PROJECT SUMMARY Alzheimer’s disease (AD) is a major unresolved public health issue. Despite decades of research on amyloid-β (Aβ) and tau, we do not fully understand the complex molecular and cellular biology of these key disease drivers. The brain reacts to the pathological accumulation of Aβ and tau with an early and profound neuroinflammatory response, which includes alterations in the profile, function, and activation of microglia, astrocytes, and oligodendrocytes. Although neuroinflammation is an integral component and putative driver of AD pathogenesis, the cell type-specific mechanisms that characterize this response and its impact on cognitive decline are incompletely understood. Biased G protein-coupled receptor (GPCR) ligands preferentially activate G protein or β-arrestin signaling pathways and are leading to the development of drugs with superior efficacy and reduced side effects in several therapeutic areas. Surprisingly, biased GPCR signaling is a largely unexplored area in AD research. In the previous funding cycle, we developed a G protein-biased GPCR 3 (GPR3) mouse model that lacks GPR3- mediated β-arrestin signaling. Using this model, we showed that biased GPR3 mice exhibit normal cognitive and behavioral functions, which are disrupted in Gpr3-deficient mice. We further determined that biased GPR3 signaling reduces Aβ levels and the Aβ plaque burden and enhances the glial response in a preclinical AD mouse model. These exciting findings indicate that GPR3 is the first GPCR to modulate both Aβ pathology and the innate immune response in AD. These studies also establish the strong scientific premise that the largely unknown cell type-specific functions of GPR3 impact the development and progression of AD. Thus, the major goal of this R01 renewal application is to test the central hypothesis that biased GPR3 signaling in microglia, astrocytes, and neurons reduces AD-related pathologies and cognitive decline by promoting protective immune responses and preserving neuronal function. Aim 1 will determine how biased GPR3 signaling alters microglia activation and function and AD-related pathways in primary human microglia and in vivo in our AD mouse models. Aim 2 will determine how biased GPR3 signaling affects the cell-autonomous and non-cell-autonomous functions of astrocytes in primary human astrocytes and in vivo in our AD mouse models. Aim 3 will determine the role of biased GPR3 signaling in neuronal function and cognition in directly converted induced neurons (iNs) from control subjects and AD patients and in vivo in our AD mouse models, respectively. Successful completion of these studies will establish the in vitro and in vivo impact of biased GPR3 signaling on neuronal- and glial- dependent pathways and provide proof of concept for the development of safer and more selective GPCR- targeting therapeutics with more directed pharmacological action for AD.