Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy
Aims/hypothesis Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of mole...
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| Veröffentlicht in: | Diabetologia 2020-10, Vol.63 (10), p.2235-2248 |
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| creator | Van Hove, Inge De Groef, Lies Boeckx, Bram Modave, Elodie Hu, Tjing-Tjing Beets, Karen Etienne, Isabelle Van Bergen, Tine Lambrechts, Diether Moons, Lieve Feyen, Jean H. M. Porcu, Michaël |
| description | Aims/hypothesis
Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by systematically mapping the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina.
Methods
Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (
Ins2
Akita
×
Vegfa
+/
–
) mice, which are known to replicate features of clinical diabetic retinopathy. This resulted in transcriptome data for 9474 retinal cells, which could be annotated to eight distinct retinal cell types. Using STRING analysis, we studied differentially expressed gene networks in neuronal, glial and immune cell compartments to create a comprehensive view on the pathological changes that occur in the Akimba retina. Using subclustering analysis, we further characterised macroglial and inflammatory cell subpopulations. Prominent findings were confirmed at the protein level using immunohistochemistry, western blotting and ELISA.
Results
At 12 weeks, the Akimba retina was found to display degeneration of rod photoreceptors and presence of inflammatory cells, identified by subclustering analysis as monocyte, macrophage and microglial populations. Analysis of differentially expressed genes in the rod, cone, bipolar cell and macroglial compartments indicated changes in cell metabolism and ribosomal gene expression, gliosis, activation of immune system pathways and redox and metal ion dyshomeostasis. Experiments at the protein level supported a metabolic shift from glycolysis to oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase), activation of microglia/macrophages (isolectin-B4), metal ion and oxidative stress response (metallothionein and haem oxygenase-1) and reactive macroglia (glial fibrillary acidic protein and S100) in the Akimba retina, compared with wild-type mice. Our single-cell approach also indicates macroglial subpopulations with distinct fibrotic, inflammatory and gliotic profiles.
Conclusions/interpretation
Our study identifies molecular pathways underlying inflammatory, metabolic and oxidative stress-mediated changes in the Akimba mouse model of diabetic retinopathy and distinguishes distinct functional s |
| doi_str_mv | 10.1007/s00125-020-05218-0 |
| format | Article |
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Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by systematically mapping the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina.
Methods
Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (
Ins2
Akita
×
Vegfa
+/
–
) mice, which are known to replicate features of clinical diabetic retinopathy. This resulted in transcriptome data for 9474 retinal cells, which could be annotated to eight distinct retinal cell types. Using STRING analysis, we studied differentially expressed gene networks in neuronal, glial and immune cell compartments to create a comprehensive view on the pathological changes that occur in the Akimba retina. Using subclustering analysis, we further characterised macroglial and inflammatory cell subpopulations. Prominent findings were confirmed at the protein level using immunohistochemistry, western blotting and ELISA.
Results
At 12 weeks, the Akimba retina was found to display degeneration of rod photoreceptors and presence of inflammatory cells, identified by subclustering analysis as monocyte, macrophage and microglial populations. Analysis of differentially expressed genes in the rod, cone, bipolar cell and macroglial compartments indicated changes in cell metabolism and ribosomal gene expression, gliosis, activation of immune system pathways and redox and metal ion dyshomeostasis. Experiments at the protein level supported a metabolic shift from glycolysis to oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase), activation of microglia/macrophages (isolectin-B4), metal ion and oxidative stress response (metallothionein and haem oxygenase-1) and reactive macroglia (glial fibrillary acidic protein and S100) in the Akimba retina, compared with wild-type mice. Our single-cell approach also indicates macroglial subpopulations with distinct fibrotic, inflammatory and gliotic profiles.
Conclusions/interpretation
Our study identifies molecular pathways underlying inflammatory, metabolic and oxidative stress-mediated changes in the Akimba mouse model of diabetic retinopathy and distinguishes distinct functional subtypes of inflammatory and macroglial cells.
Data availability
RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (
www.ebi.ac.uk/arrayexpress
) under accession number E-MTAB-9061.
