bims-mitmed Biomed News
on Mitochondrial medicine
Issue of 2023–02–26
fifty-five papers selected by
Dario Brunetti, Fondazione IRCCS Istituto Neurologico
  1. Cell lineage-specific mitochondrial resilience during mammalian organogenesis.
  2. TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.
  3. Mitochondrial optic neuropathies.
  4. Experimental therapy for mitochondrial diseases.
  5. Genetics of mitochondrial diseases: Current approaches for the molecular diagnosis.
  6. Mitochondrial disease in neurology-Past, present, and future.
  7. Reproductive options in mitochondrial disease.
  8. Complex neurological and multisystem presentations in mitochondrial disease.
  9. Peripheral neuropathy in mitochondrial disease.
  10. Mitochondrial Neurodegeneration: Lessons from Drosophila melanogaster Models.
  11. Clinical trials in mitochondrial diseases.
  12. Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.
  13. Cardiac Involvement in Mitochondrial Disorders.
  14. Studying Mitochondrial Nucleic Acid Synthesis Utilizing Intact Isolated Mitochondria.
  15. Blood biomarkers of mitochondrial disease-One for all or all for one?
  16. Creation of Mitochondrial Disease Models Using Mitochondrial DNA Editing.
  17. Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing.
  18. Neuroimaging in mitochondrial disease.
  19. Manipulation of Murine Mitochondrial DNA Heteroplasmy with mtZFNs.
  20. Investigation of oxidative phosphorylation activity and complex composition in mitochondrial disease.
  21. Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies.
  22. Genomic Strategies in Mitochondrial Diagnostics.
  23. Ataxia and spastic paraplegia in mitochondrial disease.
  24. Leigh syndrome.
  25. Progressive external ophthalmoplegia.
  26. Single Cell Analysis of Mitochondrial DNA Deletions.
  27. The role of programmed mitophagy in germline mitochondrial DNA quality control.
  28. Currently available therapies in mitochondrial disease.
  29. Stroke-like episodes in adult mitochondrial disease.
  30. Laboratory and metabolic investigations.
  31. Measurement of Nucleoid Size Using STED Microscopy.
  32. Functional Assessment of Mitochondrial DNA Maintenance by Depletion and Repopulation Using 2',3'-Dideoxycytidine in Cultured Cells.
  33. Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region.
  34. Role of human HSPE1 for OPA1 processing independent of HSPD1.
  35. Localization of Mitochondrial Nucleoids by Transmission Electron Microscopy Using the Transgenic Expression of the Mitochondrial Helicase Twinkle and APEX2.
  36. Reduced insulin signaling in neurons induces sex-specific health benefits.
  37. Mitochondria-Targeted Gene Silencing Facilitated by Mito-CPDs.
  38. Detection of UV-Induced Deletions in Mitochondrial DNA.
  39. Visualization of mtDNA Using FISH.
  40. In Vitro Assays of TWINKLE Function.
  41. The SLC25A47 locus controls gluconeogenesis and energy expenditure.
  42. Stress-dependent macromolecular crowding in the mitochondrial matrix.
  43. Quantitative Analysis of Nucleoside Triphosphate Pools in Mouse Muscle Using Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection.
  44. Stem Cell Therapies in Movement Disorders: Lessons from Clinical Trials.
  45. Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels.
  46. From mitochondria to cells to humans: Targeting bioenergetics in aging and disease.
  47. Mitochondrial Modulators: The Defender.
  48. Isolation of Functional Mitochondria and Pure mtDNA from Murine Tissues.
  49. Delivery of mitochondria confers cardioprotection through mitochondria replenishment and metabolic compliance.
  50. Marine Natural and Nature-Inspired Compounds Targeting Peroxisome Proliferator Activated Receptors (PPARs).
  51. Phenotypic Characterization of Male Tafazzin-Knockout Mice at 3, 6, and 12 Months of Age.
  52. Mitochondrial DNA variants in a cohort from Argentina with suspected Leber's hereditary optic neuropathy (LHON).
  53. Early-onset caloric restriction alleviates ageing-associated steatohepatitis in male mice via restoring mitochondrial homeostasis.
  54. Mitochondrial DNA copy number dynamics and associations with the prenatal environment from birth through adolescence in a population of Dominican and African American children.
