bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022‒11‒13
67 papers selected by
Catalina Vasilescu
Helmholz Munich
  1. AMPK-dependent phosphorylation of MTFR1L regulates mitochondrial morphology.
  2. Mitochondrial Dysfunction as an Underlying Cause of Skeletal Muscle Disorders.
  3. Mitochondrial Epilepsy, a Challenge for Neurologists.
  4. Heteroplasmic Mutant Load Differences in Mitochondrial DNA-Associated Leigh Syndrome.
  5. Neuropathological hallmarks of antenatal mitochondrial diseases with a corpus callosum defect.
  6. Mitochondrial dysfunction, aging, and the mitochondrial unfolded protein response in Caenorhabditis elegans.
  7. REC drives recombination to repair double-strand breaks in animal mtDNA.
  8. Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA.
  9. Mitochondrial Cardiomyopathy: The Roles of mt-tRNA Mutations.
  10. Selective degradation of tRNASer(AGY) is the primary driver for mitochondrial seryl-tRNA synthetase-related disease.
  11. Cardiolipin Alterations during Obesity: Exploring Therapeutic Opportunities.
  12. Metabolic Regulation of Mitochondrial Protein Biogenesis from a Neuronal Perspective.
  13. Mitochondrial dynamics, Leydig cell function, and age-related testosterone deficiency.
  14. Ubiquitin-mediated mitochondrial regulation by MITOL/MARCHF5 at a glance.
  15. ITCH deficiency clinical phenotype expansion and mitochondrial dysfunction.
  16. Mitochondrial dynamics involves molecular and mechanical events in motility, fusion and fission.
  17. Defective PTEN-induced kinase 1/Parkin mediated mitophagy and neurodegenerative diseases.
  18. Mitochondrial haplogroups and cognitive progression in Parkinson's disease.
  19. Denervation induces mitochondrial decline and exacerbates lysosome dysfunction in middle-aged mice.
  20. Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy.
  21. Resistin impairs mitochondrial homeostasis via cyclase-associated protein 1-mediated fission, leading to obesity-induced metabolic diseases.
  22. HapCUT2: A Method for Phasing Genomes Using Experimental Sequence Data.
  23. Impaired mitochondrial oxidative metabolism in skeletal progenitor cells leads to musculoskeletal disintegration.
  24. Lipopolysaccharide induces placental mitochondrial dysfunction in murine and human systems by reducing MNRR1 levels via a TLR4-independent pathway.
  25. Specialist multidisciplinary input maximises rare disease diagnoses from whole genome sequencing.
  26. Mitochondrial Cardiomyopathy: Molecular Epidemiology, Diagnosis, Models, and Therapeutic Management.
  27. Two neuronal peptides encoded from a single transcript regulate mitochondrial complex III in Drosophila.
  28. Hybridization drives mitochondrial DNA degeneration and metabolic shift in a species with biparental mitochondrial inheritance.
  29. Adenosine monophosphate activated protein kinase contributes to skeletal muscle health through the control of mitochondrial function.
  30. Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.
  31. Full-Length Transcript Phasing with Third-Generation Sequencing.
  32. Molecular etiology of defective nuclear and mitochondrial ribosome biogenesis: Clinical phenotypes and therapy.
  33. Conservation and Diversification of tRNA t6A-Modifying Enzymes across the Three Domains of Life.
  34. Approaches to Mitigate Mitochondrial Dysfunction in Sensorineural Hearing Loss.
  35. Diagnosing, discarding, or de-VUSsing: A practical guide to (un)targeted metabolomics as variant-transcending functional tests.
  36. Haplotyping-Assisted Diploid Assembly and Variant Detection with Linked Reads.
  37. The function of prohibitins in mitochondria and the clinical potentials.
  38. De novo and inherited variants in coding and regulatory regions in genetic cardiomyopathies.
  39. Odd- and even-numbered medium-chained fatty acids protect against glutathione depletion in very long-chain acyl-CoA dehydrogenase deficiency.
  40. FAVOR: functional annotation of variants online resource and annotator for variation across the human genome.
  41. The phenotypic landscape of essential human genes.
  42. Phasing DNA Methylation.
  43. Randomized trial of bilateral gene therapy injection for m.11778G > A MT-ND4 Leber optic neuropathy.
  44. Low-Cost Genome-Scale Phasing with Barcode-Linked Sequencing.
  45. Mitochondrial Transplantation Moderately Ameliorates Retinal Degeneration in Royal College of Surgeons Rats.
  46. Nuclear Interactions: A Spotlight on Nuclear Mitochondrial Membrane Contact Sites.
  47. Reduced mitochondria provide an essential function for the cytosolic methionine cycle.
  48. Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.
  49. Endosymbiotic selective pressure at the origin of eukaryotic cell biology.
  50. Fatty Acid Transport and Signaling: Mechanisms and Physiological Implications.
  51. InterPro in 2022.
  52. Rare and common genetic determinants of metabolic individuality and their effects on human health.
  53. Connexin 43 hemichannels regulate mitochondrial ATP generation, mobilization, and mitochondrial homeostasis against oxidative stress.
  54. Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer.
  55. A Simple Cost-Effective Method for Whole-Genome Sequencing, Haplotyping, and Assembly.
  56. Pooled genetic screens with image-based profiling.
  57. Peripheral blood mononuclear cell mitochondrial copy number and adenosine triphosphate inhibition test in NAFLD.
  58. Bridging clinical care and research in Ontario, Canada: Maximizing diagnoses from reanalysis of clinical exome sequencing data.
  59. Primary carnitine deficiency is a life-long disease.
  60. Amino acid variability, tradeoffs and optimality in human diet.
  61. Integration of O-GlcNAc into Stress Response Pathways.
  62. Processes in DNA damage response from a whole-cell multi-omics perspective.
  63. Prime editing for precise and highly versatile genome manipulation.
  64. Splitting up differentiation and cell cycle exit.
  65. Quantitative Proteomics Analysis Reveals That Cyclooxygenase-2 Modulates Mitochondrial Respiratory Chain Complex IV in Cardiomyocytes.
  66. RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function.
  67. Brain-Derived Neurotrophic Factor stimulates the retrograde pathway for axonal autophagy.
  1. Sci Adv. 2022 Nov 11. 8(45): eabo7956
    AMPK-dependent phosphorylation of MTFR1L regulates mitochondrial morphology.
    Lisa Tilokani, Fiona M Russell, Stevie Hamilton, Daniel M Virga, Mayuko Segawa, Vincent Paupe, Anja V Gruszczyk, Margherita Protasoni, Luis-Carlos Tabara, Mark Johnson, Hanish Anand, Michael P Murphy, D Grahame Hardie, Franck Polleux, Julien Prudent.
      Mitochondria are dynamic organelles that undergo membrane remodeling events in response to metabolic alterations to generate an adequate mitochondrial network. Here, we investigated the function of mitochondrial fission regulator 1-like protein (MTFR1L), an uncharacterized protein that has been identified in phosphoproteomic screens as a potential AMP-activated protein kinase (AMPK) substrate. We showed that MTFR1L is an outer mitochondrial membrane-localized protein modulating mitochondrial morphology. Loss of MTFR1L led to mitochondrial elongation associated with increased mitochondrial fusion events and levels of the mitochondrial fusion protein, optic atrophy 1. Mechanistically, we show that MTFR1L is phosphorylated by AMPK, which thereby controls the function of MTFR1L in regulating mitochondrial morphology both in mammalian cell lines and in murine cortical neurons in vivo. Furthermore, we demonstrate that MTFR1L is required for stress-induced AMPK-dependent mitochondrial fragmentation. Together, these findings identify MTFR1L as a critical mitochondrial protein transducing AMPK-dependent metabolic changes through regulation of mitochondrial dynamics.
    DOI:  https://doi.org/10.1126/sciadv.abo7956
  2. Int J Mol Sci. 2022 Oct 26. pii: 12926. [Epub ahead of print]23(21):
    Mitochondrial Dysfunction as an Underlying Cause of Skeletal Muscle Disorders.
    Tsung-Hsien Chen, Kok-Yean Koh, Kurt Ming-Chao Lin, Chu-Kuang Chou.
      Mitochondria are an important energy source in skeletal muscle. A main function of mitochondria is the generation of ATP for energy through oxidative phosphorylation (OXPHOS). Mitochondrial defects or abnormalities can lead to muscle disease or multisystem disease. Mitochondrial dysfunction can be caused by defective mitochondrial OXPHOS, mtDNA mutations, Ca2+ imbalances, mitochondrial-related proteins, mitochondrial chaperone proteins, and ultrastructural defects. In addition, an imbalance between mitochondrial fusion and fission, lysosomal dysfunction due to insufficient biosynthesis, and/or defects in mitophagy can result in mitochondrial damage. In this review, we explore the association between impaired mitochondrial function and skeletal muscle disorders. Furthermore, we emphasize the need for more research to determine the specific clinical benefits of mitochondrial therapy in the treatment of skeletal muscle disorders.
    Keywords:  OXPHOS; mitochondrial chaperone protein; mitochondrial dynamics; mitochondrial dysfunction; mitophagy; mtDNA mutation; skeletal muscle disorders
    DOI:  https://doi.org/10.3390/ijms232112926
  3. Int J Mol Sci. 2022 Oct 30. pii: 13216. [Epub ahead of print]23(21):
    Mitochondrial Epilepsy, a Challenge for Neurologists.
    Piervito Lopriore, Fábio Gomes, Vincenzo Montano, Gabriele Siciliano, Michelangelo Mancuso.
      Primary mitochondrial diseases are relatively common inborn errors of energy metabolism, with a combined prevalence of 1 in 4300. These disorders typically affect tissues with high energy requirements, including the brain. Epilepsy affects >1% of the worldwide population, making it one of the most common neurological illnesses; it may be the presenting feature of a mitochondrial disease, but is often part of a multisystem clinical presentation. The major genetic causes of mitochondrial epilepsy are mutations in mitochondrial DNA and in the nuclear-encoded gene POLG. Treatment of mitochondrial epilepsy may be challenging, often representing a poor prognostic feature. This narrative review will cover the most recent advances in the field of mitochondrial epilepsy, from pathophysiology and genetic etiologies to phenotype and treatment options.
    Keywords:  Leigh syndrome; MELAS; MERRF; POLG-related disorders; epilepsy; mitochondrial epilepsy; primary mitochondrial disease; stroke-like episode
    DOI:  https://doi.org/10.3390/ijms232113216
  4. Pediatr Neurol. 2022 Oct 05. pii: S0887-8994(22)00207-7. [Epub ahead of print]138 27-32
    Heteroplasmic Mutant Load Differences in Mitochondrial DNA-Associated Leigh Syndrome.
    Ji-Hoon Na, Young-Mock Lee.
      BACKGROUND: Mitochondrial DNA (mtDNA)-associated Leigh syndrome is influenced by mutant pathogenicity and corresponding heteroplasmic loads; however, the manner in which heteroplasmic mutant load affects patient phenotypes and the relationship between mutant types and heteroplasmic mutant loads remain unknown. We aimed to investigate the distribution of the mutant load of different mtDNA mutations in a single-center cohort.METHODS: We used next-generation sequencing to confirm mtDNA mutations in 31 patients with Leigh syndrome. Subsequently, we counted the number of mtDNA reads to quantitatively analyze the heteroplasmic mutant load and categorize the patients according to the mtDNA mutations they harbored. Confirmed cases of mtDNA-associated Leigh syndrome were classified according to the mutations observed in six genes and 10 nucleotides.
    RESULTS: Of the 31 patients with Leigh syndrome, 27 harbored known pathogenic mutations. We discovered that MT-ATP6 was the most commonly mutated gene (n = 13 patients), followed by MT-ND3 (n = 7) and MT-ND5 (n = 4). MT-ATP6 had a significantly higher mutant load than MT-ND3 and MT-ND5 (P < 0.001, each). By contrast, MT-ND5 had a significantly lower mutant load than MT-ND3 (P = 0.007). Notably, the mutation loads varied significantly among patients carrying the MT-ATP6, MT-ND3, and MT-ND5 mutations.
    CONCLUSIONS: Our study illustrated the heteroplasmic diversity and phenotypic expression threshold of mutated mitochondrial genes in mtDNA-associated Leigh syndrome. The results provide promising insights into the genotype-phenotype correlation in mtDNA-associated Leigh syndrome that are expected to guide the development of tailored treatments for Leigh syndrome.
    Keywords:  Heteroplasmic mutation; Heteroplasmy; Leigh syndrome; Mitochondrial DNA; Mitochondrial diseases
    DOI:  https://doi.org/10.1016/j.pediatrneurol.2022.09.006
  5. Brain. 2022 Nov 09. pii: awac417. [Epub ahead of print]
    Neuropathological hallmarks of antenatal mitochondrial diseases with a corpus callosum defect.
    Lucile Boutaud, Benedetta Ruzzenente, Aude Tessier, Olivia Anselem, Emmanuelle Pannier, Sarah Grotto, Naïma Talhi, Daniel Amram, Marjolaine Willems, Constance Wells, Patricia Blanchet, Yuri Musizzano, Clémence Jauny, Patrick Nitschke, Christine Bole-Feysot, Bettina Bessières, Houria Salhi, Amale Achaiaa, Metodi D Metodiev, Ferechte Razavi, Agnès Rötig, Laurence Loeuilllet, Tania Attié-Bitach.
      Corpus callosum defects are frequent congenital cerebral disorders caused by mutations in more than 300 genes. These include genes implicated in corpus callosum development or function, as well as genes essential for mitochondrial physiology. However, in utero corpus callosum anomalies rarely raise a suspicion of mitochondrial disease and are characterized by a very large clinical heterogeneity. Here, we report a detailed pathological and neuro-histopathological investigation of 9 fetuses from 4 unrelated families with prenatal onset of corpus callosum anomalies, sometimes associated with other cerebral or extra-cerebral defects. Next generation sequencing allowed the identification of novel pathogenic variants in 3 different nuclear genes previously reported in mitochondrial diseases: TIMMDC1, encoding a complex I assembly factor never involved before in corpus callosum defect; MRPS22, a protein of the small mitoribosomal subunit, and EARS2, the mitochondrial tRNA-glutamyl synthetase. The present report describes the antenatal histopathological findings in mitochondrial diseases and expands the genetic spectrum of antenatal corpus callosum anomalies establishing OXPHOS function as an important factor for corpus callosum biogenesis. We propose that, when observed, antenatal corpus callosum anomalies should raise suspicion of mitochondrial disease and prenatal genetic counseling should be considered.
