Mechanisms of cell fate determination and aging onset upon telomere dysfunction

描述（由申请人提供）：DNA损伤是人类衰老的主要原因之一。我们体内的细胞不断暴露于由外部损伤引起的DNA损伤，例如暴露于辐射或化学品，以及由正常细胞活动产生的内在压力，例如呼吸，复制和进行性端粒侵蚀。尽管进行了深入的研究，但DNA损伤累积导致衰老的机制仍然知之甚少。为了研究体内DNA损伤的影响，我们开发了一种小鼠模型，其中DNA损伤可以以诱导的方式专门传递到成体干细胞。通过消耗关键的端粒相关蛋白，我们可以诱导选定细胞类型中染色体末端的去保护。这导致"脱帽"染色体末端，其被识别为DNA损伤位点并启动DNA损伤反应。这种反应与在其他基因组区域发生DNA损伤的细胞中观察到的反应无法区分。该提案的重点是端粒功能障碍诱导衰老的机制，使用小鼠肠道，结肠和毛囊作为模型系统，其广泛的长期目标是定义衰老生物体中观察到的再生潜力下降所涉及的关键机制和途径。目标#1中的实验将定义干细胞在诱导端粒功能障碍后的细胞命运。在这个目标中，我们将测试的假设，即成人干细胞是固有的耐DNA损伤，他们积累突变，由于一个积极的错误倾向的DNA修复机制，NHEJ途径。目标#2中的实验将研究端粒功能障碍对组织稳态的生理后果。在这一目标中，我们利用谱系追踪方法，这将使我们能够确定DNA损伤是否导致受损细胞的积累，或者替代地，关键祖细胞的逐渐耗尽。目标#3中的实验将定义端粒功能障碍在检查点抑制的背景下的影响。这将使我们能够确定受损的组织再生是否是检查点激活（如DNA损伤）或端粒功能障碍的结果。端粒侵蚀和DNA损伤的进行性积累已被证明在人类衰老的开始中起重要作用。确定导致组织再生潜力下降的细胞和分子机制将为衰老过程提供重要的见解。

DESCRIPTION (provided by applicant): DNA damage is one of the major causes of the onset of aging in humans. Cells in our body are constantly exposed to DNA damage caused by external insults, such as exposure to radiation or chemicals, and by intrinsic stress generated by normal cellular activities such respiration, replication and progressive telomere erosion. Despite intensive study, the mechanisms by which the accumulation of DNA damage results in aging remain poorly understood. To investigate the effects of DNA damage in vivo we developed a mouse model in which DNA damage can be delivered exclusively to adult stem cells in an inducible manner. By depletion of critical telomere- associated proteins we can induce de-protection of chromosome ends in selected cell types. This results in "uncapped" chromosome ends, which are recognized as sites of DNA damage and initiate a DNA damage response. This response is indistinguishable from that observed in cells that have incurred DNA damage in other genomic regions. This proposal focuses on the mechanism of telomere dysfunction-induced aging, using as a model system the mouse intestine, colon and hair follicles, with the broad long-term objective of defining the critical mechanisms and pathways involved in the decline of regenerative potential observed in aging organisms. The experiments in Aim#1 will define the cell fate of stem cells upon the induction of telomere dysfunction. In this Aim we will test the hypothesis that adult stem cells are intrinsically resistant to DNA damage and that they accumulate mutations due to an active error prone DNA repair mechanism, the NHEJ pathway. The experiments in Aim #2 will investigate the physiological consequences of telomere dysfunction on tissue homeostasis. In this aim we take advantage of a lineage tracing approach that will allow us to define whether DNA damage results in the accumulation of damaged cells or alternatively, to the progressive depletion of critical progenitor cells. The experiments in Aim #3 will define the impact of telomere dysfunction in the context of checkpoint inhibition. This will allow us to define whether impaired tissue regeneration is the result of checkpoint activation (as from DNA damage) or from telomere dysfunction. Telomere erosion and progressive accumulation of DNA damage have been shown to play a significant role in the onset of human aging. Identifying the cellular and molecular mechanisms responsible for the decline in regenerative potential of tissues will provide crucial insight into the aging process.