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

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 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 . We crossed the AD mouse model with our GPR3 and G protein‐biased GPR3 mice. We then performed bulk RNAseq on cortical brain samples from , GPR3HA/HA (AD KI), and AppNL‐G‐F, G protein‐biased GPR3 () mice. To characterize the microglia phenotype, we are conducting Flow cytometry (FC) of relative to 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 mice is consistent with a disease‐associated microglia (DAM) profile. The DAM profile includes upregulation of phagocytosis‐ and endolysosomal‐related genes, such as , , , , and . Specifically, we observe an upregulation of and (DAP12), congruent with our observed increase in the total area occupied by microglia, Aβ plaque coverage area, and Aβ plaque compaction in mice. Preliminary FC analysis also indicates differential expression of DAM markers in microglia isolated from our 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.