bims-mitmed Biomed News
on Mitochondrial medicine
Issue of 2022‒05‒15
sixty-one papers selected by
Dario Brunetti
Fondazione IRCCS Istituto Neurologico
  1. Endocrine Manifestations and New Developments in Mitochondrial Disease.
  2. Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction.
  3. Renal Phenotype in Mitochondrial Diseases: A Multicenter Study.
  4. Roles for Mitochondrial Complex I Subunits in Regulating Synaptic Transmission and Growth.
  5. SUMO 2 the rescue: how SUMO2 regulates the mitochondria via Drp1 modification.
  6. Mitochondrial Fission is Essential to Maintain Cristae Morphology and Bioenergetics.
  7. Investigating Mitochondrial Dysfunction in Barth Syndrome.
  8. Mitochondrial Uncoupling Decreases Sarcopenic Obesity By Activation of Skeletal Muscle Mitochondrial Quality Control and Attenuated ER Stress.
  9. Hitchhiker's guide through the axon: transport and local translation of Pink1 mRNA support axonal mitophagy.
  10. Progressive Parkinsonism With Features of Mitochondrial Disease.
  11. Next-Generation Sequencing to Characterize Mitochondrial Genomic DNA Heteroplasmy.
  12. Neuroligin-mediated neurodevelopmental defects are induced by mitochondrial dysfunction and prevented by lutein in C. elegans.
  13. Respiratory defects caused by mutations affecting the Endoplasmic Reticulum-Mitochondria Encounter Structure (ERMES) can be rescued by the deletion of COQ11.
  14. Gitelman-like syndrome caused by pathogenic variants in mitochondrial DNA.
  15. A Reduction in Tafazzin Decreases Mitochondrial Function in C2C12 Myotubes.
  16. Research Priorities for Mitochondrial Disorders: Current Landscape and Patient and Professional Views.
  17. Targeted Mitochondrial Delivery to Hepatocytes: A Review.
  18. The primary astrocytic mitochondrial transplantation ameliorates ischemic stroke.
  19. Single Cell Assessment of Mitochondrial Function.
  20. Understanding the Tissue-Specific Transcriptional Effects of Mitochondrial Introgression in Drosophila.
  21. Phosphorylation-based regulation of mitochondrial metabolism.
  22. The Role of ATF5 in Mitochondrial Maintenance, Biogenesis and UPRmt Signaling Following Acute Exercise in Skeletal Muscle.
  23. AGK regulates the progression to NASH by affecting mitochondria complex I function.
  24. Intrinsic Mitochondrial Function Impacts the Outcomes of Metformin Treatment on Skeletal Muscle Mitochondrial Morphology in Aged Rat.
  25. A Novel Peroxin is Required for Mitochondrial Morphology: Implications for Resolving a New Peroxisome-Mitochondrial Contact Site.
  26. CYB5R3 overexpression preserves skeletal muscle mitochondria and autophagic signaling in aged transgenic mice.
  27. Mitochondrial Transplantation as a Novel Therapeutic for Retinal Metabolic Dysfunction-Driven Disease.
  28. 3D mitochondrial remodeling during postnatal heart development.
  29. Machinery in motion: New insights into mitochondrial proteostasis.
  30. Mitochondrial GRK2 is a Novel Regulator of Cardiac Energetics.
  31. Determining the role of ATF4 in the regulation of mitochondrial remodeling during myotube differentiation and contractile activity.
  32. Leber's hereditary optic neuropathy (LHON)-associated ND6 14 484 T > C mutation caused pleiotropic effects on the complex I, RNA homeostasis, apoptosis and mitophagy.
  33. Expression profiles of protein markers regulating the mitochondrial lifecycle in skeletal muscle of acute spinal cord transected rats.
  34. Generation of homozygous PRKN, PINK1 and double PINK1/PRKN knockout cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing.
  35. The Role of the Optic Atrophy 1 (OPA1) Protein in Drug-Induced Liver Injury.
  36. Role of TFE3 in Mitochondrial Adaptations to Skeletal Muscle Disuse.
  37. Characterisation of a novel OPA1 splice variant resulting in cryptic splice site activation and mitochondrial dysfunction.
  38. Decreased Mitochondrial Unfolded Protein Response (UPRmt) in HFpEF.
  39. Evidence of Mitochondrial Impairment in a Mouse Model of Down Syndrome.
  40. Novel Regulatory Role of SIRT3 on Cardiomyocyte Mitochondrial Frataxin and Ferroptosis.
  41. Dysregulation of Skeletal Muscle Mitochondrial Turnover and Dynamics Occurs During Late Symptomatic Stages of Spinal Muscular Atrophy.
  42. Impact of Branched Chain Amino Acid Supplementation on Muscle Lipid Accumulation and Mitochondrial Metabolism.
  43. Frataxin deficiency lowers lean mass and triggers the integrated stress response in skeletal muscle.
  44. The Effect of Leucine and Doxorubicin Treatment on Skeletal Muscle Mitochondrial Function.
  45. Nucleolar function is regulated by the mitochondrial protein sulfite oxidase (SUOX).
  46. Implications of DNA Polymerase Gamma in the Repair of the Mitochondrial Genome.
  47. Urolithin A attenuates auditory cell senescence by activating mitophagy.
  48. Potential Drug Therapy For Fragile X Tremor/Ataxia Syndrome.
  49. Aging Selectively Reduces Mitochondrial Volume Density and Respiratory Capacity in Type IIx/IIb Diaphragm Muscle Fibers.
  50. Spatial Compartmentation of mTORC1 Signaling at the Mitochondria.
  51. Mechanical instability generated by Myosin 19 contributes to mitochondria cristae architecture and OXPHOS.
  52. Mitochondrial DNA content decreases during in vitro human embryo development: insights into mitochondrial DNA variation in preimplantation embryos donated for research.
  53. Mass Spectrometry Based Subcellular Coenzyme Analysis.
  54. Harnessing NAD+ Metabolism as Therapy for Cardiometabolic Diseases.
  55. The combination of NRF1 and Nrf2 activators in myoblasts stimulate mechanisms of proteostasis without changes in mitochondrial respiration.
  56. Mitochondrial Transfer Between Mesenchymal Stem Cells and Cardiac H9c2 Cells.
  57. Exploring selective autophagy events in multiple biologic models using LC3-interacting regions (LIR)-based molecular traps.
  58. Neurological Recovery with Interferon-gamma Treatment in Friedreich's Ataxia.
  59. Mitochondrial base editor induces substantial nuclear off-target mutations.
  60. Impaired Proteostasis, not Protein Synthesis, Limits Recovery of Aged Skeletal Muscle After Disuse Atrophy.
  61. The SARS-CoV2 and mitochondria: the impact on cell fate.
  1. Endocr Rev. 2022 May 12. 43(3): 583-609
    Endocrine Manifestations and New Developments in Mitochondrial Disease.
    Yi Shiau Ng, Albert Zishen Lim, Grigorios Panagiotou, Doug M Turnbull, Mark Walker.
      Mitochondrial diseases are a group of common inherited diseases causing disruption of oxidative phosphorylation. Some patients with mitochondrial disease have endocrine manifestations, with diabetes mellitus being predominant but also include hypogonadism, hypoadrenalism, and hypoparathyroidism. There have been major developments in mitochondrial disease over the past decade that have major implications for all patients. The collection of large cohorts of patients has better defined the phenotype of mitochondrial diseases and the majority of patients with endocrine abnormalities have involvement of several other systems. This means that patients with mitochondrial disease and endocrine manifestations need specialist follow-up because some of the other manifestations, such as stroke-like episodes and cardiomyopathy, are potentially life threatening. Also, the development and follow-up of large cohorts of patients means that there are clinical guidelines for the management of patients with mitochondrial disease. There is also considerable research activity to identify novel therapies for the treatment of mitochondrial disease. The revolution in genetics, with the introduction of next-generation sequencing, has made genetic testing more available and establishing a precise genetic diagnosis is important because it will affect the risk for involvement for different organ systems. Establishing a genetic diagnosis is also crucial because important reproductive options have been developed that will prevent the transmission of mitochondrial disease because of mitochondrial DNA variants to the next generation.
    Keywords:  MIDD; clinical management; diabetes mellitus; genomic testing; mitochondrial DNA; reproductive options
    DOI:  https://doi.org/10.1210/endrev/bnab036
  2. PLoS Genet. 2022 May 09. 18(5): e1010190
    Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction.
    Dusanka Milenkovic, Adrián Sanz-Moreno, Julia Calzada-Wack, Birgit Rathkolb, Oana Veronica Amarie, Raffaele Gerlini, Antonio Aguilar-Pimentel, Jelena Misic, Marie-Lune Simard, Eckhard Wolf, Helmut Fuchs, Valerie Gailus-Durner, Martin Hrabě de Angelis, Nils-Göran Larsson.
      Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.
    DOI:  https://doi.org/10.1371/journal.pgen.1010190
  3. Kidney Dis (Basel). 2022 Mar;8(2): 148-159
    Renal Phenotype in Mitochondrial Diseases: A Multicenter Study.
    Maria Parasyri, Per Brandström, Johanna Uusimaa, Elsebet Ostergaard, Omar Hikmat, Pirjo Isohanni, Karin Naess, I F M de Coo, Andrés Nascimento Osorio, Matti Nuutinen, Christopher Lindberg, Laurence A Bindoff, Már Tulinius, Niklas Darin, Kalliopi Sofou.
      Aims: This study aimed to investigate associations between renal and extrarenal manifestations of mitochondrial diseases and their natural history as well as predictors of renal disease severity and overall disease outcome. The secondary aim was to generate a protocol of presymptomatic assessment and monitoring of renal function in patients with a defined mitochondrial disease.Methods: A multicenter, retrospective cohort study was performed by the Mitochondrial Clinical and Research Network (MCRN). Patients of any age with renal manifestations associated with a genetically verified mitochondrial disease were included from 8 expert European centers specializing in mitochondrial diseases: Gothenburg, Oulu, Copenhagen, Bergen, Helsinki, Stockholm, Rotterdam, and Barcelona.
    Results: Of the 36 patients included, two-thirds had mitochondrial DNA-associated disease. Renal manifestations were the first sign of mitochondrial disease in 19%, and renal involvement was first identified by laboratory tests in 57% of patients. Acute kidney injury occurred in 19% of patients and was the first sign of renal disease in the majority of these. The most common renal manifestation was chronic kidney disease (75% with stage 2 or greater), followed by tubulopathy (44.4%), the latter seen mostly among patients with single large-scale mitochondrial DNA deletions. Acute kidney injury and tubulopathy correlated with worse survival outcome. The most common findings on renal imaging were increased echogenicity and renal dysplasia/hypoplasia. Renal histology revealed focal segmental glomerulosclerosis, nephrocalcinosis, and nephronophthisis.
    Conclusion: Acute kidney injury is a distinct renal phenotype in patients with mitochondrial disease. Our results highlight the importance to recognize renal disease as a sign of an underlying mitochondrial disease. Acute kidney injury and tubulopathy are 2 distinct indicators of poor survival in patients with mitochondrial diseases.
    Keywords:  Acute kidney injury; Mitochondrial DNA; Mitochondrial disease; Renal manifestations
    DOI:  https://doi.org/10.1159/000521148
  4. Front Neurosci. 2022 ;16 846425
    Roles for Mitochondrial Complex I Subunits in Regulating Synaptic Transmission and Growth.
    Bhagaban Mallik, C Andrew Frank.
      To identify conserved components of synapse function that are also associated with human diseases, we conducted a genetic screen. We used the Drosophila melanogaster neuromuscular junction (NMJ) as a model. We employed RNA interference (RNAi) on selected targets and assayed synapse function and plasticity by electrophysiology. We focused our screen on genetic factors known to be conserved from human neurological or muscle functions (300 Drosophila lines screened). From our screen, knockdown of a Mitochondrial Complex I (MCI) subunit gene (ND-20L) lowered levels of NMJ neurotransmission. Due to the severity of the phenotype, we studied MCI function further. Knockdown of core MCI subunits concurrently in neurons and muscle led to impaired neurotransmission. We localized this neurotransmission function to the muscle. Pharmacology targeting MCI phenocopied the impaired neurotransmission phenotype. Finally, MCI subunit knockdowns or pharmacological inhibition led to profound cytological defects, including reduced NMJ growth and altered NMJ morphology. Mitochondria are essential for cellular bioenergetics and produce ATP through oxidative phosphorylation. Five multi-protein complexes achieve this task, and MCI is the largest. Impaired Mitochondrial Complex I subunits in humans are associated with disorders such as Parkinson's disease, Leigh syndrome, and cardiomyopathy. Together, our data present an analysis of Complex I in the context of synapse function and plasticity. We speculate that in the context of human MCI dysfunction, similar neuronal and synaptic defects could contribute to pathogenesis.
    Keywords:  Drosophila; Mitochondrial Complex I; NMJ – neuromuscular junction; neurodevelopment; neurotransmission; synapse; synaptic development; synaptic dysfunction
    DOI:  https://doi.org/10.3389/fnins.2022.846425
  5. FASEB J. 2022 May;36 Suppl 1
    SUMO 2 the rescue: how SUMO2 regulates the mitochondria via Drp1 modification.
    Allison H DeHaas, Michael J Matunis.
      Mitochondria are dynamic powerhouses of cells and their fission and fusion must be tightly regulated for normal cell function. Mitochondrial fission is mediated by the GTPase, Drp1, and mis-regulation of Drp1 leads to mitochondrial hyper-fragmentation, a known marker of disease. It has been reported that Drp1 is modified by SUMO1 and SUMO2/3, however, the mechanisms of Drp1 regulation by individual SUMO paralogs remain to be fully understood. Here, we have used CRISPR/Cas9 derived SUMO1 and SUMO2 knockout (KO) cell lines, to perform a systematic investigation of paralog-specific effects on mitochondrial maintenance and function. In contrast to expectations, we observed multiple mitochondrial defects specifically in SUMO2 KO, but not SUMO1 KO, cells. SUMO2 KO cells had reduced mitochondrial activity based on results of MTT assays. By immunofluorescence microscopy, we also observed an increase in mitochondrial fragmentation in SUMO2 KO cells. Paradoxically, increased fragmentation occurred despite reduced levels of Drp1 protein expression and sequestration of Drp1 in large cytosolic foci. Taken together, our findings indicate that SUMO2 plays a non-redundant, paralog-specific role in regulating mitochondrial function, in part through effects on Drp1. We anticipate that a more detailed understanding of the molecular effects of SUMO2 on Drp1 may lead to novel therapeutic approaches to treat or prevent ischemic injury, neurodegeneration, and other mitochondrial-related maladies.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3750
  6. FASEB J. 2022 May;36 Suppl 1
    Mitochondrial Fission is Essential to Maintain Cristae Morphology and Bioenergetics.
    Gabriella Robertson, Mira Patel, Stellan Riffle, Andrea Marshall, Heather Beasley, Edgar Garza-Lopez, Antentor O Hinton, Maria Stoll, Jason Mears, Vivian Gama.
      Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission and fusion are known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin-related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause developmental defects, we are using patient-derived fibroblasts and iPSC-derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. Using super resolution imaging, we find that patient cells, in addition to displaying elongated mitochondrial and peroxisomal morphology, present with aberrant cristae structure. Given the direct link between cristae morphology and oxidative phosphorylation efficiency, we explored the impact of these mutations on cellular energy production. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and Complex III deficiency. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in hyperpolarized mitochondrial membrane potential. Intriguingly, human fibroblasts are capable of cellular reprogramming into iPSCs and appear to display peroxisome-mediated mitochondrial adaptations that could help sustain these cell fate transitions. Understanding the mechanism by which DRP1 mutations cause cellular dysfunction will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3665
  7. FASEB J. 2022 May;36 Suppl 1
    Investigating Mitochondrial Dysfunction in Barth Syndrome.