    Keywords:   EARS2 ; MRSP22 ; TIMMDC1 ; corpus callosum defect; mitochondrial diseases
    DOI:  https://doi.org/10.1093/brain/awac417
  6. Genetics. 2022 Nov 02. pii: iyac160. [Epub ahead of print]
    Mitochondrial dysfunction, aging, and the mitochondrial unfolded protein response in Caenorhabditis elegans.
    Cole M Haynes, Siegfried Hekimi.
      We review the findings that establish that perturbations of various aspects of mitochondrial function, including oxidative phosphorylation, can promote lifespan extension, with different types of perturbations acting sometimes independently and additively on extending lifespan. We also review the great variety of processes and mechanisms that together form the mitochondrial unfolded protein response. We then explore the relationships between different types of mitochondrial dysfunction-dependent lifespan extension and the mitochondrial unfolded protein response. We conclude that, although several ways that induce extended lifespan through mitochondrial dysfunction require a functional mitochondrial unfolded protein response, there is no clear indication that activation of the mitochondrial unfolded protein response is sufficient to extend lifespan, despite the fact that the mitochondrial unfolded protein response impacts almost every aspect of mitochondrial function. In fact, in some contexts, mitochondrial unfolded protein response activation is deleterious. To explain this pattern, we hypothesize that, although triggered by mitochondrial dysfunction, the lifespan extension observed might not be the result of a change in mitochondrial function.
    Keywords:  UPRmt; WormBook; aging; mitochondria
    DOI:  https://doi.org/10.1093/genetics/iyac160
  7. J Cell Biol. 2023 Jan 02. pii: e202201137. [Epub ahead of print]222(1):
    REC drives recombination to repair double-strand breaks in animal mtDNA.
    Anna Klucnika, Peiqiang Mu, Jan Jezek, Matthew McCormack, Ying Di, Charles R Bradshaw, Hansong Ma.
      Mechanisms that safeguard mitochondrial DNA (mtDNA) limit the accumulation of mutations linked to mitochondrial and age-related diseases. Yet, pathways that repair double-strand breaks (DSBs) in animal mitochondria are poorly understood. By performing a candidate screen for mtDNA repair proteins, we identify that REC-an MCM helicase that drives meiotic recombination in the nucleus-also localizes to mitochondria in Drosophila. We show that REC repairs mtDNA DSBs by homologous recombination in somatic and germline tissues. Moreover, REC prevents age-associated mtDNA mutations. We further show that MCM8, the human ortholog of REC, also localizes to mitochondria and limits the accumulation of mtDNA mutations. This study provides mechanistic insight into animal mtDNA recombination and demonstrates its importance in safeguarding mtDNA during ageing and evolution.
    DOI:  https://doi.org/10.1083/jcb.202201137
  8. Nat Commun. 2022 Nov 07. 13(1): 6704
    Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA.
    Ayesha Sen, Sebastian Kallabis, Felix Gaedke, Christian Jüngst, Julia Boix, Julian Nüchel, Kanjanamas Maliphol, Julia Hofmann, Astrid C Schauss, Marcus Krüger, Rudolf J Wiesner, David Pla-Martín.
      Understanding the mechanisms governing selective turnover of mutation-bearing mtDNA is fundamental to design therapeutic strategies against mtDNA diseases. Here, we show that specific mtDNA damage leads to an exacerbated mtDNA turnover, independent of canonical macroautophagy, but relying on lysosomal function and ATG5. Using proximity labeling and Twinkle as a nucleoid marker, we demonstrate that mtDNA damage induces membrane remodeling and endosomal recruitment in close proximity to mitochondrial nucleoid sub-compartments. Targeting of mitochondrial nucleoids is controlled by the ATAD3-SAMM50 axis, which is disrupted upon mtDNA damage. SAMM50 acts as a gatekeeper, influencing BAK clustering, controlling nucleoid release and facilitating transfer to endosomes. Here, VPS35 mediates maturation of early endosomes to late autophagy vesicles where degradation occurs. In addition, using a mouse model where mtDNA alterations cause impairment of muscle regeneration, we show that stimulation of lysosomal activity by rapamycin, selectively removes mtDNA deletions without affecting mtDNA copy number, ameliorating mitochondrial dysfunction. Taken together, our data demonstrates that upon mtDNA damage, mitochondrial nucleoids are eliminated outside the mitochondrial network through an endosomal-mitophagy pathway. With these results, we unveil the molecular players of a complex mechanism with multiple potential benefits to understand mtDNA related diseases, inherited, acquired or due to normal ageing.
    DOI:  https://doi.org/10.1038/s41467-022-34205-9
  9. J Clin Med. 2022 Oct 30. pii: 6431. [Epub ahead of print]11(21):
    Mitochondrial Cardiomyopathy: The Roles of mt-tRNA Mutations.
    Yu Ding, Beibei Gao, Jinyu Huang.
      Mitochondria are important organelles whose primary role is generating energy through the oxidative phosphorylation (OXPHOS) system. Cardiomyopathy, a common clinical disorder, is frequently associated with pathogenic mutations in nuclear and mitochondrial genes. To date, a growing number of nuclear gene mutations have been linked with cardiomyopathy; however, knowledge about mitochondrial tRNAs (mt-tRNAs) mutations in this disease remain inadequately understood. In fact, defects in mt-tRNA metabolism caused by pathogenic mutations may influence the functioning of the OXPHOS complexes, thereby impairing mitochondrial translation, which plays a critical role in the predisposition of this disease. In this review, we summarize some basic knowledge about tRNA biology, including its structure and function relations, modification, CCA-addition, and tRNA import into mitochondria. Furthermore, a variety of molecular mechanisms underlying tRNA mutations that cause mitochondrial dysfunctions are also discussed in this article.
    Keywords:  OXPHOS system; cardiomyopathy; mt-tRNA; mutations; tRNA biology
    DOI:  https://doi.org/10.3390/jcm11216431
  10. Nucleic Acids Res. 2022 Nov 09. pii: gkac1028. [Epub ahead of print]
    Selective degradation of tRNASer(AGY) is the primary driver for mitochondrial seryl-tRNA synthetase-related disease.
    Tingting Yu, Yi Zhang, Wen-Qiang Zheng, Siqi Wu, Guoqiang Li, Yong Zhang, Niu Li, Ruen Yao, Pengfei Fang, Jian Wang, Xiao-Long Zhou.
      Mitochondrial translation is of high significance for cellular energy homeostasis. Aminoacyl-tRNA synthetases (aaRSs) are crucial translational components. Mitochondrial aaRS variants cause various human diseases. However, the pathogenesis of the vast majority of these diseases remains unknown. Here, we identified two novel SARS2 (encoding mitochondrial seryl-tRNA synthetase) variants that cause a multisystem disorder. c.654-14T > A mutation induced mRNA mis-splicing, generating a peptide insertion in the active site; c.1519dupC swapped a critical tRNA-binding motif in the C-terminus due to stop codon readthrough. Both mutants exhibited severely diminished tRNA binding and aminoacylation capacities. A marked reduction in mitochondrial tRNASer(AGY) was observed due to RNA degradation in patient-derived induced pluripotent stem cells (iPSCs), causing impaired translation and comprehensive mitochondrial function deficiencies. These impairments were efficiently rescued by wild-type SARS2 overexpression. Either mutation caused early embryonic fatality in mice. Heterozygous mice displayed reduced muscle tissue-specific levels of tRNASers. Our findings elucidated the biochemical and cellular consequences of impaired translation mediated by SARS2, suggesting that reduced abundance of tRNASer(AGY) is a key determinant for development of SARS2-related diseases.
    DOI:  https://doi.org/10.1093/nar/gkac1028
  11. Biology (Basel). 2022 Nov 09. pii: 1638. [Epub ahead of print]11(11):
    Cardiolipin Alterations during Obesity: Exploring Therapeutic Opportunities.
    Alexandre Prola, Fanny Pilot-Storck.
      Cardiolipin is a specific phospholipid of the mitochondrial inner membrane that participates in many aspects of its organization and function, hence promoting proper mitochondrial ATP production. Here, we review recent data that have investigated alterations of cardiolipin in different tissues in the context of obesity and the related metabolic syndrome. Data relating perturbations of cardiolipin content or composition are accumulating and suggest their involvement in mitochondrial dysfunction in tissues from obese patients. Conversely, cardiolipin modulation is a promising field of investigation in a search for strategies for obesity management. Several ways to restore cardiolipin content, composition or integrity are emerging and may contribute to the improvement of mitochondrial function in tissues facing excessive fat storage. Inversely, reduction of mitochondrial efficiency in a controlled way may increase energy expenditure and help fight against obesity and in this perspective, several options aim at targeting cardiolipin to achieve a mild reduction of mitochondrial coupling. Far from being just a victim of the deleterious consequences of obesity, cardiolipin may ultimately prove to be a possible weapon to fight against obesity in the future.
    Keywords:  OXPHOS; exercise; mitochondrial inner membrane; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis; respiratory coupling
    DOI:  https://doi.org/10.3390/biology11111638
  12. Biomolecules. 2022 Oct 29. pii: 1595. [Epub ahead of print]12(11):
    Metabolic Regulation of Mitochondrial Protein Biogenesis from a Neuronal Perspective.
    Jara Tabitha Hees, Angelika Bettina Harbauer.
      Neurons critically depend on mitochondria for ATP production and Ca2+ buffering. They are highly compartmentalized cells and therefore a finely tuned mitochondrial network constantly adapting to the local requirements is necessary. For neuronal maintenance, old or damaged mitochondria need to be degraded, while the functional mitochondrial pool needs to be replenished with freshly synthesized components. Mitochondrial biogenesis is known to be primarily regulated via the PGC-1α-NRF1/2-TFAM pathway at the transcriptional level. However, while transcriptional regulation of mitochondrial genes can change the global mitochondrial content in neurons, it does not explain how a morphologically complex cell such as a neuron adapts to local differences in mitochondrial demand. In this review, we discuss regulatory mechanisms controlling mitochondrial biogenesis thereby making a case for differential regulation at the transcriptional and translational level. In neurons, additional regulation can occur due to the axonal localization of mRNAs encoding mitochondrial proteins. Hitchhiking of mRNAs on organelles including mitochondria as well as contact site formation between mitochondria and endolysosomes are required for local mitochondrial biogenesis in axons linking defects in any of these organelles to the mitochondrial dysfunction seen in various neurological disorders.
    Keywords:  AMPK; PGC-1α; insulin; mTORC1; mitochondrial biogenesis; neurons; transcription; translation
    DOI:  https://doi.org/10.3390/biom12111595
  13. FASEB J. 2022 Dec;36(12): e22637
    Mitochondrial dynamics, Leydig cell function, and age-related testosterone deficiency.
    Samuel Garza, Liting Chen, Melanie Galano, Garett Cheung, Chantal Sottas, Lu Li, Yuchang Li, Barry R Zirkin, Vassilios Papadopoulos.
      The mitochondrial translocator protein (18 kDa; TSPO) is a high-affinity cholesterol-binding protein that is an integral component of the cholesterol trafficking scaffold responsible for determining the rate of cholesterol import into the mitochondria for steroid biosynthesis. Previous studies have shown that TSPO declines in aging Leydig cells (LCs) and that its decline is associated with depressed circulating testosterone levels in aging rats. However, TSPO's role in the mechanistic decline in LC function is not fully understood. To address the role of TSPO depletion in LC function, we first examined mitochondrial quality in Tspo knockout mouse tumor MA-10 nG1 LCs compared to wild-type MA-10 cells. Tspo deletion caused a disruption in mitochondrial function and membrane dynamics. Increasing mitochondrial fusion via treatment with the mitochondrial fusion promoter M1 or by optic atrophy 1 (OPA1) overexpression resulted in the restoration of mitochondrial function and mitochondrial morphology as well as in steroid formation in TSPO-depleted nG1 LCs. LCs isolated from aged rats form less testosterone than LCs isolated from young rats. Treatment of aging LCs with M1 improved mitochondrial function and increased androgen formation, suggesting that aging LC dysfunction may stem from compromised mitochondrial dynamics caused by the age-dependent LC TSPO decline. These results, taken together, suggest that maintaining or enhancing mitochondrial fusion may provide therapeutic strategies to maintain or restore testosterone levels with aging.
    Keywords:  aging; bioenergetics; hypogonadism; mitochondrial fusion; optic atrophy 1; steroidogenesis; testosterone; translocator protein
    DOI:  https://doi.org/10.1096/fj.202201026R
  14. J Biochem. 2022 Nov 08. pii: mvac092. [Epub ahead of print]
    Ubiquitin-mediated mitochondrial regulation by MITOL/MARCHF5 at a glance.
    Shun Nagashima, Naoki Ito, Isshin Shiiba, Hiroki Shimura, Shigeru Yanagi.
      Mitochondria are involved in various cellular processes, such as energy production, inflammatory responses, and cell death. Mitochondrial dysfunction is associated with many age-related diseases, including neurological disorders and heart failure. Mitochondrial quality is strictly maintained by mitochondrial dynamics linked to an adequate supply of phospholipids and other substances from the endoplasmic reticulum (ER). The outer mitochondrial membrane-localized E3 ubiquitin ligase MITOL/MARCHF5 is responsible for mitochondrial quality control through the regulation of mitochondrial dynamics, formation of mitochondria-ER contacts, and mitophagy. MITOL deficiency has been shown to impair mitochondrial function, cause an excessive inflammatory response, and increase vulnerability to stress, resulting in the exacerbation of the disease. In this study, we overview the ubiquitin-mediated regulation of mitochondrial function by MITOL and the relationship between MITOL and diseases.
