Alzheimer's disease (AD) is the most common neurodegenerative disease in which neurons and microglia play causal and distinctive roles in its pathogenesis. These cellular mechanisms of AD need to be resolved at the level of individual brain cell types, especially at the protein (proteomic) level to best guide therapeutic and biomarker discovery. Our recent proteomic studies of human post-mortem brains have identified a signaling pathway called the MAPK/ERK pathway, as a strong predictor of AD pathology and cognitive decline. We have also found that activation of the ERK pathway is characteristic of activated microglia in a mouse model of AD pathology, and inhibition of ERK in microglia reduces their pro-inflammatory and detrimental responses. Based on these findings, our overall hypothesis is that excessive ERK activation is a central mechanism of AD pathogenesis that uniquely impacts the proteomic phenotypes of neurons and microglia, leading to neurodegeneration. In order to determine how MAPK/ERK signaling changes with aging and AD pathology in a mouse model, we will apply a novel in-vivo labeling approach called CIBOP-MS that enables us to define the dynamics of proteomic changes occurring specifically in microglia or neurons without the need for cell type isolation (Aim 1). Using a small molecule inhibitor of ERK activity, we will then determine how ERK inhibition impacts neuronal and microglial proteomes and identify novel biofluid biomarkers in an amyloid beta mouse model (Aim 2). Lastly, we will manipulate ERK activity leading to either over-activation or attenuation specifically in either neurons or microglia using genetic approaches (Aim 3). Collectively, our comprehensive studies focusing on ERK signaling in neurons and microglia, will validate CIBOP-MS as a novel approach to resolve brain cell type-specific proteome dynamics in aging and neurodegeneration, with biomarker and therapeutic implications. Our multidisciplinary expertise in in-vivo cell type specific proteomic labeling approaches, systems biology, neuroinflammation, biofluid biomarker discovery and mouse models of AD pathology uniquely positions us to execute this innovative R01 proposal.