bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022–05–08
67 papers selected by
Catalina Vasilescu, University of Helsinki
  1. Novel NARS2 variant causing leigh syndrome with normal lactate levels.
  2. An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines.
  3. Regulation of mitochondrial network homeostasis by O-GlcNAcylation.
  4. The role of mitochondrial fission in cardiovascular health and disease.
  5. HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation.
  6. High-content high-throughput imaging reveals distinct connections between mitochondrial morphology and functionality for OXPHOS complex I, III, and V inhibitors.
  7. Prospects of mitochondrial transplantation in clinical medicine: aspirations and challenges.
  8. Pantothenate kinase 2 interacts with PINK1 to regulate mitochondrial quality control via acetyl-CoA metabolism.
  9. A genome on shaky ground: exploring the impact of mitochondrial DNA integrity on Parkinson's disease by highlighting the use of cybrid models.
  10. Extracellular Flux Analysis of Mitochondrial Function in Pluripotent Stem Cells.
  11. IFNγ Regulates NAD+ Metabolism to Promote the Respiratory Burst in Human Monocytes.
  12. Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.
  13. Differential mitochondrial protein interaction profile between human translocator protein and its A147T polymorphism variant.
  14. Mechanistic insights into multiple-step transport of mitochondrial ADP/ATP carrier.
  15. Novel roles of RTN4 and CLIMP-63 in regulating mitochondrial structure, bioenergetics and apoptosis.
  16. The activator/repressor Hap1 binds to the yeast eIF5A-encoding gene TIF51A to adapt its expression to the mitochondrial functional status.
  17. Protein import in mitochondria biogenesis: guided by targeting signals and sustained by dedicated chaperones.
  18. How to proceed after "negative" exome: a review on genetic diagnostics, limitations, challenges and emerging new multi-omics techniques.
  19. Photoreceptor Manifestations of Primary Mitochondrial Optic Nerve Disorders.
  20. Transient absorption spectroscopy and imaging of redox in muscle mitochondria.
  21. Circulating FGF21 and GDF15 as Biomarkers for Screening, Diagnosis, and Severity Assessment of Primary Mitochondrial Disorders in Children.
  22. Mitochondrial genome variations, mitochondrial-nuclear compatibility, and their association with metabolic diseases.
  23. Kearns-Sayre Syndrome Minus: Two Cases of Identical Large-Scale Mitochondrial DNA Deletions with Presentations outside the Classical Triad.
  24. An explanation for the decreased severity of liver malfunction in Niemann-Pick C1 disease with age.
  25. Regulation of Kidney Mitochondrial Function by Caloric Restriction.
  26. The mitoXplorer 2.0 update: integrating and interpreting mitochondrial expression dynamics within a cellular context.
  27. Influence of environmental factors and genetic variation on mitochondrial DNA copy number.
  28. Focus on Molecular Functions of Anti-Aging Deacetylase SIRT3.
  29. Comment on "Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD".
  30. Coping with starvation: Cysteine keeps mTORC1 suppressed to ensure survival.
  31. Protocol for enhanced proliferation of human pluripotent stem cells in tryptophan-fortified media.
  32. The functional role of OGDH for maintaining mitochondrial respiration and identity of primed human embryonic stem cells.
  33. Further description of two patients with biallelic variants in NADSYN1 in association with cardiac and vertebral anomalies.
  34. Differentiation of Human Induced Pluripotent Stem Cells into Cortical Neurons to Advance Precision Medicine.
  35. Diagnosis and Discovery: Insights from the NIH Undiagnosed Diseases Program.
  36. Loss of mitochondrial ATPase ATAD3A contributes to non-alcoholic fatty liver disease through accumulation of lipids and damaged mitochondria.
  37. Diet, Trimethylamine Metabolism and Mitochondrial DNA: An Observational Study.
  38. Purification of cardiomyocytes and neurons derived from human pluripotent stem cells by inhibition of de novo fatty acid synthesis.
  39. An integrated toolbox to profile macrophage immunometabolism.
  40. Purification of Ribosome-Nascent-Chain Complex for Ribosome Profiling and Selective Ribosome Profiling.
  41. Hepatocyte Mitochondrial Dynamics and Bioenergetics in Obesity-Related Non-Alcoholic Fatty Liver Disease.
  42. Integration of Protein Structure and Population-Scale DNA Sequence Data for Disease Gene Discovery and Variant Interpretation.
  43. Structural basis for feedforward control in the PINK1/Parkin pathway.
  44. Stress Responses Elicited by Misfolded Proteins Targeted to Mitochondria.
  45. The role of mitochondria in the pathophysiology and treatment of common metabolic diseases in humans.
  46. Capturing the pattern of transition from carrier to affected in Leber's hereditary optic neuropathy.
  47. Differentiation of iPS-Cells into Peripheral Sensory Neurons.
  48. Culture of Human iPSC-Derived Motoneurons in Compartmentalized Microfluidic Devices and Quantitative Assays for Studying Axonal Phenotypes.
  49. How to ensure the Human Cell Atlas benefits humanity.
  50. MFN2 Deficiency Impairs Mitochondrial Functions and PPAR Pathway During Spermatogenesis and Meiosis in Mice.
  51. Detection of Soluble and Insoluble Protein Species in Patient-Derived iPSCs.
  52. Direct Sequencing of RNA and RNA Modification Identification Using Nanopore.
  53. Investigation of apoptosis based on fluorescence lifetime imaging microscopy with a mitochondria-targeted viscosity probe.
  54. Controlling Batch Effect in Epigenome-Wide Association Study.
  55. Letting go of damaged mitochondria.
  56. Mechanisms of autophagy and mitophagy in skeletal development, diseases and therapeutics.
  57. The sex-specific role of sensory neuron LKB1 on metabolic stress-induced mechanical hypersensitivity and mitochondrial respiration.
  58. Glycine decarboxylase maintains mitochondrial protein lipoylation to support tumor growth.
  59. DIALOGUE maps multicellular programs in tissue from single-cell or spatial transcriptomics data.
  60. Melatonin: A mitochondrial resident with a diverse skill set.
  61. Dynamic O-GlcNAcylation coordinates ferritinophagy and mitophagy to activate ferroptosis.
  62. Mitochondria-derived peptides in aging and healthspan.
  63. In Vitro Generation of Heart Field Specific Cardiomyocytes.
  64. Preventing erosion of X-chromosome inactivation in human embryonic stem cells.
  65. Making sense of the ageing methylome.
  66. Cell Type-Specific Signal Analysis in Epigenome-Wide Association Studies.
  67. FORK-seq: Single-Molecule Profiling of DNA Replication.
  1. Hum Genome Var. 2022 May 04. 9(1): 12
    Novel NARS2 variant causing leigh syndrome with normal lactate levels.
    Ryosuke Tanaka, Ryo Takeguchi, Mami Kuroda, Nao Suzuki, Yoshio Makita, Kumiko Yanagi, Tadashi Kaname, Satoru Takahashi.
      Leigh syndrome is the most genetically heterogenous phenotype of mitochondrial disease. We describe a patient with Leigh syndrome whose diagnosis had not been confirmed because of normal metabolic screening results at the initial presentation. Whole-exome sequencing identified pathogenic variants in NARS2, the gene encoding a mitochondrial asparaginyl-tRNA synthetase. One of the biallelic variants was novel. This highlights the essential role of genetic testing for a definite diagnosis of Leigh syndrome.
    DOI:  https://doi.org/10.1038/s41439-022-00191-z
  2. STAR Protoc. 2022 Jun 17. 3(2): 101288
    An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines.
    Bibekananda Kar, Ankit Sabharwal, Santiago Restrepo-Castillo, Brandon W Simone, Karl J Clark, Stephen C Ekker.
      The FusX TALE Based Editor (FusXTBE) is a programmable base editing platform that can introduce specific TC-to-TT variations in the mitochondrial DNA (mtDNA). Here, we provide a protocol describing the synthesis and testing of the FusXTBE plasmids in cultured human cell lines. This tool is designed to be easily modified to work in diverse applications where editing of mitochondrial DNA is desired. For complete details on the use and execution of this protocol, please refer to Sabharwal et al. (2021) and Ma et al. (2016).
    Keywords:  CRISPR; Cell Biology; Genetics; Molecular Biology; Sequencing
    DOI:  https://doi.org/10.1016/j.xpro.2022.101288
  3. Mitochondrion. 2022 May 02. pii: S1567-7249(22)00041-1. [Epub ahead of print]
    Regulation of mitochondrial network homeostasis by O-GlcNAcylation.
    Qiu Xue, Ru Yan, Shengtao Ji, Shu Yu.
      O-GlcNAcylation, a ubiquitous post-translational modification, rapidly modulates protein activity through the reversible addition and removal of O-GlcNAc groups from serine or threonine residues in target proteins, and is involved in multiple metabolic pathways. With the discovery of enzymes and substrates for O-GlcNAc cycling in mitochondria, mitochondrial O-GlcNAc modification and its regulatory role in mitochondrial function deserve extensive attention. Adaptive regulation of the O-GlcNAc cycling in response to energy perturbations is demonstrated to be important in maintaining mitochondrial homeostasis. Dysregulation of O-GlcNAcylation in mitochondria has been associated with various mitochondrial dysfunctions, such as abnormal mitochondrial dynamics, reduced mitochondrial biosynthesis, disruption of the electron transport chain, oxidative stress and the calcium paradox, as well as activation of mitochondrial apoptosis pathways. Here, we outline the current understanding of O-GlcNAc modification in mitochondria and the key discovery of O-GlcNAcylation in regulating mitochondrial network homeostasis. This review will provide insights into targeting mitochondrial O-GlcNAcylation, as well as the mechanisms linking mitochondrial dysfunction and disease.
    Keywords:  Cellular bioenergetics; Metabolism; Mitochondrial homeostasis; Nutrient sensing; O-GlcNAcylation
    DOI:  https://doi.org/10.1016/j.mito.2022.04.007
  4. Nat Rev Cardiol. 2022 May 06.
    The role of mitochondrial fission in cardiovascular health and disease.
    Justin M Quiles, Åsa B Gustafsson.
      Mitochondria are organelles involved in the regulation of various important cellular processes, ranging from ATP generation to immune activation. A healthy mitochondrial network is essential for cardiovascular function and adaptation to pathological stressors. Mitochondria undergo fission or fusion in response to various environmental cues, and these dynamic changes are vital for mitochondrial function and health. In particular, mitochondrial fission is closely coordinated with the cell cycle and is linked to changes in mitochondrial respiration and membrane permeability. Another key function of fission is the segregation of damaged mitochondrial components for degradation by mitochondrial autophagy (mitophagy). Mitochondrial fission is induced by the large GTPase dynamin-related protein 1 (DRP1) and is subject to sophisticated regulation. Activation requires various post-translational modifications of DRP1, actin polymerization and the involvement of other organelles such as the endoplasmic reticulum, Golgi apparatus and lysosomes. A decrease in mitochondrial fusion can also shift the balance towards mitochondrial fission. Although mitochondrial fission is necessary for cellular homeostasis, this process is often aberrantly activated in cardiovascular disease. Indeed, strong evidence exists that abnormal mitochondrial fission directly contributes to disease development. In this Review, we compare the physiological and pathophysiological roles of mitochondrial fission and discuss the therapeutic potential of preventing excessive mitochondrial fission in the heart and vasculature.
    DOI:  https://doi.org/10.1038/s41569-022-00703-y
  5. J Clin Invest. 2022 May 02. pii: e153153. [Epub ahead of print]132(9):
    HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation.
    Yanxin Fan, Marta Murgia, Monika I Linder, Yoko Mizoguchi, Cong Wang, Marcin Łyszkiewicz, Natalia Ziȩtara, Yanshan Liu, Stephanie Frenz, Gabriela Sciuccati, Armando Partida-Gaytan, Zahra Alizadeh, Nima Rezaei, Peter Rehling, Sven Dennerlein, Matthias Mann, Christoph Klein.
      The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1-associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1-/- cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.
    Keywords:  Cell Biology; Immunology; Mitochondria; Neutrophils
    DOI:  https://doi.org/10.1172/JCI153153
  6. Cell Biol Toxicol. 2022 May 04.
    High-content high-throughput imaging reveals distinct connections between mitochondrial morphology and functionality for OXPHOS complex I, III, and V inhibitors.
