Manganese (Mn) is both an essential nutrient and a toxic redox metal. Hence its in vivo concentration has to be carefully maintained. Yet, little is known about how ionic Mn (i-Mn) is acquired, handled, and stored. In several unicellular organisms, i-Mn has been shown to compensate for the lack of the antioxidant enzyme, superoxide dismutase. In the simple multi-cellular organism, Caenorhabditis elegans, we have observed that i-Mn supplementation increased resistance to reactive oxygen species (ROS) and extended lifespan. It is unclear how i-Mn exerts this antioxidant-like activity. Our working hypothesis is that i-Mn exerts this beneficial activity primarily through the activation of the Fork head- related transcription factor, DAF-16, which regulates the expression of several genes that confer resistance to ROS in C. elegans. Additionally, we postulate that when DAF-16 pathway is disrupted, i- Mn binds to suitable ligands in vivo to form a small molecule with ROS neutralizing ability, which can act as a backup defense system. We will use proteomics to identify what proteins are differentially expressed with i-Mn treatment and carry out phenotypic analysis using daf-16 mutant to test the importance of this transcription factor for the observed i-Mn function. We will also carry out a functional genomics screen to identify genes required for the protective antioxidant-like function of i- Mn. Additionally, we will fractionate worms to examine if, non-protein, Mn-containing low molecular weight species form in vivo, and test if this fraction or the isolated species provide protection against ROS in C. elegans. The proposed studies will provide mechanistic details for the antioxidant-like activity of i- Mn. These studies will also pinpoint key targets of i-Mn along with identifying proteins involved in Mn acquisition, trafficking and storage. Detailed picture of Mn metabolism is likely to emerge. In addition, the proposed studies will provide outstanding training to several undergraduates and master's level students at California State University, Fullerton, and better prepare them for future careers in Biomedical Sciences. PUBLIC HEALTH RELEVANCE: Free radical mediated damage is associated with over two hundred human diseases and aging. The proposed project utilizes a simple, living, multi-cellular eukaryote, which is comparable to humans, to investigate how a potential free radical scavenger, manganese, works inside a cell. Learning how manganese gets into the cells to exert its beneficial antioxidant-like function is relevant to human health, as it can lead to novel drugs or supplements that could potentially slow the aging process and improve quality of life.