bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2024‒11‒03
63 papers selected by
Catalina Vasilescu, Helmholz Munich
  1. Monitoring mitochondrial protein import by live cell imaging.
  2. Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.
  3. The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency.
  4. In organello silencing of mitochondrial gene expression.
  5. Analysis of mitochondrial translation using click chemistry.
  6. Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.
  7. Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry.
  8. Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria.
  9. In vitro import of mitochondrial precursor proteins into yeast mitochondria.
  10. Quantifying mitochondrial protein import by mePRODmt proteomics.
  11. Assays to monitor mitochondrial translation.
  12. Methods to monitor mitochondrial disulfide bonds.
  13. Editorial: Role of mitochondrial stress response in metabolic health.
  14. Comprehensive phenotypic assessment of nonsense mutations in mitochondrial ND5 in mice.
  15. Analysis of targeting signals for mitochondrial intermembrane space import.
  16. Nifuroxazide rescues the deleterious effects due to CHCHD10-associated MICOS defects in disease models.
  17. Monitoring mitochondrial precursor processing and presequence peptide degradation.
  18. Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.
  19. Mitochondrial fission controls astrocyte morphogenesis and organization in the cortex.
  20. The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.
  21. Unraveling the molecular determinants of a rare human mitochondrial disorder caused by the P144L mutation of FDX2.
  22. Isolation of yeast mitochondria by differential centrifugation.
  23. Analysis and prediction of internal mitochondrial targeting signals.
  24. A hierarchical hepatic de novo lipogenesis substrate supply network utilizing pyruvate, acetate, and ketones.
  25. A novel m.5906G > a variant in MT-CO1 causes MELAS/Leigh overlap syndrome.
  26. The Mysteries of LETM1 Pleiotropy.
  27. Isolation of yeast mitochondria by affinity purification using magnetic beads.
  28. Mass Spectrometry Probe Combined with Machine Learning to Capture the Relationship between Metabolites and Mitochondrial Complex Activity at the Whole-Cell Level.
  29. Structure of the turnover-ready state of an ancestral respiratory complex I.
  30. Signaling Pathways Concerning Mitochondrial Dysfunction: Implications in Neurodegeneration and Possible Molecular Targets.
  31. Membrane potential stimulates ADP import and ATP export by the mitochondrial ADP/ATP carrier due to its positively charged binding site.
  32. Mitochondrial inhibitors reveal roles of specific respiratory chain complexes in CRY-dependent degradation of TIM.
  33. COX15 deficiency causes oocyte ferroptosis.
  34. Prediction of mitochondrial targeting signals and their cleavage sites.
  35. Import of mitochondrial precursor proteins into mitochondria of semi-intact yeast cells.
  36. TFAM as a sensor of UVC-induced mitochondrial DNA damage.
  37. Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis.
  38. Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.
  39. The mitochondrial citrate carrier SLC25A1 regulates metabolic reprogramming and morphogenesis in the developing heart.
  40. Subcellular fractionation by differential centrifugation for mitochondrial studies.
  41. Genetic family studies and prospective evaluation for multisystem involvement are needed in LHON patients.
  42. The multifaceted role of mitochondria in cardiac function: insights and approaches.
  43. Biallelic GGGCC repeat expansion leading to NAXE-related mitochondrial encephalopathy.
  44. Mitophagy in Cell Death Regulation: Insights into Mechanisms and Disease Implications.
  45. Phospholipid Scramblase Activity of VDAC Dimers: New Implications for Cell Death, Autophagy and Ageing.
  46. High mitochondrial DNA levels accelerate lung adenocarcinoma progression.
  47. Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP.
  48. Ortholog of autism candidate gene RBM27 regulates mitoribosomal assembly factor MALS-1 to protect against mitochondrial dysfunction and axon degeneration during neurodevelopment.
  49. Association of LONP1 gene with epilepsy and the sub-regional effect.
  50. Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.
  51. Mitochondria are absent from microglial processes performing surveillance, chemotaxis, and phagocytic engulfment.
  52. Analysis of mitochondrial biogenesis and protein localization by genetic screens and automated imaging.
  53. Reducing the mitochondrial oxidative burden alleviates lipid-induced muscle insulin resistance in humans.
  54. Sepsis-induced changes in pyruvate metabolism: insights and potential therapeutic approaches.
  55. miR-124 coordinates metabolic regulators acting at early stages of human neurogenesis.
  56. Bempedoic acid suppresses diet-induced hepatic steatosis independently of ATP-citrate lyase.
  57. Some brain disorders are "chaperonopathies".
  58. Advancements in mitochondrial-targeted nanotherapeutics: overcoming biological obstacles and optimizing drug delivery.
  59. A modified procedure for separating yeast peroxisomes from mitochondria.
  60. EMC1 Is Required for the Sarcoplasmic Reticulum and Mitochondrial Functions in the Drosophila Muscle.
  61. Exceptional longevity of mammalian ovarian and oocyte macromolecules throughout the reproductive lifespan.
  62. Loss of long-chain acyl-CoA dehydrogenase protects against acute kidney injury.
  63. Human iPSCs from Aged Donors Retain Their Mitochondrial Aging Signature.
  1. Methods Enzymol. 2024 ;pii: S0076-6879(24)00363-X. [Epub ahead of print]706 437-447
    Monitoring mitochondrial protein import by live cell imaging.
    F X Reymond Sutandy.
      The majority of mitochondrial proteins are synthesized in the cytosol and must be imported into mitochondria to attain their mature forms and execute their functions. Disruption of mitochondrial functions, whether caused by external or internal stress, may compromise mitochondrial protein import. Therefore, monitoring mitochondrial protein import has become a standard approach to assess mitochondrial health and gain insights into mitochondrial biology, especially during stress. This chapter describes a detailed protocol for monitoring mitochondrial import in live cells using microscopy. Co-localization between mitochondria and a genetic reporter of mitochondrially targeted enhanced GFP (eGFP) is employed to evaluate mitochondrial protein import efficiency under different physiological conditions. Overall, this technique provides a simple and robust approach to assess mitochondrial protein import efficiency within its native cellular environment.
    Keywords:  MTS; mitochondria; protein import; stress response
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.027
  2. Methods Enzymol. 2024 ;pii: S0076-6879(24)00370-7. [Epub ahead of print]706 365-390
    Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.
    Jordan J Crameri, Diana Stojanovski.
      Mitochondrial protein import is a complex process governing the delivery of the organelle's proteome. This process, in turn, is essential for maintaining mitochondrial function and cellular homeostasis. Initiated by protein synthesis in the cytoplasm, precursor proteins destined for the mitochondria possess targeting signals that guide them to the mitochondrial surface. At mitochondria, the translocation of proteins across the mitochondrial membranes involves an intricate interplay between translocases, chaperones, and receptors. The mitochondrial import assay offers researchers the opportunity to recapitulate the process of protein import in vitro. The assay has served as an indispensable tool in helping decipher the intricacies of protein translocation into mitochondria, first in fungal models, and subsequently in higher eukaryotic models. In this chapter, we will describe how protein import can be assayed using mammalian mitochondria and provide insight into the types of questions that can be addressed in mammalian mitochondrial biology using this experimental approach.
    Keywords:  in vitro; mitochondria; protein import; translocase
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.034
  3. bioRxiv. 2024 Oct 17. pii: 2024.10.16.617214. [Epub ahead of print]
    The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency.
    Mingchong Yang, Zengshuo Mo, Kelly Walsh, Wen Liu, Xiaoyan Guo.
      Mitophagy is crucial for maintaining mitochondrial health, but how its levels adjust to different stress conditions remains unclear. In this study, we investigated the role of the DELE1-HRI axis of integrated stress response (ISR) in regulating mitophagy, a key mitochondrial stress pathway. Our findings show that the ISR suppresses mitophagy under non-depolarizing mitochondrial stress by positively regulating mitochondrial protein import, independent of ATF4 activation. Mitochondrial protein import is regulated by the rate of protein synthesis under both depolarizing and non-depolarizing stress. Without ISR, increased protein synthesis overwhelms the mitochondrial import machinery, reducing its efficiency. Under depolarizing stress, mitochondrial import is heavily impaired even with active ISR, leading to significant PINK1 accumulation. In contrast, non-depolarizing stress allows more efficient protein import in the presence of ISR, resulting in lower mitophagy. Without ISR, mitochondrial protein import becomes severely compromised, causing PINK1 accumulation to reach the threshold necessary to trigger mitophagy. These findings reveal a novel link between ISR-regulated protein synthesis, mitochondrial import, and mitophagy, offering potential therapeutic targets for diseases associated with mitochondrial dysfunction.
    DOI:  https://doi.org/10.1101/2024.10.16.617214
  4. Methods Enzymol. 2024 ;pii: S0076-6879(24)00371-9. [Epub ahead of print]706 501-518
    In organello silencing of mitochondrial gene expression.
    Mats Koschel, Luis Daniel Cruz-Zaragoza.
      Mitochondria contain proteins from two genetic origins. Most mitochondrial proteins are encoded in the nuclear genome, translated in the cytosol, and subsequently imported into the different mitochondrial sub-compartments. A small number is encoded in the mitochondrial DNA (mtDNA). The manipulation of the mtDNA gene expression represents a challenge. Here, we present an in vitro approach using morpholinos chemically linked to a precursor protein to silence gene expression in purified human mitochondria. The protocol is demonstrated with a Jac1-morpholino chimera specifically targeting COX1 mRNA. The chimera import and mitochondrial translation requirements are described in a step-by-step procedure, where the dose-dependent effect of reducing COX1 translation is observed. The affinity and specificity of chimera-mRNA binding also show great applicability to purify transcript-associated proteins by using the imported chimera construct as bait for immunoprecipitation. This new strategy opens up the possibility to address mechanistic questions about gene expression and physiology in mitochondria.
    Keywords:  Gene expression; In vitro; Mitochondria; Morpholino; Silencing
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.035
  5. Methods Enzymol. 2024 ;pii: S0076-6879(24)00384-7. [Epub ahead of print]706 533-547
    Analysis of mitochondrial translation using click chemistry.
    Roya Yousefi, Sven Dennerlein.
      Mitochondria contain their own gene expression machinery, which synthesizes core subunits of the oxidative phosphorylation system. Monitoring mitochondrial translation within spatial compartments of cells is difficult. Here we describe a method to visualize mitochondrial translation within defined parts of cells, using a click chemistry approach. This method can be applied to different cell types such as neurons and allows detection of newly synthesized mitochondrial proteins in spatial resolution using microscopy techniques. Furthermore, using click chemistry, mitochondrial translation can also be monitored by standard SDS-PAGE. The described method avenues the analysis of newly synthesized mitochondrial encoded proteins in the cellular context, by avoiding the usage of radioactive components.
    Keywords:  Microscopy; Mitochondria; Mitochondrial translation
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.044
  6. Methods Enzymol. 2024 ;pii: S0076-6879(24)00356-2. [Epub ahead of print]706 287-311
    Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.
    Klaudia K Maruszczak, Agnieszka Chacinska.
      The vast majority of mitochondrial precursor proteins is synthesized in the cytosol and subsequently imported into the organelle with the help of targeting signals that are present within these proteins. Disruptions in mitochondrial import will result in the accumulation of the organellar precursors in the cytosol of the cell. If mislocalized proteins exceed their critical concentrations, they become prone to aggregation. Under certain circumstances, protein aggregation becomes an irreversible process, which eventually endangers cellular health. Impairment in mitochondrial biogenesis and its effect on cellular protein homeostasis were recently linked to neurodegeneration, therefore placing this process in the center of attention. In this chapter, we are presenting a set of techniques that allows to monitor and study mitochondrial precursor protein aggregates upon mitochondrial dysfunction in the cytosol of both yeast and human cells.
    Keywords:  Mitochondria; Mitochondrial dysfunction; Mitochondrial import; Protein aggregates
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.020
  7. Methods Enzymol. 2024 ;pii: S0076-6879(24)00361-6. [Epub ahead of print]706 215-242
    Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry.
    Fabian Stockert, Henrique Baeta, Pitter F Huesgen.
