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Madhwani KR, Sayied S, Ogata CH, Hogan CA, Lentini JM, Mallik M, Dumouchel JL, Storkebaum E, Fu D, O’Connor-Giles KM. tRNA modification enzyme-dependent redox homeostasis regulates synapse formation and memory. Proc Natl Acad Sci U S A 2024; 121:e2317864121. [PMID: 39495910 PMCID: PMC11572970 DOI: 10.1073/pnas.2317864121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2023] [Accepted: 03/26/2024] [Indexed: 11/06/2024] Open
Abstract
Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA-modifying enzyme that methylates wobble uridines in a subset of tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Through in vivo studies in Drosophila, we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory. ALKBH8 null animals lack wobble uridine methylation and exhibit reduced protein synthesis in the nervous system, including a specific decrease in selenoprotein levels. Either loss of ALKBH8 or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth in ALKBH8 null animals, confirming oxidative stress as the underlying cause of dysregulation. ALKBH8 null animals also exhibit associative memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA wobble uridine modification in redox homeostasis in the developing nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability.
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Affiliation(s)
| | - Shanzeh Sayied
- Department of Neuroscience, Brown University, Providence, RI02912
| | | | - Caley A. Hogan
- Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI53706
| | - Jenna M. Lentini
- Department of Biology, Center for RNA Biology, University of Rochester, Rochester, NY14627
| | - Moushami Mallik
- Molecular Neurobiology Laboratory, Donders Institute for Brain, Cognition, and Behaviour, Radboud University, Nijmegen6525 AJ, The Netherlands
| | | | - Erik Storkebaum
- Molecular Neurobiology Laboratory, Donders Institute for Brain, Cognition, and Behaviour, Radboud University, Nijmegen6525 AJ, The Netherlands
| | - Dragony Fu
- Department of Biology, Center for RNA Biology, University of Rochester, Rochester, NY14627
| | - Kate M. O’Connor-Giles
- Department of Neuroscience, Brown University, Providence, RI02912
- Carney Institute for Brain Sciences, Brown University, Providence, RI02912
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2
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Xu F, Chen A, Pan S, Wu Y, He H, Han Z, Lu L, Orgil B, Chi X, Yang C, Jia S, Yu C, Mi J. Systems genetics analysis reveals the common genetic basis for pain sensitivity and cognitive function. CNS Neurosci Ther 2024; 30:e14557. [PMID: 38421132 PMCID: PMC10850811 DOI: 10.1111/cns.14557] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Revised: 10/31/2023] [Accepted: 11/25/2023] [Indexed: 03/02/2024] Open
Abstract
BACKGROUND There is growing evidence of a strong correlation between pain sensitivity and cognitive function under both physiological and pathological conditions. However, the detailed mechanisms remain largely unknown. In the current study, we sought to explore candidate genes and common molecular mechanisms underlying pain sensitivity and cognitive function with a transcriptome-wide association study using recombinant inbred mice from the BXD family. METHODS The pain sensitivity determined by Hargreaves' paw withdrawal test and cognition-related phenotypes were systematically analyzed in 60 strains of BXD mice and correlated with hippocampus transcriptomes, followed by quantitative trait locus (QTL) mapping and systems genetics analysis. RESULTS The pain sensitivity showed significant variability across the BXD strains and co-varies with cognitive traits. Pain sensitivity correlated hippocampual genes showed a significant involvement in cognition-related pathways, including glutamatergic synapse, and PI3K-Akt signaling pathway. Moreover, QTL mapping identified a genomic region on chromosome 4, potentially regulating the variation of pain sensitivity. Integrative analysis of expression QTL mapping, correlation analysis, and Bayesian network modeling identified Ring finger protein 20 (Rnf20) as the best candidate. Further pathway analysis indicated that Rnf20 may regulate the expression of pain sensitivity and cognitive function through the PI3K-Akt signaling pathway, particularly through interactions with genes Ppp2r2b, Ppp2r5c, Col9a3, Met, Rps6, Tnc, and Kras. CONCLUSIONS Our study demonstrated that pain sensitivity is associated with genetic background and Rnf20-mediated PI3K-Akt signaling may involve in the regulation of pain sensitivity and cognitive functions.
