GPR3‐biased signaling is involved in microglial activation in a preclinical Alzheimer’s disease mouse model

Abstract Background Microglia play a crucial role in clearing amyloid‐beta (Aβ) plaques, one of the primary pathological hallmarks of AD. We previously showed that G protein‐biased signaling by the G protein‐coupled receptor GPR3 reduces soluble Aβ levels and leads to an increase in Aβ plaque compaction and a reduction in Aβ plaque area in the preclinical App NL‐G‐F AD mouse model. These results suggest a protective microglial response that may limit Aβ plaque formation in G protein‐biased GPR3 AD mice. However, the precise mechanism by which biased GPR3 signaling in microglia restricts the growth of Aβ plaques remains unknown. Method We utilized a CRISPR/Cas9 genome editing strategy to mutate six serine/threonine residues in the GPR3 C‐terminus to phosphorylation‐deficient alanine residues to generate a G protein‐biased GPR3 mouse model. We also introduced a hemagglutinin (HA) tag in the N‐terminus of GPR3 to determine the expression and localization of the GPR3 protein in vivo . We crossed the App NL‐G‐F AD mouse model with our GPR3 HA/HA and G protein‐biased GPR3 HA/Ala mice. We then performed bulk RNAseq on cortical brain samples from App NL‐G‐F , GPR3HA/HA (AD KI), and AppNL‐G‐F, G protein‐biased GPR3 ( Biased AD KI ) mice. To characterize the microglia phenotype, we are conducting Flow cytometry (FC) of Biased AD KI relative to AD KI brains at 6, 12, and 18 months of age (early‐, mid‐, and late‐stage pathology). Result The bulk RNAseq data reveal that microglial activation in Biased AD KI mice is consistent with a disease‐associated microglia (DAM) profile. The DAM profile includes upregulation of phagocytosis‐ and endolysosomal‐related genes, such as Trem2 , Lyz2 , Itgax, Cst7 , Clec7a , and Cd68 . Specifically, we observe an upregulation of Trem2 and Tyrobp (DAP12), congruent with our observed increase in the total area occupied by microglia, Aβ plaque coverage area, and Aβ plaque compaction in Biased AD KI mice. Preliminary FC analysis also indicates differential expression of DAM markers in microglia isolated from our Biased AD KI mice. Conclusion Our findings provide compelling evidence for the involvement of biased GPR3 signaling in regulating microglial function and the response to the accumulation of Aβ pathology in AD.