PROJECT SUMMARY Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is the most common cause of dementia in the United States. Variants in the apolipoprotein E gene (APOE) is strongly associated with development of AD. In the brain, APOE is mostly synthesized in the astrocytes. The primary role of APOE is to deliver lipids and cholesterol from astrocytes to other cell types. Most humans have one of the major allele of APOE, APOE ε2 (R176C), APOE ε3 (reference allele), and APOE ε4 (C130R). These missense variants are associated with different levels of Alzheimer disease risks where APOE ε4 increases the risk of disease whereas APOE ε2 decrease risk of AD. A recent analysis that genotyped over 100,000 patients showed that rare missense variants were associated with different levels of Alzheimer disease risk. The molecular mechanism of how variants in APOE changes astrocyte physiology that could lead to Alzheimer disease is not well known. To attempt to understand this, our previous work adapted mass spectrometry-based thermal proteome profiling to determine changes in protein interaction as a result of missense variants. Using yeast as simplified model, we were able to show mutant thermal proteome profiling to resolve subcomplex level protein-protein interaction disruptions of a protein complexes due to missense variants. To study the role of these rare missense variants on APOE in astrocytes, we will employ an mouse primary astrocyte culture that will generate human APOE with the specific missense variant. First, we will determine the effect of the rare missense variants on the lipoprotein functions and astrocyte physiology. We will determine the differences between the APOE variants of uptake of Aβ peptides, lipidation status, and the effects on the astrocyte transcriptome. Second, we will determine the changes in protein interactions as a result in APOE variants using mutant thermal proteome profiling to detect thermal stability changes. Taken together, these data will expand our understanding of how missense variants change the functions of APOE and help give insight into the molecular mechanisms that result in Alzheimer disease.