PROJECT ABSTRACT Alzheimer’s disease (AD) is a common and burdensome neurodegenerative disease. Apolipoprotein E (APOE) is the most prevalent risk factor for developing AD. APOE genotype may contribute to disease pathogenesis and risk by impacting astrocyte molecular profiles and subsequent biological functions. Data from my lab in human post-mortem AD and control brain proteomes identified a module of co-expressed astrocytic and microglial proteins in APOE ε4 individuals that were involved in sugar metabolism and inflammation and strongly correlated with the MAPK/ERK pathway. Because astrocytes are abundant throughout the brain, produce high levels of apoE protein, and play a critical role in brain homeostasis, they are more likely to contribute to disease vulnerability and pathogenesis of AD in the context of APOE risk. To address how APOE genotype impacts astrocyte function, my laboratory recently developed cell type-specific in vivo biotinylation of proteins (CIBOP) as a novel approach to quantify the total and phospho-proteomes of astrocytes in their native state without the need for cell isolation. In this approach, the biotin ligase, TurboID, is selectively expressed in the cell type of interest using a conditional Cre/lox genetic strategy. Using astrocyte-CIBOP in our preliminary studies, we have successfully obtained native-state proteomes of astrocytes from mouse brain, and quantified astrocyte- derived cytokines and MAPK/ERK signaling phospho-proteins. Leveraging this highly innovative methodology, my goal in the current study is to for the first time define the astrocyte-like immune response to APOE ε4 genotype and determine if the MAPK/ERK pathway is a mechanism of this response. My central hypothesis is that APOE ε4 genotype augments pro-inflammatory profiles of astrocytes via increased cytokine and complement production in an ERK signaling-dependent manner. I will use the astrocyte-CIBOP mice derived on homozygous human APOE 4/4 and APOE 3/3 knock-in genetic backgrounds to determine the differential impacts of APOE genetic risk on molecular signatures of astrocytes in vivo. In Aim 1, I will test the hypothesis that APOE 4/4 astrocytes, compared to APOE 3/3, will exhibit elevated levels of pro-inflammatory cytokines, increased complement protein production, amplified MAPK/ERK signaling, and aberrant changes in lipid metabolism. In Aim 2, I will test the hypothesis that the exaggerated pro-inflammatory response of APOE4 astrocytes, initiated by systemic inflammatory challenge (LPS), is mediated via ERK signaling. Using bioinformatic approaches on proteomic and transcriptomic data and validation IHC approaches in mouse and human brain tissues, I will identify molecular changes in reactivity states of APOE 4/4 and 3/3 astrocytes in vivo and identify ERK- dependent and ERK-independent mechanisms of APOE. My work will lay the foundation to further investigate astrocyte-mediated mechanisms of AD pathogenesis to employ CIBOP in future stu...