Project Summary Ischemic brain damage remains a leading cause of long-term disability and death with limited treatment options. Cerebral ischemic injury is strongly associated with excessive production of reactive oxygen species (ROS) that contributes to endothelial dysfunction, blood brain barrier disruption, neuronal cell death, and worsened brain damage. Thus, efforts to curtail ROS have major impact on improving stroke outcome. Dual oxidases (Duox) are novel members of reduced nicotinamide dinucleotide phosphate oxidases family. The main function of Duox1 is to generate hydrogen peroxide (H2O2)/ROS. Duox1 at low levels, is involved in essential cellular functions, antimicrobial host defense, thyroid hormone production, and airway epithelial cell migration, and injury. However, excessive production and activation of Duox1 may contribute to pathological events including inflammation, apoptosis, hypertension, cancer, and tissue damage. The direct link between Duox1 and ROS in airway epithelial cells was shown in our previous work. Pro-inflammatory cytokines and deranged calcium signaling increase the activity and expression of Duox in airway epithelial cells and thyroid tissues. Interestingly, ischemic stroke causes aberrant Ca2+ influx. Despite these compelling observations, the specific roles of Duox in the brain and cerebral ischemia are largely unknown. We have recently identified that focal cerebral ischemia in rodents, and in-vitro oxygen glucose deprivation rapidly induce the expression of Duox1 in endothelial and neuronal cells in association with increased ROS production. However, pre-treatment of neuronal cells with Duox1 specific small interfering RNA decreased Duox1 expression and ROS levels. These data led us to hypothesize that cerebral ischemia evokes Duox1 over-expression which in turn increases ROS in the brain, leading to exacerbation of pro-inflammatory and apoptotic processes that worsen the brain injury. We further propose that Duox1 inhibition has a great potential to mitigate post-ischemic brain damage and neurological dysfunction. Accordingly, our specific Aims are; To determine the spatiotemporal changes in the expression of Duox1 in brain following focal cerebral ischemia/reperfusion; To determine if Duox1 inhibition or genetic loss of Duox1 decreases ROS, and reduces ischemic brain damage, and thus improves post-stroke functional recovery; To investigate the role of Duox1 as a key driver of inflammatory and apoptotic processes in ischemic brain. We will address these Aims using a wide array of molecular, cellular, and biochemical approaches in both in vivo animal, and in vitro cell culture models. Overall, this ‘proof of concept’ study will determine a previously unidentified role for Duox1 in ischemic brain. These studies pave the way towards better understanding of the role of Duox1 mediated ROS and neuro-inflammatory mechanisms in brain and may open up a promising new approach for treating cerebro- vascular diseases...