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Title: Microglial phagocytosis of bacteria and synapses
Author: Cockram, Thomas
ISNI:       0000 0004 9348 3928
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Microglia, the resident brain macrophage, are essential for maintaining normal brain homeostasis through removal of pathogens - including bacteria and fungi - via phagocytosis. However, the mechanisms that regulate these phagocytic events are not clear. In this work, involvement of calreticulin, galectin-3, apolipoprotein E and β-amyloid in microglial phagocytosis of bacteria were studied. Microglia activated with bacterial endotoxin upregulated and released extracellular calreticulin and galectin-3 in culture, and both proteins could bind the surface of the gram-negative bacteria E. coli. Moreover, association of these proteins with the bacteria opsonised them for phagocytosis by microglia. This opsonisation was inhibitable by sugars, or by blocking microglial receptors MerTK or LRP1. LPS-activated microglia phagocytosed more E. coli than unactivated microglia, and this phagocytic induction was inhibited by an antibody against calreticulin, sugars, blocking MerTK or LRP1, or simply exchanging the cellular media. Thus, calreticulin and galectin-3 are bacterial opsonins, which may play an important role in immune responses to bacteria in the brain. Apolipoprotein E and β-amyloid – both of which are known to circulate extracellularly in the brain - also bound and opsonised E. coli for phagocytosis by microglia, and may further contribute to bacterial clearance in the brain. Extracellular nucleotides mediate a variety of microglial functions by binding receptors exposed by the microglia, including the phagocytic receptor P2Y6R and the chemotactic receptor P2Y12R. Both E. coli and inflammatory-activated microglia released extracellular agonist of P2Y6R, as did non-activated microglia, astrocytes and pheochromocytoma under specific experimental conditions. Exogenous UDP induced microglial phagocytosis of the bacteria in a time-sensitive manner, and this effect was inhibitable by blocking P2Y6R, suggesting that P2Y6R signalling regulates bacterial phagocytosis. Furthermore, LPS-induction of phagocytosis was inhibited by apyrase, or by blocking P2Y6R, indicating that LPS-induced phagocytosis is mediated by microglial P2Y6R. Moreover, exogenous ADP (which activates P2Y12R) inhibited E. coli phagocytosis by microglia, as did blocking P2Y12R, consistent with a role for ADP- P2Y12R signalling in microglial chemotaxis toward bacteria. In addition to eliminating pathogens, microglial phagocytosis of synapses is a crucial facilitator of the ‘synaptic pruning’ that occurs in the developing brain, but this regulation is poorly understood. Here, the mechanisms that regulate microglial phagocytosis of synapses were studied. Calreticulin and galectin-3 opsonised isolated synapses, or ‘synaptosomes’, in a similar fashion to the bacteria, suggesting diverse regulatory functions for these proteins. Microglial phagocytosis of synaptosomes was also enhanced by apolipoprotein E and extracellular tau, which may play a role in pathological synaptic loss during neurodegeneration. In addition, blocking P2Y6R or P2Y12R inhibited microglial phagocytosis of synaptosomes. Through a novel model of inflammatory synaptic loss in cerebellar neuronal cultures, LPS was found to induce synaptic loss without neuronal loss, which was mediated by microglia and consistent with microglial phagocytosis of the synapses. Such synaptic loss was absent in cultures lacking P2Y6R, and in cultures treated with a P2Y12R antagonist – further implicating both P2Y6R and P2Y12R in microglial phagocytosis of synapses. Taken together, these findings provide insights into novel mechanisms through which microglial phagocytosis of two diverse targets – bacteria and synapses - is regulated.
Supervisor: Brown, Guy Charles ; Lummis, Sarah Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Microglia ; Phagocytosis ; Bacteria ; Synapse