afflicted in central nervous system (CNS) disorders with minimal understanding on how to improve mitochondrial homeostasis to promote neuroprotection. Here we have used human stem cell differentiated retinal ganglion cells (hRGCs) of the CNS, which are highly sensitive towards mitochondrial dysfunctions due to their unique structure and function, to identify mechanisms for improving mitochondrial quality control (MQC). We show that hRGCs are efficient in maintaining mitochondrial homeostasis through rapid degradation and biogenesis of mitochondria under acute damage. Using a glaucomatous Optineurin mutant (E50K) stem cell line, we show that at basal level mutant hRGCs possess less mitochondrial mass and suffer mitochondrial swelling due to excess ATP production load. Activation of mitochondrial biogenesis through pharmacological inhibition of the Tank binding kinase 1 (TBK1) restores energy homeostasis, mitigates mitochondrial swelling with neuroprotection against acute mitochondrial damage for glaucomatous E50K hRGCs, revealing a novel neuroprotection mechanism.
    DOI:  https://doi.org/10.1038/s42003-023-04576-w
  86. Cell Signal. 2023 Feb 17. pii: S0898-6568(23)00045-1. [Epub ahead of print] 110631
    Parkin and mitochondrial signalling.
    Elizabeth M Connelly, Karling S Frankel, Gary S Shaw.
      Aging, toxic chemicals and changes to the cellular environment are sources of oxidative damage to mitochondria which contribute to neurodegenerative conditions including Parkinson's disease. To counteract this, cells have developed signalling mechanisms to identify and remove select proteins and unhealthy mitochondria to maintain homeostasis. Two important proteins that work in concert to control mitochondrial damage are the protein kinase PINK1 and the E3 ligase parkin. In response to oxidative stress, PINK1 phosphorylates ubiquitin present on proteins at the mitochondrial surface. This signals the translocation of parkin, accelerates further phosphorylation, and stimulates ubiquitination of outer mitochondrial membrane proteins such as Miro1/2 and Mfn1/2. The ubiquitination of these proteins is the key step needed to target them for degradation via the 26S proteasomal machinery or eliminate the entire organelle through mitophagy. This review highlights the signalling mechanisms used by PINK1 and parkin and presents several outstanding questions yet to be resolved.
    Keywords:  Mitophagy; PINK1; Phosphorylation; Protein structure; Ubiquitination
    DOI:  https://doi.org/10.1016/j.cellsig.2023.110631
  87. Handb Clin Neurol. 2023 ;pii: B978-0-323-85555-6.00007-2. [Epub ahead of print]193 53-66
    Disease mechanisms as Subtypes: Mitochondrial and bioenergetic dysfunction.
    Patricia Gonzalez-Rodriguez, Enrico Zampese, D James Surmeier.
      Parkinson disease (PD) is the second most common neurodegenerative disease in the world. Despite its enormous human and societal cost, there is no disease-modifying therapy for PD. This unmet medical need reflects our limited understanding of PD pathogenesis. One of the most important clues comes from the recognition that PD motor symptoms arises from the dysfunction and degeneration of a very select group of neurons in the brain. These neurons have a distinctive set of anatomic and physiologic traits that reflect their role in brain function. These traits elevate mitochondrial stress, potentially making them particularly vulnerable to age, as well as to genetic mutations and environmental toxins linked to PD incidence. In this chapter, the literature supporting this model is outlined, along with gaps in our knowledge base. The translational implications of this hypothesis are then discussed, with a focus on why disease-modification trials have failed to date and what this means for the development of new strategies for altering disease course.
    Keywords:  Aging; Calcium; Mitochondria; Neurodegeneration; Selective vulnerability
    DOI:  https://doi.org/10.1016/B978-0-323-85555-6.00007-2
  88. J Cell Biol. 2023 Mar 06. pii: e202206008. [Epub ahead of print]222(3):
    PERK recruits E-Syt1 at ER-mitochondria contacts for mitochondrial lipid transport and respiration.
    Maria Livia Sassano, Alexander R van Vliet, Ellen Vervoort, Sofie Van Eygen, Chris Van den Haute, Benjamin Pavie, Joris Roels, Johannes V Swinnen, Marco Spinazzi, Leen Moens, Kristina Casteels, Isabelle Meyts, Paolo Pinton, Saverio Marchi, Leila Rochin, Francesca Giordano, Blanca Felipe-Abrio, Patrizia Agostinis.
