Summary: Chronic neuroinflammation and metabolic dysfunction characterize neurodegenerative diseases, including Alzheimer's disease (AD). One of the earliest known biomarkers of AD is an altered pattern of regional glucose metabolism in the brain. Carriers of the ε4 allele of apolipoprotein E (APOE) exhibit this pattern of altered cerebral glucose metabolism from a young age and suffer from a dramatically increased risk of acquiring AD. Meanwhile, ε4 individuals are also considered to have a maladaptive and detrimental neuroinflammatory phenotype, with increased pro-inflammatory signaling in ε4 microglia. Additionally, it is well- established that microglial inflammatory phenotypes are driven by precise metabolic reprogramming, with pro- inflammatory cytokine production requiring increased aerobic glycolysis – a phenomenon known as `immunometabolism'. This proposal aims to investigate whether the neuroinflammatory phenotype in ε4 microglia could be intrinsically driven by ε4’s effects on glucose metabolism. Aim 1 will investigate the hypothesis that enhanced pro-glycolytic gene expression drives neuroinflammatory ε4 transcriptional phenotypes by leveraging the power of targeted single-cell RNA sequencing to interrogate gene expression of a panel of pre-selected metabolic, neuroinflammatory, and disease-associated genes, with results validated at the protein and post-translational level. Aim 2 will address the hypothesis that ε4 microglia show an increased pro-inflammatory phenotype due to underlying impairments in central carbon metabolism. We will use Stable Isotope Resolved Metabolomics (SIRM) with a 13C-glucose tracer to measure metabolic flux in ε2, ε3, and ε4 primary microglia responding to pro- and anti-inflammatory treatments. We will compare oxidative phosphorylation and glycolytic activity in response to these treatments. These metabolic measures will then be linked to readouts of microglial function by assessing cytokine release and phagocytic activity in parallel. Aim 3 will evaluate the efficacy of targeting key metabolic nodes to reprogram the ε4 inflammatory response. We hypothesize that pharmacological inhibition or viral overexpression of specific metabolic enzymes identified in our preliminary data as being perturbed by APOE genotype will rebalance cytokine production in ε4 microglia and improve phagocytosis of amyloid beta peptide. Our ultimate goals for this research are to: (1) understand how APOE genotype influences the metabolic response to inflammatory stimuli in microglia, (2) determine whether altered metabolic gene expression drives a transcriptional profile that favors neuroinflammation and increased AD risk, and (3) identify immunometabolic pathways altered by APOE genotype that could represent avenues to target in the treatment of neurodegenerative disease. This proposal integrates innovative transcriptomic and metabolomic techniques with functional readouts of microglia to unify our understanding of two major facets ...