    Wanda van der Stel, Huan Yang, Sylvia E le Dévédec, Bob van de Water, Joost B Beltman, Erik H J Danen.
      Cells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation, changes in mitochondrial function, and altered mitochondrial morphology is not well understood. Here, we made use of high-throughput high-content confocal microscopy to assess the effects of distinct classes of oxidative phosphorylation (OXPHOS) complex inhibitors on mitochondrial parameters in a concentration and time resolved manner. Mitochondrial morphology phenotypes were clustered based on machine learning algorithms and mitochondrial integrity patterns were mapped. In parallel, changes in mitochondrial membrane potential (MMP), mitochondrial and cellular ATP levels, and viability were microscopically assessed. We found that inhibition of MMP, mitochondrial ATP production, and oxygen consumption rate (OCR) using sublethal concentrations of complex I and III inhibitors did not trigger mitochondrial fragmentation. Instead, complex V inhibitors that suppressed ATP and OCR but increased MMP provoked a more fragmented mitochondrial morphology. In agreement, complex V but not complex I or III inhibitors triggered proteolytic cleavage of the mitochondrial fusion protein, OPA1. The relation between increased MMP and fragmentation did not extend beyond OXPHOS complex inhibitors: increasing MMP by blocking the mPTP pore did not lead to OPA1 cleavage or mitochondrial fragmentation and the OXPHOS uncoupler FCCP was associated with OPA1 cleavage and MMP reduction. Altogether, our findings connect vital mitochondrial functions and phenotypes in a high-throughput high-content confocal microscopy approach that help understanding of chemical-induced toxicity caused by OXPHOS complex perturbing chemicals.
    Keywords:  ATP; Machine learning; Membrane potential; Mitochondria; Morphology
    DOI:  https://doi.org/10.1007/s10565-022-09712-6
  7. Mitochondrion. 2022 Apr 30. pii: S1567-7249(22)00040-X. [Epub ahead of print]
    Prospects of mitochondrial transplantation in clinical medicine: aspirations and challenges.
    Sina Hosseinian, Paria Ali Pour, Arash Kheradvar.
      Mitochondria, known as the powerhouse of the cell, are at the center of healthy physiology and provide cells with energy in the form of ATP. These unique organelles are also implicated in many pathological conditions affecting a variety of organs in various systems. Recently, mitochondrial transplantation, inspired by mitochondria's endosymbiotic origin, has been attempted as a potential biotherapy in mitigating a variety of pathological conditions. Mitochondrial transplantation consists of the process of isolation, transfer, and uptake of exogenous, intact mitochondria into damaged cells. Here, we discuss mitochondrial transplantation in the context of clinical medicine practiced in neurology, cardiology, pulmonary medicine, and oncology, among others. We outline the role of mitochondria in various pathologies and discuss the state-of-the-art research that potentially form the basis of new therapeutics for the treatment of a variety of diseases due to mitochondrial dysfunction. Lastly, we explore some of the challenges associated with mitochondrial transplantation that must be addressed before mitochondrial transplantation becomes a viable therapeutic option in clinical settings.
    Keywords:  Mitochondrial transplantation; clinical medicine; mitochondrial transfer; mitochondrial transplantation in medicine
    DOI:  https://doi.org/10.1016/j.mito.2022.04.006
  8. Nat Commun. 2022 May 03. 13(1): 2412
    Pantothenate kinase 2 interacts with PINK1 to regulate mitochondrial quality control via acetyl-CoA metabolism.
    Yunpeng Huang, Zhihui Wan, Yinglu Tang, Junxuan Xu, Bretton Laboret, Sree Nallamothu, Chenyu Yang, Boxiang Liu, Rongze Olivia Lu, Bingwei Lu, Juan Feng, Jing Cao, Susan Hayflick, Zhihao Wu, Bing Zhou.
      Human neurodegenerative disorders often exhibit similar pathologies, suggesting a shared aetiology. Key pathological features of Parkinson's disease (PD) are also observed in other neurodegenerative diseases. Pantothenate Kinase-Associated Neurodegeneration (PKAN) is caused by mutations in the human PANK2 gene, which catalyzes the initial step of de novo CoA synthesis. Here, we show that fumble (fbl), the human PANK2 homolog in Drosophila, interacts with PINK1 genetically. fbl and PINK1 mutants display similar mitochondrial abnormalities, and overexpression of mitochondrial Fbl rescues PINK1 loss-of-function (LOF) defects. Dietary vitamin B5 derivatives effectively rescue CoA/acetyl-CoA levels and mitochondrial function, reversing the PINK1 deficiency phenotype. Mechanistically, Fbl regulates Ref(2)P (p62/SQSTM1 homolog) by acetylation to promote mitophagy, whereas PINK1 regulates fbl translation by anchoring mRNA molecules to the outer mitochondrial membrane. In conclusion, Fbl (or PANK2) acts downstream of PINK1, regulating CoA/acetyl-CoA metabolism to promote mitophagy, uncovering a potential therapeutic intervention strategy in PD treatment.
    DOI:  https://doi.org/10.1038/s41467-022-30178-x
  9. Cell Mol Life Sci. 2022 May 05. 79(5): 283
    A genome on shaky ground: exploring the impact of mitochondrial DNA integrity on Parkinson's disease by highlighting the use of cybrid models.
    Martin Lang, Anne Grünewald, Peter P Pramstaller, Andrew A Hicks, Irene Pichler.
      Mitochondria play important roles in the regulation of key cellular processes, including energy metabolism, oxidative stress response, and signaling towards cell death or survival, and are distinguished by carrying their own genome (mtDNA). Mitochondrial dysfunction has emerged as a prominent cellular mechanism involved in neurodegeneration, including Parkinson's disease (PD), a neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons and the occurrence of proteinaceous Lewy body inclusions. The contribution of mtDNA variants to PD pathogenesis has long been debated and is still not clearly answered. Cytoplasmic hybrid (cybrid) cell models provided evidence for a contribution of mtDNA variants to the PD phenotype. However, conclusive evidence of mtDNA mutations as genetic cause of PD is still lacking. Several models have shown a role of somatic, rather than inherited mtDNA variants in the impairment of mitochondrial function and neurodegeneration. Accordingly, several nuclear genes driving inherited forms of PD are linked to mtDNA quality control mechanisms, and idiopathic as well as familial PD tissues present increased mtDNA damage. In this review, we highlight the use of cybrids in this PD research field and summarize various aspects of how and to what extent mtDNA variants may contribute to the etiology of PD.
    Keywords:  Cybrids; Mitochondria; Mitochondrial genome; Parkinson’s disease; mtDNA
    DOI:  https://doi.org/10.1007/s00018-022-04304-3
  10. Methods Mol Biol. 2022 ;2429 85-102
    Extracellular Flux Analysis of Mitochondrial Function in Pluripotent Stem Cells.
    Enkhtuul Tsogtbaatar, Katherine Minter-Dykhouse, Alicia Saarinen, Clifford D L Folmes.
      Mitochondrial function and energy metabolism are increasingly recognized not only as regulators of pluripotent stem cell function and fate, but also as critical targets in disease pathogenesis and aging. Therefore across the downstream applications of pluripotent stem cells, including development and disease modeling, drug screening, and cell-based therapies, it is crucial to be able to measure mitochondrial function and metabolism in a high-throughput, real-time and label-free manner. Here we describe the application of Seahorse extracellular flux analysis to measure mitochondrial function in pluripotent stem cells and their derivatives. Specifically, we highlight two assays, the Mitochondrial Stress Test, which quantifies overall mitochondrial function including basal, maximal and ATP-couple oxygen consumption rates, and the Electron Transport Chain Complex Specific assay, that quantifies function of individual complexes within the electron transport chain.
    Keywords:  Differentiation; Embryonic stem cells; Induced pluripotent stem cells; Mitochondrial respiration; Oxidative metabolism; Oxidative phosphorylation
    DOI:  https://doi.org/10.1007/978-1-0716-1979-7_7
  11. Blood Adv. 2022 May 02. pii: bloodadvances.2021005776. [Epub ahead of print]
    IFNγ Regulates NAD+ Metabolism to Promote the Respiratory Burst in Human Monocytes.
    Katelyn J McCann, Stephen M Christensen, Devon H Colby, Peter J McGuire, Ian A Myles, Christa S Zerbe, Clifton L Dalgard, Gauthaman Sukumar, Warren J Leonard, Beth A McCormick, Steven M Holland.
      IFNγ is an essential and pleiotropic activator of human monocytes, but little is known about the changes in cellular metabolism required for IFNγ-induced activation. We sought to elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. We found that IFNγ increased oxygen consumption rates (OCR) in monocytes, indicative of reactive oxygen species generation by both mitochondria and NADPH oxidase. Transcriptional profiling revealed that this oxidative phenotype was driven by IFNγ-induced reprogramming of NAD+ metabolism, which is dependent on nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. Consistent with this pathway, monocytes from patients with gain-of-function mutations in STAT1 demonstrated higher than normal OCR. Whereas chemical or genetic disruption of mitochondrial complex I (rotenone treatment or Leigh Syndrome patient monocytes) or NADPH oxidase (DPI treatment or chronic granulomatous disease (CGD) patient monocytes) reduced OCR. Interestingly, inhibition of NAMPT in healthy monocytes completely abrogated the IFNγ-induced oxygen consumption, comparable to levels observed in CGD monocytes. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for IFNγ activation of human monocytes.
    DOI:  https://doi.org/10.1182/bloodadvances.2021005776
  12. Nat Commun. 2022 May 05. 13(1): 2483
    Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.
    Xiaojian Shi, Bryn Reinstadler, Hardik Shah, Tsz-Leung To, Katie Byrne, Luanna Summer, Sarah E Calvo, Olga Goldberger, John G Doench, Vamsi K Mootha, Hongying Shen.
      The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states - glucose, galactose, OXPHOS inhibition, and absence of pyruvate - designed to unmask the inter-dependence of these genes. In total, we screen 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recover 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrates that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells is genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. GxG analysis highlights a buffering interaction between the iron transporter SLC25A37 (A37) and the poorly characterized SLC25A39 (A39). Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis identify A39 as critical for mitochondrial glutathione (GSH) import. Functional studies reveal that A39-mediated glutathione homeostasis and A37-mediated mitochondrial iron uptake operate jointly to support mitochondrial OXPHOS. Our work underscores the value of studying family-wide genetic interactions across different metabolic environments.
    DOI:  https://doi.org/10.1038/s41467-022-30126-9
  13. PLoS One. 2022 ;17(5): e0254296
    Differential mitochondrial protein interaction profile between human translocator protein and its A147T polymorphism variant.
    Prita R Asih, Anne Poljak, Michael Kassiou, Yazi D Ke, Lars M Ittner.
      The translocator protein (TSPO) has been implicated in mitochondrial transmembrane cholesterol transport, brain inflammation, and other mitochondrial functions. It is upregulated in glial cells during neuroinflammation in Alzheimer's disease. High affinity TSPO imaging radioligands are utilized to visualize neuroinflammation. However, this is hampered by the common A147T polymorphism which compromises ligand binding. Furthermore, this polymorphism has been linked to increased risk of neuropsychiatric disorders, and possibly reduces TSPO protein stability. Here, we used immunoprecipitation coupled to mass-spectrometry (IP-MS) to establish a mitochondrial protein binding profile of wild-type (WT) TSPO and the A147T polymorphism variant. Using mitochondria from human glial cells expressing either WT or A147T TSPO, we identified 30 WT TSPO binding partners, yet only 23 for A147T TSPO. Confirming that A147T polymorphism of the TSPO might confer loss of function, we found that one of the identified interactors of WT TSPO, 14-3-3 theta (YWHAQ), a protein involved in regulating mitochondrial membrane proteins, interacts much less with A147T TSPO. Our data presents a network of mitochondrial interactions of TSPO and its A147T polymorphism variant in human glial cells and indicate functional relevance of A147T in mitochondrial protein networks.
    DOI:  https://doi.org/10.1371/journal.pone.0254296
  14. Comput Struct Biotechnol J. 2022 ;20 1829-1840
    Mechanistic insights into multiple-step transport of mitochondrial ADP/ATP carrier.
    Shihao Yao, Qiuzi Yi, Boyuan Ma, Xiaoting Mao, Ye Chen, Min-Xin Guan, Xiaohui Cang.
