Role of DNA damage and cellular senescence in osteoarthritis pathophysiology

项目摘要 NIH的一个关键优先事项是限制骨关节炎（OA）和其他慢性疾病造成的残疾， 随着年龄的增长而出现。关节组织内的衰老细胞有助于OA，但关于OA的知识存在空白。 几十年的衰老引发细胞衰老的触发因素。另一种衰老的关键介质 持续的DNA损伤和随后激活的一组信号通路被称为 DNA损伤反应（DDR）。DDR可以驱动炎症和基质降解的产生， 这些分子统称为衰老相关分泌表型（SASP），具有很强的 与已知导致OA的分解代谢分子重叠。正如通过使用单细胞 凝胶电泳“彗星”测定，软骨细胞在整个老化过程中积累显著水平的DNA损伤 在OA这种损伤主要以单链断裂（SSB）的形式出现，但也有一部分细胞 具有双链断裂（DSB）。这些不同形式的损伤可以在年轻的细胞中启动 尸体供体和小鼠，以模拟老年供体/小鼠中发现的水平，使用甲磺酸甲酯（MMS） 用于SSB，用于DSB的椭圆树碱，以及照射以产生SSB和DSB。相反，DNA的负担 老年尸体捐赠者和老年小鼠的损伤可以通过激活 Sirtuin 6（SIRT 6）使用小分子MDL-800。这项工作的长期目标是催化更有效的 通过确定关节细胞衰老的机制来治疗OA。中央 一种假说认为，关节组织中DNA损伤的积累在驱动衰老中起着因果作用， SASP和随后的OA。第一个目标是确定SSBs和DSB对衰老的贡献 通过对尸体人软骨细胞施加不同形式的DNA损伤（辐射、MMS、椭圆树碱）， 滑膜细胞第二个目的是确定DNA损伤在多大程度上驱动衰老和OA， 小鼠我们还使用关节内注射剂（MMS或椭圆形）增加或减少损伤 （MDL-800）在p16 tdTom报告基因小鼠的关节中的作用，以评估衰老和功能性/组织学OA。第三 目的是使用多重抗体确定有助于向SASP进展的蛋白质特征 称为迭代间接免疫荧光成像（4 i）的染色方法来追踪 在DNA损伤时被激活。这项工作的预期成果包括更好地了解 导致关节组织衰老的DNA损伤类型以及连接关节组织的信号通路， DDR到SASP。这项工作是创新的，因为采用了量身定制的干预措施来改变DNA水平。 损伤，具有复杂的衰老读数，包括用于评估蛋白质特征的4 i， 衰老报告小鼠。预计这些捐款将对社会产生积极影响， 更有效的策略，以靶向衰老细胞预防和治疗OA。

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 positive impact on society by stimulating more effective strategies to target senescent cells for the prevention and treatment of OA.