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Affiliation(s)
- Fuyi Xu
- Shandong Technology Innovation Center of Molecular Targeting and Intelligent Diagnosis and TreatmentBinzhou Medical UniversityYantaiChina
| | - Anran Chen
- The Affiliated Yantai Yuhuangding Hospital of Qingdao UniversityYantaiChina
| | - Shuijing Pan
- Shandong Technology Innovation Center of Molecular Targeting and Intelligent Diagnosis and TreatmentBinzhou Medical UniversityYantaiChina
| | - Yingying Wu
- Shandong Technology Innovation Center of Molecular Targeting and Intelligent Diagnosis and TreatmentBinzhou Medical UniversityYantaiChina
| | - Hongjie He
- Shandong Technology Innovation Center of Molecular Targeting and Intelligent Diagnosis and TreatmentBinzhou Medical UniversityYantaiChina
| | - Zhe Han
- Shandong Technology Innovation Center of Molecular Targeting and Intelligent Diagnosis and TreatmentBinzhou Medical UniversityYantaiChina
| | - Lu Lu
- University of Tennessee Health Science CenterMemphisTennesseeUSA
| | | | - XiaoDong Chi
- Shandong Technology Innovation Center of Molecular Targeting and Intelligent Diagnosis and TreatmentBinzhou Medical UniversityYantaiChina
| | - Cunhua Yang
- Shandong Technology Innovation Center of Molecular Targeting and Intelligent Diagnosis and TreatmentBinzhou Medical UniversityYantaiChina
| | - Shushan Jia
- Department of AnesthesiologyYanTai Affiliated Hospital of BinZhou Medical UniversityYantaiChina
| | - Cuicui Yu
- The Affiliated Yantai Yuhuangding Hospital of Qingdao UniversityYantaiChina
| | - Jia Mi
- Shandong Technology Innovation Center of Molecular Targeting and Intelligent Diagnosis and TreatmentBinzhou Medical UniversityYantaiChina
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3
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Madhwani KR, Sayied S, Ogata CH, Hogan CA, Lentini JM, Mallik M, Dumouchel JL, Storkebaum E, Fu D, O’Connor-Giles KM. tRNA modification enzyme-dependent redox homeostasis regulates synapse formation and memory. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.14.566895. [PMID: 38014328 PMCID: PMC10680711 DOI: 10.1101/2023.11.14.566895] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/29/2023]
Abstract
Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA modifying enzyme that methylates wobble uridines in specific tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Through in vivo studies in Drosophila, we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory. ALKBH8 null animals lack wobble uridine methylation and exhibit a global reduction in protein synthesis, including a specific decrease in selenoprotein levels. Loss of ALKBH8 or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth in ALKBH8 null animals, confirming oxidative stress as the underlying cause of dysregulation. ALKBH8 animals also exhibit associative learning and memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA modification in redox homeostasis in the nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability.
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Affiliation(s)
| | - Shanzeh Sayied
- Department of Neuroscience, Brown University, Providence, RI, USA
| | | | - Caley A. Hogan
- Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, USA
| | - Jenna M. Lentini
- Department of Biology, Center for RNA Biology, University of Rochester, Rochester, NY, USA
| | - Moushami Mallik
- Molecular Neurobiology Laboratory, Donders Institute for Brain, Cognition, and Behaviour, Radboud University, Nijmegen, NL
| | | | - Erik Storkebaum
- Molecular Neurobiology Laboratory, Donders Institute for Brain, Cognition, and Behaviour, Radboud University, Nijmegen, NL
| | - Dragony Fu
- Department of Biology, Center for RNA Biology, University of Rochester, Rochester, NY, USA
| | - Kate M. O’Connor-Giles
- Department of Neuroscience, Brown University, Providence, RI, USA
- Carney Institute for Brain Sciences, Brown University, Providence, RI, USA
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Patkar OL, Caruso M, Teakle N, Keshvari S, Bush SJ, Pridans C, Belmer A, Summers KM, Irvine KM, Hume DA. Analysis of homozygous and heterozygous Csf1r knockout in the rat as a model for understanding microglial function in brain development and the impacts of human CSF1R mutations. Neurobiol Dis 2021; 151:105268. [PMID: 33450391 PMCID: PMC7941205 DOI: 10.1016/j.nbd.2021.105268] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2020] [Revised: 01/06/2021] [Accepted: 01/07/2021] [Indexed: 12/11/2022] Open
Abstract
