PROJECT SUMMARY The Role of the FOXO Pathway in the Control of Transposons in Aging Understanding aging and the processes that effect lifespan at the molecular level remains an important problem in modern biology and medicine. The Forkhead-box, family O (FOXO) transcription factors have been identified as factors that can extend lifespan and stave off aging in a number of model systems including worms, flies and mice. In general, increasing FOXO activity increases lifespan in these models. FOXO activity can be increased by overexpressing FOXO or by downregulating the insulin signaling pathway, a major negative regulator of FOXO. Complementing the findings in model organisms, SNPs associated with longevity in humans have been identified in the FOXO1, FOXO3 and components of the insulin signaling pathway in multiple cohorts. This strongly suggests that the FOXO pathway influences lifespan in humans as well. Therefore, an important goal is to identify the molecular targets and pathways controlled by FOXO transcription factors to better understand the multiple mechanisms that influence aging. Mammals have four FOXO genes (FOXO1, FOXO3, FOXO4 and FOXO6) with some division of labor between the four factors often complicating interpretation of experiments. Invertebrates each have a single FOXO gene, daf-16 in C. elegans and foxo in Drosophila. Given the conserved role in affecting lifespan, the simple invertebrate system offers a powerful tool to understand the FOXO regulon in molecular detail. FOXO factors are typically regulated as downstream components of insulin and insulin-like factor signaling. Because of this, much attention has been paid to the role of FOXO on metabolic influences driving aging. However, there are certainly other drivers of aging in addition to the metabolic. One area that is underexplored is FOXO's role in the maintenance of genome integrity as factor increasing longevity. Genome integrity decreases with age and it has been argued that this may be a driver of ageing phenotypes in somatic cells. While genome integrity is controlled by multiple mechanisms there is a growing appreciation that transposon activation occurs as a function of age and may be a driver of genome integrity loss. This age related transposon activation has been observed in both Drosophila and mammals. In Drosophila transposon mobilization has been shown to lead to neurodegeneration and increase mortality. We recently identified the small RNA pathways as downstream direct targets of foxo in Drosophila. We also identified a physiological role for this control in viral innate immunity through the siRNA pathway. Increasing foxo activity led to an increase in the efficacy of the RNAi response. This effect both increased post- transcriptional silencing of endogenous loci and led to viral clearance in infected cells. This is the first pathway identified that can modulate RNA interference in somatic cells. Although viral resistance could be one of the ways foxo enhances lifespan this would require an acute encounter with an RNA virus and would not be a generalizable effect. However, the siRNA pathway is also known to control transposons in somatic cells. This role could potentially provide a broader effect on lifespan as transposon mobilization has been identified as increasing with age, and has been shown to be a potential cause of shortened lifespan. We are seeking to extend these findings in a new direction. We hypothesize that foxo regulates the small RNA pathways and aids in the prevention of transposon mobilization and thus helps to protect genome integrity leading to increased lifespan. In order to begin to test this hypothesis we propose to determine the role of foxo in transposon regulation as a function of age using our Drosophila model.