      The majority of mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol and imported into mitochondria mediated by an N-terminal mitochondrial targeting sequences (MTS). After import, the MTS is cleaved off by the mitochondrial processing peptidase (MPP) and subsets of the imported proteins are further processed by the aminopeptidase intermediate cleaving peptidase 55 (ICP55), the mitochondrial intermediate peptidase (MIP), octapeptidyl aminopeptidase 1 (Oct1) or other proteolytic enzymes. Mutations that impair the mitochondrial processing machinery or mitochondrial protein degradation result in rare but severe human diseases. In addition, aging and various stress conditions are associated with altered proteolysis of mitochondrial proteins. Enrichment of protein terminal peptides in combination with mass spectrometry-based identification and quantification has become the method of choice to study proteolytic processing. Here, we describe an updated step-by-step protocol for the enrichment of N-terminal peptides by Hypersensitive Undecanal-mediated Enrichment of N-Terminal peptides (HUNTER). We describe analysis of mass spectrometry data acquired for HUNTER samples and present a suite of dedicated Python and R scripts for HUNTER quality control, classification of the enriched peptides, annotation of mitochondrial processing sites and quantitative evaluation. The scripts are freely available at https://github.com/FabianStockert/mito_annotation.
    Keywords:  Data analysis; Degradomics; Mass spectrometry; Mitochondria; N-terminome; Peptide quantification; Positional annotation; Protein N-termini; Proteolytic processing
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.025
  8. bioRxiv. 2024 Oct 17. pii: 2024.10.15.618543. [Epub ahead of print]
    Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria.
    Yi Fu, Max Land, Ruobing Cui, Tamar Kavlashvili, Minsoo Kim, Toby Lieber, Keun Woo Ryu, Emily DeBitetto, Ignas Masilionis, Rahul Saha, Meril Takizawa, Daphne Baker, Marco Tigano, Ed Reznik, Roshan Sharma, Ronan Chaligne, Craig B Thompson, Dana Pe'er, Agnel Sfeir.
      Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial myopathies and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a ∼3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of ∼75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies.Highlights: Combining prokaryotic end-joining with targeted endonucleases generates specific mtDNA deletions in human cellsEngineering a panel of cell lines with a large-scale deletion that spans the full spectrum of heteroplasmy75% heteroplasmy is the threshold that triggers mitochondrial and cellular dysfunctionTwo distinct nuclear transcriptional programs in response to mtDNA deletions: threshold-triggered and heteroplasmy-sensing.
    DOI:  https://doi.org/10.1101/2024.10.15.618543
  9. Methods Enzymol. 2024 ;pii: S0076-6879(24)00342-2. [Epub ahead of print]706 347-363
    In vitro import of mitochondrial precursor proteins into yeast mitochondria.
    Soraya Badrie, Julian Alexander Draken, Dejana Mokranjac.
      Mitochondria contain about 1000 different proteins, only a handful of which are encoded in the mitochondrial genome. The remaining c. 99% of mitochondrial proteins are encoded in the nuclear genome, synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals and are subsequently imported into the organelle. Mitochondrial targeting signals are very diverse and mitochondria therefore also have a number of very sophisticated molecular machines that recognize, import and sort mitochondrial precursor proteins to the different mitochondrial subcompartments. The ability to synthesize mitochondrial precursor proteins in vitro and subsequently import them into isolated mitochondria has revolutionized our understanding of mitochondrial protein import pathways. Here, we describe the basic protocol for synthesis of mitochondrial precursor proteins in vitro and their subsequent import into isolated mitochondria from yeast Saccharomyces cerevisiae, the method which was used to elucidate and characterize the vast majority of mitochondrial protein import pathways.
    Keywords:  (35)S-methionine; In vitro import; In vitro transcription and translation; Isolated mitochondria; Protein translocation; Saccharomyces cerevisiae
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.016
  10. Methods Enzymol. 2024 ;pii: S0076-6879(24)00347-1. [Epub ahead of print]706 449-474
    Quantifying mitochondrial protein import by mePRODmt proteomics.
    Süleyman Bozkurt, Bhavesh S Parmar, Christian Münch.
      Mitochondrial protein import is crucial for maintaining cellular health and homeostasis. Disruptions in this process have been linked to various diseases. Traditional methods for studying mitochondrial protein import predominantly focus on individual proteins and lack the dynamic resolution needed to fully appreciate the complexity of mitochondrial proteostasis and protein trafficking. To address these limitations, we developed a technique called mitochondria-specific multiplexed enhanced protein dynamics (mePRODmt). This method is a novel application of the mePROD methodology and utilizes pulsed stable isotope labeling with amino acids in cell culture (pSILAC)-based proteomics approach to study transient mitochondrial protein import. This chapter outlines the mePRODmt protocol, which includes the preparation of heavy SILAC-labeled peptides for boosting overall mitochondrial peptide signals (booster), SILAC labeling of cultured cells under experimental conditions, mitochondria isolation, sample preparation for multiplex proteomics using tandem mass tags (TMT) for isobaric labeling, recommended liquid chromatography-mass spectrometry (LC-MS) settings for reporter ion quantitation and a data analysis pipeline to analyze pSILAC-TMT data.
    Keywords:  Mass spectrometry; Mitochondria; Mitochondrial protein import; Proteomics; SILAC; TMT multiplex; Translation; mePROD; pSILAC
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.017
  11. Methods Enzymol. 2024 ;pii: S0076-6879(24)00385-9. [Epub ahead of print]706 519-532
    Assays to monitor mitochondrial translation.
    Sung-Jun Jung, Martin Ott.
      The complexes of the oxidative phosphorylation (OXPHOS) system found in the mitochondrial inner membrane comprises nuclear and mitochondrial-encoded proteins. The mitochondrial-encoded subunits of the OXPHOS complexes play vital catalytic roles for OXPHOS. These subunits are inserted co-translationally into the inner membrane, where they are matured and assembled with nuclear encoded subunits, requiring a set of OXPHOS assembly and quality control factors. Hence, monitoring the fate of newly synthesized mitochondrial-encoded polypeptides is a basic and essential approach for exploring OXPHOS biogenesis and the related protein quality control processes. Here, we describe a detailed protocol for labeling mitochondrial encoded proteins with 35S-methionine for pulse and pulse/chase experiments, both in vivo and in organello, using the yeast Saccharomyces cerevisiae as the model. These methods enable analyses of the early steps during the biogenesis and turnover of mitochondrial-encoded proteins.
    Keywords:  35S-methionine; Mitochondrial translation; isolated mitochondria; protein stability; protein synthesis; yeast
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.045
  12. Methods Enzymol. 2024 ;pii: S0076-6879(24)00379-3. [Epub ahead of print]706 125-158
    Methods to monitor mitochondrial disulfide bonds.
    Ben Hur Marins Mussulini, Michal Wasilewski, Agnieszka Chacinska.
      Mitochondria contain numerous proteins that utilize the chemistry of cysteine residues, which can be reversibly oxidized. These proteins are involved in mitochondrial biogenesis, protection against oxidative stress, metabolism, energy transduction to adenosine triphosphate, signaling and cell death among other functions. Many proteins located in the mitochondrial intermembrane space are imported by the mitochondrial import and assembly pathway the activity of which is based on the reversible oxidation of cysteine residues and oxidative trapping of substrates. Oxidative modifications of cysteine residues are particularly difficult to study because of their labile character. Here we present techniques that allow for monitoring the oxidative state of mitochondrial proteins as well as to investigate the mitochondrial import and assembly pathway. This chapter conveys basic concepts on sample preparation and techniques to monitor the redox state of cysteine residues in mitochondrial proteins as well as the strategies to study mitochondrial import and assembly pathway.
    Keywords:  Direct thiol trap; Import assay; Indirect thiol trap; MIA40; Mitochondria
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.039
  13. Front Endocrinol (Lausanne). 2024 ;15 1504718
    Editorial: Role of mitochondrial stress response in metabolic health.
    Renata O Pereira, Susanne Keipert.
      
    Keywords:  FGF21; GDF15; MOTS-c; integrated stress response (ISR); metabolic health; mitochondria; mitohormesis; mitokine
    DOI:  https://doi.org/10.3389/fendo.2024.1504718
  14. Exp Mol Med. 2024 Nov 01.
    Comprehensive phenotypic assessment of nonsense mutations in mitochondrial ND5 in mice.
    Sanghun Kim, Seul Gi Park, Jieun Kim, Seongho Hong, Sang-Mi Cho, Soo-Yeon Lim, Eun-Kyoung Kim, Sungjin Ju, Su Bin Lee, Sol Pin Kim, Tae Young Jeong, Yeji Oh, Seunghun Han, Hae-Rim Kim, Taek Chang Lee, Hyoung-Chin Kim, Won Kee Yoon, Tae Hyeon An, Kyoung-Jin Oh, Ki-Hoan Nam, Seonghyun Lee, Kyoungmi Kim, Je Kyung Seong, Hyunji Lee.
      Mitochondrial dysfunction induced by mitochondrial DNA (mtDNA) mutations has been implicated in various human diseases. A comprehensive analysis of mitochondrial genetic disorders requires suitable animal models for human disease studies. While gene knockout via premature stop codons is a powerful method for investigating the unique functions of target genes, achieving knockout of mtDNA has been rare. Here, we report the genotypes and phenotypes of heteroplasmic MT-ND5 gene-knockout mice. These mutant mice presented damaged mitochondrial cristae in the cerebral cortex, hippocampal atrophy, and asymmetry, leading to learning and memory abnormalities. Moreover, mutant mice are susceptible to obesity and thermogenetic disorders. We propose that these mtDNA gene-knockdown mice could serve as valuable animal models for studying the MT-ND5 gene and developing therapies for human mitochondrial disorders in the future.
    DOI:  https://doi.org/10.1038/s12276-024-01333-9
  15. Methods Enzymol. 2024 ;pii: S0076-6879(24)00366-5. [Epub ahead of print]706 243-262
    Analysis of targeting signals for mitochondrial intermembrane space import.
    Kostas Tokatlidis, Amiyo Haider.
      The mitochondrial intermembrane space (IMS) is the smallest sub-mitochondrial compartment, containing only 5%-10% of mitochondrial proteins. Despite its size, it exhibits the most diverse array of protein import mechanisms. These are underpinned by several different types of targeting signals that are quite distinct from targeting signals for other mitochondrial sub-compartments. In this chapter we outlined our current understanding of some of the main IMS import pathways, the primary oxidative protein folding targeting signal, and explore the remarkable variety of alternative import methods. Unlike proteins destined for the matrix or inner membrane (IM), IMS proteins need only traverse the outer mitochondrial membrane. This process doesn't require energy from ATP hydrolysis in the matrix or the IM electrochemical potential. We also examine unconventional IMS import pathways that remain poorly understood, often guided by ill-defined or unknown targeting peptides. Many IMS proteins are implicated in human diseases, making it crucial to comprehend how they reach their functional location within the IMS. The chapter concludes by discussing current insights into how understanding IMS targeting pathways can contribute to improved understanding of a wide range of human disorders.
    Keywords:  Chaperones; Disulfide bonds; In vitro protein import; Intermembrane space; MIA pathway; Oxidative folding; Redox; Targeting
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.030
  16. Brain. 2024 Oct 30. pii: awae348. [Epub ahead of print]
    Nifuroxazide rescues the deleterious effects due to CHCHD10-associated MICOS defects in disease models.
    Baptiste Ropert, Sylvie Bannwarth, Emmanuelle C Genin, Loan Vaillant-Beuchot, Sandra Lacas-Gervais, Blandine Madji Hounoum, Aurore Bernardin, Nhu Dinh, Alessandra Mauri-Crouzet, Marc-Alexandre D'Elia, Gaelle Augé, Françoise Lespinasse, Audrey Di Giorgio, Willian Meira, Nathalie Bonnefoy, Laurent Monassier, Manuel Schiff, Laila Sago, Devrim Kilinc, Frédéric Brau, Virginie Redeker, Delphine Bohl, Déborah Tribouillard-Tanvier, Vincent Procaccio, Stéphane Azoulay, Jean-Ehrland Ricci, Agnès Delahodde, Véronique Paquis-Flucklinger.
