PROJECT SUMMARY With rapid improvements to the quality of our healthcare, there is an urgency to better our understanding of age- related diseases like Alzheimer’s Disease (AD). Clear connections between AD progression and glucose- dependent bioenergetic deficits in the brain have motivated studies to uncover key biomarkers in AD diagnosis; apolipoprotein E4 (ApoE4) has proven to be a hallmark indicator for patients at risk of developing AD. Astrocytes are the primary manufacturer of ApoE4 in the brain, motivating most AD-relevant studies to focus on defining the relationship between glial cells and ApoE4. Despite evidence connecting neuronal metabolic dysfunction to the expression of ApoE4, the underlying biology of the metabolic changes is not understood. Our primary interest with this project is to define the mechanisms through which ApoE4 expression and reduced neuronal glucose metabolism are connected. We hypothesize that correcting disrupted mechanisms in ApoE4-expressing neurons will result in a recovery of metabolic phenotypes to more closely resemble neurons expressing ApoE3. To test our hypothesis, we have developed a paradigm that combines the targeted metabolomic analysis of human induced pluripotent stem cell (iPSC) derived neurons in vitro with the spatial transcriptomic analysis of mice in vivo. The in vivo model will be C57BL/6 mice with a whole-body knock in of human ApoE3 or ApoE4 on an amyloid precursor protein (APP) background with the Swedish, Iberian, and Arctic mutations (APPNL-G-F). We will perform spatial transcriptomics on the hippocampus, one of the first regions affected by AD progression, to longitudinally track the most differentially expressed genes due to ApoE4. The human iPSCs expressing either the E3 or E4 isoform of ApoE allows for a nearly pure (>99%) neuronal culture. The whole neuronal metabolome is probed with uniformly 13C labeled glucose ([U-13C] glucose) to quantify which metabolites in neurons are being derived from glucose. The metabolome of the ApoE3 and ApoE4 expressing neurons will be compared to define the primary differences in how glucose is metabolized between neurons based on their ApoE isoform. We will use CRISPR inhibition or activation (CRISPRi/a) to alter the expression of our genes of interest in a targeted metabolomics study to evaluate the functionality of each gene in relation to glucose metabolism in neurons. We will then evaluate whether metabolic recovery is achieved through comparing ApoE4 neurons to ApoE3 neurons based on equivalence of glucose-derived metabolites, cytosolic ATP and glucose levels, and cell survival. Successful completion of these aims will inform future studies focusing on metabolic recovery in neurons as well as providing candidates for therapeutic targets against neurodegenerative disease. The research will be conducted at Gladstone Institutes and UCSF under the mentorship of Dr. Ken Nakamura along with key facilities such as the Stem Cell, Genomics, and Bioinfor...