Graphical abstract</description><identifier>ISSN: 0012-186X</identifier><identifier>EISSN: 1432-0428</identifier><identifier>DOI: 10.1007/s00125-020-05218-0</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Bipolar cells ; Blindness ; Cell activation ; Diabetes ; Diabetes mellitus ; Diabetic retinopathy ; Enzyme-linked immunosorbent assay ; Gene expression ; Glia ; Glial fibrillary acidic protein ; Gliosis ; Glyceraldehyde ; Glyceraldehyde 3-phosphate ; Glyceraldehyde-3-phosphate dehydrogenase ; Glycolysis ; Heme oxygenase (decyclizing) ; Human Physiology ; Immune system ; Immunohistochemistry ; Inflammation ; Internal Medicine ; Macrophages ; Medicine ; Medicine & Public Health ; Metabolic Diseases ; Metabolism ; Microglia ; Neuronal-glial interactions ; Oxidative phosphorylation ; Oxidative stress ; Photoreceptors ; Proteins ; Retina ; Ribonucleic acid ; RNA ; Transcription ; Western blotting</subject><ispartof>Diabetologia, 2020-10, Vol.63 (10), p.2235-2248</ispartof><rights>Springer-Verlag GmbH Germany, part of Springer Nature 2020</rights><rights>Springer-Verlag GmbH Germany, part of Springer Nature 2020.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c424t-928b365e4ea5125d50de00d3766e2b38e7c5eca7f9730d2322b2f10c0fafdb1b3</citedby><cites>FETCH-LOGICAL-c424t-928b365e4ea5125d50de00d3766e2b38e7c5eca7f9730d2322b2f10c0fafdb1b3</cites><orcidid>0000-0002-9369-5547</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00125-020-05218-0$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00125-020-05218-0$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>315,781,785,27926,27927,41490,42559,51321</link.rule.ids></links><search><creatorcontrib>Van Hove, Inge</creatorcontrib><creatorcontrib>De Groef, Lies</creatorcontrib><creatorcontrib>Boeckx, Bram</creatorcontrib><creatorcontrib>Modave, Elodie</creatorcontrib><creatorcontrib>Hu, Tjing-Tjing</creatorcontrib><creatorcontrib>Beets, Karen</creatorcontrib><creatorcontrib>Etienne, Isabelle</creatorcontrib><creatorcontrib>Van Bergen, Tine</creatorcontrib><creatorcontrib>Lambrechts, Diether</creatorcontrib><creatorcontrib>Moons, Lieve</creatorcontrib><creatorcontrib>Feyen, Jean H. M.</creatorcontrib><creatorcontrib>Porcu, Michaël</creatorcontrib><title>Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy</title><title>Diabetologia</title><addtitle>Diabetologia</addtitle><description>Aims/hypothesis
Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by systematically mapping the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina.
Methods
Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (
Ins2
Akita
×
Vegfa
+/
–
) mice, which are known to replicate features of clinical diabetic retinopathy. This resulted in transcriptome data for 9474 retinal cells, which could be annotated to eight distinct retinal cell types. Using STRING analysis, we studied differentially expressed gene networks in neuronal, glial and immune cell compartments to create a comprehensive view on the pathological changes that occur in the Akimba retina. Using subclustering analysis, we further characterised macroglial and inflammatory cell subpopulations. Prominent findings were confirmed at the protein level using immunohistochemistry, western blotting and ELISA.
Results
At 12 weeks, the Akimba retina was found to display degeneration of rod photoreceptors and presence of inflammatory cells, identified by subclustering analysis as monocyte, macrophage and microglial populations. Analysis of differentially expressed genes in the rod, cone, bipolar cell and macroglial compartments indicated changes in cell metabolism and ribosomal gene expression, gliosis, activation of immune system pathways and redox and metal ion dyshomeostasis. Experiments at the protein level supported a metabolic shift from glycolysis to oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase), activation of microglia/macrophages (isolectin-B4), metal ion and oxidative stress response (metallothionein and haem oxygenase-1) and reactive macroglia (glial fibrillary acidic protein and S100) in the Akimba retina, compared with wild-type mice. Our single-cell approach also indicates macroglial subpopulations with distinct fibrotic, inflammatory and gliotic profiles.
Conclusions/interpretation
Our study identifies molecular pathways underlying inflammatory, metabolic and oxidative stress-mediated changes in the Akimba mouse model of diabetic retinopathy and distinguishes distinct functional subtypes of inflammatory and macroglial cells.
Data availability
RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (
www.ebi.ac.uk/arrayexpress
) under accession number E-MTAB-9061.
