PROJECT SUMMARY The objective of this application is to determine how tissue nonspecific alkaline phosphatase (TNAP) enzymatic activity maintains cerebrovascular function within the neurovascular unit (NVU). Brain microvascular endothelial cells (BMECs) comprise the cerebral microvasculature and serve as the structural foundation of the blood-brain barrier (BBB) and. Increased permeability and diminished integrity of BMECs are two common mechanisms through which cerebrovascular function is compromised in human disease. TNAP is a highly enriched enzyme in cerebral microvessels whose function in brain BMECs is poorly understood. Our preliminary data demonstrate that TNAP activity stimulates a novel signaling mechanism which protects against the loss of cerebral microvascular integrity and permeability. These intriguing findings led us to propose the central hypothesis that TNAP maintains NVU homeostasis during cerebral ischemia by preserving BBB integrity. We will utilize mice with a VE-cadherin-Cre driven conditional deletion of TNAP in endothelial cells (VEcKO) and its wild type littermates to interrogate the role of TNAP in BMECs. We will compare responses in young (4-5 months) and aged (18-20 months) mice to assess the age-dependent effects of brain endothelial TNAP on indices of brain endothelial barrier permeability combined with vascular network analysis and functional behavioral outcomes. Aim 1 will elucidate the contribution of brain endothelial cell TNAP to NVU dysfunction in ischemic stroke in young mice. We will employ the transient middle artery occlusion model to assess quantitative differences in cerebrovascular outcomes and behavioral indices. Aim 2 will determine the impact of brain endothelial cell TNAP on age-dependent impairment of the NVU. This aim will assess the impact TNAP on BMEC function in aging and the putative age-dependent interactions in ischemic stroke. Aim 3 will determine how the TNAP-Rho associated kinase (ROCK) pathway regulates the barrier function and whether pharmacological inhibition of the ROCK pathway protects against the loss of barrier integrity and functional deficits associated ischemic stroke in both young and aged mice. Taken together, the studies in this proposal will delineate a novel mechanism through which brain endothelial cell TNAP enzyme activity preserves NVU function in ischemic stroke and improves functional recovery post-stroke. The overall results will contribute to our limited understanding of the basic biology of TNAP’s role at the BBB and its contribution to NVU homeostasis in human health and disease.

PROJECT NARRATIVE There is a critical need to identify the cellular and molecular mechanisms underlying cerebrovascular function in both healthy and disease states associated with aging. The studies in this proposal will investigate how the brain endothelial cell enriched enzyme, tissue nonspecific alkaline phosphatase (TNAP), protects against the loss of cerebral function. The results will identify a potential therapeutic target that may be used improve cerebrovascular function and functional recovery in age-related disorders.