Abstract Recent advances in human genetics and genomics provide novel opportunities for the identification and validation of drug targets. Studies in model organisms have demonstrated that the rate of aging and the frequency and severity of age-related pathologies are influenced by conserved genetic pathways and factors which, when targeted genetically, pharmacologically, or through dietary modulation, can extend lifespan and healthspan. This overwhelming evidence raises hopes for new drugs that slow the aging process and attenuate age-related disease in humans by modulating these conserved pathways of aging. In Project 2 (old Project 1), we have been conducting the experiments aimed at exactly this goal: To identify and functionally characterize genetic variants in the conserved pathways of aging that are associated with human healthy aging and extreme longevity for therapeutic modulation to improve human healthspan and lifespan. We demonstrated through cell models that longevity-associated rare coding variants in SIRT6, USP35, and UBE3C alter protein function and/or expression which antagonize age-related deleterious changes in human tissues and during cellular senescence. We found the same aging-antagonizing effects of longevity-associated non-coding variants in SMAD3 and the 3 genes (NFKBIA, CLU and PRKCH) involved in the PKC/NF-B signaling pathways. In the renewal application, Project 2 proposes to confirm and extend our observations by taking a systematic multidisciplinary approach. The objective of Project 2 is to identify and directly test the impact of causal variants, whose genetic perturbations underlie the association with longevity. Our approach is to use high-throughput screening methods, such as INtegrated PrOtein INteractome perTurbation screening (InPOINT) for coding variants and Massively Parallel Reporter Assay (MPRA) for non-coding variants, and to investigate the underlying mechanisms using CRISPR- engineered human pluripotent stem cells (hPSC), multiple cellular differentiation paradigms, multiomics approaches, and functional analysis composed of molecular, biochemical, and cellular assays. Our functional analyses of longevity-associated variants in SIRT6, USP35, and UBE3C point to their potential role in protection against age-related cognitive decline and risk of Alzheimer’s disease (AD) and on the phenotypes of cellular senescence, which are increasingly implicated in neurodegenerative disease. Thus, we will focus on the aging- antagonizing effects of the SIRT6, UBE3C, and USP35 coding variants in the context of cognitive health and protection from AD. The ultimate impact of Project 2 lies in its potential to reveal conserved pathways as fundamental mechanisms of aging in humans and as therapeutic targets for healthy aging, in close collaboration with Projects 1, 3, 4 and Core B.