Graphical abstract</description><subject>Bipolar cells</subject><subject>Blindness</subject><subject>Cell activation</subject><subject>Diabetes</subject><subject>Diabetes mellitus</subject><subject>Diabetic retinopathy</subject><subject>Enzyme-linked immunosorbent assay</subject><subject>Gene expression</subject><subject>Glia</subject><subject>Glial fibrillary acidic protein</subject><subject>Gliosis</subject><subject>Glyceraldehyde</subject><subject>Glyceraldehyde 3-phosphate</subject><subject>Glyceraldehyde-3-phosphate dehydrogenase</subject><subject>Glycolysis</subject><subject>Heme oxygenase (decyclizing)</subject><subject>Human Physiology</subject><subject>Immune system</subject><subject>Immunohistochemistry</subject><subject>Inflammation</subject><subject>Internal Medicine</subject><subject>Macrophages</subject><subject>Medicine</subject><subject>Medicine & Public Health</subject><subject>Metabolic Diseases</subject><subject>Metabolism</subject><subject>Microglia</subject><subject>Neuronal-glial interactions</subject><subject>Oxidative phosphorylation</subject><subject>Oxidative stress</subject><subject>Photoreceptors</subject><subject>Proteins</subject><subject>Retina</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>Transcription</subject><subject>Western blotting</subject><issn>0012-186X</issn><issn>1432-0428</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><recordid>eNp9kctq3DAUhkVpodNJXqArQTfdqD262Z5lCL1BIIu0kJ2Q5eMZpbblSpqCnyKvXDkuFLrI6gjO939w9BPylsMHDlB_TABcaAYCGGjBGwYvyI4rKRgo0bwku3XPeFPdvyZvUnoAAKlVtSOPd346DsgcDgPN0U7JRT_nMCK1kx2W5BMNPc0npFc__dhaOoZzQhox-8mW8RvtkOgaZ3mZkaUZne-9o35K_njKqTxyeBLMNp9C68MQjssq7bxti8ZtsrCulwvyqi9CvPw79-TH50_fr7-ym9sv366vbphTQmV2EE0rK40KrS6Hdxo6BOhkXVUoWtlg7TQ6W_eHWkInpBCt6Dk46G3ftbyVe_J-884x_Dpjymb0ab3CTlgONEKJQ5HJkt-Td_-hD-Ecy-cUSuuKaxAH_iylFGjFKykLJTbKxZBSxN7M0Y82LoaDWZs0W5OmNGmemjRQQnILpQJPR4z_1M-k_gAlYaMF</recordid><startdate>20201001</startdate><enddate>20201001</enddate><creator>Van Hove, Inge</creator><creator>De Groef, Lies</creator><creator>Boeckx, Bram</creator><creator>Modave, Elodie</creator><creator>Hu, Tjing-Tjing</creator><creator>Beets, Karen</creator><creator>Etienne, Isabelle</creator><creator>Van Bergen, Tine</creator><creator>Lambrechts, Diether</creator><creator>Moons, Lieve</creator><creator>Feyen, Jean H. M.</creator><creator>Porcu, Michaël</creator><general>Springer Berlin Heidelberg</general><general>Springer Nature B.V</general><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7T5</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>H94</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-9369-5547</orcidid></search><sort><creationdate>20201001</creationdate><title>Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy</title><author>Van Hove, Inge ; De Groef, Lies ; Boeckx, Bram ; Modave, Elodie ; Hu, Tjing-Tjing ; Beets, Karen ; Etienne, Isabelle ; Van Bergen, Tine ; Lambrechts, Diether ; Moons, Lieve ; Feyen, Jean H. M. ; Porcu, Michaël</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c424t-928b365e4ea5125d50de00d3766e2b38e7c5eca7f9730d2322b2f10c0fafdb1b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Bipolar cells</topic><topic>Blindness</topic><topic>Cell activation</topic><topic>Diabetes</topic><topic>Diabetes mellitus</topic><topic>Diabetic retinopathy</topic><topic>Enzyme-linked immunosorbent assay</topic><topic>Gene expression</topic><topic>Glia</topic><topic>Glial fibrillary acidic protein</topic><topic>Gliosis</topic><topic>Glyceraldehyde</topic><topic>Glyceraldehyde 3-phosphate</topic><topic>Glyceraldehyde-3-phosphate dehydrogenase</topic><topic>Glycolysis</topic><topic>Heme oxygenase (decyclizing)</topic><topic>Human Physiology</topic><topic>Immune system</topic><topic>Immunohistochemistry</topic><topic>Inflammation</topic><topic>Internal Medicine</topic><topic>Macrophages</topic><topic>Medicine</topic><topic>Medicine & Public Health</topic><topic>Metabolic Diseases</topic><topic>Metabolism</topic><topic>Microglia</topic><topic>Neuronal-glial interactions</topic><topic>Oxidative phosphorylation</topic><topic>Oxidative stress</topic><topic>Photoreceptors</topic><topic>Proteins</topic><topic>Retina</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>Transcription</topic><topic>Western blotting</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Van Hove, Inge</creatorcontrib><creatorcontrib>De Groef, Lies</creatorcontrib><creatorcontrib>Boeckx, Bram</creatorcontrib><creatorcontrib>Modave, Elodie</creatorcontrib><creatorcontrib>Hu, Tjing-Tjing</creatorcontrib><creatorcontrib>Beets, Karen</creatorcontrib><creatorcontrib>Etienne, Isabelle</creatorcontrib><creatorcontrib>Van Bergen, Tine</creatorcontrib><creatorcontrib>Lambrechts, Diether</creatorcontrib><creatorcontrib>Moons, Lieve</creatorcontrib><creatorcontrib>Feyen, Jean H. M.