PROJECT SUMMARY/ABSTRACT The Alzheimer’s Association describes Alzheimer’s disease (AD) as “the most common type of dementia”, a term that generally describes the loss of memory and some forms of cognitive skills that interfere with everyday activities. AD is one of the top three causes of death in aged individuals in the United States. It was first described by Alois Alzheimer in 1906 as an accumulation of extracellular amyloid plaques and intracellular neurofibrillary tau tangles; yet, more than a century later, there is still no effective treatment or cure to the disease. Although an abundance of research has focused on understanding AD, there are still many unknowns to the mechanisms of disease pathogenesis. Microglia, the primary immune cell in the brain, have multiple activation phenotypes that are involved in broad and complex functions in the brain, including in neurotoxicity and release of inflammatory cytokines, in repair and regeneration, in immune regulation, in neuroprotection and release of anti-inflammatory cytokines, in cell survival/proliferation/migration, and in phagocytosis. TREM2 and TYROBP are known to form a complex in microglia that can lead to complex intracellular signaling networks, and these proteins have recently emerged as important regulators of the transition between resting (homeostatic) microglia and its activation states. Recent findings from our group and others have shown a TYROBP-dependent and TREM2-independent molecular signature that exhibits involvement in the early transition step from homeostatic microglia to disease-associated microglia (DAM). Interestingly, the sequential step of DAM activation is TREM2-dependent. However, the underlying mechanisms of how TREM2 or TYROBP regulate these downstream cellular phenotypes are largely unknown. In this proposal, we aim to systematically test whether, and to what extent, AD-relevant stimuli, such as amyloid beta (A) oligomers or physiological Apolipoprotein E 3 or 4 isoforms are able to activate microglia, in the absence of TYROBP or TREM2 or in the presence of the AD-associated Trem2 R47H knockin mutation. Here, we will utilize a thorough approach to investigate not only signal transduction and various cellular activation pathways, but also we will use state-of-the-art mass spectrometry-based phosphoproteomics to unbiasedly examine global changes in phosphopeptides, in order to specifically elucidate differences in microglial homeostasis and activation states in our models. The overall goal of the study is to understand the differential roles of TREM2 and TYROBP in the mechanisms underlying microglial activation in the context of disease-like stimuli, and to ultimately identify potential therapeutic targets that specifically focus on microglia activation phenotypes, and that could contribute to the delay or treatment of AD pathology.