    Olivia L Sniezek, Arianna Anzmann, Nanami Senoo, Grant Butschek, Steven Claypool, Hilary Vernon.
      Barth syndrome (BTHS) is a rare, X-linked disorder of mitochondrial phospholipid metabolism caused by variants in the gene TAFAZZIN.TAFAZZIN is a transacylase involved in the remodeling of cardiolipin (CL), a dimeric phospholipid localized to the inner mitochondrial membrane. Lack of TAFAZZIN-based remodeling results in irregular cardiolipin content, characterized by increased unremodeled CL, increased monoloysocardiolipin (the CL remodeling intermediate), and a decrease in remodeled CL, which is enriched in polyunsaturated fatty acyl chains that are tissue-specific in composition. BTHS is clinically characterized by cardiomyopathy, neutropenia, and myopathy, with a high morbidity and mortality. There are no approved disease-specific therapies. To investigate the cellular pathology and to identify new areas of potential therapeutic intervention, we developed two CRISPR-edited cell lines: TAFAZZIN-knockout (KO) HEK293 cells and iPSCs with which to perform broad-based discovery experiments and to study tissue-specific disease effects, respectively. A combined multi-omics approach including proteomics, lipidomics, and metabolomics in TAZ-KO HEK293 cells revealed diverse mitochondrial abnormalities, including defects in complex I of the respiratory chain, abnormal PDK2 expression, and dysregulation of proteins involved in mitochondrial quality control including PARL and PGAM5. Importantly, we discovered that molecules that bind to cardiolipin (SS-31) or inhibit nascent cardiolipin deacylation (bromoenol lactone), partially remediate these mitochondrial defects. We next explored cell-type specific dysfunction in iPSC-derived TAZ-KO and wild-type cardiomyocytes and neurons via lipidomics, RNA-seq, and functional studies. We identified disturbances in cellular lipid content including an expected increase in the monolysocardiolipin content and a reduction that exhibited cell-type specificity. RNAseq identified dysregulation in pathways regulated by PARL and PGAM5 including Wnt signaling, apoptosis, and autophagy in the undifferentiated state, with differentiated cell types highlighting pathways such as glucose metabolism and response to cellular stimuli. Oxygen consumption studies show impaired maximal respiratory capacity in TAZ-KO cardiomyocytes and neurons. Ongoing investigations aim to address cell type specific mitochondrial dysfunction by characterizing PARL abundance, PGAM5 cleavage, and mitochondrial morphology in TAZ-KO iPSC derived cell types. Additionally, we are currently targeting cardiolipin metabolism in differentiated TAZ-deficient cells with the goal of remediating cellular lipids, mitochondrial gene expression, and oxygen consumption abnormalities.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R389
  8. FASEB J. 2022 May;36 Suppl 1
    Mitochondrial Uncoupling Decreases Sarcopenic Obesity By Activation of Skeletal Muscle Mitochondrial Quality Control and Attenuated ER Stress.
    Wagner S Dantas, Elizabeth R M Zunica, Elizabeth C Heintz, Charles L Hoppel, Christopher L Axelrod, John P Kirwan.
      BACKGROUND: Sarcopenic obesity is a highly prevalent disease with poor survival and ineffective medical interventions. Mitochondrial dysfunction is purported to be central in the pathogenesis of sarcopenic obesity by impairing both organelle biogenesis and quality control. We have previously identified an orally available mitochondrial-targeted furazano[3,4-b]pyrazine named BAM15 that selectively lowers respiratory coupling efficiency and protects against diet-induced obesity in mice. Here, we tested the hypothesis that mitochondrial uncoupling simultaneously attenuates loss of muscle function and weight gain in a mouse model of sarcopenic obesity.METHODS: 80-week-old male C57BL/6J mice with obesity were randomized to 10 weeks of high fat diet (CTRL) or BAM15 (BAM15; 0.1% w/w in high fat diet) treatment. Body composition, muscle function, energy expenditure, and locomotor activity were determined after treatment. Skeletal muscle was harvested and evaluated for histology, gene expression, protein signaling, and mitochondrial structure and function.
    RESULTS: BAM15 decreased body weight (~25% reduction, P<0.001) which was attributable to increased energy expenditure (~20% increment, P<0.001). BAM15 increased muscle mass (~13% increment, P<0.001), strength (~37% increment, P<0.0001), and locomotor activity (~25% increment, P<0.001). Improvements in physical function were mediated in part by reductions in skeletal muscle inflammation (IL-6 and gp130, both P<0.05), enhanced mitochondrial function, and improved endoplasmic reticulum homeostasis and reduced inflammation. Specifically, BAM15 activated mitochondrial quality control through AMPK (PINK1-ubiquitin binding and LC3II, P<0.01), increased electron transport chain activity (citrate synthase and complex II activity, all P<0.05), restricted endoplasmic reticulum (ER) misfolding (decreased oligomer A11 insoluble/soluble ratio, P<0.0001) while limiting ER stress (decreased PERK signaling, P<0.0001), apoptotic signaling (decreased cytochrome C release and Caspase-3/9 activation, all P<0.001), and muscle protein degradation (decreased 14-kDa actin fragment insoluble/soluble ratio, P<0.001).
    CONCLUSIONS: Mitochondrial uncoupling agents such as BAM15 may mitigate age-related decline in muscle mass and function by molecular and cellular bioenergetic adaptations that confer protection against sarcopenic obesity through activation of mitochondrial quality control and attenuation of ER stress.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.L7532
  9. Autophagy. 2022 May 09. 1-2
    Hitchhiker's guide through the axon: transport and local translation of Pink1 mRNA support axonal mitophagy.
    Shree Padma Metur, Daniel J Klionsky.
      The unique cellular organization and metabolic demands of neurons pose a challenge in the maintenance of neuronal homeostasis. A critical element in maintaining neuronal health and homeostasis is mitochondrial quality control via replacement and rejuvenation at the axon. Dysregulation of mitochondrial quality control mechanisms such as mitophagy has been implicated in neurodegenerative diseases including Parkinson disease and amyotrophic lateral sclerosis. To sustain mitophagy at the axon, a continuous supply of PINK1 is required; however, how do neurons maintain a steady supply of this protein at the distal axons? In the study highlighted here, Harbauer et al. show that axonal mitophagy is supported by local translation of Pink1 mRNA that is co-transported with mitochondria to the distal ends of the neuron. This neuronal-specific pathway provides a continuous supply of PINK1 to sustain mitophagy.
    Keywords:  Autophagy; mitochondria; neurodegeneration; neuron; stress
    DOI:  https://doi.org/10.1080/15548627.2022.2071081
  10. JAMA Neurol. 2022 May 09.
    Progressive Parkinsonism With Features of Mitochondrial Disease.
    Tjerk J Lagrand, Mehri Salari, Alexander C Lehn.
      
    DOI:  https://doi.org/10.1001/jamaneurol.2022.0920
  11. Curr Protoc. 2022 May;2(5): e412
    Next-Generation Sequencing to Characterize Mitochondrial Genomic DNA Heteroplasmy.
    Zeyu Yang, Jesse Slone, Taosheng Huang.
      Mitochondria play a very important role in many crucial cellular functions. Each eukaryotic cell contains hundreds of mitochondria with hundreds of mitochondrial genomes. Mutant and wild-type mitochondrial DNA (mtDNA) may co-exist as heteroplasmy and cause human disease. The purpose of the protocols in this article is to simultaneously determine the mtDNA sequence and quantify the heteroplasmy level using parallel sequencing. The protocols include mitochondrial genomic DNA PCR amplification of two full-length products using two distinct sets of PCR primers. The PCR products are mixed at an equimolar ratio, and the samples are then barcoded and sequenced with high-throughput next-generation sequencing technology. This technology is highly sensitive, specific, and accurate in determining mtDNA mutations and the degree/level of heteroplasmy. © 2022 Wiley Periodicals LLC. Basic Protocol 1: PCR amplification of mitochondrial DNA Basic Protocol 2: Analysis of next-generation sequencing of mitochondrial DNA Basic Protocol 3: Mutect2 pipeline for automated sample processing and large-scale data analysis.
    Keywords:  DNA sequence analysis; PCR; heteroplasmy; mitochondria; mitochondrial DNA; next-generation sequencing
    DOI:  https://doi.org/10.1002/cpz1.412
  12. Nat Commun. 2022 May 12. 13(1): 2620
    Neuroligin-mediated neurodevelopmental defects are induced by mitochondrial dysfunction and prevented by lutein in C. elegans.
    Silvia Maglioni, Alfonso Schiavi, Marlen Melcher, Vanessa Brinkmann, Zhongrui Luo, Anna Laromaine, Nuno Raimundo, Joel N Meyer, Felix Distelmaier, Natascia Ventura.
      Complex-I-deficiency represents the most frequent pathogenetic cause of human mitochondriopathies. Therapeutic options for these neurodevelopmental life-threating disorders do not exist, partly due to the scarcity of appropriate model systems to study them. Caenorhabditis elegans is a genetically tractable model organism widely used to investigate neuronal pathologies. Here, we generate C. elegans models for mitochondriopathies and show that depletion of complex I subunits recapitulates biochemical, cellular and neurodevelopmental aspects of the human diseases. We exploit two models, nuo-5/NDUFS1- and lpd-5/NDUFS4-depleted animals, for a suppressor screening that identifies lutein for its ability to rescue animals' neurodevelopmental deficits. We uncover overexpression of synaptic neuroligin as an evolutionarily conserved consequence of mitochondrial dysfunction, which we find to mediate an early cholinergic defect in C. elegans. We show lutein exerts its beneficial effects by restoring neuroligin expression independently from its antioxidant activity, thus pointing to a possible novel pathogenetic target for the human disease.
    DOI:  https://doi.org/10.1038/s41467-022-29972-4
  13. FASEB J. 2022 May;36 Suppl 1
    Respiratory defects caused by mutations affecting the Endoplasmic Reticulum-Mitochondria Encounter Structure (ERMES) can be rescued by the deletion of COQ11.
    Noelle Alexa Novales, Catherine Clarke.
      Coenzyme Q (CoQ) is an essential redox-active lipid that plays a major role in the electron transport chain, driving mitochondrial ATP synthesis. Deficiency of CoQ causes a wide range of clinical deficiencies, highlighting the need to study the biosynthesis of this lipid to design therapeutics to treat these symptoms. In Saccharomyces cerevisiae, CoQ biosynthesis takes place exclusively in the mitochondrial matrix using a multi-subunit protein-lipid complex, the CoQ Synthome, that includes the polypeptides Coq3-Coq9 and Coq11. A recently identified regulator of CoQ Synthome assembly and CoQ production is the ER-mitochondria encounter structure (ERMES). ERMES is a tethering complex that bridges the ER and mitochondria, and the CoQ Synthome resides in specific membrane niches or domains directly adjacent to this complex. Loss of ERMES results in transcriptionally upregulated expression of COQ genes, yet inefficient synthesis of CoQ due to a destabilized CoQ Synthome. In this work, ERMESΔcoq11Δ mutants have been generated in an effort to correct this defect. Deletion of COQ11 has been shown to promote mitochondrial CoQ content, enhance CoQ Synthome stability, and rescue the respiratory deficiency of the coq10Δ mutant. We seek to investigate the functional roles of Coq11 and ERMES to better understand the regulation of CoQ biosynthesis and aid in the development of more effective therapeutics for diseases linked to CoQ deficiencies.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2208
  14. FASEB J. 2022 May;36 Suppl 1
    Gitelman-like syndrome caused by pathogenic variants in mitochondrial DNA.
    Jeroen H F de Baaij, Daan Viering, Karl P Schlingmann, Marguerite Hureaux, Tom Nijenhuis, Nine V Knoers, Rosa Vargas Poussou, Detlef Bockenhauer.
      Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl- cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants in CLCNKB, HNF1B, FXYD2 or KCNJ10 may result in renal phenocopies of Gitelman syndrome, as they can lead to reduced NCC activity. Nevertheless, ±10% of patients with a Gitelman syndrome phenotype remain genetically unsolved. After identification of mitochondrial DNA (mtDNA) variants in three families with Gitelman syndrome-like electrolyte abnormalities, 156 families were investigated for variants in MT-TI and MT-TF, encoding the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. In NCC-expressing HEK293 cells, mitochondrial dysfunction was induced to assess the effect on thiazide-sensitive 22Na+ transport. Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF) and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Maximal mitochondrial respiratory capacity was reduced in patient fibroblasts, caused by dysfunction of oxidative phosphorylation complex IV. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, demonstrated an inhibitory effect on NCC phosphorylation and NCC-mediated sodium uptake. Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman syndrome-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2172
  15. FASEB J. 2022 May;36 Suppl 1
    A Reduction in Tafazzin Decreases Mitochondrial Function in C2C12 Myotubes.
    Brandon J Richards, Ashley N Oliviera, David A Hood.
      Mitochondria, which are often regarded as the "powerhouse of the cell", are labile organelles that regulate cellular metabolism, determine cell fate, and act as important signaling hubs. A phospholipid that is exclusively found within mitochondria that serves numerous roles is cardiolipin (CL). In healthy mitochondria, CL is predominately localized in the inner mitochondrial membrane (IMM) and binds to several membrane proteins including those in the electron transport chain (ETC) and in the protein import machinery (PIM). Similarly, CL has also been shown to regulate cell death and mitophagy in dysfunctional mitochondria. Following the biosynthesis of nascent CL in the inner membrane, it is remodeled into its mature form by a transacylase, tafazzin (Taz). The absence of this enzyme is associated with Barth Syndrome, a disease characterized by cardiomyopathy and skeletal myopathy. Interestingly, little is known about the role of mature CL in skeletal muscle. Therefore, the objective of this study was to identify whether Taz deficiency diminishes mitochondrial function in the presence or absence of changes in organelle volume and function. We hypothesized that a reduction in Taz will attenuate mitochondrial function, while lysosomal and mitochondrial content will be elevated. To test this hypothesis, C2C12 myotubes were transfected on day 3 of differentiation with either a scrambled or Taz siRNA vector. Cells were harvested for analysis on day 7 of differentiation. Organelle volume was assessed using immunohistochemistry and Western blot techniques, whereas Seahorse technology was used to measure mitochondrial respiration. In comparison to C2C12 myotubes that were treated with a scrambled vector, Taz protein content was reduced by 85% (P<0.05) in siRNA-treated cells. Maximal oxygen consumption and ATP production were attenuated in Taz-deficient cells by 18% and 14%, respectively, while basal respiration remained unaffected. Lysosome and mitochondrial content were increased 20% and 24%, respectively, in Taz-deficient cells. Similarly, myotubes lacking Taz exhibited a 7% increase (P=0.056) in Complex IV protein content, but lysosomal-associated membrane protein 1 (LAMP1) decreased 33% (P<0.05). Myosin heavy chain (MHC) IIX was also elevated by 32% in mature CL depleted myotubes. CL-deficient myotubes were 32% smaller in diameter and 10% longer when compared to scrambled treated myotubes. These data suggest that a reduction in mature CL impairs skeletal muscle mitochondrial function. Moreover, although lysosomal content was elevated, lysosomes appear to have a reduced capacity to fuse with autophagosomes, which may in part explain why there is an increase in dysfunctional mitochondria. Future research should evaluate mitophagy flux and mitochondrial membrane potential in the presence of reduced mature CL in C2C12 myotubes, and whether exercise has the ability to rescue mitochondrial function.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3954
  16. J Inherit Metab Dis. 2022 May 11.
    Research Priorities for Mitochondrial Disorders: Current Landscape and Patient and Professional Views.