  55. Essential role for albumin in preserving liver cells from TNFα-induced mitochondrial injury.
  1. 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
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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
  11. 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
  12. 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
  13. 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
  14. 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
  15. 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
  16. 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
  17. 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
  18. 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
  19. 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
  20. 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
  21. 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
  22. 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
  23. 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
  24. 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
  25. 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
  26. 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
  27. 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
  28. 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
  29. 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
  30. 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
  31. 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
  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. J Biol Chem. 2023 Feb 21. pii: S0021-9258(23)00189-8. [Epub ahead of print] 103057
    Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region.
    MorganA Gingerich, Jie Zhu, Biaoxin Chai, MichaelP Vincent, Nuli Xie, Vaibhav Sidarala, NicholasA Kotov, Debashish Sahu, DanielJ Klionsky, Santiago Schnell, ScottA Soleimanpour.
      CLEC16A is an E3 ubiquitin ligase that regulates mitochondrial quality control through mitophagy and is associated with over 20 human diseases. CLEC16A forms a complex with another E3 ligase, RNF41, and a ubiquitin-specific peptidase, USP8; however, regions that regulate CLEC16A activity or the assembly of the tripartite mitophagy regulatory complex are unknown. Here, we report that CLEC16A contains an internal intrinsically disordered protein region (IDPR) that is crucial for CLEC16A function and turnover. IDPRs lack a fixed secondary structure and possess emerging, yet still equivocal roles in protein stability, interactions, and enzymatic activity. We find that the internal IDPR of CLEC16A is crucial for its degradation. CLEC16A turnover was promoted by RNF41, which binds and acts upon the internal IDPR to destabilize CLEC16A. Loss of this internal IDPR also destabilized the ubiquitin-dependent tripartite CLEC16A-RNF41-USP8 complex. Finally, the presence of an internal IDPR within CLEC16A was confirmed using NMR and circular dichroism spectroscopy. Together, our studies reveal that an IDPR is essential to control the reciprocal regulatory balance between CLEC16A and RNF41, which could be targeted to improve mitochondrial health in disease.
    DOI:  https://doi.org/10.1016/j.jbc.2023.103057
  34. 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
  35. 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
  36. Sci Adv. 2023 Feb 22. 9(8): eade8137
    Reduced insulin signaling in neurons induces sex-specific health benefits.
    Maarouf Baghdadi, Tobias Nespital, Andrea Mesaros, Sandra Buschbaum, Dominic J Withers, Sebastian Grönke, Linda Partridge.
      Reduced activity of insulin/insulin-like growth factor signaling (IIS) extends health and life span in mammals. Loss of the insulin receptor substrate 1 (Irs1) gene increases survival in mice and causes tissue-specific changes in gene expression. However, the tissues underlying IIS-mediated longevity are currently unknown. Here, we measured survival and health span in mice lacking IRS1 specifically in liver, muscle, fat, and brain. Tissue-specific loss of IRS1 did not increase survival, suggesting that lack of IRS1 in more than one tissue is required for life-span extension. Loss of IRS1 in liver, muscle, and fat did not improve health. In contrast, loss of neuronal IRS1 increased energy expenditure, locomotion, and insulin sensitivity, specifically in old males. Neuronal loss of IRS1 also caused male-specific mitochondrial dysfunction, activation of Atf4, and metabolic adaptations consistent with an activated integrated stress response at old age. Thus, we identified a male-specific brain signature of aging in response to reduced IIS associated with improved health at old age.
    DOI:  https://doi.org/10.1126/sciadv.ade8137
  37. 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
  38. 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
  39. 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
  40. 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
  41. 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
  42. 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
  43. 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
  44. Biomedicines. 2023 Feb 09. pii: 505. [Epub ahead of print]11(2):
    Stem Cell Therapies in Movement Disorders: Lessons from Clinical Trials.
    Luca Marsili, Jennifer Sharma, Tiago Fleming Outeiro, Carlo Colosimo.
