PROJECT 3 - The role of Cellular Senescence in brain aging -SUMMARY This Project investigates the relationship between cellular senescence and brain aging. Cellular senescence is a well-established driver of tissue and organismal aging, a process partly mediated via a chronic Senescence- associated secretory phenotype (SASP). Consequently, there is great interest in selectively targeting senescent cells as a strategy to promote healthy aging. We have found that senescence-associated markers p21Cip1 and p16Ink4a accumulate in microglia and Blood Brain Barrier (BBB) endothelial cells of aged mice. Importantly, we showed that clearance of senescent cells, using both genetic and pharmacological approaches improved cognitive function and neuroinflammation in aged mice. This led us to hypothesize that senescence in the brain, exacerbates age-related cognitive decline and contributes to age-related loss of BBB integrity. Importantly, we found that there is little overlap between p21Cip1 and p16Ink4a senescent sub-types in the aged hippocampus and that they have a different SASP profile, suggesting they may play different physiological roles. In this Project, innovative transgenic mouse models developed by the PPG, along with advancements in single- cell molecular phenotyping and systems biology, will be leveraged to thoroughly investigate the heterogeneity of cellular senescence in the brain and its functional consequences. Collaboration and integration between various Projects and Cores will enable the exploration of the interplay between senescence in different organs, such as the brain, bone, and muscle. To achieve our goals, we will use common PPG mouse models (generated in Core B) to investigate the relative impact of p16Ink4a vs p21Cip1 in driving age-related cognitive decline. Additionally, the efficacy of novel senotherapeutic drugs discovered by Core C will be tested to determine their potential to target either p16Ink4a- driven or p21Cip1-driven senescence across different tissues. Finally, we will use innovative mouse models developed by the PPG capable of targeting specific senescent cell-types and sub-types in the brain (p16Ink4a vs p21Cip1), to investigate if in situ clearance of senescent microglia and BBB endothelial cells impacts age-related cognitive decline. The project will benefit from the expertise of the molecular phenotyping core (Core D) and computational biology (Core A) to conduct comprehensive analyses of cellular senescence and its heterogeneity using emerging single-cell multiomics technologies. Our ultimate goal is to identify new, more targeted interventions to alleviate cognitive decline during aging.