    Rhys H Thomas, Amy Hunter, Lyndsey Butterworth, Catherine Feeney, Tracey D Graves, Sarah Holmes, Pushpa Hossain, Jo Lowndes, Jenny Sharpe, Sheela Upadhyaya, Kristin N Varhaug, Marcela Votruba, Russell Wheeler, Kristina Staley, Shamima Rahman.
      Primary mitochondrial disorders encompass a wide range of clinical presentations and a spectrum of severity. They currently lack effective disease-modifying therapies and have a high mortality and morbidity rate. It is therefore essential to know that competitively-funded research designed by academics meets core needs of people with mitochondrial disorders and their clinicians. The Priority Setting Partnerships are an established collaborative methodology that brings patients, carers and families, charity representatives and clinicians together to try to establish the most pressing and unanswered research priorities for a particular disease. We developed a web-based questionnaire, requesting all patients affected by primary mitochondrial disease, their carers, and clinicians to pose their research questions. This yielded 709 questions from 147 participants. These were grouped into overarching themes including basic biology, causation, health services, clinical management, social impacts, prognosis, prevention, symptoms, treatment, and psychological impact. Following the removal of 'answered questions' the process resulted in a list of 42 discrete, answerable questions. This was further refined by web-based ranking by the community to 24 questions. These were debated at a face-to-face workshop attended by a diverse range of patients, carers, charity representatives and clinicians to create a definitive 'Top Ten of unanswered research questions for primary mitochondrial disorders'. These Top Ten questions related to understanding biological processes, including triggers of disease onset, mechanisms underlying progression and reasons for differential symptoms between individuals with identical genetic mutations; new treatments; biomarker discovery; psychological support; and optimal management of stroke-like episodes and fatigue. This article is protected by copyright. All rights reserved.
    Keywords:  Primary mitochondrial disease; gene therapy; patient involvement; priority setting partnership; treatment
    DOI:  https://doi.org/10.1002/jimd.12521
  17. J Clin Transl Hepatol. 2022 Apr 28. 10(2): 321-328
    Targeted Mitochondrial Delivery to Hepatocytes: A Review.
    Brent D Heineman, Xiaocong Liu, George Y Wu.
      Defects in mitochondria are responsible for various genetic and acquired diseases. Mitochondrial transplantation, a method that involves introduction of healthy donor mitochondria into cells with dysfunctional mitochondria, could offer a novel approach to treat such diseases. Some studies have demonstrated the therapeutic benefit of mitochondrial transplantation and targeted delivery in vivo and in vitro within hepatocytes and the liver. This review discusses the issues regarding isolation and delivery of mitochondria to hepatocytes and the liver, and examines the existing literature in order to elucidate the utility and practicality of mitochondrial transplantation in the treatment of liver disease. Studies reviewed demonstrate that mitochondrial uptake could specifically target hepatocytes, address the challenge of non-specific localization of donor mitochondria, and provide evidence of changes in liver function following injection of mitochondria into mouse and rat disease models. While potential benefits and advantages of mitochondrial transplantation are evident, more research is needed to determine the practicality of mitochondrial transplantation for the treatment of genetic and acquired liver diseases.
    Keywords:  Hepatocytes; In vitro techniques; Liver; Mitochondria; Transplantation
    DOI:  https://doi.org/10.14218/JCTH.2021.00093
  18. FASEB J. 2022 May;36 Suppl 1
    The primary astrocytic mitochondrial transplantation ameliorates ischemic stroke.
    Eun-Hye Lee, Minkyung Kim, Seung Hwan Ko, Minhyung Lee, Chang-Hwan Park.
      Mitochondria are important organelle which regulate adenosine triphosphate (ATP) production, intracellular calcium buffering, cell survival and apoptosis. They are known to deliver the potential therapeutic role in injured cells through transcellular transfer via extracellular vesicles (EVs), gap junctions, and tunneling nanotubes (TNTs). Astrocytes secrete numerous factors that promote neuron survival, synapse formation, and plasticity. Recent studies have demonstrated that astrocytes transfer mitochondria into damaged neurons to enhance cell viability and recovery. In this study, we observed that treatment of isolated mitochondria from rat primary astrocytes enhance cell viability and ameliorate H2 O2 -damaged neurons. Interestingly, the isolated astrocytic mitochondria increased cell number in damaged neurons but not normal neurons, even though the mitochondrial transfer efficiency was no difference between them. Furthermore, this effect showed in astrocytic mitochondrial transplantation to rat middle cerebral artery occlusion (MCAO) models. These findings suggest that mitochondrial transfer therapy can be used to acute ischemic stroke and other diseases treatment.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R802
  19. FASEB J. 2022 May;36 Suppl 1
    Single Cell Assessment of Mitochondrial Function.
    Sanjana Mahadev Bhat, Jane Q Yap, Oscar A Ramirez Ramirez, Philippe Delmotte, Gary C Sieck.
      Cellular mitochondrial function can be assessed using high resolution respirometry that measures O2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDHmax ) in isolated human airway smooth muscle (hASM) cells using 1-methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDHmax , the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil-immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDHmax was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP-mediated disruption of the mitochondrial proton gradient does not affect SDHmax , while SDHmax is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDHmax was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDHmax can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3283
  20. FASEB J. 2022 May;36 Suppl 1
    Understanding the Tissue-Specific Transcriptional Effects of Mitochondrial Introgression in Drosophila.
    Alexandra Burgess, John Santiago, Jennifer Sanders, David Rand.
      Mitochondrial DNA (mtDNA) mutations are an important cause of inherited disease. According to the United Mitochondrial Disease Foundation, "every 30 minutes a child is born who will develop a mitochondrial disease by age 10". Mitochondrial Replacement Therapy can be used as a form of in-vitro fertilization in which mitochondria are moved from a third party donor to a recipient oocyte or embryo. This new therapy enables women with mtDNA mutations to have healthy children. However, little is known about the effects of placing mtDNA into a "foreign" nuclear background. This study uses Drosophila as a model system to investigate how an alternative mitochondrial genotype (Sm21;OreR) effects the nuclear and mitochondrial transcriptome in 3 tissues (head, abdomen, and thorax). The effect of sex on RNA expression in these samples is also investigated. Drosophila melanogaster and Drosophila simulans flies were mated and extensively backcrossed to produce a progeny with isogenic D. melanogaster nuclear genome and D. simulans mtDNA (away team). The transcriptional response of tissues was then examined by performing RNA-Seq on triplicate biological replicates of male and female Sm21;OreR (away team) and control OreR;OreR (home team) flies. Using bash in Oscar systems RNA-seq reads from these tissues were aligned to a D. melanogaster genomic map. RStudio was used to perform statistical tests (PCA, Volcano plots, Heatmaps) to measure transcriptional variation within the samples. Our preliminary findings indicate that tissue type and sex are the main drivers of transcriptional difference within these samples.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3094
  21. FASEB J. 2022 May;36 Suppl 1
    Phosphorylation-based regulation of mitochondrial metabolism.
    Nataile Niemi.
      Phosphorylation has long been appreciated to influence mitochondrial metabolism via the regulation of pyruvate dehydrogenase. However, the extent to which phosphorylation broadly influences mitochondrial function remains unclear, despite the presence of multiple protein phosphatases within the organelle. We recently demonstrated that deletion of the mitochondrial matrix phosphatase Pptc7 unexpectedly caused perinatal lethality in mice, suggesting that the regulation of mitochondrial phosphorylation is essential in mammalian development. Pptc7-/- mice exhibit severe metabolic deficiencies, including hypoglycemia and lactic acidosis, and die within one day of birth. Biochemical and proteomic approaches revealed that Pptc7-/- tissues have decreased mitochondrial function concomitant with a post-transcriptional downregulation of mitochondrial proteins. Multiple elevated mitochondrial protein phosphorylation sites in Pptc7-/- tissues suggest novel functional connections between Pptc7-mediated dephosphorylation and these observed metabolic consequences. Interestingly, these modifications occur on components of the import machinery of the mitochondria and within the mitochondrial targeting sequences of select nuclear-encoded precursor proteins. Collectively, our data reveal an unappreciated role for a matrix-localized phosphatase in the post-translational regulation of the mitochondrial proteome and organismal metabolic homeostasis.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R6264
  22. FASEB J. 2022 May;36 Suppl 1
    The Role of ATF5 in Mitochondrial Maintenance, Biogenesis and UPRmt Signaling Following Acute Exercise in Skeletal Muscle.
    Mikhaela Slavin, Rita Kumari, David Hood.
      Maintenance of the mitochondrial protein folding environment is essential for organellar and cellular homeostasis. Over 99% of mitochondrial proteins require import into mitochondria, followed by their folding and intraorganellar sorting. Mitochondrial stress can result in the accretion of misfolded proteins, establishing a requirement for mitochondrial protein quality control (MQC) strategies. The Mitochondrial Unfolded Protein Response (UPRmt ) is a compartment-specific MQC mechanism that increases the expression of protective enzymes by Activating Transcription Factor 5 (ATF5) to restore mitochondrial function. Contractile activity during acute exercise is a stressor that has the potential to temporarily disrupt organellar protein homeostasis. However, the roles of ATF5 and the UPRmt in basal mitochondrial maintenance and exercise-induced UPRmt signaling in skeletal muscle are not known. To investigate this, we subjected WT and whole-body ATF5 KO mice to a bout of acute exercise and collected skeletal muscle tissues immediately after. ATF5 KO animals exhibited 2-fold increases in phosphorylated JNK protein levels, indicative of enhanced stress signaling. Interestingly, in KO muscle, PGC-1a protein was enhanced by 50% and 40% in nuclear and cytosolic compartments, respectively, suggesting an increased drive toward mitochondrial biogenesis in the absence of ATF5. Muscle from these animals also displayed a more abundant, but dysfunctional, mitochondrial pool, with a 20% increase in mitochondrial content, 30-40% reductions in respiration, and a 20% increase in ROS emissions, corresponding with no changes in exercise performance. The UPRmt proteins mtHSP70 and LONP were upregulated 20-30% in KO muscle, while ATF4 mRNA was upregulated 2.5-3.7-fold, along with an 8% increase in its nuclear localization. Furthermore, KO muscle showed an impaired UPRmt mRNA response to acute exercise, suggesting a regulatory role for ATF5 in the maintenance of a high-quality mitochondrial pool, and in mediating the transcription of UPRmt genes during exercise.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2945
  23. Theranostics. 2022 ;12(7): 3237-3250
    AGK regulates the progression to NASH by affecting mitochondria complex I function.
    Nan Ding, Kang Wang, Haojie Jiang, Mina Yang, Lin Zhang, Xuemei Fan, Qiang Zou, Jianxiu Yu, Hui Dong, Shuqun Cheng, Yanyan Xu, Junling Liu.
      Background: Impaired mitochondrial function contributes to non-alcoholic steatohepatitis (NASH). Acylglycerol kinase (AGK) is a subunit of the translocase of the mitochondrial inner membrane 22 (TIM22) protein import complex. AGK mutation is the leading cause of Sengers syndrome, characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, lactic acidosis, and liver dysfunction. The potential roles and mechanisms of AGK in NASH are not yet elucidated. Methods: Hepatic-specific AGK-deficient mice and AGK G126E mutation (AGK kinase activity arrest) mice were on a choline-deficient and high-fat diet (CDAHFD) and a methionine choline-deficient diet (MCD). The mitochondrial function and the molecular mechanisms underlying AGK were investigated in the pathogenesis of NASH. Results: The levels of AGK were significantly downregulated in human NASH liver samples. AGK deficiency led to severe liver damage and lipid accumulation in mice. Aged mice lacking hepatocyte AGK spontaneously developed NASH. AGK G126E mutation did not affect the structure and function of hepatocytes. AGK deficiency, but not AGK G126E mice, aggravated CDAHFD- and MCD-induced NASH symptoms. AGK deficiency-induced liver damage could be attributed to hepatic mitochondrial dysfunction. The mechanism revealed that AGK interacts with mitochondrial respiratory chain complex I subunits, NDUFS2 and NDUFA10, and regulates mitochondrial fatty acid metabolism. Moreover, the AGK DGK domain might directly interact with NDUFS2 and NDUFA10 to maintain the hepatic mitochondrial respiratory chain complex I function. Conclusions: The current study revealed the critical roles of AGK in NASH. AGK interacts with mitochondrial respiratory chain complex I to maintain mitochondrial integrity via the kinase-independent pathway.
    Keywords:  NDUFS2; fatty acid metabolism; mitochondrial ROS; mitochondrial respiratory chain
    DOI:  https://doi.org/10.7150/thno.69826
  24. FASEB J. 2022 May;36 Suppl 1
    Intrinsic Mitochondrial Function Impacts the Outcomes of Metformin Treatment on Skeletal Muscle Mitochondrial Morphology in Aged Rat.
    Arik Davidyan, Matthew P Bubak, Lauren G Koch, Tommy L Lewis, Benjamin F Miller.
      Aging is the main risk factor for many costly, irreversible, and comorbid chronic diseases. The anti-diabetic drug Metformin is being considered as an anti-aging treatment although little is known about its effects in healthy individuals. Previous data from our lab suggest that metformin is beneficial to individuals that are relatively insulin resistant and may be detrimental to those who are insulin sensitive. We aimed to understand how intrinsic function of mitochondria affects metformin treatment outcomes in skeletal muscle. We hypothesized that the effects of metformin on skeletal muscle mitochondrial morphology and remodeling will differ based on intrinsic mitochondrial function. To test our hypothesis, we used the High-Capacity Runner/Low-Capacity Runner (HCR/LCR) rat model system which comprises two selectively bred lines of genetically heterogeneous rats that diverge for exercise capacity, cardiovascular risk factors, lifespan, and healthspan. In drinking water, we treated six HCR and four LCR male rats (age 18 months) with a dose of 100 mg/kg/day metformin for a week, followed by three weeks of 200 mg/kg/day. We included HCR and LCR control groups (n=5) for a total of four groups. A week prior to sacrifice, we initiated deuterium oxide (D2O) labeling to assess protein turnover and electroporated DNA constructs that encode for mitochondrial matrix-targeted YFP and tdTomato-tagged TOM20, a protein located on the mitochondrial outer membrane. We analyzed protein turnover in hindlimb muscles using GC-MS, and prepared histological sections of the Tibialis Anterior (TA) muscle for further analysis of mitochondrial morphology. Normalized to tibial length, the mass of the gastrocnemius (-16.6%) and plantaris (-20.8%) muscles were lower in metformin treated HCR rats compared to untreated, while the normalized mass of the TA muscle was lower in both HCR (-15%) and LCR (-20.1%) metformin treated rats compared to control. There were no significant differences in mean fiber cross sectional area (CSA) of the TA muscle. We detected no significant differences in bulk protein synthesis of both the myofibrillar and mitochondrial fractions of the TA, gastrocnemius, and soleus muscles. The number of mitochondrial branches observed in a cross section of TA fibers from metformin treated LCR rats were 30.5% lower compared to control LCR rats. However, mean CSA of each mitochondrial branch in metformin treated LCR rats increased by 82.8% compared to control LCR rats, which, together with the decrease in branch number, resulted in maintenance of the cumulative mitochondrial CSA in each fiber. Analysis based on mitochondrial sub-populations showed that the subsarcolemmal mitochondrial population was more impacted than intermyofibrillar mitochondria. There were no significant differences in mitochondrial morphology in control vs metformin treated HCR rats. Our data supports our hypothesis that metformin has differential effects in skeletal muscle based on intrinsic mitochondrial function prior to treatment. Additional studies are needed to determine what impact these disparities have on the aging process.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4693
  25. FASEB J. 2022 May;36 Suppl 1
    A Novel Peroxin is Required for Mitochondrial Morphology: Implications for Resolving a New Peroxisome-Mitochondrial Contact Site.