      The identification of a point mutation (p.Ser59Leu) in the CHCHD10 gene was the first genetic evidence that mitochondrial dysfunction can trigger motor neuron disease. Since then, we have shown that this mutation leads to the disorganization of the MItochondrial contact site and Cristae Organizing System (MICOS) complex that maintains the mitochondrial cristae structure. Here, we generated yeast mutant strains mimicking MICOS instability and used them to test the ability of more than 1600 compounds from 2 repurposed libraries to rescue the growth defect of those cells. Among the hits identified, we selected nifuroxazide, a broad-spectrum antibacterial molecule. We show that nifuroxazide rescues mitochondrial network fragmentation and cristae abnormalities in CHCHD10S59L/+ patient fibroblasts. This molecule also decreases caspase-dependent death of human CHCHD10S59L/+ iPSC-derived motor neurons. Its benefits involve KIF5B-mediated mitochondrial transport enhancement, evidenced by increased axonal movement and syntaphilin degradation in patient-derived motor neurons. Our findings strengthen the MICOS-mitochondrial transport connection. Nifuroxazide and analogues emerge as potential therapeutics for MICOS-related disorders like motor neuron disease. Its impact on syntaphilin hints at broader neurological disorder applicability for nifuroxazide.
    Keywords:  ALS; MICOS; mitochondrial disease; nifuroxazide
    DOI:  https://doi.org/10.1093/brain/awae348
  17. Methods Enzymol. 2024 ;pii: S0076-6879(24)00354-9. [Epub ahead of print]706 193-213
    Monitoring mitochondrial precursor processing and presequence peptide degradation.
    Cansu Kücükköse, F-Nora Vögtle, Annette Flotho.
      The maturation of mitochondrial presequence precursor proteins after their import into the organelle is a complex process that requires the interaction of several mitochondrial proteases. Precursor processing by the mitochondrial presequence proteases is directly coupled to the proteolytic turnover of the cleaved targeting signal by mitochondrial presequence peptidases. Dysfunction of these enzymes is associated with a variety of human diseases, including neurological disorders, cardiomyopathies and renal diseases. In this chapter, we describe experimental approaches to study the activity of the major mitochondrial presequence protease (MPP) and of the presequence peptidases. In vitro assays and soluble mitochondrial extracts allow the assessment and experimental manipulation of peptidase and protease activity using immunoblotting, fluorescence measurements and autoradiography as readouts. In particular, the assays allow manipulation at multiple levels including in vivo, in organello or in soluble extracts/in vitro. Purification of the yeast heterodimeric MPP allows in vitro reconstitution of the initial presequence processing step using radiolabeled precursors as substrates. Application of soluble mitochondrial extracts enables direct assessment of MPP processing and presequence peptide turnover which can be easily manipulated and is uncoupled from protein translocation across the mitochondrial membranes. The techniques presented in this chapter allow in-depth analysis of precursor processing and presequence turnover as well as direct assessment of the impact of patient mutations on the activity of the presequence processing machinery.
    Keywords:  Mitochondrial precursor processing; Mitochondrial protein import; Presequence degradation; Presequences; Targeting peptides
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.018
  18. J Cell Sci. 2024 Oct 28. pii: jcs.263548. [Epub ahead of print]
    Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.
    Marta Lopez-Nieto, Zhaozhi Sun, Emily Relton, Rahme Safakli, Brian D Freibaum, J Paul Taylor, Alessia Ruggieri, Ioannis Smyrnias, Nicolas Locker.
      To rapidly adapt to harmful changes to their environment, cells activate the integrated stress response (ISR). This results in an adaptive transcriptional and translational rewiring, and the formation of biomolecular condensates named stress granules (SGs), to resolve stress. In addition to this first line of defence, the mitochondrial unfolded protein response (UPRmt) activates a specific transcriptional programme to maintain mitochondrial homeostasis. We present evidence that SGs and UPRmt pathways are intertwined and communicate. UPRmt induction results in eIF2a phosphorylation and the initial and transient formation of SGs, which subsequently disassemble. The induction of GADD34 during late UPRmt protects cells from prolonged stress by impairing further assembly of SGs. Furthermore, mitochondrial functions and cellular survival are enhanced during UPRmt activation when SGs are absent, suggesting that UPRmt-induced SGs have an adverse effect on mitochondrial homeostasis. These findings point to a novel crosstalk between SGs and the UPRmt that may contribute to restoring mitochondrial functions under stressful conditions.
    Keywords:  GADD34; Integrated stress response; Mitochondrial stress response; Stress granules; UPRmt
    DOI:  https://doi.org/10.1242/jcs.263548
  19. bioRxiv. 2024 Oct 22. pii: 2024.10.22.619706. [Epub ahead of print]
    Mitochondrial fission controls astrocyte morphogenesis and organization in the cortex.
    Maria Pia Rodriguez Salazar, Sprihaa Kolanukuduru, Valentina Ramirez, Boyu Lyu, Gabrielle Sejourne, Hiromi Sesaki, Guoqiang Yu, Cagla Eroglu.
      Dysfunctional mitochondrial dynamics are a hallmark of devastating neurodevelopmental disorders such as childhood refractory epilepsy. However, the role of glial mitochondria in proper brain development is not well understood. We show that astrocyte mitochondria undergo extensive fission while populating astrocyte distal branches during postnatal cortical development. Loss of mitochondrial fission regulator, Dynamin-related protein 1 (Drp1), decreases mitochondrial localization to distal astrocyte processes, and this mitochondrial mislocalization reduces astrocyte morphological complexity. Functionally, astrocyte-specific conditional deletion of Drp1 induces astrocyte reactivity and disrupts astrocyte organization in the cortex. These morphological and organizational deficits are accompanied by loss of astrocytic gap junction protein Connexin 43. These findings uncover a crucial role for mitochondrial fission in coordinating astrocytic morphogenesis and organization, revealing the regulation of astrocytic mitochondria dynamics as a critical step in neurodevelopment.Summary: During cortical astrocyte morphogenesis, mitochondria decrease in size to populate distal astrocyte processes. Drp1-mediated mitochondrial fission is necessary for peripheral astrocyte process formation. Astrocyte-specific Drp1 loss induces astrocyte reactivity, disrupts cortical astrocyte organization, and dysregulates gap-junction protein Connexin 43 abundance.
    DOI:  https://doi.org/10.1101/2024.10.22.619706
  20. Methods Enzymol. 2024 ;pii: S0076-6879(24)00369-0. [Epub ahead of print]706 407-436
    The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.
    Hope I Needs, Youmian Yan, Natalie M Niemi, Ian Collinson.
      The NanoLuc split luciferase assay has proven to be a powerful tool for the analysis of protein translocation. Its flexibility has enabled in vivo, ex vivo, and in vitro studies-including systems reconstituting protein transport from pure components. The assay has been particularly useful in the characterization of bacterial secretion and mitochondrial protein import. In the latter case, MitoLuc has been developed for the investigation of the TIM23-pathway via import into the matrix of isolated yeast mitochondria. Subsequent analysis identified three distinct phases of import, rather than in a single continuous step. The assay has also been developed to monitor import into the mitochondrial matrix of intact cultured cells. This latter innovation has laid the foundations for further analysis of the import process in humans, including the consequences of interactions with cytosolic factors and neighboring organelles. The versatility of the MitoLuc assay is conducive for its adaptation to also monitor import into the inter-membrane space (MIA-pathway), and into the inner-membrane via the TIM22- and TIM23-complexes. Here, we present detailed protocols for the application of MitoLuc to mitochondria isolated from yeast and to those within cultured human cells.
    Keywords:  Cell culture; Luciferase; MitoLuc assay; Mitochondrial biogenesis; Mitochondrial protein import; NanoLuc; Protein translocation; Yeast
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.033
  21. Protein Sci. 2024 Nov;33(11): e5197
    Unraveling the molecular determinants of a rare human mitochondrial disorder caused by the P144L mutation of FDX2.
    Deborah Grifagni, Davide Doni, Bianca Susini, Bruno M Fonseca, Ricardo O Louro, Paola Costantini, Simone Ciofi-Baffoni.
      Episodic mitochondrial myopathy with or without optic atrophy and reversible leukoencephalopathy (MEOAL) is a rare, orphan autosomal recessive disorder caused by mutations in ferredoxin-2 (FDX2), which is a [2Fe-2S] cluster-binding protein participating in the formation of iron-sulfur clusters in mitochondria. In this biosynthetic pathway, FDX2 works as electron donor to promote the assembly of both [2Fe-2S] and [4Fe-4S] clusters. A recently identified missense mutation of MEOAL is the homozygous mutation c.431C>T (p.P144L) described in six patients from two unrelated families. This mutation alters a highly conserved proline residue located in a loop of FDX2 that is distant from the [2Fe-2S] cluster. How this Pro to Leu substitution damages iron-sulfur cluster biosynthesis is unknown. In this work, we have first compared the structural, dynamic, cluster binding and redox properties of WT and P144L [2Fe-2S] FDX2 to have clues on how the pathogenic P144L mutation can perturb the FDX2 function. Then, we have investigated the interaction of both WT and P144L [2Fe-2S] FDX2 with its physiological electron donor, ferredoxin reductase FDXR, comparing their electron transfer efficiency and protein-protein recognition patterns. Overall, the data indicate that the pathogenic P144L mutation negatively affects the FDXR-dependent electron transfer pathway from NADPH to FDX2, thereby reducing the capacity of FDX2 in assembling both [2Fe-2S] and [4Fe-4S] clusters. Our study also provided solid molecular evidences on the functional role of the C-terminal tail of FDX2 in the electron transfer between FDX2 and FDXR.
    Keywords:  FDX2; ISC machinery; MEOAL; NMR; P144L; iron–sulfur protein; rare disease
    DOI:  https://doi.org/10.1002/pro.5197
  22. Methods Enzymol. 2024 ;pii: S0076-6879(24)00360-4. [Epub ahead of print]706 3-18
    Isolation of yeast mitochondria by differential centrifugation.
    Kuo Song, Heike Rampelt.
      The isolation of intact and functional mitochondria is a powerful approach to characterize and study this organelle. The classical biochemical method of differential centrifugation is routinely used to isolate mitochondria. This method has several advantages, such as a high yield and easy adaptability. The isolated mitochondria are physiologically active and can be used for a variety of follow-up experiments, for example protein import and respiration measurements. Here, we describe the procedure to purify mitochondria from the budding yeast Saccharomyces cerevisiae. In addition, two approaches are introduced to assess the quality of isolated mitochondria, by limited proteinase K digestion or measurement of the membrane potential.
    Keywords:  Mitochondrial preparation; fractionation; limited proteolysis; membrane potential; organelle isolation
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.024
  23. Methods Enzymol. 2024 ;pii: S0076-6879(24)00378-1. [Epub ahead of print]706 263-283
    Analysis and prediction of internal mitochondrial targeting signals.
    Felix Jung, Saskia Rödl, Johannes M Herrmann, Timo Mühlhaus.
      Mitochondria consist of several hundreds of proteins, the vast majority of which are synthesized in the cytosol as precursor proteins from where they are targeted to and imported into mitochondria. The transport of proteins into mitochondria relies on specific targeting information encoded within the protein sequence, known as mitochondrial targeting sequences (MTSs). These N-terminal extensions are usually between 8 and 80 residues long and form amphipathic helices with one hydrophobic and one positively charged surface. Receptors on the mitochondrial surface recognize the MTSs and direct precursors through protein-conducting channels in the outer and inner membrane to the mitochondrial matrix, where presequences are often removed by proteases. In addition to these MTSs, many mitochondrial proteins contain internal matrix targeting sequences (iMTSs) which share the same structural features with MTSs. These iMTSs are neither necessary nor sufficient for mitochondrial targeting, however, they help to increase the import-competence of precursor proteins as they bind to the TOM receptors and presumably facilitate the unfolding of precursors on the mitochondrial surface. Prediction algorithms allow the identification of iMTSs in protein sequences. In this chapter, we present iMLP, an agnostic algorithm for the prediction of iMTS propensity profiles. This iMTS prediction tool is provided via an iMLP webservice at http://iMLP.bio.uni-kl.de and is also available as a BioFSharp application that can be executed locally. We describe and explain the usage of this prediction algorithm and how to interpret the results of this valuable tool.
    Keywords:  IMTS; Machine learning; Mitochondria; Presequence; Protein targeting; Sequence analysis; TargetP; Targeting prediction; Tom70; Webservice
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.038
  24. Cell Metab. 2024 Oct 25. pii: S1550-4131(24)00409-1. [Epub ahead of print]
    A hierarchical hepatic de novo lipogenesis substrate supply network utilizing pyruvate, acetate, and ketones.
    Adam J Rauckhorst, Ryan D Sheldon, Daniel J Pape, Adnan Ahmed, Kelly C Falls-Hubert, Ronald A Merrill, Reid F Brown, Kshitij Deshmukh, Thomas A Vallim, Stanislaw Deja, Shawn C Burgess, Eric B Taylor.
