bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023–05–14
fifty-nine papers selected by
Catalina Vasilescu, Helmholz Munich
  1. Sucrose Gradient Analysis of Human Mitochondrial Ribosomes and RNA.
  2. Discovery of Mitochondrial Ribosomes.
  3. Human Mitoribosome Profiling: A Re-engineered Approach Tailored to Study Mitochondrial Translation.
  4. Assembly of the Mitochondrial Translation Initiation Complex.
  5. Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation.
  6. Newly imported proteins in mitochondria are particularly sensitive to aggregation.
  7. Severe neonatal onset neuroregression with paroxysmal dystonia and apnoea: Expanding the phenotypic and genotypic spectrum of CARS2-related mitochondrial disease.
  8. C. elegans as a model to study mitochondrial biology and disease.
  9. Protein transport along the presequence pathway.
  10. Reconstitution of Mammalian Mitochondrial Translation System Capable of Long Polypeptide Synthesis.
  11. Mitoribosome Biogenesis.
  12. Biallelic variants in COQ7 cause distal hereditary motor neuropathy with upper motor neuron signs.
  13. A two-step mitochondrial import pathway couples the disulfide relay with matrix complex I biogenesis.
  14. FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.
  15. Fuel oxidation under the control of mitochondrial shape and dynamics.
  16. Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.
  17. Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles.
  18. Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria.
  19. Messenger RNA rescues medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts from patients and a murine model.
  20. Dead-Seq: Discovering Synthetic Lethal Interactions from Dead Cells Genomics.
  21. Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes.
  22. A draft human pangenome reference.
  23. TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.
  24. Translation in Mitochondrial Ribosomes.
  25. The ARG8m Reporter for the Study of Yeast Mitochondrial Translation.
  26. Cryo-EM for Structure Determination of Mitochondrial Ribosome Samples.
  27. Mitochondrial translational defect extends lifespan in C. elegans by activating UPRmt.
  28. Deficiency of the mitochondrial ribosomal subunit, MRPL50, causes autosomal recessive syndromic premature ovarian insufficiency.
  29. Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.
  30. Mitochondria-SR interaction and mitochondrial fusion/fission in the regulation of skeletal muscle metabolism.
  31. [Couples with risk of a child with a mitochondrial disease: wat are the reproductive options?]
  32. Insights into mitoribosomal biogenesis from recent structural studies.
  33. Mitochondrial Dysfunction as a Signaling Target for Therapeutic Intervention in Major Neurodegenerative Disease.
  34. Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation.
  35. Fission-Fueled Nucleoid Dispersion, a Novel Mitochondrial Metabolic Activation Mechanism.
  36. APOE expression and secretion are modulated by mitochondrial dysfunction.
  37. The trap of genetic tag: The importance of pathogenicity prediction tools in the correct interpretation of variants of uncertain significance in the era of high-throughput genome sequencing.
  38. Metabolic impact of genetic and chemical ADP/ATP carrier inhibition in renal proximal tubule epithelial cells.
  39. Mitochondrial Ion Channels.
  40. The mitochondrial calcium signaling, regulation, and cellular functions: A novel target for therapeutic medicine in neurological disorders.
  41. Mitochondrial citrate metabolism and efflux regulate BeWo differentiation.
  42. High-throughput deep learning variant effect prediction with Sequence UNET.
  43. [Optic atrophy in a patient with axonal Charcot-Marie-Tooth disease 2A2A due to MFN2 gene mutations].
  44. Skeletal Muscle Mitochondrial Dysfunction and Exercise Intolerance in Heart Failure With Preserved Ejection Fraction.
  45. Mitochondrial Myopathy in a 21-Year-Old Man Presenting With Bilateral Lower Extremity Weakness and Swelling.
  46. Secondary structure of the human mitochondrial genome affects formation of deletions.
  47. Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.
  48. Maintenance of mitochondrial homeostasis for Alzheimer's disease: Strategies and challenges.
  49. Reduced mitophagy is an early feature of NAFLD and liver-specific PARKIN knockout hastens the onset of steatosis, inflammation and fibrosis.
  50. Rapid Cryopurification of the Yeast Mitochondrial Ribosome.
  51. Mitochondria in human reproduction: novel paradigm in the onset of neurodegenerative disorders.
  52. In situ architecture of the ER-mitochondria encounter structure.
  53. The impact of metabolic stressors on mitochondrial homeostasis in a renal epithelial cell model of methylmalonic aciduria.
  54. Dodecamer assembly of a metazoan AAA+ chaperone couples substrate extraction to refolding.
  55. GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity.
  56. Mind the GAP(43) for mitochondria transfer to glioblastomas.
  57. Evolutionary genetics of the mitochondrial genome: insights from Drosophila.
  58. FGF21-FGFR1 controls mitochondrial homeostasis in cardiomyocytes by modulating the degradation of OPA1.
  59. Misrouting to mitochondria of renin carrying dominant mutations in the leader peptide or pro-segment.
  1. Methods Mol Biol. 2023 ;2661 101-117
    Sucrose Gradient Analysis of Human Mitochondrial Ribosomes and RNA.
    Kah Ying Ng, Brendan J Battersby.
      Faithful expression of the mitochondrial genome is required for the synthesis of the oxidative phosphorylation complexes and cell fitness. In humans, mitochondrial DNA (mtDNA) encodes 13 essential subunits of four oxidative phosphorylation complexes along with tRNAs and rRNAs needed for the translation of these proteins. Protein synthesis occurs on unique ribosomes within the organelle. Over the last decade, the revolution in genetic diagnostics has identified disruptions to the faithful synthesis of these 13 mitochondrial proteins as the largest group of inherited human mitochondrial pathologies. All of the molecular steps required for mitochondrial protein synthesis can be affected, from the genome to protein, including cotranslational quality control. Here, we describe methodologies for the biochemical separation of mitochondrial ribosomes from cultured human cells for RNA and protein analysis. Our method has been optimized to facilitate analysis for low-level sample material and thus does not require prior organelle enrichment.
    Keywords:  Human disease; Mitochondria; Mitochondrial disease; RNA; Ribosomes; Sucrose gradient
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_7
  2. Methods Mol Biol. 2023 ;2661 3-5
    Discovery of Mitochondrial Ribosomes.
    Thomas W O'Brien.
      In this introductory chapter, I will briefly describe how I came to discover the mammalian mitoribosome and will add a few notes on my contribution to the field.
    Keywords:  Human MRP genes; Human ribosome purification; Mitochondrial disease; Mitochondrial ribosomal proteins; Mitochondrial translation
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_1
  3. Methods Mol Biol. 2023 ;2661 257-280
    Human Mitoribosome Profiling: A Re-engineered Approach Tailored to Study Mitochondrial Translation.
    Iliana Soto, Mary Couvillion, L Stirling Churchman.
      To understand the human mitochondrial translation process, tools are required to dissect this system at a global scale. The mechanisms and regulation of translation in mitochondria are different from those in the cytosol, and mitochondrial ribosomes have distinct biochemical properties. In this chapter, we describe in detail the modifications we have made to the ribosome profiling approach to adapt it to the unique characteristics of the human mitochondrial ribosome. This approach maximizes the fraction of mitochondrial ribosomes recovered, providing a snapshot of the mitochondrial translation landscape with minimal bias. We also describe the use of mouse lysate as an internal spike-in control for normalization, allowing quantification of global changes in translation across samples. Finally, we outline the bioinformatic pipelines to process the raw reads and identify mitoribosome A sites in the absence of untranslated regions flanking open reading frames. This method offers a subcodon-resolution time-sensitive global approach to explore the mitochondrial translation process in human cells.
    Keywords:  Human mitochondrial translation; Mitochondrial ribosome profiling
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_15
  4. Methods Mol Biol. 2023 ;2661 217-232
    Assembly of the Mitochondrial Translation Initiation Complex.
    Cristina Remes, Minh Duc Nguyen, Henrik Spahr, Martin Ng, Clark Fritsch, Arpan Bhattacharya, Hong Li, Barry Cooperman, Joanna Rorbach.
      Mitochondria maintain their own translational machinery that is responsible for the synthesis of essential components of the oxidative phosphorylation system. The mammalian mitochondrial translation system differs significantly from its cytosolic and bacterial counterparts. Here, we describe detailed protocols for efficient in vitro reconstitution of the mammalian mitochondrial translation initiation complex, which can be further used for mechanistic analyses of different aspects of mitochondrial translation.
    Keywords:  Mitoribosome; Translation; Translation factors; Translation initiation; tRNA purification
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_13
  5. Methods Mol Biol. 2023 ;2661 119-132
    Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation.
    Andreas Aufschnaiter, Andreas Carlström, Martin Ott.
      Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners.
    Keywords:  Immunoprecipitation; Mitochondria; Mitoribosome; Sucrose cushion; Sucrose gradient; Yeast
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_8
  6. Acta Physiol (Oxf). 2023 May 12. e13985
    Newly imported proteins in mitochondria are particularly sensitive to aggregation.
    Carmela Vazquez-Calvo, Verena Kohler, Johanna L Höög, Sabrina Büttner, Martin Ott.
       AIM: A functional proteome is essential for life and maintained by protein quality control (PQC) systems in the cytosol and organelles. Protein aggregation is an indicator of a decline of PQC linked to aging and disease. Mitochondrial PQC is critical to maintain mitochondrial function and thus cellular fitness. How mitochondria handle aggregated proteins is not well understood. Here we tested how the metabolic status impacts on formation and clearance of aggregates within yeast mitochondria and assessed which proteins are particularly sensitive to denaturation.
    METHODS: Confocal microscopy, electron microscopy, immunoblotting and genetics were applied to assess mitochondrial aggregate handling in response to heat shock and ethanol, using the mitochondrial disaggregase Hsp78 as a marker for protein aggregates.