    Keywords:  MITOL/MARCHF5; mitochondria; mitochondria-ER contacts; mitochondrial dynamics; ubiquitin
    DOI:  https://doi.org/10.1093/jb/mvac092
  15. Mol Genet Metab Rep. 2022 Dec;33 100932
    ITCH deficiency clinical phenotype expansion and mitochondrial dysfunction.
    Rachel Wolfe, Paige Heiman, Olivia D'Annibale, Anuradha Karunanidhi, Alyssa Powers, Marianne Mcguire, Bianca Seminotti, Steven F Dobrowolski, Miguel Reyes-Múgica, Kathryn S Torok, Al-Walid Mohsen, Jerry Vockley, Lina Ghaloul-Gonzalez.
      Autoimmune Disease, Multisystem, with Facial Dysmorphism (ADMFD) is an autosomal recessive disorder due to pathogenic variants in the ITCH gene. It is characterized by failure to thrive, dysmorphic facial features, developmental delay, and systemic autoimmunity that can manifest variably with autoimmune hepatitis, thyroiditis, and enteropathy, among other organ manifestations. It was originally described in 10 consanguineous Old Order Amish patients, and more recently in two patients of White British and Black German ethnicities. While the role of ITCH protein in apoptosis and inflammation has previously been characterized, a defect in cellular bioenergetics has not yet been reported in ITCH deficiency. Here we present a Caucasian female originally evaluated for possible mitochondrial respiratory chain deficiency, who ultimately was found to have two novel variants in ITCH with absence of ITCH protein in patient derived fibroblasts. Clinical studies of patient muscle showed mitochondrial DNA copy number of 57% compared to controls. Functional studies in skin fibroblasts revealed decreased activity of mitochondrial fatty acid oxidation and oxidative phosphorylation, and decreased overall ATP production. Our findings confirm mitochondrial energy dysfunction in a patient with ITCH deficiency offering the opportunity to assess alternative therapeutic options.
    Keywords:  Apoptosis; Autoimmune disease; DMEM, Dulbecco's Modified Eagle Medium; E3 ligase; ETC, Mitochondrial electron transport chain; FAO, Fatty acid oxidation; FOXP3, Forkhead box P3 protein; HECT, Homologous to the E6-Associated Protein C-Terminus; IBD, Inflammatory Bowel Disease; IF, Immunofluorescence analysis; ITCH; MAVS, Mitochondrial antiviral signaling protein; Mitochondrial dysfunction; NOTCH1, Notch receptor 1 protein; OCR, Oxygen consumption rate; OXPHOS, Oxidative Phosphorylation; TAX1BP1, TAX1-binding protein 1; TFP, Trifunctional protein; TXNIP, Thioredoxin Interacting Protein; Tregs, T regulatory cells; UPS, Ubiquitin proteasome system; Ubiquitination; VLCAD, Very long-chain acyl-CoA dehydrogenase protein
    DOI:  https://doi.org/10.1016/j.ymgmr.2022.100932
  16. Front Cell Dev Biol. 2022 ;10 1010232
    Mitochondrial dynamics involves molecular and mechanical events in motility, fusion and fission.
    Adam Green, Tanvir Hossain, David M Eckmann.
      Mitochondria are cell organelles that play pivotal roles in maintaining cell survival, cellular metabolic homeostasis, and cell death. Mitochondria are highly dynamic entities which undergo fusion and fission, and have been shown to be very motile in vivo in neurons and in vitro in multiple cell lines. Fusion and fission are essential for maintaining mitochondrial homeostasis through control of morphology, content exchange, inheritance of mitochondria, maintenance of mitochondrial DNA, and removal of damaged mitochondria by autophagy. Mitochondrial motility occurs through mechanical and molecular mechanisms which translocate mitochondria to sites of high energy demand. Motility also plays an important role in intracellular signaling. Here, we review key features that mediate mitochondrial dynamics and explore methods to advance the study of mitochondrial motility as well as mitochondrial dynamics-related diseases and mitochondrial-targeted therapeutics.
    Keywords:  disease; fission; fusion; live-cell imaging; mitochondria; mitochondrial DNA; motility; therapeutics
    DOI:  https://doi.org/10.3389/fcell.2022.1010232
  17. Front Cell Neurosci. 2022 ;16 1031153
    Defective PTEN-induced kinase 1/Parkin mediated mitophagy and neurodegenerative diseases.
    Megan M Braun, Luigi Puglielli.
      The selective degradation of mitochondria through mitophagy is a crucial process for maintaining mitochondrial function and cellular health. Mitophagy is a specialized form of selective autophagy that uses unique machinery to recognize and target damaged mitochondria for mitophagosome- and lysosome-dependent degradation. This process is particularly important in cells with high metabolic activity like neurons, and the accumulation of defective mitochondria is a common feature among neurodegenerative disorders. Here, we describe essential steps involved in the induction and progression of mitophagy, and then highlight the various mechanisms that specifically contribute to defective mitophagy in highly prevalent neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and Amyotrophic Lateral Sclerosis.
    Keywords:  Alzheimer’s disease; Huntington’s disease; Parkinson’s disease; amyotrophic lateral sclerosis; mitochondria; mitochondrial dysfunction; mitophagy; neurodegeneration
    DOI:  https://doi.org/10.3389/fncel.2022.1031153
  18. Brain. 2022 Nov 08. pii: awac327. [Epub ahead of print]
    Mitochondrial haplogroups and cognitive progression in Parkinson's disease.
    International Genetics of Parkinson Disease Progression (IGPP) Consortium
      Mitochondria are a culprit in the onset of Parkinson's disease, but their role during disease progression is unclear. Here we used Cox proportional hazards models to exam the effect of variation in the mitochondrial genome on longitudinal cognitive and motor progression over time in 4064 patients with Parkinson's disease. Mitochondrial macro-haplogroup was associated with reduced risk of cognitive disease progression in the discovery and replication population. In the combined analysis, patients with the super macro-haplogroup J, T, U# had a 41% lower risk of cognitive progression with P = 2.42 × 10-6 compared to those with macro-haplogroup H. Exploratory analysis indicated that the common mitochondrial DNA variant, m.2706A>G, was associated with slower cognitive decline with a hazard ratio of 0.68 (95% confidence interval 0.56-0.81) and P = 2.46 × 10-5. Mitochondrial haplogroups were not appreciably linked to motor progression. This initial genetic survival study of the mitochondrial genome suggests that mitochondrial haplogroups may be associated with the pace of cognitive progression in Parkinson's disease over time.
    Keywords:  Parkinson’s disease; cognitive progression; mitochondrial haplogroups
    DOI:  https://doi.org/10.1093/brain/awac327
  19. Aging (Albany NY). 2022 Nov 04. 14
    Denervation induces mitochondrial decline and exacerbates lysosome dysfunction in middle-aged mice.
    Matthew Triolo, Debasmita Bhattacharya, David A Hood.
      With age, skeletal muscle undergoes a progressive decline in size and quality. Imbalanced mitochondrial turnover and the resultant dysfunction contribute to these phenotypic alterations. Motor neuron denervation (Den) is a contributor to the etiology of muscle atrophy associated with age. Further, aged muscle exhibits reduced plasticity to both enhanced and suppressed contractile activity. It remains unclear when the onset of this blunted response occurs, and how middle-aged muscle adapts to denervation. The purpose of this study was to compare mitochondrial turnover pathways in young (Y, ~5months) and middle-aged (MA, ~15months) mice, and determine the influence of Den. Transgenic mt-Keima mice were subjected to 1,3 or 7 days of Den. Muscle mass, mitochondrial content, and PGC-1α protein were not different between Y and MA mice. However, indications of enhanced mitochondrial fission and mitophagy were evident in MA muscle which were supported by a greater abundance of lysosome proteins. Den resulted in muscle atrophy and reductions in mitochondrial protein content by 7-days. These changes occurred concomitant with modest decreases in PGC-1α protein, but without further elevations in mitophagy. Although both autophagosomal and lysosomal proteins were elevated, evidence of lysosome dysfunction was present following Den in MA mice. These data suggest that increases in fission drive an acceleration of mitophagy in muscle of MA mice to preserve mitochondrial quality. Den exacerbates the aging phenotype by reducing biogenesis in the absence of a change in mitophagy, perhaps limited by lysosomal capacity, leading to an accumulation of dysfunctional mitochondria with an age-related loss of neuromuscular innervation.
    Keywords:  autophagy; lysosomes; mitochondrial biogenesis; mitophagy; muscle
    DOI:  https://doi.org/10.18632/aging.204365
  20. Cell Death Dis. 2022 Nov 10. 13(11): 947
    Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy.
    Nur Mehpare Kocaturk, Nesibe Peker, Karin Eberhart, Yunus Akkoc, Gamze Deveci, Joern Dengjel, Devrim Gozuacik.
      Autophagy is an evolutionarily conserved eukaryotic cellular mechanism through which cytosolic fragments, misfolded/aggregated proteins and organelles are degraded and recycled. Priming of mitochondria through ubiquitylation is required for the clearance the organelle by autophagy (mitophagy). Familial Parkinson's Disease-related proteins, including the E3-ligase PARK2 (PARKIN) and the serine/threonine kinase PARK6 (PINK1) control these ubiquitylation reactions and contribute to the regulation of mitophagy. Here we describe, novel protein complexes containing autophagy protein ATG5 and ubiquitin-proteasome system (UPS) components. We discovered that ATG5 interacts with PSMA7 and PARK2 upon mitochondrial stress. Results suggest that all three proteins translocate mitochondria and involve in protein complexes containing autophagy, UPS and mitophagy proteins. Interestingly, PARK2 and ATG5 recruitment onto mitochondria requires proteasome components PSMA7 and PSMB5. Strikingly, we discovered that subunit of 20 S proteasome, PSMA7, is required for the progression of PARK2-PARK6-mediated mitophagy and the proteasome activity following mitochondrial stress. Our results demonstrate direct, dynamic and functional interactions between autophagy and UPS components that contribute to the regulation of mitophagy.
    DOI:  https://doi.org/10.1038/s41419-022-05339-x
  21. Metabolism. 2022 Nov 07. pii: S0026-0495(22)00221-9. [Epub ahead of print] 155343
    Resistin impairs mitochondrial homeostasis via cyclase-associated protein 1-mediated fission, leading to obesity-induced metabolic diseases.
    Han-Mo Yang, Joonoh Kim, Dasom Shin, Ju-Young Kim, Jihye You, Hyun-Chae Lee, Hyun-Duk Jang, Hyo-Soo Kim.
      OBJECTIVE: One of the suggested mechanisms of obesity-induced insulin resistance is mitochondrial dysfunction in target tissues such as skeletal muscle. In our study, we examined whether resistin, an adipokine associated with obesity-mediated insulin resistance, induced metabolic disorders by impairing mitochondrial homeostasis.METHODS: The morphology and function of mitochondria of skeletal muscle were examined in resistin-knockout and humanized resistin mice that were subjected to high-fat diet for 3 months. Morphology was examined by transmission electron microscopy. Mitochondria bioenergetics of skeletal muscle were evaluated using a Seahorse XF96 analyzer. Human skeletal myoblasts were used for in vitro studies on signaling mechanisms in responses to resistin.
    RESULTS: A high-fat diet in humanized resistin mice increased fragmented and shorter mitochondria in the skeletal muscle, whereas resistin-knockout mice had healthy normal mitochondria. In vitro studies showed that human resistin treatment impaired mitochondrial homeostasis by inducing mitochondrial fission, leading to a decrease in ATP production and mitochondrial dysfunction. Induction of mitochondrial fission by resistin was accompanied by increased formation of mitochondria-associated ER membranes (MAM). At the same time, resistin induced up-regulation of the protein kinase A (PKA) pathway. This activation of PKA induced phosphorylation of Drp1 at serine 616, leading to Drp1 activation and subsequent induction of mitochondrial fission. The key molecule that mediated human resistin-induced mitochondrial fission was adenylyl cyclase-associated protein 1 (CAP1), which was reported as a bona fide receptor for human resistin. Moreover, our newly developed biomimetic selective blocking peptide could repress human resistin-mediated mitochondrial dysfunction. High-fat diet-fed mice showed lower exercise capacity and higher insulin resistance, which was prevented by a novel peptide to block the binding of resistin to CAP1 or in the CAP1-knockdown mice.
    CONCLUSIONS: Our study demonstrated that human resistin induces mitochondrial dysfunction by inducing abnormal mitochondrial fission. This result suggests that the resistin-CAP1 complex could be a potential therapeutic target for the treatment of obesity-related metabolic diseases such as diabetes and cardiometabolic diseases.
    Keywords:  Cyclase-associate protein 1; Fission; Mitochondria; Obesity; Resistin
    DOI:  https://doi.org/10.1016/j.metabol.2022.155343
  22. Methods Mol Biol. 2023 ;2590 139-147
    HapCUT2: A Method for Phasing Genomes Using Experimental Sequence Data.
    Vikas Bansal.
      Rapid advances in high-throughput DNA sequencing technologies have enabled variant discovery from whole-genome sequencing (WGS) datasets; however linking variants on a chromosome together into haplotypes, also known as haplotype phasing, remains difficult. Human genomes are diploid and haplotype phasing is crucial for the complete interpretation and analysis of genetic variation.Hapcut2 ( https://github.com/vibansal/HapCUT2 ) is an open-source software for phasing diploid genomes using sequence data generated using different sequencing technologies and experimental methods. In this article, we give an overview of the algorithm used by Hapcut2 and describe how to use Hapcut2 for haplotype phasing of individual genomes using different types of sequence data.