      Hepatic de novo lipogenesis (DNL) is a fundamental physiologic process that is often pathogenically elevated in metabolic disease. Treatment is limited by incomplete understanding of the metabolic pathways supplying cytosolic acetyl-CoA, the obligate precursor to DNL, including their interactions and proportional contributions. Here, we combined extensive 13C tracing with liver-specific knockout of key mitochondrial and cytosolic proteins mediating cytosolic acetyl-CoA production. We show that the mitochondrial pyruvate carrier (MPC) and ATP-citrate lyase (ACLY) gate the major hepatic lipogenic acetyl-CoA production pathway, operating in parallel with acetyl-CoA synthetase 2 (ACSS2). Given persistent DNL after mitochondrial citrate carrier (CiC) and ACSS2 double knockout, we tested the contribution of exogenous and leucine-derived acetoacetate to acetoacetyl-CoA synthetase (AACS)-dependent DNL. CiC knockout increased acetoacetate-supplied hepatic acetyl-CoA production and DNL, indicating that ketones function as mitochondrial-citrate reciprocal DNL precursors. By delineating a mitochondrial-cytosolic DNL substrate supply network, these findings may inform strategies to therapeutically modulate DNL.
    Keywords:  AACS; ACLY; ACSS2; ATP-citrate lyase; CiC; DNL; MPC; acetoacetyl-CoA synthetase; acetyl-CoA synthetase 2; de novo lipogenesis; liver; metabolomics; mitochondrial citrate carrier; mitochondrial pyruvate carrier; stable isotope tracers
    DOI:  https://doi.org/10.1016/j.cmet.2024.10.013
  25. Mol Genet Genomics. 2024 Oct 26. 299(1): 102
    A novel m.5906G > a variant in MT-CO1 causes MELAS/Leigh overlap syndrome.
    Zhimei Liu, Yaojun Xie, Xiaoting Lou, Xiaofei Zeng, Luyi Zhang, Meng Yu, Junling Wang, Jiuwei Li, Danmin Shen, Hua Li, Suzhou Zhao, Yuwei Zhou, Hezhi Fang, Jianxin Lyu, Yun Yuan, Zhaoxia Wang, Liqin Jin, Fang Fang.
      The MELAS/Leigh overlap syndrome manifests with a blend of clinical and radiographic traits from both MELAS and LS. However, the association of MELAS/Leigh overlap syndrome with MT-CO1 gene variants has not been previously reported. In this study, we report a patient diagnosed with MELAS/Leigh overlap syndrome harboring the m.5906G > A variant in MT-CO1, with biochemical evidence supporting the pathogenicity of the variant. The variant m.5906G > A that led to a synonymous variant in the start codon of MT-CO1 was filtered as the candidate disease-causing variant of the patient. Patient-derived fibroblasts were used to generate a series of monoclonal cells carrying different m.5906G > A variant loads for further functional assays. The oxygen consumption rate, ATP production, mitochondrial membrane potential and lactate assay indicated an impairment of cellular bioenergetics due to the m.5906G > A variant. Blue native PAGE analysis revealed that the m.5906G > A variant caused a deficiency in the content of mitochondrial oxidative phosphorylation complexes. Furthermore, molecular biology assays performed for the pathogenesis, mtDNA copy number, mtDNA-encoded subunits, and recovery capacity of mtDNA were all deficient due to the m.5906G > A variant, which might be caused by mtDNA replication deficiency. Overall, our findings demonstrated the pathogenicity of m.5906G > A variant and proposed a potential pathogenic mechanism, thereby expanding the genetic spectrum of MELAS/Leigh overlap syndrome.
    Keywords:  M.5906G > A; MT-CO1 ; MELAS/Leigh overlap syndrome; MtDNA replication
    DOI:  https://doi.org/10.1007/s00438-024-02181-y
  26. Pharmacol Res. 2024 Oct 29. pii: S1043-6618(24)00430-4. [Epub ahead of print] 107485
    The Mysteries of LETM1 Pleiotropy.
    Sami E M Mohammed, Karin Nowikovsky.
      LETM1 is a nuclear-encoded protein located in the inner mitochondrial membrane, playing a critical role in regulating mitochondrial cation and volume homeostasis. However, numerous studies on functional features, molecular interactions, and disease-associated effects of LETM1 revealed that LETM1 is also involved in other metabolic functions including glucose utilization, mitochondrial DNA and ribosome organization, cristae architecture and respiratory complex stability. Undisputedly, osmoregulatory processes are essential for mitochondrial functionality, but the pleiotropic aspects of LETM1 challenges us to understand the core function of LETM1, which still remains elusive. In this review, we provide an overview of the current knowledge and latest developments regarding the activities involving LETM1. We highlight various findings that offer different functional perspectives and ideas on the core function of LETM1. Specifically, we emphasize data supporting LETM1's role as a mitochondrial translational factor, K+/H+ exchanger, or Ca2+/H+ exchanger, along with recent findings on its interaction with ATAD3A and TMBIM5. We also present the severe clinical implications of LETM1 deficiency. Finally, we discuss emerging questions raised by the different views on LETM1, which need to be addressed to guide future research directions and ultimately resolve the function of this essential protein and develop targeted therapeutic strategies.
    Keywords:  ATAD3A; Ca(2+)/H(+) exchanger; K(+)/H(+) exchanger; LETM1; TMBIM5; mitochondria
    DOI:  https://doi.org/10.1016/j.phrs.2024.107485
  27. Methods Enzymol. 2024 ;pii: S0076-6879(24)00368-9. [Epub ahead of print]706 19-36
    Isolation of yeast mitochondria by affinity purification using magnetic beads.
    Tzu-Ying Lin, Shih-Hung Chien, Liza A Pon, Pin-Chao Liao.
      Isolated mitochondria have been widely utilized in various model organisms to investigate the diverse functions of the organelle. Techniques such as differential centrifugation, density gradient ultracentrifugation and antibody-coated magnetic beads are employed for isolation of the organelle from whole cells. However, mitochondria isolated using differential centrifugation are often contaminated with other organelles; isolation using density gradient ultracentrifugation can reduce contamination but is time-intensive and requires large amounts of starting materials; and mitochondria isolated using antibody-coated magnetic beads are irreversibly bound to the beads. Here, we provide a step-by-step protocol for the isolation of highly pure mitochondria from Saccharomyces cerevisiae using a magnetic bead affinity purification method that overcomes these limitations. This protocol describes how to isolate mitochondria, tagged by insertion of 6 histidines (6xHis) into the chromosomal copy of the TOM70 (Translocase of outer membrane 70) gene using Ni-NTA (nickel(II) nitrilotriacetic acid) paramagnetic beads, and the subsequent release of mitochondria from the beads using a buffer containing imidazole. We provide examples of expected results, highlighting the purity, integrity and import activity of isolated mitochondria. These affinity-purified mitochondria are intact and functional, containing less contamination with cytosol and other organelles compared to mitochondria isolated by other methods. Our method is adaptable and can be applied to other model organisms that can be genetically manipulated using CRISPR or other methods.
    Keywords:  Affinity purification; Budding yeast; Magnetic beads; Mitochondrial import; Mitochondrial isolation
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.032
  28. Anal Chem. 2024 Nov 01.
    Mass Spectrometry Probe Combined with Machine Learning to Capture the Relationship between Metabolites and Mitochondrial Complex Activity at the Whole-Cell Level.
    Jia-Yi Zheng, Xiao-Yuan Ji, An-Qi Zhao, Fang-Yuan Sun, Li-Fang Liu, Gui-Zhong Xin.
      Mitochondrial complex activity controls a multitude of physiological processes by regulating the cellular metabolism. Current methods for evaluating mitochondrial complex activity mainly focus on single metabolic reactions within mitochondria. These methods often require fresh samples in large quantities for mitochondria purification or intact mitochondrial membranes for real-time monitoring. Confronting these limitations, we shifted the analytical perspective toward interactive metabolic networks at the whole-cell level to reflect mitochondrial complex activity. To this end, we compiled a panel of mitochondrial respiratory chain-mapped metabolites (MRCMs), whose perturbations theoretically provide an overall reflection on mitochondrial complex activity. By introducing N-dimethyl-p-phenylenediamine and N-methyl-p-phenylenediamine as a pair of mass spectrometry probes, an ultraperformance liquid chromatography-tandem mass spectrometry method with high sensitivity (LLOQ as low as 0.2 fmol) was developed to obtain accurate quantitative data of MRCMs. Machine learning was then combined to capture the relationship between MRCMs and mitochondrial complex activity. Using Complex I as a proof-of-concept, we identified NADH, alanine, and phosphoenolpyruvate as metabolites associated with Complex I activity based on the whole-cell level. The effectiveness of using their concentrations to reflect Complex I activity was further validated in external data sets. Hence, by capturing the relationship between metabolites and mitochondrial complex activity at the whole-cell level, this study explores a novel analytical paradigm for the interrogation of mitochondrial complex activity, offering a favorable complement to existing methods particularly when sample quantities, type, and treatment timeliness pose challenges. More importantly, it shifts the focus from individual metabolic reactions within mitochondria to a more comprehensive view of an interactive metabolic network, which should serve as a promising direction for future research into the functional architecture between mitochondrial complexes and metabolites.
    DOI:  https://doi.org/10.1021/acs.analchem.4c04376
  29. Nat Commun. 2024 Oct 29. 15(1): 9340
    Structure of the turnover-ready state of an ancestral respiratory complex I.
    Bozhidar S Ivanov, Hannah R Bridges, Owen D Jarman, Judy Hirst.
      Respiratory complex I is pivotal for cellular energy conversion, harnessing energy from NADH:ubiquinone oxidoreduction to drive protons across energy-transducing membranes for ATP synthesis. Despite detailed structural information on complex I, its mechanism of catalysis remains elusive due to lack of accompanying functional data for comprehensive structure-function analyses. Here, we present the 2.3-Å resolution structure of complex I from the α-proteobacterium Paracoccus denitrificans, a close relative of the mitochondrial progenitor, in phospholipid-bilayer nanodiscs. Three eukaryotic-type supernumerary subunits (NDUFS4, NDUFS6 and NDUFA12) plus a novel L-isoaspartyl-O-methyltransferase are bound to the core complex. Importantly, the enzyme is in a single, homogeneous resting state that matches the closed, turnover-ready (active) state of mammalian complex I. Our structure reveals the elements that stabilise the closed state and completes P. denitrificans complex I as a unified platform for combining structure, function and genetics in mechanistic studies.
    DOI:  https://doi.org/10.1038/s41467-024-53679-3
  30. J Mol Neurosci. 2024 Oct 28. 74(4): 101
    Signaling Pathways Concerning Mitochondrial Dysfunction: Implications in Neurodegeneration and Possible Molecular Targets.
    Yati Sharma, Jeetendra Kumar Gupta, M Arockia Babu, Sumitra Singh, Rakesh K Sindhu.
      Mitochondrion is an important organelle present in our cells responsible for meeting energy requirements. All higher organisms rely on efficient mitochondrial bioenergetic machinery to sustain life. No other respiratory process can produce as much power as generated by mitochondria in the form of ATPs. This review is written in order to get an insight into the magnificent working of mitochondrion and its implications in cellular homeostasis, bioenergetics, redox, calcium signaling, and cell death. However, if this machinery gets faulty, it may lead to several disease states. Mitochondrial dysfunctioning is of growing concern today as it is seen in the pathogenesis of several diseases which includes neurodegenerative disorders, cardiovascular disorders, diabetes mellitus, skeletal muscle defects, liver diseases, and so on. To cover all these aspects is beyond the scope of this article; hence, our study is restricted to neurodegenerative disorders only. Moreover, faulty functioning of this organelle can be one of the causes of early ageing in individuals. This review emphasizes mutations in the mitochondrial DNA, defects in oxidative phosphorylation, generation of ROS, and apoptosis. Researchers have looked into new approaches that might be able to control mitochondrial failure and show a lot of promise as treatments.
    Keywords:  Mitochondrial dysfunction; Neurodegeneration; Neurotoxicity; Oxidative stress
    DOI:  https://doi.org/10.1007/s12031-024-02269-5
  31. Sci Adv. 2024 Nov;10(44): eadp7725
    Membrane potential stimulates ADP import and ATP export by the mitochondrial ADP/ATP carrier due to its positively charged binding site.