      The ADP/ATP carrier (AAC) is crucial for mitochondrial functions by importing ADP and exporting ATP across the inner mitochondrial membrane. However, the mechanism of highly specific ADP recognition and transport by AAC remains largely elusive. In this work, spontaneous ADP binding process to the ground c-state AAC was investigated through rigorous molecular dynamics simulations of over 31 microseconds in total. With improved simulation strategy, we have successfully identified a highly specific ADP binding site in the upper region of the cavity, and this site exhibits selectivity for ADP over ATP based on free-energy calculations. Sequence analyses on adenine nucleotide transporters also suggest that this subgroup uses the upper region of the cavity, rather than the previously proposed central binding site located at the bottom of the cavity to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC and explains the dependence of transport on the flexibility between anti and syn glycosidic conformers of ADP. Moreover, this new site together with the central site supports early biochemical findings. In light of these early findings, our simulations described a multi-step model in which the carrier uses different sites for substrate attraction, recognition and conformational transition. These results provide new insights into the transport mechanism of AAC and other adenine nucleotide transporters.
    Keywords:  AAC, ADP/ADP carrier; ATP translocases; CATR, carboxyatractyloside; CoA, coenzyme A; GDC, Graves disease carrier protein, or SLC25A16; MCF, mitochondrial carrier family; MD simulation, molecular dynamics simulation PCA, Principal component analysis; Mitochondrial ADP; OXPHOS, oxidative phosphorylation; SCaMCs, short Ca2+-binding mitochondrial carrier, or Mg-ATP/Pi carrier; Solute carrier family 25, molecular dynamics simulation; Substrate recognition; Transporter; c-state, cytosol-open state; m-state, matrix-open state
    DOI:  https://doi.org/10.1016/j.csbj.2022.03.032
  15. Cell Death Dis. 2022 May 04. 13(5): 436
    Novel roles of RTN4 and CLIMP-63 in regulating mitochondrial structure, bioenergetics and apoptosis.
    Rachel J Carter, Mateus Milani, Alison J Beckett, Shiyu Liu, Ian A Prior, Gerald M Cohen, Shankar Varadarajan.
      The recruitment of DRP1 to mitochondrial membranes prior to fission is facilitated by the wrapping of endoplasmic reticulum (ER) membranes around the mitochondria. To investigate the complex interplay between the ER membranes and DRP1 in the context of mitochondrial structure and function, we downregulate two key ER shaping proteins, RTN4 and CLIMP-63, and demonstrate pronounced mitochondrial hyperfusion and reduced ER-mitochondria contacts, despite their differential regulation of ER architecture. Although mitochondrial recruitment of DRP1 is unaltered in cells lacking RTN4 or CLIMP-63, several aspects of mitochondrial function, such as mtDNA-encoded translation, respiratory capacity and apoptosis are significantly hampered. Further mechanistic studies reveal that CLIMP-63 is required for cristae remodeling (OPA1 proteolysis) and DRP1-mediated mitochondrial fission, whereas both RTN4 and CLIMP-63 regulate the recruitment of BAX to ER and mitochondrial membranes to enable cytochrome c release and apoptosis, thereby performing novel and distinct roles in the regulation of mitochondrial structure and function.
    DOI:  https://doi.org/10.1038/s41419-022-04869-8
  16. FEBS Lett. 2022 May 01.
    The activator/repressor Hap1 binds to the yeast eIF5A-encoding gene TIF51A to adapt its expression to the mitochondrial functional status.
    Marina Barba-Aliaga, Paula Alepuz.
      Mitochondrial activity adapts to cellular energetic and metabolic demands; its dysfunction is a hallmark of aging and many human diseases. The evolutionarily conserved translation elongation factor eIF5A is involved in maintaining mitochondrial function. In humans, eIF5A is encoded by two highly homologous but differentially expressed genes; in yeast, these are TIF51A and TIF51B. We show that yeast transcription factor Hap1 constitutively binds to the TIF51A promoter to activate its expression under respiration, but represses its expression under non-respiration conditions by recruiting the co-repressor Tup1. Hap1 indirectly regulates TIF51B expression by binding to and activating the TIF51B-repressor genes ROX1 and MOT3 under respiration and repressing them under non-respiration. Thus, the levels of eIF5A isoforms are adapted to the mitochondrial functional status.
    Keywords:   TIF51A ; TIF51B ; Hap1; Tup1; eIF5A; mitochondrial respiration; translation; yeast
    DOI:  https://doi.org/10.1002/1873-3468.14366
  17. RSC Adv. 2021 Sep 27. 11(51): 32476-32493
    Protein import in mitochondria biogenesis: guided by targeting signals and sustained by dedicated chaperones.
    Anna-Roza Dimogkioka, Jamie Lees, Erik Lacko, Kostas Tokatlidis.
      Mitochondria have a central role in cellular metabolism; they are responsible for the biosynthesis of amino acids, lipids, iron-sulphur clusters and regulate apoptosis. About 99% of mitochondrial proteins are encoded by nuclear genes, so the biogenesis of mitochondria heavily depends on protein import pathways into the organelle. An intricate system of well-studied import machinery facilitates the import of mitochondrial proteins. In addition, folding of the newly synthesized proteins takes place in a busy environment. A system of folding helper proteins, molecular chaperones and co-chaperones, are present to maintain proper conformation and thus avoid protein aggregation and premature damage. The components of the import machinery are well characterised, but the targeting signals and how they are recognised and decoded remains in some cases unclear. Here we provide some detail on the types of targeting signals involved in the protein import process. Furthermore, we discuss the very elaborate chaperone systems of the intermembrane space that are needed to overcome the particular challenges for the folding process in this compartment. The mechanisms that sustain productive folding in the face of aggregation and damage in mitochondria are critical components of the stress response and play an important role in cell homeostasis.
    DOI:  https://doi.org/10.1039/d1ra04497d
  18. J Inherit Metab Dis. 2022 May 04.
    How to proceed after "negative" exome: a review on genetic diagnostics, limitations, challenges and emerging new multi-omics techniques.
    Saskia B Wortmann, Machteld M Oud, Mariëlle Alders, Karlien L M Coene, Saskia N van der Crabben, René G Feichtinger, Alejandro Garanto, Alex Hoischen, Mirjam Langeveld, Dirk Lefeber, Johannes A Mayr, Charlotte W Ockeloen, Holger Prokisch, Richard Rodenburg, Hans R Waterham, Ron A Wevers, Bart P C van de Warrenburg, Michel A A P Willemsen, Nicole I Wolf, Lisenka E L M Vissers, Clara D M van Karnebeek.
      Exome sequencing (ES) in the clinical setting of inborn metabolic disease (IMDs) has created tremendous improvement in achieving an accurate and timely molecular diagnosis for a greater number of patients, but it still leaves the majority of patients without a diagnosis. In parallel, (personalized) treatment strategies are increasingly available, but this requires the availability of a molecular diagnosis. IMDs comprise an expanding field with the ongoing identification of novel disease genes and the recognition of multiple inheritance patterns, mosaicism, variable penetrance and expressivity for known disease genes. The analysis of trio ES is preferred over singleton ES as information on the allelic origin (paternal, maternal, 'de novo') reduces the number of variants that require interpretation. All ES data and interpretation strategies should be exploited including CNV and mitochondrial DNA analysis. The constant advancements in available techniques and knowledge necessitates the close exchange of clinicians and molecular geneticists about genotypes and phenotypes, as well as knowledge of the challenges and pitfalls of ES to initiate proper further diagnostic steps. Functional analyses (transcriptomics, proteomics, metabolomics) can be applied to characterize and validate the impact of identified variants, or to guide the genomic search for a diagnosis in unsolved cases. Future diagnostic techniques (genome sequencing (GS), optical genome mapping, long-read sequencing, epigenetic profiling) will further enhance the diagnostic yield. We provide an overview of the challenges and limitations inherent to ES followed by an outline of solutions and a clinical checklist, focused on establishing a diagnosis to eventually achieve (personalized) treatment.
    Keywords:  diagnostic yield; exome sequencing; exome-negative; genome sequencing; inborn metabolic disease; treatment
    DOI:  https://doi.org/10.1002/jimd.12507
  19. Invest Ophthalmol Vis Sci. 2022 May 02. 63(5): 5
    Photoreceptor Manifestations of Primary Mitochondrial Optic Nerve Disorders.
    Yin-Hsi Chang, Eugene Yu-Chuan Kang, Pei-Kang Liu, Sarah R Levi, Hung-Hsuan Wang, Yun-Ju Tseng, Go Hun Seo, Hane Lee, Lung-Kun Yeh, Kuan-Jen Chen, Wei-Chi Wu, Chi-Chun Lai, Laura Liu, Nan-Kai Wang.
       Purpose: To compare the manifestations of photoreceptors (PRs) in three hereditary optic neuropathies affected by primary mitochondrial dysfunction and discuss whether the retinal ganglion cells (RGCs) or the PRs are preferentially affected.
    Methods: A retrospective analysis of patients with genetically confirmed diagnoses of optic neuropathies associated with mitochondrial dysfunction was performed. This cohort included Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy type 1 (OPA1), and optic atrophy type 13 (OPA13). Patient chart evaluations included clinical characteristics, best-corrected visual acuity (BCVA), fundus photography, spectral-domain optical coherence tomography (SD-OCT), electroretinogram (ERG), and visual evoked potential data.
    Results: This analysis included seven patients with LHON, six with OPA1, and one with OPA13 from a tertiary medical center. Thirteen of the 14 individuals were male. The average BCVA at diagnosis was 20/285 and 20/500 in the right and left eyes, respectively. Five of the seven patients with LHON, and three of the six patients with OPA1 also showed a mild amplitude reduction or delayed latency on light-adapted ERG and 30-Hz flicker responses; however, SD-OCT imaging did not show correlated PR abnormalities. Notably, a 7-year follow-up of a patient with OPA13 revealed degeneration of RGCs prior to the degeneration of PRs. Follow-up data also demonstrated continuous loss of cone outer segment tips on SD-OCT imaging.
    Conclusions: RGCs are, in general, affected by mitochondrial dysfunction, whereas variable PR dysfunction exists in patients with LHON and OPA1, especially with respect to the cone responses. Involvement of PRs is particularly evident in OPA13 after RGC degenerations.
    DOI:  https://doi.org/10.1167/iovs.63.5.5
  20. Biomed Opt Express. 2022 Apr 01. 13(4): 2103-2116
    Transient absorption spectroscopy and imaging of redox in muscle mitochondria.
    Erkang Wang, Luke A Whitcomb, Adam J Chicco, Jesse W Wilson.
      Mitochondrial redox is an important indicator of cell metabolism and health, with implications in cancer, diabetes, aging, neurodegenerative diseases, and mitochondrial disease. The most common method to observe redox of individual cells and mitochondria is through fluorescence of NADH and FAD+, endogenous cofactors serve as electron transport inputs to the mitochondrial respiratory chain. Yet this leaves out redox within the respiratory chain itself. To a degree, the missing information can be filled in by exogenous fluorophores, but at the risk of disturbed mitochondrial permeability and respiration. Here we show that variations in respiratory chain redox can be detected up by visible-wavelength transient absorption microscopy (TAM). In TAM, the selection of pump and probe wavelengths can provide multiphoton imaging contrast between non-fluorescent molecules. Here, we applied TAM with a pump at 520nm and probe at 450nm, 490nm, and 620nm to elicit redox contrast from mitochondrial respiratory chain hemeproteins. Experiments were performed with reduced and oxidized preparations of isolated mitochondria and whole muscle fibers, using mitochondrial fuels (malate, pyruvate, and succinate) to set up physiologically relevant oxidation levels. TAM images of muscle fibers were analyzed with multivariate curve resolution (MCR), revealing that the response at 620nm probe provides the best redox contrast and the most consistent response between whole cells and isolated mitochondria.
    DOI:  https://doi.org/10.1364/BOE.452559
  21. Front Pediatr. 2022 ;10 851534
    Circulating FGF21 and GDF15 as Biomarkers for Screening, Diagnosis, and Severity Assessment of Primary Mitochondrial Disorders in Children.
    Yi Li, Shengrui Li, Yinfeng Qiu, Maobin Zhou, Min Chen, Yue Hu, Siqi Hong, Li Jiang, Yi Guo.
