title: Store-Operated Ca2+Entry (SOCE) and Purinergic Receptor-Mediated Ca2+Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation.
abstract: Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+entry (ROCE). Although store-operated Ca2+entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+imaging. After depleting internal Ca2+stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]iwhereas lower concentrations (≤100 μM) also induced Ca2+oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+oscillations (P2X4) and sustained [Ca2+]iincreases. We demonstrate specific drug effects on purinergic Ca2+pathways and provide new pharmacological insights into Ca2+oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca2+-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammationin vitro.
authors.affiliation: Department of Chemical and Biological Engineering, Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-NürnbergErlangen, Germany; Erlangen Graduate School in Advanced Optical Technologies, Friedrich-Alexander-Universität Erlangen-NürnbergErlangen, Germany.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Gilbert
authors.affiliation: Health Innovations Research Institute, Royal Melbourne Institute of Technology University, Melbourne VIC, Australia.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stebbing
authors.affiliation: Department of Chemical and Biological Engineering, Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-NürnbergErlangen, Germany; Erlangen Graduate School in Advanced Optical Technologies, Friedrich-Alexander-Universität Erlangen-NürnbergErlangen, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Kuenzel
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne VIC, Australia.
authors.forename: Robyn M
authors.initials: RM
authors.lastname: Murphy
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne VIC, Australia.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Zacharewicz
authors.affiliation: Department of Chemical and Biological Engineering, Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Buttgereit
authors.affiliation: Health Innovations Research Institute, Royal Melbourne Institute of Technology University, Melbourne VIC, Australia.
authors.forename: Leanne
authors.initials: L
authors.lastname: Stokes
authors.affiliation: Health Innovations Research Institute, Royal Melbourne Institute of Technology University, Melbourne VIC, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
authors.affiliation: Department of Chemical and Biological Engineering, Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-NürnbergErlangen, Germany; Erlangen Graduate School in Advanced Optical Technologies, Friedrich-Alexander-Universität Erlangen-NürnbergErlangen, Germany; Health Innovations Research Institute, Royal Melbourne Institute of Technology University, MelbourneVIC, Australia.
authors.forename: Oliver
authors.initials: O
authors.lastname: Friedrich
keywords: ATP; BV2 microglia; calcium; high-content screening; minocycline; multiphoton imaging; store-operated calcium entry