    Vasiliki Mavridou, Martin S King, Andre Bazzone, Roger Springett, Edmund R S Kunji.
      The mitochondrial adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier imports ADP into the mitochondrion and exports ATP to the cell. Here, we demonstrate that 3.3 positive charges are translocated with the negatively charged substrate in each transport step. They can be assigned to three positively charged residues of the central substrate-binding site and two asparagine/arginine pairs. In this way, the membrane potential stimulates not only the ATP4- export step, as a net -0.7 charge is transported, but also the ADP3- import step, as a net +0.3 charge is transported with the electric field. These positive charge movements also inhibit the import of ATP and export of ADP in the presence of a membrane potential, allowing these nucleotides to be maintained at high concentrations in the cytosol and mitochondrial matrix to drive the hydrolysis and synthesis of ATP, respectively. Thus, this is the mechanism by which the membrane potential drives adenine nucleotide exchange with high directional fluxes to fuel the cellular processes.
    DOI:  https://doi.org/10.1126/sciadv.adp7725
  32. Sci Rep. 2024 10 30. 14(1): 26051
    Mitochondrial inhibitors reveal roles of specific respiratory chain complexes in CRY-dependent degradation of TIM.
    Xiangzhong Zheng, Dechun Chen, Brian Zoltowski, Amita Sehgal.
      Drosophila Cryptochrome (CRY) is an essential photoreceptor that mediates the resetting of the circadian clock by light. in vitro studies demonstrated a critical role of redox cycling of the FAD cofactor for CRY activation by light. However, it is unknown if CRY responds to cellular redox environment to modulate the circadian clock. We report here that the mitochondrial respiratory chain impinges on CRY activity. Inhibition of complex III and V blocks CRY-mediated degradation of TIMELESS (TIM) in response to light, and also blocks light-induced CRY degradation. On the other hand, inhibition of complex I facilitates TIM degradation even in the dark. Mutations of critical residues of the CRY C-terminus promote TIM degradation in the dark, even in the presence of complex III and V inhibitors. We propose that complex III and V activities are important for activation of CRY in response to light. Interestingly, we found that transcriptional repressor functions of Drosophila and mammalian CRY proteins are not affected by mitochondrial inhibitors. Together these data suggest that the two functions of CRY have different sensitivity to disruptions of the mitochondrial respiratory chain: one is sensitive to mitochondrial activities that enable resetting, the other is insensitive so as to sustain the molecular oscillator.
    Keywords:  Circadian clock; Cryptochrome; Mitochondria; Respiratory chain; Timeless
    DOI:  https://doi.org/10.1038/s41598-024-77692-0
  33. Proc Natl Acad Sci U S A. 2024 Nov 05. 121(45): e2406174121
    COX15 deficiency causes oocyte ferroptosis.
    Zhihua Zhang, Ran Yu, Qiuwen Shi, Zhi-Jing Wu, Qingchun Li, Jian Mu, Biaobang Chen, Juanzi Shi, Renmin Ni, Ling Wu, Qiaoli Li, Jing Fu, Rong Li, Xiaoxi Sun, Jiucun Wang, Lin He, Yanping Kuang, Qing Sang, Lei Wang.
      Mitochondria play diverse roles in mammalian physiology. The architecture, activity, and physiological functions of mitochondria in oocytes are largely different from those in somatic cells, but the mitochondrial proteins related to oocyte quality and reproductive longevity remain largely unknown. Here, using whole-exome sequencing data from 1,024 women (characterized by oocyte maturation arrest and degenerated or morphologically abnormal oocytes) and 2,868 healthy controls, we performed a population and gene-based burden test for mitochondrial genes and identified a candidate gene, cytochrome c oxidase assembly protein 15 (COX15). We report that biallelic COX15 pathogenic variants cause human oocyte ferroptosis and female infertility in a recessive inheritance pattern. COX15 variants impaired mitochondrial respiration in Saccharomyces cerevisiae and led to reduced protein levels in HeLa cells. Oocyte-specific deletion of Cox15 led to impaired Fe2+ and reactive oxygen species homeostasis that caused mitochondrial dysfunction and ultimately sensitized oocytes to ferroptosis. In addition, ferrostatin-1 (an inhibitor of ferroptosis) could rescue the oocyte ferroptosis phenotype in vitro and ex vivo. Our findings not only provide a genetic diagnostic marker for oocyte development defects but also expand the spectrum of mitochondrial disorders to female infertility and contribute to unique insights into the role of ferroptosis in human oocyte defects.
    Keywords:  COX15 deficiency; female infertility; ferroptosis; mitochondrial disorders; oocyte defects
    DOI:  https://doi.org/10.1073/pnas.2406174121
  34. Methods Enzymol. 2024 ;pii: S0076-6879(24)00362-8. [Epub ahead of print]706 161-192
    Prediction of mitochondrial targeting signals and their cleavage sites.
    Fukasawa Yoshinori, Kenichiro Imai, Paul Horton.
      In this chapter we survey prediction tools and computational methods for the prediction of amino acid sequence elements which target proteins to the mitochondria. We will primarily focus on the prediction of N-terminal mitochondrial targeting signals (MTSs) and their N-terminal cleavage sites by mitochondrial peptidases. We first give practical details useful for using and installing some prediction tools. Then we describe procedures for preparing datasets of MTS containing proteins for statistical analysis or development of new prediction methods. Following that we lightly survey some of the computational techniques used by prediction tools. Finally, after discussing some caveats regarding the reliability of such methods to predict the effects of mutations on MTS function; we close with a discussion of possible future directions of computer prediction methods related to mitochondrial proteins.
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.026
  35. Methods Enzymol. 2024 ;pii: S0076-6879(24)00359-8. [Epub ahead of print]706 391-405
    Import of mitochondrial precursor proteins into mitochondria of semi-intact yeast cells.
    Johannes M Herrmann, Svenja Lenhard, Katja G Hansen.
      Mitochondria import hundreds of different precursor proteins from the cytosol and direct each of these to its specific mitochondrial subcompartment. The import routes and mechanisms by which precursors are transported into the outer membrane, the intermembrane space (IMS), the inner membrane and the matrix have been characterized in depth by use of very powerful in vitro assays. In the 'classical' import assays, radiolabeled precursor proteins are incubated with isolated mitochondria and the protein uptake is monitored by one or more of the following observations: intramitochondrial processing, resistance to externally added proteases, or the formation of disulfide bonds. In this chapter, we describe an alternative import assay which employs semi-intact yeast cells. This assay uses spheroplasts from which the cell wall had been removed by enzymatic digestion before the plasma membrane was partially permeabilized by a freeze-thawing step. Since the organellar architecture is largely maintained in semi-intact cells, this in vitro import assay allows to elucidate the targeting of precursor proteins from the cytoplasm to the mitochondrial surface. Thereby the contribution of other compartments such as the endoplasmic reticulum (ER) can be assessed. Here we describe how semi-intact cells are prepared and used in the in vitro import assay and discuss the pros and cons of this approach.
    Keywords:  Intracellular targeting; Mitochondria; Organellar contact sites; Protein import; Protein targeting; Radiolabeled precursor proteins; Spheroplasts
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.023
  36. bioRxiv. 2024 Oct 24. pii: 2024.10.24.620005. [Epub ahead of print]
    TFAM as a sensor of UVC-induced mitochondrial DNA damage.
    Dillon E King, Emily E Beard, Matthew J Satusky, Ian Ryde, Alex George, Caitlin Johnson, Emma L Dolan, Yuning Zhang, Wei Zhu, Hunter Wilkins, Evan Corden, Susan K Murphy, Dorothy Erie, Raluca Gordân, Joel N Meyer.
      Mitochondria lack nucleotide excision DNA repair; however, mitochondrial DNA (mtDNA) is resistant to mutation accumulation following DNA damage. These observations suggest additional damage sensing or protection mechanisms. Transcription Factor A, Mitochondrial (TFAM) compacts mtDNA into nucleoids. As such, TFAM has emerged as a candidate for protecting DNA or sensing damage. To examine these possibilities, we used live-cell imaging, cell-based assays, atomic force microscopy, and high-throughput protein-DNA binding assays to characterize the binding properties of TFAM to UVC-irradiated DNA and cellular consequences of UVC irradiation. Our data indicate an increase in mtDNA degradation and turnover, without a loss in mitochondrial membrane potential that might trigger mitophagy. We identified a reduction in sequence specificity of TFAM associated with UVC irradiation and a redistribution of TFAM binding throughout the mitochondrial genome. Our AFM data show increased compaction of DNA by TFAM in the presence of damage. Despite the TFAM-mediated compaction of mtDNA, we do not observe any protective effect on DNA damage accumulation in cells or in vitro . Taken together, these studies indicate that UVC-induced DNA damage promotes compaction by TFAM, suggesting that TFAM may act as a damage sensor, sequestering damaged genomes to prevent mutagenesis by direct removal or suppression of replication.
    DOI:  https://doi.org/10.1101/2024.10.24.620005
  37. Sci Adv. 2024 Nov;10(44): eadk8801
    Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis.
    Spencer D Shelton, Sara House, Luiza Martins Nascentes Melo, Vijayashree Ramesh, Zhenkang Chen, Tao Wei, Xun Wang, Claire B Llamas, Siva Sai Krishna Venigalla, Cameron J Menezes, Gabriele Allies, Jonathan Krystkiewicz, Jonas Rösler, Sven W Meckelmann, Peihua Zhao, Florian Rambow, Dirk Schadendorf, Zhiyu Zhao, Jennifer G Gill, Ralph J DeBerardinis, Sean J Morrison, Alpaslan Tasdogan, Prashant Mishra.
      Mitochondrial DNA (mtDNA) mutations are frequent in cancer, yet their precise role in cancer progression remains debated. To functionally evaluate the impact of mtDNA variants on tumor growth and metastasis, we developed an enhanced cytoplasmic hybrid (cybrid) generation protocol and established isogenic human melanoma cybrid lines with wild-type mtDNA or pathogenic mtDNA mutations with partial or complete loss of mitochondrial oxidative function. Cybrids with homoplasmic levels of pathogenic mtDNA reliably established tumors despite dysfunctional oxidative phosphorylation. However, these mtDNA variants disrupted spontaneous metastasis from primary tumors and reduced the abundance of circulating tumor cells. Migration and invasion of tumor cells were reduced, indicating that entry into circulation is a bottleneck for metastasis amid mtDNA dysfunction. Pathogenic mtDNA did not inhibit organ colonization following intravenous injection. In heteroplasmic cybrid tumors, single-cell analyses revealed selection against pathogenic mtDNA during melanoma growth. Collectively, these findings experimentally demonstrate that functional mtDNA is favored during melanoma growth and supports metastatic entry into the blood.
    DOI:  https://doi.org/10.1126/sciadv.adk8801
  38. J Cachexia Sarcopenia Muscle. 2024 Oct 31.
    Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.
    Makayla S Lancaster, Paul Hafen, Andrew S Law, Catalina Matias, Timothy Meyer, Kathryn Fischer, Marcus Miller, Chunhai Hao, Patrick Gillespie, David McKinzie, Jeffrey J Brault, Brett H Graham.
      BACKGROUND: Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel in vivo model of mitochondrial myopathy.METHODS: The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model in vivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, ex vivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes.
    RESULTS: Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls.
    CONCLUSIONS: SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in ex vivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.
    Keywords:  contractility; extensor digitorum longus; fibre‐type switching; mitochondrial myopathy; soleus; succinyl‐CoA synthetase
    DOI:  https://doi.org/10.1002/jcsm.13617
  39. Commun Biol. 2024 Oct 31. 7(1): 1422
    The mitochondrial citrate carrier SLC25A1 regulates metabolic reprogramming and morphogenesis in the developing heart.
    Chiemela Ohanele, Jessica N Peoples, Anja Karlstaedt, Joshua T Geiger, Ashley D Gayle, Nasab Ghazal, Fateemaa Sohani, Milton E Brown, Michael E Davis, George A Porter, Victor Faundez, Jennifer Q Kwong.
