PROJECT SUMMARY The levels of fructose are higher in the brains of patients with Alzheimer’s disease, and diets rich in high- fructose corn syrup are associated with an increased risk of developing Alzheimer’s disease. However, the mechanism by which fructose metabolism may accelerate Alzheimer’s disease onset and progression is unknown. Microglia—the innate immune cells of the brain—protect against or promote the development of AD, depending on their function. Notably, microglial function is regulated by metabolism. When activated, microglia shift towards glucose utilization and away from oxidative phosphorylation. Chronic activation eventually leads to ‘metabolic reprogramming’ and microglial dysfunction, which drives AD pathology. Although strategies targeting microglia hold enormous potential for treating AD, molecular targets that can help reverse the metabolic programming of microglia have not yet been identified. The fructose transporter GLUT5 (also known as solute carrier family 2-member 5 [Slc2A5]) is predominantly expressed by microglia of the mammalian brain and has recently been identified as one of several genes within a ‘microglia-specific’ signature linked to late- onset-AD pathology. Our compelling preliminary data has also shown that GLUT5 is increased in the brains of aged and 5xFAD mice and that down-regulating fructose metabolism improves age-associated changes in microglial morphology. Therefore, we hypothesize that GLUT5-mediated fructose metabolism may drive the metabolic reprogramming of microglia associated with AD pathology. However, due to the lack of available genetic tools, the role of fructose metabolism and microglial GLUT5 in AD pathology has not been empirically determined. This R21 proposal brings together a scientific team uniquely positioned to interrogate this critical question: Dr. Bruce (microglia, metabolism, neurodegenerative disease); Dr. Lanaspa (fructose metabolism, cardiometabolic disease); Dr. D’Alessandro (metabolomics): and Dr. Frietze (bioinformatics, single-cell omics). In Aim 1, we will characterize the first conditional GLUT5flox/flox mice and generate the first microglia-specific GLUT5 KO mouse to empirically determine the role of GLUT5 in microglial metabolism and function. In Aim 2, we will utilize AD susceptible transgenic mice and mice lacking GLUT5 and Ketohexokinase (KKH, rate-limiting enzyme in future kinase) to determine whether limiting fructose metabolism prevents metabolic programming and microglial function in AD. In Aim 3, we will use human microglia-like cells derived from peripheral blood mononucleocytes to test whether pharmacologically inhibiting GLUT5 can restore age- associated dysfunction in human microglia. The impact of this study is threefold: 1. To develop novel genetic tools of great interest to AD researchers and beyond; 2. To understand the mechanisms driving fructose- mediated metabolic reprogramming; 3. To test interventions that limit fructose metabolism, validatin...