PROJECT SUMMARY This application is being submitted in response to NOT-AG-21-018. Alzheimer's disease (AD) is a progressive neurodegenerative brain disease and the most common type of dementia affecting nearly 47 million people worldwide. AD cases (∼98 %) are prevalent in aging subjects and often involve various other co-morbidities including obesity and diabetes. Accumulating evidence shows that diabetes is an independent risk factor of AD. Obesity and diabetes are known to cause damage to vascular cells including endothelial cells and pericytes, and various studies have demonstrated that cellular homeostasis of these cells are essential for the integrity of the blood-brain-barrier (BBB), the disruption of which has been observed in many CNS pathologies, including AD and vascular dementia. However, due to the relative inaccessibility of functional human brain tissues for research, the underlying mechanisms of diabetes-induced cognitive impairment linked to vascular damage remain poorly understood. To that end, in this Alzheimer’s-focused administrative supplement, we aim to use diabetic patient- specific induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) and iPSC- derived pericytes in 2D and 3D cultures to model this diseased phenotype in a dish. In Aim 1, we will generate iPSC-BMECs and iPSC-pericytes from healthy and diabetic patients, and test whether a novel RAB5/7- dependent endosomal-mediated mitochondrial degradation pathway is disrupted in these cells during hyperglycemia. We will functionally characterize how impairment of this degradation pathway in the presence of hyperglycemia will affect the phenotype of iPSC-BMECs and iPSC-pericytes. This is based on our parent grant R01 showing that this pathway was affected in cardiomyocytes who are at heightened risk against diabetes. In Aim 2, we will use a novel Brain-Chip platform to generate an in vitro 3D blood-brain barrier-like structure (the neurovascular unit) comprising of isogenic CRISPR RAB7 KO iPSC-BMECs and iPSC-pericytes, along with isogenic healthy iPSC-astrocytes, and iPSC-neurons as a more physiological relevant platform to investigate the effects of cell-cell interaction on hyperglycemia/RAB7-KO induced BBB integrity disruption. Single cell RNA sequencing to identify cross-talk between dysfunctional vascular cells and neurons and glial cells will also be performed. Overall, the platform we will be establishing can serve as a proof-of-principle to test druggable targets that can ameliorate cognitive deficits or various types of neurodegenerative diseases caused by diabetes.