    Keywords:  Haplotype; Haplotype assembly; Long reads; Next-generation sequencing; Phasing; Proximity-ligation; Variants
    DOI:  https://doi.org/10.1007/978-1-0716-2819-5_9
  23. Nat Commun. 2022 Nov 11. 13(1): 6869
    Impaired mitochondrial oxidative metabolism in skeletal progenitor cells leads to musculoskeletal disintegration.
    Chujiao Lin, Qiyuan Yang, Dongsheng Guo, Jun Xie, Yeon-Suk Yang, Sachin Chaugule, Ngoc DeSouza, Won-Taek Oh, Rui Li, Zhihao Chen, Aijaz A John, Qiang Qiu, Lihua Julie Zhu, Matthew B Greenblatt, Sankar Ghosh, Shaoguang Li, Guangping Gao, Cole Haynes, Charles P Emerson, Jae-Hyuck Shim.
      Although skeletal progenitors provide a reservoir for bone-forming osteoblasts, the major energy source for their osteogenesis remains unclear. Here, we demonstrate a requirement for mitochondrial oxidative phosphorylation in the osteogenic commitment and differentiation of skeletal progenitors. Deletion of Evolutionarily Conserved Signaling Intermediate in Toll pathways (ECSIT) in skeletal progenitors hinders bone formation and regeneration, resulting in skeletal deformity, defects in the bone marrow niche and spontaneous fractures followed by persistent nonunion. Upon skeletal fracture, Ecsit-deficient skeletal progenitors migrate to adjacent skeletal muscle causing muscle atrophy. These phenotypes are intrinsic to ECSIT function in skeletal progenitors, as little skeletal abnormalities were observed in mice lacking Ecsit in committed osteoprogenitors or mature osteoblasts. Mechanistically, Ecsit deletion in skeletal progenitors impairs mitochondrial complex assembly and mitochondrial oxidative phosphorylation and elevates glycolysis. ECSIT-associated skeletal phenotypes were reversed by in vivo reconstitution with wild-type ECSIT expression, but not a mutant displaying defective mitochondrial localization. Collectively, these findings identify mitochondrial oxidative phosphorylation as the prominent energy-driving force for osteogenesis of skeletal progenitors, governing musculoskeletal integrity.
    DOI:  https://doi.org/10.1038/s41467-022-34694-8
  24. iScience. 2022 Nov 18. 25(11): 105342
    Lipopolysaccharide induces placental mitochondrial dysfunction in murine and human systems by reducing MNRR1 levels via a TLR4-independent pathway.
    Neeraja Purandare, Yusef Kunji, Yue Xi, Roberto Romero, Nardhy Gomez-Lopez, Andrew Fribley, Lawrence I Grossman, Siddhesh Aras.
      Mitochondria play a key role in placental growth and development, and mitochondrial dysfunction is associated with inflammation in pregnancy pathologies. However, the mechanisms whereby placental mitochondria sense inflammatory signals are unknown. Mitochondrial nuclear retrograde regulator 1 (MNRR1) is a bi-organellar protein responsible for mitochondrial function, including optimal induction of cellular stress-responsive signaling pathways. Here, in a lipopolysaccharide-induced model of systemic placental inflammation, we show that MNRR1 levels are reduced both in mouse placental tissues in vivo and in human trophoblastic cell lines in vitro. MNRR1 reduction is associated with mitochondrial dysfunction, enhanced oxidative stress, and activation of pro-inflammatory signaling. Mechanistically, we uncover a non-conventional pathway independent of Toll-like receptor 4 (TLR4) that results in ATM kinase-dependent threonine phosphorylation that stabilizes mitochondrial protease YME1L1, which targets MNRR1. Enhancing MNRR1 levels abrogates the bioenergetic defect and induces an anti-inflammatory phenotype. We therefore propose MNRR1 as an anti-inflammatory therapeutic in placental inflammation.
    Keywords:  Cell biology; Human metabolism; Immunity
    DOI:  https://doi.org/10.1016/j.isci.2022.105342
  25. Nat Commun. 2022 Nov 07. 13(1): 6324
    Specialist multidisciplinary input maximises rare disease diagnoses from whole genome sequencing.
    Genomics England Research Consortium
      Diagnostic whole genome sequencing (WGS) is increasingly used in rare diseases. However, standard, semi-automated WGS analysis may overlook diagnoses in complex disorders. Here, we show that specialist multidisciplinary analysis of WGS, following an initial 'no primary findings' (NPF) report, improves diagnostic rates and alters management. We undertook WGS in 102 adults with diagnostically challenging primary mitochondrial disease phenotypes. NPF cases were reviewed by a genomic medicine team, thus enabling bespoke informatic approaches, co-ordinated phenotypic validation, and functional work. We enhanced the diagnostic rate from 16.7% to 31.4%, with management implications for all new diagnoses, and detected strong candidate disease-causing variants in a further 3.9% of patients. This approach presents a standardised model of care that supports mainstream clinicians and enhances diagnostic equity for complex disorders, thereby facilitating access to the potential benefits of genomic healthcare. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project: http://www.genomicsengland.co.uk .
    DOI:  https://doi.org/10.1038/s41467-022-32908-7
  26. Cells. 2022 Nov 06. pii: 3511. [Epub ahead of print]11(21):
    Mitochondrial Cardiomyopathy: Molecular Epidemiology, Diagnosis, Models, and Therapeutic Management.
    Jinjuan Yang, Shaoxiang Chen, Fuyu Duan, Xiuxiu Wang, Xiaoxian Zhang, Boonxuan Lian, Meng Kou, Zhixin Chiang, Ziyue Li, Qizhou Lian.
      Mitochondrial cardiomyopathy (MCM) is characterized by abnormal heart-muscle structure and function, caused by mutations in the nuclear genome or mitochondrial DNA. The heterogeneity of gene mutations and various clinical presentations in patients with cardiomyopathy make its diagnosis, molecular mechanism, and therapeutics great challenges. This review describes the molecular epidemiology of MCM and its clinical features, reviews the promising diagnostic tests applied for mitochondrial diseases and cardiomyopathies, and details the animal and cellular models used for modeling cardiomyopathy and to investigate disease pathogenesis in a controlled in vitro environment. It also discusses the emerging therapeutics tested in pre-clinical and clinical studies of cardiac regeneration.
    Keywords:  animal model; cellular model; diagnosis; gene therapy; mitochondrial cardiomyopathy; mitochondrial transfer/transplantation; molecular epidemiology; pharmacological approach
    DOI:  https://doi.org/10.3390/cells11213511
  27. Elife. 2022 Nov 08. pii: e82709. [Epub ahead of print]11
    Two neuronal peptides encoded from a single transcript regulate mitochondrial complex III in Drosophila.
    Justin A Bosch, Berrak Ugur, Israel Pichardo-Casas, Jordan Rabasco, Felipe Escobedo, Zhongyuan Zuo, Ben Brown, Susan Celniker, David A Sinclair, Hugo J Bellen, Norbert Perrimon.
      Naturally produced peptides (<100 amino acids) are important regulators of physiology, development, and metabolism. Recent studies have predicted that thousands of peptides may be translated from transcripts containing small open reading frames (smORFs). Here, we describe two peptides in Drosophila encoded by conserved smORFs, Sloth1 and Sloth2. These peptides are translated from the same bicistronic transcript and share sequence similarities, suggesting that they encode paralogs. Yet, Sloth1 and Sloth2 are not functionally redundant, and loss of either peptide causes animal lethality, reduced neuronal function, impaired mitochondrial function, and neurodegeneration. We provide evidence that Sloth1/2 are highly expressed in neurons, imported to mitochondria, and regulate mitochondrial complex III assembly. These results suggest that phenotypic analysis of smORF genes in Drosophila can provide a wealth of information on the biological functions of this poorly characterized class of genes.
    Keywords:  D. melanogaster; genetics; genomics; neuroscience
    DOI:  https://doi.org/10.7554/eLife.82709
  28. Genome Res. 2022 Nov 09. pii: gr.276885.122. [Epub ahead of print]
    Hybridization drives mitochondrial DNA degeneration and metabolic shift in a species with biparental mitochondrial inheritance.
    Mathieu Hénault, Souhir Marsit, Guillaume Charron, Christian R Landry.
      Mitochondrial DNA (mtDNA) is a cytoplasmic genome that is essential for respiratory metabolism. While uniparental mtDNA inheritance is most common in animals and plants, distinct mtDNA haplotypes can coexist in a state of heteroplasmy, either because of paternal leakage or de novo mutations. mtDNA integrity and the resolution of heteroplasmy have important implications, notably for mitochondrial genetic disorders, speciation, and genome evolution in hybrids. However, the impact of genetic variation on the transition to homoplasmy from initially heteroplasmic backgrounds remains largely unknown. Here, we use Saccharomyces yeasts, fungi with constitutive biparental mtDNA inheritance, to investigate the resolution of mtDNA heteroplasmy in a variety of hybrid genotypes. We previously designed 11 crosses along a gradient of parental evolutionary divergence using undomesticated isolates of Saccharomyces paradoxus and Saccharomyces cerevisiae Each cross was independently replicated 48 to 96 times, and the resulting 864 hybrids were evolved under relaxed selection for mitochondrial function. Genome sequencing of 446 MA lines revealed extensive mtDNA recombination, but recombination rate was not predicted by parental divergence level. We found a strong positive relationship between parental divergence and the rate of large-scale mtDNA deletions, which lead to the loss of respiratory metabolism. We also uncovered associations between mtDNA recombination, mtDNA deletion, and genome instability that were genotype-specific. Our results show that hybridization in yeast induces mtDNA degeneration through large-scale deletion and loss of function, with deep consequences for mtDNA evolution, metabolism and the emergence of reproductive isolation.
    DOI:  https://doi.org/10.1101/gr.276885.122
  29. Front Pharmacol. 2022 ;13 947387
    Adenosine monophosphate activated protein kinase contributes to skeletal muscle health through the control of mitochondrial function.
    Yan Yan, Ming Li, Jie Lin, Yanan Ji, Kexin Wang, Dajun Yan, Yuntian Shen, Wei Wang, Zhongwei Huang, Haiyan Jiang, Hualin Sun, Lei Qi.
      Skeletal muscle is one of the largest organs in the body and the largest protein repository. Mitochondria are the main energy-producing organelles in cells and play an important role in skeletal muscle health and function. They participate in several biological processes related to skeletal muscle metabolism, growth, and regeneration. Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor and regulator of systemic energy balance. AMPK is involved in the control of energy metabolism by regulating many downstream targets. In this review, we propose that AMPK directly controls several facets of mitochondrial function, which in turn controls skeletal muscle metabolism and health. This review is divided into four parts. First, we summarize the properties of AMPK signal transduction and its upstream activators. Second, we discuss the role of mitochondria in myogenesis, muscle atrophy, regeneration post-injury of skeletal muscle cells. Third, we elaborate the effects of AMPK on mitochondrial biogenesis, fusion, fission and mitochondrial autophagy, and discuss how AMPK regulates the metabolism of skeletal muscle by regulating mitochondrial function. Finally, we discuss the effects of AMPK activators on muscle disease status. This review thus represents a foundation for understanding this biological process of mitochondrial dynamics regulated by AMPK in the metabolism of skeletal muscle. A better understanding of the role of AMPK on mitochondrial dynamic is essential to improve mitochondrial function, and hence promote skeletal muscle health and function.
    Keywords:  AMPK; mitochondria; muscle atrophy; muscle regeneration; skeletal muscle
    DOI:  https://doi.org/10.3389/fphar.2022.947387
  30. Cell. 2022 Nov 02. pii: S0092-8674(22)01318-6. [Epub ahead of print]
    Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.
    Haopeng Xiao, Luiz H M Bozi, Yizhi Sun, Christopher L Riley, Vivek M Philip, Mandy Chen, Jiaming Li, Tian Zhang, Evanna L Mills, Margo P Emont, Wenfei Sun, Anita Reddy, Ryan Garrity, Jiani Long, Tobias Becher, Laura Potano Vitas, Dina Laznik-Bogoslavski, Martha Ordonez, Xinyue Liu, Xiong Chen, Yun Wang, Weihai Liu, Nhien Tran, Yitong Liu, Yang Zhang, Aaron M Cypess, Andrew P White, Yuchen He, Rebecca Deng, Heiko Schöder, Joao A Paulo, Mark P Jedrychowski, Alexander S Banks, Yu-Hua Tseng, Paul Cohen, Linus T Tsai, Evan D Rosen, Samuel Klein, Maria Chondronikola, Fiona E McAllister, Nick Van Bruggen, Edward L Huttlin, Bruce M Spiegelman, Gary A Churchill, Steven P Gygi, Edward T Chouchani.
      Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.
    DOI:  https://doi.org/10.1016/j.cell.2022.10.003
  31. Methods Mol Biol. 2023 ;2590 49-57
    Full-Length Transcript Phasing with Third-Generation Sequencing.
    Nenad Svrzikapa, Ramakrishna Boyanapalli.
      Haplotyping individual full-length transcripts can be important in diagnosis and treatment of certain genetic diseases. One set of diseases, repeat expansions of simple tandem repeat sequences are the cause of over 40 neurological disorders. In many of these conditions, expanding a polymorphic repeat beyond a given threshold has been strongly associated with disease onset and severity. Given that most repeat expansions are inherited in an autosomal dominant pattern, repeat expansion disorders are typically characterized by a heterozygous expansion locus associated with a single haplotype. Precision genetic medicines can be used to selectively target expansion-containing sequences in a haplotype-specific manner.However, repeat expansion lengths often exceed the capacity of next-generation sequencing (NGS) reads. Therefore, the accurate length and haplotype determination of repeat expansions requires special considerations and requires the development of custom methods. Here we highlight a method for targeted haplotype phasing of the HTT gene, which can be adopted for use with other full-length transcripts and in other repeat expansion disorders.
    Keywords:  Allele-selective targeting; Antisense oligonucleotides; Clinical haplotyping; Full-length transcript; Genetic medicines; Haplotype phasing; Long-read sequencing; Repeat expansion disorders; Repeat expansions
    DOI:  https://doi.org/10.1007/978-1-0716-2819-5_3
  32. Biochimie. 2022 Nov 03. pii: S0300-9084(22)00287-5. [Epub ahead of print]
    Molecular etiology of defective nuclear and mitochondrial ribosome biogenesis: Clinical phenotypes and therapy.
