Project Summary Genome-wide association studies have identified many Alzheimer’s disease (AD) risk genes related to immune response. Among these are the microglial receptors CD33 and TREM2. We previously showed that knock-out of CD33 attenuated amyloid beta (Aβ) pathology and improved cognition in 5XFAD mice, both of which were abrogated by additional Trem2 knock-out. Knocking out Trem2 in 5XFAD mice exacerbated Aβ pathology and neurodegeneration but reduced microglia numbers. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are downregulated in 5XFAD;Trem2-/- mice. Differential gene expression in 5XFAD;CD33-/- microglia depended on the presence of Trem2, suggesting TREM2 acts downstream of CD33. Moreover, we reported that the D87N and T96K variants of TREM2 increased AD risk in the NIMH family-based genetic data and ADSP case-control samples and showed that these variants increased binding to TREM2 ligands. In our preliminary study, we found that the gain-of-function Trem2T96K variant increased levels of insoluble Aβ42 but reduced both microglia numbers and clustering of microglia around Aβ plaques in 5XFAD mice. Furthermore, the Trem2T96K variant reduced secretion of sTrem2 in 5XFAD mice. Finally, the T96K variant led to reduced cell surface expression and secretion of TREM2 in human microglial cell lines. Here, we propose to 1) assess the impact of TREM2 gain-of-function mutations on AD pathogenesis, 2) investigate the effects of these mutations on microglial activation and gene expression, and 3) identify and characterize novel AD-associated TREM2 gain-of-function mutations as well as target TREM2 for therapeutic purposes. In Aim 1, we will examine the effects of the gain-of-function Trem2T96K mutation on Aβ pathology, neurodegeneration and plaque associated-neuritic dystrophy in 5XFAD mice. We will also determine whether Trem2T96K impacts cognitive function in 5XFAD mice. In Aim 2, we will investigate the impact of the gain-of-function Trem2T96K mutation on microglial cell activation and recruitment to Aβ plaques as well as immune response to lipopolysaccharide stimulation in 5XFAD mice. RNA-seq profiling of microglia will reveal the effects of Trem2T96K on microglial gene expression signature and how they compare to disease-associated microglia. In Aim 3, we will analyze whole genome sequencing data from the NIMH family sample and NIA ADSP to identify novel AD-associated TREM2 mutations that will be characterized for their effects on ligand-dependent activation of TREM2. We will then introduce novel TREM2 gain-of-function mutations into iPSC-derived human microglia-like cells using CRISPR/Cas9 system and investigate underlying molecular mechanisms. Finally, we will predict and validate novel targeted drugs that mimic TREM2 activation and/or oppose TREM2 loss-of-function using computational approaches. The major goals of this proposal are to comprehensively assess the...