Phagocytic clearance of apoptotic neurons by microglia/brain macrophages in vitro: Involvement of lectin-, integrin-, and phosphatidylserine-mediatedrecognition

Microglia, the tissue macrophages of the brain, play a crucial role in reco gnition and phagocytic removal of apoptotic neurons, The microglial recepto rs for recognition of apoptotic neurons are not yet characterized. Here we established a co-culture model of primary microglia and cerebellar granule neurons to examine the receptor systems involved in recognition/uptake of a poptotic neurons. Treatment with 100 mu M S-nitrosocysteine induced apoptos is of cerebellar neurons as indicated by nuclear condensation and phosphati dylserine exposure to the exoplasmic leaflet of the plasma membrane. Microg lial cells were added to neurons 2 h after apoptosis induction and co-cultu red for 6 h in the presence of ligands that inhibit recognition by binding to respective receptors. Binding/phagocytosis was determined after combined 4',6-diamidino-2-phenylindole/ propidium iodide (for apoptotic/necrotic ne urons) and lectin staining (for microglia). Uptake of apoptotic neurons was reduced by N-acetylglucosamine or galactose, suggesting that recognition i nvolves asialoglycoprotein-like lectins, Furthermore, the inhibition of mic roglial binding/uptake of apoptotic neurons by RGDS peptide suggests a role of microglial vitronectin receptor. As microglia selectively bind lipid ve sicles enriched in phosphatidylserine and O-phospho-L-serine interfered wit h the uptake of apoptotic neurons, an involvement of phosphatidylserine rec eptor is rather likely, Apoptotic neurons do not release soluble signals th at serve to attract or activate microglia, Collectively, these results sugg est that apoptotic neurons generate a complex surface signal recognized by different receptor systems on microglia.