    Emily W Knight, Andrew Gianos, Logan Crowe, Meredith T Morris.
      Peroxisomes are dynamic and ubiquitous organelles that house many metabolic pathways and interact with other organelles such as the endoplasmic reticulum, lipid droplets, and mitochondria. One mechanism for organelle interaction is through membrane contact sites. While contact sites between multiple organelles have been identified, little is known about the proteins that serve as molecular tethers in such sites. We study organelle dynamics using peroxisome-like organelles called glycosomes in the early diverging organism Trypanosoma brucei and have identified a novel peroxin (protein involved in peroxisome biogenesis) that is essential for mitochondrial morphology. Silencing this protein leads to a significant growth defect and swollen mitochondria. Multiple mitochondrial membrane transport channels have been identified in immunoprecipitation studies. Based on these findings, we hypothesize that this protein that we have named a putative peroxisome-mitochondrial contact protein (PPMCP), localizes to glycosomes and mitochondria at contact points, which facilitate the transfer of metabolites between the two organelles. Disruption of this connection results in "leaky" mitochondria and cell death. Current work is focused on resolving the metabolic defects in PPMCP-deficient cells and identifying additional molecular components of these contact sites. This work forwards our understanding of how contact sites are established and the role they play in interorganelle communication.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2441
  26. Geroscience. 2022 May 09.
    CYB5R3 overexpression preserves skeletal muscle mitochondria and autophagic signaling in aged transgenic mice.
    Sara López-Bellón, Sandra Rodríguez-López, José A González-Reyes, M Isabel Burón, Rafael de Cabo, José M Villalba.
      Cytochrome b5 reductase 3 (CYB5R3) overexpression activates respiratory metabolism and exerts prolongevity effects in transgenic mice, mimicking some of the salutary effects of calorie restriction. The aim of our study was to understand how CYB5R3 overexpression targets key pathways that modulate the rate of aging in skeletal muscle, a postmitotic tissue with a greater contribution to resting energy expenditure. Mitochondrial function, autophagy and mitophagy markers were evaluated in mouse hind limb skeletal muscles from young-adult (7 months old) and old (24 months old) males of wild-type and CYB5R3-overexpressing genotypes. Ultrastructure of subsarcolemmal and intermyofibrillar mitochondria was studied by electron microscopy in red gastrocnemius. CYB5R3, which was efficiently overexpressed and targeted to skeletal muscle mitochondria regardless of age, increased the abundance of complexes I, II, and IV in old mice and prevented the age-related decrease of complexes I, III, IV, and V and the mitofusin MFN-2. ATP was significantly decreased by aging, which was prevented by CYB5R3 overexpression. Coenzyme Q and the mitochondrial biogenesis markers TFAM and NRF-1 were also significantly diminished by aging, but CYB5R3 overexpression did not protect against these declines. Both aging and CYB5R3 overexpression upregulated SIRT3 and the mitochondrial fission markers FIS1 and DRP-1, although with different outcomes on mitochondrial ultrastructure: old wild-type mice exhibited mitochondrial fragmentation whereas CYB5R3 overexpression increased mitochondrial size in old transgenic mice concomitant with an improvement of autophagic recycling. Interventions aimed at stimulating CYB5R3 could represent a valuable strategy to counteract the deleterious effects of aging in skeletal muscle.
    Keywords:  Aging; Autophagy; Cytochrome b 5 reductase; Mitochondria; Skeletal muscle
    DOI:  https://doi.org/10.1007/s11357-022-00574-8
  27. FASEB J. 2022 May;36 Suppl 1
    Mitochondrial Transplantation as a Novel Therapeutic for Retinal Metabolic Dysfunction-Driven Disease.
    Erik R Butcher, Daisy Y Shu, Scott Frank, Magali Saint-Geniez.
      Metabolic dysfunction and mitochondria defect are implicated in several age-associated diseases including age-related macular degeneration (AMD), a leading cause of blindness in the elderly. In AMD, mitochondrial oxidative stress in the retinal pigment epithelium (RPE) drives disease progression and growth of atrophic lesions. Mitochondria release and uptake has been recently identified as a novel mechanism of intercellular communication but its implication in ocular diseases such as AMD has never been investigated. Here, we examined the role of mitochondrial transfer as a new mechanism of metabolic crosstalk between RPE cells. Diseased mitochondria were purified from RPE cells treated with the AMD-associated cytokine TNFα (10 ng/mL), administered to host RPE cells and the effects of MitoTNFA compared to MitoCtrl , isolated from control RPE, or exposure to exogenous TNFα. We showed that treatment of healthy RPE with MitoTNFA , and not MitoCtrl , triggers mitochondrial network fragmentation and transcriptional upregulation of inflammatory factors RelB, IL6, IL8, and repression of PGC1α, mirroring the effect of direct TNFα treatment. ELISA assay confirmed that the effects observed were not caused by presence of soluble TNFα within the mitochondrial fraction. Metabolic profiling using the Seahorse XFe96 bioanalyzer further validated the ability of MitoTNFA to phenocopy TNFα-induced metabolic reprograming in RPE. Finally, we demonstrated that transfer of MitoCtrl improved the bioenergetic functions of both healthy and diseased RPE with significant increases in basal, maximal, and spare respiratory functions. Our results showed that mitochondria transfer recapitulates the context-specific phenotype from donor to host cells in RPE. This new paradigm in RPE biology may not only explain the centrifugal expansion of RPE lesions in AMD but also presents a promising therapeutic avenue for mitochondria-driven disorders such as AMD.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2669
  28. FASEB J. 2022 May;36 Suppl 1
    3D mitochondrial remodeling during postnatal heart development.
    Yuho Kim, Christopher K Bleck, Brian Glancy.
      The heart is featured by high mitochondrial volume, by which continuous contractile activity can be maintained. However, it is unclear how mitochondrial structures are matured during postnatal development. Using FIB-SEM, we thus sought to identify 3D mitochondrial structural characteristics in the heart of mice at postnatal (P) day 1, 7, 14, and 42, respectively. Here, matured mitochondria (P14-42) were shown to have larger and more spherical structures and were highly connected with adjacent mitochondria as compared to early postnatal days (P1-7). Nevertheless, the immature mitochondria appeared to have more intense interaction with other subcellular organelles including lipid droplets and sarcotubular structures (i.e., SR/T-tubules). Thus, our study suggests that initial mitochondrial development can be augmented by crosstalk with adjacent mitochondria as well as other subcellular components and that it would be important for the further mitochondrial maturation process in the heart muscle.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R6223
  29. FASEB J. 2022 May;36 Suppl 1
    Machinery in motion: New insights into mitochondrial proteostasis.
    Gabriel Lander, Jeff Mindrebo.
      ATP-dependent AAA+ proteases are critical regulators of mitochondrial functions, playing crucial roles in the mitochondrial quality control response system. The past years have provided much structural insight into the molecular mechanisms associated with degradation of substrates by these proteolytic machines. Recent cryo-electron microscopy (cryo-EM) studies have provided critical insights into a conserved, AAA+-mediated hand-over-hand substrate translocation mechanism required to processively engage, unfold, and degrade proteolytic substrates. However, the underlying mechanisms regulating their various activities are not well understood. Numerous prior studies suggest that AAA+ protease have evolved numerous layers of regulation to control or tune proteolytic activity to meet cellular needs. Herein, we present compelling biochemical and structural data that support a long-range allosteric model linking substrate binding to proteolytic activity.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0I120
  30. FASEB J. 2022 May;36 Suppl 1
    Mitochondrial GRK2 is a Novel Regulator of Cardiac Energetics.
    Kimberly M Ferrero, Kurt Chuprun, Jessica Pfleger, Douglas Tilley, Erhe Gao, Walter J Koch.
      G protein-coupled receptor (GPCR) kinase 2 (GRK2) is highly expressed in the heart, where during injury or heart failure (HF), both its levels and activity increase. GRK2 is canonically studied in the context of GPCR phosphorylation; however, noncanonical activities of GRK2 have emerged and it is now appreciated that GRK2 has a large non-GPCR interactome. For example, in cardiac myocytes, GRK2 translocates from the cytosol to mitochondria (mtGRK2) following oxidative stress or ischemia injury, and this pool of mtGRK2 is associated with negative effects on metabolism and also induces myocyte cell death. However, the mechanisms by which mtGRK2 contributes to cardiac dysfunction and HF are not fully understood. We hypothesized that mtGRK2 could have novel substrates and phosphorylate proteins involved in mitochondrial bioenergetics, thus contributing to our previously established post-injury phenotype. Stress-induced mitochondrial translocation of cytosolic GRK2 was validated in cell and animal models and the mtGRK2 interactome was identified using liquid chromatography-mass spectroscopy (LCMS). Proteomics analysis identified mtGRK2 interacting proteins which were involved in mitochondrial dysfunction, bioenergetics, and OXPHOS, particularly complexes I, II, IV and V of the electron transport chain (ETC). Specifically, mtGRK2 interactions with Complex V (ATP synthase) subunits were particularly increased following stress. We established that mtGRK2 phosphorylates ATP synthase on the F1 catalytic barrel, which is critical for oxidative phosphorylation and ATP production. We have also determined that alterations in either the levels or activity of GRK2 appear to alter ATP synthase enzymatic activity in vitro. Excitingly, in vivo data suggest that reducing levels of GRK2 in a mouse model of myocardial infarction prevents the post-injury reduction in ATP synthesis. We are currently assessing the ability of the SSRI drug paroxetine, a GRK2 inhibitor, to preserve mitochondrial bioenergetics in a transgenic GRK2 mouse model. Thus, phosphorylation of the ATP synthesis machinery by mtGRK2 may contribute to the impaired mitochondrial phenotype observed in injured or failing heartssuch as reduced fatty acid metabolism and substrate utilization. These data uncover a druggable, novel target for rescuing cardiac function in patients with injured and/or failing hearts.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2991
  31. FASEB J. 2022 May;36 Suppl 1
    Determining the role of ATF4 in the regulation of mitochondrial remodeling during myotube differentiation and contractile activity.
    Jonathan M Memme, Victoria C Sanfrancesco, David A Hood.
      ATF4 is a transcriptional regulator that is selectively induced in response to cellular stress conditions, such as exercise, through the activation of the integrated stress response (ISR). Specifically, in the context of mitochondrial-specific stress, ATF4 is induced as a key component of the mitochondrial unfolded protein response (UPRmt ) and is suggested to upregulate various organellar chaperones and proteases that both preserve, and promote, mitochondrial function. In response to the stress brought about by contractile activity, ATF4 has been implicated in regulating skeletal muscle health by mediating the various signaling events associated with mitochondrial quality control (MQC), including i) mitochondrial biogenesis (expansion), ii) the mitophagy-lysosomal clearance of damaged and thus potentially harmful organelles, or iii) by activating the mitochondrial unfolded protein response (UPRmt ) as an intermediate response to acute cellular stress. However, it remains to be determined whether ATF4 is necessary for mitochondrial adaptations in skeletal muscle. Therefore, our aim was to determine whether ATF4 is required for the maintenance of mitochondrial function and adaptation following an acute 3h bout of contractile activity (ACA), or after repeated bouts (4 days; CCA) in C2C12 myotubes in which ATF4 was either overexpressed (OE) or knocked down (KD) via lentiviral transduction of plasmids containing the ATF4 open reading frame, or siRNA, respectively. Knockdown of ATF4 promoted elongated myotube formation following 5 days of differentiation, whereas ATF4 OE contributed to the opposite effect in which shorter myotubes were observed relative to the control condition. Induction of PGC-1α mRNA following ACA in ATF4 KD myotubes was attenuated, suggesting diminished drive for mitochondrial biogenesis in the absence of ATF4. The mRNA expression of ATF5, a downstream target of ATF4, was induced both in response to ACA as well as with ATF4 OE, and was reduced in the absence of ATF4. Mitophagy flux, measured by mitochondrial-localized LC3-II, was upregulated in ATF4 OE and KD myotubes basally, and was augmented in both control and ATF4 OE cells following ACA, but not in the absence of ATF4. Furthermore, ATF4 OE revealed decrements in mitochondrial content indicated by 20%, and 40%, reductions in VDAC and COX I protein expression relative to control, while 1.2-1.5-fold increases in these markers were observed when ATF4 was knocked down. However, following CCA, COX I and VDAC protein content were increased 3-4-fold in ATF4 OE cells, while there was no observable increase in mitochondrial content in ATF4 KD myotubes. Together, these data highlight a potential role of ATF4 in regulating basal mitochondrial content and further suggest that ATF4 may be required for contractile activity-induced increases in mitochondrial content.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4060
  32. Hum Mol Genet. 2022 May 14. pii: ddac109. [Epub ahead of print]
    Leber's hereditary optic neuropathy (LHON)-associated ND6 14 484 T > C mutation caused pleiotropic effects on the complex I, RNA homeostasis, apoptosis and mitophagy.
    Min Liang, Chun Ji, Liyao Zhang, Xuan Wang, Cuifang Hu, Juanjuan Zhang, Yiwei Zhu, Jun Q Mo, Min-Xin Guan.
      Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mitochondrial DNA (mtDNA) mutations. LHON-linked ND6 14 484 T > C (p.M64V) mutation affected structural components of complex I but its pathophysiology is poorly understood. The structural analysis of complex I revealed that the M64 forms a nonpolar interaction Y59 in the ND6, Y59 in the ND6 interacts with E34 of ND4L, and L60 of ND6 interacts with the Y114 of ND1. These suggested that the m.14484 T > C mutation may perturb the structure and function of complex I. Mutant cybrids constructed by transferring mitochondria from lymphoblastoid cell lines of one Chinese LHON family into mtDNA-less (ρo) cells revealed decreases in the levels of ND6, ND1 and ND4L. The m.14484 T > C mutation may affect mitochondrial mRNA homeostasis, supported by reduced levels of SLIRP and SUPV3L1 involved in mRNA degradation and increasing expression of ND6, ND1 and ND4L genes. These alterations yielded decreased activity of complex I, respiratory deficiency, diminished mitochondrial ATP production and reduced membrane potential, and increased production of reactive oxygen species in the mutant cybrids. Furthermore, the m.14484 T > C mutation promoted apoptosis, evidenced by elevating Annexin V-positive cells, release of cytochrome c into cytosol, levels in apoptotic proteins BAX, caspases 3, 7, 9 and decreasing levels in anti-apoptotic protein Bcl-xL in the mutant cybrids. Moreover, the cybrids bearing the m.14484 T > C mutation exhibited the reduced levels of autophagy protein LC3, increased levels of substrate P62 and impaired PINK1/Parkin-dependent mitophagy. Our findings highlighted the critical role of m.14484 T > C mutation in the pathogenesis of LHON.