Mutations in the human CSF1R gene have been associated with dominant and recessive forms of neurodegenerative disease. Here we describe the impacts of Csf1r mutation in the rat on development of the brain. Diffusion imaging indicated small reductions in major fiber tracts that may be associated in part with ventricular enlargement. RNA-seq profiling revealed a set of 105 microglial markers depleted in all brain regions of the Csf1rko rats. There was no evidence of region or sex-specific expression of microglia-associated transcripts. Other than the microglial signature, Csf1rko had no effect on any neuronal or region-specific transcript cluster. Expression of markers of oligodendrocytes, astrocytes, dopaminergic neurons and Purkinje cells was minimally affected. However, there were defects in dendritic arborization of doublecortin-positive neurogenic precursors and expression of poly-sialylated neural cell adhesion molecule (PS-NCAM) in the dentate gyrus of the hippocampus. Heterozygous Csf1rko rats had no detectable brain phenotype. We conclude that most brain developmental processes occur normally in the absence of microglia and that CSF1R haploinsufficiency is unlikely to cause leukoencephalopathy.
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Affiliation(s)
- Omkar L Patkar
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Melanie Caruso
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Ngari Teakle
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Sahar Keshvari
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Stephen J Bush
- Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK
| | - Clare Pridans
- University of Edinburgh Centre for Inflammation Research, Edinburgh, UK and Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, UK
| | - Arnauld Belmer
- School of Clinical Sciences, Queensland University of Technology, Brisbane, Australia
| | - Kim M Summers
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Katharine M Irvine
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - David A Hume
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia.
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5
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Haas Bueno R, Recamonde-Mendoza M. Meta-analysis of Transcriptomic Data Reveals Pathophysiological Modules Involved with Atrial Fibrillation. Mol Diagn Ther 2020; 24:737-751. [PMID: 33095430 DOI: 10.1007/s40291-020-00497-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/30/2020] [Indexed: 02/06/2023]
Abstract
BACKGROUND Atrial fibrillation (AF) is a complex disease and affects millions of people around the world. The biological mechanisms that are involved with AF are complex and still need to be fully elucidated. Therefore, we performed a meta-analysis of transcriptome data related to AF to explore these mechanisms aiming at more sensitive and reliable results. METHODS Ten public transcriptomic datasets were downloaded, analyzed for quality control, and individually pre-processed. Differential expression analysis was carried out for each dataset, and the results were meta-analytically aggregated using the rth ordered p value method. We analyzed the final list of differentially expressed genes through network analysis, namely topological and modularity analysis, and functional enrichment analysis. RESULTS The meta-analysis of transcriptomes resulted in 1197 differentially expressed genes, whose protein-protein interaction network presented 39 hubs-bottlenecks and four main identified functional modules. These modules were enriched for 39, 20, 64, and 10 biological pathways involved with the pathophysiology of AF, especially with the disease's structural and electrical remodeling processes. The stress of the endoplasmic reticulum, protein catabolism, oxidative stress, and inflammation are some of the enriched processes. Among hub-bottlenecks genes, which are highly connected and probably have a key role in regulating these processes, HSPA5, ANK2, CTNNB1, and MAPK1 were identified. CONCLUSION Our approach based on transcriptome meta-analysis revealed a set of key genes that demonstrated consistent overall changes in expression patterns associated with AF despite data heterogeneity related, among others, to type of tissue. Further experimental investigation of our findings may shed light on the pathophysiology of the disease and contribute to the identification of new therapeutic targets.
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Affiliation(s)
- Rodrigo Haas Bueno
- Experimental and Molecular Cardiovascular Laboratory, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS, Brazil
- Bioinformatics Core, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS, Brazil
| | - Mariana Recamonde-Mendoza
- Experimental and Molecular Cardiovascular Laboratory, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS, Brazil.
- Bioinformatics Core, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS, Brazil.
- Institute of Informatics, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.