       Background: Primary mitochondrial disorders (PMDs) are a diagnostic challenge for paediatricians, and identification of reliable and easily measurable biomarkers has become a high priority. This study aimed to investigate the role of serum fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) in children with PMDs.
    Methods: We analysed serum FGF21 and GDF15 concentrations by enzyme-linked immunosorbent assay (ELISA) in children with PMDs, patients with non-mitochondrial neuromuscular disorders (NMDs), and aged-matched healthy children, and compared them with serum lactate and ratio of lactate and pyruvate (L/P). We also evaluated correlations between these biomarkers and the phenotype, genotype, and severity of PMDs.
    Results: The median serum GDF15 and FGF21 concentrations were significantly elevated in fifty-one patients with PMDs (919.46 pg/ml and 281.3 pg/ml) compared with those of thirty patients with NMDs (294.86 pg/ml and 140.51 pg/ml, both P < 0.05) and fifty healthy controls (221.21 pg/ml and 85.02 pg/ml, both P < 0.05). The area under the curve of GDF15 for the diagnosis of PMDs was 0.891, which was higher than that of the other biomarkers, including FGF21 (0.814), lactate (0.863) and L/P ratio (0.671). Calculated by the maximum Youden index, the critical value of GDF15 was 606.369 pg/ml, and corresponding sensitivity and specificity were 74.5and 100%. In the PMD group, FGF21 was significantly correlated with International Paediatric Mitochondrial Disease Scale (IPMDS) score. The levels of GDF15 and FGF21 were positively correlated with age, critical illness condition, and multisystem involvement but were not correlated with syndromic/non-syndromic PMDs, different mitochondrial syndromes, nuclear DNA/mitochondrial DNA pathogenic variants, gene functions, or different organ/system involvement.
    Conclusion: Regardless of clinical phenotype and genotype, circulating GDF15 and FGF21 are reliable biomarkers for children with PMDs. GDF15 can serve as a screening biomarker for diagnosis, and FGF21 can serve as a severity biomarker for monitoring.
    Keywords:  FGF21; GDF15; biomarkers; childhood; primary mitochondrial disorders
    DOI:  https://doi.org/10.3389/fped.2022.851534
  22. Obesity (Silver Spring). 2022 May 02.
    Mitochondrial genome variations, mitochondrial-nuclear compatibility, and their association with metabolic diseases.
    Agnieszka H Ludwig-Słomczyńska, Markus Rehm.
      Two genomes regulate the energy metabolism of eukaryotic cells: the nuclear genome, which codes for most cellular proteins, and the mitochondrial genome, which, together with the nuclear genome, coregulates cellular bioenergetics. Therefore, mitochondrial genome variations can affect, directly or indirectly, all energy-dependent cellular processes and shape the metabolic state of the organism. This review provides a current and up-to-date overview on how codependent these two genomes are, how they appear to have coevolved, and how variations within the mitochondrial genome might be associated with the manifestation of metabolic diseases. This review summarizes and structures results obtained from epidemiological studies that identified links between mitochondrial haplogroups and individual risks for developing obesity and diabetes. This is complemented by findings on the compatibility of mitochondrial and nuclear genomes and cellular bioenergetic fitness, which have been acquired from well-controlled studies in conplastic animal models. These elucidate, more mechanistically, how single-nucleotide variants can influence cellular metabolism and physiology. Overall, it seems that certain mitochondrial genome variations negatively affect mitochondrial-nuclear compatibility and are statistically linked with the onset of metabolic diseases, whereas, for others, greater uncertainty exists, and additional research into this exciting field is required.
    DOI:  https://doi.org/10.1002/oby.23424
  23. Case Rep Genet. 2022 ;2022 4153357
    Kearns-Sayre Syndrome Minus: Two Cases of Identical Large-Scale Mitochondrial DNA Deletions with Presentations outside the Classical Triad.
    Shir Wey Gloria Pang, Hencher Han Chih Lee, Carol Ng Wing Kei, Eric Kin Cheong Yau, Joannie Hui.
      A curious triad of retinitis pigmentosa, external ophthalmoplegia, and complete heart block was presented by Sayre et al. in 1958. Since then, the disorder named Kearns-Sayre syndrome (KSS) has come to represent patients with mitochondrial DNA deletions presenting before adulthood, primarily with chronic progressive external ophthalmoplegia (CPEO) and pigmentary retinopathy. However, it is increasingly noted that the presentations can well be variable despite similar genetic deletions. Here, we present two cases with identical large-scale mitochondrial DNA deletions but very dissimilar outlook.
    DOI:  https://doi.org/10.1155/2022/4153357
  24. J Appl Genet. 2022 May 05.
    An explanation for the decreased severity of liver malfunction in Niemann-Pick C1 disease with age.
    Robert P Erickson, Lawrence I Grossman, Siddhesh Aras.
      Niemann-Pick C disease frequently presents as severe cholestatic disease in infants. However, it progressively becomes less of a problem as children age. We have found that, in an appropriate mouse model, liver cholesterol levels, which are initially very high, decrease while mitochondrial function, initially quite compromised, increases with age. The key mitochondrial regulator, MNRR1, increases in parallel with the increase in mitochondrial function. These changes appear to explain the amelioration of the liver disease that occurs with time in this disorder.
    Keywords:  Cholesterol; Infantile hepatic storage disease; Liver; MNRR1; Nieman Pick C1 disease
    DOI:  https://doi.org/10.1007/s13353-022-00695-y
  25. Am J Physiol Renal Physiol. 2022 May 02.
    Regulation of Kidney Mitochondrial Function by Caloric Restriction.
    Julian D C Serna, Andressa G Amaral, Camille C Caldeira da Silva, Ana Cláudia Munhoz, Eloisa Aparecida Vilas-Boas, Sergio L Menezes-Filho, Alicia J Kowaltowski.
      Caloric restriction (CR) prevents obesity and increases resilience against pathological stimuli in laboratory rodents. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher calcium uptake rates and capacities, avoiding Ca2+-induced mitochondrial permeability transition. Dietary restriction has also been shown to increase kidney resistance against damaging stimuli, but if these effects are related to similar mitochondrial adaptations has not been uncovered. Here, we characterized changes in mitochondrial function in response to six months CR in rats, measuring bioenergetic parameters, redox balance and calcium homeostasis. CR promoted an increase in succinate-supported mitochondrial oxygen consumption rates. While CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney we found that mitochondrial H2O2 release was enhanced in a succinate-dependent manner. Surprisingly, and opposite to the effects observed in brain and liver, mitochondria from CR animals are more prone to Ca2+-induced mitochondrial permeability transition, in a manner reversed by antioxidant dithiothreitol. CR mitochondria also displayed higher calcium uptake rates, which were not accompanied by changes in calcium efflux rates, nor related to altered inner mitochondrial membrane potentials or amounts of the mitochondrial calcium uniporter (MCU). Instead, increased mitochondrial calcium uptake rates in CR kidneys correlate with a loss of MICU2, an MCU modulator. Interestingly, MICU2 is also modulated by CR in liver, suggesting it has a broader diet-sensitive regulatory role controlling mitochondrial calcium homeostasis. Together, our results highlight the organ-specific bioenergetic, redox, and ionic transport effects of CR, with some unexpected deleterious effects in kidney.
    Keywords:  Calorie restriction; Mitochondria; Reactive oxygen species; calcium; kidney
    DOI:  https://doi.org/10.1152/ajprenal.00461.2021
  26. Nucleic Acids Res. 2022 May 07. pii: gkac306. [Epub ahead of print]
    The mitoXplorer 2.0 update: integrating and interpreting mitochondrial expression dynamics within a cellular context.
    Fabio Marchiano, Margaux Haering, Bianca Hermine Habermann.
      Mitochondria are subcellular organelles present in almost all eukaryotic cells, which play a central role in cellular metabolism. Different tissues, health and age conditions are characterized by a difference in mitochondrial structure and composition. The visual data mining platform mitoXplorer 1.0 was developed to explore the expression dynamics of genes associated with mitochondrial functions that could help explain these differences. It, however, lacked functions aimed at integrating mitochondria in the cellular context and thus identifying regulators that help mitochondria adapt to cellular needs. To fill this gap, we upgraded the mitoXplorer platform to version 2.0 (mitoXplorer 2.0). In this upgrade, we implemented two novel integrative functions, network analysis and transcription factor enrichment, to specifically help identify signalling or transcriptional regulators of mitochondrial processes. In addition, we implemented several other novel functions to allow the platform to go beyond simple data visualization, such as an enrichment function for mitochondrial processes, a function to explore time-series data, the possibility to compare datasets across species and an IDconverter to help facilitate data upload. We demonstrate the usefulness of these functions in three specific use cases. mitoXplorer 2.0 is freely available without login at http://mitoxplorer2.ibdm.univ-mrs.fr.
    DOI:  https://doi.org/10.1093/nar/gkac306
  27. J Anim Sci. 2022 May 01. pii: skac103. [Epub ahead of print]100(5):
    Influence of environmental factors and genetic variation on mitochondrial DNA copy number.
      
    DOI:  https://doi.org/10.1093/jas/skac103
  28. Biochemistry (Mosc). 2022 Jan;87(1): 21-34
    Focus on Molecular Functions of Anti-Aging Deacetylase SIRT3.
    Jarmila Nahálková.
      SIRT3 is a protein lysine deacetylase with a prominent role in the maintenance of mitochondrial integrity, which is a vulnerable target in many diseases. Intriguingly, cellular aging is reversible just by SIRT3 overexpression, which raises many questions about the role of SIRT3 in the molecular anti-aging mechanisms. Therefore, functions of SIRT3 were analyzed through the interaction network of 407 substrates collected by data mining. Results of the pathway enrichment and gene function prediction confirmed functions in the primary metabolism and mitochondrial ATP production. However, it also suggested involvement in thermogenesis, brain-related neurodegenerative diseases Alzheimer's (AD), Parkinson's, Huntington's disease (HD), and non-alcoholic fatty liver disease. The protein node prioritization analysis identified subunits of the complex I of the mitochondrial respiratory chain (MRC) as the nodes with the main regulatory effect within the entire interaction network. Additional high-ranked nodes were succinate dehydrogenase subunit B (SDHB), complex II, and ATP5F1, complex V of MRC. The analysis supports existence of the NADH/NAD+ driven regulatory feedback loop between SIRT3, complex I (MRC), and acetyl-CoA synthetases, and existence of the nuclear substrates of SIRT3. Unexplored functions of SIRT3 substrates such as LMNA and LMNB; HIF-1a, p53, DNA-PK, and PARK7 are highlighted for further scientific advances. SIRT3 acts as a repressor of BACE1 through the SIRT3-LKB1-AMPK-CREB-PGC1A-PPARG-BACE1 (SIRT3-BACE1), which functions are fitted the best by the Circadian Clock pathway. It forms a new working hypothesis as the therapeutical target for AD treatment. Other important pathways linked to SIRT3 activity are highlighted for therapeutical interventions.
    Keywords:  NAD+-dependent protein deacetylase; SIRT3; age-related disease; aging; mitochondria; pathway enrichment analysis; protein interaction network; respiratory electron transport chain
    DOI:  https://doi.org/10.1134/S0006297922010035
  29. Hepatology. 2022 May 01.
    Comment on "Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD".
    Yuan Qin, Anqi Li, Bilin Liu, Meng Gao, Guohua Gong.
      
    DOI:  https://doi.org/10.1002/hep.32551
  30. Mol Cell. 2022 May 05. pii: S1097-2765(22)00375-6. [Epub ahead of print]82(9): 1613-1615
    Coping with starvation: Cysteine keeps mTORC1 suppressed to ensure survival.
    Dohun Kim, Gerta Hoxhaj.
      Jouandin et al. (2022) show that lysosomal-derived cysteine serves as a signal to promote the tricarboxylic acid (TCA) cycle and suppress TORC1 signaling for Drosophila to endure starvation periods.
    DOI:  https://doi.org/10.1016/j.molcel.2022.04.018
  31. STAR Protoc. 2022 Jun 17. 3(2): 101341
    Protocol for enhanced proliferation of human pluripotent stem cells in tryptophan-fortified media.
    Kotaro Kameda, Shota Someya, Jun Fujita, Keiichi Fukuda, Shugo Tohyama.
