PROJECT SUMMARY Alzheimer’s disease (AD)-associated pathological alterations are universally present in individuals with Down syndrome (DS) by 40 years of age, and the majority of people (88%) with DS are negatively affected by AD at 65 years of age. Because of this high disease burden, effective treatments for AD would be particularly beneficial to the DS population, but the complex molecular events that underlie the pathogenesis of AD in DS (AD-DS)— the key to developing targeted therapies—remain poorly understood. In the proposed research, we will use CRISPR technology to engineer mutant mice to probe the regulatory roles of MS4A proteins on TREM2 and microglial activation, which are known to play important roles in the development of AD in both DS and the general population. TREM2 is a transmembrane protein that is expressed in microglia, and TREM2 functions as a pattern recognition receptor that detects and binds to Aβ oligomers and induces an innate immune response. Soluble TREM2 (sTREM2) inhibits Aβ oligomerization and disaggregates preformed Aβ oligomers and protofibrils, thus inhibiting Aβ fibrillization, and consequently, Aβ-induced neurotoxicity is blocked. Experimental support for this cascade has been obtained in mouse models, and different levels of TREM2 in cerebrospinal fluid (CSF) are known to be associated with different disease states in patients with AD. MS4A proteins are considered among the most important regulators of TREM2 and microglial activation, and MS4A mutations are closely associated with different levels of TREM2 in CSF and activation of microglia l cells. However, the functional relationships among TREM2, MS4As, and microglial activation were established mainly through genome-wide association studies. Presently, in vivo data on how TREM2 and microglial activation are regulated by MS4As and the consequence in AD-DS are lacking. To fill this important knowledge gap, we plan to examine Ms4a-mediated regulation of TREM2 in AD-DS in vivo in two engineered mouse models. Afterward, we will pursue the following phenotyping experiments for each aim: (a) analysis of TREM2 levels in CSF; (b) analysis of the activation of microglial cells; (c) measurement of the levels of Aβ in mouse brain tissue; (d) examination of AD-associated neuropathology; and (e) analysis of cognitive deficits. Attainment of these objectives will allow us to directly test the hypothesis that Ms4a-mediated TREM2 regulation and microglial activation play an important role in AD-DS for the first time by using our mutant mice. Furthermore, achievement of our goals will open up a major area for new AD research, with promise for those with or without DS, and bring us closer to developing effective treatments for AD and other medical conditions associated with TREM2, MS4A, and microglia.