PROJECT SUMMARY Iron delivery to the brain is essential for multiple neurological processes such as myelination, neurotransmitter synthesis and, as it is for all organs, for the utilization of oxygen for energy production. Previously, we introduced and established the concept that brain iron uptake is regulated at the level of the endothelial cells of the blood-brain barrier (BBB), which is contrary to the widely held concept that the BBB endothelial cells serve as a simple conduit for the delivery of transferrin. We also identified H-ferritin, classically considered an intracellular iron storage protein, as a significant iron delivery protein for the brain. Both of these new concepts start to address the question of how iron is acquired by the brain in a timely manner and in adequate amounts. The importance of timely iron delivery during development is clinically manifested in the long term neurological and cognitive impact of developmental iron deficiency. The importance of regulation to adequately manage the amounts of iron delivered in the adult brain is clinically manifested in neurological disorders such as Restless Legs Syndrome (too little iron) and neurodegenerative diseases (too much iron). Despite the prevailing opinion that the BBB was a simple conduit with no apparent mechanism for regulation of brain iron uptake, we identified age, genotype, sex, and systemic iron status as physiological factors that are associated with altered brain iron acquisition. The immediate translational relevance of our findings will relate to treatment of systemic iron deficiency which is treated, often aggressively, with intravenous iron injections or oral iron supplementation. These strategies have led to considerable public health debate over concerns that these treatments could override brain uptake mechanisms and increase brain iron accumulation leading to oxidative stress and neurodegenerative diseases. Therefore, the most significant knowledge gap addressed in this application is how brain iron acquisition is regulated. The scientific premise for this proposal is that both transferrin and H-ferritin serve as iron carriers and are taken up into endothelial cells of the BBB by different receptors but the release of these two proteins and their iron cargo into the brain are coordinated by signals from the cerebrospinal fluid and extracellular fluid in the brain. The major objectives for this current proposal will be addressed using a human endothelial cell culture model of the blood-brain-barrier (Aim 1) and clinically relevant animal models (Aim 2) to interrogate the underlying mechanisms for brain iron acquisition and regulation, as well as to identify clinically indices of iron status on brain iron acquisition. The deliverables from the proposed studies are: 1) how brain iron uptake is regulated which is relevant to whether a therapy can be anticipated to work or not, 2) whether H-ferritin represents a novel iron delivery system for repleting brain iron, and 3) what conditions affect iron uptake and thus repletion strategies.