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Panula S, Kurek M, Kumar P, Albalushi H, Padrell Sánchez S, Damdimopoulou P, Olofsson JI, Hovatta O, Lanner F, Stukenborg JB. Human induced pluripotent stem cells from two azoospermic patients with Klinefelter syndrome show similar X chromosome inactivation behavior to female pluripotent stem cells. Hum Reprod 2020; 34:2297-2310. [PMID: 31743397 PMCID: PMC6894010 DOI: 10.1093/humrep/dez134] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2019] [Revised: 06/15/2019] [Indexed: 12/21/2022] Open
Abstract
STUDY QUESTION Does the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype? SUMMARY ANSWER Our results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features. WHAT IS KNOWN ALREADY So far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture. STUDY DESIGN, SIZE, DURATION The study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor. PARTICIPANTS/MATERIALS, SETTING, METHODS In this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives. MAIN RESULTS AND THE ROLE OF CHANCE Applying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS. LIMITATIONS, REASONS FOR CAUTION Detailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments. WIDER IMPLICATIONS OF THE FINDINGS As KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI ‘escapee’ genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2–388/2016–40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program ‘Growsperm’ (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest.
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Affiliation(s)
- Sarita Panula
- Department of Clinical Sciences, Intervention and Technology, Division of Gynecology and Reproductive Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden.,Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Stockholm, Sweden
| | - Magdalena Kurek
- NORDFERTIL Research Laboratory Stockholm, Childhood Cancer Research Unit, Bioclinicum J9:30, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, Solna, Sweden
| | - Pankaj Kumar
- Department of Clinical Sciences, Intervention and Technology, Division of Gynecology and Reproductive Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden.,Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Stockholm, Sweden
| | - Halima Albalushi
- NORDFERTIL Research Laboratory Stockholm, Childhood Cancer Research Unit, Bioclinicum J9:30, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, Solna, Sweden.,College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
| | - Sara Padrell Sánchez
- Department of Clinical Sciences, Intervention and Technology, Division of Gynecology and Reproductive Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden.,Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Stockholm, Sweden
| | - Pauliina Damdimopoulou
- Department of Clinical Sciences, Intervention and Technology, Division of Gynecology and Reproductive Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
| | - Jan I Olofsson
- Division of Obstetrics and Gynecology, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, Solna, Sweden
| | - Outi Hovatta
- Department of Clinical Sciences, Intervention and Technology, Division of Gynecology and Reproductive Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
| | - Fredrik Lanner
- Department of Clinical Sciences, Intervention and Technology, Division of Gynecology and Reproductive Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden.,Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Stockholm, Sweden
| | - Jan-Bernd Stukenborg
- NORDFERTIL Research Laboratory Stockholm, Childhood Cancer Research Unit, Bioclinicum J9:30, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, Solna, Sweden
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Tsang HG, Clark EL, Markby GR, Bush SJ, Hume DA, Corcoran BM, MacRae VE, Summers KM. Expression of Calcification and Extracellular Matrix Genes in the Cardiovascular System of the Healthy Domestic Sheep ( Ovis aries). Front Genet 2020; 11:919. [PMID: 33101359 PMCID: PMC7506100 DOI: 10.3389/fgene.2020.00919] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2020] [Accepted: 07/23/2020] [Indexed: 12/31/2022] Open
Abstract
The maintenance of a healthy cardiovascular system requires expression of genes that contribute to essential biological activities and repression of those that are associated with functions likely to be detrimental to cardiovascular homeostasis. Vascular calcification is a major disruption to cardiovascular homeostasis, where tissues of the cardiovascular system undergo ectopic calcification and consequent dysfunction, but little is known about the expression of calcification genes in the healthy cardiovascular system. Large animal models are of increasing importance in cardiovascular disease research as they demonstrate more similar cardiovascular features (in terms of anatomy, physiology and size) to humans than do rodent species. We used RNA sequencing results from the sheep, which has been utilized extensively to examine calcification of prosthetic cardiac valves, to explore the transcriptome of the heart and cardiac valves in this large animal, in particular looking at expression of calcification and extracellular matrix genes. We then examined genes implicated in the process of vascular calcification in a wide array of cardiovascular tissues and across multiple developmental stages, using RT-qPCR. Our results demonstrate that there is a balance between genes that promote and those that suppress mineralization during development and across cardiovascular tissues. We show extensive expression of genes encoding proteins involved in formation and maintenance of the extracellular matrix in cardiovascular tissues, and high expression of hematopoietic genes in the cardiac valves. Our analysis will support future research into the functions of implicated genes in the development of valve calcification, and increase the utility of the sheep as a large animal model for understanding ectopic calcification in cardiovascular disease. This study provides a foundation to explore the transcriptome of the developing cardiovascular system and is a valuable resource for the fields of mammalian genomics and cardiovascular research.