      The developing mammalian heart undergoes an important metabolic shift from glycolysis towards mitochondrial oxidation that is critical to support the increasing energetic demands of the maturing heart. Here, we describe a new mechanistic link between mitochondria and cardiac morphogenesis, uncovered by studying mitochondrial citrate carrier (SLC25A1) knockout mice. Slc25a1 null embryos displayed impaired growth, mitochondrial dysfunction and cardiac malformations that recapitulate the congenital heart defects observed in 22q11.2 deletion syndrome, a microdeletion disorder involving the SLC25A1 locus. Importantly, Slc25a1 heterozygous embryos, while overtly indistinguishable from wild type, exhibited an increased frequency of these defects, suggesting Slc25a1 haploinsuffiency and dose-dependent effects. Mechanistically, SLC25A1 may link mitochondria to transcriptional regulation of metabolism through epigenetic control of gene expression to promote metabolic remodeling in the developing heart. Collectively, this work positions SLC25A1 as a novel mitochondrial regulator of cardiac morphogenesis and metabolic maturation, and suggests a role in congenital heart disease.
    DOI:  https://doi.org/10.1038/s42003-024-07110-8
  40. Methods Enzymol. 2024 ;pii: S0076-6879(24)00377-X. [Epub ahead of print]706 61-73
    Subcellular fractionation by differential centrifugation for mitochondrial studies.
    Conny Steiert, Jon V Busto, Laura Melchionda, Nils Wiedemann.
      In addition to fluorescence microscopy, the subcellular fractionation of eukaryotic cells remains one of the central methods for the basic characterization of proteins. Here we describe an optimized procedure for the subcellular fractionation of yeast cells, specifically for mitochondrial studies. Major recommendations are to separate the fractions immediately after each centrifugation step, to carefully discard a significant part of the supernatant fractions which is in the direct vicinity to the pellets and, in addition, to perform an extra homogenization step of the post nuclear supernatant fraction. These principles help to collect supernatant fractions with less cross-contaminations from the corresponding pellets. These approaches are scalable and adaptable for the fractionation of other cell types and are also useful for the characterization of other organelles.
    Keywords:  Cell organelles; Cytosol; Endoplasmic reticulum; Microsomes; Mitochondria; Nucleus; Post nuclear supernatant; Saccharomyces cerevisiae; Yeast
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.037
  41. Rom J Ophthalmol. 2024 Jul-Sep;68(3):68(3): 338-339
    Genetic family studies and prospective evaluation for multisystem involvement are needed in LHON patients.
    Josef Finsterer.
      
    Keywords:  LHON; idebenone; mitochondrial disorder; mtDNA; visual acuity
    DOI:  https://doi.org/10.22336/rjo.2024.62
  42. Cell Commun Signal. 2024 Oct 29. 22(1): 525
    The multifaceted role of mitochondria in cardiac function: insights and approaches.
    Sriram Ravindran, Christoph D Rau.
      Cardiovascular disease (CVD) remains a global economic burden even in the 21st century with 85% of deaths resulting from heart attacks. Despite efforts in reducing the risk factors, and enhancing pharmacotherapeutic strategies, challenges persist in early identification of disease progression and functional recovery of damaged hearts. Targeting mitochondrial dysfunction, a key player in the pathogenesis of CVD has been less successful due to its role in other coexisting diseases. Additionally, it is the only organelle with an agathokakological function that is a remedy and a poison for the cell. In this review, we describe the origins of cardiac mitochondria and the role of heteroplasmy and mitochondrial subpopulations namely the interfibrillar, subsarcolemmal, perinuclear, and intranuclear mitochondria in maintaining cardiac function and in disease-associated remodeling. The cumulative evidence of mitochondrial retrograde communication with the nucleus is addressed, highlighting the need to study the genotype-phenotype relationships of specific organelle functions with CVD by using approaches like genome-wide association study (GWAS). Finally, we discuss the practicality of computational methods combined with single-cell sequencing technologies to address the challenges of genetic screening in the identification of heteroplasmy and contributory genes towards CVD.
    Keywords:  Cardiovascular disease; Cell-organelle communication; Computational biology; Mitochondrial genetics; Mitochondrial subpopulations; Organellogenesis
    DOI:  https://doi.org/10.1186/s12964-024-01899-x
  43. NPJ Genom Med. 2024 Oct 25. 9(1): 48
    Biallelic GGGCC repeat expansion leading to NAXE-related mitochondrial encephalopathy.
    Kokoro Ozaki, Yukiko Yatsuka, Yoshinobu Oyazato, Atsushi Nishiyama, Kazuhiro R Nitta, Yoshihito Kishita, Takuya Fushimi, Masaru Shimura, Shohei Noma, Yohei Sugiyama, Michihira Tagami, Moe Fukunaga, Hiroko Kinoshita, Tomoko Hirata, Wataru Suda, Yasuhiro Murakawa, Piero Carninci, Akira Ohtake, Kei Murayama, Yasushi Okazaki.
      Repeat expansions cause at least 50 hereditary disorders, including Friedreich ataxia and other diseases known to cause mitochondrial dysfunction. We identified a patient with NAXE-related mitochondrial encephalopathy and novel biallelic GGGCC repeat expansion as long as ~200 repeats in the NAXE promoter region using long-read sequencing. In addition to a marked reduction in the RNA and protein, we found a marked reduction in nascent RNA in the promoter using native elongating transcript-cap analysis of gene expression (NET-CAGE), suggesting transcriptional suppression. Accordingly, CpG hypermethylation was observed in the repeat region. Genetic analyses determined that homozygosity in the patient was due to maternal chromosome 1 uniparental disomy (UPD). We assessed short variants within NAXE including the repeat region in the undiagnosed mitochondrial encephalopathy cohort of 242 patients. This study identified the GGGCC repeat expansion causing a mitochondrial disease and suggests that UPD could significantly contribute to homozygosity for rare repeat-expanded alleles.
    DOI:  https://doi.org/10.1038/s41525-024-00429-5
  44. Biomolecules. 2024 Oct 09. pii: 1270. [Epub ahead of print]14(10):
    Mitophagy in Cell Death Regulation: Insights into Mechanisms and Disease Implications.
    Jiani Lin, Xinyao Chen, Yuyang Du, Jiapeng Li, Tingting Guo, Sai Luo.
      Mitophagy, a selective form of autophagy, plays a crucial role in maintaining optimal mitochondrial populations, normal function, and intracellular homeostasis by monitoring and removing damaged or excess mitochondria. Furthermore, mitophagy promotes mitochondrial degradation via the lysosomal pathway, and not only eliminates damaged mitochondria but also regulates programmed cell death-associated genes, thus preventing cell death. The interaction between mitophagy and various forms of cell death has recently gained increasing attention in relation to the pathogenesis of clinical diseases, such as cancers and osteoarthritis, neurodegenerative, cardiovascular, and renal diseases. However, despite the abundant literature on this subject, there is a lack of understanding regarding the interaction between mitophagy and cell death. In this review, we discuss the main pathways of mitophagy, those related to cell death mechanisms (including apoptosis, ferroptosis, and pyroptosis), and the relationship between mitophagy and cell death uncovered in recent years. Our study offers potential directions for therapeutic intervention and disease diagnosis, and contributes to understanding the molecular mechanism of mitophagy.
    Keywords:  cell death; intracellular homeostasis; mitochondria; mitophagy
    DOI:  https://doi.org/10.3390/biom14101270
  45. Biomolecules. 2024 Sep 26. pii: 1218. [Epub ahead of print]14(10):
    Phospholipid Scramblase Activity of VDAC Dimers: New Implications for Cell Death, Autophagy and Ageing.
    Patrick Rockenfeller.
      Voltage-dependent anion channels (VDACs) are important proteins of the outer mitochondrial membrane (OMM). Their beta-barrel structure allows for efficient metabolite exchange between the cytosol and mitochondria. VDACs have further been implicated in the control of regulated cell death. Historically, VDACs have been pictured as part of the mitochondrial permeability transition pore (MPTP). New concepts of regulated cell death involving VDACs include its oligomerisation to form a large pore complex in the OMM; however, alternative VDAC localisation to the plasma membrane has been suggested in the literature and will be discussed regarding its potential role during cell death. Very recently, a phospholipid scramblase activity has been attributed to VDAC dimers, which explains the manifold lipidomic changes observed in VDAC-deficient yeast strains. In this review, I highlight the recent advances regarding VDAC's phospholipid scramblase function and discuss how this new insight sheds new light on VDAC's implication in regulated cell death, autophagy, and ageing.
    Keywords:  VDAC; ageing; apoptosis; autophagy; cell death; porin; scramblase
    DOI:  https://doi.org/10.3390/biom14101218
  46. Sci Adv. 2024 Nov;10(44): eadp3481
    High mitochondrial DNA levels accelerate lung adenocarcinoma progression.
    Mara Mennuni, Stephen E Wilkie, Pauline Michon, David Alsina, Roberta Filograna, Markus Lindberg, David E Sanin, Florian Rosenberger, Alina Schaaf, Erik Larsson, Erika L Pearce, Nils-Göran Larsson.
      Lung adenocarcinoma is a common aggressive cancer and a leading cause of mortality worldwide. Here, we report an important in vivo role for mitochondrial DNA (mtDNA) copy number during lung adenocarcinoma progression in the mouse. We found that lung tumors induced by KRASG12D expression have increased mtDNA levels and enhanced mitochondrial respiration. To experimentally assess a possible causative role in tumor progression, we induced lung cancer in transgenic mice with a general increase in mtDNA copy number and found that they developed a larger tumor burden, whereas mtDNA depletion in tumor cells reduced tumor growth. Immune cell populations in the lung and cytokine levels in plasma were not affected by increased mtDNA levels. Analyses of large cancer databases indicate that mtDNA copy number is also important in human lung cancer. Our study thus reports experimental evidence for a tumor-intrinsic causative role for mtDNA in lung cancer progression, which could be exploited for development of future cancer therapies.
    DOI:  https://doi.org/10.1126/sciadv.adp3481
  47. Methods Enzymol. 2024 ;pii: S0076-6879(24)00364-1. [Epub ahead of print]706 75-95
    Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP.
    Solène Zuttion, Bruno Senger, Chiranjit Panja, Sylvie Friant, Róża Kucharczyk, Hubert Dominique Becker.
      Even if a myriad of approaches has been developed to identify the subcellular localization of a protein, the easiest and fastest way remains to fuse the protein to Green Fluorescent Protein (GFP) and visualize its location using fluorescence microscopy. However, this strategy is not well suited to visualize the organellar pools of proteins that are simultaneously localized both in the cytosol and in organelles because the GFP signal of a cytosolic pool of the protein (cytosolic echoform) will inevitably mask or overlay the GFP signal of the organellar pool of the protein (organellar echoform). To solve this issue, we engineered a dedicated yeast strain expressing a Bi-Genomic Mitochondrial-Split-GFP. This split-GFP is bi-genomic because the first ten ß-strands of GFP (GFPß1-10) are encoded by the mitochondrial genome and translated by mitoribosomes whereas the remaining ß-strand of GFP (GFPß11) is fused to the protein of interest encoded by the nucleus and expressed by cytosolic ribosomes. Consequently, if the GFPß11-tagged protein localizes into mitochondria, GFP will be reconstituted by self-assembly GFPß1-10 and GFPß11 thereby generating a GFP signal restricted to mitochondria and detectable by regular fluorescence microscopy. In addition, because mitochondrial translocases and import mechanisms are evolutionary well conserved, the BiG Mito-Split-GFP yeast strain can be used to probe mitochondrial importability of proteins regardless of their organismal origins and can thus serve to identify unsuspected mitochondrial echoforms readily from any organism.
    Keywords:  Dual-localized; Microscopy; Mitochondria; Protein import; Saccharomyces cerevisiae; Split-GFP
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.028
  48. PLoS Biol. 2024 Oct 31. 22(10): e3002876
    Ortholog of autism candidate gene RBM27 regulates mitoribosomal assembly factor MALS-1 to protect against mitochondrial dysfunction and axon degeneration during neurodevelopment.
    Tamjid A Chowdhury, David A Luy, Garrett Scapellato, Dorian Farache, Amy S Y Lee, Christopher C Quinn.
