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Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia
Activated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring th...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9133589/ https://www.ncbi.nlm.nih.gov/pubmed/35545052 http://dx.doi.org/10.1016/j.celrep.2022.110791 |
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author | Ruan, Zhi Takamatsu-Yukawa, Kayo Wang, Yuzhi Ushman, Margaret L. Labadorf, Adam Thomas Ericsson, Maria Ikezu, Seiko Ikezu, Tsuneya |
author_facet | Ruan, Zhi Takamatsu-Yukawa, Kayo Wang, Yuzhi Ushman, Margaret L. Labadorf, Adam Thomas Ericsson, Maria Ikezu, Seiko Ikezu, Tsuneya |
author_sort | Ruan, Zhi |
collection | PubMed |
description | Activated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato(+) cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP(+) tdTomato(+) cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1β release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders. |
format | Online Article Text |
id | pubmed-9133589 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
record_format | MEDLINE/PubMed |
spelling | pubmed-91335892022-05-26 Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia Ruan, Zhi Takamatsu-Yukawa, Kayo Wang, Yuzhi Ushman, Margaret L. Labadorf, Adam Thomas Ericsson, Maria Ikezu, Seiko Ikezu, Tsuneya Cell Rep Article Activated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato(+) cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP(+) tdTomato(+) cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1β release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders. 2022-05-10 /pmc/articles/PMC9133589/ /pubmed/35545052 http://dx.doi.org/10.1016/j.celrep.2022.110791 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) ). |
spellingShingle | Article Ruan, Zhi Takamatsu-Yukawa, Kayo Wang, Yuzhi Ushman, Margaret L. Labadorf, Adam Thomas Ericsson, Maria Ikezu, Seiko Ikezu, Tsuneya Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia |
title | Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia |
title_full | Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia |
title_fullStr | Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia |
title_full_unstemmed | Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia |
title_short | Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia |
title_sort | functional genome-wide short hairpin rna library screening identifies key molecules for extracellular vesicle secretion from microglia |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9133589/ https://www.ncbi.nlm.nih.gov/pubmed/35545052 http://dx.doi.org/10.1016/j.celrep.2022.110791 |
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