The genetic contribution to longevity in humans is estimated to be >25% and may be much higher in populations with larger numbers of exceptional survivors. Activation of the DAF-16/FoxO3 transcription factor was required in experiments that increased the lifespan of flies by >50% and worms by six-fold. Environmental signals such as caloric restriction and oxidative stress trigger FoxO3 to upregulate target genes involved in stress resistance, metabolism, cell cycle arrest, and apoptosis which may play a preventative role for age-related diseases. Protective single-nucleotide polymorphisms (SNPs) in FOXO3 are strongly associated with exceptional longevity in humans. Surprisingly, mouse studies incorporating extra copies of FoxO3 have not yet been performed. It is currently unclear if FoxO3 is capable of increasing lifespan in mice analogous to what has been observed for other known longevity genes. There is a need to correct this gap in knowledge because identifying FoxO3 as a target gene in mice would be an important advance for developing future aging interventions. The long-term research goal is to identify candidate targets for enhancing longevity in model systems so that these discoveries can be translated to human therapies. The objective for the proposal is to demonstrate a clear relation between FoxO3 and longevity in mouse models. The central hypothesis is that increased dosage of FoxO3 will improve healthspan in mice and mimic aging phenotypes seen in humans with the protective genotype. This hypothesis has been formed on the basis of studies demonstrating FoxO3's role in human longevity, as well as reports showing that FoxO3 provides resistance to oxidative stress, protects against cancer, and is required for the life- extending effect of dietary restriction in mice, among other longevity-supporting effects. This hypothesis will be tested by pursuing three specific aims: 1) Generate mouse models for longevity-associated genes and assay gain and loss of function of FoxO3 at key timepoints in the mouse lifespan; 2) Determine if increased expression of FoxO3 improves aging phenotypes in mouse models; and 3) Establish correlations between FoxO3 mouse models and the Kuakini Honolulu Heart Program (Kuakini HHP) human cohort. FOXO3 is one of only two genes (the other being APOE) that have been replicated consistently for association with longevity in humans. The proposed research is innovative because the applicants will analyze novel mouse lines capable of activating or deactivating FoxO3 when desired. This will be the first time that genetic enhancement of FoxO3 will be assayed for improvements to healthspan and lifespan in mice. The approach is significant because studying FoxO3 could extend lifespan and healthspan in mice, and therefore lead to a better understanding of human longevity. Ultimately, this may lead to potential therapeutic approaches to help humans live longer and healthier lives.