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地暴露在DNA损伤中，这些损伤是由外部损伤引起的，比如暴露在辐射或化学物质中，以及由正常细胞活动产生的内在压力，比如呼吸、复制和渐进的端粒侵蚀。尽管进行了深入的研究，但DNA损伤积累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，在这种模型中，DNA损伤可以以诱导的方式完全传递给成体干细胞。通过消耗关键的端粒相关蛋白，我们可以在选定的细胞类型中诱导染色体末端的去保护。这导致“无帽”染色体末端，这被认为是DNA损伤的位点，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难以区分。本研究以小鼠肠、结肠和毛囊为模型系统，重点研究端粒功能障碍诱导衰老的机制，以确定衰老生物体中观察到的再生潜力下降的关键机制和途径。Aim#1中的实验将在端粒功能障碍诱导下确定干细胞的细胞命运。在本研究中，我们将验证一个假设，即成体干细胞本质上对DNA损伤具有抗性，并且由于活跃的易出错DNA修复机制（NHEJ途径）而积累突变。Aim #2的实验将研究端粒功能障碍对组织稳态的生理影响。在这个目标中，我们利用谱系追踪方法，使我们能够确定DNA损伤是导致受损细胞的积累，还是导致关键祖细胞的逐渐消耗。Aim #3中的实验将定义在检查点抑制的背景下端粒功能障碍的影响。这将使我们能够确定受损的组织再生是检查点激活（如DNA损伤）还是端粒功能障碍的结果。端粒侵蚀和DNA损伤的逐渐积累已被证明在人类衰老的开始中起着重要作用。确定导致组织再生潜力下降的细胞和分子机制将为了解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.