The novel phosphorylation site PLCγ2 Y1217 facilitates the TREM2 amyloid-β sensing pathway in microglia

Abstract

Alzheimer’s disease (AD) is the most common neurodegenerative disease and manifests itself in the brain as aggregations of amyloid-β containing extracellular plaques and intracellular aggregates of hyperphosphorylated tau. The majority of AD cases are sporadic, have no clear single cause and have been termed as late-onset Alzheimer’s disease (LOAD), where symptoms manifest at 65-75 years of age. A significant portion of loci linked to AD risk modulation identified in genome wide association studies have been confirmed to map to SNPs in genes highly enriched within microglia and include BIN1, TREM2, PLCG2 and CLU. The TREM2 R47H mutation carries the second highest genetic risk factor for LOAD and one allele increases risk of AD three-fold. TREM2 is an immunoreceptor expressed on microglia and detects anionic damage associated molecular patterns such as phosphatidylserine and amyloid-β. In this study I aimed to identify differentially expressed genes (DEGs) in microglia isolated from the amyloidogenic TgCRND8 mouse line crossed with AD risk mutant TREM2 R47H or TREM2 knockout (KO) mice. I then sought to identify the functional pathways and signalling cascades the DEGs were components of. I selected two differentially expressed genes, ATP6V0C and ABCG1, which are involved in significantly altered molecular pathways identified by gene ontology analysis to validate the findings of my scRNAseq study. While the single cell RNA sequencing element of my project was not sufficiently powered, I was able to find similarities to a larger dataset comparing amyloidogenic TREM2WT/WT and TREM2KO/KO mice. I confirmed alterations in the reduction in expression of two genes that failed to be upregulated in TREM2 R47H and TREM2 KO mice in BV2 cells stimulated with a TREM2 activating antibody. I then sought to evaluate the effects of TREM2 knockout on the activation of downstream responses, such as phosphorylation of PLCγ2 and intracellular calcium release. PLCγ2 was shown to be phosphorylated at tyrosine 1217, not the canonical residues Y753 or Y759 as they are in the B cell receptor signalling pathway, in response to oligomeric Aβ. Additionally, I found that intracellular Ca2+ release in THP1 cells in response to Aβ oligomers was impaired when TREM2 was silenced by siRNA, indicating the TREM2 pathway is reliant on calcium flux to facilitate its signalling response to Aβ oligomers. Next, I evaluated microglial density and plaque localisation in the brains of TgCRND8 containing homozygous R47H or TREM2 knockout. There were reduced overall numbers of microglia, and they displayed reduced association with amyloid plaques. I additionally quantified pY1217 PLCγ2 signal in microglia as a function of distance to place centre and found that this phosphorylation motif was proportional to plaque distance in wild-type TREM2 mice, but not R47H or TREM2 knockout mice. Finally, in isogenic human iPSC-derived macrophages I found that the P522R PLCγ2 variant has increased duration of the phosphorylation event at Y1217 but not quantities, increases phagocytosis of apoptotic neurons, and reduces pro-inflammatory cytokine secretion in response to LPS challenge.