Primary phagocytosis of viable neurons by microglia activated with LPS or Aβ is dependent on calreticulin/LRP phagocytic signalling

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.

Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis

“…Calreticulin exposure on neurons can act as another 'eat-me' signal, which induces phagocytosis through the low-density lipoprotein receptorrelated proteins (LRPs) on microglia (Fricker et al, 2012b). UDP release from stressed neurons can act as a final engulfment signal by activating P2Y 6 receptors on microglia (Koizumi et al, 2007;Neher et al, 2014).…”

“…PC12 cells are a rat pheochromocytoma cell line that can be differentiated into cells of a neuronal phenotype (Greene and Tischler, 1976). Apoptotic PC12 cells can be phagocytosed by BV-2 cells; this process can require LRPmediated recognition of exposed calreticulin (Fricker et al, 2012b) and/or extracellular annexin A1 binding both PtdSer on PC12 cells and formyl peptide receptor 2 on the BV-2 cells (McArthur et al, 2010). Moreover, BV-2 cells have been suggested to phagocytose 'live' PC12 cells when activated with LPS (McArthur et al, 2010) or PMA , although it was not tested whether the PC12 were in fact alive when phagocytosed, or whether blocking phagocytosis prevented cell death.…”

Branched Sialylated <i>N</i>-glycans Are Accumulated in Brain Synaptosomes and Interact with Siglec-H

“…Microglia also express Siglec-E, and Siglec-E binds not only to α2,3-and α2,8-sialylated glycans but also to 6SLeC (Redelinghuys et al, 2011). Several studies have identified calreticulin on the cell surface of target cells where it acts as an 'eat-me' signal to induce phagocytosis of these cells (Arosa et al, 1999;Fricker et al, 2012;Raghavan et al, 2013;White et al, 1995). Thus, calreticulin may also play a role as an eat-me signal to induce the removal of synapses.…”

Morphological, immunocytochemical and functional characterization of the microglial phenotype in culture

“…LPS activation renders microglia ameboid, induces several pro-and anti-inflammatory signaling molecules (Lim et al, 2015; and neurotoxic substances through binding to the CD14/MD-2/Toll-like receptor 4-complex (Fricker et al, 2012;Tokes et al, 2011), and gives rise, among others, to cell spreading by interfering with the organization of the actin cytoskeleton through the alteration of integrin clustering .…”

“…Selected CaM inhibitors such as CALMID and TFP, previously reported to have different modes of action (Matsushima et al, 2000;, were quantitatively tested for their ability to modify the microglial morphology, lamellipodia, filopodia and podosome formation, and specific functions such as cell proliferation and survival, protein expression and phagocytosis in unchallenged (control) and lipopolysaccharide (LPS)-challenged cells. Stimulation with LPS was used to evaluate the ability of microglial cells to respond to activation (Fricker et al, 2012;Tokes et al, 2011). …”