Abstract Diet and nutrition influence our cognitive abilities, how our brains age, and vulnerability to neurodegeneration. However, the interaction of diet with the human brain is complex and influenced by every person’s unique genetic composition. Clinical studies found that ketogenic diets and supplements improve cognition and protect against Alzheimer's disease (AD) in most individuals but have no beneficial effect on individuals with the strongest genetic risk factor for AD, APOE4. How APOE4 interacts with diet at the cellular and molecular level to influence AD is unknown. The primary metabolite of ketogenic diets beta-hydroxybutyrate freely passes across the blood-brain barrier into the human brain where it is converted to acetyl-CoA. Our data demonstrate that cholesterol transport is impaired in APOE4 glia leading to intracellular cholesterol accumulation which triggers inflammation, AD pathogenesis, and cognitive decline. As a compensatory mechanism to impaired cholesterol trafficking, APOE4 glia upregulates cholesterol biosynthesis, which uses acetyl-CoA to generate cholesterol. We hypothesize that impaired cholesterol trafficking and upregulation of cholesterol biosynthesis in APOE4 glia adversely interact with high-fat/ketogenic diets to exacerbate and accelerate AD pathogenesis. We established methods to mimic ketogenic diets in vitro. This revealed in APOE4 glia ketones increase aberrant intracellular cholesterol deposits and promote neuroinflammation and hypomyelination. We developed an in vitro model of human brain tissue that contains all the major cell types and tissues including cerebrovasculature, neurocircuits, myelination, and neuro-immune cells. Aim 1 will employ this system (miBrain) to further investigate the interaction of APOE genotype with high-fat/ketogenic diets and its contribution to AD pathogenesis in human brain tissue. Using transcriptomic and biochemical approaches we will discover the underlying mechanisms that we will modulate via chemical and genetic approaches to identify therapeutic targets for promoting beneficial APOE4-diet interactions. We will complement this with studies in APOE3/3 and APOE4/4 humanized mice in Aim 2 to mechanistically dissect the interaction between APOE genotype and high-fat/ketogenic diets at the organismal level. Together aims 1 and 2 will provide holistic insight into the peripheral and central interactions of APOE4 with ketogenic diets. Several other AD risk variants also have functional roles in cholesterol and lipid homeostasis. We further hypothesize that cholesterol dysregulation and its interaction with diet is a central pathogenic mechanism of AD. In Aim 3, we will investigate using isogenic human brain tissue generated from iPSCs harboring genetic risk factors in SORL1, TREM2, ABCA7, and APOE. We will determine how each risk variant interacts with ketogenic diets to influence pathogenic outcomes in AD. Collectively, this study will pioneer approaches and technology that wi...