Establishment and application of a microglial phagocytosis/cell health high content assay for Alzheimer’s disease drug discovery targeting microglia

Abstract

AbstractBackgroundTargeting microglial phagocytosis has been proposed as a therapeutic strategy for Alzheimer’s disease drug discovery. A reliable cellular assay capable of measuring drug‐regulated phagocytosis activity while simultaneously measuring treatment‐caused cell stress and toxicity is needed for drug discovery research targeting microglia. This need motivated us to develop and establish a high content microglial phagocytosis/cell health assay to meet the challenge.MethodThe assay uses 384‐well cellular imaging plates, microglial cell lines (HMC3 and BV2) and mouse primary microglia, pHrodo‐myelin/membrane debris as ligand, and a high content imaging system for imaging and analysis. Assay procedures include 1) plate cells on day 1 and incubate cell plates overnight in 37oC; 2) treat cells with compounds on day 2; 3) add pHrodo‐myelin/membrane debris to cell plates on day 3 and incubate in 37oC for 20 hours; 4) stain cell nuclei with Hoechst‐33342 for 30 min before performing high content imaging and analysis on day 4. Three selected parameters are measured from cell images: 1) mean total fluorescence intensity per cell of pHrodo‐myelin/membrane debris in phagocytosis vesicles to quantify phagocytosis; 2) cell counts per well (assessing effects on proliferation and cell death); and 3) average nuclear intensity (detecting induced apoptosis).ResultVarious treatments targeting different mechanisms regulating microglial phagocytosis were tested; we observed that Cytochalasin D inhibited phagocytosis, LPS stimulated phagocytosis in BV2 cells but not HMC3 cells, Idelalisib (PI3Kd inhibitor) inhibited HMC3 cell phagocytosis, and Saracatinib (Src/abl family of kinases inhibitor) stimulated HMC3 phagocytosis. These results validated the assay for use in drug discovery targeting microglial phagocytosis. With the assay, we identified some INPP5D inhibitors and some PLCG2 activators stimulated phagocytosis in a concentration range without significant cell stress/toxicity with all three cell types tested.ConclusionA microglial phagocytosis/cell health assay was established. The simultaneous measurement of phagocytosis and cell health allows for the distinction of drug effects on regulation of phagocytosis from cellular stress/toxicity‐related changes, a distinguishing feature of the assay, which helped compound selection of drug discovery projects targeting INPP5D and PLCG2 respectively.