    DOI:  https://doi.org/10.1093/hmg/ddac109
  33. FASEB J. 2022 May;36 Suppl 1
    Expression profiles of protein markers regulating the mitochondrial lifecycle in skeletal muscle of acute spinal cord transected rats.
    Jon-Philippe K Hyatt, Beau R Pullman.
      Spinal cord transection (ST) inactivates the neuromuscular complex and triggers progressive muscular atrophy of the affected skeletal muscles. Disruption in neural input also leads to a reduction in mitochondrial volume. The mitochondria lifecycle can be generally divided into processes of biogenesis, expression of OXPHOS proteins, fusion, fission, and mito/autophagy; specifically, the balance between biogenesis vs. mitophagy dictates the rate of formation and destruction, respectively. Here, we examined the expression profiles of the markers associated with the mitochondrial lifecycle up to one month following ST in phenotypically slow (soleus; SOL) and mixed (plantaris; PLT) skeletal muscle. We hypothesized that ST would induce a reduction in mitochondrial DNA (mtDNA) copy number, decrease expression in markers associated with biogenesis, and increased proteins that regulate mito/autophagy. Adult female Sprague Dawley rats were randomly divided into control (CON; n=6), 1 day (1dST; n=5), 8 day (8dST; n=8), and 28 day post-ST (28dST; n=8). Compared to CON relative SOL and PLT muscle masses (absolute mass / body mass) were 98, 65, and 62% and 85, 60, and 73% at 1, 8 and 28d post-ST, respectively (p<0.05). Compared to CON, no significant changes were observed in mtDNA copy number in ST muscles following amplification using standard end-point PCR protocols for mtDNA (ND1, COX1, or ATP6) and nuclear (β2M) genes (p>0.05). Total protein was isolated from SOL and PLT, separated using standard SDS-PAGE protocol, and probed for markers associated with various stages of the mitochondrial lifecycle using standard western blot protocols. Upstream markers known to influence mitochondrial lifecycle signaling (Rev-Erbα, AMPK) showed a varied temporal and muscle-specific response: in 1dST SOL muscles, Rev-Erbα increased 74% from CON then decreased 43% at 28dST, whereas 8dST PLT muscle exhibited a 44% increase (p<0.05). In 8dST SOL and PLT muscles, the pAMPK:AMPK ratio was elevated 366 and 85% from CON, respectively (p<0.05). Markers for mitochondrial biogenesis (PGC-1α, NRF1, NRF2) in SOL and PLT muscles were largely unchanged, although a ~50% decrease in Tfam protein expression was observed at each ST time point compared to CON (p<0.05). Mitofusion2, a marker for mitochondrial fusion, was unchanged (p>0.05). However, for markers detecting mitochondrial fission (MFF, Drp1, Fis1), we observed a ~60% increase in MFF expression in 1dST SOL and PLT muscles (p<0.05). As expected markers for mitophagy (Parkin, PINK, p62, LC3 I/II) were significantly elevated: Parkin increased ~70% at 28dST (p<0.05), whereas LC3 I/II increased 65% and 112% at 1dST in in SOL and PLT muscles, respectively (p<0.05), and remained significantly elevated in PLT muscles at 8dST (27%) and 28d (37%) (p<0.05). Taken together, these data suggest that mitochondrial volume and expression of markers regulating mitochondrial biogenesis and fusion are not influenced by acute ST despite a decrease in muscle mass. However, selective markers for fission and mitophagy are significantly elevated in both SOL and PLT muscles, suggesting that mitochondrial destruction mechanism are activated acutely following ST injury. Lastly, selective proteins of the OXPHOS protein complex significantly increased in 1dST SOL and PLT muscles suggesting possible activity-dependent regulation of mitochondrial gene expression.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R5789
  34. Stem Cell Res. 2022 May 04. pii: S1873-5061(22)00155-6. [Epub ahead of print]62 102806
    Generation of homozygous PRKN, PINK1 and double PINK1/PRKN knockout cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing.
    Carol X-Q Chen, Zhipeng You, Narges Abdian, Julien Sirois, Irina Shlaifer, Mahdieh Tabatabaei, Marie-Noëlle Boivin, Lydiane Gaborieau, Jason Karamchandani, Lenore K Beitel, Edward A Fon, Thomas M Durcan.
      Autosomal recessive mutations in either PRKN or PINK1 are associated with early-onset Parkinson's disease. The corresponding proteins, PRKN, an E3 ubiquitin ligase, and the mitochondrial serine/threonine-protein kinase PINK1 play a role in mitochondrial quality control. Using CRISPR/CAS9 technology we generated three human iPSC lines from the well characterized AIW002-02 control line. These isogenic iPSCs contain homozygous knockouts of PRKN (PRKN-KO, CBIGi001-A-1), PINK1 (PINK1-KO, CBIGi001-A-2) or both PINK1 and PRKN (PINK1-KO/PRKN-KO, CBIGi001-A-3). The knockout lines display normal karyotypes, express pluripotency markers and upon differentiation into relevant brain cells or midbrain organoids may be valuable tools to model Parkinson's disease.
    DOI:  https://doi.org/10.1016/j.scr.2022.102806
  35. FASEB J. 2022 May;36 Suppl 1
    The Role of the Optic Atrophy 1 (OPA1) Protein in Drug-Induced Liver Injury.
    Hakjoo Lee, Hiromi Sesaki, Yisang Yoon.
      The liver is the metabolic hub, and is responsible for the myriad of processes including the nutrient homeostasis and detoxification. Mitochondria of liver are critical for these functions. The detoxification process in liver, when severe, often results in liver damage through causing oxidative stress. Mitochondria are the main source of ROS and are also vulnerable to oxidant damage. Therefore, mitochondrial dysfunction is one of the prominent causes for drug-induced liver injury. Mitochondrial fission and fusion, the main processes of mitochondrial dynamics, determine mitochondrial shape, and are important for functional maintenance of mitochondria. However, the role of mitochondrial dynamics in drug-induced liver injury is poorly understood. In the current study, we examined the role of the optic atrophy 1 (OPA1) protein in drug-induced liver injury. OPA1 is associated with mitochondrial inner membrane (IM) and mediates IM fusion. OPA1 also regulates cristate structure, and is required for proper electron transport and ATP production. To investigate the OPA1's role, we used liver-specific OPA1-knockout (OPA1-LKO) mice with acetaminophen (APAP) administration as a model for drug-induced liver injury. We generated OPA1-LKO mice by crossing OPA1 flox mice with mice carrying the Cre recombinase under the albumin promoter. Whereas whole body KO of OPA1 causes embryonic lethality, OPA1-LKO mice appeared healthy and showed normal growth and behavior. Although the OPA1 gene in the liver was disrupted, OPA1-LKO mice showed approximately 30% of OPA1 remaining in the liver, presumably due to less efficient albumin promoter-mediated Cre expression and from other cell types of the liver. Liver histology revealed that OPA1-KO livers have disorganized hepatic cords with enlarged hepatocytes. Despite a reduced OPA1 level, mitochondria in OPA1-KO liver show near intact cristae structure and respiration, suggesting that a low level of OPA1 would support mitochondrial function in liver. We then tested the effect of OPA1 LKO on liver function under APAP stress. In APAP overdose, excess APAP metabolite depletes GSH in hepatocytes, which causes mitochondrial oxidative stress, leading to mitochondrial permeability transition, mitochondrial dysfunction, ATP depletion, and ultimately necrotic cell death. Upon administration of excess APAP, we found that OPA1-LKO mice were more sensitive to APAP-induced liver injury compared with the control mice. Histological analyses showed significantly more expanded focal centrilobular necrosis with vacuolization, cell swelling, and nuclear disintegration in OPA1-KO livers. Alanine aminotransferase levels, as a clinical chemistry parameter, were higher in OPA1-LKO mice than in control mice with APAP overdose. Furthermore, phospho-JNK levels were higher in OPA1-KO livers, indicating increased initial oxidative stress. However, depletion of hepatic glutathione (GSH) contents and reduction of GSH/GSSG ratio upon APAP treatment were similar between the LKO and control liver. Together, our experimental results indicate that although liver is tolerant to a reduced level of OPA1, OPA1 depletion lowers the stress threshold and makes hepatocytes more sensitive to APAP-induced liver injury.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3945
  36. FASEB J. 2022 May;36 Suppl 1
    Role of TFE3 in Mitochondrial Adaptations to Skeletal Muscle Disuse.
    Ashley N Oliveira, Jonathan M Memme, Jenna Wong, David A Hood.
      Prolonged muscle disuse is accompanied by a phenotypic shift characterized by declines in mitochondrial content and function within skeletal muscle. This loss in mitochondrial content can be partially attributed to elevations in various catabolic processes that occur with atrophy. One of these is termed mitophagy, a selective form of cellular recycling (autophagy) whereby dysfunctional mitochondria are degraded via lysosomes. TFEB and TFE3 are key transcription factors that regulate lysosomes by activating the expression of various lysosomal genes. Loss of TFE3 has been associated with depressed levels of autophagy and with mitochondrial dysfunction. It is speculated that these functional impairments are due to diminished clearance via the lysosomes, however to date this has not been examined. We hypothesized that the loss of TFE3 would amplify the mitochondrial dysfunction associated with muscle disuse, while paradoxically preserving muscle mass and mitochondrial content. Using a severe model of muscle disuse, sciatic denervation, we observed a 10% loss in hindlimb muscle mass within 7 days of denervation. In the absence of TFE3, a trend for muscle preservation was observed, as these animals lost 20% less muscle mass than WT counterparts. Reduced rates of respiration supported by complex I and II were observed irrespective of genotype, concurrent with elevated ROS emissions, suggesting an impaired oxidative capacity following 7 days of denervation. Surprisingly, TFE3 KO animals did exhibit lower levels of oxidative stress basally, but this too increased following 7 days of denervation, and taken relative to baseline the fold induction of ROS emission was 3 times greater than WT animals. Higher levels of mitophagy flux were observed in the absence of TFE3 basally, which supports the observed 35% decline in mitochondrial content as measured by COX activity, as well as the lower levels of ROS emissions. Following only 1 day of denervation, increases in mitophagy flux were observed in WT animals, while the response in KO animals was clearly attenuated. In the WT animals, denervation led to a 30% reduction in mitochondrial content, however no change was observed in the absence of TFE3. Finally, increases in a number of autophagy-related markers such Beclin-1 and ATG7 were observed following denervation irrespective of genotype. However, the mature form of Cathepsin B, a hydrolytic enzyme, was markedly reduced by 55% in the absence of TFE3 and did not increase to the same extent as WT following 7 days of denervation suggesting an impairment in lysosomal function. Together, our results suggest that TFE3 exerts multiple roles in skeletal muscle plasticity, as a partial mediator of muscle mass, and in the control of lysosomal function and mitophagy in response to the acute stress of muscle disuse.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4019
  37. Eur J Hum Genet. 2022 May 09.
    Characterisation of a novel OPA1 splice variant resulting in cryptic splice site activation and mitochondrial dysfunction.
    Joshua Paul Harvey, Patrick Yu-Wai-Man, Michael Edward Cheetham.
      Autosomal dominant optic atrophy (DOA) is an inherited optic neuropathy that results in progressive, bilateral visual acuity loss and field defects. OPA1 is the causative gene in around 60% of cases of DOA. The majority of patients have a pure ocular phenotype, but 20% have extra-ocular features (DOA +). We report on a patient with DOA + manifesting as bilateral optic atrophy, spastic paraparesis, urinary incontinence and white matter changes in the central nervous system associated with a novel heterozygous splice variant NM_015560.2(OPA1):c.2356-1 G > T. Further characterisation, which was performed using fibroblasts obtained from a skin biopsy, demonstrated that this variant altered mRNA splicing of the OPA1 transcript, specifically a 21 base pair deletion at the start of exon 24, NM_015560.2(OPA1):p.Cys786_Lys792del. The majority of variant transcripts were shown to escape nonsense-mediated decay and modelling of the predicted protein structure suggests that the in-frame 7 amino acid deletion may affect OPA1 oligomerisation. Fibroblasts carrying the c.2356-1 G > T variant demonstrated impaired mitochondrial bioenergetics, membrane potential, increased cell death, and disrupted and fragmented mitochondrial networks in comparison to WT cells. This study suggests that the c.2356-1 G > T OPA1 splice site variant leads to a cryptic splice site activation and may manifest in a dominant-negative manner, which could account for the patient's severe syndromic phenotype.
    DOI:  https://doi.org/10.1038/s41431-022-01102-0
  38. FASEB J. 2022 May;36 Suppl 1
    Decreased Mitochondrial Unfolded Protein Response (UPRmt) in HFpEF.
    Shyam S Nandi, Gerald M Maresh, Kaushik P Patel.
      Mitochondrial dysfunction is a feature of heart failure with preserve ejection fraction (HFpEF). Central infusion of Ang II causes sympatho-excitation and hypertension with consequent HFpEF. The UPRmt activation is a retrograde mitochondrial stress response that promote recovery of defective mitochondria. UPRmt is activated by misfolded accumulation of nuclear-encoded mitochondrial proteins in cytosol and mitochondria. Despite this information, the mechanisms of UPRmt and mitochondrial dysfunction are largely unknown in the HFpEF heart. We hypothesized that concomitant sympathoexcitation with hypertension downregulates cardiac UPRmt leads to cardiac remodeling in HFpEF. Male Sprague-Drawly rats (250-300g) were subjected to central infusion of either Ang II (at 20 ng/min, 0.5 μl/h, ICV) or isotonic saline (0.5 μl/h, ICV, control) through osmotic mini-pumps for 14 days. This leads to concomitant sympathetic overstimulation and systemic hypertension with myriad features of HFpEF. Transthoracic echocardiography and haemodynamic recordings were performed at day 14 post ICV infusion. UPRmt , mitochondrial injury, and cardiac remodeling were assessed using, whole-tissue, cytosolic, and isolated mitochondrial protein Western immunoblots, cytochemistry, and histology. Sympatho-excitatory effect on UPRmt was examined in vitro using norepinephrine (NE) and H9c2 cardiomyocytes. The HFpEF rats showed significant diastolic dysfunction indicated by reduced E/A (HFpEF: 1.2 ± 0.1 vs Con: 1.5 ± 0.2) with preserved left ventricular ejection fraction (HFpEF: 75 ± 3% vs Con: 77 ± 4%). Histological evaluation showed increased cardiomyocyte hypertrophy (HFpEF: 46.3 ± 5 vs Con: 36.9 ± 6) and cardiac fibrosis (HFpEF: 4.3 ± 0.2 vs Con: 2.1 ± 0.3). Measurement of ATF5 (Activating Transcription Factor 5), a key UPRmt activation marker was decreased in HFpEF cytosol and mitochondria, while mitochondrial chaperonin HSP60 (heat-shock protein 60) was decreased in cytosol but increased in HFpEF mitochondria. Simultaneously, there was increased accumulation of oxidative phosphorylation (OXPH) Complex I, IV, and V misfolded subunits in the mitochondrial matrix. Furthermore, YME1L1, a mitochondrial matrix metalloprotease of misfolded protein degradation is reduced in HFpEF mitochondria. Concomitantly, there was increased mitochondrial ROS, reduced Mn-SOD, and increased autophagy markers p62-SQSTM1 and LC3B-II in HFpEF hearts. Notably, a reduced mitochondrial biogenesis and fusion, but increased fission, indicates a lack of UPRmt stress activated mitochondrial recovery in HFpEF heart. Our in vitro data corroborated a reduced UPRmt in response to NE treatment associated with mitochondrial depolarization and increased mitochondrial ROS level. In conclusion, concomitant sympatho-excitation and systemic hypertension contributes to reduced UPRmt , which is a unique feature of HFpEF heart. A lack of UPRmt activation fails to execute defective mitochondrial recovery in HFpEF heart conceivably driving HFpEF cardiac remodeling. This study identifies the key intermediary links of UPRmt downregulation in a potential HFpEF rat model. Therefore, boosting UPRmt can potentially have a therapeutic benefit for clinical HFpEF.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R451
  39. FASEB J. 2022 May;36 Suppl 1
    Evidence of Mitochondrial Impairment in a Mouse Model of Down Syndrome.
