@article{88e12fc9510a4b058b27cae5b4b161ea,
title = "Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia",
abstract = "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.",
keywords = "CP: Neuroscience, extracellular vesicle, interleukin-1β, microglia, microtubule-associated protein tau, neuroinflammation, shRNA library screening",
author = "Zhi Ruan and Kayo Takamatsu-Yukawa and Yuzhi Wang and Ushman, {Margaret L.} and Labadorf, {Adam Thomas} and Maria Ericsson and Seiko Ikezu and Tsuneya Ikezu",
note = "Funding Information: We thank Michelle Li, Hana Do, Shuiqiao Hu, and other members of the Laboratory of Molecular NeuroTherapeutics for technical assistance. We also thank Brian Tilton at the Flow Cytometry Core Facility at Boston University School of Medicine, Dr. Yuriy Alekseyev and Ashley LeClerc at the Microarray & Sequencing Resource Core Facility at Boston University School of Medicine, and Dr. Laura J. Lewis-Tuffin at the Cytometry and Imaging Laboratory at the Mayo Clinic Florida. This work was funded in part by Ono Pharmaceutical (to T.I.), NIH R01 AG066429 (to T.I.), RF1 AG054199 (to T.I.), R01 AG054672 (to T.I.), R01 AG067763-01A1 (to T.I.), R01 AG072719 (to T.I.), the Cure Alzheimer's Fund (to T.I. and S.I.), and BU ADC NIH P30 AG013846 (to S.I. and Z.R.). Conceptualization, Z.R. and T.I.; methodology, Z.R. K.T.-Y. Y.W. M.L.U. and M.E.; bioinformatics, Z.R. and A.T.L.; data analysis, Z.R. and T.I.; manuscript writing and editing, Z.R. S.I. and T.I. All authors have read and approved the final version of the manuscript. T.I. had a sponsored research agreement with Ono Pharmaceutical and consults with Takeda. Funding Information: We thank Michelle Li, Hana Do, Shuiqiao Hu, and other members of the Laboratory of Molecular NeuroTherapeutics for technical assistance. We also thank Brian Tilton at the Flow Cytometry Core Facility at Boston University School of Medicine, Dr. Yuriy Alekseyev and Ashley LeClerc at the Microarray & Sequencing Resource Core Facility at Boston University School of Medicine, and Dr. Laura J. Lewis-Tuffin at the Cytometry and Imaging Laboratory at the Mayo Clinic Florida. This work was funded in part by Ono Pharmaceutical (to T.I.), NIH R01 AG066429 (to T.I.), RF1 AG054199 (to T.I.), R01 AG054672 (to T.I.), R01 AG067763-01A1 (to T.I.), R01 AG072719 (to T.I.), the Cure Alzheimer{\textquoteright}s Fund (to T.I. and S.I.), and BU ADC NIH P30 AG013846 (to S.I. and Z.R.). Publisher Copyright: {\textcopyright} 2022 The Author(s)",
year = "2022",
month = may,
day = "10",
doi = "10.1016/j.celrep.2022.110791",
language = "English (US)",
volume = "39",
journal = "Cell Reports",
issn = "2211-1247",
publisher = "Cell Press",
number = "6",
}