Izquierdo Garrudo, Pablo; (2021) Regulation of microglial surveillance and phagocytosis in health and disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Microglia are the resident immune cells of the central nervous system. They survey the brain for redundant synapses, debris and dying cells, which they remove through phagocytosis. This thesis examines factors which regulate this microglial behaviour, in health and in Alzheimer’s disease (AD). Applying pharmacological manipulations to microglial uptake of fluorescent microbeads in rodent brain slices, I demonstrated that membrane potential, purinergic receptors and intracellular calcium regulated phagocytosis. Buffering intracellular Ca2+ inhibited bead phagocytosis, whereas removing external Ca2+ did not, suggesting that Ca2+ release from internal stores controls phagocytosis. I therefore characterised spontaneous and damage-evoked Ca2+ transients in microglia in brain slices. A K+ channel blocker that depolarises microglia inhibited bead uptake, and our lab has shown that microglial ramification is regulated by membrane voltage, set by the tonically-active plasma membrane K+ channel THIK-1. I showed that THIK-1 is also required for phagocytosis of fluorescent microbeads. Using immunohistochemistry and electrophysiology, I found that mice lacking THIK-1 have an altered number of glutamatergic synapses, suggesting synapse development is regulated by THIK-1. Surveillance and phagocytosis by microglia in AD were assessed using (i) a fluorescent form of amyloid beta (Aβ), (ii) AppNL-G-F knock-in mice to mimic the disease, and (iii) knockouts for the AD-linked phagocytic receptor TREM2. I showed that, while astrocytes also contributed, microglia were the main Aβ phagocytes in situ. Lack of TREM2 increased surveillance in aged but not young mice, independently of THIK-1. In the AD mouse model, Aβ plaque coverage was unaffected by THIK-1 expression, but THIK-1 knockout mice exhibited a higher density of dendritic spines suggesting that Aβ-induced synapse loss depends on THIK-1. Microglia from fresh human biopsies confirmed that blocking THIK-1 has similar effects as in rodents, suggesting that manipulating THIK-1 could regulate microglial surveillance and phagocytosis in ageing humans.

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