    Cynthia Anyaoku, Lara R DeRuisseau, Josef Brandauer.
      INTRODUCTION: Down Syndrome (DS) is typically the result of triplication of the 21st chromosome. DS causes widespread developmental and physical changes to the body. One commonly observed physical change of DS is muscle weakness, which can contribute to difficulty in executing activities of daily living. Nicotinamide adenine dinucleotide (NAD) is an important determinant of skeletal muscle function and mitochondrial activity. Currently, it is unknown whether muscle weakness in those with DS is at least partially related to an imbalance of NAD or a lack of mitochondrial NAD usage. Nicotinamide phosphoribosyl transferase (Nampt) catalyzes the rate-limiting step in the mammalian NAD recycling pathway, while SIRT3 is an NAD-dependent protein deacetylase which regulates mitochondrial content and activity. We hypothesized the expression of these proteins would be decreased in Ts65Dn animals, an established mouse model of DS, relative to wild-type (WT) animals.METHODS: Diaphragm and left ventricle samples from 12- or 18-month-old male Ts65Dn mice and WT animals were pulverized and homogenized in ice-cold lysis buffer. Total lysate protein concentration was determined by Bradford assay. Samples were resolved in SDS gels, transferred to nitrocellulose membranes, and blotted for Nampt and SIRT3. Lysates were also separately analyzed for citrate synthase activity.
    RESULTS: Both Nampt (p = 0.02) and SIRT3 (p = 0.002) expression levels were significantly higher in WT than Ts65Dn ventricles at 12 months. In addition, SIRT3 levels were higher in diaphragm of 12-month-old WT animals (p = 0.02). Citrate synthase levels were similar in all tissues.
    CONCLUSION: Our findings of reduced Nampt protein suggest that reduced NAD recycling takes place in DS muscle tissue despite similar observed mitochondrial (citrate synthase) activity. These results suggest a role of diminished peripheral mitochondrial function and ATP synthesis in Down Syndrome muscle.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2003
  40. FASEB J. 2022 May;36 Suppl 1
    Novel Regulatory Role of SIRT3 on Cardiomyocyte Mitochondrial Frataxin and Ferroptosis.
    Aubrey Cantrell, Han Su, Heng Zeng, Jian-Xiong Chen.
      AIMS: Sirtuin 3 (SIRT3) has been shown to contribute to the mitochondrial cardiomyopathy of Friedreich's Ataxia (FRDA), which is characterized by a deficiency in mitochondrial frataxin along with mitochondrial hyperacetylation. Using a cardiomyocyte-specific SIRT3 knockout (SIRT3cKO) mouse model, we addressed two key questions: (1) whether SIRT3-induced acetylation of proteins is specific to the mitochondria; and (2) if mitochondrial SIRT3 is a key regulator of mitochondrial frataxin which impacts mitochondrial iron homeostasis and ferroptosis.METHODS AND RESULTS: The mitochondrial and cytosolic fractions were isolated from the ventricles of the hearts of SIRT3cKO mice. The mitochondrial and cytosolic proteins and mitochondrial iron levels were analyzed by comparison to SIRT3-Loxp wild-type (WT) control mice. Cardiac function study showed that SIRT3cKO mice developed heart failure as evidenced by reduction of ejection fraction (EF) and fraction shortening (FS) and increased isovolumic relaxation time (IVRT) and myocardial performance index (MPI) when compared to WT controls. Comparison of the mitochondrial and cytosolic fractions of the SIRT3cKO model to those of the WT control shows that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased in the heart. Similarly, acetylated p53 (p53ace) was significantly upregulated only in the mitochondria, while levels of p53 were not altered in either compartment. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase, while acetylation in the cytosol is independent of SIRT3 in cardiomyocytes. Most importantly, loss of SIRT3 in the mitochondria resulted in significant reduction of the protein frataxin and the Iron (II) export protein ferroportin (FPN). Furthermore, levels of glutathione peroxidase 4 (GPX4) were also downregulated in the mitochondria. This was accompanied by a significant increase in levels of 4-hydroxynonenal (4-HNE), an indicator of lipid peroxidation, and suggestive of upregulated mitochondrial ferroptosis in SIRT3cKO mouse hearts. Additionally, mitophagy marker beclin-1 expression was significantly reduced in the mitochondria of SIRT3cKO mice. Levels of glucose transporter-1 (GLUT1), which functions to deliver the antioxidant Vitamin C to the mitochondria and reduce ROS production, were also diminished in the mitochondria. Mechanistically, mitochondrial levels of hypoxia inducible factor-2α (HIF-2α) were downregulated, while those of HIF-1α remained unchanged in SIRT3cKO mouse hearts. Treatment with ferroptosis inhibitor ferrostatin-1 for 14 days significantly reduced 4-HNE levels and rescued preexisting impaired cardiac function in SIRT3cKO mice.
    CONCLUSIONS: For the first time, we have demonstrated that cardiomyocyte SIRT3 deficiency causes mitochondrion-specific acetylation and impairment of frataxin and ferroportin potentially via downregulation of HIF-2α. Our results suggest that the SIRT3-ferroptosis pathway may be a novel target for the mitochondrial cardiomyopathy of FRDA.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2008
  41. FASEB J. 2022 May;36 Suppl 1
    Dysregulation of Skeletal Muscle Mitochondrial Turnover and Dynamics Occurs During Late Symptomatic Stages of Spinal Muscular Atrophy.
    Andrew I Mikhail, Sean Y Ng, Aislin K Osborne, Abbas K Canteenwala, Vladimir Ljubicic.
      Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder caused by a mutation in the survival motor neuron 1 (SMN1) gene and it is the leading genetic cause of infant mortality. Recently approved genetic therapies designed to augment full length SMN protein in the central nervous system fail to ameliorate abnormal skeletal muscle features, which strongly suggests an important role for SMN protein in skeletal muscle homeostasis. This study aimed to characterize key modifiers of skeletal muscle mitochondrial turnover and dynamics during disease progression in a pre-clinical murine model of SMA in vivo. Additionally, we investigated the effects of a single dose of exercise on these mitochondrial biology-regulating pathways in the skeletal muscle of SMA mice. Muscle samples were collected from wild-type (WT) and Smn2B/- (SMA) mice at postnatal day 9 (P9), P13, and P21 to examine skeletal muscle-specific disease progression. Mitochondrial content did not differ between genotypes at all timepoints as evident by similar levels of mitochondrial oxidative phosphorylation proteins, succinate dehydrogenase staining, and citrate synthase content. However, mRNA content of key genes responsible for regulating mitochondrial quality such as nuclear respiratory factor 2, mitochondrial transcription factor A, and p53 were significantly higher in SMA mice versus WT at P21. Additionally, we observed elevated mitochondrial fission activity as indicated by a 2-fold increase (p < 0.05) in dynamin related protein 1 (DRP1) activation. Concomitantly, the expression of several mitophagy proteins including BLC2 interacting protein 3, parkin, and PTEN-induced kinase 1 were significantly increased by 2.9-, 2.7-, and 2-fold, respectively, compared to WT animals at P21. Interestingly, we observed blunted (p < 0.05) inclusion of optic atrophy 1 exon 4b, a key exon in mitochondrial biogenesis, between WT and SMA mice at P21. Acute exercise significantly increased the inhibition of DRP1-mediated mitochondrial fission activity in the muscles of SMA mice relative to sedentary SMA mice. However, a single exercise dose failed to alter the expression of mitophagy proteins up to three hours after running. Our data demonstrate that skeletal muscle mitochondrial health is compromised in SMA mice due to elevated fission and mitophagy processes. Furthermore, SMA mice display aberrant alternative splicing of Opa1 transcripts that may in turn hinder mitochondrial biogenesis in later disease stages. We also highlight that an acute bout of treadmill running elicits pro-fusion signaling to potentially improve the mitochondrial reticulum. Collectively, this study is the first to reveal mitochondrial dysfunction in SMA skeletal muscle and outlines signalling associated with mitochondrial plasticity following a single dose of exercise.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2886
  42. FASEB J. 2022 May;36 Suppl 1
    Impact of Branched Chain Amino Acid Supplementation on Muscle Lipid Accumulation and Mitochondrial Metabolism.
    Caitlin E Ryan, Kruti Patel, Vaishna Muralidaran, Chaitra Surugihalli, Parama Bhattacharjee, Nishanth E Sunny.
      Cross talk between branched-chain amino acids (BCAAs; valine, leucine and isoleucine), mitochondrial metabolism and lipid accumulation have been shown to occur in several tissues, including skeletal muscle. In fact, defects in BCAA metabolism and their degradation networks coexist with defects in lipid metabolism in insulin resistant muscle. Here, we hypothesized that the relationship between BCAAs and lipid metabolism is due to the ability of BCAAs to modulate mitochondrial oxidative metabolism and lipid accumulation in the muscle. Mice (C57BL/6N) were randomly assigned to a low-fat diet (LFD; 10% fat kcal), LFD supplemented with BCAAs (LFBA; 150% BCAA), high-fat diet (HFD; 60% fat kcal) or HFD supplemented with BCAAs (HFBA; 150% BCAA) for 12 or 26 weeks (n = 8-11 per group). Following the dietary treatments, leg muscle tissue were collected and flash frozen in liquid nitrogen, under feeding and overnight fasting conditions. Lipid accumulation was evaluated by analyzing muscle triglycerides, Oil-Red-O staining, and gene expression profiles. Mitochondrial metabolism and BCAA degradation were evaluated using a combination of gas-chromatography/mass-spectroscopy based targeted metabolomics and western blot analysis. Levels of BCAAs in the muscle showed significant correlations to their corresponding keto-acids (p ≤ 0.01). However, supplementation of BCAAs did not affect the levels of mitochondrial tricarboxylic acid (TCA) cycle intermediates or amino acids in the muscle. Further, mitochondrial proteins involved in oxidative phosphorylation also remained similar between non-supplemented and BCAA supplemented muscle. Muscle tissue from HFD mice had higher lipid accumulation (mg/g ± SEM) (LFD; 8.22 ± 0.91, HFD; 11.03 ± 1.08; p ≤ 0.05), compared to their LFD counterparts. Interestingly, BCAA supplementation for 12-weeks along with the HFD blunted muscle lipid accumulation (LFBA; 8.62 ± 1.12, HFBA; 9.76 ± 0.75), which was also evidenced from Oil Red-O staining of the muscle (p = 0.07). A similar lowering of lipid accumulation (p ≤ 0.05) was evident between the HFD vs. HFBA groups, following 26-weeks on the diets, as quantified by Oil Red-O staining. Further, the expression of genes involved in lipid accumulation/synthesis Srebp1c (p ≤ 0.05) and Acc1 (p ≤ 0.1) were lower in the 12-week LFBA vs. their LF counterparts, under fed conditions. Taken together, our results show that BCAAs have the ability to blunt muscle lipid accumulation without altering mitochondrial oxidative metabolism in the muscle.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3621
  43. JCI Insight. 2022 May 09. pii: e155201. [Epub ahead of print]7(9):
    Frataxin deficiency lowers lean mass and triggers the integrated stress response in skeletal muscle.
    César Vásquez-Trincado, Julia Dunn, Ji In Han, Briyanna Hymms, Jaclyn Tamaroff, Monika Patel, Sara Nguyen, Anna Dedio, Kristin Wade, Chinazo Enigwe, Zuzana Nichtova, David R Lynch, Gyorgy Csordas, Shana E McCormack, Erin L Seifert.
      Friedreich's ataxia (FRDA) is an inherited disorder caused by reduced levels of frataxin (FXN), which is required for iron-sulfur cluster biogenesis. Neurological and cardiac comorbidities are prominent and have been a major focus of study. Skeletal muscle has received less attention despite indications that FXN loss affects it. Here, we show that lean mass is lower, whereas body mass index is unaltered, in separate cohorts of adults and children with FRDA. In adults, lower lean mass correlated with disease severity. To further investigate FXN loss in skeletal muscle, we used a transgenic mouse model of whole-body inducible and progressive FXN depletion. There was little impact of FXN loss when FXN was approximately 20% of control levels. When residual FXN was approximately 5% of control levels, muscle mass was lower along with absolute grip strength. When we examined mechanisms that can affect muscle mass, only global protein translation was lower, accompanied by integrated stress response (ISR) activation. Also in mice, aerobic exercise training, initiated prior to the muscle mass difference, improved running capacity, yet, muscle mass and the ISR remained as in untrained mice. Thus, FXN loss can lead to lower lean mass, with ISR activation, both of which are insensitive to exercise training.
    Keywords:  Cell stress; Mitochondria; Muscle Biology; Skeletal muscle
    DOI:  https://doi.org/10.1172/jci.insight.155201
  44. FASEB J. 2022 May;36 Suppl 1
    The Effect of Leucine and Doxorubicin Treatment on Skeletal Muscle Mitochondrial Function.
    Austin E Carlen, Erik D Marchant, Richard W Evans, Chad R Hancock.
      Doxorubicin (DOX) is a common, effective chemotherapy drug. However, DOX treatment causes the negative side-effects of skeletal muscle atrophy and weakness. One of the key mechanisms by which DOX can lead to increased atrophy is through increased mitochondrial damage and inhibition of protein synthesis via down-regulation of mTOR. Leucine (LEU), an essential branched-chain amino acid, activates mTOR, increases mitochondrial biogenesis, and muscle protein synthesis. It is not clear if enhanced activation of mTOR by LEU might moderate the damaging effects of DOX in skeletal muscle. The purpose of this study is to determine if LEU supplementation attenuates the loss of mitochondrial function caused by DOX. C2C12 myoblast cells were grown to confluence and then treated with 10 mM LEU, 0.5 μM DOX, or both for 24 hours. High-resolution respirometry was performed on cells using the Oroboros O2K respirometer to assess mitochondrial function in leak, coupled, and uncoupled states. Total cells used for respiration were assessed by aliquoting a portion of cells before respirometry and performing a BCA protein assay on this representative aliquot. Citrate synthase activity was also evaluated on cells used for respirometry as a marker of mitochondrial content. We report that DOX caused reduced leak, maximum coupled respiration, and uncoupled respiration by 33%, 42%, and 38%, respectively (p<0.05). DOX also lowered citrate synthase activity by 44% (p<0.05). Total protein, measured for total cell homogenates was reduced by 63% with DOX treatment (p<0.05). Contrary to our hypothesis that LEU may mitigate the increase in cellular degradation seen with DOX treatment, there was no significant difference between DOX treatment and DOX + LEU treatment. These findings suggest DOX interferes with LEU's ability to upregulate mTOR activity. Interestingly, the total protein content was reduced significantly more by DOX (63%) than either reductions seen in mitochondrial respiration (~40%) and citrate synthase activity (44%). These findings suggest that the effects of DOX on general cellular protein content is far greater than the negative effects observed on mitochondrial content or function.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.L8117
  45. FASEB J. 2022 May;36 Suppl 1
    Nucleolar function is regulated by the mitochondrial protein sulfite oxidase (SUOX).