      Mitochondrial dysfunction is thought to be a key component of neurodevelopmental disorders such as autism, intellectual disability, and attention-deficit hyperactivity disorder (ADHD). However, little is known about the molecular mechanisms that protect against mitochondrial dysfunction during neurodevelopment. Here, we address this question through the investigation of rbm-26, the Caenorhabditis elegans ortholog of the RBM27 autism candidate gene, which encodes an RNA-binding protein whose role in neurons is unknown. We report that RBM-26 (RBM26/27) protects against axonal defects by negatively regulating expression of the MALS-1 (MALSU1) mitoribosomal assembly factor. Autism-associated missense variants in RBM-26 cause a sharp decrease in RBM-26 protein expression along with defects in axon overlap and axon degeneration that occurs during larval development. Using a biochemical screen, we identified the mRNA for the MALS-1 mitoribosomal assembly factor as a binding partner for RBM-26. Loss of RBM-26 function causes a dramatic overexpression of mals-1 mRNA and MALS-1 protein. Moreover, genetic analysis indicates that this overexpression of MALS-1 is responsible for the mitochondrial and axon degeneration defects in rbm-26 mutants. These observations reveal a mechanism that regulates expression of a mitoribosomal assembly factor to protect against axon degeneration during neurodevelopment.
    DOI:  https://doi.org/10.1371/journal.pbio.3002876
  49. Sci Rep. 2024 10 26. 14(1): 25575
    Association of LONP1 gene with epilepsy and the sub-regional effect.
    Si-Xiu Li, Na He, Jian-Xiang Liao, Xin-Guo Lu, Wen-Guang Hu, Xiao-Rong Liu, Wei-Ping Liao, Xing-Wang Song, Bin Li.
      The LONP1 gene encodes Lon protease, which is responsible for degrading damaged or misfolded proteins and binding mitochondrial DNA. Previously, LONP1 variants have been identified in patients with cerebral, ocular, dental, auricular, and skeletal anomalies (CODAS syndrome) and mitochondrial diseases. Seizures were occasionally observed. However, the association between LONP1 and epilepsy remains elusive. In this study, we performed trio-based whole-exome sequencing in a cohort of 450 patients with unexplained epilepsy and identified four pairs of compound heterozygous LONP1 variants in four unrelated cases. All patients exhibited good responses to anti-seizure medications and demonstrated no developmental delay or intellectual disabilities. The variant allele frequencies observed in this study were absent or low in the general population and were significantly lower than those of benign variants. At least one variant in each biallelic pair affected hydrogen bonding and/or altered protein stability. The CODAS syndrome-associated variants were concentrated in the AAA+ module, especially the α domain. Four of the five mitochondrial disease-associated variants were located in the AAA + domain and the NTD5H and NTD3H subdomains. In contrast, each of the biallelic variants from the patients with pure epilepsy had one variant located in the linker domain, and the other variant located in the mitochondrial targeting sequence or P domain. This study suggested that LONP1 gene is potentially a novel candidate gene for pure epilepsy. The phenotypic variation is associated with the sub-regional effects of variants.
    Keywords:   LONP1 gene; Epilepsy; Genotype-phenotype correlation; Molecular sub-regional effect
    DOI:  https://doi.org/10.1038/s41598-024-77039-9
  50. bioRxiv. 2024 Oct 14. pii: 2024.10.14.618123. [Epub ahead of print]
    Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.
    Michael G Atser, Chelsea D Wenyonu, Elyn M Rowe, Connie L K Leung, Haoning Howard Cen, Eric D Queathem, Leo T Liu, Renata Moravcova, Jason Rogalski, David Perrin, Peter Crawford, Leonard J Foster, Armando Alcazar, James D Johnson.
      Pyruvate dehydrogenase kinase (PDK) 1 is one of four isozymes that inhibit the oxidative decarboxylation of pyruvate to acetyl-CoA via pyruvate dehydrogenase. PDK activity is elevated in fasting or starvation conditions to conserve carbohydrate reserves. PDK has also been shown to increase mitochondrial fatty acid utilization. In cardiomyocytes, metabolic flexibility is crucial for the fulfillment of high energy requirements. The PDK1 isoform is abundant in cardiomyocytes, but its specific contribution to cardiomyocyte metabolism is unclear. Here we show that PDK1 regulates cardiomyocyte fuel preference by mediating triacylglycerol turnover in differentiated H9c2 myoblasts using lentiviral shRNA to knockdown Pdk1. Somewhat surprisingly, PDK1 loss did not affect overall PDH activity, basal glycolysis, or glucose oxidation revealed by oxygen consumption rate experiments and 13C6 glucose labelling. On the other hand, we observed decreased triacylglycerol turnover in H9c2 cells with PDK1 knockdown, which was accompanied by decreased mitochondrial fatty acid utilization following nutrient deprivation. 13C16 palmitate tracing of uniformly labelled acyl chains revealed minimal acyl chain shuffling within triacylglycerol, indicating that the triacylglycerol hydrolysis, and not re-esterification, was dysfunctional in PDK1 suppressed cells. Importantly, PDK1 loss did not significantly impact the cellular lipidome or triacylglycerol accumulation following palmitic acid treatment, suggesting that effects of PDK1 on lipid metabolism were specific to the nutrient-deprived state. We validated that PDK1 loss decreased triacylglycerol turnover in Pdk1 knockout mice. Together, these findings implicate a novel role for PDK1 in lipid metabolism in cardiomyocytes, independent of its canonical roles in glucose metabolism.
    Keywords:  carbohydrate metabolism; cardiac metabolism; lipid metabolism; pyruvate dehydrogenase kinase; triacylglycerol
    DOI:  https://doi.org/10.1101/2024.10.14.618123
  51. bioRxiv. 2024 Oct 18. pii: 2024.10.15.618505. [Epub ahead of print]
    Mitochondria are absent from microglial processes performing surveillance, chemotaxis, and phagocytic engulfment.
    Alicia N Pietramale, Xhoela Bame, Megan E Doty, Robert A Hill.
      Microglia continually surveil the brain allowing for rapid detection of tissue damage or infection. Microglial metabolism is linked to tissue homeostasis, yet how mitochondria are subcellularly partitioned in microglia and dynamically reorganize during surveillance, injury responses, and phagocytic engulfment in the intact brain are not known. Here, we performed intravital imaging of microglia mitochondria, revealing that microglial processes diverge, with some containing multiple mitochondria while others are completely void. Microglial processes that engage in minute-to-minute surveillance typically do not have mitochondria. Moreover, unlike process surveillance, mitochondrial motility does not change with animal anesthesia. Likewise, the processes that acutely chemoattract to a lesion site or initially engage with a neuron undergoing programmed cell death do not contain mitochondria. Rather, microglia mitochondria have a delayed arrival into the responding cell processes. Thus, there is subcellular heterogeneity of mitochondrial partitioning and asymmetry between mitochondrial localization and cell process motility or acute damage responses.
    DOI:  https://doi.org/10.1101/2024.10.15.618505
  52. Methods Enzymol. 2024 ;pii: S0076-6879(24)00358-6. [Epub ahead of print]706 97-123
    Analysis of mitochondrial biogenesis and protein localization by genetic screens and automated imaging.
    Yury S Bykov, Maya Schuldiner.
      Budding yeast is a laboratory model of a simple eukaryotic cell. Its compact genome is very easy to edit. This allowed to create systematic collections (libraries) of yeast strains where every gene is either perturbed or tagged. Here we review how such collections were used to study mitochondrial biology by doing genetic screens. First, we introduce the principles of yeast genome editing and the basics of its life cycle that are useful for genetic experiments. Then we overview what yeast strain collections were created over the past years. We also describe the creation and the usage of the new generation of SWAP-Tag (SWAT) collections that allow to create custom libraries. We outline the principles of changing the genetic background of whole collections in parallel, and the basics of synthetic genetic array (SGA) approach. Then we review the discoveries that were made using different types of genetic screens focusing on general mitochondrial functions, proteome, and protein targeting pathways. The development of new collections and screening techniques will continue to bring valuable insight into the function of mitochondria and other organelles.
    Keywords:  High-throughput genetics; Mitochondria; Protein targeting; Proteome; Swap-Tag (SWAT); Synthetic genetic array; Yeast; Yeast collections
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.022
  53. Sci Adv. 2024 Nov;10(44): eadq4461
    Reducing the mitochondrial oxidative burden alleviates lipid-induced muscle insulin resistance in humans.
    Matteo Fiorenza, Johan Onslev, Carlos Henríquez-Olguín, Kaspar W Persson, Sofie A Hesselager, Thomas E Jensen, Jørgen F P Wojtaszewski, Morten Hostrup, Jens Bangsbo.
      Preclinical models suggest mitochondria-derived oxidative stress as an underlying cause of insulin resistance. However, it remains unknown whether this pathophysiological mechanism is conserved in humans. Here, we used an invasive in vivo mechanistic approach to interrogate muscle insulin action while selectively manipulating the mitochondrial redox state in humans. To this end, we conducted insulin clamp studies combining intravenous infusion of a lipid overload with intake of a mitochondria-targeted antioxidant (mitoquinone). Under lipid overload, selective modulation of mitochondrial redox state by mitoquinone enhanced insulin-stimulated glucose uptake in skeletal muscle. Mechanistically, mitoquinone did not affect canonical insulin signaling but augmented insulin-stimulated glucose transporter type 4 (GLUT4) translocation while reducing the mitochondrial oxidative burden under lipid oversupply. Complementary ex vivo studies in human muscle fibers exposed to high intracellular lipid levels revealed that mitoquinone improves features of mitochondrial bioenergetics, including diminished mitochondrial H2O2 emission. These findings provide translational and mechanistic evidence implicating mitochondrial oxidants in the development of lipid-induced muscle insulin resistance in humans.
    DOI:  https://doi.org/10.1126/sciadv.adq4461
  54. EMBO Mol Med. 2024 Oct 28.
    Sepsis-induced changes in pyruvate metabolism: insights and potential therapeutic approaches.
    Louise Nuyttens, Jolien Vandewalle, Claude Libert.
      Sepsis is a heterogeneous syndrome resulting from a dysregulated host response to infection. It is considered as a global major health priority. Sepsis is characterized by significant metabolic perturbations, leading to increased circulating metabolites such as lactate. In mammals, pyruvate is the primary substrate for lactate production. It plays a critical role in metabolism by linking glycolysis, where it is produced, with the mitochondrial oxidative phosphorylation pathway, where it is oxidized. Here, we provide an overview of all cytosolic and mitochondrial enzymes involved in pyruvate metabolism and how their activities are disrupted in sepsis. Based on the available data, we also discuss potential therapeutic strategies targeting these pyruvate-related enzymes leading to enhanced survival.
    Keywords:  Lactate; Metabolism; Mitochondria; Pyruvate; Sepsis
    DOI:  https://doi.org/10.1038/s44321-024-00155-6
  55. Commun Biol. 2024 Oct 25. 7(1): 1393
    miR-124 coordinates metabolic regulators acting at early stages of human neurogenesis.
    Geurim Son, Yongwoo Na, Yongsung Kim, Ji-Hoon Son, Gregory D Clemenson, Simon T Schafer, Jong-Yeon Yoo, Sarah L Parylak, Apua Paquola, Hyunsu Do, Dayeon Kim, Insook Ahn, Mingyu Ju, Chanhee S Kang, Younghee Ju, Eunji Jung, Aidan H McDonald, Youngjin Park, Gilhyun Kim, Se-Bum Paik, Junho Hur, Joon Kim, Yong-Mahn Han, Seung-Hee Lee, Fred H Gage, Jong-Seo Kim, Jinju Han.
      Metabolic dysregulation of neurons is associated with diverse human brain disorders. Metabolic reprogramming occurs during neuronal differentiation, but it is not fully understood which molecules regulate metabolic changes at the early stages of neurogenesis. In this study, we report that miR-124 is a driver of metabolic change at the initiating stage of human neurogenesis. Proteome analysis has shown the oxidative phosphorylation pathway to be the most significantly altered among the differentially expressed proteins (DEPs) in the immature neurons after the knockdown of miR-124. In agreement with these proteomics results, miR-124-depleted neurons display mitochondrial dysfunctions, such as decreased mitochondrial membrane potential and cellular respiration. Moreover, morphological analyses of mitochondria in early differentiated neurons after miR-124 knockdown result in smaller and less mature shapes. Lastly, we show the potential of identified DEPs as novel metabolic regulators in early neuronal development by validating the effects of GSTK1 on cellular respiration. GSTK1, which is upregulated most significantly in miR-124 knockdown neurons, reduces the oxygen consumption rate of neural cells. Collectively, our data highlight the roles of miR-124 in coordinating metabolic maturation at the early stages of neurogenesis and provide insights into potential metabolic regulators associated with human brain disorders characterized by metabolic dysfunctions.
    DOI:  https://doi.org/10.1038/s42003-024-07089-2
  56. Cell Metab. 2024 Oct 26. pii: S1550-4131(24)00410-8. [Epub ahead of print]
    Bempedoic acid suppresses diet-induced hepatic steatosis independently of ATP-citrate lyase.
