ABSTRACT: Developing neurons have high iron requirements to support their metabolism, growth, and differentiation. Yet, free iron can produce oxidative stress and be cytotoxic. To avoid neurological damage from iron deficiency (ID) and overload, neuronal iron levels must be tightly regulated. Ferritin protein complexes play a critical role in regulating intracellular iron availability by storing iron that is not immediately used. During times of high iron demand (e.g., development), ferritin iron release must be controlled to prevent ID. Ferritinophagy, the process by which iron is released from ferritin and delivered to sites of high iron demand (e.g., mitochondria), was recently characterized in developing red blood cells (RBCs). Nuclear receptor coactivator 4 (NCOA4) is the specific cargo receptor that initiates mobilization of ferritin iron by directing ferritin to lysosomes via selective autophagy. Ferritinophagy is critical for maintaining the supply of iron required for mitochondrial heme synthesis in developing RBCs. There are currently no data on the role of NCOA4 or ferritinophagy during neuron development, causing a significant gap in our understanding of how the release of iron stored in ferritin is regulated during this highly iron-sensitive process. Dysregulation of neuronal ferritinophagy could result in severe iron underload or overload with significant clinical ramifications. This proposal focuses on early-life ID because it is prevalent throughout the world and permanently impairs neurobehavioral function (e.g., learning and memory) in children. ID specifically within the developing hippocampal neuron accounts for a significant portion of the learning/memory deficits. Basic principles of ferritin iron regulation discovered in this neuronal subtype will likely apply to all rapidly developing neurons. We hypothesize that, similar to iron handling during RBC development, iron released through NCOA4-mediated ferritinophagy forms an iron pool that is that is essential for normal neuron development and function. Aim 1 uses our unique in vitro model of chronic early-life hippocampal neuronal ID to test whether NCOA4 and ferritinophagy are required for optimal neuronal development by regulating iron availability. We hypothesize that loss of NCOA4 will disrupt neuronal iron homeostasis and impair critical neurodevelopmental processes (i.e., mitochondrial respiration, neuronal dendrite and synapse formation). Aim 2 translates Aim 1’s in vitro findings to the in vivo brain to reveal the developmental age-dependent role of NCOA4 and ferritinophagy in regulating hippocampal neuron iron utilization. We hypothesize that NCOA4-mediated ferritinophagy provides a source of iron that is required during the postnatal switch from iron storage to utilization and when neuronal iron supply is restricted (i.e., ID). We will test this using two unique hippocampal-specific transgenic mouse lines that model disruptions to neuronal iron uptake (Slc11a2 KO) ...