    RESULTS: We show that aggregates formed upon heat or ethanol stress with different dynamics depending on the metabolic state. While fermenting cells displayed numerous small aggregates that coalesced into one large foci that was resistant to clearance, respiring cells showed less aggregates and cleared these aggregates more efficiently. Acute inhibition of mitochondrial translation had no effect, while preventing protein import into mitochondria by inhibition of cytosolic translation prevented aggregate formation.
    CONCLUSION: Collectively, our data show that the metabolic state of the cells impacts the dynamics of aggregate formation and clearance, and that mainly newly imported and not yet assembled proteins are prone to form aggregates. Because mitochondrial functionality is crucial for cellular metabolism, these results highlight the importance of efficient protein biogenesis to maintain the mitochondrial proteome operational during metabolic adaptations and cellular stress.
    Keywords:  Ageing; Aggregates; Cellular stress; Hsp78; Metabolism; Mitochondria; Protein quality control; Proteostasis
    DOI:  https://doi.org/10.1111/apha.13985
  7. JIMD Rep. 2023 May;64(3): 223-232
    Severe neonatal onset neuroregression with paroxysmal dystonia and apnoea: Expanding the phenotypic and genotypic spectrum of CARS2-related mitochondrial disease.
    Jessie Poquérusse, Melinda Nolan, David R Thorburn, Johan L K Van Hove, Marisa W Friederich, Donald R Love, Juliet Taylor, Christopher A Powell, Michal Minczuk, Russell G Snell, Klaus Lehnert, Emma Glamuzina, Jessie C Jacobsen.
      Disorders of mitochondrial function are a collectively common group of genetic diseases in which deficits in core mitochondrial translation machinery, including aminoacyl tRNA synthetases, are key players. Biallelic variants in the CARS2 gene (NM_024537.4), which encodes the mitochondrial aminoacyl-tRNA synthetase for cysteine (CARS2, mt-aaRScys; MIM*612800), result in childhood onset epileptic encephalopathy and complex movement disorder with combined oxidative phosphorylation deficiency (MIM#616672). Prior to this report, eight unique pathogenic variants in the CARS2 gene had been reported in seven individuals. Here, we describe a male who presented in the third week of life with apnoea. He rapidly deteriorated with paroxysmal dystonic crises and apnoea resulting in death at 16 weeks. He had no evidence of seizure activity or multisystem disease and had normal brain imaging. Skeletal muscle biopsy revealed a combined disorder of oxidative phosphorylation. Whole-exome sequencing identified biallelic variants in the CARS2 gene: one novel (c.1478T>C, p.Phe493Ser), and one previously reported (c.655G>A, p.Ala219Thr; rs727505361). Northern blot analysis of RNA isolated from the patient's fibroblasts confirmed a clear defect in aminoacylation of the mitochondrial tRNA for cysteine (mt-tRNACys). To our knowledge, this is the earliest reported case of CARS2 deficiency with severe, early onset dystonia and apnoea, without epilepsy.
    Keywords:  CARS2; mitochondrial disorders; neurodevelopmental disorder; tRNA synthetases; whole‐exome sequencing
    DOI:  https://doi.org/10.1002/jmd2.12360
  8. Semin Cell Dev Biol. 2023 May 04. pii: S1084-9521(23)00101-5. [Epub ahead of print]
    C. elegans as a model to study mitochondrial biology and disease.
    Tessa Onraet, Steven Zuryn.
      Mitochondria perform a myriad of essential functions that ensure organismal homeostasis, including maintaining bioenergetic capacity, sensing and signalling the presence of pathogenic threats, and determining cell fate. Their function is highly dependent on mitochondrial quality control and the appropriate regulation of mitochondrial size, shape, and distribution during an entire lifetime, as well as their inheritance across generations. The roundworm Caenorhabditis elegans has emerged as an ideal model organism through which to study mitochondria. The remarkable conservation of mitochondrial biology has allowed C. elegans researchers to investigate complex processes that are challenging to study in higher organisms. In this review, we explore the key recent contributions of C. elegans to mitochondrial biology through the lens of mitochondrial dynamics, organellar removal, and mitochondrial inheritance, as well as their involvement in immune responses, various types of stress, and transgenerational signalling.
    Keywords:  Aging; Biogenesis; Fission; Fusion; Mitochondrial disease; Mitophagy; MtDNA; Neurodegeneration; Proteotoxicity; UPRmt
    DOI:  https://doi.org/10.1016/j.semcdb.2023.04.006
  9. Biol Chem. 2023 May 09.
    Protein transport along the presequence pathway.
    Abhijith Makki, Peter Rehling.
      Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via the presequence import pathway that involves the translocases of the outer and inner membranes - TOM and TIM23 complexes. In this article, we discuss how mitochondrial matrix and inner membrane precursor proteins are imported along the presequence pathway in Saccharomyces cerevisiae with a focus on the dynamics of the TIM23 complex, and further update with some of the key findings that advanced the field in the last few years.
    Keywords:  PAM; TIM23 complex; TOM complex; mitochondria; presequence translocase; protein translocation
    DOI:  https://doi.org/10.1515/hsz-2023-0133
  10. Methods Mol Biol. 2023 ;2661 233-255
    Reconstitution of Mammalian Mitochondrial Translation System Capable of Long Polypeptide Synthesis.
    Muhoon Lee, Nono Takeuchi-Tomita.
      Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. Here, we describe the in vitro reconstitution of the mammalian mitochondrial translation system, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a heterologous yeast tRNA mixture. The system is capable of translating leaderless mRNAs encoding model proteins, such as nanoluciferase with a molecular weight of 19 kDa, and is readily applicable for in vitro evaluations of mRNAs and nascent peptide chain sequences, as well as factors and small molecules that affect mitochondrial translation.
    Keywords:  55S ribosome; In vitro translation; Leaderless mRNA; Mammalian mitochondria; Reconstituted translation system
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_14
  11. Methods Mol Biol. 2023 ;2661 23-51
    Mitoribosome Biogenesis.
    J Conor Moran, Samuel Del'Olio, Austin Choi, Hui Zhong, Antoni Barrientos.
      Mitoribosome biogenesis is a complex and energetically costly process that involves RNA elements encoded in the mitochondrial genome and mitoribosomal proteins most frequently encoded in the nuclear genome. The process is catalyzed by extra-ribosomal proteins, nucleus-encoded assembly factors that act in all stages of the assembly process to coordinate the processing and maturation of ribosomal RNAs with the hierarchical association of ribosomal proteins. Biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided hints regarding their assembly. In this general concept chapter, we will briefly describe the current knowledge, mainly regarding the mammalian mitoribosome biogenesis pathway and factors involved, and will emphasize the biological sources and approaches that have been applied to advance the field.
    Keywords:  Mitochondrial disease; Mitochondrial ribosome; Mitochondrial translation; Mitoribosome assembly; OXPHOS deficiency
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_3
  12. Brain. 2023 May 12. pii: awad158. [Epub ahead of print]
    Biallelic variants in COQ7 cause distal hereditary motor neuropathy with upper motor neuron signs.
    Adriana P Rebelo, Pedro J Tomaselli, Jessica Medina, Ying Wang, Maike Dohrn, Eva Nyvltova, Matt Denzi, Mark Garrett, Sean Smith, Alan Pestronk, ChengCheng Li, Ariel Ruiz, Elizabeth Jacobs, Shawna M E Feely, Marcondes C França, Marcus V Gomes, Diogo Santos, Surinder Kumar, David B Lombard, Mario Saporta, Siegfried Hekimi, Antonio Barrientos, Conrad Weihl, Michael Shy, Wilson Marques, Stephan Zuchner.
      COQ7 encodes a hydroxylase responsible for the penultimate step of coenzyme Q10 (CoQ10) biosynthesis in mitochondria. CoQ10 is essential for multiple cellular functions, including mitochondrial oxidative phosphorylation, lipid metabolism, and reactive oxygen species homeostasis. Mutations in COQ7 have been previously associated with primary coenzyme Q10 deficiency, a clinically heterogeneous multisystemic mitochondrial disorder. We identified COQ7 biallelic variants in nine families diagnosed with distal hereditary motor neuropathy (dHMN) with upper neuron involvement, expending the clinical phenotype associated with defects in this gene. A recurrent p.Met1? change was identified in five families from Brazil with evidence of a founder effect. Fibroblasts isolated from patients revealed a substantial depletion of COQ7 protein levels, indicating protein instability leading to loss of enzyme function. HPLC assay showed that fibroblasts from patients had reduced levels of CoQ10, and abnormal accumulation of the biosynthetic precursor DMQ10. Accordingly, fibroblasts from patients displayed significantly decreased oxygen consumption rates in patients, suggesting mitochondrial respiration deficiency. iPSC-derived motor neurons from patient fibroblasts showed significantly increased levels of extracellular neurofilament light protein, indicating axonal degeneration. Our findings indicate a molecular pathway involving CoQ10 biosynthesis deficiency and mitochondrial dysfunction in patients with dHMN. Further studies will be important to evaluate the potential benefits of CoQ10 supplementation in the clinical outcome of the disease.
    Keywords:  Charcot-Marie-Tooth disease; CoQ10; hereditary motor neuropathy; mitochondria; motor neuron
    DOI:  https://doi.org/10.1093/brain/awad158
  13. J Cell Biol. 2023 Jul 03. pii: e202210019. [Epub ahead of print]222(7):
    A two-step mitochondrial import pathway couples the disulfide relay with matrix complex I biogenesis.
    Esra Peker, Konstantin Weiss, Jiyao Song, Christine Zarges, Sarah Gerlich, Volker Boehm, Aleksandra Trifunovic, Thomas Langer, Niels H Gehring, Thomas Becker, Jan Riemer.
