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The P522R variant of PLCG2, expressed by microglia, is associated with reduced risk of Alzheimer's disease (AD). Yet, the impact of this protective mutation on microglial responses to AD pathology remains unknown. Chimeric AD and wild‐type mice were generated by transplanting PLCG2‐P522R or isogenic wild‐type human induced pluripotent stem cell microglia. At 7 months of age, single‐cell and bulk RNA sequencing, and histological analyses were performed. The PLCG2‐P522R variant induced a significant increase in microglial human leukocyte antigen (HLA) expression and the induction of antigen presentation, chemokine signaling, and T cell proliferation pathways. Examination of immune‐intact AD mice further demonstrated that the PLCG2‐P522R variant promotes the recruitment of CD8+ T cells to the brain. These data provide the first evidence that the PLCG2‐P522R variant increases the capacity of microglia to recruit T cells and present antigens, promoting a microglial transcriptional state that has recently been shown to be reduced in AD patient brains. [ABSTRACT FROM AUTHOR]

Using a novel microfluidic chamber that allows the isolation of axons without contamination by nonaxonal material, we have for the first time purified mRNA from naive, matured CNS axons, and identified the presence of >300 mRNA transcripts. We demonstrate that the transcripts are axonal in nature, and that many of the transcripts present in uninjured CNS axons overlap with those previously identified in PNS injury-conditioned DRG axons. The axonal transcripts detected in matured cortical axons are enriched for protein translational machinery, transport, cytoskeletal components, and mitochondrial maintenance. We next investigated how the axonal mRNA pool changes after axotomy, revealing that numerous gene transcripts related to intracellular transport, mitochondria and the cytoskeleton show decreased localization 2 d after injury. In contrast, gene transcripts related to axonal targeting and synaptic function show increased localization in regenerating cortical axons, suggesting that there is an increased capacity for axonal outgrowth and targeting, and increased support for synapse formation and presynaptic function in regenerating CNS axons after injury. Our data demonstrate that CNS axons contain many mRNA species of diverse functions, and suggest that, like invertebrate and PNS axons, CNS axons synthesize proteins locally, maintaining a degree of autonomy from the cell body. [ABSTRACT FROM AUTHOR]

Background: Genetic studies have uncovered several Alzheimer's Disease (AD) risk genes that are highly expressed in microglia, suggesting that immune dysregulation plays a critical role in the development and progression of AD. In line with this, a recently discovered polymorphism (P522R) in the microglia‐expressed gene Phospholipase C‐Gamma 2 (PLCG2) was shown to be protective for AD. Interestingly, PLCG2 codes for a protein that regulates the release of calcium from ER stores and is also a component of the downstream signaling pathway of TREM2, another microglial AD risk gene. This underscores the need to improve our understanding of microglial calcium signaling, the effects of AD risk genes on this pathway, and the impacts of PLCG2 and calcium signaling on human microglial function. Methods: CRISPR gene editing was used to produce isogenic iPSCs that express the AD protective mutation in PLCG2. Several in vitro assays were then performed to examine calcium signaling pathways and phagocytosis. PLCG2‐P522R and WT HPCs were also transplanted directly into xenotransplantation‐compatible mouse pups (MITRG‐5xfAD mice), where they readily differentiated into human microglia, exhibiting typical markers and morphology.. Xenotransplanted human microglia (x‐MG) were then examined by confocal microscopy and isolated from the brain for single‐cell RNA sequencing. Results: Thus far our in vitro studies suggest that the P522R coding mutation increases both basal cytosolic calcium concentrations and amyloid phagocytosis. Additional functional experiments are further examining the interactions between PLCG2 mutations and calcium signaling during the uptake of AD‐relevant substrates. In addition, single cell RNA sequencing studies of chimeric mice reveal that the P522R mutation leads to an increased percentage of human microglia that have adopted a disease‐associated microglial (DAM) phenotype in vivo. Conclusions: By combining iPSC‐microglia, CRISPR gene editing, and chimeric AD mice these studies have begun to uncover the impact of PLCG2 mutations on human microglial function and reveal that the protective P522R mutation increases Ab phagocytosis and promotes the adoption of a DAM‐like microglial response. [ABSTRACT FROM AUTHOR]