    Joyce Y Liu, Ramya S Kuna, Laura V Pinheiro, Phuong T T Nguyen, Jaclyn E Welles, Jack M Drummond, Nivitha Murali, Prateek Sharma, Julianna G Supplee, Mia Shiue, Steven Zhao, Aimee T Farria, Avi Kumar, Mauren L Ruchhoeft, Christina Demetriadou, Daniel S Kantner, Adam Chatoff, Emily Megill, Paul M Titchenell, Nathaniel W Snyder, Christian M Metallo, Kathryn E Wellen.
      ATP citrate lyase (ACLY) synthesizes acetyl-CoA for de novo lipogenesis (DNL), which is elevated in metabolic dysfunction-associated steatotic liver disease. Hepatic ACLY is inhibited by the LDL-cholesterol-lowering drug bempedoic acid (BPA), which also improves steatosis in mice. While BPA potently suppresses hepatic DNL and increases fat catabolism, it is unclear if ACLY is its primary molecular target in reducing liver triglyceride. We show that on a Western diet, loss of hepatic ACLY alone or together with the acetyl-CoA synthetase ACSS2 unexpectedly exacerbates steatosis, linked to reduced PPARα target gene expression and fatty acid oxidation. Importantly, BPA treatment ameliorates Western diet-mediated triacylglyceride accumulation in both WT and liver ACLY knockout mice, indicating that its primary effects on hepatic steatosis are ACLY independent. Together, these data indicate that hepatic ACLY plays an unexpected role in restraining diet-dependent lipid accumulation and that BPA exerts substantial effects on hepatic lipid metabolism independently of ACLY.
    Keywords:  ACLY; ACSS2; PPARα; bempedoic acid; lipid metabolism; metabolic dysfunction-associated steatotic liver disease
    DOI:  https://doi.org/10.1016/j.cmet.2024.10.014
  57. Science. 2024 Nov;386(6721): 496-497
    Some brain disorders are "chaperonopathies".
    Manu Sharma.
      Mutations that impair a protein-folding chaperone can lead to brain malformations.
    DOI:  https://doi.org/10.1126/science.adt0039
  58. Front Immunol. 2024 ;15 1451989
    Advancements in mitochondrial-targeted nanotherapeutics: overcoming biological obstacles and optimizing drug delivery.
    Yang Li, Xiao-Meng Li, Li-Si Wei, Jun-Feng Ye.
      In recent decades, nanotechnology has significantly advanced drug delivery systems, particularly in targeting subcellular organelles, thus opening new avenues for disease treatment. Mitochondria, critical for cellular energy and health, when dysfunctional, contribute to cancer, neurodegenerative diseases, and metabolic disorders. This has propelled the development of nanomedicines aimed at precise mitochondrial targeting to modulate their function, marking a research hotspot. This review delves into the recent advancements in mitochondrial-targeted nanotherapeutics, with a comprehensive focus on targeting strategies, nanocarrier designs, and their therapeutic applications. It emphasizes nanotechnology's role in enhancing drug delivery by overcoming biological barriers and optimizing drug design for specific mitochondrial targeting. Strategies exploiting mitochondrial membrane potential differences and specific targeting ligands improve the delivery and mitochondrial accumulation of nanomedicines. The use of diverse nanocarriers, including liposomes, polymer nanoparticles, and inorganic nanoparticles, tailored for effective mitochondrial targeting, shows promise in anti-tumor and neurodegenerative treatments. The review addresses the challenges and future directions in mitochondrial targeting nanotherapy, highlighting the need for precision, reduced toxicity, and clinical validation. Mitochondrial targeting nanotherapy stands at the forefront of therapeutic strategies, offering innovative treatment perspectives. Ongoing innovation and research are crucial for developing more precise and effective treatment modalities.
    Keywords:  biocompatibility; drug delivery systems; mitochondrial targeting; nanotechnology; subcellular organelles; therapeutic nanocarriers
    DOI:  https://doi.org/10.3389/fimmu.2024.1451989
  59. Methods Enzymol. 2024 ;pii: S0076-6879(24)00386-0. [Epub ahead of print]706 37-57
    A modified procedure for separating yeast peroxisomes from mitochondria.
    Nitya Aravindan, Doron Rapaport.
      Mitochondria and peroxisomes are mutually dependent organelles that share several membrane proteins that carry out the same function in both organelles. To study the unique features of these dually localized proteins in each of the two organelles, it is essential to separate mitochondria from peroxisomes. Isolating organelles from cells of Baker's yeast, Saccharomyces cerevisiae, is crucial for our understanding of the biogenesis and functions of proteins. Traditionally, subcellular fractionation and isolation of individual organelles by differential centrifugation benefit from the specific and unique density of each organelle. However, when yeast cells are grown under normal conditions, certain organelles like mitochondria and peroxisomes share strikingly similar densities. This similarity challenges the separation of these organelles from one another. In this chapter, we describe an optimized procedure to address this task. We depict growth conditions that would favor stimulation of peroxisomes to increase their number and density, and portray organellar isolation followed by gradient centrifugation, enabling an improved separation of both organelles. Additionally, we illustrate the advantage of the procedure to study the dual localization of the membrane protein Fis1.
    Keywords:  Density gradient; Fis1; Mitochondria; Peroxisomes; Subcellular fractionation; Yeast
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.046
  60. Biomolecules. 2024 Oct 05. pii: 1258. [Epub ahead of print]14(10):
    EMC1 Is Required for the Sarcoplasmic Reticulum and Mitochondrial Functions in the Drosophila Muscle.
    Carlos Antonio Couto-Lima, Maiaro Cabral Rosa Machado, Lucas Anhezini, Marcos Túlio Oliveira, Roberto Augusto da Silva Molina, Rodrigo Ribeiro da Silva, Gabriel Sarti Lopes, Vitor Trinca, David Fernando Colón, Pablo M Peixoto, Nadia Monesi, Luciane Carla Alberici, Ricardo Guelerman P Ramos, Enilza Maria Espreafico.
      EMC1 is part of the endoplasmic reticulum (ER) membrane protein complex, whose functions include the insertion of transmembrane proteins into the ER membrane, ER-mitochondria contact, and lipid exchange. Here, we show that the Drosophila melanogaster EMC1 gene is expressed in the somatic musculature and the protein localizes to the sarcoplasmic reticulum (SR) network. Muscle-specific EMC1 RNAi led to severe motility defects and partial late pupae/early adulthood lethality, phenotypes that are rescued by co-expression with an EMC1 transgene. Motility impairment in EMC1-depleted flies was associated with aberrations in muscle morphology in embryos, larvae, and adults, including tortuous and misaligned fibers with reduced size and weakness. They were also associated with an altered SR network, cytosolic calcium overload, and mitochondrial dysfunction and dysmorphology that impaired membrane potential and oxidative phosphorylation capacity. Genes coding for ER stress sensors, mitochondrial biogenesis/dynamics, and other EMC components showed altered expression and were mostly rescued by the EMC1 transgene expression. In conclusion, EMC1 is required for the SR network's mitochondrial integrity and influences underlying programs involved in the regulation of muscle mass and shape. We believe our data can contribute to the biology of human diseases caused by EMC1 mutations.
    Keywords:  endoplasmic reticulum membrane protein complex; mitochondria; musculature
    DOI:  https://doi.org/10.3390/biom14101258
  61. Elife. 2024 Oct 31. pii: RP93172. [Epub ahead of print]13
    Exceptional longevity of mammalian ovarian and oocyte macromolecules throughout the reproductive lifespan.
    Ewa K Bomba-Warczak, Karen M Velez, Luhan T Zhou, Christelle Guillermier, Seby Edassery, Matthew L Steinhauser, Jeffrey N Savas, Francesca E Duncan.
      The mechanisms contributing to age-related deterioration of the female reproductive system are complex, however aberrant protein homeostasis is a major contributor. We elucidated exceptionally stable proteins, structures, and macromolecules that persist in mammalian ovaries and gametes across the reproductive lifespan. Ovaries exhibit localized structural and cell-type-specific enrichment of stable macromolecules in both the follicular and extrafollicular environments. Moreover, ovaries and oocytes both harbor a panel of exceptionally long-lived proteins, including cytoskeletal, mitochondrial, and oocyte-derived proteins. The exceptional persistence of these long-lived molecules suggest a critical role in lifelong maintenance and age-dependent deterioration of reproductive tissues.
    Keywords:  cell biology; long-lived proteins; mass spectrometry imaging; mouse; oocyte; ovaries; proteomics; reproductive aging
    DOI:  https://doi.org/10.7554/eLife.93172
  62. bioRxiv. 2024 Oct 25. pii: 2024.10.22.619640. [Epub ahead of print]
    Loss of long-chain acyl-CoA dehydrogenase protects against acute kidney injury.
    Takuto Chiba, Akira Oda, Yuxun Zhang, Joanna Bons, Sivakama S Bharathi, Katherine E Pfister, Bob B Zhang, Adam C Richert, Birgit Schilling, Eric S Goetzman, Sunder Sims-Lucas.
      Proximal tubular epithelial cells (PTECs) are particularly vulnerable to acute kidney injury (AKI). While fatty acids are the preferred energy source for PTECs via fatty acid oxidation (FAO), FAO-mediated H 2 O 2 production in mitochondria has been shown to be a major source of oxidative stress. We have previously shown that a mitochondrial flavoprotein, long-chain acyl-CoA dehydrogenase (LCAD), which catalyzes a key step in mitochondrial FAO, directly produces H 2 O 2 in vitro . Further we have established that loss of a lysine deacylase, Sirtuin 5 ( Sirt5 -/- ), induces hypersuccinylation and inhibition of mitochondrial FAO genes to stimulate peroxisomal FAO and to protect against AKI. However, the role of LCAD has yet to be determined. Mass spectrometry data acquisition revealed that LCAD is hypersuccinylated in Sirt5 -/- kidneys after AKI. Following two distinct models of AKI, cisplatin treatment or renal ischemia/reperfusion (IRI), LCAD knockout mice ( LCAD -/- ) demonstrated renoprotection against AKI. Specifically, LCAD -/- kidneys displayed mitigated renal tubular injury, decreased oxidative stress, preserved mitochondrial function, enhanced peroxisomal FAO, and decreased ferroptotic cell death. LCAD deficiency confers protection against two distinct models of AKI. This suggests a therapeutically attractive mechanism whereby preserved mitochondrial respiration as well as enhanced peroxisomal FAO by loss of LCAD mediates renoprotection against AKI.
    DOI:  https://doi.org/10.1101/2024.10.22.619640
  63. Int J Mol Sci. 2024 Oct 18. pii: 11199. [Epub ahead of print]25(20):
    Human iPSCs from Aged Donors Retain Their Mitochondrial Aging Signature.
    Imane Lejri, Zameel Cader, Amandine Grimm, Anne Eckert.
      Aging represents the leading risk factor for developing neurodegenerative disorders. One of the nine hallmarks of aging is mitochondrial dysfunction. Age-related mitochondrial alterations have been shown to affect mitochondrial energy metabolism, reduction-oxidation homeostasis, and mitochondrial dynamics. Previous reports have shown that induced pluripotent stem cells (iPSCs) from aged donors do not keep the aging signature at the transcriptomic level. However, not all aspects of aging have been investigated, and especially not the mitochondria-related aging signature. Therefore, the present study compared the mitochondrial function in iPSCs from healthy aged donors compared to those of young donors. We addressed whether aged iPSCs may be used as drug-screening models of "aging in a dish" to identify therapies alleviating mitochondria aging. Compared to iPSCs from young donors, we demonstrate that iPSCs from aged donors show impaired mitochondrial bioenergetics and exhibit a rise in reactive oxygen species generation. Furthermore, aged iPSCs present a lower mitochondrial mass and alterations in the morphology of the mitochondrial network when compared to iPSCs from young donors. This study provides the first evidence that the aging phenotype is present at the mitochondrial level in iPSCs from aged donors, ranging from bioenergetics to mitochondrial network morphology. This model might be used to screen mitochondria-targeting drugs to promote healthy aging at the mitochondrial level.
    Keywords:  aging; bioenergetics; human-induced pluripotent stem cells; mitochondria; mitochondrial morphology; oxidative stress
    DOI:  https://doi.org/10.3390/ijms252011199
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