Phagocytic Clearance of Apoptotic Neurons by Microglia/Brain Macrophages In Vitro

Abstract

Microglia, the tissue macrophages of the brain, play a crucial role in recognition and phagocytic removal of apoptotic neurons. The microglial receptors 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 apoptotic neurons. Treatment with 100 M S-nitrosocysteine induced apoptosis of cerebellar neurons as indicated by nuclear condensation and phosphatidylserine exposure to the exoplasmic leaflet of the plasma membrane. Microglial cells were added to neurons 2 h after apoptosis induction and co-cultured for 6 h in the presence of ligands that inhibit recognition by binding to respective receptors. Binding/phagocytosis was determined after combined 4Ј,6-diamidino-2-phenylindole/ propidium iodide (for apoptotic/necrotic neurons) and lectin staining (for microglia). Uptake of apoptotic neurons was reduced by N-acetylglucosamine or galactose, suggesting that recognition involves asialoglycoprotein-like lectins. Furthermore, the inhibition of microglial binding/uptake of apoptotic neurons by RGDS peptide suggests a role of microglial vitronectin receptor. As microglia selectively bind lipid vesicles enriched in phosphatidylserine and O-phospho-Lserine interfered with the uptake of apoptotic neurons, an involvement of phosphatidylserine receptor is rather likely. Apoptotic neurons do not release soluble signals that serve to attract or activate microglia. Collectively, these results suggest that apoptotic neurons generate a complex surface signal recognized by different receptor systems on microglia. Key Words: Cerebellar neurons-Apoptosis-Phagocytosis-Microglia-Phosphatidylserine -VitronectinLectins-Brain macrophages. J. Neurochem. 75, 1060Neurochem. 75, -1070Neurochem. 75, (2000.Apoptosis is abundant in both the developing central and the peripheral nervous system (Oppenheim, 1991;Blaschke et al., 1996). Neuronal cell death as a result of apoptosis is also associated with various neurodegenerative disorders and cerebrovascular stroke (Li et al., 1995;Nicotera et al., 1999). Although large populations of neurons undergo apoptosis, only few degenerating neurons are observed at a given time within the tissue. This is a consequence of the rapid phagocytosis of apoptotic cells by phagocytes prior to cell lysis. Microglial cells represent the tissue macrophages of the CNS. By virtue of their phagocytic and cytotoxic properties, they contribute to the defense system of the CNS (Perry, 1994;Kreutzberg, 1996). Microglial cells develop from hematogenous precursors that invade the brain during embryonic development (Ling and Wong, 1993). In early postnatal mice, clusters of amoeboid phagocytic microglia can be observed in regions where programmed cell death of neurons and glial cells occurs (Ashwell, 1990;Brockhaus et al., 1996). In the adult, intact brain microglia are characterized by their ramified morphology and a downregulated immunophenotype. ...

Microglia as a critical player in both developmental and late-life CNS pathologies

“…Phagocytosis of apoptotic cells is a necessary part of normal tissue development [101], and is thus integral to the role of microglia as tissue-resident macrophage. During the process of brain maturation, millions of neurons undergo apoptosis, yet leave barely a trace—thanks to vigilant corpse removal attributable largely to microglia [126]. Similarly, in experiments involving induced massive ablation of cells within the brain parenchyma, there is little—if any—detectable debris to be found even days after [95].…”

MFG-E8 Regulates Microglial Phagocytosis of Apoptotic Neurons

“…Microglia, which are similar to macrophages in the periphery of the body, act as the phagocytic cells in the brain to remove harmful material and dying cells (Garden and Moller 2006; Stolzing and Grune 2004; Witting et al 2000), but little is known about MFG-E8 and microglia function. MFG-E8 is expressed in the brain (Boddaert et al 2007; Hanayama et al 2004), and one report indicates fractalkine (CX3CL1) treatment of cultured microglia increases MFG-E8 mRNA levels (Leonardi-Essmann et al 2005).…”

Prix Fixe: Efferocytosis as a Four-Course Meal

“…In the brain, however, microglia act as resident macrophages to accomplish this function. Microglia have phenotypical similarities to peripheral macrophages, in that they express and utilize PS receptors for the recognition and uptake of dead cells (Witting et al 2000). Multiple neurodegenerative disorders have been associated with defective efferocytosis, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Mattson 2000).…”