      The integrity of ER-mitochondria appositions ensures transfer of ions and phospholipids (PLs) between these organelles and exerts crucial effects on mitochondrial bioenergetics. Malfunctions within the ER-mitochondria contacts altering lipid trafficking homeostasis manifest in diverse pathologies, but the molecular effectors governing this process remain ill-defined. Here, we report that PERK promotes lipid trafficking at the ER-mitochondria contact sites (EMCS) through a non-conventional, unfolded protein response-independent, mechanism. PERK operates as an adaptor for the recruitment of the ER-plasma membrane tether and lipid transfer protein (LTP) Extended-Synaptotagmin 1 (E-Syt1), within the EMCS. In resting cells, the heterotypic E-Syt1-PERK interaction endorses transfer of PLs between the ER and mitochondria. Weakening the E-Syt1-PERK interaction or removing the lipid transfer SMP-domain of E-Syt1, compromises mitochondrial respiration. Our findings unravel E-Syt1 as a PERK interacting LTP and molecular component of the lipid trafficking machinery of the EMCS, which critically maintains mitochondrial homeostasis and fitness.
    DOI:  https://doi.org/10.1083/jcb.202206008
  89. Nat Genet. 2023 Feb 23.
    Network expansion of genetic associations defines a pleiotropy map of human cell biology.
    Inigo Barrio-Hernandez, Jeremy Schwartzentruber, Anjali Shrivastava, Noemi Del-Toro, Asier Gonzalez, Qian Zhang, Edward Mountjoy, Daniel Suveges, David Ochoa, Maya Ghoussaini, Glyn Bradley, Henning Hermjakob, Sandra Orchard, Ian Dunham, Carl A Anderson, Pablo Porras, Pedro Beltrao.
      Interacting proteins tend to have similar functions, influencing the same organismal traits. Interaction networks can be used to expand the list of candidate trait-associated genes from genome-wide association studies. Here, we performed network-based expansion of trait-associated genes for 1,002 human traits showing that this recovers known disease genes or drug targets. The similarity of network expansion scores identifies groups of traits likely to share an underlying genetic and biological process. We identified 73 pleiotropic gene modules linked to multiple traits, enriched in genes involved in processes such as protein ubiquitination and RNA processing. In contrast to gene deletion studies, pleiotropy as defined here captures specifically multicellular-related processes. We show examples of modules linked to human diseases enriched in genes with known pathogenic variants that can be used to map targets of approved drugs for repurposing. Finally, we illustrate the use of network expansion scores to study genes at inflammatory bowel disease genome-wide association study loci, and implicate inflammatory bowel disease-relevant genes with strong functional and genetic support.
    DOI:  https://doi.org/10.1038/s41588-023-01327-9
  90. JCI Insight. 2023 Feb 21. pii: e158429. [Epub ahead of print]
    Lipogenesis promotes mitochondrial fusion and maintains cancer stemness in human NSCLC.
    Zhen Liu, Jiaxin Lei, Tong Wu, Weijie Hu, Ming Zheng, Ying Wang, Jingdong Song, Hang Ruan, Lin Xu, Tao Ren, Wei Xu, Zhenke Wen.
      Cancer stem-like cells (CSCs) are critically involved in cancer metastasis and chemoresistance, acting as one major obstacle in clinical practice. While accumulating studies have implicated the metabolic reprogramming of CSCs, mitochondrial dynamics in such cells remain poorly understood. Here we pinpointed OPA1high with mitochondrial fusion as a metabolic feature of human lung CSCs, licensing their stem-like properties. Specifically, human lung CSCs exerted enhanced lipogenesis, inducing OPA1 expression via transcription factor SPDEF. In consequence, OPA1high promoted mitochondrial fusion and stemness of CSCs. Such lipogenesishigh, SPDEFhigh, and OPA1high metabolic adaptions were verified with primary CSCs from lung cancer patients. Accordingly, blocking lipogenesis and mitochondrial fusion efficiently impeded CSC expansion and growth of lung cancer patient-derived organoids. Together, lipogenesis regulates mitochondrial dynamics via OPA1 for controlling CSCs in human lung cancer.
