Role of PLCG2 in Aβ clearance and synaptic pruning.

Abstract

The microglial mediated neuroinflammatory system has emerged as a major contributor to the molecular and phenotypic changes observed in the AD brain, including excessive synaptic loss (synaptic pruning) and decreased Aβ clearance (Heneka et al., 2015). GWAS studies have identified a number of novel rare coding variants for late-onset AD (LOAD) in microglial-associated genes (Sims et al., 2017). Notably, a single nucleotide polymorphism in the phospholipase C-gamma 2 gene (PLCγ2) (Pro522Arg) was found to be protective against LOAD (Magno et al., 2019). PLCγ2 hydrolyses the membrane phospholipid PIP2 (1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate) to the secondary messengers IP3 (myo-inositol 1,4,5-trisphosphate) and DAG (diacyl-glycerol) activating a wide range of downstream signalling cascades (Bunney and Katan, 2011). However, how the P522R variant of PLCg2 attenuates pathologic insult in AD remains unknown. This study investigates the role of PLCg2 in Ab clearance and synaptic pruning using BV2 (mouse microglia) cells. The P552R variant was inserted into the human PLCγ2-myc-DDK (WT) construct (OriGene; RC200442), using Q5 site-directed mutagenesis (NEB, E0554S). BV2 cells were transfected with control (scrambled), WT (522P) or 522R variant PLCg2 using lipofectamine-2000. 48hrs post transfection cells were incubated with fluorescently labelled Ab or synaptosomes (purified from mouse brain) for 1hr. After 10 min of membrane and DAPI staining, cells were imaged using a Nikon A1R confocal microscope. Relative mRNA and protein expression levels were measured using qPCR and western blot. Over-expression of PLCg2 resulted in a robust increase in Ab uptake compared to the control (Fig 1F,H&I), and this was mildly enhanced in cells expressing the P552R variant. Conversely, synaptosome uptake was significantly reduced in BV2 cells over expressing PLCg2 and this reduction was again mildly enhanced in cells expressing the P552R variant (Fig 1E,G&I). Our study highlights the protective capacity of microglia in AD to modulate Ab clearance and synaptic pruning. We show that PLCg2 plays a key role in differentially regulating Ab and synaptosome uptake, and this regulation may be modified by the P552R variant. Increasing Ab uptake while minimising synaptosome loss is a key goal in the treatment of AD, hence PLCg2 may be an ideal therapeutic target.