      Stem cell-based therapies (SCT) to treat neurodegenerative disorders have promise but clinical trials have only recently begun, and results are not expected for several years. While most SCTs largely lead to a symptomatic therapeutic effect by replacing lost cell types, there may also be disease-modifying therapeutic effects. In fact, SCT may complement a multi-drug, subtype-specific therapeutic approach, consistent with the idea of precision medicine, which matches molecular therapies to biological subtypes of disease. In this narrative review, we examine published and ongoing trials in SCT in Parkinson's Disease, atypical parkinsonian disorders, Huntington's disease, amyotrophic lateral sclerosis, and spinocerebellar ataxia in humans. We discuss the benefits and pitfalls of using this treatment approach within the spectrum of disease-modification efforts in neurodegenerative diseases. SCT may hold greater promise in the treatment of neurodegenerative disorders, but much research is required to determine the feasibility, safety, and efficacy of these complementary aims of therapeutic efforts.
    Keywords:  disease-modifying therapies; movement disorders; neurodegeneration; precision medicine; stem cell therapies
    DOI:  https://doi.org/10.3390/biomedicines11020505
  45. 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
  46. 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
  47. Biomolecules. 2023 Jan 24. pii: 226. [Epub ahead of print]13(2):
    Mitochondrial Modulators: The Defender.
    Emmanuel Makinde, Linlin Ma, George D Mellick, Yunjiang Feng.
      Mitochondria are widely considered the "power hub" of the cell because of their pivotal roles in energy metabolism and oxidative phosphorylation. However, beyond the production of ATP, which is the major source of chemical energy supply in eukaryotes, mitochondria are also central to calcium homeostasis, reactive oxygen species (ROS) balance, and cell apoptosis. The mitochondria also perform crucial multifaceted roles in biosynthetic pathways, serving as an important source of building blocks for the biosynthesis of fatty acid, cholesterol, amino acid, glucose, and heme. Since mitochondria play multiple vital roles in the cell, it is not surprising that disruption of mitochondrial function has been linked to a myriad of diseases, including neurodegenerative diseases, cancer, and metabolic disorders. In this review, we discuss the key physiological and pathological functions of mitochondria and present bioactive compounds with protective effects on the mitochondria and their mechanisms of action. We highlight promising compounds and existing difficulties limiting the therapeutic use of these compounds and potential solutions. We also provide insights and perspectives into future research windows on mitochondrial modulators.
    Keywords:  compounds; mitochondria diseases; mitochondria dysfunction; mitochondria health
    DOI:  https://doi.org/10.3390/biom13020226
  48. 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
  49. 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
  50. Mar Drugs. 2023 Jan 26. pii: 89. [Epub ahead of print]21(2):
    Marine Natural and Nature-Inspired Compounds Targeting Peroxisome Proliferator Activated Receptors (PPARs).
    Enrico D'Aniello, Pietro Amodeo, Rosa Maria Vitale.
      Peroxisome proliferator-activated receptors α, γ and β/δ (PPARα, PPARγ, and PPARβ/δ) are a family of ligand-activated transcriptional factors belonging to the superfamily of nuclear receptors regulating the expression of genes involved in lipid and carbohydrate metabolism, energy homeostasis, inflammation, and the immune response. For this reason, they represent attractive targets for the treatment of a variety of metabolic diseases and, more recently, for neurodegenerative disorders due to their emerging neuroprotective effects. The degree of activation, from partial to full, along with the selectivity toward the different isoforms, greatly affect the therapeutic efficacy and the safety profile of PPAR agonists. Thus, there is a high interest toward novel scaffolds with proper combinations of activity and selectivity. This review intends to provide an overview of the discovery, optimization, and structure-activity relationship studies on PPAR modulators from marine sources, along with the structural and computational studies that led to their identification and/or elucidation, and rationalization of their mechanisms of action.
    Keywords:  PPAR modulators; PPARs; drug discovery; marine natural products
    DOI:  https://doi.org/10.3390/md21020089
  51. Biomedicines. 2023 Feb 20. pii: 638. [Epub ahead of print]11(2):
    Phenotypic Characterization of Male Tafazzin-Knockout Mice at 3, 6, and 12 Months of Age.
    Michelle V Tomczewski, John Z Chan, Zurie E Campbell, Douglas Strathdee, Robin E Duncan.
