PROJECT SUMMARY Stroke is one of the leading causes of dementia. However, the mechanisms underlying neurovascular dysfunction and cognitive impairment post-stroke that lead to Alzheimer’s disease and Alzheimer’s disease related dementias (AD/ADRD) remain unclear. Vascular cognitive impairment and dementia a type of ADRD and is the second leading cause of dementia in older adults after AD. Although post-stroke cognitive impairment (PSCI) and eventual post-stroke dementia (PSD) are now recognized as distinct upstream events that are independent of VCID, it is also widely accepted that stroke initiates a series of neurovascular events that either independently or dependently, contribute to the onset of VCID. The objective this supplement application is to employ the novel mouse model in in our funded R01 application to elucidate mechanisms underlying the pathophysiology of stroke-induced VCID. The goal of our funded R01 was to determine the role of endothelial tissue-nonspecific alkaline phosphatase (TNAP) in neurovascular dysfunction through the lens of sex and age in our VEcKO mouse. This mouse contains a conditional deletion of Alpl, the gene that encodes for TNAP, under the control of the VE-cadherin promoter, i.e.VEcKO mouse. In both young (6 months) and aged (20-24 months) mice, we have identified a novel phenotype following the induction of experimental ischemic stroke that mimics multiple neuropathological features of VCID that include: microhemorrhages, white matter lesions, and blood-brain barrier dysfunction. This supplement will investigate the central hypothesis that endothelial deletion of TNAP is a novel model of VCID that results from ischemic stroke. The aims of this R01 supplement application will employ a comprehensive approach to assess the neuropathological, cognitive, and mechanistic underpinnings of VEcKO mice as a novel model of VCID following ischemic stroke. The proposed aims are within the scope of the funded R01 application and will require the addition of approximately 100 mice, along mice already included in the funded application, to complete the supplement aims. Aim 1 will expand the neuropathological characterization of VCID mice to include the detection of microinfarcts using magnetic resonance imaging (MRI) and cerebral amyloid angiopathy. Aim 2 will employ a comprehensive cognitive behavioral assessment to evaluate the long-term impact of loss of endothelial TNAP on VCID. Aim 3 will a mechanistic approach to investigate a novel role for circulating (plasma) extracellular vesicles on brain endothelial cell function. Aim 4 will use spatial transcriptomics to identify cortical gene expression pathways that are implicated in the pathophysiology of VCID. This supplement will provide a framework for the development and characterization of a novel mouse model to better understand the stroke-induced neurovascular, cognitive, and pathophysiological mechanisms that contribute to VCID.