      Mitochondria critically rely on protein import and its tight regulation. Here, we found that the complex I assembly factor NDUFAF8 follows a two-step import pathway linking IMS and matrix import systems. A weak targeting sequence drives TIM23-dependent NDUFAF8 matrix import, and en route, allows exposure to the IMS disulfide relay, which oxidizes NDUFAF8. Import is closely surveyed by proteases: YME1L prevents accumulation of excess NDUFAF8 in the IMS, while CLPP degrades reduced NDUFAF8 in the matrix. Therefore, NDUFAF8 can only fulfil its function in complex I biogenesis if both oxidation in the IMS and subsequent matrix import work efficiently. We propose that the two-step import pathway for NDUFAF8 allows integration of the activity of matrix complex I biogenesis pathways with the activity of the mitochondrial disulfide relay system in the IMS. Such coordination might not be limited to NDUFAF8 as we identified further proteins that can follow such a two-step import pathway.
    DOI:  https://doi.org/10.1083/jcb.202210019
  14. EMBO J. 2023 May 10. e112767
    FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.
    Giang Thanh Nguyen-Dien, Keri-Lyn Kozul, Yi Cui, Brendan Townsend, Prajakta Gosavi Kulkarni, Soo Siang Ooi, Antonio Marzio, Nissa Carrodus, Steven Zuryn, Michele Pagano, Robert G Parton, Michael Lazarou, S Sean Millard, Robert W Taylor, Brett M Collins, Mathew Jk Jones, Julia K Pagan.
      To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.
    Keywords:  BNIP3; FBXL4; NIX/BNIP3L; mitochondria; mitophagy
    DOI:  https://doi.org/10.15252/embj.2022112767
  15. EMBO J. 2023 May 08. e114129
    Fuel oxidation under the control of mitochondrial shape and dynamics.
    Erin L Seifert, György Hajnóczky.
      How mitochondrial shape and substrate-specific metabolism are related has been a difficult question to address. Here, new work by Ngo et al (2023) reports that mitochondrial shape-long versus fragmented-determines the activity of β-oxidation of long-chain fatty acids, supporting a novel role for mitochondrial fission products as β-oxidation hubs.
    DOI:  https://doi.org/10.15252/embj.2023114129
  16. Methods Mol Biol. 2023 ;2661 143-161
    Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.
    Lea Bertgen, Tamara Flohr, Johannes M Herrmann.
      The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as a powerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don'ts will enable beginners and experienced scientists to study the import and assembly of MRPs.
    Keywords:  Immunoprecipitation; Isolation of mitochondria; Mitochondrial protein import; Mitoribosomal protein (MRP); Sample preparation for mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_10
  17. Methods Mol Biol. 2023 ;2661 193-215
    Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles.
    Priyanka Maiti, Flavia Fontanesi.
      Mitochondria retain their own genome and translational apparatus that is highly specialized in the synthesis of a handful of proteins, essential components of the oxidative phosphorylation system. During evolution, the players and mechanisms involved in mitochondrial translation have acquired some unique features, which we have only partially disclosed. The study of the mitochondrial translation process has been historically hampered by the lack of an in vitro translational system and has largely relied on the analysis of the incorporation rate of radiolabeled amino acids into mitochondrial proteins in cellulo or in organello. In this chapter, we describe methods to monitor mitochondrial translation by labeling newly synthesized mitochondrial polypeptides with [S35]-methionine in either yeast or mammalian whole cells or isolated mitochondria.
    Keywords:  Human cells; Mitochondrial translation; Newly synthesized polypeptides; Protein synthesis; Pulse-chase labeling; Yeast; [S35]-methionine
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_12
  18. Methods Mol Biol. 2023 ;2661 317-328
    Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria.
    Danielle L Rudler, Stefan J Siira, Oliver Rackham, Aleksandra Filipovska.
      RNA-binding proteins and mitochondrial ribosomes have been found to be linchpins of mitochondrial gene expression in health and disease. The expanding repertoire of proteins that bind and regulate the mitochondrial transcriptome has necessitated the development of new tools and methods to examine their molecular functions. Next-generation sequencing technologies have advanced the RNA biology field through application of high-throughput methods to study RNA-protein interactions. Here we describe a digital RNase footprinting method to analyze protein and ribosome interactions with mitochondrially encoded transcripts that provides insight into their mechanisms and minimal binding sites. We provide details on RNase digestion and next-generation sequencing, along with computational analyses and visualization of the binding targets within the mitochondrial transcriptome.
    Keywords:  Bioinformatics; Footprinting; Mitoribosome; RNA-Seq; RNA-binding proteins; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_18
  19. Hum Mol Genet. 2023 May 10. pii: ddad076. [Epub ahead of print]
    Messenger RNA rescues medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts from patients and a murine model.
    Xue-Jun Zhao, A I-Walid Mohsen, Stephanie Mihalik, Keaton Solo, Shakuntala Basu, Ermal Aliu, Huifang Shi, Catherine Kochersberger, Anuradha Karunanidhi, Clinton Van't Land, Kimberly A Coughlan, Summar Siddiqui, Lisa M Rice, Shawn Hillier, Eleonora Guadagnin, Christine DeAntonis, Paloma H Giangrande, Paolo G V Martini, Jerry Vockley.
      Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm-/- mice. Administration of multiple intravenous doses of hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm-/- mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart, and skeletal muscle homogenates. Treated Acadm-/- mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in liver from treated Acadm-/- mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.
    Keywords:   Acadm−/− mouse; fatty acid oxidation disorders; fibroblasts; mRNA therapy; medium-chain acyl-CoA dehydrogenase deficiency; mitochondria
    DOI:  https://doi.org/10.1093/hmg/ddad076
  20. Methods Mol Biol. 2023 ;2661 329-342
    Dead-Seq: Discovering Synthetic Lethal Interactions from Dead Cells Genomics.
    Joan Blanco-Fernandez, Alexis A Jourdain.
      Pooled genetic screens have revolutionized the field of functional genomics, yet perturbations that decrease fitness, such as those leading to synthetic lethality, have remained difficult to quantify at the genomic level. We and colleagues previously developed "death screening," a protocol based on the purification of dead cells in genetic screens, and used it to identify a set of genes necessary for mitochondrial gene expression, translation, and oxidative phosphorylation (OXPHOS), thus offering new possibilities for the diagnosis of mitochondrial disorders. Here, we describe Dead-Seq, a refined protocol for death screening that is compatible with most pooled screening protocols, including genome-wide CRISPR/Cas9 screening. Dead-Seq converts negative-selection screens into positive-selection screens and generates high-quality data directly from dead cells, at limited sequencing costs.
    Keywords:  Annexin V; Apoptosis; Auxotrophy; Drop-out screen; Galactose; Genome-wide screening; MACS; Metabolism; Mitochondria; Mitochondrial translation; Necroptosis; ORFeome; RNAi; Synthetic lethality; Systems genetics; sgRNA; shRNA
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_19
  21. Methods Mol Biol. 2023 ;2661 303-316
    Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes.
    Christin A Albus, Rolando Berlinguer-Palmini, Zofia M Chrzanowska-Lightowlers, Robert N Lightowlers.
      High-resolution imaging has enabled scientists to explore the mitochondrial network at remarkable resolution. This has been exploited to help increase our knowledge of how mitochondrial gene expression is compartmentalized in cultured cells. Here, we provide detailed methodology to simultaneously visualize up to four components including mtDNA-encoded transcripts, submitochondrial marker proteins, mitoribosomal subunits, or core members of the translational apparatus using STED super-resolution nanoscopy.
    Keywords:  Mitochondrial mRNA; RNA in situ hybridization; STED super-resolution; Super-resolution nanoscopy
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_17
  22. Nature. 2023 May;617(7960): 312-324
    A draft human pangenome reference.
    Wen-Wei Liao, Mobin Asri, Jana Ebler, Daniel Doerr, Marina Haukness, Glenn Hickey, Shuangjia Lu, Julian K Lucas, Jean Monlong, Haley J Abel, Silvia Buonaiuto, Xian H Chang, Haoyu Cheng, Justin Chu, Vincenza Colonna, Jordan M Eizenga, Xiaowen Feng, Christian Fischer, Robert S Fulton, Shilpa Garg, Cristian Groza, Andrea Guarracino, William T Harvey, Simon Heumos, Kerstin Howe, Miten Jain, Tsung-Yu Lu, Charles Markello, Fergal J Martin, Matthew W Mitchell, Katherine M Munson, Moses Njagi Mwaniki, Adam M Novak, Hugh E Olsen, Trevor Pesout, David Porubsky, Pjotr Prins, Jonas A Sibbesen, Jouni Sirén, Chad Tomlinson, Flavia Villani, Mitchell R Vollger, Lucinda L Antonacci-Fulton, Gunjan Baid, Carl A Baker, Anastasiya Belyaeva, Konstantinos Billis, Andrew Carroll, Pi-Chuan Chang, Sarah Cody, Daniel E Cook, Robert M Cook-Deegan, Omar E Cornejo, Mark Diekhans, Peter Ebert, Susan Fairley, Olivier Fedrigo, Adam L Felsenfeld, Giulio Formenti, Adam Frankish, Yan Gao, Nanibaa' A Garrison, Carlos Garcia Giron, Richard E Green, Leanne Haggerty, Kendra Hoekzema, Thibaut Hourlier, Hanlee P Ji, Eimear E Kenny, Barbara A Koenig, Alexey Kolesnikov, Jan O Korbel, Jennifer Kordosky, Sergey Koren, HoJoon Lee, Alexandra P Lewis, Hugo Magalhães, Santiago Marco-Sola, Pierre Marijon, Ann McCartney, Jennifer McDaniel, Jacquelyn Mountcastle, Maria Nattestad, Sergey Nurk, Nathan D Olson, Alice B Popejoy, Daniela Puiu, Mikko Rautiainen, Allison A Regier, Arang Rhie, Samuel Sacco, Ashley D Sanders, Valerie A Schneider, Baergen I Schultz, Kishwar Shafin, Michael W Smith, Heidi J Sofia, Ahmad N Abou Tayoun, Françoise Thibaud-Nissen, Francesca Floriana Tricomi, Justin Wagner, Brian Walenz, Jonathan M D Wood, Aleksey V Zimin, Guillaume Bourque, Mark J P Chaisson, Paul Flicek, Adam M Phillippy, Justin M Zook, Evan E Eichler, David Haussler, Ting Wang, Erich D Jarvis, Karen H Miga, Erik Garrison, Tobias Marschall, Ira M Hall, Heng Li, Benedict Paten.
      Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals1. These assemblies cover more than 99% of the expected sequence in each genome and are more than 99% accurate at the structural and base pair levels. Based on alignments of the assemblies, we generate a draft pangenome that captures known variants and haplotypes and reveals new alleles at structurally complex loci. We also add 119 million base pairs of euchromatic polymorphic sequences and 1,115 gene duplications relative to the existing reference GRCh38. Roughly 90 million of the additional base pairs are derived from structural variation. Using our draft pangenome to analyse short-read data reduced small variant discovery errors by 34% and increased the number of structural variants detected per haplotype by 104% compared with GRCh38-based workflows, which enabled the typing of the vast majority of structural variant alleles per sample.
    DOI:  https://doi.org/10.1038/s41586-023-05896-x
  23. Cell Rep. 2023 Apr 30. pii: S2211-1247(23)00465-5. [Epub ahead of print] 112454
    TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.
    Shiori Akabane, Kiyona Watanabe, Hidetaka Kosako, Shun-Ichi Yamashita, Kohei Nishino, Masahiro Kato, Shiori Sekine, Tomotake Kanki, Noriyuki Matsuda, Toshiya Endo, Toshihiko Oka.
      PINK1 is activated by autophosphorylation and forms a high-molecular-weight complex, thereby initiating the selective removal of damaged mitochondria by autophagy. Other than translocase of the outer mitochondrial membrane complexes, members of PINK1-containing protein complexes remain obscure. By mass spectrometric analysis of PINK1 co-immunoprecipitates, we identify the inner membrane protein TIM23 as a component of the PINK1 complex. TIM23 downregulation decreases PINK1 levels and significantly delays autophosphorylation, indicating that TIM23 promotes PINK1 accumulation in response to depolarization. Moreover, inactivation of the mitochondrial protease OMA1 not only enhances PINK1 accumulation but also represses the reduction in PINK1 levels induced by TIM23 downregulation, suggesting that TIM23 facilitates PINK1 activation by safeguarding against degradation by OMA1. Indeed, deficiencies of pathogenic PINK1 mutants that fail to interact with TIM23 are partially restored by OMA1 inactivation. These findings indicate that TIM23 plays a distinct role in activating mitochondrial autophagy by protecting PINK1.
    Keywords:  CP: Cell biology; OMA1; PINK1; TIM23; mitochondrial quality control
    DOI:  https://doi.org/10.1016/j.celrep.2023.112454
  24. Methods Mol Biol. 2023 ;2661 53-72
    Translation in Mitochondrial Ribosomes.
    Zofia M Chrzanowska-Lightowlers, Robert N Lightowlers.
      Mitochondrial protein synthesis is essential for the life of aerobic eukaryotes. Without it, oxidative phosphorylation cannot be coupled. Evolution has shaped a battery of factors and machinery that are key to production of just a handful of critical proteins. In this general concept chapter, we attempt to briefly summarize our current knowledge of the overall process in mitochondria from a variety of species, breaking this down to the four parts of translation: initiation, elongation, termination, and recycling. Where appropriate, we highlight differences between species and emphasize gaps in our understanding. Excitingly, with the current revolution in cryoelectron microscopy and mitochondrial genome editing, it is highly likely that many of these gaps will be resolved in the near future. However, the absence of a faithful in vitro reconstituted system to study mitochondrial translation is still problematic.
    Keywords:  Elongation; Initiation; Mitochondria; Mitoribosomes; Protein synthesis; Recycling; Termination; Translation; mt-mRNAs
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_4
  25. Methods Mol Biol. 2023 ;2661 281-301
    The ARG8m Reporter for the Study of Yeast Mitochondrial Translation.
    Daniel Flores-Mireles, Yolanda Camacho-Villasana, Xochitl Pérez-Martínez.
      Mitochondrial translation is an intricate process involving both general and mRNA-specific factors. In addition, in the yeast Saccharomyces cerevisiae, translation of mitochondrial mRNAs is coupled to assembly of nascent polypeptides into the membrane. ARG8m is a reporter gene widely used to study the mechanisms of yeast mitochondrial translation. This reporter is a recodified gene that uses the mitochondrial genetic code and is inserted at the desired locus in the mitochondrial genome. After deletion of the endogenous nuclear gene, this reporter produces Arg8, an enzyme necessary for arginine biosynthesis. Since Arg8 is a soluble protein with no relation to oxidative phosphorylation, it is a reliable reporter to study mitochondrial mRNAs translation and dissect translation form assembly processes. In this chapter, we explain how to insert the ARG8m reporter in the desired spot in the mitochondrial DNA, how to analyze Arg8 synthesis inside mitochondria, and how to follow steady-state levels of the protein. We also explain how to use it to find spontaneous suppressors of translation defects.
    Keywords:  ARG8m; ATP synthase; Cytochrome c oxidase; Mitochondria; Mitochondrial DNA; Respiratory complexes; Suppressor; Translation; Yeast; bc1 complex
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_16
  26. Methods Mol Biol. 2023 ;2661 89-100
    Cryo-EM for Structure Determination of Mitochondrial Ribosome Samples.
    Hauke S Hillen.
      Single-particle cryoelectron microscopy (cryo-EM) allows structure determination of large macromolecular complexes from conformationally and compositionally heterogeneous mixtures of particles. This technique has been used to reveal the architecture of the mitochondrial ribosome and to visualize transient states that occur during the translation cycle or during mitoribosome biogenesis. Here, we outline an exemplary workflow for the analysis of single-particle cryo-EM data of human mitoribosome samples. In addition, we provide an example dataset which can be used for training purposes alongside the protocol.
    Keywords:  Mitochondrial ribosome; image processing; sample preparation; single-particle cryo-EM
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_6
  27. Redox Biol. 2023 May 07. pii: S2213-2317(23)00123-4. [Epub ahead of print]63 102722
    Mitochondrial translational defect extends lifespan in C. elegans by activating UPRmt.
    Miaomiao Guo, Xinhua Qiao, Yuanyuan Wang, Zi-Han Li, Chang Shi, Yun Chen, Lu Kang, Chang Chen, Xiao-Long Zhou.
      Aminoacyl-tRNA synthetases (aaRSs) are indispensable players in translation. Usually, two or three genes encode cytoplasmic and mitochondrial threonyl-tRNA synthetases (ThrRSs) in eukaryotes. Here, we reported that Caenorhabditis elegans harbors only one tars-1, generating cytoplasmic and mitochondrial ThrRSs via translational reinitiation. Mitochondrial tars-1 knockdown decreased mitochondrial tRNAThr charging and translation and caused pleotropic phenotypes of delayed development, decreased motor ability and prolonged lifespan, which could be rescued by replenishing mitochondrial tars-1. Mitochondrial tars-1 deficiency leads to compromised mitochondrial functions including the decrease in oxygen consumption rate, complex Ⅰ activity and the activation of the mitochondrial unfolded protein response (UPRmt), which contributes to longevity. Furthermore, deficiency of other eight mitochondrial aaRSs in C. elegans and five in mammal also caused activation of the UPRmt. In summary, we deciphered the mechanism of one tars-1, generating two aaRSs, and elucidated the biochemical features and physiological function of C. elegans tars-1. We further uncovered a conserved connection between mitochondrial translation deficiency and UPRmt.
    Keywords:  Aminoacyl-tRNA synthetases; Lifespan; Mitochondrial translation; UPR(mt); tars-1(ora1) Ⅱ/wt
    DOI:  https://doi.org/10.1016/j.redox.2023.102722
  28. Hum Genet. 2023 May 06.
    Deficiency of the mitochondrial ribosomal subunit, MRPL50, causes autosomal recessive syndromic premature ovarian insufficiency.
    Shabnam Bakhshalizadeh, Daniella H Hock, Nicole A Siddall, Brianna L Kline, Rajini Sreenivasan, Katrina M Bell, Franca Casagranda, Sadishkumar Kamalanathan, Jayaprakash Sahoo, Niya Narayanan, Dukhabandhu Naik, Varun Suryadevara, Alison G Compton, Sumudu S C Amarasekera, Ridam Kapoor, Sylvie Jaillard, Andrea Simpson, Gorjana Robevska, Jocelyn van den Bergen, Svenja Pachernegg, Katie L Ayers, David R Thorburn, David A Stroud, Gary R Hime, Andrew H Sinclair, Elena J Tucker.
      Premature ovarian insufficiency (POI) is a common cause of infertility in women, characterised by amenorrhea and elevated FSH under the age of 40 years. In some cases, POI is syndromic in association with other features such as sensorineural hearing loss in Perrault syndrome. POI is a heterogeneous disease with over 80 causative genes known so far; however, these explain only a minority of cases. Using whole-exome sequencing (WES), we identified a MRPL50 homozygous missense variant (c.335T > A; p.Val112Asp) shared by twin sisters presenting with POI, bilateral high-frequency sensorineural hearing loss, kidney and heart dysfunction. MRPL50 encodes a component of the large subunit of the mitochondrial ribosome. Using quantitative proteomics and western blot analysis on patient fibroblasts, we demonstrated a loss of MRPL50 protein and an associated destabilisation of the large subunit of the mitochondrial ribosome whilst the small subunit was preserved. The mitochondrial ribosome is responsible for the translation of subunits of the mitochondrial oxidative phosphorylation machinery, and we found patient fibroblasts have a mild but significant decrease in the abundance of mitochondrial complex I. These data support a biochemical phenotype associated with MRPL50 variants. We validated the association of MRPL50 with the clinical phenotype by knockdown/knockout of mRpL50 in Drosophila, which resulted abnormal ovarian development. In conclusion, we have shown that a MRPL50 missense variant destabilises the mitochondrial ribosome, leading to oxidative phosphorylation deficiency and syndromic POI, highlighting the importance of mitochondrial support in ovarian development and function.