    Emily J McFadden, Susan J Baserga.
      Ribosome biogenesis occurs in the nucleolus and relies upon RNA Polymerase I (Pol I) transcription of the rDNA into rRNA. This process is critical for normal cells but is dysregulated in diseases such as Alzheimer's disease and cancer. To date, however, we have not yet explored the potential relationship between nucleoli and mitochondria. Mitochondria are critical for cellular energy and mitochondrial disorders exhibit dysregulation of nutrient and energy homeostasis. Like nucleoli, mitochondria are also dysregulated in Alzheimer's disease and cancer, suggesting we may have overlooked a regulatory relationship between these two organelles. In pursuing novel regulators of nucleoli number and function, we have identified a mitochondrial protein that when individually depleted by siRNA in breast epithelial cells (MCF10As) causes a change in nucleolar number - sulfite oxidase (SUOX). Upon SUOX depletion, the nucleolar number decreases from the typical 2-3 nucleoli to 1 nucleolus. SiRNA-mediated SUOX depletion reduces nucleolar rRNA levels in a high-throughput 5-EU assay (Bryant et al. 2021) and reduces global protein translation by a puromycin incorporation assay, indicating defective nucleolar function. Furthermore, combined metabolomic and transcriptomic analyses following siRNA-mediated SUOX depletion reveal disruption in the activated methyl cycle, consistent with aberrant ribosome biogenesis. Experiments are underway to validate defective methylation experimentally at specific steps in ribosome biogenesis. The glutamate cycle is also disrupted, congruent with the presentation of the recessive nervous system disorder Isolated Sulfite Oxidase Deficiency (ISOD). Thus, we have defined a novel mitochondrial regulator of nucleolar number and function, nucleolar rRNA biogenesis, and protein translation with implications in human disease. Our work will open up a new avenue of nucleolus-mitochondria relationships and regulation, and potentially connect mitochondrial disorders with ribosome biogenesis as well as identify how mitochondrial homeostasis is critical for nucleolar function.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4250
  46. FASEB J. 2022 May;36 Suppl 1
    Implications of DNA Polymerase Gamma in the Repair of the Mitochondrial Genome.
    Carolina de Bovi Pontes, Cody Jefferys, Muhamad Bedwan, Elena J Ciesielska, Grzegorz L Ciesielski.
      Mitochondrial DNA (mtDNA) encodes thirteen essential proteins of the oxidative phosphorylation system, responsible for the major production of ATP in the cell. Therefore, damages to the mitochondrial genome result in energy deprivation, which may in turn onset human diseases. Notably, due to its proximity to the electron transport chain, mtDNA remains exposed to damage by reactive oxygen species, thus the maintenance of its integrity requires a robust repair system. Until recently, DNA polymerase gamma (Pol γ) has been the only polymerase identified in mitochondria, bearing responsibility for efficient replication as well as post-replication repair of the genome. We have previously suggested that the division of the roles of Pol γ may be controlled by the association of its catalytic subunit, Pol γA, with the accessory subunit Pol γB, such that the holoenzyme is engaged in the processive mtDNA replication, whereas, alone, Pol γA may serve the repair processes. Recently, the major repair polymerase of the nucleus, Pol β, has been discovered to also localize in mitochondria, which raises the question for its competition or cooperation with Pol γ in the mtDNA repair processes. To address this, we have tested in vitro the efficiency of DNA synthesis by the two polymerases, separately and in combination, using various DNA substrates. In agreement with previous reports, we did not observe any indication of a functional interaction between the Pol γ holoenzyme and Pol β. We did, however, observe a cooperative activity of Pol β with the Pol γA subunit. In conclusion, our results suggest that the repair of mtDNA may entail a synergistic activity of the catalytic subunit of Pol γ and Pol β.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4339
  47. Sci Rep. 2022 May 11. 12(1): 7704
    Urolithin A attenuates auditory cell senescence by activating mitophagy.
    Sung Il Cho, Eu-Ri Jo, Hansoo Song.
      Aging of sensory organs is associated with a decline in mitochondrial function and the accumulation of dysfunctional mitochondria. Impaired mitophagy blocks the turnover of dysfunctional mitochondria and leads to their accumulation. Urolithin A (UA) induces mitophagy in various mammalian cells. This study was aimed at investigating the effect of the mitophagy activator, UA, on premature senescent auditory cells. The levels of cellular senescence-associated p53 and p21 significantly increased in H2O2-induced senescent House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear explants. However, the levels of mitophagy-related molecules significantly decreased. UA significantly decreased the expression of senescence-associated p53 and p21, and increased the expression of mitophagy-related proteins, in H2O2-induced senescent cells and cochlear explants. The percentage of β-galactosidase-stained senescent cells also reduced in H2O2-treated cells and cochlear explants upon UA pre-treatment. The formation of mitophagosomes and mitophagolysosomes was restored upon UA pre-treatment of H2O2-induced senescent cells. The knockdown of mitophagy-related genes (Parkin and Bnip3) resulted in annulment of UA-induced anti-senescent activity. UA significantly increased the ATP content, mitochondrial DNA (mtDNA) integrity, and mitochondrial membrane potential in senescent HEI-OC1 cells. These findings indicate that UA counteracted mitophagy decline and prevented premature senescence in auditory cells. Hence, UA administration might be a promising strategy for preventing mitochondrial dysfunction in patients with age-related hearing loss.
    DOI:  https://doi.org/10.1038/s41598-022-11894-2
  48. FASEB J. 2022 May;36 Suppl 1
    Potential Drug Therapy For Fragile X Tremor/Ataxia Syndrome.
    Collis Brown, Sonya Sobrian, Tamaro Hudson.
      Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurological disorder that affects carriers of the alleles for the Fragile X gene, Fragile X Mental Retardation (FMR1) due to the CGG triplet repeat expansion within the 5'UTR of FMR1. Clinical symptoms that are likely to occur are progressive ataxia, intention tremor, intranuclear inclusions, and Parkinsonian-like motor symptoms. The goal of this research is to assess the biological efficacy of Curcumin, Muscadine Grape Seed Extract (MSKE) on the neuroprotective capacity with respect to mitochondrial damage, and their ability to restore mitochondrial health in patient with FXTAS. To establish mitochondrial dysfunction, normal human cell lines and human-induced pluripotent cells were treated with multiple concentrations of glucose/ glucose oxidase (GluOx) at 2,12,and 24 hour time points to induce varying intensities of oxidative stress. The degrees of oxidative stress were measured by apoptosis and mitochondrial reactive oxygen species (ROS) production. Curcumin and MSKE, compounds effective against oxidative damage in mitochondria, were used to rescue glucose oxidase induced oxidative damage in both cell lines. To test the ability of these drugs to restore mitochondrial health, cell viability and cellular superoxide production were assessed by propidium iodide and the MitoSox fluorescence assay, respectively. We anticipated that GluOx at varying concentrations and time points would proportionally increase levels of apoptosis and mitochondrial ROS, reflective of mitochondrial damage, with the most severe dysfunction occurring at a dose of 25 nM and the longest duration of 24-hr exposure. Administration of MSKE in concentrations ranging from 10-8 to 10-5 M in half log increments, did not reverse the oxidative defects induced in the cell lines. However, curcumin concentrations increased cell viability at the 2, 12, and 24 hour time period. Results indicate that the research design should be modified by increasing concentration of both glucose and MSKE to provide a reliable test of the hypothesis. Although, the research design will be modified to better assessed the hypothesis, the overall research is translational in that it lends flexibility to testing therapeutics in neuronal FXTAS models and expands the discovery of mitochondrial markers for the syndrome.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R6346
  49. FASEB J. 2022 May;36 Suppl 1
    Aging Selectively Reduces Mitochondrial Volume Density and Respiratory Capacity in Type IIx/IIb Diaphragm Muscle Fibers.
    Leah A Davis, Alyssa D Brown, Matthew J Fogarty, Carlso B Mantilla, Gary C Sieck.
      Diaphragm muscle (DIAm) sarcopenia involves selective atrophy of type IIx/IIb DIAm fibers. In addition, there is an age-related decrease in the maximum velocity of the succinate dehydrogenase reaction (SDHmax ) in type IIx/IIb fibers. SDH is a key enzyme of the tricarboxylic acid (TCA) cycle as well as complex II of the electron transport chain (ETC). These results suggest that sarcopenia may reduce mitochondrial volume density in type IIx/IIb DIAm fibers and thereby reduce SDHmax . Alternatively, sarcopenia may reduce the intrinsic respiratory capacity of mitochondria in type IIx/IIb DIAm fibers. We hypothesized that there is an age-related selective reduction in mitochondrial volume density and SDHmax per mitochondrial volume in type IIx/IIb DIAm fibers. In 24 Fischer 344 rats (6-mo: n=6 female, n=6 male; 24-mo: n=6 female, n=6 male), DIAm samples were dissected from the mid-costal region and rapidly frozen at optimal sarcomere length. In 10 mm cross-sections, DIAm fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. Mitochondria were labelled using Mitotracker Green and imaged using 3D confocal microscopy (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. Each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI=SA3 /16µ2 V2 ) using ImageJ and NIS elements. In alternate 6 mm sections, SDHmax was measured using a quantitative histochemical assay in which accumulation of nitro blue tetrazolium diformazan (NBTdfz ) was measured in DIAm fibers every 15 s for 10 min. Consistent with previous findings, type IIx/IIb DIAm fibers were ~36% smaller in 24-mo compared to 6-mo rats (P<0.0001), whereas there were no age-related differences in cross-sectional areas (CSA) of type I and IIa fibers. Mitochondrial volume density was lower in IIx/IIb fibers of 24-mo compared to 6-mo rats (P<0.0001), whereas there were no age-related differences in type I and IIa fibers. The MCI in type IIx/IIb fibers was lower (greater fragmentation) in 24-mo compared to 6-mo rats (P<0.0001), whereas the MCIs of type I and IIa fibers were similar across ages. Finally, SDHmax per fiber volume was lower in type IIx/IIb DIAm fibers in 24-mo compared with 6-mo rats (P<0.0001), whereas there were no age-related differences in type I and IIa fibers. When SDHmax was normalized for mitochondrial volume, there was no age-related difference in the intrinsic mitochondrial respiratory capacity in type IIx/IIb fibers. Therefore, aging results in reduced mitochondrial volume density and increased fragmentation in type IIx/IIb DIAm fibers, but no change in the intrinsic respiratory capacity of mitochondria.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3573
  50. FASEB J. 2022 May;36 Suppl 1
    Spatial Compartmentation of mTORC1 Signaling at the Mitochondria.
    Ayse Z Sahan, Yanghao Zhong, Xin Zhou, Danielle L Schmitt, Jin Zhang.
      The mechanistic target of rapamycin complex 1 (mTORC1) senses diverse signals to regulate cell growth and metabolism. The complex is present at the plasma membrane, nucleus, lysosomes, and the outer mitochondrial membrane. Such spatial compartmentation has been suggested to enhance signaling efficiency and specificity. For instance, we recently discovered nuclear mTORC1 activity, which is distinctly regulated from the canonical lysosomal mTORC1 (Zhou et al., 2020). Previous studies have shown that mTOR is present at the outer mitochondrial membrane (OMM), but it is not clear whether mTORC1 is active at this location and what the functional consequences are. To investigate this, we targeted our FRET-based mTORC1 activity reporter, TORCAR (Zhou et al., 2015), to the OMM and probed the subcellular activity of mTORC1. We found that platelet-derived growth factor (PDGF) stimulation increases mTORC1 activity at the OMM in addition to at the lysosome and in the nucleus, whereas insulin specifically stimulates mTORC1 activity at the OMM without affecting the lysosomal and nuclear activities. We further dissected the regulation of mitochondrial mTORC1 activity and applied a novel approach of identifying new mTORC1 substrates. Elucidating the signaling events that lead to subcellular mTORC1 activity at mitochondria and its downstream functions will increase our understanding of the roles that mTORC1 may play in diseases associated with altered metabolism or mitochondrial dysfunction, such as diabetes and cancer.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4944
  51. Nat Commun. 2022 May 13. 13(1): 2673
    Mechanical instability generated by Myosin 19 contributes to mitochondria cristae architecture and OXPHOS.
    Peng Shi, Xiaoyu Ren, Jie Meng, Chenlu Kang, Yihe Wu, Yingxue Rong, Shujuan Zhao, Zhaodi Jiang, Ling Liang, Wanzhong He, Yuxin Yin, Xiangdong Li, Yong Liu, Xiaoshuai Huang, Yujie Sun, Bo Li, Congying Wu.
      The folded mitochondria inner membrane-cristae is the structural foundation for oxidative phosphorylation (OXPHOS) and energy production. By mechanically simulating mitochondria morphogenesis, we speculate that efficient sculpting of the cristae is organelle non-autonomous. It has long been inferred that folding requires buckling in living systems. However, the tethering force for cristae formation and regulation has not been identified. Combining electron tomography, proteomics strategies, super resolution live cell imaging and mathematical modeling, we reveal that the mitochondria localized actin motor-myosin 19 (Myo19) is critical for maintaining cristae structure, by associating with the SAM-MICOS super complex. We discover that depletion of Myo19 or disruption of its motor activity leads to altered mitochondria membrane potential and decreased OXPHOS. We propose that Myo19 may act as a mechanical tether for effective ridging of the mitochondria cristae, thus sustaining the energy homeostasis essential for various cellular functions.
    DOI:  https://doi.org/10.1038/s41467-022-30431-3
  52. F S Sci. 2020 Aug;pii: S2666-335X(20)30004-5. [Epub ahead of print]1(1): 36-45
    Mitochondrial DNA content decreases during in vitro human embryo development: insights into mitochondrial DNA variation in preimplantation embryos donated for research.
    Marta Pérez-Sánchez, Antonio Díez-Juan, Diana Beltrán, Amparo Mifsud, Amparo Mercader, Carmina Vidal, Elena Labarta, Antonio Pellicer, Emre Seli, María José De Los Santos.
      OBJECTIVE: To assess the mitochondrial DNA (mtDNA) load and variation in human oocytes and during preimplantation embryo development using specimens donated for research.DESIGN: Prospective cohort study.
    SETTING: Not applicable.
    PATIENTS: A total of 50 in vitro fertilization patients and 11 oocyte donors whose specimens were obtained between July 2017 and July 2018.
    INTERVENTIONS: None.
    MAIN OUTCOME MEASURES: All specimens were separately collected. Quantitative polymerase chain reaction was performed with SurePlex DNA Amplification System (Illumina). Primers for the adenosine triphosphate 8 mitochondrial gene and the β-actin were used. Data were statistically analyzed by analysis of variance with the Scheffé multiple pairwise comparison for categorical variables and by linear regression for numerical variables.
    RESULTS: Human metaphase II (MII) oocytes had significantly more total mtDNA copy number than day 3 embryos, and day 3 embryos had more total and per-cell mtDNA copy number than aneuploid blastocysts. There was a significant decrease in mtDNA content associated with failed-fertilized oocytes compared to noninseminated metaphase II oocytes.
