The role of AD protective variant PLCγ2P522R in modulating microglia mediated clearance and synaptic pruning

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

PLCG2-P522R, a rare coding variant in the Phospholipase C gamma-2 (PLCG2) gene, has been found to be protective against late onset Alzheimer's disease (AD). Within the central nervous system, PLCγ2 is most abundantly expressed in microglia, and microglial mediated neuroinflammatory system has emerged as a major contributor to the molecular and phenotypic changes observed in the AD brain. However, the mechanism by which the P522R variant of PLCγ2 reduces AD pathology is still unknown. BV2 (mouse microglia) cells and human induced pluripotent stem-cells (hiPSC) derived microglia were used in this thesis work to evaluate the role of PLCγ2 in modifying various disease-relevant microglia functions.

PLCγ2WT and PLCγ2P522R expression constructs were transfected into BV2 cells to examine the effects of PLCγ2 overexpression on various microglia functions including amyloid beta (Aβ) clearance and synaptic targeting, and various transcriptional changes linked to AD. hiPSCs were genome edited using CRISPR/Cas9 to generate both heterozygous and homozygous forms of the PLCG2_P522R variant in healthy controls. These hiPSC derived microglia were used to explore the effects of the PLCγ2P522R basal level on disease-relevant processes, such as microglial capacity to uptake Aβ and synapses. Microglia transcriptional changes were examined using targeted qPCR analysis to investigate changes in expression of key microglial genes. Mitochondrial function and calcium level changes were also investigated in these microglia cells to determine their metabolic fitness. In addition, the microglia were subjected to acute and chronic treatment of oligomeric Aβ to examine the impact of PLCγ2P522R on microglia's ability to respond to acute and chronic stress. As a result, the effects of Aβ oligomers on lysosomal biogenesis and phagocytic capacities of these microglia were examined further. As a result of PLCγ2 overexpression, Aβ uptake and other immune- provoking cargoes like zymosan were significantly increased. In contrast, the uptake of synaptosomes in BV2 cells overexpressing PLCγ2 was considerably reduced. Similarly, microglia generated from hiPSCs also showed enhanced clearance of Aβ and preservation of synapses by PLCγ2P522R variants. In the PLCγ2P522R microglia variants, the expression of multiple genes, including IL-10 and CX3CR1, as well as mitochondrial function, cytoplasmic calcium flux, and cellular motility were all increased. It was found that the protective effect of PLCγ2P522R was vitally dependent on 'allelic-dose', as homozygous cells displayed a lower preservation of synapse and a distinct gene expression profile compared to heterozygous cells.

Similarly, microglia with the protective mutation PLCγ2P522R displayed higher inflammatory cytokine IL-1β level, and better response to acute treatment with Aβ oligomers. PLCγ2P522R appeared to resist the quiescence that was seen in WT microglia variants, by increasing cytokine production and lysosomal biogenesis. My findings suggest that the P522R variant in PLCγ2 increases microglia capacity to clear toxic aggregates such as Aβ while preserving synapses. Furthermore, my findings suggests that PLCγ2P522R contributes to greater microglial surveillance, as well as microglia priming towards a pro-inflammatory state, along with an increased capacity to adapt to growing energy demands. This, however, also shows the delicate balance of this system, as increasing the 'dosage' of PLCγ2P522R may result in diminished favourable benefits.
Date of Award1 Dec 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorMarc-David Ruepp (Supervisor), Anthony Vernon (Supervisor) & Jackie Mitchell (Supervisor)

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