    DOI:  https://doi.org/10.1007/s00439-023-02563-z
  29. Stem Cell Reports. 2023 May 09. pii: S2213-6711(23)00136-4. [Epub ahead of print]18(5): 1090-1106
    Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.
    Malgorzata Zatyka, Tatiana R Rosenstock, Congxin Sun, Adina M Palhegyi, Georgina W Hughes, Samuel Lara-Reyna, Dewi Astuti, Alessandro di Maio, Axel Sciauvaud, Miriam E Korsgen, Vesna Stanulovic, Gamze Kocak, Malgorzata Rak, Sandra Pourtoy-Brasselet, Katherine Winter, Thiago Varga, Margot Jarrige, Hélène Polvèche, Joao Correia, Eva-Maria Frickel, Maarten Hoogenkamp, Douglas G Ward, Laetitia Aubry, Timothy Barrett, Sovan Sarkar.
      Mitochondrial dysfunction involving mitochondria-associated ER membrane (MAM) dysregulation is implicated in the pathogenesis of late-onset neurodegenerative diseases, but understanding is limited for rare early-onset conditions. Loss of the MAM-resident protein WFS1 causes Wolfram syndrome (WS), a rare early-onset neurodegenerative disease that has been linked to mitochondrial abnormalities. Here we demonstrate mitochondrial dysfunction in human induced pluripotent stem cell-derived neuronal cells of WS patients. VDAC1 is identified to interact with WFS1, whereas loss of this interaction in WS cells could compromise mitochondrial function. Restoring WFS1 levels in WS cells reinstates WFS1-VDAC1 interaction, which correlates with an increase in MAMs and mitochondrial network that could positively affect mitochondrial function. Genetic rescue by WFS1 overexpression or pharmacological agents modulating mitochondrial function improves the viability and bioenergetics of WS neurons. Our data implicate a role of WFS1 in regulating mitochondrial functionality and highlight a therapeutic intervention for WS and related rare diseases with mitochondrial defects.
    Keywords:  Cyclosporin A; Human induced pluripotent stem cell-derived neurons; Mitochondria-associated ER membrane; Mitochondrial dysfunction; Mitochondrial membrane potential; MnTBAP; Neurodegeneration; VDAC1; WFS1; Wolfram syndrome
    DOI:  https://doi.org/10.1016/j.stemcr.2023.04.002
  30. Metabolism. 2023 May 08. pii: S0026-0495(23)00181-6. [Epub ahead of print] 155578
    Mitochondria-SR interaction and mitochondrial fusion/fission in the regulation of skeletal muscle metabolism.
    Mauricio Castro-Sepulveda, Rodrigo Fernández-Verdejo, Hermann Zbinden-Foncea, Jennifer Rieusset.
      Mitochondria-endoplasmic/sarcoplasmic reticulum (ER/SR) interaction and mitochondrial fusion/fission are critical processes that influence substrate oxidation. This narrative review summarizes the evidence on the effects of substrate availability on mitochondrial-SR interaction and mitochondria fusion/fission dynamics to modulate substrate oxidation in human skeletal muscle. Evidence shows that an increase in mitochondria-SR interaction and mitochondrial fusion are associated with elevated fatty acid oxidation. In contrast, a decrease in mitochondria-SR interaction and an increase in mitochondrial fission are associated with an elevated glycolytic activity. Based on the evidence reviewed, we postulate two hypotheses for the link between mitochondrial dynamics and insulin resistance in human skeletal muscle. First, glucose and fatty acid availability modifies mitochondria-SR interaction and mitochondrial fusion/fission to help the cell to adapt substrate oxidation appropriately. Individuals with an impaired response to these substrate challenges will accumulate lipid species and develop insulin resistance in skeletal muscle. Second, a chronically elevated substrate availability (e.g. overfeeding) increases mitochondrial production of reactive oxygen species and induced mitochondrial fission. This decreases fatty acid oxidation, thus leading to the accumulation of lipid species and insulin resistance in skeletal muscle. Altogether, we propose mitochondrial dynamics as a potential target for disturbances associated with low fatty acid oxidation.
    Keywords:  Fatty acid oxidation; Metabolic flexibility; Mitochondria dynamics; Mitochondria-associated membranes; Organelle dynamics
    DOI:  https://doi.org/10.1016/j.metabol.2023.155578
  31. Ned Tijdschr Geneeskd. 2023 May 10. pii: D7360. [Epub ahead of print]167
    [Couples with risk of a child with a mitochondrial disease: wat are the reproductive options?]
    Debby M E I Hellebrekers, Christine E M de Die-Smulders, Mirian C H Janssen.
      Mitochondrial diseases are the most common inborn errors of metabolism. These severe multisystem disorders cause serious morbidity and mortality. Generally no treatment is available. This underlines the importance of counseling about the reproductive options to prevent the transmission of mitochondrial disorders. The majority of mitochondrial disorders is caused by a defect in a nuclear gene, in which cases the standard reproductive options can be applied, such as prenatal diagnosis (PND) and preimplantation genetic testing (PGT). For mitochondrial disorders caused by a mitochondrial DNA (mtDNA) mutation, reproductive options are determined by the recurrence risk, requiring specific reproductive counseling. For de novomtDNA mutations and inherited mtDNA mutations with a low recurrence risk, PND is possible. In case of a moderate or higher recurrence risk, PGT is the best option. In case the risk of a healthy embryo is (very) low, mitochondrial replacement therapy (MRT) may be a possibility in the future.
  32. Trends Biochem Sci. 2023 May 09. pii: S0968-0004(23)00086-5. [Epub ahead of print]
    Insights into mitoribosomal biogenesis from recent structural studies.
    Anas Khawaja, Miriam Cipullo, Annika Krüger, Joanna Rorbach.
      The mitochondrial ribosome (mitoribosome) is a multicomponent machine that has unique structural features. Biogenesis of the human mitoribosome includes correct maturation and folding of the mitochondria-encoded RNA components (12S and 16S mt-rRNAs, and mt-tRNAVal) and their assembly together with 82 nucleus-encoded mitoribosomal proteins. This complex process requires the coordinated action of multiple assembly factors. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have provided detailed insights into the specific functions of several mitoribosome assembly factors and have defined their timing. In this review we summarize mitoribosomal small (mtSSU) and large subunit (mtLSU) biogenesis based on structural findings, and we discuss potential crosstalk between mtSSU and mtLSU assembly pathways as well as coordination between mitoribosome biogenesis and other processes involved in mitochondrial gene expression.
    Keywords:  assembly factors; mitochondrial ribosome; mitoribosome assembly; ribosomal RNA
    DOI:  https://doi.org/10.1016/j.tibs.2023.04.002
  33. Neurotox Res. 2023 May 10.
    Mitochondrial Dysfunction as a Signaling Target for Therapeutic Intervention in Major Neurodegenerative Disease.
    Shubhada V Mangrulkar, Nitu L Wankhede, Mayur B Kale, Aman B Upaganlawar, Brijesh G Taksande, Milind J Umekar, Md Khalid Anwer, Hamad Ghaleb Dailah, Syam Mohan, Tapan Behl.
      Neurodegenerative diseases (NDD) are incurable and the most prevalent cognitive and motor disorders of elderly. Mitochondria are essential for a wide range of cellular processes playing a pivotal role in a number of cellular functions like metabolism, intracellular signaling, apoptosis, and immunity. A plethora of evidence indicates the central role of mitochondrial functions in pathogenesis of many aging related NDD. Considering how mitochondria function in neurodegenerative diseases, oxidative stress, and mutations in mtDNA both contribute to aging. Many substantial reports suggested the involvement of numerous contributing factors including, mitochondrial dysfunction, oxidative stress, mitophagy, accumulation of somatic mtDNA mutations, compromised mitochondrial dynamics, and transport within axons in neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Amyotrophic Lateral Sclerosis. Therapies therefore target fundamental mitochondrial processes such as energy metabolism, free-radical generation, mitochondrial biogenesis, mitochondrial redox state, mitochondrial dynamics, mitochondrial protein synthesis, mitochondrial quality control, and metabolism hold great promise to develop pharmacological based therapies in NDD. By emphasizing the most efficient pharmacological strategies to target dysfunction of mitochondria in the treatment of neurodegenerative diseases, this review serves the scientific community engaged in translational medical science by focusing on the establishment of novel, mitochondria-targeted treatment strategies.
    Keywords:  Fission; Fusion; Mitochondrial dysfunction; Mitochondrial medicine; Mitochondrial quality control; Oxidative stress
    DOI:  https://doi.org/10.1007/s12640-023-00647-2
  34. Methods Mol Biol. 2023 ;2661 75-88
    Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation.
    Lena Thärichen, Robert Englmeier, Friedrich Förster.
      Cryoelectron tomography is a method to image biological samples three-dimensionally at molecular resolution. This modality provides insights into intracellular processes in their physiological settings. Obtaining a high-quality sample for cryoelectron tomography on mitochondria, however, can be challenging. In this chapter, we describe the crucial steps from sample preparation to data acquisition enabling studies of mitochondrial translation in situ by cryoelectron tomography. We provide detailed protocols for yeast and human mitochondria preparations yielding a high concentration of intact mitochondrial vesicles on cryo-EM grids. In addition, we describe a workflow for particle identification and spatial mapping in context of the organelle.
    Keywords:  Cryoelectron tomography; Mitochondria; Ribosome; Translation
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_5
  35. Mol Cells. 2023 May 31. 46(5): 278-280
    Fission-Fueled Nucleoid Dispersion, a Novel Mitochondrial Metabolic Activation Mechanism.
    Jeongah Kim.
      
    DOI:  https://doi.org/10.14348/molcells.2023.0043
  36. Elife. 2023 May 12. pii: e85779. [Epub ahead of print]12
    APOE expression and secretion are modulated by mitochondrial dysfunction.