      Barth syndrome (BTHS) is an X-linked mitochondrial disease caused by mutations in the gene encoding for tafazzin (TAZ), a key enzyme in the remodeling of cardiolipin. Mice with a germline deficiency in Taz have been generated (Taz-KO) but not yet fully characterized. We performed physiological assessments of 3-, 6-, and 12-month-old male Taz-KO mice, including measures of perinatal survival, growth, lifespan, gross anatomy, whole-body energy and substrate metabolism, glucose homeostasis, and exercise capacity. Taz-KO mice displayed reduced viability, with lower-than-expected numbers of mice recorded at 4 weeks of age, and a shortened lifespan due to disease progression. At all ages, Taz-KO mice had lower body weights compared with wild-type (Wt) littermates despite similar absolute food intakes. This finding was attributed to reduced adiposity and diminutive organs and tissues, including heart and skeletal muscles. Although there were no differences in basal levels of locomotion between age-matched genotypes, indirect calorimetry studies showed higher energy expenditure measures and respiratory exchange ratios in Taz-KO mice. At the youngest age, Taz-KO mice had comparable glucose tolerance and insulin action to Wt mice, but while these measures indicated metabolic impairments in Wt mice with advancing age that were likely associated with increasing adiposity, Taz-KO mice were protected. Comparisons across the three age-cohorts revealed a significant and more severe deterioration of exercise capacity in Taz-KO mice than in their Wt littermate controls. The Taz-KO mouse model faithfully recapitulates important aspects of BTHS, and thus provides an important new tool to investigate pathophysiological mechanisms and potential therapies.
    Keywords:  Barth syndrome; cardiolipin; energy metabolism; exercise capacity; mice; mitochondria; tafazzin
    DOI:  https://doi.org/10.3390/biomedicines11020638
  52. 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
  53. Biogerontology. 2023 Feb 20.
    Early-onset caloric restriction alleviates ageing-associated steatohepatitis in male mice via restoring mitochondrial homeostasis.
    Chun-Hsien Chiang, Sin-Jin Li, Yu-Han Lin, Pei-Yu Wang, Pu-Sheng Hsu, Shau-Ping Lin, Ting-Chia Chiang, Ching-Yi Chen.
      Non-alcoholic fatty liver disease is associated with ageing, and impaired mitochondrial homeostasis is the main cause for hepatic ageing. Caloric restriction (CR) is a promising therapeutic approach for fatty liver. The purpose of the present study was to investigate the possibility of early-onset CR in decelerating the progression of ageing-related steatohepatitis. The putative mechanism associated with mitochondria was further determined. C57BL/6 male mice at 8 weeks of age were randomly assigned to one of three treatments: Young-AL (AL, ad libitum), Aged-AL, or Aged-CR (60% intake of AL). Mice were sacrificed when they were 7 months old (Young) or 20 months old (Aged). Aged-AL mice displayed the greatest body weight, liver weight, and liver relative weight among treatments. Steatosis, lipid peroxidation, inflammation, and fibrosis coexisted in the aged liver. Mega mitochondria with short, randomly organized crista were noticed in the aged liver. The CR ameliorated these unfavourable outcomes. The level of hepatic ATP decreased with ageing, but this was reversed by CR. Ageing caused a decrease in mitochondrial-related protein expressions of respiratory chain complexes (NDUFB8 and SDHB) and fission (DRP1), but an increase in proteins related to mitochondrial biogenesis (TFAM), and fusion (MFN2). CR reversed the expression of these proteins in the aged liver. Both Aged-CR and Young-AL revealed a comparable pattern of protein expression. To summarize, this study demonstrated the potential of early-onset CR in preventing ageing-associated steatohepatitis, and maintaining mitochondrial functions may contribute to CR's protection during hepatic ageing.
    Keywords:  Ageing; Caloric restriction; Energy deficit; Mitochondrial functions; Steatohepatitis
    DOI:  https://doi.org/10.1007/s10522-023-10023-4
  54. Mitochondrion. 2023 Feb 16. pii: S1567-7249(23)00017-X. [Epub ahead of print]69 140-146
    Mitochondrial DNA copy number dynamics and associations with the prenatal environment from birth through adolescence in a population of Dominican and African American children.
    Allison Kupsco, Tessa R Bloomquist, Heng Hu, Aalekhya Reddam, Deliang Tang, Jeff Goldsmith, Andrew G Rundle, Andrea A Baccarelli, Julie B Herbstman.