      We describe a protocol for the efficient culture of human pluripotent stem cells (hPSCs) by supplementing conventional culture medium with L-tryptophan (TRP). TRP is an essential amino acid that is widely available at an affordable cost, thereby allowing cost-effective proliferation of hPSCs compared to using a conventional medium alone. Here, we describe the steps for enhanced proliferation of hPSCs from dermal fibroblasts or peripheral blood cells, but the protocol can be applied to any hPSCs. For complete details on the use and execution of this protocol, please refer to Someya et al. (2021).
    Keywords:  Cell culture; Metabolism; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2022.101341
  32. Biochem Biophys Res Commun. 2022 Apr 15. pii: S0006-291X(22)00582-4. [Epub ahead of print]612 30-36
    The functional role of OGDH for maintaining mitochondrial respiration and identity of primed human embryonic stem cells.
    Yujie Liu, Han Wang, Min Shao, Ying Jin, Bing Liao.
      Human embryonic stem cells (hESCs) can self-renew infinitely and differentiate into the cell types of all lineages of our body, holding great promise for investigating early human embryo development and providing functional cells for disease treatment. For the full application of hESCs, it is necessary to elucidate how hESCs maintain their identity. Recent studies have shown that glycolysis and mitochondrial respiration are linked to pluripotency states. However, the function of mitochondrial respiration in hESCs has not been fully understood. Herein, we report that the adenosine triphosphate (ATP) production rate is comparable between mitochondrial respiration and glycolysis, suggesting an important contribution of mitochondrial respiration to ATP production in conventionally cultured hESCs. To investigate the function of mitochondrial respiration, we silence OGDH expression in hESCs by the inducible CRISPRi method, and find that OGDH knockdown (KD) results in disrupted TCA (tricarboxylic acid) cycle, and diminished mitochondrial respiration activity and total ATP level. Moreover, OGDH KD leads to hESC death and aberrant transcriptional program. Interestingly, blockage of the electron transport chain (ETC) by small molecule inhibitors gives rise to the phenotype similar to that observed in OGDH deficient hESCs. Therefore, genetic and pharmacological perturbations of the mitochondrial respiration impair identity of hESCs. Collectively, our study highlights the pivotal role of the mitochondrial respiration activity for the stemness maintenance of primed hESCs, and unveils OGDH as a key regulator for the proper production of ATP and TCA cycle metabolites in primed hESCs.
    Keywords:  ATP production; Human embryonic stem cells; Mitochondrial respiration; OGDH; Primed pluripotency
    DOI:  https://doi.org/10.1016/j.bbrc.2022.04.059
  33. Am J Med Genet A. 2022 May 02.
    Further description of two patients with biallelic variants in NADSYN1 in association with cardiac and vertebral anomalies.
    Hannah Kortbawi, Elizabeth Ames, Amanda Pritchard, Patrick Devine, Jessica van Ziffle, Anne Slavotinek.
      Congenital nicotinamide adenine dinucleotide (NAD) deficiency disorders are associated with pathogenic variants in the genes NADSYN1, HAAO, and KYNU. These disorders overlap with the anomalies present in vertebral, anal, cardiac, tracheoesophageal, radial and renal, and limb anomalies (VATER/VACTERL) association and often result in premature death. Children who survive typically have developmental delays or intellectual disability. Here, we describe two patients with compound heterozygous variants in NADSYN1 who presented with cardiac and vertebral defects overlapping with the VATER/VACTERL association, although the patients did not satisfy criteria for the diagnosis of VATER/VACTERL due to their lack of limb anomalies and significant renal anomalies. One patient survived into childhood with developmental delays and may represent an expansion of the survival data for NADSYN1-associated NAD deficiency disorders. Interestingly, one patient had hypoplastic left heart syndrome (HLHS) and one had an aortic coarctation and transverse hypoplasia of the aortic arch, suggesting that NADSYN1 sequencing should be performed in children presenting with congenital anomalies related to VATER/VACTERL association and with HLHS and aortic arch abnormalities.
    Keywords:  NADSYN1; VATER/VACTERL; congenital NAD deficiency disorder; kynurenine; niacin
    DOI:  https://doi.org/10.1002/ajmg.a.62765
  34. Methods Mol Biol. 2022 ;2429 143-174
    Differentiation of Human Induced Pluripotent Stem Cells into Cortical Neurons to Advance Precision Medicine.
    M Catarina Silva, Ghata Nandi, Stephen J Haggarty.
      A major obstacle in studying human central nervous system (CNS) diseases is inaccessibility to the affected tissue and cells. Even in limited cases where tissue is available through surgical interventions, differentiated neurons cannot be maintained for extended time frames, which is prohibitive for experimental repetition and scalability. Advances in methodologies for reprogramming human somatic cells into induced pluripotent stem cells (iPSC) and directed differentiation of human neurons in culture now allow access to physiological and disease relevant cell types. In particular, patient iPSC-derived neurons represent unique ex vivo neuronal networks that allow investigating disease genetic and molecular pathways in physiologically accurate cellular microenvironments, importantly recapitulating molecular and cellular phenotypic aspects of disease. Generation of functional neural cells from iPSCs relies on manipulation of culture formats in the presence of specific factors that promote the conversion of pluripotent stem cells into neurons. To this end, several experimental protocols have been developed. Direct differentiation of stem cells into post-mitotic neurons is usually associated with low throughput, low yield, and high technical variability. Instead, methods relying on expansion of the intermediate neural progenitor cells (NPCs) show incredible potential for posterior generation of suitable neuronal cultures for cellular and biochemical assays, as well as drug screening. NPCs are expandable, self-renewable multipotent cells that can differentiate into astrocytes, oligodendrocytes, and electrically active neurons. Here, we describe a protocol for generating iPSC-derived NPCs via formation of neural aggregates and selection of NPC precursor neural rosettes, followed by a simple and reproducible method for generating a mixed population of cortical-like neurons through growth factor withdrawal. Implementation of this protocol has the potential to advance the goals of precision medicine research for both neurological and psychiatric disorders.
    Keywords:  Drug discovery; Human neurons; Induced pluripotent stem cells; Neural differentiation; Neural progenitor cells; Neural rosettes; Precision medicine
    DOI:  https://doi.org/10.1007/978-1-0716-1979-7_10
  35. J Inherit Metab Dis. 2022 Apr 30.
    Diagnosis and Discovery: Insights from the NIH Undiagnosed Diseases Program.
    Carolina Montano, Thomas Cassini, Shira G Ziegler, Manfred Boehm, Elena-Raluca Nicoli, Joseph A Mindell, Ariane G Soldatos, Irini Manoli, Lynne Wolfe, Ellen F Macnamara, May Christine V Malicdan, David R Adams, Cynthia J Tifft, Camilo Toro, William A Gahl.
      Living with an undiagnosed medical condition places a tremendous burden on patients, their families, and their healthcare providers. The Undiagnosed Diseases Program (UDP) was established at the National Institutes of Health (NIH) in 2008 with the primary goals of providing a diagnosis for patients with mysterious conditions and advancing medical knowledge about rare and common diseases. The program reviews applications from referring clinicians for cases that are considered undiagnosed despite a thorough evaluation. Those that are accepted receive clinical evaluations involving deep phenotyping and genetic testing that includes exome and genomic sequencing. Selected candidate gene variants are evaluated by collaborators using functional assays. Since its inception, the UDP has received more than 4500 applications and has completed evaluations on nearly 1300 individuals. Here we present six cases that exemplify the discovery of novel disease mechanisms, the importance of deep phenotyping for rare diseases, and how genetic diagnoses have led to appropriate treatment. The creation of the Undiagnosed Diseases Network (UDN) in 2014 has substantially increased the number of patients evaluated and allowed for greater opportunities for data sharing. Expansion to the Undiagnosed Diseases Network International (UDNI) has the possibility to extend this reach even farther. Together, networks of undiagnosed diseases programs are powerful tools to advance our knowledge of pathophysiology, accelerate accurate diagnoses and improve patient care for patients with rare conditions. This article is protected by copyright. All rights reserved.
    Keywords:  CHST14; CLCN7; KMT2B; NT5E; SNORD118; SNP microarray; Undiagnosed Diseases Program; amyloidosis; exome sequencing; whole genome sequencing
    DOI:  https://doi.org/10.1002/jimd.12506
  36. J Biol Chem. 2022 May 02. pii: S0021-9258(22)00448-3. [Epub ahead of print] 102008
    Loss of mitochondrial ATPase ATAD3A contributes to non-alcoholic fatty liver disease through accumulation of lipids and damaged mitochondria.
    Liting Chen, Yuchang Li, Chantal Sottas, Anthoula Lazaris, Stephanie K Petrillo, Peter Metrakos, Lu Li, Yuji Ishida, Takeshi Saito, Samuel Garza, Vassilios Papadopoulos.
      Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and non-alcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced non-alcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells, and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells, and also that triglyceride (TG) levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, TG, and damaged mitochondria in hepatocytes.
    Keywords:  ATAD3A; NAFLD; autophagy; cholesterol; fatty acid oxidation; free fatty acid; mitochondrial respiration; mitophagy; triglyceride
    DOI:  https://doi.org/10.1016/j.jbc.2022.102008
  37. Mol Nutr Food Res. 2022 May 01. e2200003
    Diet, Trimethylamine Metabolism and Mitochondrial DNA: An Observational Study.
    Laura Bordoni, Anna M Malinowska, Irene Petracci, Artur Szwengiel, Rosita Gabbianelli, Agata Chmurzynska.
       SCOPE: Mitochondrial DNA copy number (mtDNAcn) and its methylation level in the D-loop area have been correlated with metabolic health and are suggested to vary in response to environmental stimuli, including diet. Circulating levels of trimethylamine-n-oxide (TMAO), which is an oxidative derivative of the trimethylamine (TMA) produced by the gut microbiome from dietary precursors, have been associated with chronic diseases and are suggested to have an impact on mitochondrial dynamics. This study aimed to investigate the relationship between diet, TMA, TMAO, and mtDNAcn, as well as methylation.
    METHODS AND RESULTS: 200 subjects with extreme (healthy and unhealthy) dietary patterns were recruited. Dietary records were collected to assess their diets' quality (Healthy Eating Index). Blood levels of TMA and TMAO, circulating levels of TMA precursors and their dietary intakes were measured. MtDNAcn, nuclear DNA methylation (LINE-1) and strand-specific D-loop methylation levels were assessed. There was no association between dietary patterns and mtDNAcn. The TMAO/TMA ratio was negatively correlated with D-loop methylation levels but positively with mtDNAcn.
    CONCLUSIONS: These findings suggest a potential association between TMA metabolism and mitochondrial dynamics (and mtDNA), indicating a new avenue for further research.  This article is protected by copyright. All rights reserved.
    Keywords:  DNA methylation; epigenetics; mitochondrial DNA; nutrigenomics; trimethylamine-n-oxide
    DOI:  https://doi.org/10.1002/mnfr.202200003
  38. STAR Protoc. 2022 Jun 17. 3(2): 101360
    Purification of cardiomyocytes and neurons derived from human pluripotent stem cells by inhibition of de novo fatty acid synthesis.
    Sho Tanosaki, Tomohiko Akiyama, Sayaka Kanaami, Jun Fujita, Minoru S H Ko, Keiichi Fukuda, Shugo Tohyama.
      Here we describe a protocol to obtain highly pure cardiomyocytes and neurons from human induced pluripotent stem cells (hiPSCs) via metabolic selection processes. Compared to conventional purification protocols, this approach is easier to perform and scale up and more cost-efficient. The protocol can be applied to hiPSCs and human embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Tohyama et al. (2016) and Tanosaki et al. (2020).
    Keywords:  Cell Differentiation; Cell culture; Cell isolation; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2022.101360
  39. Cell Rep Methods. 2022 Apr 25. 2(4): 100192
    An integrated toolbox to profile macrophage immunometabolism.
    Sanne G S Verberk, Kyra E de Goede, Friederike S Gorki, Xanthe A M H van Dierendonck, Rafael J Argüello, Jan Van den Bossche.