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Affiliation(s)
- Hiu-Gwen Tsang
- The Roslin Institute and R(D)SVS, The University of Edinburgh, Edinburgh, United Kingdom
| | - Emily L. Clark
- The Roslin Institute and R(D)SVS, The University of Edinburgh, Edinburgh, United Kingdom
| | - Greg R. Markby
- The Roslin Institute and R(D)SVS, The University of Edinburgh, Edinburgh, United Kingdom
| | - Stephen J. Bush
- The Roslin Institute and R(D)SVS, The University of Edinburgh, Edinburgh, United Kingdom
- Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
| | - David A. Hume
- Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia
| | - Brendan M. Corcoran
- The Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Edinburgh, United Kingdom
| | - Vicky E. MacRae
- The Roslin Institute and R(D)SVS, The University of Edinburgh, Edinburgh, United Kingdom
| | - Kim M. Summers
- The Roslin Institute and R(D)SVS, The University of Edinburgh, Edinburgh, United Kingdom
- Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia
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8
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Summers KM, Bush SJ, Wu C, Su AI, Muriuki C, Clark EL, Finlayson HA, Eory L, Waddell LA, Talbot R, Archibald AL, Hume DA. Functional Annotation of the Transcriptome of the Pig, Sus scrofa, Based Upon Network Analysis of an RNAseq Transcriptional Atlas. Front Genet 2020; 10:1355. [PMID: 32117413 PMCID: PMC7034361 DOI: 10.3389/fgene.2019.01355] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2019] [Accepted: 12/11/2019] [Indexed: 12/15/2022] Open
Abstract
The domestic pig (Sus scrofa) is both an economically important livestock species and a model for biomedical research. Two highly contiguous pig reference genomes have recently been released. To support functional annotation of the pig genomes and comparative analysis with large human transcriptomic data sets, we aimed to create a pig gene expression atlas. To achieve this objective, we extended a previous approach developed for the chicken. We downloaded RNAseq data sets from public repositories, down-sampled to a common depth, and quantified expression against a reference transcriptome using the mRNA quantitation tool, Kallisto. We then used the network analysis tool Graphia to identify clusters of transcripts that were coexpressed across the merged data set. Consistent with the principle of guilt-by-association, we identified coexpression clusters that were highly tissue or cell-type restricted and contained transcription factors that have previously been implicated in lineage determination. Other clusters were enriched for transcripts associated with biological processes, such as the cell cycle and oxidative phosphorylation. The same approach was used to identify coexpression clusters within RNAseq data from multiple individual liver and brain samples, highlighting cell type, process, and region-specific gene expression. Evidence of conserved expression can add confidence to assignment of orthology between pig and human genes. Many transcripts currently identified as novel genes with ENSSSCG or LOC IDs were found to be coexpressed with annotated neighbouring transcripts in the same orientation, indicating they may be products of the same transcriptional unit. The meta-analytic approach to utilising public RNAseq data is extendable to include new data sets and new species and provides a framework to support the Functional Annotation of Animals Genomes (FAANG) initiative.