    CONCLUSIONS: During preimplantation development, before implantation, human embryos undergo a significant decrease in total mtDNA content and no increase in mtDNA content at the blastocyst stage. Oocytes need to carry a correct threshold of mitochondrial load in the oocyte in order to successfully fertilize. An active degradation of mtDNA before implantation occurs after fertilization takes place. These findings could be used to improve knowledge about the best embryo culture conditions and would serve as a basis for further studies addressing again the use of mtDNA content as an embryo viability marker.
    Keywords:  Mitochondria; failed-fertilized oocyte; human embryo development; mtDNA load; mtDNA variation
    DOI:  https://doi.org/10.1016/j.xfss.2020.05.001
  53. FASEB J. 2022 May;36 Suppl 1
    Mass Spectrometry Based Subcellular Coenzyme Analysis.
    Nathaniel W Snyder, Anna Bostwick, Hannah Pepper, Sophie Trefely.
      Metabolism in eukaryotes relies on compartmentalization of processes between sub-cellular compartments. Our objective was to develop, test, and apply methods that can quantitatively measure families of metabolites within distinct sub-cellular compartments in eukaryotic cells. We created Stable Isotope Labeling of Essential nutrients in Cell culture - Subcellular Fractionation (SILEC-SF) with the essential precursors of the major cellular coenzymes, Coenzyme A and NAD to incorporate a 13 C,15 N-label into the families of each coenzyme present within cells. Using multiple fractionation techniques coupled to liquid chromatography-high resolution mass spectrometry we quantify distinct cytoplasmic, mitochondrial, and nuclear pools within eukaryotic cells. We successfully applied these methods to cells and human tissue demonstrating distinct compartmental metabolic changes by pathway in genetic models of compartmentalized metabolism, in adipocyte differentiation and in changing oxygen tension. This confirmed orthogonal measurements of subcellular metabolism but revealed unexpected localizations and enrichments of certain metabolite pools.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R6113
  54. Curr Heart Fail Rep. 2022 May 13.
    Harnessing NAD+ Metabolism as Therapy for Cardiometabolic Diseases.
    Akash Chakraborty, Keaton E Minor, Hina Lateef Nizami, Ying Ann Chiao, Chi Fung Lee.
      PURPOSE OF THE REVIEW: This review summarizes current understanding on the roles of nicotinamide adenine dinucleotide (NAD+) metabolism in the pathogeneses and treatment development of metabolic and cardiac diseases.RECENT FINDINGS: NAD+ was identified as a redox cofactor in metabolism and a co-substrate for a wide range of NAD+-dependent enzymes. NAD+ redox imbalance and depletion are associated with many pathologies where metabolism plays a key role, for example cardiometabolic diseases. This review is to delineate the current knowledge about harnessing NAD+ metabolism as potential therapy for cardiometabolic diseases. The review has summarized how NAD+ redox imbalance and depletion contribute to the pathogeneses of cardiometabolic diseases. Therapeutic evidence involving activation of NAD+ synthesis in pre-clinical and clinical studies was discussed. While activation of NAD+ synthesis shows great promise for therapy, the field of NAD+ metabolism is rapidly evolving. Therefore, it is expected that new mechanisms will be discovered as therapeutic targets for cardiometabolic diseases.
    Keywords:  Cardiometabolic diseases; Heart failure; NAD+ metabolism; Redox balance
    DOI:  https://doi.org/10.1007/s11897-022-00550-5
  55. FASEB J. 2022 May;36 Suppl 1
    The combination of NRF1 and Nrf2 activators in myoblasts stimulate mechanisms of proteostasis without changes in mitochondrial respiration.
    Maureen A Walsh, Qian Zhang, Robert V Musci, Karyn L Hamilton.
      Persistent oxidative stress contributes to hallmarks of aging, including impaired proteostasis and mitochondrial dysfunction, while acute oxidative challenges resolved swiftly contribute to beneficial adaptations. Adaptive homeostasis is where acute exposures to sub-toxic stimuli kindle transient expansion of responses necessary to reestablish homeostasis. Elucidating mechanisms underlying adaptive homeostasis will provide novel targets for healthspan extension. Nrf2 is a key regulator of cytoprotective gene transcription for redox homeostasis; NRF1 is a transcription factor that regulates expression of genes necessary for mitochondrial function. Regulation of both is compromised with advancing age. We hypothesized that NRF1 (NRF1a) and Nrf2 (Nrf2a) activators might improve adaptive homeostasis in response to an oxidative challenge by promoting mitochondrial proteome maintenance and function in C2C12 myoblasts. Using deuterium stable isotope tracing, we assessed protein synthesis over a 16-hr treatment with a CON (DMSO), NRF1a, Nrf2a, or both, with and without a hydrogen peroxide (H2 O2 ) stress. We assessed mitochondrial function using high-resolution respirometry. Co-treatment of NRF1a and Nrf2a under H2 O2 stress favored proteostatic maintenance (p<0.05) through maintained protein synthesis (p>0.05), but a decrease in cell proliferation (p<0.05). H2 O2 stress significantly decreased mitochondrial maximal respiration, but this decrease was not rescued by NRF1a/Nrf2a co-treatment indicating the same ATP availability. Interestingly, there were no differences in submaximal ADP stimulated respiration or ADP sensitivity between CON, H2 O2 , or the NRF1a/Nrf2a co-treatment (p>0.05). Further, there were no differences in endogenous redox capacity or mitochondrial protein content between treatment groups. These results suggest that simultaneously targeting NRF1 and Nrf2 may be a viable approach for reestablishing mitochondrial protein homeostasis following a stress, but that this adaptation may not improve respiratory capacity.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R239
  56. FASEB J. 2022 May;36 Suppl 1
    Mitochondrial Transfer Between Mesenchymal Stem Cells and Cardiac H9c2 Cells.
    Miranda Kortenhoeven, Robert Boomsma.
      Mesenchymal stem cells (MSC) have been shown to improve heart function after myocardial infarction, but the exact mechanisms are not completely understood. Studies have shown that stem cells are able to transfer mitochondria through cytosol extensions to change the abilities and programming of surrounding cells. The purpose of this study was to determine whether mitochondrial transfer takes place between MSCs and cardiac H9c2 cells, and the effect of hypoxic conditions on this process. Mouse bone marrow MSC were cultured in Mesencult + 10% mouse supplement (Stem Cell Technologies) + 10% FBS and H9c2 cells were cultured in DMEM + 10% FBS. MSC mitochondria were stained using MitoTracker Red CMX Ros (Invitrogen), while H9C2 cells were stained using CellTracker Green CMFDA (Invitrogen) according to the manufacturer's instructions. Following staining, the cells were co-cultured for 24 hours in Fluorobrite DMEM (Gibco) + 10% FBS in 4-well glass culture slides. After washing with PBS and mounting with ProLong Live Antifade Reagent (Invitrogen), cells were observed using an Olympus BH2 fluorescent microscope. Our results showed close interactions between MSC and H9c2 cells with mitochondria in long filamentous extensions that made contact with H9c2. There was some evidence that mitochondria were transferred from MSC to H9c2 cells. Experiments are underway to determine whether mitochondria transfer from H9c2 cells to MSC, and the effect of hypoxia. These results continue to suggest that mitochondrial transfer may be one mechanism used by MSC to improve heart function after myocardial infarction.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4095
  57. Sci Rep. 2022 May 10. 12(1): 7652
    Exploring selective autophagy events in multiple biologic models using LC3-interacting regions (LIR)-based molecular traps.
    Grégoire Quinet, Pierre Génin, Oznur Ozturk, Naima Belgareh-Touzé, Lilas Courtot, Renaud Legouis, Robert Weil, Mickael M Cohen, Manuel S Rodriguez.
      Autophagy is an essential cellular pathway that ensures degradation of a wide range of substrates including damaged organelles or large protein aggregates. Understanding how this proteolytic pathway is regulated would increase our comprehension on its role in cellular physiology and contribute to identify biomarkers or potential drug targets to develop more specific treatments for disease in which autophagy is dysregulated. Here, we report the development of molecular traps based in the tandem disposition of LC3-interacting regions (LIR). The estimated affinity of LC3-traps for distinct recombinant LC3/GABARAP proteins is in the low nanomolar range and allows the capture of these proteins from distinct mammalian cell lines, S. cerevisiae and C. elegans. LC3-traps show preferences for GABARAP/LGG1 or LC3/LGG2 and pull-down substrates targeted to proteaphagy and mitophagy. Therefore, LC3-traps are versatile tools that can be adapted to multiple applications to monitor selective autophagy events in distinct physiologic and pathologic circumstances.
    DOI:  https://doi.org/10.1038/s41598-022-11417-z
  58. J Coll Physicians Surg Pak. 2022 May;32(5): 671-673
    Neurological Recovery with Interferon-gamma Treatment in Friedreich's Ataxia.
    Hande Gazeteci Tekin, Erturk Levent.
      Friedreich's ataxia (FA) is a rare, progressive, and degenerative hereditary disorder caused by a deficiency of frataxin protein. This disease is characterised by severe neurological dysfunction and life-threatening cardiomyopathy. Various drugs are used to slow down / stop the neurodegenerative progress. However, recent clinical trials and animal experiments demonstrate that interferon-gamma (IFN-ɣ) treatment might improve signs of FA as well. A 9-year-old girl was admitted to our hospital with gait instability, mild dysarthria, and sensorimotor polyneuropathy. Her genetic examination was consistent with FA. IFN-ɣ treatment was started 3 times a week. The treatment was evaluated by physical examination and side effects assessment. Friedreich Ataxia Rating Scale (FARS), 9-hole peg test (9HPT), and time of 25-foot walk (T25FW) were measured. Ataxia and cerebellar findings improved within 9 months. Although clinical neurological improvement was achieved, there was no improvement in cardiomyopathy. Key Words: Interferon-gamma, Friedreich ataxia, FARS, Children, Cardiomyopathy.
    DOI:  https://doi.org/10.29271/jcpsp.2022.05.671
  59. Nature. 2022 May 12.
    Mitochondrial base editor induces substantial nuclear off-target mutations.
    Zhixin Lei, Haowei Meng, Lulu Liu, Huanan Zhao, Xichen Rao, Yongchang Yan, Hao Wu, Min Liu, Aibin He, Chengqi Yi.
      DddA-derived cytosine base editors (DdCBEs), which are fusions of the split-DddA halves and transcription activator-like effector (TALE) array proteins, enable targeted C·G-to- T·A conversions in mitochondrial DNA1. However, its genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or -independent. TAS-dependent off-target sites in the nuclear DNA (nDNA) are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by a paired TALE proteins positioned in close proximity. TAS-independent nDNA off-target sites are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with CTCF-binding sites and are enriched in TAD boundaries. We also engineered DdCBE to alleviate such off-target effect. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base editing tools.
    DOI:  https://doi.org/10.1038/s41586-022-04836-5
  60. FASEB J. 2022 May;36 Suppl 1
    Impaired Proteostasis, not Protein Synthesis, Limits Recovery of Aged Skeletal Muscle After Disuse Atrophy.
    Jordan D Fuqua, Marcus M Lawrence, Zachary Hettinger, Agnieszka K Borowik, Frederick F Peelor, Amy L Confides, Esther E Dupont-Versteegden, Benjamin F Miller.
      Older individuals undergo more frequent inpatient hospitalizations and bedrest compared to younger persons, causing rapid losses of muscle mass and strength. Evidence suggests that only aged muscle fails to completely recover following disuse despite aged and adult muscle having similar levels of muscle loss. Current efforts to improve muscle recovery in older individuals are commonly aimed at increasing myofibrillar protein synthesis via mTOR stimulation despite recent evidence demonstrating that old muscle has chronically elevated levels of mTORC1 activity. We hypothesized that old muscle fails to fully recover muscle mass due to impaired proteostasis and not due limitations in protein synthesis. METHODS: Adult (10 month) and old (30 month) F344BN rats were hindlimb unloaded (HU) for 14-days to induce atrophy, followed by subsequent reloading (RE) to study muscle regrowth. During RE the rats were labeled with deuterium oxide (D2 O) for 7, 15, 30, 45 or 60 days (n=4-6 per timepoint per group) to determine bulk and individual protein synthesis and RNA synthesis. We also assessed muscle mass, fiber size by immunohistochemistry, mTORC1 signaling by western blot, and aggregate formation by tissue fractionation. RESULTS: Adult and old gastrocnemius (GA) muscle had significant losses of muscle mass (359 mg ±55 and 724 mg ±60 loss respectively,p<0.05) and fiber size (1296 um2 ±202 and 1480 um2 ±142 loss respectively, p<0.01) with HU. While adult muscle fully recovered GA mass and fiber size by day 15, old GA muscle did not fully recover during the entire 60 days of RE (18% CSA and 11% mass loss still present). Despite limited mass and fiber size recovery in old muscle, GA myofibrillar protein synthesis rates (1.45 ±0.18 vs 2.28 ±0.24, FSR%, p<0.05) and mTORC1-related signaling were higher in old muscle compared to adult. Additionally, GA RNA synthesis rates (2.79 ±0.139 vs 5.18 ±0.684, p<0.05) and RNA concentration (183 ng ±3.6 vs 249 ng ±13.9, p<0.05), which are markers of translational capacity, were also elevated in old muscle relative to adult during RE. Old muscle had higher levels of insoluble protein aggregates, a marker of impaired proteostasis, during RE compared to adult (1.2 ±0.09 vs 1.5 ±012 fold increase, p<0.05). Lastly, we assessed individual protein synthesis rates of the whole muscle proteome and discovered that old GA muscle had a larger number of proteins with increased synthesis rates compared to adult. In conclusion, these data strongly suggest that limitations in old muscle to recover after disuse are not due to limitations in protein synthesis but are instead due to impaired proteostasis with age. Therefore, understanding how proteostasis responds during these periods surrounding unloading in old muscle are critical to improve muscle recovery after disuse.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3524
  61. Acta Biomed. 2022 May 11. 93(2): e2022199
    The SARS-CoV2 and mitochondria: the impact on cell fate.
    Eleonora Madeddu, Barbara Maniga, Marco Zaffanello, Vassilios Fanos, Antonietta Marcialis.
      Coronavirus infection causes endoplasmic reticulum stress inside the cells, which inhibits protein folding. Prolonged endoplasmic reticulum stress causes an apoptotic process of unfolded protein response-induced cell death. Endoplasmic reticulum stress rapidly induces the activation of mTORC1, responsible for the induction of the IRE1-JNK pathway. IRE1-JNK stands out for its dual nature: pro-apoptotic in the first stage of infection, anti-apoptotic in persistently infected cells. Once penetrated the cells, the virus can deflect the mitochondrial function by implementing both waterfalls pro-apoptotic and anti-apoptotic response. The virus prevents, through Open Reading Frame 9b (ORF-9b) interacting with mitochondria, the response of the type I interferon of the cells affected by the infection and is fundamental for generating an antiviral cellular state. ORF-9b has effects on mitochondrial dynamics, inducing fusion and autophagy and promoting cell survival. The recognition of ORF-9b has made it possible to identify it as a molecular target of some existing potentially effective drugs (Midostaurin and Ruxolitinib). Other drugs, with the same target, are currently being tested. Given the great importance of mitochondria in virus-host interaction, in-depth knowledge of the actors and pathways involved is essential to continue developing new therapeutic strategies against SARS CoV2.
    DOI:  https://doi.org/10.23750/abm.v93i2.10327
This page is being maintained by Thomas Krichel. It was last updated on 2022‒05‒15 at 07:17:34.