Project Abstract Changes to cerebral microvasculature that impact blood flow and blood brain barrier function are increasingly recognized as a key feature of early Alzheimer’s disease (AD) and cognitive impairment. The underlying cause of this dysfunction is largely unknown, though data from transgenic mice indicates pathways related to microvascular stress (inflammation, senescence, and angiogenesis) are upregulated with increasing amyloid beta and tau deposition. This research will make use of human tissue samples collected in the Massachusetts Alzheimer’s Disease Research Center (ADRC) and mouse models to examine the contribution of microvascular stress to AD. In Aim 1, we will determine when and where microvascular stress appears in relation to accumulation of amyloid beta plaques and tangles. In Aim 2, we will determine if microvascular stress contributes to functional weakening of the blood brain barrier (BBB) in AD by measuring a panel of serum proteins and by microdissecting “leaky” and “non-leaky” vessels to identify a mechanistic basis for dysfunction at the level of individual vessel segments. In Aim 3 we will manipulate gene expression in endothelial cells and test the causal role of specific microvascular stressors to pathological AD changes. This work will assess human vascular transcriptomes from multiple brain regions in Alzheimer’s, providing unparalleled information about how vascular cells are affected by AD pathology and insight into variability of human brain vasculature that might underlie regional differences in disease susceptibility. Further, we will make use of innovative methods including multiplex immunofluorescence to measure up to 18 proteins in tissue sections at once, clear brain preparations to assess spatial relationships between stress markers and AD pathology, and a novel adeno-associated virus that can specifically target endothelial cells in vasculature and that may be an exciting tool for future therapeutic gene delivery. Overall, these studies will significantly contribute to our knowledge of the biological mechanisms of impaired microvascular function in AD and will help uncover biomarkers to identify patient populations that would benefit from vascular-directed therapeutics arising from this project.