Project Summary The causes of Alzheimer’s disease (AD) are thought to include a combination of age-related changes in the brain, genetic factors (risk and deterministic genes), and environmental and lifestyle factors (e.g. diet, exercise, sleep). Age-related changes in the brain include atrophy, inflammation, generation of reactive oxygen species, and vascular dysfunction. A central question in AD research is whether vascular dysfunction (specifically in small vessels in the brain) is a primary cause of AD etiologies (vascular hypothesis) or a secondary consequence of other processes (e.g. amyloid cascade hypothesis). Studies in humans and transgenic mice have established that blood-brain barrier (BBB) dysfunction can occur prior to amyloid beta deposition, cerebral amyloid angiopathy (CAA), and the onset of cognitive impairment. The onset of AD is thought to occur 20 years or more before symptoms such as memory loss and cognitive deficits. While this window provides opportunities for intervention, it also poses challenges in uncovering the underlying causes of the disease. Identifying the mechanisms of BBB dysfunction is crucial to understanding disease progression and identifying targets for intervention. The objective of this supplement project is to identify the mechanisms of BBB dysfunction occurring within a tissue-engineered brain arteriole model caused by APP and PSEN1 mutations, and by amyloid beta (Aβ) exposure. The objective will be addressed in two aims. In Aim 1 brain microvascular endothelial cells (iBMECs) and smooth muscle cells (iSMCs) differentiated from an isogenic panel of iPSCs harboring PSEN1(M146V) or APP(Swe) mutations, along with wild type controls, will be incorporated into a brain-specific arteriole model developed in our laboratory. From analysis of the differential gene expression profiles of iBMECs and iSMCs, biological pathways altered by AD mutations and Aβ exposure that may underlie cerebrovascular dysfunction will be identified. In Aim 2 a selected library of functional assays will be used to determine mutation-dependent and amyloid beta-dependent mechanisms of cerebrovascular dysfunction. Functional measurements will include a core assays associated with barrier function, as well as assays developed based on pathways identified in Aim 1. This hybrid approach will enable us to focus on cerebrovascular functions that are most likely to be altered by mutational status and Aβ exposure.