Bull, Daniel Anthony; (2022) Investigating the effect that disease-linked PLCγ2 variants have on enzymatic activity and exploring PLCγ2’s role within microglia cell function. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Phospholipase C-gamma 2 (PLCγ2) is highly expressed in immune cells, such as microglia. PLCγ2 hydrolyses phosphatidylinositol 4,5-bisphosphate into inositol trisphosphate (IP3) and diacylglycerol. Variants of PLCγ2 have been described in several diseases, such as late onset Alzheimer’s disease (LOAD), inflammatory bowel disease, as well as rare immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function. To characterise the enzymatic activity of disease-linked PLCγ2 variants, an assay measuring inositol monophosphate (IP1), a downstream metabolite of IP3, was adapted and optimised. The S707Y, Δ845-848 and M1141K PLCγ2 variants, linked to a complex immune disorder, have strong hypermorphic activity, whereas the PLCγ2 V1103I variant shows a mild increase in PLC activity. In the context of LOAD, microglia have been implicated as key mediators of disease pathophysiology. However, the role of PLCγ2 within microglia is still not fully understood. To address this issue, the hypermorphic PLCγ2 S707Y disease linked-variant was introduced into human inducible pluripotent stem cells (hiPSCs) to explore the role that strong gain-of-function PLCγ2 variants have within hiPSC-derived microglia. Based on the IP1 measurements, PLCγ2 S707Y hiPSC-derived microglia exhibited hypermorphic enzymatic activity under both basal and stimulated conditions, which in turn resulted in increased calcium flux. However, when challenged with apoptotic neuronal cells, a reduction in phagocytosis was observed. Additionally, secretion of IL-1β, IL-8 and TNF-α was shown to be elevated in basal conditions. However, when challenged with lipopolysaccharide, PLCγ2 S707Y hiPSC-derived microglia exhibited a reduction in IL-10, IL-6 and TNF-α secretion, likely due to decreased NF-κB activation and translocation. RNA sequencing of the PLCγ2 S707Y hiPSC-derived microglia revealed a downregulation of genes related to innate immunity and response. Therefore, this thesis demonstrates that despite the increase in PLCγ2 enzymatic activity, the PLCγ2 S707Y hiPSC-derived microglia display a loss of function for key microglial processes.

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