PROJECT SUMMARY/ABSTRACT This application investigates the relationship between mitochondrial RNA, inflammation, senescence, and aging. Cellular senescence is a well-established driver of tissue and organismal aging, a process thought to be partly mediated via the induction of a chronic Senescence-associated secretory phenotype (SASP). Consequently, there is great interest in selectively targeting senescent cells as a strategy to promote healthy aging. Our work has demonstrated that mitochondrial dysfunction is a hallmark of cellular senescence and a driver of the SASP and that by targeting them we may be able to suppress the detrimental SASP known to contribute to aging. Mitochondrial double-stranded RNA (mtdsRNA) when present in the cytosol is known to be especially immunogenic and trigger an inflammatory response. We observed that senescent cells contain increased levels of cytosolic mtdsRNA together with increased expression of RNA sensors. Furthermore, we found that transfection of cells with mtdsRNA triggers an inflammatory response and inhibition of mitochondrial DNA transcription in senescent cells, decreases the SASP. Finally, we observed that mtdsRNA requires the cytosolic DNA sensor cGAS to induce inflammation suggesting an interplay between RNA and DNA sensing pathways in the process. This led us to hypothesize that cytosolic mdsRNA plays a role in the regulation of the SASP and may be a novel target for interventions to improve healthspan during aging. In this project, we will dissect the mechanisms by which mtdsRNA increases in the cytosol of senescent cells as well as investigate if activation of RNA sensing pathways contributes to the SASP. Additionally, we will explore the mechanisms mediating the interplay between mitochondrial DNA and RNA in the development of the SASP. Our ultimate goal is to identify new interventions that target senescent cells to alleviate age-related dysfunction.