      Macrophages are dynamic immune cells that can adopt several activation states. Fundamental to these functional activation states is the regulation of cellular metabolic processes. Especially in mice, metabolic alterations underlying pro-inflammatory or homeostatic phenotypes have been assessed using various techniques. However, researchers new to the field may encounter ambiguity in choosing which combination of techniques is best suited to profile immunometabolism. To address this need, we have developed a toolbox to assess cellular metabolism in a semi-high-throughput 96-well-plate-based format. Application of the toolbox to activated mouse and human macrophages enables fast metabolic pre-screening and robust measurement of extracellular fluxes, mitochondrial mass and membrane potential, and glucose and lipid uptake. Moreover, we propose an application of SCENITH technology for ex vivo metabolic profiling. We validate established activation-induced metabolic rewiring in mouse macrophages and report new insights into human macrophage metabolism. By thoroughly discussing each technique, we hope to guide readers with practical workflows for investigating immunometabolism.
    Keywords:  immunometabolism; macrophages; metabolism; semi-high throughput screening; toolbox
    DOI:  https://doi.org/10.1016/j.crmeth.2022.100192
  40. Methods Mol Biol. 2022 ;2477 179-193
    Purification of Ribosome-Nascent-Chain Complex for Ribosome Profiling and Selective Ribosome Profiling.
    Hagit Bar-Yosef, Johannes Venezian, Kevin Klann, Ayala Shiber.
      Selective Ribosome Profiling (SeRP) is an emerging methodology, developed to capture cotranslational interactions in vivo. To date, SeRP is the only method that can directly capture, in near-codon resolution, ribosomes in action. Thus, SeRP allows us to study the mechanisms of protein synthesis and the network of protein-protein interactions that are formed already during synthesis. Here we report, in detail, the protocol for purification of ribosome- and Nascent-Chain associated factors, followed by isolation of ribosome-protected mRNA footprints, cDNA library generation and subsequent data analysis.
    Keywords:  Affinity purification; Cotranslational interactions; Nascent-chain; Ribosome; Ribosome occupancy
    DOI:  https://doi.org/10.1007/978-1-0716-2257-5_11
  41. Curr Obes Rep. 2022 May 02.
    Hepatocyte Mitochondrial Dynamics and Bioenergetics in Obesity-Related Non-Alcoholic Fatty Liver Disease.
    Aigli-Ioanna Legaki, Ioannis I Moustakas, Michalina Sikorska, Grigorios Papadopoulos, Rallia-Iliana Velliou, Antonios Chatzigeorgiou.
       PURPOSE OF THE REVIEW: Mitochondrial dysfunction has long been proposed to play a crucial role in the pathogenesis of a considerable number of disorders, such as neurodegeneration, cancer, cardiovascular, and metabolic disorders, including obesity-related insulin resistance and non-alcoholic fatty liver disease (NAFLD). Mitochondria are highly dynamic organelles that undergo functional and structural adaptations to meet the metabolic requirements of the cell. Alterations in nutrient availability or cellular energy needs can modify their formation through biogenesis and the opposite processes of fission and fusion, the fragmentation, and connection of mitochondrial network areas respectively. Herein, we review and discuss the current literature on the significance of mitochondrial adaptations in obesity and metabolic dysregulation, emphasizing on the role of hepatocyte mitochondrial flexibility in obesity and NAFLD.
    RECENT FINDINGS: Accumulating evidence suggests the involvement of mitochondrial morphology and bioenergetics dysregulations to the emergence of NAFLD and its progress to non-alcoholic steatohepatitis (NASH). Most relevant data suggests that changes in liver mitochondrial dynamics and bioenergetics hold a key role in the pathogenesis of NAFLD. During obesity and NAFLD, oxidative stress occurs due to the excessive production of ROS, leading to mitochondrial dysfunction. As a result, mitochondria become incompetent and uncoupled from respiratory chain activities, further promoting hepatic fat accumulation, while leading to liver inflammation, insulin resistance, and disease's deterioration. Elucidation of the mechanisms leading to dysfunctional mitochondrial activity of the hepatocytes during NAFLD is of predominant importance for the development of novel therapeutic approaches towards the treatment of this metabolic disorder.
    Keywords:  Energy metabolism; Liver; Mitochondrial bioenergetics; Mitochondrial dysfunction; NAFLD; NASH
    DOI:  https://doi.org/10.1007/s13679-022-00473-1
  42. Annu Rev Biomed Data Sci. 2022 May 04.
    Integration of Protein Structure and Population-Scale DNA Sequence Data for Disease Gene Discovery and Variant Interpretation.
    Bian Li, Bowen Jin, John A Capra, William S Bush.
      The experimental and computational techniques for capturing information about protein structures and genetic variation within the human genome have advanced dramatically in the past 20 years, generating extensive new data resources. In this review, we discuss these advances, along with new approaches for determining the impact a genetic variant has on protein function. We focus on the potential of new methods that integrate human genetic variation into protein structures to discover relationships to disease, including the discovery of mutational hotspots in cancer-related proteins, the localization of protein-altering variants within protein regions for common complex diseases, and the assessment of variants of unknown significance for Mendelian traits. We expect that approaches that integrate these data sources will play increasingly important roles in disease gene discovery and variant interpretation. Expected final online publication date for the Annual Review of Biomedical Data Science, Volume 5 is August 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biodatasci-122220-112147
  43. EMBO J. 2022 May 02. e109460
    Structural basis for feedforward control in the PINK1/Parkin pathway.
    Véronique Sauvé, George Sung, Emma J MacDougall, Guennadi Kozlov, Anshu Saran, Rayan Fakih, Edward A Fon, Kalle Gehring.
      PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observed that phospho-ubiquitin can bind to two distinct sites on parkin, a high-affinity site on RING1 that controls parkin localization and a low-affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho-ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.
    Keywords:  Parkinson's disease; autophagy; mitophagy; open-loop control; ubiquitin
    DOI:  https://doi.org/10.15252/embj.2021109460
  44. J Mol Biol. 2022 Apr 29. pii: S0022-2836(22)00198-X. [Epub ahead of print] 167618
    Stress Responses Elicited by Misfolded Proteins Targeted to Mitochondria.
    Kannan Boosi Narayana Rao, Pratima Pandey, Rajasri Sarkar, Asmita Ghosh, Shemin Mansuri, Mudassar Ali, Priyanka Majumder, K Ranjith Kumar, Arjun Ray, Swasti Raychaudhuri, Koyeli Mapa.
      The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and discrete protein quality control (PQC) machineries resulting inherent differences in their protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to these sub-mitochondrial compartments, we took misfolding and aggregation-prone stressor proteins and fused it to well described signal sequences to specifically target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, by employing transcriptomics and proteomics, we report a comprehensive stress response elicited by stressor proteins specifically targeted to mitochondrial matrix or IMS. A general response to proteotoxic stress by mitochondria-targeted misfolded proteins is mitochondrial fragmentation, and an adaptive abrogation of mitochondrial respiration with concomitant upregulation of glycolysis. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that stress-imparted by bipartite signal sequence-fused stressor proteins to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.
    Keywords:  Mitochondrial Unfolded Protein Response; Molecular Chaperone; Protein misfolding; Proteostasis; Proteotoxic stress; Ribosome Quality Control; Stress Response; TOM complex; Vms1
    DOI:  https://doi.org/10.1016/j.jmb.2022.167618
  45. Am J Physiol Cell Physiol. 2022 May 04.
    The role of mitochondria in the pathophysiology and treatment of common metabolic diseases in humans.
    Asen Georgiev, Cesare Granata, Michael Roden.
      Common metabolic diseases such as obesity, type 2 diabetes mellitus and non-alcoholic fatty liver disease significantly contribute to morbidity and mortality worldwide. They frequently associate with insulin resistance and altered mitochondrial functionality. Insulin-responsive tissues can show changes in mitochondrial features such as oxidative capacity, mitochondrial content and turnover, which do not necessarily reflect abnormalities but rather adaption to a certain metabolic condition. Lifestyle modifications and classic or novel drugs can modify these alterations and help treating these metabolic diseases. This review addresses the role of mitochondria in human metabolic diseases and discusses potential future research directions.
    Keywords:  adipose tissue; mitophagy; oxidative phosphorylation; skeletal muscle; steatosis
    DOI:  https://doi.org/10.1152/ajpcell.00035.2022
  46. Am J Ophthalmol. 2022 May 02. pii: S0002-9394(22)00166-0. [Epub ahead of print]
    Capturing the pattern of transition from carrier to affected in Leber's hereditary optic neuropathy.
    Michele Carbonelli, Chiara La Morgia, Martina Romagnoli, Giulia Amore, Pietro D'Agati, Maria Lucia Valentino, Leonardo Caporali, Maria Lucia Cascavilla, Marco Battista, Enrico Borrelli, Antonio Di Renzo, Vincenzo Parisi, Nicole Balducci, Valerio Carelli, Piero Barboni.
       PURPOSE: to capture the key features patterning the transition from unaffected mutation carriers to clinically affected Leber's hereditary optic neuropathy (LHON), as investigated by Optical Coherence Tomography.
    DESIGN: Observational case series.
    METHODS: four unaffected eyes of four LHON patients with first eye affected, followed across conversion to affected, from 60 days before up to 170 days after conversion.
    MAIN OUTCOME MEASURES: multiple timepoints measurements of peripapillary retinal nerve fiber layer (RNFL) thickness for temporal emi-side of the optic nerve in (six sectors from 6 to 11, clockwise for the right eye and counterclockwise for the left eye) in all patients and nasal emi-macular RNFL and Ganglion cell layer thickness in two patients.
    RESULTS: while the pre-symptomatic stage was characterized by a dynamic thickening of sector 8, the beginning of the conversion coincided with an increase in the thickness of the sectors bordering the papillo-macular bundle (6 and 7 for the inferior sectors, 10 and 11 for the superior) synchronous with the thinning of sectors 8 and then 9. Conversely, the GCL did not undergo significant changes until the onset of visual loss when a significant reduction of thickness became evident.
    CONCLUSION: In this study we demonstrated that the thinning of sector 8 can be considered the structural hallmark of the conversion from pre-symptomatic to affected state in LHON. It is preceded by its own progressive thickening extending from the optic nerve head towards the macula and occurs regardless of the amount of swelling of the rest of the peripapillary fibers.
    Keywords:  Leber's hereditary optic neuropathy; Optical coherence tomography; Retinal ganglion cells; Retinal nerve fiber layer
    DOI:  https://doi.org/10.1016/j.ajo.2022.04.016
  47. Methods Mol Biol. 2022 ;2429 175-188
    Differentiation of iPS-Cells into Peripheral Sensory Neurons.
    Anika Neureiter, Esther Eberhardt, Angelika Lampert.
      Induced pluripotent stem cells (iPS-cells) have significantly expanded our experimental possibilities, by creating new strategies for the molecular study of human disease and drug development. Treatment of pain has not seen much improvement over the past decade, likely due to species differences in preclinical models. Thus, iPS-cell derived sensory neurons offer a highly welcome translational approach for research and drug development. Although central neuronal differentiation is relatively straightforward, the successful and reliable generation of peripheral neurons requires more complex measures. Here, we describe a small molecule-based protocol for the differentiation of human sensory neurons from iPS-cells which renders functional nociceptor-like cells within several weeks.
    Keywords:  Differentiation; Disease modeling; Induced pluripotent stem cells; Neural crest cells; Pain; Peripheral neurons; Sensory neurons; Sensory neuropathy; iPS-cells
    DOI:  https://doi.org/10.1007/978-1-0716-1979-7_11
  48. Methods Mol Biol. 2022 ;2429 189-199
    Culture of Human iPSC-Derived Motoneurons in Compartmentalized Microfluidic Devices and Quantitative Assays for Studying Axonal Phenotypes.
    Maria Giovanna Garone, Chiara D'Antoni, Alessandro Rosa.
      In order to use induced Pluripotent Stem Cells (iPSCs) to model neurodegenerative diseases, efficient and homogeneous generation of neurons in vitro represents a key step. Here we describe a method to obtain and characterize functional human spinal and cranial motoneurons using a combined approach of microfluidic chips and programs designed for scientific multidimensional imaging. We have used this approach to analyze axonal phenotypes. These tools are useful to investigate the cellular and molecular bases of neuromuscular diseases, including amyotrophic lateral sclerosis and spinal muscular atrophy.
    Keywords:  Amyotrophic lateral sclerosis; Axon; Axotomy; Cranial motoneuron; Differentiation protocol; Microfluidic device; Spinal motoneuron; iPSC; piggyBac
    DOI:  https://doi.org/10.1007/978-1-0716-1979-7_12
  49. Nature. 2022 May;605(7908): 30
    How to ensure the Human Cell Atlas benefits humanity.
