The hypermorphic PLC?2 S707Y variant dysregulates microglial cell function – Insight into PLC?2 activation in brain health and disease, and opportunities for therapeutic modulation

Phospholipase C-gamma 2 (PLC?2) is highly expressed in hematopoietic and immune cells, where it is a key signalling node enabling diverse cellular functions. Within the periphery, gain-of-function (GOF) PLC?2 variants, such as the strongly hypermorphic S707Y, cause severe immune dysregulation. The milder hypermorphic mutation PLC?2 P522R increases longevity and confers protection in central nervous system (CNS) neurodegenerative disorders, implicating PLC?2 as a novel therapeutic target for treating these CNS indications. Currently, nothing is known about what consequences strong PLC?2 GOF has on CNS functionality, and more precisely on the specific biological functions of microglia. Using the PLC?2 S707Y variant as a model of chronic activation we investigated the functional consequences of strong PLC?2 GOF on human microglia. PLC?2 S707Y expressing human inducible pluripotent stem cells (hiPSC)-derived microglia exhibited hypermorphic enzymatic activity under both basal and stimulated conditions, compared to PLC?2 wild type. Despite the increase in PLC?2 enzymatic activity, the PLC?2 S707Y hiPSC-derived microglia display diminished functionality for key microglial processes including phagocytosis and cytokine secretion upon inflammatory challenge. RNA sequencing revealed a downregulation of genes related to innate immunity and response, providing molecular support for the phenotype observed. Our data suggests that chronic activation of PLC?2 elicits a detrimental phenotype that is contributing to unfavourable CNS functions, and informs on the therapeutic window for targeting PLC?2 in the CNS. Drug candidates targeting PLC?2 will need to precisely mimic the effects of the PLC?2 P522R variant on microglial function, but not those of the PLC?2 S707Y variant. Overall design: RNA was extracted from PLC?2 WT and S707Y hiPSC-derived microglia from three separately generated hematopoietic inductions, at different myeloid precursor differentiation ages (early, middle and late). Libraries were prepared using the KAPA RNA HyperPrep Kit and sequenced on an Illumina HiSeq 4000 sequencer at a minimum of 25 million paired-end reads (75 bp) per sample performed by UCL Genomics (London, England).