Abstract
BackgroundMicroglia are resident brain macrophages that can phagocytose dead, dying or viable neurons, which may be beneficial or detrimental in inflammatory, ischaemic and neurodegenerative brain pathologies. Cell death caused by phagocytosis of an otherwise viable cell is called ‘primary phagocytosis’ or ‘phagoptosis’. Calreticulin (CRT) exposure on the surface of cancer cells can promote their phagocytosis via LRP (low-density lipoprotein receptor-related protein) on macrophages, but it is not known whether this occurs with neurons and microglia.MethodsWe used primary cultures of cerebellar neurons, astrocytes and microglia to investigate the potential role of CRT/LRP phagocytic signalling in the phagocytosis of viable neurons by microglia stimulated with lipopolysaccharide (LPS) or nanomolar concentrations of amyloid-β peptide1-42 (Aβ). Exposure of CRT on the neuronal surface was investigated using surface biotinylation and western blotting. A phagocytosis assay was also developed using BV2 and PC12 cell lines to investigate CRT/LRP signalling in microglial phagocytosis of apoptotic cells.ResultsWe found that BV2 microglia readily phagocytosed apoptotic PC12 cells, but this was inhibited by a CRT-blocking antibody or LRP-blocking protein (receptor-associated protein: RAP). Activation of primary rat microglia with LPS or Aβ resulted in loss of co-cultured cerebellar granule neurons, and this was blocked by RAP or antibodies against CRT or against LRP, preventing all neuronal loss and death. CRT was present on the surface of viable neurons, and this exposure did not change in inflammatory conditions. CRT antibodies prevented microglia-induced neuronal loss when added to neurons, while LRP antibodies prevented neuronal loss when added to the microglia. Pre-binding of CRT to neurons promoted neuronal loss if activated microglia were added, but pre-binding of CRT to microglia or both cell types prevented microglia-induced neuronal loss.ConclusionsCRT exposure on the surface of viable or apoptotic neurons appears to be required for their phagocytosis via LRP receptors on activated microglia, but free CRT can block microglial phagocytosis of neurons by acting on microglia. Phagocytosis of CRT-exposing neurons by microglia can be a direct cause of neuronal death during inflammation, and might therefore contribute to neurodegeneration and be prevented by blocking the CRT/LRP pathway.
Highlights
Microglia are resident brain macrophages that can phagocytose dead, dying or viable neurons, which may be beneficial or detrimental in inflammatory, ischaemic and neurodegenerative brain pathologies
Phagocytosis is normally thought to occur after the target cell has undergone cell death, but we found that in inflammatory conditions inhibition of phagocytic signalling rescues neurons both in vitro and in vivo, demonstrating that phagocytosis can be a direct cause of neuronal death in models of inflammatory neurodegeneration [5,6,7]
CRT/LRP signalling in phagocytosis of apoptotic PC12 cells by BV2 microglial cells Cell-surface-exposed CRT has been demonstrated to play an important role in mediating phagocytosis in various cancer cell lines, either as an eat-me signal on the target cell or as a co-receptor on the phagocytic cell surface
Summary
Microglia are resident brain macrophages that can phagocytose dead, dying or viable neurons, which may be beneficial or detrimental in inflammatory, ischaemic and neurodegenerative brain pathologies. We have recently described a novel form of neuronal death mediated by inflammatory-activated microglia in which microglia phagocytose viable neurons, referred to as ‘primary phagocytosis’ or ‘phagoptosis’ [3,4]. Upon induction of cell death by apoptosis or necrosis, PS becomes exposed on the cell surface due to inactivation of the translocase or activation of a scramblase, which randomises phospholipid distribution between the inner and outer leaflets resulting in net PS exposure. PS exposure occurs on the surface of viable cells when ‘activated’, usually as a result of calcium stimulation of the scramblase and inhibition of the translocase, for example during activation of all leucocytes [9,10,11], and on neurons exposed to oxidants from activated microglia [5]. We have shown that primary phagocytosis of viable neurons by inflammatory-activated microglia is mediated by microglia-induced PS exposure on viable neurons, evoking microglial phagocytosis via MFG-E8 and the vitronectin receptor [5,7]
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