    Maria Sona Jerome, Dechamma Pandyanda Nanjappa, Anirban Chakraborty, Sanjiban Chakrabarty.
      Ribosomopathies are rare congenital disorders associated with defective ribosome biogenesis due to pathogenic variations in genes that encode proteins related to ribosome function and biogenesis. Defects in ribosome biogenesis result in a nucleolar stress response involving the TP53 tumor suppressor protein and impaired protein synthesis leading to a deregulated translational output. Despite the accepted notion that ribosomes are omnipresent and essential for all cells, most ribosomopathies show tissue-specific phenotypes affecting blood cells, hair, spleen, or skin. On the other hand, defects in mitochondrial ribosome biogenesis are associated with a range of clinical manifestations affecting more than one organ. Intriguingly, the deregulated ribosomal function is also a feature in several human malignancies with a selective upregulation or downregulation of specific ribosome components. Here, we highlight the clinical conditions associated with defective ribosome biogenesis in the nucleus and mitochondria with a description of the affected genes and the implicated pathways, along with a note on the treatment strategies currently available for these disorders.
    Keywords:  Diamond-Blackfan anemia; MELAS; Mitochondria; Ribosome; Ribosomopathies
    DOI:  https://doi.org/10.1016/j.biochi.2022.11.001
  33. Int J Mol Sci. 2022 Nov 06. pii: 13600. [Epub ahead of print]23(21):
    Conservation and Diversification of tRNA t6A-Modifying Enzymes across the Three Domains of Life.
    Chenchen Su, Mengqi Jin, Wenhua Zhang.
      The universal N6-threonylcarbamoyladenosine (t6A) modification occurs at position 37 of tRNAs that decipher codons starting with adenosine. Mechanistically, t6A stabilizes structural configurations of the anticodon stem loop, promotes anticodon-codon pairing and safeguards the translational fidelity. The biosynthesis of tRNA t6A is co-catalyzed by two universally conserved protein families of TsaC/Sua5 (COG0009) and TsaD/Kae1/Qri7 (COG0533). Enzymatically, TsaC/Sua5 protein utilizes the substrates of L-threonine, HCO3-/CO2 and ATP to synthesize an intermediate L-threonylcarbamoyladenylate, of which the threonylcarbamoyl-moiety is subsequently transferred onto the A37 of substrate tRNAs by the TsaD-TsaB -TsaE complex in bacteria or by the KEOPS complex in archaea and eukaryotic cytoplasm, whereas Qri7/OSGEPL1 protein functions on its own in mitochondria. Depletion of tRNA t6A interferes with protein homeostasis and gravely affects the life of unicellular organisms and the fitness of higher eukaryotes. Pathogenic mutations of YRDC, OSGEPL1 and KEOPS are implicated in a number of human mitochondrial and neurological diseases, including autosomal recessive Galloway-Mowat syndrome. The molecular mechanisms underscoring both the biosynthesis and cellular roles of tRNA t6A are presently not well elucidated. This review summarizes current mechanistic understandings of the catalysis, regulation and disease implications of tRNA t6A-biosynthetic machineries of three kingdoms of life, with a special focus on delineating the structure-function relationship from perspectives of conservation and diversity.
    Keywords:  ATPase activity; KEOPS; N6-threonylcarbamoyladenosine synthetases; TsaC/Sua5; TsaD/Kae1/Qri7; TsaD–TsaB–TsaE; human disease; protein–protein interactions; structure–function relationship; tRNA binding; tRNA modification
    DOI:  https://doi.org/10.3390/ijms232113600
  34. Ann Biomed Eng. 2022 Nov 11.
    Approaches to Mitigate Mitochondrial Dysfunction in Sensorineural Hearing Loss.
    Mustafa Nazir Okur, Hamid R Djalilian.
      Mitochondria are highly dynamic multifaceted organelles with various functions including cellular energy metabolism, reactive oxygen species (ROS) generation, calcium homeostasis, and apoptosis. Because of these diverse functions, mitochondria are key regulators of cell survival and death, and their dysfunction is implicated in numerous diseases, particularly neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, and Huntington's Disease. One of the most common neurodegenerative disorders is sensorineural hearing loss (SNHL). SNHL primarily originates from the degenerative changes in the cochlea, which is the auditory portion of the inner ear. Many cochlear cells contain an abundance of mitochondria and are metabolically highly active, rendering them susceptible to mitochondrial dysfunction. Indeed, the causal role of mitochondrial dysfunction in SNHL progression is well established, and therefore, targeted for treatment. In this review, we aim to compile the emerging findings in the literature indicating the role of mitochondrial dysfunction in the progression of sensorineural hearing loss and highlight potential therapeutics targeting mitochondrial dysfunction for hearing loss treatment.
    Keywords:  Hearing loss; Mitochondria; Mitochondrial dysfunction; Sensorineural hearing loss
    DOI:  https://doi.org/10.1007/s10439-022-03103-y
  35. Genet Med. 2022 Nov 08. pii: S1098-3600(22)00970-4. [Epub ahead of print]
    Diagnosing, discarding, or de-VUSsing: A practical guide to (un)targeted metabolomics as variant-transcending functional tests.
    Elise A Ferreira, Annemarijne R J Veenvliet, Udo F H Engelke, Leo A J Kluijtmans, Marleen C D G Huigen, Brechtje Hoegen, Lonneke de Boer, Maaike C de Vries, Bregje W van Bon, Erika Leenders, Elisabeth A M Cornelissen, Charlotte A Haaxma, Jolanda H Schieving, M Estela Rubio-Gozalbo, Irene M L W Körver-Keularts, Lara M Marten, Susann Diegmann, Jeroen Mourmans, Alexander J M Rennings, Clara D M van Karnebeek, Richard J Rodenburg, Karlien L M Coene.
      PURPOSE: For patients with inherited metabolic disorders (IMDs), any diagnostic delay should be avoided because early initiation of personalized treatment could prevent irreversible health damage. To improve diagnostic interpretation of genetic data, gene function tests can be valuable assets. For IMDs, variant-transcending functional tests are readily available through (un)targeted metabolomics assays. To support the application of metabolomics for this purpose, we developed a gene-based guide to select functional tests to either confirm or exclude an IMD diagnosis.METHODS: Using information from a diagnostic IMD exome panel, Kyoto Encyclopedia of Genes and Genomes, and Inborn Errors of Metabolism Knowledgebase, we compiled a guide for metabolomics-based gene function tests. From our practical experience with this guide, we retrospectively selected illustrative cases for whom combined metabolomic/genomic testing improved diagnostic success and evaluated the effect hereof on clinical management.
    RESULTS: The guide contains 2047 metabolism-associated genes for which a validated or putative variant-transcending gene function test is available. We present 16 patients for whom metabolomic testing either confirmed or ruled out the presence of a second pathogenic variant, validated or ruled out pathogenicity of variants of uncertain significance, or identified a diagnosis initially missed by genetic analysis.
    CONCLUSION: Metabolomics-based gene function tests provide additional value in the diagnostic trajectory of patients with suspected IMD by enhancing and accelerating diagnostic success.
    Keywords:  Exome sequencing; Functional test; Genome sequencing; Inborn errors of metabolism; Metabolomics
    DOI:  https://doi.org/10.1016/j.gim.2022.10.002
  36. Methods Mol Biol. 2023 ;2590 161-182
    Haplotyping-Assisted Diploid Assembly and Variant Detection with Linked Reads.
    Yunfei Hu, Chao Yang, Lu Zhang, Xin Zhou.
      Phasing is essential for determining the origins of each set of alleles in the whole-genome sequencing data of individuals. As such, it provides essential information for the causes of hereditary diseases and the sources of individual variability. Recent technical breakthroughs in linked-read (referred to as co-barcoding in other chapters of the book) and long-read sequencing and downstream analysis have brought the goal of accurate and complete phasing within reach. Here we review recent progress related to the assembly and phasing of personal genomes based on linked-reads and related applications. Motivated by current limitations in generating high-quality diploid assemblies and detecting variants, a new suite of software tools, Aquila, was developed to fully take advantage of linked-read sequencing technology. The overarching goal of Aquila is to exploit the strengths of linked-read technology including long-range connectivity and inherent phasing of variants for reference-assisted local de novo assembly at the whole-genome scale. The diploid nature of the assemblies facilitates detection and phasing of genetic variation, including single nucleotide variations (SNVs), small insertions and deletions (indels), and structural variants (SVs). An extension of Aquila, Aquila_stLFR, focuses on another newly developed linked-reads sequencing technology, single-tube long-fragment read (stLFR). AquilaSV, a region-based diploid assembly approach, is used to characterize structural variants and can achieve diploid assembly in one target region at a time. Lastly, we introduce HAPDeNovo, a program that exploits phasing information from linked-read sequencing to improve detection of de novo mutations. Use of these tools is expected to harness the advantages of linked-reads technology, improve phasing, and advance variant discovery.
    Keywords:  Aquila; Diploid assembly; Haplotype estimation; Linked-read sequencing; Structural variants; Variant phasing
    DOI:  https://doi.org/10.1007/978-1-0716-2819-5_11
  37. Cancer Cell Int. 2022 Nov 08. 22(1): 343
    The function of prohibitins in mitochondria and the clinical potentials.
    Linda Oyang, Jian Li, Xianjie Jiang, Jinguan Lin, Longzheng Xia, Lixia Yang, Shiming Tan, Nayiyuan Wu, Yaqian Han, Yiqing Yang, Xia Luo, Jinyun Li, Qianjin Liao, Yingrui Shi, Yujuan Zhou.
      Prohibitins (PHBs) are a class of highly evolutionarily conserved proteins that widely distribute in prokaryotes and eukaryotes. PHBs function in cell growth and proliferation or differentiation, regulating metabolism and signaling pathways. PHBs have different subcellular localization in eukaryotes, but they are mainly located in mitochondria. In the mitochondria, PHBs stabilize the structure of the mitochondrial membrane and regulate mitochondrial autophagy, mitochondrial dynamics, mitochondrial biogenesis and quality control, and mitochondrial unfolded protein response. PHBs has shown to be associated with many diseases, such as mitochondria diseases, cancers, infectious diseases, and so on. Some molecule targets of PHBs can interfere with the occurrence and development of diseases. Therefore, this review clarifies the functions of PHBs in mitochondria, and provides a summary of the potential values in clinics.
    Keywords:  Cancer; Mitochondria; Mitochondria disease; Mitochondrial biogenesis and quality control; Mitochondrial dynamics; Mitophagy; Prohibitin
    DOI:  https://doi.org/10.1186/s12935-022-02765-x
  38. Hum Genomics. 2022 Nov 10. 16(1): 55
    De novo and inherited variants in coding and regulatory regions in genetic cardiomyopathies.
    Genomics England Research Consortium
      BACKGROUND: Cardiomyopathies are a leading cause of progressive heart failure and sudden cardiac death; however, their genetic aetiology remains poorly understood. We hypothesised that variants in noncoding regulatory regions and oligogenic inheritance mechanisms may help close the diagnostic gap.METHODS: We first analysed whole-genome sequencing data of 143 parent-offspring trios from Genomics England 100,000 Genomes Project. We used gene panel testing and a phenotype-based, variant prioritisation framework called Exomiser to identify candidate genes in trios. To assess the contribution of noncoding DNVs to cardiomyopathies, we intersected DNVs with open chromatin sequences from single-cell ATAC-seq data of cardiomyocytes. We also performed a case-control analysis in an exome-negative cohort, including 843 probands and 19,467 controls, to assess the association between noncoding variants in known cardiomyopathy genes and disease.
    RESULTS: In the trio analysis, a definite or probable genetic diagnosis was identified in 21 probands according to the American College of Medical Genetics guidelines. We identified novel DNVs in diagnostic-grade genes (RYR2, TNNT2, PTPN11, MYH7, LZR1, NKX2-5), and five cases harbouring a combination of prioritised variants, suggesting that oligogenic inheritance and genetic modifiers contribute to cardiomyopathies. Phenotype-based ranking of candidate genes identified in noncoding DNV analysis revealed JPH2 as the top candidate. Moreover, a case-control analysis revealed an enrichment of rare noncoding variants in regulatory elements of cardiomyopathy genes (p = .035, OR = 1.43, 95% Cl = 1.095-1.767) versus controls. Of the 25 variants associated with disease  (p< 0.5), 23 are novel and nine are predicted to disrupt transcription factor binding motifs.
    CONCLUSION: Our results highlight complex genetic mechanisms in cardiomyopathies and reveal novel genes for future investigations.
    Keywords:  Cardiomyopathy; De novo; Noncoding; Oligogenic; Regulome; Single-cell
    DOI:  https://doi.org/10.1186/s40246-022-00420-0
  39. Biochim Biophys Acta Mol Cell Biol Lipids. 2022 Nov 07. pii: S1388-1981(22)00138-X. [Epub ahead of print] 159248
    Odd- and even-numbered medium-chained fatty acids protect against glutathione depletion in very long-chain acyl-CoA dehydrogenase deficiency.
    Martin Lund, Robert Heaton, Iain P Hargreaves, Niels Gregersen, Rikke K J Olsen.
