ABSTRACT. The central focus of this application to is to mechanistically phenotype development of cognitive impairment and Alzheimer’s disease in pulmonary hypertension (PH) using both in vitro and in vivo model systems. Reports by NIA indicate that 6 million Americans have dementia caused by Alzheimer’s disease, which ranks as the 6th leading cause of death. Alzheimer’s is characterized by progressive cognitive decline, which is pathophysiologically featured by presence of extracellular amyloid β plaques and intraneural tau neurofibrillary tangles. Cognitive impairment and Alzheimer’s have been linked to hypoxaemia/hypoxia, and blood-brain barrier (BBB) dysfunction that involves endothelial barrier dysfunction. Hypoxia contributes to accumulation of amyloid β, hyperphosphorylation of tau, BBB dysfunction and degeneration of neurons. Of note, respiratory limitation due to primary PH or secondary PH (e.g. consequent to COPD), can lead to hypoxaemia/hypoxia in the brain, which would consequently cause cognitive decline and phenotypes of Alzheimer’s. Elevated cytokine levels in PH patients (e.g. TNF-α, IL-6), can also induce neuroinflammation and BBB dysfunction, hence potentially leading to development of Alzheimer’s. In preliminary experiments we found that TNF-α and IL-6 induced dose-dependent downregulation of junctional proteins ZO-1 and Occudin in human brain microvascular endothelial cells (HBMECs). Of note, NADPH oxidase 4 (NOX4) can be activated by TNF-α in HBMECs. Our recent work indicates that NOX4 is selectively responsible for increased oxidative stress and endothelial barrier dysfunction to mediate sepsis induced acute lung injury. We have also recently identified a novel role of STUB1, in functioning as the specific E3 ligase responsible for regulating NOX4 protein stability. Therefore, in Aim 1 we will examine whether and how hypoxia and PH related cytokines induce endothelial barrier dysfunction in HBMECs (changes in junctional proteins, endothelial permeability, and transendothelial cell electrical resistance/TEER), which can in turn lead to BBB dysfunction and development of cognitive impairment and Alzheimer’s. In the parent award, we have established a novel human disease like murine model of PH with which the animals displayed dose-dependent increases in mPAP and RVSP, a full spectrum of human disease like vascular lesions in the lung, and similar gene regulatory profiles as in human PH patients. In Aim 2, we will employ this novel model of PH, and the classical hypoxia model of PH, to examine whether and how induction of PH in aged mice will lead to cognitive impairment and features of Alzheimer’s. NOX4 knockout mice and AAV-mediated STUB1 overexpression in vivo will be employed to examine if it will attenuate observed phenotypes in PH mice. Disease-targeted treatment of PH will be employed to alleviate hypoxaemia/hypoxia to examine role of hypoxia alongside measurements of cerebral tissue oxygenation and blood flow. Accomplis...