      Mitochondrial DNA copy number (mtDNAcn) dynamics throughout childhood are poorly understood. We profiled mtDNAcn from birth through adolescence and evaluated how the prenatal environment influences mtDNAcn across childhood. Data were collected from children from New York City followed through 18 years. Using duplexed qRT-PCR, we quantified mtDNAcn relative to nuclear DNA in blood collected from the umbilical cord (n = 450), children aged 5-7 (n = 510), and adolescents aged 15-18 (n = 278). We examined mtDNAcn across childhood with linear mixed-effects models (LMM). Relative mtDNAcn was lowest at birth (mean ± SD: 0.67 ± 0.35) and increased in childhood (1.24 ± 0.50) then slightly declined in adolescence (1.13 ± 0.44). We observed no differences in mtDNAcn by sex or race/ethnicity. mtDNAcn was positively associated with prenatal environmental tobacco smoke exposure (0.077 [ 0.01, 0.14] change in relative mtDNAcn) but negatively associated with maternal completion of high school (-0.066 [-0.13, 0.00]), with the receipt of public assistance at birth (-0.074 [-0.14, -0.01]), and when mother born outside the U.S (-0.061 [-0.13, 0.003]). Infant birth outcomes were not associated with mtDNAcn. MtDNAcn levels were dynamic through childhood and associated with some prenatal factors, underscoring the need for the investigation of longitudinal mtDNAcn for human health research.
    Keywords:  Biomarkers; Children’s health; Mitochondria
    DOI:  https://doi.org/10.1016/j.mito.2023.02.008
  55. FASEB J. 2023 Mar;37(3): e22817
    Essential role for albumin in preserving liver cells from TNFα-induced mitochondrial injury.
    Marta Duran-Güell, Glòria Garrabou, Roger Flores-Costa, Mireia Casulleras, Cristina López-Vicario, Ingrid W Zhang, Judith Cantó-Santos, Bryan J Contreras, María B Sánchez-Rodríguez, Berta Romero-Grimaldo, Raquel Horrillo, Montserrat Costa, Vicente Arroyo, Joan Clària.
      Cytokine-induced inflammation and mitochondrial oxidative stress are key drivers of liver tissue injury. Here, we describe experiments modeling hepatic inflammatory conditions in which plasma leakage leads to large amounts of albumin to reach the interstitium and parenchymal surfaces to explore whether this protein plays a role in preserving hepatocyte mitochondria against the damaging actions of the cytotoxic cytokine tumor necrosis factor alpha (TNFα). Hepatocytes and precision-cut liver slices were cultured in the absence or presence of albumin in the cell media and then exposed to mitochondrial injury with the cytokine TNFα. The homeostatic role of albumin was also investigated in a mouse model of TNFα-mediated liver injury induced by lipopolysaccharide and D-galactosamine (LPS/D-gal). Mitochondrial ultrastructure, oxygen consumption, ATP and reactive oxygen species (ROS) generation, fatty acid β-oxidation (FAO), and metabolic fluxes were assessed by transmission electron microscopy (TEM), high-resolution respirometry, luminescence-fluorimetric-colorimetric assays and NADH/FADH2 production from various substrates, respectively. TEM analysis revealed that in the absence of albumin, hepatocytes were more susceptible to the damaging actions of TNFα and showed more round-shaped mitochondria with less intact cristae than hepatocytes cultured with albumin. In the presence of albumin in the cell media, hepatocytes also showed reduced mitochondrial ROS generation and FAO. The mitochondria protective actions of albumin against TNFα damage were associated with the restoration of a breakpoint between isocitrate and α-ketoglutarate in the tricarboxylic acid cycle and the upregulation of the antioxidant activating transcription factor 3 (ATF3). The involvement of ATF3 and its downstream targets was confirmed in vivo in mice with LPS/D-gal-induced liver injury, which showed increased hepatic glutathione levels, indicating a reduction in oxidative stress after albumin administration. These findings reveal that the albumin molecule is required for the effective protection of liver cells from mitochondrial oxidative stress induced by TNFα. These findings emphasize the importance of maintaining the albumin levels in the interstitial fluid within the normal range to protect the tissues against inflammatory injury in patients with recurrent hypoalbuminemia.
    Keywords:  hepatocytes; liver injury; mitochondrial dysfunction; mitochondrial oxidative stress; mitochondrial respiration; tricarboxylic acid cycle
    DOI:  https://doi.org/10.1096/fj.202201526R
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