    Partha Majumder, Musa Mhlanga, Alex Shalek, Roderic Guigó, Bartha Maria Knoppers, Barbara Wold.
      
    Keywords:  Cell biology; Diseases; Ethics; Genomics
    DOI:  https://doi.org/10.1038/d41586-022-01186-0
  50. Front Cell Dev Biol. 2022 ;10 862506
    MFN2 Deficiency Impairs Mitochondrial Functions and PPAR Pathway During Spermatogenesis and Meiosis in Mice.
    Tianren Wang, Yuan Xiao, Zhe Hu, Jingkai Gu, Renwu Hua, Zhuo Hai, Xueli Chen, Jian V Zhang, Zhiying Yu, Ting Wu, William S B Yeung, Kui Liu, Chenxi Guo.
      Mitochondria are highly dynamic organelles and their activity is known to be regulated by changes in morphology via fusion and fission events. However, the role of mitochondrial dynamics on cellular differentiation remains largely unknown. Here, we explored the molecular mechanism of mitochondrial fusion during spermatogenesis by generating an Mfn2 (mitofusin 2) conditional knock-out (cKO) mouse model. We found that depletion of MFN2 in male germ cells led to disrupted spermatogenesis and meiosis during which the majority of Mfn2 cKO spermatocytes did not develop to the pachytene stage. We showed that in these Mfn2 cKO spermatocytes, oxidative phosphorylation in the mitochondria was affected. In addition, RNA-Seq analysis showed that there was a significantly altered transcriptome profile in the Mfn2 deficient pachytene (or pachytene-like) spermatocytes, with a total of 262 genes up-regulated and 728 genes down-regulated, compared with wild-type (control) mice. Pathway enrichment analysis indicated that the peroxisome proliferator-activated receptor (PPAR) pathway was altered, and subsequent more detailed analysis showed that the expression of PPAR α and PPAR γ was up-regulated and down-regulated, respectively, in the MFN2 deficient pachytene (or pachytene-like) spermatocytes. We also demonstrated that there were more lipid droplets in the Mfn2 cKO cells than in the control cells. In conclusion, our study demonstrates a novel finding that MFN2 deficiency negatively affects mitochondrial functions and alters PPAR pathway together with lipid metabolism during spermatogenesis and meiosis.
    Keywords:  lipid metabolism; meiosis; mitochondrial dynamics; mitofusin 2; spermatogenesis
    DOI:  https://doi.org/10.3389/fcell.2022.862506
  51. Methods Mol Biol. 2022 ;2429 73-84
    Detection of Soluble and Insoluble Protein Species in Patient-Derived iPSCs.
    Stephanie Santarriaga, Ian Luecke, Allison D Ebert.
      Protein aggregation is one of the hallmarks of many neurodegenerative diseases. While protein aggregation is a heavily studied aspect of neurodegenerative disease, methods of detection vary from one model system to another. Induced pluripotent stem cells (iPSCs) present an opportunity to model disease using patient-specific cells. However, iPSC-derived neurons are fetal-like in maturity, making it a challenge to detect key features such as protein aggregation that are often exacerbated with age. Nevertheless, we have previously found abnormal soluble and insoluble protein burden in motor neurons generated from amyotrophic lateral sclerosis (ALS) iPSCs, though protein aggregation has not been readily detected in iPSC-derived neurons from other neurodegenerative diseases. Therefore, here we present an ultracentrifugation method that detects insoluble protein species in various models of neurodegenerative disease, including Huntington's disease, Alzheimer's disease, and ALS. This method is able to detect soluble, insoluble, and SDS-resistant species in iPSC-derived neurons and is designed to be flexible for optimal detection of various aggregation-prone proteins.
    Keywords:  Alzheimer’s Disease; Amyotrophic Lateral Sclerosis; Huntington’s Disease; Induced Pluripotent Stem Cells; Neurodegeneration; Neurons; Protein Aggregation
    DOI:  https://doi.org/10.1007/978-1-0716-1979-7_6
  52. Methods Mol Biol. 2022 ;2477 71-77
    Direct Sequencing of RNA and RNA Modification Identification Using Nanopore.
    Thidathip Wongsurawat, Piroon Jenjaroenpun, Intawat Nookaew.
      Direct RNA sequencing (dRNA-seq) simultaneously enables the detection of RNA modifications and characterization of full-length transcripts. In principle, full-length native RNA molecule is translocated through the nanopore by a motor protein while a sensor measures ionic current shifts. Then, the current shifts are interpreted by an algorithm that turn out to RNA sequence. Currently, the standard protocol of dRNA-seq provided by Oxford Nanopore Technologies (ONT) allows to directly ligate and sequence only polyadenylated RNA (poly(A) RNA). Here, we describe a method of dRNA-seq that can be applied for both poly(A) RNA and non-poly(A) tailed-RNA.
    Keywords:  Direct RNA sequencing; Epitranscriptome; Long-read; Methylation; Modification; Nanopore; Native; RNA; Sequencing Technology; Transcriptome
    DOI:  https://doi.org/10.1007/978-1-0716-2257-5_5
  53. RSC Adv. 2021 Nov 29. 11(61): 38750-38758
    Investigation of apoptosis based on fluorescence lifetime imaging microscopy with a mitochondria-targeted viscosity probe.
    Gengjin Zou, Wenhui Yu, Yunjian Xu, Yanping Li, Rui Hu, Junle Qu, Liwei Liu.
      Cell apoptosis detection based on the functionality changes of cellular organelles, such as mitochondria, offers a quantitative method compared to morphology-based detection. However, the conventional detection methods for potential variation of the mitochondrial membrane based on fluorescence spectrum changes cannot offer a precise quantification of the degree of apoptosis. Here, a mitochondria-targeted two-photon viscosity probe (TPA-Mit), which sensitively responds to viscosity variations with fluorescence lifetime changes, is designed to detect the viscosity of mitochondria. Noteworthily, the proposed phasor fluorescence lifetime imaging microscopy (phasor-FLIM) allows for more precise quantification (in terms of smaller uncertainty) when estimating the degree of apoptosis with a microviscosity probe. The experimental results of SKOV-3 cells show that the fluorescence lifetime of mitochondria-targeted TPA-Mit increased from 550 ps to 800 ps after 24 hours of paclitaxel (PTX)-induced apoptosis. We believe that our method provides a new means for the measurement of cellular microviscosity and apoptosis monitoring at early stages.
    DOI:  https://doi.org/10.1039/d1ra06697h
  54. Methods Mol Biol. 2022 ;2432 73-84
    Controlling Batch Effect in Epigenome-Wide Association Study.
    Yale Jiang, Jianjiao Chen, Wei Chen.
      Methylation data, similar to other omics data, is susceptible to various technical issues that are potentially associated with unexplained or unrelated factors. Any difference in the measurement of DNA methylation, such as laboratory operation and sequencing platform, may lead to batch effects. With the accumulation of large-scale omics data, scientists are making joint efforts to generate and analyze omics data to answer various scientific questions. However, batch effects are inevitable in practice, and careful adjustment is needed. Multiple statistical methods for controlling bias and inflation between batches have been developed either by correcting based on known batch factors or by estimating directly from the output data. In this chapter, we will review and demonstrate several popular methods for batch effect correction and make practical recommendations in epigenome-wide association studies (EWAS).
    Keywords:  Batch effects; COMBAT; DNA methylation; Linear mixed effect model
    DOI:  https://doi.org/10.1007/978-1-0716-1994-0_6
  55. Sci Signal. 2022 May 03. 15(732): eabq7456
    Letting go of damaged mitochondria.
    Wei Wong.
      Thermogenesis requires that macrophages digest damaged mitochondria released by brown adipocytes.
    DOI:  https://doi.org/10.1126/scisignal.abq7456
  56. Life Sci. 2022 Apr 30. pii: S0024-3205(22)00295-8. [Epub ahead of print] 120595
    Mechanisms of autophagy and mitophagy in skeletal development, diseases and therapeutics.
    Jyotirmaya Behera, Jessica Ison, Ashish Tyagi, Gabriel Mbalaviele, Neetu Tyagi.
      Autophagy is a highly evolutionarily conserved process in the eukaryotic cellular system by which dysfunctional organelles are selectively degraded through a series of processes of lysosomal activity and then returned to the cytoplasm for reuse. All cells require this process to maintain cellular homeostasis and promote cell survival during stress responses such as deprivation and hypoxia. Osteoblasts and osteoclasts are two cellular phenotypes in the bone that mediate bone homeostasis. However, an imbalance between osteoblastic bone formation and osteoclastic bone resorption contributes to the onset of bone diseases. A recent study suggests that autophagy, mitophagy, and selective mitochondrial autophagy may play an essential role in regulating osteoblast differentiation and osteoclast maturation. Autophagic activity dysregulation alters the equilibrium between osteoblastic bone creation and osteoclastic bone resorption, allowing bone disorders like osteoporosis to develop more easily. The current review emphasizes the role of autophagy and mitophagy and their related molecular mechanisms in bone metabolic disorders. In the current review, we emphasize the role of autophagy and mitophagy as well as their related molecular mechanism in bone metabolic disorders. Furthermore, we will discuss its potential as a new molecular target for the treating of metabolic bone disease and future application in therapeutic translational research.
    Keywords:  Autophagy; Epigenetics; Mitophagy; Osteoporosis; miRNA regulation
    DOI:  https://doi.org/10.1016/j.lfs.2022.120595
  57. Am J Physiol Regul Integr Comp Physiol. 2022 May 03.
    The sex-specific role of sensory neuron LKB1 on metabolic stress-induced mechanical hypersensitivity and mitochondrial respiration.
    Katherine Marie Garner, Michael D Burton.
      Pain disorders induce metabolic stress in peripheral sensory neurons by reducing mitochondrial output, shifting cellular metabolism, and altering energy use. These processes implicate neuronal metabolism as an avenue for creating novel therapeutics. Liver kinase B1 (LKB1) mediates the cellular response to metabolic stress by inducing the AMPK pathway. The LKB1-AMPK pathway increases energy producing processes, including mitochondrial output. These processes inhibit pain by directly or indirectly restoring energetic balance within a cell. Although the LKB1-AMPK pathway has been linked to pain relief, it is not yet known which cell is responsible for this property, as well any direct ties to cellular metabolism. To elucidate this, we developed a genetic mouse model where LKB1 is selectively removed from Nav1.8-pain sensory neurons and metabolically stressed them by fasting for 24 hours. We found females, but not males, had neuron-specific, LKB1-dependent restoration of metabolic stress-induced mitochondrial metabolism. This was reflected in mechanical hypersensitivity, where the absence of LKB1 led to hypersensitivity in female, but not male, animals. This discrepancy suggests a sex- and cell-specific contribution to LKB1-depdendent fasting-induced mechanical hypersensitivity. While our data represent a potential role for LKB1 in anti-pain pathways in a metabolic-specific manner, more must be done to investigate these sex differences.
    Keywords:  LKB1; cell metabolism; fasting; mechanical hypersensitivity; sensory neuron
    DOI:  https://doi.org/10.1152/ajpregu.00279.2021
  58. Cell Metab. 2022 May 03. pii: S1550-4131(22)00133-4. [Epub ahead of print]34(5): 775-782.e9
    Glycine decarboxylase maintains mitochondrial protein lipoylation to support tumor growth.
    Dzmitry Mukha, Mariam Fokra, Alona Feldman, Boris Sarvin, Nikita Sarvin, Keren Nevo-Dinur, Elazar Besser, Elior Hallo, Elina Aizenshtein, Zachary T Schug, Tomer Shlomi.
      The folic acid cycle mediates the transfer of one-carbon (1C) units to support nucleotide biosynthesis. While the importance of serine as a mitochondrial and cytosolic donor of folate-mediated 1C units in cancer cells has been thoroughly investigated, a potential role of glycine oxidation remains unclear. We developed an approach for quantifying mitochondrial glycine cleavage system (GCS) flux by combining stable and radioactive isotope tracing with computational flux decomposition. We find high GCS flux in hepatocellular carcinoma (HCC), supporting nucleotide biosynthesis. Surprisingly, other than supplying 1C units, we found that GCS is important for maintaining protein lipoylation and mitochondrial activity. Genetic silencing of glycine decarboxylase inhibits the lipoylation and activity of pyruvate dehydrogenase and impairs tumor growth, suggesting a novel drug target for HCC. Considering the physiological role of liver glycine cleavage, our results support the notion that tissue of origin plays an important role in tumor-specific metabolic rewiring.
