Thomas W. Bastian, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Iron deficiency (ID), with and without anemia affects an estimated 2 billion people including 40% of pregnant women. ID is particularly deleterious during early-life brain development, leading to significant neurological impairments in children (e.g. learning and memory deficits). More troubling is that the learning and memory impairments persist into adulthood despite iron treatment in infancy, implying that iron therapy alone is not sufficient for full recovery. Thus, the long-term effects of lost education and job potential are te real cost to society of early-life ID. Understanding the iron-dependent molecular/cellular mechanisms that directly cause neurological dysfunction will allow future design of more effective therapies to prevent the long-lasting effects. The newborn brain is highly metabolic, accounting for 60% of total body oxygen consumption. Iron provides the catalytic component for several mitochondrial enzymes required for energy (ATP) production. Early-life ID reduces the energy status of the developing hippocampus, a highly metabolic brain region important for learning and memory. Early-life ID also impairs hippocampal neuron maturation, a metabolically demanding process, leading to reduced dendritic complexity and blunted spine formation in adulthood despite normalization of iron status. However, the molecular/cellular mechanisms contributing to the hippocampal energy impairments and how impaired energy status causes long-term neuronal structural deficits following developmental ID remain unclear. I will test the hypothesis that impaired mitochondrial respiration causes reduced energy status, leading to structural abnormalities in developing iron-deficient and mature formerly iron- deficient mouse hippocampal neurons. I will address this hypothesis through two specific aims. In Aim 1, I will determine the effect of ID on mitochondrial respiration and intracellular trafficking in developing hippocampal neurons. Key parameters of mitochondrial oxidative phosphorylation and glycolysis and mitochondrial recruitment to growing dendrites/spines will be measured in iron-sufficient and -deficient hippocampal neurons. In Aim 2, I will test whether restoring energy status in addition to iron repletion attenuates the acute and chronic neuronal structural deficits f developmental ID. Select steps of energy metabolism will be genetically or pharmacologically manipulated in cultured mouse hippocampal neurons, with or without iron repletion, and dendrite complexity and dendritic spine density and morphology will be assessed. This proposal is significant because it defines the role of specific energy metabolic processes in normal neuronal development and determines how a clinically relevant level of ID compromises them, thus providing a mechanistic basis for energy-specific therapies for fetal/neonatal ID. These discoveries and my professional and technical training in neuronal live-cell microscopy, morphology analysis and cellular bioenergetics will form the basis for an independent research career studying nutritional-metabolic regulation of neurodevelopment.

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) or storage (Ncoa4 KO). Findings from the proposed studies will shift the current paradigm of how neuronal iron homeostasis is controlled during development, opening up a wealth of new research avenues with the potential to inform new therapeutic strategies for common iron-related brain disorders.