    Meghan E Wynne, Oluwaseun Ogunbona, Alicia R Lane, Avanti Gokhale, Stephanie A Zlatic, Chongchong Xu, Zhexing Wen, Duc M Duong, Sruti Rayaprolu, Anna Ivanova, Eric A Ortlund, Eric B Dammer, Nicholas T Seyfried, Blaine R Roberts, Amanda Crocker, Vinit Shanbhag, Michael Petris, Nanami Senoo, Selvaraju Kandasamy, Steven Michael Claypool, Antoni Barrientos, Aliza Wingo, Thomas S Wingo, Srikant Rangaraju, Allan I Levey, Erica Werner, Victor Faundez.
      Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer's disease risk factor apolipoprotein E (APOE) and other secretome components. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain, elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.
    Keywords:  cell biology; human; neuroscience
    DOI:  https://doi.org/10.7554/eLife.85779
  37. Clin Case Rep. 2023 May;11(5): e7054
    The trap of genetic tag: The importance of pathogenicity prediction tools in the correct interpretation of variants of uncertain significance in the era of high-throughput genome sequencing.
    Francesco Pellegrino.
      Although recent advancements in DNA sequencing technologies and their widely used, the interpretation of variants of uncertain significance from these large datasets is not clear-cut. Here, we present the case of a family referred to our metabolic disease department, in which three males' individuals were affected by a suspected a genetic inherited disease, resulting from next-generation sequencing results. A correct assessment of the clinical significance of the genetic variant found in our cases, with a review of the literature, the evaluation of population database and the use of computational predictive program changed the initial suspect. Despite NGS technologies have increased diagnostic sensitivity, most of these variants remains of uncertain clinical significance. An efficient systematic approach is fundamental to determine the pathogenicity of a variant, avoiding incorrect interpretation in a clinical setting.
    Keywords:  ALG13‐CDG; variants of uncertain significance; variants prediction tools
    DOI:  https://doi.org/10.1002/ccr3.7054
  38. Arch Toxicol. 2023 May 08.
    Metabolic impact of genetic and chemical ADP/ATP carrier inhibition in renal proximal tubule epithelial cells.
    Charlotte A Hoogstraten, Maaike M E Jacobs, Guido de Boer, Melissa A E van de Wal, Werner J H Koopman, Jan A M Smeitink, Frans G M Russel, Tom J J Schirris.
      Mitochondrial dysfunction is pivotal in drug-induced acute kidney injury (AKI), but the underlying mechanisms remain largely unknown. Transport proteins embedded in the mitochondrial inner membrane form a significant class of potential drug off-targets. So far, most transporter-drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC). Since it remains unknown to what extent AAC contributes to drug-induced mitochondrial dysfunction in AKI, we here aimed to better understand the functional role of AAC in the energy metabolism of human renal proximal tubular cells. To this end, CRISPR/Cas9 technology was applied to generate AAC3-/- human conditionally immortalized renal proximal tubule epithelial cells. This AAC3-/- cell model was characterized with respect to mitochondrial function and morphology. To explore whether this model could provide first insights into (mitochondrial) adverse drug effects with suspicion towards AAC-mediated mechanisms, wild-type and knockout cells were exposed to established AAC inhibitors, after which cellular metabolic activity and mitochondrial respiratory capacity were measured. Two AAC3-/- clones showed a significant reduction in ADP import and ATP export rates and mitochondrial mass, without influencing overall morphology. AAC3-/- clones exhibited reduced ATP production, oxygen consumption rates and metabolic spare capacity was particularly affected, mainly in conditions with galactose as carbon source. Chemical AAC inhibition was stronger compared to genetic inhibition in AAC3-/-, suggesting functional compensation by remaining AAC isoforms in our knockout model. In conclusion, our results indicate that ciPTEC-OAT1 cells have a predominantly oxidative phenotype that was not additionally activated by switching energy source. Genetic inhibition of AAC3 particularly impacted mitochondrial spare capacity, without affecting mitochondrial morphology, suggesting an important role for AAC in maintaining the metabolic spare respiration.
    Keywords:  ADP/ATP carrier; CRISPR/Cas9; Drug-induced mitochondrial dysfunction; Nephrotoxicity; Off-target; Oxidative metabolism
    DOI:  https://doi.org/10.1007/s00204-023-03510-7
  39. Annu Rev Biophys. 2023 05 09. 52 229-254
    Mitochondrial Ion Channels.
    Ildiko Szabo, Adam Szewczyk.
      Mitochondria are involved in multiple cellular tasks, such as ATP synthesis, metabolism, metabolite and ion transport, regulation of apoptosis, inflammation, signaling, and inheritance of mitochondrial DNA. The majority of the correct functioning of mitochondria is based on the large electrochemical proton gradient, whose component, the inner mitochondrial membrane potential, is strictly controlled by ion transport through mitochondrial membranes. Consequently, mitochondrial function is critically dependent on ion homeostasis, the disturbance of which leads to abnormal cell functions. Therefore, the discovery of mitochondrial ion channels influencing ion permeability through the membrane has defined a new dimension of the function of ion channels in different cell types, mainly linked to the important tasks that mitochondrial ion channels perform in cell life and death. This review summarizes studies on animal mitochondrial ion channels with special focus on their biophysical properties, molecular identity, and regulation. Additionally, the potential of mitochondrial ion channels as therapeutic targets for several diseases is briefly discussed.
    Keywords:  calcium channels; chloride channels; mitochondria; mitochondrial megachannel; porin; potassium channels
    DOI:  https://doi.org/10.1146/annurev-biophys-092622-094853
  40. J Cell Biochem. 2023 May 09.
    The mitochondrial calcium signaling, regulation, and cellular functions: A novel target for therapeutic medicine in neurological disorders.
    Saeid Bagheri-Mohammadi, Mohammad Farjami, Ali Jaafari Suha, Seyed Mohsen Aghaei Zarch, Sajad Najafi, Ali Esmaeili.
      Mitochondrial calcium (Ca2+ ) dynamics play critical roles in regulating vital physiological conditions in the brain. Importantly, Mitochondria-associated endoplasmic reticulum (ER) membranes serve different cellular functions including Ca2+ signaling, bioenergetics, phospholipid biosynthesis, cholesterol esterification, programmed cell death, and communication between the two organelles. Several Ca2+ -transport systems specialize at the mitochondria, ER, and their contact sites that provide tight control of mitochondrial Ca2+ signaling at the molecular level. The biological function of Ca2+ channels and transporters as well as the role of mitochondrial Ca2+ signaling in cellular homeostasis can open new perspectives for investigation and molecular intervention. Emerging evidence suggests that abnormalities in ER/mitochondrial brain functions and dysregulation of Ca2+ homeostasis are neuropathological hallmarks of neurological disorders like Alzheimer's disease, but little evidence is available to demonstrate their relationship to disease pathogenesis and therapeutic approaches. In recent years, the detection of the molecular mechanism regulating cellular Ca2+ homeostasis and also mitochondrial functions have expanded the number of targeted treatments. The main experimental data identify beneficial effects, whereas some scientific trials did not meet the expectations. Together with an overview of the important function of mitochondria, this review paper introduced the possible tested therapeutic approaches that target mitochondria in the context of neurodegenerative diseases. Since these treatments in neurological disorders have shown different degrees of progress, it is essential to perform a detailed assessment of the significance of mitochondrial deterioration in neurodegenerative diseases and of a pharmacological treatment at this stage.
    Keywords:  Alzheimer's disease; ageing; calcium signaling; cellular homeostasis; mitochondria-associated ER membrane; therapeutic strategy
    DOI:  https://doi.org/10.1002/jcb.30414
  41. Sci Rep. 2023 05 06. 13(1): 7387
    Mitochondrial citrate metabolism and efflux regulate BeWo differentiation.
    Renee M Mahr, Snehalata Jena, Sereen K Nashif, Alisa B Nelson, Adam J Rauckhorst, Ferrol I Rome, Ryan D Sheldon, Curtis C Hughey, Patrycja Puchalska, Micah D Gearhart, Eric B Taylor, Peter A Crawford, Sarah A Wernimont.
      Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established BeWo cell culture model of trophoblast differentiation. Differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.
    DOI:  https://doi.org/10.1038/s41598-023-34435-x
  42. Genome Biol. 2023 May 09. 24(1): 110
    High-throughput deep learning variant effect prediction with Sequence UNET.
    Alistair S Dunham, Pedro Beltrao, Mohammed AlQuraishi.
      Understanding coding mutations is important for many applications in biology and medicine but the vast mutation space makes comprehensive experimental characterisation impossible. Current predictors are often computationally intensive and difficult to scale, including recent deep learning models. We introduce Sequence UNET, a highly scalable deep learning architecture that classifies and predicts variant frequency from sequence alone using multi-scale representations from a fully convolutional compression/expansion architecture. It achieves comparable pathogenicity prediction to recent methods. We demonstrate scalability by analysing 8.3B variants in 904,134 proteins detected through large-scale proteomics. Sequence UNET runs on modest hardware with a simple Python package.
    Keywords:  Deep learning; Machine learning; Mutation; PSSM; Pathogenicity; Variant effect prediction
    DOI:  https://doi.org/10.1186/s13059-023-02948-3
  43. Zhonghua Yan Ke Za Zhi. 2023 May 11. 59(5): 408-410
    [Optic atrophy in a patient with axonal Charcot-Marie-Tooth disease 2A2A due to MFN2 gene mutations].
    C Y Pan, W H Bai, M M Sun, S H Wei, H F Zhou.
      A 27-year-old male patient had progressive vision loss in both eyes, which was mainly manifested by impaired ganglion cells in the macular area, accompanied by systemic muscle atrophy in limbs. A complete mitochondrial exon gene detection was performed. The final diagnosis was bilateral optic atrophy and axonal Charcot-Marie-Tooth disease 2A2A caused by mutations of the MFN2 gene. There has been no effective treatment. Applications of nutrients to restore the mitochondrial function may alleviate the clinical symptoms.
    DOI:  https://doi.org/10.3760/cma.j.cn112142-20220611-00289
  44. JAMA Cardiol. 2023 May 10.
    Skeletal Muscle Mitochondrial Dysfunction and Exercise Intolerance in Heart Failure With Preserved Ejection Fraction.