      Recent trials have reported the ability of triheptanoin to improve clinical outcomes for the severe symptoms associated with long-chain fatty acid oxidation disorders, including very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. However, the milder myopathic symptoms are still challenging to treat satisfactorily. Myopathic pathogenesis is multifactorial, but oxidative stress is an important component. We have previously shown that metabolic stress increases the oxidative burden in VLCAD-deficient cell lines and can deplete the antioxidant glutathione (GSH). We investigated whether medium-chain fatty acids provide protection against GSH depletion during metabolic stress in VLCAD-deficient fibroblasts. To investigate the effect of differences in anaplerotic capacity, we included both even-(octanoate) and odd-numbered (heptanoate) medium-chain fatty acids. Overall, we show that modulation of the concentration of medium-chain fatty acids in culture media affects levels of GSH retained during metabolic stress in VLCAD-deficient cell lines but not in controls. Lowered glutamine concentration in the culture media during metabolic stress led to GSH depletion and decreased viability in VLCAD deficient cells, which could be rescued by both heptanoate and octanoate in a dose-dependent manner. Unlike GSH levels, the levels of total thiols increased after metabolic stress exposure, the size of this increase was not affected by differences in cell culture medium concentrations of glutamine, heptanoate or octanoate. Addition of a PPAR agonist further exacerbated stress-related GSH-depletion and viability loss, requiring higher concentrations of fatty acids to restore GSH levels and cell viability. Both odd- and even-numbered medium-chain fatty acids efficiently protect VLCADdeficient cells against metabolic stress-induced antioxidant depletion.
    Keywords:  Anaplerosis; Fatty acids oxidation deficiency; Glutathione; Heptanoate; Mitochondria; Octanoate; Oxidative stress; VLCAD
    DOI:  https://doi.org/10.1016/j.bbalip.2022.159248
  40. Nucleic Acids Res. 2022 Nov 09. pii: gkac966. [Epub ahead of print]
    FAVOR: functional annotation of variants online resource and annotator for variation across the human genome.
    NHGRI Genome Sequencing Program Variant Functional Annotation Working Group
      Large biobank-scale whole genome sequencing (WGS) studies are rapidly identifying a multitude of coding and non-coding variants. They provide an unprecedented resource for illuminating the genetic basis of human diseases. Variant functional annotations play a critical role in WGS analysis, result interpretation, and prioritization of disease- or trait-associated causal variants. Existing functional annotation databases have limited scope to perform online queries and functionally annotate the genotype data of large biobank-scale WGS studies. We develop the Functional Annotation of Variants Online Resources (FAVOR) to meet these pressing needs. FAVOR provides a comprehensive multi-faceted variant functional annotation online portal that summarizes and visualizes findings of all possible nine billion single nucleotide variants (SNVs) across the genome. It allows for rapid variant-, gene- and region-level queries of variant functional annotations. FAVOR integrates variant functional information from multiple sources to describe the functional characteristics of variants and facilitates prioritizing plausible causal variants influencing human phenotypes. Furthermore, we provide a scalable annotation tool, FAVORannotator, to functionally annotate large-scale WGS studies and efficiently store the genotype and their variant functional annotation data in a single file using the annotated Genomic Data Structure (aGDS) format, making downstream analysis more convenient. FAVOR and FAVORannotator are available at https://favor.genohub.org.
    DOI:  https://doi.org/10.1093/nar/gkac966
  41. Cell. 2022 Nov 02. pii: S0092-8674(22)01359-9. [Epub ahead of print]
    The phenotypic landscape of essential human genes.
    Luke Funk, Kuan-Chung Su, Jimmy Ly, David Feldman, Avtar Singh, Brittania Moodie, Paul C Blainey, Iain M Cheeseman.
      Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.
    Keywords:  CRISPR-Cas9; cell division; essential genes; functional genomics; high-content screening; in situ sequencing; microscopy; mitosis; morphology; optical pooled screening
    DOI:  https://doi.org/10.1016/j.cell.2022.10.017
  42. Methods Mol Biol. 2023 ;2590 219-235
    Phasing DNA Methylation.
    Vahid Akbari, Steven J M Jones.
      Haplotyping enables the study of allele-specific events. Heterozygous variants, primarily single nucleotide variants (SNVs), enable the assignment of the paternal and maternal origin of the chromosomes and are widely employed to phase sequencing reads to their haplotype of origin. Certain long-read technologies enable the detection of both the DNA sequence and DNA modifications. These long reads and their inherent methylation information are suitable for genome-wide haplotyping and allele-specific DNA methylation analysis. Here, we describe the workflow to phase reads and DNA methylation using nanopore sequencing.
    Keywords:  Allele-specific methylation; DNA methylation; Haplotyping; Long read; Phasing
    DOI:  https://doi.org/10.1007/978-1-0716-2819-5_14
  43. Brain. 2022 Nov 09. pii: awac421. [Epub ahead of print]
    Randomized trial of bilateral gene therapy injection for m.11778G > A MT-ND4 Leber optic neuropathy.
    LHON REFLECT Study Group
      Leber hereditary optic neuropathy (LHON) is an important example of mitochondrial blindness with the m.11778G > A mutation in the MT-ND4 gene being the most common disease-causing mitochondrial DNA (mtDNA) variant worldwide. The REFLECT phase 3 pivotal study is a randomized, double-masked, placebo-controlled trial investigating the efficacy and safety of bilateral intravitreal injection of lenadogene nolparvovec in patients with a confirmed m.11778G > A mutation, using a recombinant adeno-associated virus vector 2, serotype 2 (rAAV2/2-ND4). The first-affected eye received gene therapy; the fellow (affected/not-yet-affected) eye was randomly injected with gene therapy or placebo. The primary endpoint was the difference in change from baseline of best-corrected visual acuity (BCVA) in second-affected/not-yet-affected eyes treated with lenadogene nolparvovec versus placebo at 1.5 years post-treatment, expressed in logarithm of the minimal angle of resolution (LogMAR). Forty-eight patients were treated bilaterally and 50 unilaterally. At 1.5 years, the change from baseline in BCVA was not statistically different between second-affected/not-yet-affected eyes receiving lenadogene nolparvovec and placebo (primary endpoint). A statistically significant improvement in BCVA was reported from baseline to 1.5 years in lenadogene nolparvovec-treated eyes: -0.23 LogMAR for the first-affected eyes of bilaterally treated patients (p < 0.01); and -0.15 LogMAR for second-affected/not-yet-affected eyes of bilaterally treated patients and the first-affected eyes of unilaterally treated patients (p < 0.05). The mean improvement in BCVA from nadir to 1.5 years was -0.38 (0.052) LogMAR and -0.33 (0.052) LogMAR in first-affected and second-affected/not-yet-affected eyes treated with lenadogene nolparvovec, respectively (bilateral treatment group). A mean improvement of -0.33 (0.051) LogMAR and -0.26 (0.051) LogMAR was observed in first-affected lenadogene nolparvovec-treated eyes and second-affected/not-yet-affected placebo-treated eyes, respectively (unilateral treatment group). The proportion of patients with one or both eyes on-chart at 1.5 years was 85.4% and 72.0% for bilaterally and unilaterally treated patients, respectively. The gene therapy was well tolerated, with no systemic issues. Intraocular inflammation, which was mostly mild and well controlled with topical corticosteroids, occurred in 70.7% of lenadogene nolparvovec-treated eyes versus 10.2% of placebo-treated eyes. Among eyes treated with lenadogene nolparvovec, there was no difference in the incidence of intraocular inflammation between bilaterally and unilaterally treated patients. Overall, the REFLECT trial demonstrated an improvement of BCVA in LHON eyes carrying the m.11778G > A mtDNA mutation treated with lenadogene nolparvovec or placebo to a degree not reported in natural history studies and supports an improved benefit/risk profile for bilateral injections of lenadogene nolparvovec relative to unilateral injections.
    Keywords:  Leber hereditary optic neuropathy; NADH dehydrogenase 4; lenadogene nolparvovec; mitochondrial DNA; recombinant adeno-associated virus vector 2
    DOI:  https://doi.org/10.1093/brain/awac421
  44. Methods Mol Biol. 2023 ;2590 85-99
    Low-Cost Genome-Scale Phasing with Barcode-Linked Sequencing.
    David Redin.
      Complete comprehension of clinically relevant variation among human genomes is likely only to come from sequencing platforms that are cost-efficient, and which feature both accurate base calling and long-range DNA phasing capability. The NGS revolution has struggled to meet the latter of these needs. Here we describe a protocol to address this limitation by preserving the molecular origin of short sequencing reads with an insignificant increase to sequencing costs. Whole haplotype-resolved genomes with megabase-scale phase blocks can be obtained with this method; offering researchers a unique opportunity to tackle the hurdles of de novo sequencing without being limited by a lack of resources.
    Keywords:  Barcode-linked reads; DNA phasing; De novo sequencing; Haplotype-resolved; High throughput barcoding; Reference-free assembly; Whole genome haplotyping
    DOI:  https://doi.org/10.1007/978-1-0716-2819-5_6
  45. Biomedicines. 2022 Nov 10. pii: 2883. [Epub ahead of print]10(11):
    Mitochondrial Transplantation Moderately Ameliorates Retinal Degeneration in Royal College of Surgeons Rats.
    Shih-Fang Wu, Chih-Yao Lin, Rong-Kung Tsai, Yao-Tseng Wen, Feng-Huei Lin, Chia-Yu Chang, Ching-I Shen, Shinn-Zong Lin, Horng-Jyh Harn, Tzyy-Wen Chiou, Chin-San Liu, Yan-Ting Chen, Hong-Lin Su.
      Retinal pigmented epithelial (RPE) cells possess high mitochondria content for energy production, which is required for phagocytosis and vision cycle metabolism. The mitochondrial integrity in RPE cells helps the homeostasis of photoreceptor turnover and prevents retina aging and degeneration. Mitochondrial transplantation benefits the recovery of several acute inflammatory diseases, leading us to investigate the effects of mitochondrial transplantation on retina degeneration. Allogeneic mitochondria were isolated and delivered into the vitreous chamber in the Royal College of Surgeons (RCS) rats, which exhibit inherited and early-onset retina degeneration. The progress of retina degeneration was examined with optical coherence tomography (OCT) and visual evoked potential (VEP) to determine the retina thickness and integrity of afferent electrical signals from affected eyes, respectively. We found that mitochondria engraftment moderately attenuated the degeneration of retinal layers in RCS rats by histological examination. This result was consistent with the OCT measurement of retina thickness around the optic disc. The VEP analysis revealed that the peak one (N1) latency, representing the arriving time of electrical impulse from the retina to cortex, was substantially maintained as the normal value after the mitochondrial transplantation. This result suggests that the intra-vitreous transplanted mitochondria ameliorate the degeneration of photoreceptors in RCS rats and might be potential for clinical application.
    Keywords:  macular degeneration; mitochondrial transplantation; retinal degeneration; retinitis pigmentosa
    DOI:  https://doi.org/10.3390/biomedicines10112883
  46. Contact (Thousand Oaks). 2022 Jan-Dec;5:5 25152564221096217
    Nuclear Interactions: A Spotlight on Nuclear Mitochondrial Membrane Contact Sites.
    Jana Ovciarikova, Shikha Shikha, Lilach Sheiner.
      Membrane contact sites (MCS) are critical for cellular functions of eukaryotes, as they enable communication and exchange between organelles. Research over the last decade unravelled the function and composition of MCS between a variety of organelles including mitochondria, ER, plasma membrane, lysosomes, lipid droplets, peroxisome and endosome, to name a few. In fact, MCS are found between any pair of organelles studied to date, with common functions including lipid exchange, calcium signalling and organelle positioning in the cell. Work in the past year has started addressing the composition and function of nuclear-mitochondrial MCS. Tether components mediating these contacts in yeast have been identified via comprehensive phenotypic screens, which also revealed a possible link between this contact and phosphatidylcholine metabolism. In human cells, and in the protozoan parasites causing malaria, proximity between these organelles is proposed to promote cell survival via a mitochondrial retrograde response. These pioneering studies should inspire the field to explore what cellular processes depend on the exchange between the nucleus and the mitochondrion, given that they play such central roles in cell biology.
    Keywords:  membrane contact sites; mitochondrion (mitochondria); nucleus; parasite
    DOI:  https://doi.org/10.1177/25152564221096217
  47. Curr Biol. 2022 Nov 02. pii: S0960-9822(22)01673-6. [Epub ahead of print]
    Reduced mitochondria provide an essential function for the cytosolic methionine cycle.
    Justyna Zítek, Zoltán Füssy, Sebastian C Treitli, Priscila Peña-Diaz, Zuzana Vaitová, Daryna Zavadska, Karel Harant, Vladimír Hampl.
      The loss of mitochondria in oxymonad protists has been associated with the redirection of the essential Fe-S cluster assembly to the cytosol. Yet as our knowledge of diverse free-living protists broadens, the list of functions of their mitochondrial-related organelles (MROs) expands. We revealed another such function in the closest oxymonad relative, Paratrimastix pyriformis, after we solved the proteome of its MRO with high accuracy, using localization of organelle proteins by isotope tagging (LOPIT). The newly assigned enzymes connect to the glycine cleavage system (GCS) and produce folate derivatives with one-carbon units and formate. These are likely to be used by the cytosolic methionine cycle involved in S-adenosyl methionine recycling. The data provide consistency with the presence of the GCS in MROs of free-living species and its absence in most endobionts, which typically lose the methionine cycle and, in the case of oxymonads, the mitochondria.
    Keywords:  LOPIT; Paratrimastix; glycine cleavage system; methionine cycle; mitochondrion-related organelle; one-carbon metabolism; proteome; spatial proteomics
    DOI:  https://doi.org/10.1016/j.cub.2022.10.028
  48. Cell. 2022 Nov 04. pii: S0092-8674(22)01367-8. [Epub ahead of print]
    Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.
    Xiaonan Fu, Li Sun, Runze Dong, Jane Y Chen, Runglawan Silakit, Logan F Condon, Yiing Lin, Shin Lin, Richard D Palmiter, Liangcai Gu.
      Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.
    Keywords:  DNA array; DNA stamping; Pixel-seq; chronic pain; microcontact printing; olfactory bulb; parabrachial nucleus; polony gel; polony sequencing; spatial transcriptomics
    DOI:  https://doi.org/10.1016/j.cell.2022.10.021
  49. Elife. 2022 Nov 10. pii: e81033. [Epub ahead of print]11
    Endosymbiotic selective pressure at the origin of eukaryotic cell biology.