</creatorcontrib><creatorcontrib>Porcu, Michaël</creatorcontrib><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Immunology Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><jtitle>Diabetologia</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Van Hove, Inge</au><au>De Groef, Lies</au><au>Boeckx, Bram</au><au>Modave, Elodie</au><au>Hu, Tjing-Tjing</au><au>Beets, Karen</au><au>Etienne, Isabelle</au><au>Van Bergen, Tine</au><au>Lambrechts, Diether</au><au>Moons, Lieve</au><au>Feyen, Jean H. M.</au><au>Porcu, Michaël</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy</atitle><jtitle>Diabetologia</jtitle><stitle>Diabetologia</stitle><date>2020-10-01</date><risdate>2020</risdate><volume>63</volume><issue>10</issue><spage>2235</spage><epage>2248</epage><pages>2235-2248</pages><issn>0012-186X</issn><eissn>1432-0428</eissn><abstract>Aims/hypothesis
Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by systematically mapping the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina.
Methods
Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (
Ins2
Akita
×
Vegfa
+/
–
) mice, which are known to replicate features of clinical diabetic retinopathy. This resulted in transcriptome data for 9474 retinal cells, which could be annotated to eight distinct retinal cell types. Using STRING analysis, we studied differentially expressed gene networks in neuronal, glial and immune cell compartments to create a comprehensive view on the pathological changes that occur in the Akimba retina. Using subclustering analysis, we further characterised macroglial and inflammatory cell subpopulations. Prominent findings were confirmed at the protein level using immunohistochemistry, western blotting and ELISA.
Results
At 12 weeks, the Akimba retina was found to display degeneration of rod photoreceptors and presence of inflammatory cells, identified by subclustering analysis as monocyte, macrophage and microglial populations. Analysis of differentially expressed genes in the rod, cone, bipolar cell and macroglial compartments indicated changes in cell metabolism and ribosomal gene expression, gliosis, activation of immune system pathways and redox and metal ion dyshomeostasis. Experiments at the protein level supported a metabolic shift from glycolysis to oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase), activation of microglia/macrophages (isolectin-B4), metal ion and oxidative stress response (metallothionein and haem oxygenase-1) and reactive macroglia (glial fibrillary acidic protein and S100) in the Akimba retina, compared with wild-type mice. Our single-cell approach also indicates macroglial subpopulations with distinct fibrotic, inflammatory and gliotic profiles.
Conclusions/interpretation
Our study identifies molecular pathways underlying inflammatory, metabolic and oxidative stress-mediated changes in the Akimba mouse model of diabetic retinopathy and distinguishes distinct functional subtypes of inflammatory and macroglial cells.
Data availability
RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (
www.ebi.ac.uk/arrayexpress
) under accession number E-MTAB-9061.
Graphical abstract</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><doi>10.1007/s00125-020-05218-0</doi><tpages>14</tpages><orcidid>https://orcid.org/0000-0002-9369-5547</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Bipolar cells Blindness Cell activation Diabetes Diabetes mellitus Diabetic retinopathy Enzyme-linked immunosorbent assay Gene expression Glia Glial fibrillary acidic protein Gliosis Glyceraldehyde Glyceraldehyde 3-phosphate Glyceraldehyde-3-phosphate dehydrogenase Glycolysis Heme oxygenase (decyclizing) Human Physiology Immune system Immunohistochemistry Inflammation Internal Medicine Macrophages Medicine Medicine & Public Health Metabolic Diseases Metabolism Microglia Neuronal-glial interactions Oxidative phosphorylation Oxidative stress Photoreceptors Proteins Retina Ribonucleic acid RNA Transcription Western blotting |
| title | Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy |
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