    Keywords:  Lung cancer; Mitochondria; Oncology
    DOI:  https://doi.org/10.1172/jci.insight.158429
  91. Aging Dis. 2023 Feb 01. 14(1): 33-45
    Skeletal Muscle Mitochondrial Dysfunction in Chronic Obstructive Pulmonary Disease: Underlying Mechanisms and Physical Therapy Perspectives.
    Yingqi Wang, Peijun Li, Yuanyuan Cao, Chanjing Liu, Jie Wang, Weibing Wu.
      Skeletal muscle dysfunction (SMD) is a prevalent extrapulmonary complication and a significant independent prognostic factor in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial dysfunction is one of the core factors that damage structure and function in COPD skeletal muscle and is closely related to smoke exposure, hypoxia, and insufficient physical activity. The currently known phenotypes of mitochondrial dysfunction are reduced mitochondrial content and biogenesis, impaired activity of mitochondrial respiratory chain complexes, and increased mitochondrial reactive oxygen species production. Significant progress has been made in research on physical therapy (PT), which has broad prospects for treating the abovementioned potential mitochondrial-function changes in COPD skeletal muscle. In terms of specific types of PT, exercise therapy can directly act on mitochondria and improve COPD SMD by increasing mitochondrial density, regulating mitochondrial biogenesis, upregulating mitochondrial respiratory function, and reducing oxidative stress. However, improvements in mitochondrial-dysfunction phenotype in COPD skeletal muscle due to different exercise strategies are not entirely consistent. Therefore, based on the elucidation of this phenotype, in this study, we analyzed the effect of exercise on mitochondrial dysfunction in COPD skeletal muscle and the regulatory mechanism thereof. We also provided a theoretical basis for exercise programs to rehabilitate this condition.
    Keywords:  chronic obstructive pulmonary disease; exercise; mechanisms; mitochondrial dysfunction; skeletal muscle dysfunction
    DOI:  https://doi.org/10.14336/AD.2022.0603
  92. Br J Nutr. 2023 Feb 23. 1-36
    Intermittent fasting promotes adipocyte mitochondrial fusion through Sirt3-mediated deacetylation of Mdh2.
    Yizhou Li, Juntong Liang, Xin Tian, Qi Chen, Longbo Zhu, Han Wang, Zunhai Liu, Xulei Dai, Chenqi Bian, Chao Sun.
      Fat deposition and lipid metabolism are closely related to the morphology, structure and function of mitochondria. The morphology of mitochondria between fusion and fission processes is mainly regulated by protein posttranslational modification (PTM). Intermittent fasting (IF) promotes high expression of Sirtuin 3 (Sirt3) and induces mitochondrial fusion in high-fat diet (HFD)-fed mice. However, the mechanism by which Sirt3 participates in mitochondrial protein acetylation during IF to regulate mitochondrial fusion and fission dynamics remains unclear. This article demonstrates that IF promotes mitochondrial fusion and improves mitochondrial function in HFD mouse inguinal white adipose tissue (iWAT). Proteomic sequencing revealed that IF increased protein deacetylation levels in HFD mice and significantly increased Sirt3 mRNA and protein expression. After transfecting with Sirt3 overexpression or interference vectors into adipocytes, we found that Sirt3 promoted adipocyte mitochondrial fusion and improved mitochondrial function. Furthermore, Sirt3 regulates the JNK-FIS1 pathway by deacetylating malate dehydrogenase 2 (MDH2) to promote mitochondrial fusion. In summary, our study indicates that IF promotes mitochondrial fusion and improves mitochondrial function by upregulating the high expression of Sirt3 in HFD mice, promoting deacetylation of MDH2, and inhibiting the JNK-FIS1 pathway. This research provides theoretical support for studies related to energy limitation and animal lipid metabolism.
    Keywords:  Sirt3; deacetylation; intermittent fasting; mitochondrial dynamics; mitochondrial function
    DOI:  https://doi.org/10.1017/S000711452300048X
  93. Nature. 2023 Feb;614(7949): 798-800
    Innovative technologies crowd the short-read sequencing market.
    Michael Eisenstein.
      
    Keywords:  Biological techniques; Epigenetics; Genomics; Transcriptomics
    DOI:  https://doi.org/10.1038/d41586-023-00512-4
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