PROJECT SUMMARY A key priority for the NIH is to limit disability caused by osteoarthritis (OA) and other chronic diseases that emerge with age. Senescent cells within joint tissues contribute to OA, but there is a knowledge gap regarding the triggers by which decades of aging initiate cellular senescence. One key mediator of senescence in other contexts is persistent DNA damage and the subsequent activation of a set of signaling pathways known as the DNA damage response (DDR). The DDR can drive the production of inflammatory and matrix-degrading molecules collectively known as the senescence-associated secretory phenotype (SASP), which has strong overlap with catabolic molecules known to contribute to OA. As demonstrated through the use of a single-cell gel electrophoresis “comet” assay, chondrocytes accumulate significant levels of DNA damage throughout aging and during OA. This damage is mostly in the form of single-strand breaks (SSBs) but a subset of cells also harbor double-strand breaks (DSBs). These distinct forms of damage can be initiated in cells from young cadaveric donors and mice to mimic the levels found in older donors/mice, with methyl methanesulfonate (MMS) for SSBs, ellipticine for DSBs, and irradiation to generate both SSBs and DSBs. Conversely, the burden of DNA damage in older cadaveric donors and older mice can be reduced by boosting DNA repair with activation of Sirtuin 6 (SIRT6) using the small molecule MDL-800. The long-term goal of this work is to catalyze more effective treatments for OA by determining the mechanisms by which joint cells become senescent. The central hypothesis is that the accumulation of DNA damage in joint tissues plays a causal role in driving senescence, the SASP, and subsequent OA. The first aim is to establish the contribution of SSBs and DSBs to senescence by applying distinct forms of DNA damage (irradiation, MMS, ellipticine) to cadaveric human chondrocytes and synovial cells. The second aim is to determine the extent to which DNA damage drives senescence and OA in mice. We also use intra-articular injection of agents to increase damage (MMS or ellipticine) or decrease damage (MDL-800) in the joints of p16tdTom reporter mice to assess senescence and functional/histologic OA. The third aim is to define the protein signatures that contribute to progression towards the SASP using a multiplex antibody staining method known as iterative indirect immunofluorescence imaging (4i) to track the signaling pathways that are activated in response to DNA damage. The expected outcomes of this work include a better understanding of the types of DNA damage that lead to senescence in joint tissues and the signaling pathways that link the DDR to SASP. This work is innovative in that tailored interventions are employed to alter the levels of DNA damage, with sophisticated readouts of senescence, including 4i for assessing protein signatures and senescence reporter mice. These contributions are expected to have a...