    Keywords:  GCS; GLDC; PDH; glycine cleavage system; glycine decarboxylase; hepatocellular carcinoma; one-carbon metabolism; protein P; protein lipoylation; pyruvate dehydrogenase
    DOI:  https://doi.org/10.1016/j.cmet.2022.04.006
  59. Nat Biotechnol. 2022 May 05.
    DIALOGUE maps multicellular programs in tissue from single-cell or spatial transcriptomics data.
    Livnat Jerby-Arnon, Aviv Regev.
      Deciphering the functional interactions of cells in tissues remains a major challenge. Here we describe DIALOGUE, a method to systematically uncover multicellular programs (MCPs)-combinations of coordinated cellular programs in different cell types that form higher-order functional units at the tissue level-from either spatial data or single-cell data obtained without spatial information. Tested on spatial datasets from the mouse hypothalamus, cerebellum, visual cortex and neocortex, DIALOGUE identified MCPs associated with animal behavior and recovered spatial properties when tested on unseen data while outperforming other methods and metrics. In spatial data from human lung cancer, DIALOGUE identified MCPs marking immune activation and tissue remodeling. Applied to single-cell RNA sequencing data across individuals or regions, DIALOGUE uncovered MCPs marking Alzheimer's disease, ulcerative colitis and resistance to cancer immunotherapy. These programs were predictive of disease outcome and predisposition in independent cohorts and included risk genes from genome-wide association studies. DIALOGUE enables the analysis of multicellular regulation in health and disease.
    DOI:  https://doi.org/10.1038/s41587-022-01288-0
  60. Life Sci. 2022 May 03. pii: S0024-3205(22)00312-5. [Epub ahead of print] 120612
    Melatonin: A mitochondrial resident with a diverse skill set.
    Russel J Reiter, Ramaswamy Sharma, Sergio Rosales-Corral, Debora Aparecida Pires de Campos Zuccari, Luiz Gustavo de Almeida Chuffa.
      Melatonin is an ancient molecule that originated in bacteria. When these prokaryotes were phagocytized by early eukaryotes, they eventually developed into mitochondria and chloroplasts. These new organelles retained the melatonin synthetic capacity of their forerunners such that all present-day animal and plant cells may produce melatonin in their mitochondria and chloroplasts. Melatonin concentrations are higher in mitochondria than in other subcellular compartments. Isolated mouse oocyte mitochondria form melatonin when they are incubated with serotonin, a necessary precursor. Oocyte mitochondria subsequently give rise to these organelles in all adult vertebrate cells where they continue to synthesize melatonin. The enzymes that convert serotonin to melatonin, i.e., arylalkylamine-N-acetyltransferase (AANAT) and acetylserotonin-O-methyltransferase, have been identified in brain mitochondria which, when incubated with serotonin, also form melatonin. Melatonin is a potent antioxidant and anti-cancer agent and is optimally positioned in mitochondria to aid in the maintenance of oxidative homeostasis and to reduce cancer cell transformation. Melatonin stimulates the transfer of mitochondria from healthy cells to damaged cells via tunneling nanotubes. Melatonin also regulates the major NAD+-dependent deacetylase, sirtuin 3, in the mitochondria. Disruptions of mitochondrial melatonin synthesis may contribute to a number of mitochondria-related diseases, as discussed in this review.
    Keywords:  Acetyl coenzyme a; Pyruvate metabolism; SIRT3; Tunneling nanotubes; cancer; sepsis
    DOI:  https://doi.org/10.1016/j.lfs.2022.120612
  61. Cell Discov. 2022 May 03. 8(1): 40
    Dynamic O-GlcNAcylation coordinates ferritinophagy and mitophagy to activate ferroptosis.
    Fan Yu, Qianping Zhang, Hanyu Liu, Jinming Liu, Song Yang, Xiaofan Luo, Wei Liu, Hao Zheng, Qiqi Liu, Yunxi Cui, Guo Chen, Yanjun Li, Xinglu Huang, Xiyun Yan, Jun Zhou, Quan Chen.
      Ferroptosis is a regulated iron-dependent cell death characterized by the accumulation of lipid peroxidation. A myriad of facets linking amino acid, lipid, redox, and iron metabolisms were found to drive or to suppress the execution of ferroptosis. However, how the cells decipher the diverse pro-ferroptotic stress to activate ferroptosis remains elusive. Here, we report that protein O-GlcNAcylation, the primary nutrient sensor of glucose flux, orchestrates both ferritinophagy and mitophagy for ferroptosis. Following the treatment of ferroptosis stimuli such as RSL3, a commonly used ferroptosis inducer, there exists a biphasic change of protein O-GlcNAcylation to modulate ferroptosis. Pharmacological or genetic inhibition of O-GlcNAcylation promoted ferritinophagy, resulting in the accumulation of labile iron towards mitochondria. Inhibition of O-GlcNAcylation resulted in mitochondria fragmentation and enhanced mitophagy, providing an additional source of labile iron and rendering the cell more sensitive to ferroptosis. Mechanistically, we found that de-O-GlcNAcylation of the ferritin heavy chain at S179 promoted its interaction with NCOA4, the ferritinophagy receptor, thereby accumulating labile iron for ferroptosis. Our findings reveal a previously uncharacterized link of dynamic O-GlcNAcylation with iron metabolism and decision-making for ferroptosis, thus offering potential therapeutic intervention for fighting disease.
    DOI:  https://doi.org/10.1038/s41421-022-00390-6
  62. J Clin Invest. 2022 May 02. pii: e158449. [Epub ahead of print]132(9):
    Mitochondria-derived peptides in aging and healthspan.
    Brendan Miller, Su-Jeong Kim, Hiroshi Kumagai, Kelvin Yen, Pinchas Cohen.
      The mechanisms that explain mitochondrial dysfunction in aging and healthspan continue to be studied, but one element has been unexplored: microproteins. Small open reading frames in circular mitochondria DNA can encode multiple microproteins, called mitochondria-derived peptides (MDPs). Currently, eight MDPs have been published: humanin, MOTS-c, and SHLPs 1-6. This Review describes recent advances in microprotein discovery with a focus on MDPs. It discusses what is currently known about MDPs in aging and how this new understanding could add to the way we understand age-related diseases including type 2 diabetes, cancer, and neurodegenerative diseases at the genomic, proteomic, and drug-development levels.
    DOI:  https://doi.org/10.1172/JCI158449
  63. Methods Mol Biol. 2022 ;2429 257-267
    In Vitro Generation of Heart Field Specific Cardiomyocytes.
    Arash Pezhouman, Ngoc B Nguyen, Allison Shevtsov, Rong Qiao, Reza Ardehali.
      Myocardial infarction (MI) can lead to irreversible loss of cardiomyocytes (CMs), primarily localized to the left ventricle (LV) of the heart. The CMs of the LV are predominantly derived from first heart field (FHF) progenitors, whereas the majority of CMs within the right ventricle originate from the second heart field (SHF) during early cardiogenesis. Human embryonic stem cells (hESCs) serve as a valuable source of CMs for understanding early cardiac development and lineage commitment of CMs within these two heart fields that ultimately enable the development of more effective candidates for cell therapy. An ideal candidate may be FHF CMs that share the same ontogeny with the LV CMs that die after MI. We previously generated a double reporter hESC line that utilizes two important cardiac transcription factors, TBX5 and NKX2-5. TBX5 marks FHF progenitors and CMs, while NKX2-5 is expressed in nearly all myocytes of the developing heart. Here, we describe a step-by-step approach to efficiently generate FHF and SHF CMs using this double reporter hESC line. In addition, this approach can be applied to any non-genetically modified hESC lines to enrich FHF and SHF CMs. Obtaining enriched populations of these two CM subtypes provides a platform for downstream comparative analyses and in vitro studies to facilitate a deeper understanding of cardiovascular lineage commitment and the development of more effective candidates for cell therapy to treat diseases or defects that affect specific regions of the heart.
    Keywords:  Cardiac differentiation; First heart field cardiomyocyte; Human embryonic stem cell; Human heart development; Second heart field cardiomyocyte
    DOI:  https://doi.org/10.1007/978-1-0716-1979-7_17
  64. Nat Commun. 2022 May 06. 13(1): 2516
    Preventing erosion of X-chromosome inactivation in human embryonic stem cells.
    Marissa Cloutier, Surinder Kumar, Emily Buttigieg, Laura Keller, Brandon Lee, Aaron Williams, Sandra Mojica-Perez, Indri Erliandri, Andre Monteiro Da Rocha, Kenneth Cadigan, Gary D Smith, Sundeep Kalantry.
      X-chromosome inactivation is a paradigm of epigenetic transcriptional regulation. Female human embryonic stem cells (hESCs) often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that culture media composition dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined xenofree medium stably maintained XIST RNA expression and coating, whereas hESCs cultured in the widely used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of lithium chloride or inhibitors of GSK-3 proteins that are targeted by lithium to the defined hESC culture medium impeded XIST RNA expression. GSK-3 inhibition in differentiating female mouse embryonic stem cells and epiblast stem cells also resulted in a loss of XIST RNA expression. Together, these data may reconcile observed variations in X-inactivation in hESCs and inform the faithful culture of pluripotent stem cells.
    DOI:  https://doi.org/10.1038/s41467-022-30259-x
  65. Nat Rev Genet. 2022 May 02.
    Making sense of the ageing methylome.
    Kirsten Seale, Steve Horvath, Andrew Teschendorff, Nir Eynon, Sarah Voisin.
      Over time, the human DNA methylation landscape accrues substantial damage, which has been associated with a broad range of age-related diseases, including cardiovascular disease and cancer. Various age-related DNA methylation changes have been described, including at the level of individual CpGs, such as differential and variable methylation, and at the level of the whole methylome, including entropy and correlation networks. Here, we review these changes in the ageing methylome as well as the statistical tools that can be used to quantify them. We detail the evidence linking DNA methylation to ageing phenotypes and the longevity strategies aimed at altering both DNA methylation patterns and machinery to extend healthspan and lifespan. Lastly, we discuss theories on the mechanistic causes of epigenetic ageing.
    DOI:  https://doi.org/10.1038/s41576-022-00477-6
  66. Methods Mol Biol. 2022 ;2432 57-71
    Cell Type-Specific Signal Analysis in Epigenome-Wide Association Studies.
    Charles E Breeze.
      Hundreds of epigenome-wide association studies (EWAS) have been performed, successfully identifying replicated epigenomic signals in processes such as aging and smoking. Despite this progress, it remains a major challenge in EWAS to detect both cell type-specific and cell type confounding effects impacting study results. One way to identify these effects is through eFORGE (experimentally derived Functional element Overlap analysis of ReGions from EWAS), a published tool that uses 815 datasets from large-scale mapping studies to detect enriched tissues, cell types, and genomic regions. Here, I show that eFORGE analysis can be extended to EWAS differentially variable positions (DVPs), identifying target cell types and tissues. In addition, I also show that eFORGE tissue-specific enrichment can be detected for sites below EWAS significance threshold. I develop on these and other analysis examples, extending our knowledge of eFORGE cell type- and tissue-specific enrichment results for different EWAS.
    Keywords:  DNA methylation; Differentially methylated position; Differentially variable position; Epigenome-wide association study
    DOI:  https://doi.org/10.1007/978-1-0716-1994-0_5
  67. Methods Mol Biol. 2022 ;2477 107-128
    FORK-seq: Single-Molecule Profiling of DNA Replication.
    Magali Hennion, Bertrand Theulot, Jean-Michel Arbona, Benjamin Audit, Olivier Hyrien.
      Most genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200 nucleotide resolution using a nanopore current interpretation tool allowing the quantification of BrdU incorporation. Along pulse-chased replication intermediates from Saccharomyces cerevisiae, we can orient replication tracks and reproduce population-based replication directionality profiles. Additionally, we can map individual initiation and termination events. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.
    Keywords:  Convolutional neural network; DNA replication; Nanopore sequencing; Replication fork direction; Replication origins; Single-molecule analysis; Termination sites; Whole-genome
    DOI:  https://doi.org/10.1007/978-1-0716-2257-5_8
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