    Ambarish Pandey, Neil Keshvani, Windy Alonso.
      
    DOI:  https://doi.org/10.1001/jamacardio.2023.0963
  45. J Prim Care Community Health. 2023 Jan-Dec;14:14 21501319231172697
    Mitochondrial Myopathy in a 21-Year-Old Man Presenting With Bilateral Lower Extremity Weakness and Swelling.
    Kavya Bharathidasan, Abbie Evans, Fabiana Monte Alegre Olmos Fernandez, Arunee Tansrisook Motes, Kenneth Nugent.
      Bilateral lower extremity weakness and swelling can have several causes. Although often underdiagnosed, mitochondrial myopathy is more prevalent in the general population than more commonly suspected diseases, such as Guillain-Barre syndrome. The clinical manifestations of mitochondrial disease can be broadly classified into 3 categories: chronic progressive external ophthalmoplegia, skeletal muscle-central nervous system syndromes, or pure myopathy. Cardiac abnormalities occur in 30% to 32% of cases, mostly in the form of hypertrophic cardiomyopathy, dilated cardiomyopathy, or conduction abnormalities. We report a case of a 21-year-old student who developed bilateral lower limb weakness, pain, and swelling diagnosed with mitochondrial myopathy on muscle biopsy. Initial laboratory tests revealed elevated creatinine kinase, brain natriuretic peptide, troponin, myoglobin, and lactic acid and reduced serum bicarbonate. Cardiac workup revealed systolic heart failure with a reduced ejection fraction. Endomyocardial biopsy revealed punctate foci of lymphocytic myocarditis. However, cardiac magnetic resonance imaging did not reveal either myocarditis or an infiltrative cardiac disease. An extensive autoimmune and infection work-up was negative. A muscle biopsy from the patient's rectus femoris revealed scattered ragged-blue fibers (stained with NADH dehydrogenase), scattered ragged-red fibers on modified Gomori trichrome stain, and cytochrome-c oxidase negative fibers with increased perimysial and endomysial connective tissue, consistent with active and chronic primary mitochondrial myopathy. The patient was treated successfully with furosemide, metoprolol, and methylprednisolone. Adult-onset mitochondrial myopathy is a rare clinical disorder, and our experience stresses the importance of using an inter-disciplinary team approach to diagnose uncommon clinical disorders with widely variable multisystem involvement.
    Keywords:  cardiomyopathy; mitochondrial disease; muscle weakness; myopathy
    DOI:  https://doi.org/10.1177/21501319231172697
  46. BMC Biol. 2023 May 08. 21(1): 103
    Secondary structure of the human mitochondrial genome affects formation of deletions.
    Victor Shamanskiy, Alina A Mikhailova, Evgenii O Tretiakov, Kristina Ushakova, Alina G Mikhailova, Sergei Oreshkov, Dmitry A Knorre, Natalia Ree, Jonathan B Overdevest, Samuel W Lukowski, Irina Gostimskaya, Valerian Yurov, Chia-Wei Liou, Tsu-Kung Lin, Wolfram S Kunz, Alexandre Reymond, Ilya Mazunin, Georgii A Bazykin, Jacques Fellay, Masashi Tanaka, Konstantin Khrapko, Konstantin Gunbin, Konstantin Popadin.
       BACKGROUND: Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions.
    RESULTS: By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a "hot spot" where one deletion breakpoint occurred within the region of 6-9 kb and another within 13-16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6-9 kb and 13-16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470-8482 bp (base pair) and a second arm at 13,447-13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging.
    CONCLUSIONS: Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.
    Keywords:  Aging; Contact zone; Deletions; Direct repeats; Global secondary structure; Inverted repeats; Mitochondrial DNA; Single-stranded DNA; mtDNA replication
    DOI:  https://doi.org/10.1186/s12915-023-01606-1
  47. Methods Mol Biol. 2023 ;2661 163-191
    Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.
    Samuel Del'Olio, Antoni Barrientos.
      Studies of yeast mitoribosome assembly have been historically hampered by the difficulty of generating mitoribosome protein-coding gene deletion strains with a stable mitochondrial genome. The identification of mitochondrial DNA-stabilizing approaches allows for the generation of a complete set of yeast deletion strains covering all mitoribosome proteins and known assembly factors. These strains can be used to analyze the integrity and assembly state of mitoribosomes by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts, coupled to mass spectrometry analysis of mitoribosome composition. Subsequent hierarchical cluster analysis of mitoribosome subassemblies accumulated in mutant strains reveals details regarding the order of protein association during the mitoribosome biogenetic process. These strains also allow the expression of truncated protein variants to probe the role of mitochondrion-specific protein extensions, the relevance of protein cofactors, or the importance of RNA-protein interactions in functional sites of the mitoribosome. In this chapter, we will detail the methodology involved in these studies.
    Keywords:  Clustering analysis; Gradient fractionation; Immunoblotting; Mass spectrometry; Mitochondrial ribosome; Mitoribosome assembly intermediate; Mitoribosome profile; Sucrose gradient; Yeast mitoribosome gene deletion strain
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_11
  48. Redox Biol. 2023 May 06. pii: S2213-2317(23)00135-0. [Epub ahead of print]63 102734
    Maintenance of mitochondrial homeostasis for Alzheimer's disease: Strategies and challenges.
    Ying Han, Daozhou Liu, Ying Cheng, Qifeng Ji, Miao Liu, Bangle Zhang, Siyuan Zhou.
      Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, and its early onset is closely related to mitochondrial energy metabolism. The brain is only 2% of body weight, but consumes 20% of total energy needs. Mitochondria are responsible for providing energy in cells, and maintaining their homeostasis ensures an adequate supply of energy to the brain. Mitochondrial homeostasis is constituted by mitochondrial quantity and quality control, which is dynamically regulated by mitochondrial energy metabolism, mitochondrial dynamics and mitochondrial quality control. Impaired energy metabolism of brain cells occurs early in AD, and maintaining mitochondrial homeostasis is a promising therapeutic target in the future. We summarized the mechanism of mitochondrial homeostasis in AD, its influence on the pathogenesis of early AD, strategies for maintaining mitochondrial homeostasis, and mitochondrial targeting strategies. This review concludes with the authors' opinions on future research and development for mitochondrial homeostasis of early AD.
    Keywords:  Alzheimer's disease; Mitochondrial dynamics; Mitochondrial quality control; Mitochondrial targeting; Oxidative phosphorylation
    DOI:  https://doi.org/10.1016/j.redox.2023.102734
  49. Sci Rep. 2023 May 10. 13(1): 7575
    Reduced mitophagy is an early feature of NAFLD and liver-specific PARKIN knockout hastens the onset of steatosis, inflammation and fibrosis.
    R Undamatla, O G Fagunloye, J Chen, L R Edmunds, A Murali, A Mills, B Xie, M M Pangburn, I Sipula, G Gibson, C St Croix, M J Jurczak.
      Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies that includes steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with insulin resistance and type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD, particularly in the transition from steatosis to NASH. Mitophagy is a mitochondrial quality control mechanism that allows for the selective removal of damaged mitochondria from the cell via the autophagy pathway. While past work demonstrated a negative association between liver fat content and rates of mitophagy, when changes in mitophagy occur during the pathogenesis of NAFLD and whether such changes contribute to the primary endpoints associated with the disease are currently poorly defined. We therefore undertook the studies described here to establish when alterations in mitophagy occur during the pathogenesis of NAFLD, as well as to determine the effects of genetic inhibition of mitophagy via conditional deletion of a key mitophagy regulator, PARKIN, on the development of steatosis, insulin resistance, inflammation and fibrosis. We find that loss of mitophagy occurs early in the pathogenesis of NAFLD and that loss of PARKIN accelerates the onset of key NAFLD disease features. These observations suggest that loss of mitochondrial quality control in response to nutritional stress may contribute to mitochondrial dysfunction and the pathogenesis of NAFLD.
    DOI:  https://doi.org/10.1038/s41598-023-34710-x
  50. Methods Mol Biol. 2023 ;2661 133-141
    Rapid Cryopurification of the Yeast Mitochondrial Ribosome.
    Hong Weng Pang, Antoni Barrientos.
      Cryogenic milling, or cryomilling, involves the use of liquid nitrogen to lower the temperature of the biological material and/or the milling process. When applied to the study of subcellular or suborganellar structures and processes, it allows for their rapid extraction from whole cells frozen in the physiological state of choice. This approach has proven to be useful for the study of yeast mitochondrial ribosomes. Following cryomilling of 100 mL of yeast culture, conveniently tagged mitochondrial ribosomes can be immunoprecipitated and purified in native conditions. These ribosomes are suitable for the application of downstream approaches. These include mitoribosome profiling to analyze the mitochondrial translatome or mass spectrometry analyses to assess the mitoribosome proteome in normal growth conditions or under stress, as described in this method.
    Keywords:  Cryomilling; Immunoblotting; Immunoprecipitation; Mass spectrometry; Yeast mitoribosome
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_9
  51. Physiol Res. 2023 Apr 30. 72(2): 137-148
    Mitochondria in human reproduction: novel paradigm in the onset of neurodegenerative disorders.
    M Shavit, M Iniesta-Cuerda, J Nevoral.
      The disease progression of neurodegenerative disorders (NDD), including Alzheimer's, Parkinson's and Huntington's disease, is inextricably tied to mitochondrial dysfunction. However, although the contribution by nuclear gene mutations is recognised for familial onset of NDD, the degree to which cytoplasmic inheritance serves as a predetermining factor for the predisposition and onset of NDD is not yet fully understood. We review the reproductive mechanisms responsible for ensuring a healthy mitochondrial population within each new generation and elucidate how advanced maternal age can constitute an increased risk for the onset of NDD in the offspring, through the increased heteroplasmic burden. On the one hand, this review draws attention to how assisted reproductive technologies (ART) can impair mitochondrial fitness in offspring. On the other hand, we consider qualified ART approaches as a significant tool for the prevention of NDD pathogenesis.