    Parth K Raval, Sriram G Garg, Sven B Gould.
      The dichotomy that separates prokaryotic from eukaryotic cells runs deep. The transition from pro- to eukaryote evolution is poorly understood due to a lack of reliable intermediate forms and definitions regarding the nature of the first host that could no longer be considered a prokaryote, the first eukaryotic common ancestor, FECA. The last eukaryotic common ancestor, LECA, was a complex cell that united all traits characterising eukaryotic biology including a mitochondrion. The role of the endosymbiotic organelle in this radical transition towards complex life forms is, however, sometimes questioned. In particular the discovery of the asgard archaea has stimulated discussions regarding the pre-endosymbiotic complexity of FECA. Here we review differences and similarities among models that view eukaryotic traits as isolated coincidental events in asgard archaeal evolution or, on the contrary, as a result of and in response to endosymbiosis. Inspecting eukaryotic traits from the perspective of the endosymbiont uncovers that eukaryotic cell biology can be explained as having evolved as a solution to housing a semi-autonomous organelle and why the addition of another endosymbiont, the plastid, added no extra compartments. Mitochondria provided the selective pressures for the origin (and continued maintenance) of eukaryotic cell complexity. Moreover, they also provided the energetic benefit throughout eukaryogenesis for evolving thousands of gene families unique to eukaryotes. Hence, a synthesis of the current data lets us conclude that traits such as the Golgi apparatus, the nucleus, autophagosomes, and meiosis and sex evolved as a response to the selective pressures an endosymbiont imposes.
    Keywords:  FECA; LECA; cell biology; endomembrane system; endosymbiosis; eukaryogenesis; mitochondria
    DOI:  https://doi.org/10.7554/eLife.81033
  50. Annu Rev Physiol. 2022 Nov 08.
    Fatty Acid Transport and Signaling: Mechanisms and Physiological Implications.
    Dmitri Samovski, Miriam Jacome-Sosa, Nada A Abumrad.
      Long-chain fatty acids (FAs) are components of plasma membranes and an efficient fuel source and also serve as metabolic regulators through FA signaling mediated by membrane FA receptors. Impaired tissue FA uptake has been linked to major complications of obesity, including insulin resistance, cardiovascular disease, and type 2 diabetes. Fatty acid interactions with a membrane receptor and the initiation of signaling can modify pathways related to nutrient uptake and processing, cell proliferation or differentiation, and secretion of bioactive factors. Here, we review the major membrane receptors involved in FA uptake and FA signaling. We focus on two types of membrane receptors for long-chain FAs: CD36 and the G protein-coupled FA receptors FFAR1 and FFAR4. We describe key signaling pathways and metabolic outcomes for CD36, FFAR1, and FFAR4 and highlight the parallels that provide insight into FA regulation of cell function. Expected final online publication date for the Annual Review of Physiology, Volume 85 is February 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-physiol-032122-030352
  51. Nucleic Acids Res. 2022 Nov 09. pii: gkac993. [Epub ahead of print]
    InterPro in 2022.
    Typhaine Paysan-Lafosse, Matthias Blum, Sara Chuguransky, Tiago Grego, Beatriz Lázaro Pinto, Gustavo A Salazar, Maxwell L Bileschi, Peer Bork, Alan Bridge, Lucy Colwell, Julian Gough, Daniel H Haft, Ivica Letunić, Aron Marchler-Bauer, Huaiyu Mi, Darren A Natale, Christine A Orengo, Arun P Pandurangan, Catherine Rivoire, Christian J A Sigrist, Ian Sillitoe, Narmada Thanki, Paul D Thomas, Silvio C E Tosatto, Cathy H Wu, Alex Bateman.
      The InterPro database (https://www.ebi.ac.uk/interpro/) provides an integrative classification of protein sequences into families, and identifies functionally important domains and conserved sites. Here, we report recent developments with InterPro (version 90.0) and its associated software, including updates to data content and to the website. These developments extend and enrich the information provided by InterPro, and provide a more user friendly access to the data. Additionally, we have worked on adding Pfam website features to the InterPro website, as the Pfam website will be retired in late 2022. We also show that InterPro's sequence coverage has kept pace with the growth of UniProtKB. Moreover, we report the development of a card game as a method of engaging the non-scientific community. Finally, we discuss the benefits and challenges brought by the use of artificial intelligence for protein structure prediction.
    DOI:  https://doi.org/10.1093/nar/gkac993
  52. Nat Med. 2022 Nov 10.
    Rare and common genetic determinants of metabolic individuality and their effects on human health.
    Praveen Surendran, Isobel D Stewart, Victoria P W Au Yeung, Maik Pietzner, Johannes Raffler, Maria A Wörheide, Chen Li, Rebecca F Smith, Laura B L Wittemans, Lorenzo Bomba, Cristina Menni, Jonas Zierer, Niccolò Rossi, Patricia A Sheridan, Nicholas A Watkins, Massimo Mangino, Pirro G Hysi, Emanuele Di Angelantonio, Mario Falchi, Tim D Spector, Nicole Soranzo, Gregory A Michelotti, Wiebke Arlt, Luca A Lotta, Spiros Denaxas, Harry Hemingway, Eric R Gamazon, Joanna M M Howson, Angela M Wood, John Danesh, Nicholas J Wareham, Gabi Kastenmüller, Eric B Fauman, Karsten Suhre, Adam S Butterworth, Claudia Langenberg.
      Garrod's concept of 'chemical individuality' has contributed to comprehension of the molecular origins of human diseases. Untargeted high-throughput metabolomic technologies provide an in-depth snapshot of human metabolism at scale. We studied the genetic architecture of the human plasma metabolome using 913 metabolites assayed in 19,994 individuals and identified 2,599 variant-metabolite associations (P < 1.25 × 10-11) within 330 genomic regions, with rare variants (minor allele frequency ≤ 1%) explaining 9.4% of associations. Jointly modeling metabolites in each region, we identified 423 regional, co-regulated, variant-metabolite clusters called genetically influenced metabotypes. We assigned causal genes for 62.4% of these genetically influenced metabotypes, providing new insights into fundamental metabolite physiology and clinical relevance, including metabolite-guided discovery of potential adverse drug effects (DPYD and SRD5A2). We show strong enrichment of inborn errors of metabolism-causing genes, with examples of metabolite associations and clinical phenotypes of non-pathogenic variant carriers matching characteristics of the inborn errors of metabolism. Systematic, phenotypic follow-up of metabolite-specific genetic scores revealed multiple potential etiological relationships.
    DOI:  https://doi.org/10.1038/s41591-022-02046-0
  53. Elife. 2022 Nov 08. pii: e82206. [Epub ahead of print]11
    Connexin 43 hemichannels regulate mitochondrial ATP generation, mobilization, and mitochondrial homeostasis against oxidative stress.
    Jingruo Zhang, Manuel A Riquelme, Rui Hua, Francisca M Acosta, Sumin Gu, Jean X Jiang.
      Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. Here, we reported a new role of mitochondrial Cx43 (mtCx43) and hemichannels (HCs) in modulating mitochondria homeostasis and function in bone osteocytes under oxidative stress. In murine long bone osteocyte-Y4 cells, the translocation of Cx43 to mitochondria was increased under H2O2-induced oxidative stress. H2O2 increased the mtCx43 level accompanied by elevated mtCx43 HC activity, determined by dye uptake assay. Cx43 knockdown (KD) by the CRISPR-Cas9 lentivirus system resulted in impairment of mitochondrial function, primarily manifested as decreased ATP production. Cx43 KD had reduced intracellular reactive oxidative species levels and mitochondrial membrane potential. Additionally, live-cell imaging results demonstrated that the proton flux was dependent on mtCx43 HCs because its activity was specifically inhibited by an antibody targeting Cx43 C-terminus. The co-localization and interaction of mtCx43 and ATP synthase subunit F (ATP5J2) were confirmed by Förster resonance energy transfer and a protein pull-down assay. Together, our study suggests that mtCx43 HCs regulate mitochondrial ATP generation by mediating K+, H+, and ATP transfer across the mitochondrial inner membrane and the interaction with mitochondrial ATP synthase, contributing to the maintenance of mitochondrial redox levels in response to oxidative stress.
    Keywords:  ATP synthase; cell biology; connexin 43; mouse; osteocytes; oxidative stress; proton
    DOI:  https://doi.org/10.7554/eLife.82206
  54. Nat Cell Biol. 2022 Nov 10.
    Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer.
    Katie Cockburn, Karl Annusver, David G Gonzalez, Smirthy Ganesan, Dennis P May, Kailin R Mesa, Kyogo Kawaguchi, Maria Kasper, Valentina Greco.
      Highly regenerative tissues continuously produce terminally differentiated cells to replace those that are lost. How they orchestrate the complex transition from undifferentiated stem cells towards post-mitotic, molecularly distinct and often spatially segregated differentiated populations is not well understood. In the adult skin epidermis, the stem cell compartment contains molecularly heterogeneous subpopulations1-4 whose relationship to the complete trajectory of differentiation remains unknown. Here we show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes with upregulation of differentiation genes preceding downregulation of typical stemness genes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit-amplifying programme but help to buffer the differentiating cell pool during heightened demand. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover.
    DOI:  https://doi.org/10.1038/s41556-022-01021-8
  55. Methods Mol Biol. 2023 ;2590 101-125
    A Simple Cost-Effective Method for Whole-Genome Sequencing, Haplotyping, and Assembly.
    Ou Wang, Xiaofang Cheng, Radoje Drmanac, Brock A Peters.
      In this chapter, we describe single-tube long fragment read (stLFR), a simple preparation method for whole-genome sequencing and physical haplotyping based on the DNA co-barcoding strategy. Similar to LFR, stLFR applies the concept of adding the same barcode to subfragments derived from the same long DNA molecule. However, instead of a 384-well plate, stLFR uses the surface of micron-sized magnetic beads to create millions of virtual compartments in a single reaction tube. This is enabled by a split and pool barcoded bead preparation process capable of generating ~500,000 copies of the same unique barcode, from a library of ~3.6 billion unique barcodes, on each bead. The instruments and devices used in the stLFR process are easily accessible in nearly all standard molecular biology laboratories, and the cost of reagents can be as low as 30 dollars per sample. stLFR libraries can be sequenced by standard second-generation sequencing instruments (e.g., MGI or Illumina devices), and the barcode sharing information enables detection and phasing of all variations, including large structural variations. In addition, stLFR data can be used to scaffold contigs and de novo assemble genomes.
    Keywords:  Co-barcoding; Combinatorial barcode beads; Diploid de novo assembly; Experimental haplotyping; Long DNA molecules; Phasing; Whole-genome sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-2819-5_7
  56. Mol Syst Biol. 2022 Nov;18(11): e10768
    Pooled genetic screens with image-based profiling.
    Russell T Walton, Avtar Singh, Paul C Blainey.
      Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to show how biological systems work in detail by cataloging many cellular phenotypes with one experimental assay. Microscopy-based cellular profiling provides information complementary to next-generation sequencing (NGS) profiling and has only recently become compatible with large-scale genetic screens. Optical screening now offers the scale needed for systematic characterization and is poised for further scale-up. We discuss how these methodologies, together with emerging technologies for genetic perturbation and microscopy-based multiplexed molecular phenotyping, are powering new approaches to reveal genotype-phenotype relationships.
    Keywords:  morphological profiling; multiplexed imaging; optical microscopy profiling; optical screening; pooled genetic screening
    DOI:  https://doi.org/10.15252/msb.202110768
  57. Front Endocrinol (Lausanne). 2022 ;13 967848
    Peripheral blood mononuclear cell mitochondrial copy number and adenosine triphosphate inhibition test in NAFLD.
    A-Hyeon Lee, Ju Hee Oh, Hyun Sung Kim, Jeong-Hun Shin, Eileen L Yoon, Dae Won Jun.
      Background and aim: Non-alcoholic fatty liver disease (NAFLD) is associated with mitochondrial dysfunction. This study aims to develop biomarkers for assessing mitochondrial dysfunction in patients with NAFLD.Methods: Mitochondrion-associated transcriptome analysis was performed. Peripheral blood mononuclear cells obtained from patients with NAFLD (69) and healthy controls (19) were used to determine the mitochondrial DNA (mtDNA) copy number. A mitochondrial inhibition substrate test (ATP assay) was performed in HepG2 cells using the patient serum.
    Results: Hepatic mRNA transcriptome analysis showed that the gene expression related to mitochondrial functions (mitochondrial fusion, apoptotic signal, and mitochondrial envelope) increased in patients with steatohepatitis, but not in those with NAFL. Gene set enrichment analysis revealed that the upregulated expression of genes is related to the pathways of the tricarboxylic (TCA) cycle and deoxyribonucleic acid (DNA) replication in patients with steatohepatitis, but not in healthy controls. The mtDNA copy number in the peripheral blood mononuclear cells was 1.28-fold lower in patients with NAFLD than that in healthy controls (P <.0001). The mitochondrial inhibition substrate test showed that the cellular adenosine triphosphate (ATP) concentration was 1.2-fold times less in NAFLD patients than that in healthy controls (P <.0001). The mtDNA copy number and mitochondrial ATP inhibition substrate test demonstrated negative correlations with the degree of hepatic steatosis, whereas the ATP concentration showed a positive correlation with the mtDNA copy number.
    Conclusion: The mitochondrial copy number of peripheral blood mononuclear cells and mitochondrial ATP inhibition substrate can be used as biomarkers for assessing the mitochondrial dysfunction in patients with NAFLD.
    Keywords:  ATP; NAFLD; biomarker; mitochondria copy number; mitochondrial dysfunction
    DOI:  https://doi.org/10.3389/fendo.2022.967848
  58. Clin Genet. 2022 Nov 10.
    Bridging clinical care and research in Ontario, Canada: Maximizing diagnoses from reanalysis of clinical exome sequencing data.
