The impact of lysosomal Phospholipase D3 deficiency on activated response microglia regulation

The high region specialization and complexity of the brain provides microglia with a multiplicity of signals, requiring different responses. One of these activation states encompasses the activated response microglia (ARM) phenotype that is acquired when microglia surround amyloid plaques in Alzheimer brains. The phenotype is characterized by an increased lipid metabolism, phagocytosis rate, lysosomal protease content and the secretion of neuroprotective agents, leaving to reason that the ARM phenotype is adapted in an attempt to restore homeostasis. One of the proteins that is systematically upregulated in ARMs, is the late-onset Alzheimer risk factor: Phospholipase D3 (PLD3). PLD3 is a lysosomal 5'-3' exonuclease that regulates inflammatory responses by degrading ssDNA; i.e. the substrate of toll-like receptor 9. Interestingly, the host lab recently detected major defects in the neuronal autophagic-lysosomal system caused by PLD3 dysfunctions. This suggests that PLD3 could be one of the endolysosomal system components operating at the crossroad of inflammation and microglial capacity to keep pace with degradation. Given the human microglial population shows a heterogeneity that is lacking in other species, the lab has developed a series of tools, including human patient iPSCs with an Alzheimer-linked V232M-PLD3 background as well as knockouts and isogenic controls. In addition, I will start from a human microglial clone 3 (HMC3) microglia cell line that has been knocked out for PLD3 and use CRISPR/Cas9 genome editing to generate isogenic controls as well as PLD3 mutant rescue lines. Together with primary microglia from the lab’s PLD3KO and PLD3KOxAPPKI mouse lines and iPSC-derived microglia, these microglia PLD3 models will be used to investigate (i) how PLD3 is involved in ARM polarization, (ii) what impact PLD3 has on the microglial endo-lysosomal system-associated functions, and (iii) if PLD3 dysfunction impacts on amyloid clearance through microglia. The knowledge I will gather will provide a foundation to functionally link a dysfunctional exonuclease/PLD3 activity to microglial activities as well as its impact on Alzheimer pathology.