  52. Nature. 2023 May 10.
    In situ architecture of the ER-mitochondria encounter structure.
    Michael R Wozny, Andrea Di Luca, Dustin R Morado, Andrea Picco, Rasha Khaddaj, Pablo Campomanes, Lazar Ivanović, Patrick C Hoffmann, Elizabeth A Miller, Stefano Vanni, Wanda Kukulski.
      The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.
    DOI:  https://doi.org/10.1038/s41586-023-06050-3
  53. Sci Rep. 2023 May 11. 13(1): 7677
    The impact of metabolic stressors on mitochondrial homeostasis in a renal epithelial cell model of methylmalonic aciduria.
    Anke Schumann, Marion Brutsche, Monique Havermans, Sarah C Grünert, Stefan Kölker, Olaf Groß, Luciana Hannibal, Ute Spiekerkoetter.
      Methylmalonic aciduria (MMA-uria) is caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). MUT deficiency hampers energy generation from specific amino acids, odd-chain fatty acids and cholesterol. Chronic kidney disease (CKD) is a well-known long-term complication. We exposed human renal epithelial cells from healthy controls and MMA-uria patients to different culture conditions (normal treatment (NT), high protein (HP) and isoleucine/valine (I/V)) to test the effect of metabolic stressors on renal mitochondrial energy metabolism. Creatinine levels were increased and antioxidant stress defense was severely comprised in MMA-uria cells. Alterations in mitochondrial homeostasis were observed. Changes in tricarboxylic acid cycle metabolites and impaired energy generation from fatty acid oxidation were detected. Methylcitrate as potentially toxic, disease-specific metabolite was increased by HP and I/V load. Mitophagy was disabled in MMA-uria cells, while autophagy was highly active particularly under HP and I/V conditions. Mitochondrial dynamics were shifted towards fission. Sirtuin1, a stress-resistance protein, was down-regulated by HP and I/V exposure in MMA-uria cells. Taken together, both interventions aggravated metabolic fingerprints observed in MMA-uria cells at baseline. The results point to protein toxicity in MMA-uria and lead to a better understanding, how the accumulating, potentially toxic organic acids might trigger CKD.
    DOI:  https://doi.org/10.1038/s41598-023-34373-8
  54. Sci Adv. 2023 May 10. 9(19): eadf5336
    Dodecamer assembly of a metazoan AAA+ chaperone couples substrate extraction to refolding.
    Arpit Gupta, Alfred M Lentzsch, Alex Siegel, Zanlin Yu, Un Seng Chio, Yifan Cheng, Shu-Ou Shan.
      Ring-forming AAA+ chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA+ chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states. High-resolution structures of Skd3 hexamers in distinct conformations capture ratchet-like motions that mediate substrate extraction. Unlike previously described disaggregases, Skd3 hexamers further assemble into dodecameric cages in which solubilized substrate proteins can attain near-native states. Skd3 mutants defective in dodecamer assembly retain disaggregase activity but are impaired in client refolding, linking the disaggregase and refolding activities to the hexameric and dodecameric states of Skd3, respectively. We suggest that Skd3 is a combined disaggregase and foldase, and this property is particularly suited to meet the complex proteostatic demands in the mitochondrial IMS.
    DOI:  https://doi.org/10.1126/sciadv.adf5336
  55. Nat Cancer. 2023 May 11.
    GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity.
    Dionysios C Watson, Defne Bayik, Simon Storevik, Shannon Sherwin Moreino, Samuel A Sprowls, Jianhua Han, Mina Thue Augustsson, Adam Lauko, Palavalasa Sravya, Gro Vatne Røsland, Katie Troike, Karl Johan Tronstad, Sabrina Wang, Katharina Sarnow, Kristen Kay, Taral R Lunavat, Daniel J Silver, Sahil Dayal, Justin Vareecal Joseph, Erin Mulkearns-Hubert, Lars Andreas Rømo Ystaas, Gauravi Deshpande, Joris Guyon, Yadi Zhou, Capucine R Magaut, Juliana Seder, Laura Neises, Sarah E Williford, Johannes Meiser, Andrew J Scott, Peter Sajjakulnukit, Jason A Mears, Rolf Bjerkvig, Abhishek Chakraborty, Thomas Daubon, Feixiong Cheng, Costas A Lyssiotis, Daniel R Wahl, Anita B Hjelmeland, Jubayer A Hossain, Hrvoje Miletic, Justin D Lathia.
      The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development.
    DOI:  https://doi.org/10.1038/s43018-023-00556-5
  56. Nat Cancer. 2023 May 11.
    Mind the GAP(43) for mitochondria transfer to glioblastomas.
    Martina Semenzato, Luca Scorrano.
      
    DOI:  https://doi.org/10.1038/s43018-023-00564-5
  57. Genetics. 2023 May 12. pii: iyad036. [Epub ahead of print]
    Evolutionary genetics of the mitochondrial genome: insights from Drosophila.
    Damian K Dowling, Jonci N Wolff.
      Mitochondria are key to energy conversion in virtually all eukaryotes. Intriguingly, despite billions of years of evolution inside the eukaryote, mitochondria have retained their own small set of genes involved in the regulation of oxidative phosphorylation (OXPHOS) and protein translation. Although there was a long-standing assumption that the genetic variation found within the mitochondria would be selectively neutral, research over the past 3 decades has challenged this assumption. This research has provided novel insight into the genetic and evolutionary forces that shape mitochondrial evolution and broader implications for evolutionary ecological processes. Many of the seminal studies in this field, from the inception of the research field to current studies, have been conducted using Drosophila flies, thus establishing the species as a model system for studies in mitochondrial evolutionary biology. In this review, we comprehensively review these studies, from those focusing on genetic processes shaping evolution within the mitochondrial genome, to those examining the evolutionary implications of interactions between genes spanning mitochondrial and nuclear genomes, and to those investigating the dynamics of mitochondrial heteroplasmy. We synthesize the contribution of these studies to shaping our understanding of the evolutionary and ecological implications of mitochondrial genetic variation.
    Keywords:  FlyBook; Mother's Curse; cytonuclear; experimental evolution; heteroplasmy; mito-nuclear; mitochondria; mitonuclear; mtDNA
    DOI:  https://doi.org/10.1093/genetics/iyad036
  58. Cell Death Dis. 2023 05 08. 14(5): 311
    FGF21-FGFR1 controls mitochondrial homeostasis in cardiomyocytes by modulating the degradation of OPA1.
    Bing Yan, Zhu Mei, Yaohan Tang, Haixu Song, Hanlin Wu, Quanmin Jing, Xiaolin Zhang, Chenghui Yan, Yaling Han.
      Fibroblast growth factor 21 (FGF21) is a pleiotropic hormone secreted primarily by the liver and is considered a major regulator of energy homeostasis. Recent research has revealed that FGF21 could play an important role in cardiac pathological remodeling effects and prevention of cardiomyopathy; however, the underlying mechanism remains largely unknown. This study aimed to determine the mechanism underlying the cardioprotective effects of FGF21. We engineered FGF21 knock out mice and subsequently elucidated the effects of FGF21 and its downstream mediators using western blotting, qRT-PCR, and mitochondrial morphological and functional analyses. FGF21 knockout mice showed cardiac dysfunction, accompanied by a decline in global longitudinal strain (GLS) and ejection fraction (EF), independent of metabolic disorders. Mitochondrial quality, quantity, and function were abnormal, accompanied by decreased levels of optic atrophy-1 (OPA1) in FGF21 KO mice. In contrast to FGF21 knockout, cardiac-specific overexpression of FGF21 alleviated the cardiac dysfunction caused by FGF21 deficiency. In an in vitro study, FGF21 siRNA deteriorated mitochondrial dynamics and impaired function induced by cobalt chloride (CoCl2). Both recombinant FGF21 and adenovirus-mediated FGF21 overexpression could alleviate CoCl2-induced mitochondrial impairment by restoring mitochondrial dynamics. FGF21 was essential for maintaining mitochondrial dynamics and function of the cardiomyocytes. As a regulator of cardiomyocyte mitochondrial homeostasis under oxidative stress, FGF21 could be an important new target for therapeutic options for patients with heart failure.
    DOI:  https://doi.org/10.1038/s41419-023-05842-9
  59. Dis Model Mech. 2023 May 09. pii: dmm.049963. [Epub ahead of print]
    Misrouting to mitochondria of renin carrying dominant mutations in the leader peptide or pro-segment.
    Céline Schaeffer, Maurizio De Fusco, Elena Pasqualetto, Caterina Scolari, Claudia Izzi, Francesco Scolari, Luca Rampoldi.
      Autosomal Dominant Tubulointerstitial Kidney Disease, a rare genetic disorder characterised by progressive chronic kidney disease, is caused by mutations in different genes including REN, encoding renin. Renin is a secreted protease composed of 3 domains: the leader peptide allowing insertion in the endoplasmic reticulum (ER), a pro-segment regulating its activity, and the mature part. Mutations in mature renin lead to ER retention of mutant protein and to late onset disease, while mutations in the leader peptide, associated with defective ER translocation, and mutations in the pro-segment, accumulating in the ER-to-Golgi compartment, lead to a more severe, early-onset disease. In this study we demonstrate a common, unprecedented effect of mutations in the leader peptide and pro-segment as they lead to full or partial mistargeting of mutated protein to mitochondria. The mutated pre-pro sequence of renin is necessary and sufficient to drive mitochondrial rerouting, mitochondrial import defect and fragmentation. Mitochondrial localisation and fragmentation are also observed for wild type renin when affecting ER translocation. These results expand the spectrum of cellular phenotypes associated with ADTKD-REN mutations providing new insight into the disease molecular pathogenesis.
    Keywords:  ADTKD; Mitochondria; Renin; Signal sequence; Trafficking defect
    DOI:  https://doi.org/10.1242/dmm.049963
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