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Affiliation(s)
- Kim M. Summers
- Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia
| | - Stephen J. Bush
- Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom
| | - Chunlei Wu
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, United States
| | - Andrew I. Su
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, United States
| | - Charity Muriuki
- The Roslin Institute, University of Edinburgh, Midlothian, United Kingdom
| | - Emily L. Clark
- The Roslin Institute, University of Edinburgh, Midlothian, United Kingdom
| | | | - Lel Eory
- The Roslin Institute, University of Edinburgh, Midlothian, United Kingdom
| | - Lindsey A. Waddell
- The Roslin Institute, University of Edinburgh, Midlothian, United Kingdom
| | - Richard Talbot
- The Roslin Institute, University of Edinburgh, Midlothian, United Kingdom
| | - Alan L. Archibald
- The Roslin Institute, University of Edinburgh, Midlothian, United Kingdom
| | - David A. Hume
- Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia
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9
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Hume DA, Caruso M, Ferrari-Cestari M, Summers KM, Pridans C, Irvine KM. Phenotypic impacts of CSF1R deficiencies in humans and model organisms. J Leukoc Biol 2019; 107:205-219. [PMID: 31330095 DOI: 10.1002/jlb.mr0519-143r] [Citation(s) in RCA: 80] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2019] [Revised: 06/20/2019] [Accepted: 07/01/2019] [Indexed: 12/12/2022] Open
Abstract
Mϕ proliferation, differentiation, and survival are controlled by signals from the Mϕ CSF receptor (CSF1R). Mono-allelic gain-of-function mutations in CSF1R in humans are associated with an autosomal-dominant leukodystrophy and bi-allelic loss-of-function mutations with recessive skeletal dysplasia, brain disorders, and developmental anomalies. Most of the phenotypes observed in these human disease states are also observed in mice and rats with loss-of-function mutations in Csf1r or in Csf1 encoding one of its two ligands. Studies in rodent models also highlight the importance of genetic background and likely epistatic interactions between Csf1r and other loci. The impacts of Csf1r mutations on the brain are usually attributed solely to direct impacts on microglial number and function. However, analysis of hypomorphic Csf1r mutants in mice and several other lines of evidence suggest that primary hydrocephalus and loss of the physiological functions of Mϕs in the periphery contribute to the development of brain pathology. In this review, we outline the evidence that CSF1R is expressed exclusively in mononuclear phagocytes and explore the mechanisms linking CSF1R mutations to pleiotropic impacts on postnatal growth and development.
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Affiliation(s)
- David A Hume
- Mater Research Institute, University of Queensland, Woolloongabba, Queensland, Australia
| | - Melanie Caruso
- Mater Research Institute, University of Queensland, Woolloongabba, Queensland, Australia
| | | | - Kim M Summers
- Mater Research Institute, University of Queensland, Woolloongabba, Queensland, Australia
| | - Clare Pridans
- Centre for Inflammation Research, The University of Edinburgh, Edinburgh, United Kingdom
| | - Katharine M Irvine
- Mater Research Institute, University of Queensland, Woolloongabba, Queensland, Australia
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Combination of novel and public RNA-seq datasets to generate an mRNA expression atlas for the domestic chicken. BMC Genomics 2018; 19:594. [PMID: 30086717 PMCID: PMC6081845 DOI: 10.1186/s12864-018-4972-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2017] [Accepted: 07/31/2018] [Indexed: 12/20/2022] Open
Abstract
Background The domestic chicken (Gallus gallus) is widely used as a model in developmental biology and is also an important livestock species. We describe a novel approach to data integration to generate an mRNA expression atlas for the chicken spanning major tissue types and developmental stages, using a diverse range of publicly-archived RNA-seq datasets and new data derived from immune cells and tissues. Results Randomly down-sampling RNA-seq datasets to a common depth and quantifying expression against a reference transcriptome using the mRNA quantitation tool Kallisto ensured that disparate datasets explored comparable transcriptomic space. The network analysis tool Graphia was used to extract clusters of co-expressed genes from the resulting expression atlas, many of which were tissue or cell-type restricted, contained transcription factors that have previously been implicated in their regulation, or were otherwise associated with biological processes, such as the cell cycle. The atlas provides a resource for the functional annotation of genes that currently have only a locus ID. We cross-referenced the RNA-seq atlas to a publicly available embryonic Cap Analysis of Gene Expression (CAGE) dataset to infer the developmental time course of organ systems, and to identify a signature of the expansion of tissue macrophage populations during development. Conclusion Expression profiles obtained from public RNA-seq datasets – despite being generated by different laboratories using different methodologies – can be made comparable to each other. This meta-analytic approach to RNA-seq can be extended with new datasets from novel tissues, and is applicable to any species. Electronic supplementary material The online version of this article (10.1186/s12864-018-4972-7) contains supplementary material, which is available to authorized users.
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