    Taila Hartley, Élisabeth Soubry, Meryl Acker, Matthew Osmond, Madeline Couse, Meredith K Gillespie, Yoko Ito, Aren E Marshall, Gabrielle Lemire, Lijia Huang, Caitlin Chisholm, Alison J Eaton, E Magda Price, James J Dowling, Arun K Ramani, Roberto Mendoza-Londono, Gregory Costain, Michelle M Axford, Anna Szuto, Vanda McNiven, Nadirah Damseh, Rebekah Jobling, Leanne de Kock, Bahareh A Mojarad, Ted Young, Zhuo Shao, Robin Hayeems, Ian Graham, Mark Tarnopolsky, Lauren Brady, Christine M Armour, Michael Geraghty, Julie Richer, Sarah Sawyer, Matthew Lines, Saadet Mercimek-Andrews, Melissa T Carter, Gail Graham, Peter Kannu, Joanna Lazier, Chumei Li, Ritu B Aul, Tugce B Balci, Nomazulu Dlamini, Lauren Badalato, Andrea Guerin, Jagdeep Walia, David Chitayat, Ronald Cohn, Hanna Faghfoury, Cynthia Forster-Gibson, Hernan Gonorazky, Eyal Grunebaum, Michal Inbar-Feigenberg, Natalya Karp, Chantal Morel, Alison Rusnak, Neal Sondheimer, Jodi Warman-Chardon, Priya T Bhola, Danielle K Bourque, Inara J Chacon, Lauren Chad, Pranesh Chakraborty, Karen Chong, Asif Doja, Elaine Suk-Ying Goh, Maha Saleh, Beth Potter, Christian R Marshall, David A Dyment, Kristin Kernohan, Kym M Boycott.
      We examined the utility of clinical and research processes in the reanalysis of publicly-funded clinical exome sequencing data in Ontario, Canada. In partnership with 8 sites, we recruited 287 families with suspected rare genetic diseases tested between 2014 and 2020. Data from 7 laboratories was reanalyzed with the referring clinicians. Reanalysis of clinically relevant genes identified diagnoses in 4% (13/287); 4 were missed by clinical testing. Translational research methods, including analysis of novel candidate genes, identified candidates in 21% (61/287). Of these, 24 families have additional evidence through data sharing to support likely diagnoses (8% of cohort). This study indicates few diagnoses are missed by clinical laboratories, the incremental gain from reanalysis of clinically-relevant genes is modest, and the highest yield comes from validation of novel disease-gene associations. Future implementation of translational research methods, including continued reporting of compelling genes of uncertain significance by clinical laboratories, should be considered to maximize diagnoses. This article is protected by copyright. All rights reserved.
    Keywords:  Reanalysis; exome sequencing; healthcare system; matchmaking; rare disease
    DOI:  https://doi.org/10.1111/cge.14262
  59. JIMD Rep. 2022 Nov;63(6): 524-528
    Primary carnitine deficiency is a life-long disease.
    Loek L Crefcoeur, Mireille C Melles, Tobias A Bruning, Rob Rodrigues Pereira, Janneke G Langendonk.
      Primary carnitine deficiency is a rare autosomal recessive disease associated with acute hypoketotic hypoglycaemia, cardiomyopathy and sudden cardiac death. Effective treatment with carnitine supplementation is available. An 18 months old boy, who presented with cardiomyopathy was diagnosed with primary carnitine deficiency, and carnitine supplementation resulted in a full recovery. At age 13 years, he discontinued his medication and at 20 years, he discontinued clinical monitoring. Nine years later, age 29, he presented with heart failure and atrial fibrillation and was admitted to an intensive care unit, where he was treated with furosemide, enoximone and intravenous carnitine supplementation, this lead to improved cardiac function within 2 weeks, and with continued oral carnitine supplements, his left ventricular ejection fraction normalised. The last 8 years were uneventful and he continued to attend his regular follow-up visits at a specialised metabolic outpatient clinic. We report recurrent reversible severe heart failure in a patient with primary carnitine deficiency; it was directly related to non-compliance to carnitine supplementation (and monitoring). This case report emphasises first, the importance of continued monitoring of metabolic disease patients, second, the potential reversibility of cardiomyopathy in an adult patient, and third, the potential risks in the period of transition from the paediatric to adult care. This is an age where young adults desire to be healthy and ignore the need for ongoing medical treatment, even as simple as oral suppletion. Before they reach this age, adequate disease insight and self-management of the disease should be promoted.
    Keywords:  OCTN2 deficiency; cardiomyopathy; case report; heart failure; primary carnitine deficiency
    DOI:  https://doi.org/10.1002/jmd2.12319
  60. Nat Commun. 2022 Nov 05. 13(1): 6683
    Amino acid variability, tradeoffs and optimality in human diet.
    Ziwei Dai, Weiyan Zheng, Jason W Locasale.
      Studies at the molecular level demonstrate that dietary amino acid intake produces substantial effects on health and disease by modulating metabolism. However, how these effects may manifest in human food consumption and dietary patterns is unknown. Here, we develop a series of algorithms to map, characterize and model the landscape of amino acid content in human food, dietary patterns, and individual consumption including relations to health status, covering over 2,000 foods, ten dietary patterns, and over 30,000 dietary profiles. We find that the type of amino acids contained in foods and human consumption is highly dynamic with variability far exceeding that of fat and carbohydrate. Some amino acids positively associate with conditions such as obesity while others contained in the same food negatively link to disease. Using linear programming and machine learning, we show that these health trade-offs can be accounted for to satisfy biochemical constraints in food and human eating patterns to construct a Pareto front in dietary practice, a means of achieving optimality in the face of trade-offs that are commonly considered in economic and evolutionary theories. Thus this study may enable the design of human protein quality intake guidelines based on a quantitative framework.
    DOI:  https://doi.org/10.1038/s41467-022-34486-0
  61. Cells. 2022 Nov 05. pii: 3509. [Epub ahead of print]11(21):
    Integration of O-GlcNAc into Stress Response Pathways.
    Kamau M M Fahie, Kyriakos N Papanicolaou, Natasha E Zachara.
      The modification of nuclear, mitochondrial, and cytosolic proteins by O-linked βN-acetylglucosamine (O-GlcNAc) has emerged as a dynamic and essential post-translational modification of mammalian proteins. O-GlcNAc is cycled on and off over 5000 proteins in response to diverse stimuli impacting protein function and, in turn, epigenetics and transcription, translation and proteostasis, metabolism, cell structure, and signal transduction. Environmental and physiological injury lead to complex changes in O-GlcNAcylation that impact cell and tissue survival in models of heat shock, osmotic stress, oxidative stress, and hypoxia/reoxygenation injury, as well as ischemic reperfusion injury. Numerous mechanisms that appear to underpin O-GlcNAc-mediated survival include changes in chaperone levels, impacts on the unfolded protein response and integrated stress response, improvements in mitochondrial function, and reduced protein aggregation. Here, we discuss the points at which O-GlcNAc is integrated into the cellular stress response, focusing on the roles it plays in the cardiovascular system and in neurodegeneration.
    Keywords:  O-GlcNAc; chaperone; glycosylation; stress
    DOI:  https://doi.org/10.3390/cells11213509
  62. iScience. 2022 Nov 18. 25(11): 105341
    Processes in DNA damage response from a whole-cell multi-omics perspective.
    James C Pino, Alexander L R Lubbock, Leonard A Harris, Danielle B Gutierrez, Melissa A Farrow, Nicole Muszynski, Tina Tsui, Stacy D Sherrod, Jeremy L Norris, John A McLean, Richard M Caprioli, John P Wikswo, Carlos F Lopez.
      Technological advances have made it feasible to collect multi-condition multi-omic time courses of cellular response to perturbation, but the complexity of these datasets impedes discovery due to challenges in data management, analysis, visualization, and interpretation. Here, we report a whole-cell mechanistic analysis of HL-60 cellular response to bendamustine. We integrate both enrichment and network analysis to show the progression of DNA damage and programmed cell death over time in molecular, pathway, and process-level detail using an interactive analysis framework for multi-omics data. Our framework, Mechanism of Action Generator Involving Network analysis (MAGINE), automates network construction and enrichment analysis across multiple samples and platforms, which can be integrated into our annotated gene-set network to combine the strengths of networks and ontology-driven analysis. Taken together, our work demonstrates how multi-omics integration can be used to explore signaling processes at various resolutions and demonstrates multi-pathway involvement beyond the canonical bendamustine mechanism.
    Keywords:  Bioinformatics; Complex system biology; Data processing in systems biology; Systems biology
    DOI:  https://doi.org/10.1016/j.isci.2022.105341
  63. Nat Rev Genet. 2022 Nov 07.
    Prime editing for precise and highly versatile genome manipulation.
    Peter J Chen, David R Liu.
      Programmable gene-editing tools have transformed the life sciences and have shown potential for the treatment of genetic disease. Among the CRISPR-Cas technologies that can currently make targeted DNA changes in mammalian cells, prime editors offer an unusual combination of versatility, specificity and precision. Prime editors do not require double-strand DNA breaks and can make virtually any substitution, small insertion and small deletion within the DNA of living cells. Prime editing minimally requires a programmable nickase fused to a polymerase enzyme, and an extended guide RNA that both specifies the target site and templates the desired genome edit. In this Review, we summarize prime editing strategies to generate programmed genomic changes, highlight their limitations and recent developments that circumvent some of these bottlenecks, and discuss applications and future directions.
    DOI:  https://doi.org/10.1038/s41576-022-00541-1
  64. Nat Cell Biol. 2022 Nov 10.
    Splitting up differentiation and cell cycle exit.
    Albert Herms, Philip H Jones.
      
    DOI:  https://doi.org/10.1038/s41556-022-01022-7
  65. Int J Mol Sci. 2022 Nov 03. pii: 13476. [Epub ahead of print]23(21):
    Quantitative Proteomics Analysis Reveals That Cyclooxygenase-2 Modulates Mitochondrial Respiratory Chain Complex IV in Cardiomyocytes.
    Maria Soledad Alvarez, Estefanía Núñez, Marina Fuertes-Agudo, Carme Cucarella, Maria Fernandez-Velasco, Lisardo Boscá, Jesús Vázquez, Rodrigue Rossignol, Paloma Martin-Sanz, Marta Casado.
      The biochemical mechanisms of cell injury and myocardial cell death after myocardial infarction remain unresolved. Cyclooxygenase 2 (COX-2), a key enzyme in prostanoid synthesis, is expressed in human ischemic myocardium and dilated cardiomyopathy, but it is absent in healthy hearts. To assess the role of COX-2 in cardiovascular physiopathology, we developed transgenic mice that constitutively express functional human COX-2 in cardiomyocytes under the control of the α-myosin heavy chain promoter. These animals had no apparent phenotype but were protected against ischemia-reperfusion injury in isolated hearts, with enhanced functional recovery and diminished cellular necrosis. To further explore the phenotype of this animal model, we carried out a differential proteome analysis of wild-type vs. transgenic cardiomyocytes. The results revealed a tissue-specific proteomic profile dominated by mitochondrial proteins. In particular, an increased expression of respiratory chain complex IV proteins was observed. This correlated with increased catalytic activity, enhanced respiratory capacity, and increased ATP levels in the heart of COX-2 transgenic mice. These data suggest a new link between COX-2 and mitochondria, which might contribute to the protective cardiac effects of COX-2 against ischemia-reperfusion injury.
    Keywords:  COX-2; mitochondria; prostaglandins; respiratory capacity; transgenic animals
    DOI:  https://doi.org/10.3390/ijms232113476
  66. Circ Res. 2022 Nov 11.
    RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function.
    Alexander A Akerberg, Michael Trembley, Vincent Butty, Asya Schwertner, Long Zhao, Manu Beerens, Xujie Liu, Mohammed Mahamdeh, Shiaulou Yuan, Laurie Boyer, Calum MacRae, Christopher Nguyen, William T Pu, Caroline E Burns, C Geoffrey Burns.
      BACKGROUND: RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. Methods: We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells.RESULTS: We identified 1848 genes enriched in nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3.
    CONCLUSIONS: Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.
    Keywords:  RNA-binding proteins; alternative splicing; induced pluripotent stem cell; myocytes, cardiac; zebrafish
    DOI:  https://doi.org/10.1161/CIRCRESAHA.122.321728
  67. J Biol Chem. 2022 Nov 03. pii: S0021-9258(22)01116-4. [Epub ahead of print] 102673
    Brain-Derived Neurotrophic Factor stimulates the retrograde pathway for axonal autophagy.
    David Kader Sidibe, Vineet Vinay Kulkarni, Audrey Dong, Jessica Brandt Herr, Maria Chalokh Vogel, Max Henry Stempel, Sandra Maday.
      Autophagy is a lysosomal degradative pathway important for neuronal development, function, and survival. How autophagy in axons is regulated by neurotrophins to impact neuronal viability and function is poorly understood. Here, we use live-cell imaging in primary neurons to investigate the regulation of axonal autophagy by the neurotrophin Brain-Derived Neurotrophic Factor (BDNF), and elucidate whether autophagosomes carry BDNF-mediated signaling information. We find that BDNF induces autophagic flux in primary neurons by stimulating the retrograde pathway for autophagy in axons. We observed an increase in autophagosome density and retrograde flux in axons, and a corresponding increase in autophagosome density in the soma. However, we find little evidence of autophagosomes co-migrating with BDNF. In contrast, BDNF effectively engages its cognate receptor TrkB to undergo retrograde transport in the axon. These compartments, however, are distinct from LC3-positive autophagic organelles in the axon. Together, we find that BDNF stimulates autophagy in the axon, but retrograde autophagosomes do not appear to carry BDNF cargo. Thus, autophagosomes likely do not play a major role in relaying neurotrophic signaling information across the axon in the form of active BDNF/TrkB complexes. Rather, BDNF likely stimulates autophagy as a consequence of BDNF-induced processes that require canonical roles for autophagy in degradation.
    Keywords:  Autophagy; Axon; BDNF; Neurons; Neurotrophin
    DOI:  https://doi.org/10.1016/j.jbc.2022.102673
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