端粒组件参与氧化应激介导有丝分裂后细胞老化

Some cells can progressively lose their functional capacities with age without exhibiting any clear sign of senescence. This is for instance the case of long-lived post-mitotic cells (collectively named LLPMCs), such as myofibers and neurons. Among the ageing hallmarks, the instability and dynamics of telomeres act as a mitotic clock of cellular senescence. However, evidence for changes in telomeres during LLPMCs aging remains limited. .Our unpublished results showed that telomeric protective protein TRF2 regulates the expresssion and the activity of the longevity genes SIRT3 and FoxO3a during LLPMC aging, suggesting that telomeres are involved in LLPMCs aging through redox. The ambition of this program is to investigate the telomere-dependent mechanisms that lead to the gradual cellular decline of LLPMCs function(s). Based on a wealth of recent published and unpublished data, we hypothesized that: .1)Telomeres are structurally and functionally reorganized in aging LLPMCs;.2)The telomere changes in aging LLPMCs contribute to their progressive functional decline. We will investigate this hypothesis by focusing on the role of the key telomeric capping protein TRF2 in neurons and skeletal myofibers. .This will include four tasks:1)We will use ChIP, 3D-FISH to investigate the changes of telomere structure and the activity of FoxO3a bound and protective telomeres during LLPMCs aging; 2) We will use 3C and CAS9 knockout technique to study the long distance chromatin interaction loop induced by TRF2 for the transcriptional regulation of SIRT3; 3) RNA-Seq and ChIP-Seq will be analyzed to study the activation of FoxO3a function in telomere protection during LLPMC aging in the case of lacking TRF2-SIRT3 regulatory pathway; 4)specific TRF2 knockout mice and muscle or neural degenerative mice models will be used to study the TRF2-SIRT3-FoxO3a axis involved in redox balance, telomere protection and LLPMCs aging. This study will demonstrate a new concept of telomere dysfunction driving LLPMCs aging and provide an original approach to explore mechanisms of aging.

长寿的有丝分裂后细胞（LLPMC）随年龄上升出现功能下降和细胞减少，但老化机制不详。传统观念认为端粒动力学不参与LLPMC老化。我们前期结果发现LLPMC老化时端粒保护蛋白TRF2调控长寿基因SIRT3和FoxO3a，提示端粒通过氧化还原参与LLPMC老化。本项目在神经和肌肉LLPMC中：⑴用ChIP、3D-FISH证明LLPMC老化存在端粒结构改变和FoxO3a结合端粒能力下降；⑵3C和CAS9敲除技术证明TRF2形成长距离染色质互动环调控SIRT3转录表达，抑制LLPMC老化；⑶RNA和ChIP-Seq探索TRF2-SIRT3通路缺失，能激活FoxO3a抗氧化稳定端粒；⑷选择性TRF2敲除鼠和神经、肌肉退行性变鼠模型共同证明TRF2-SIRT3-FoxO3a轴参与氧化还原平衡、端粒保护和LLPMC老化。该研究将证明端粒损伤驱动LLPMC老化的新概念，为探索延缓衰老的途径提供原创性思路。

虽然大量研究关注复制性衰老，但端粒在如肌纤维等有丝分裂后细胞的衰老过程中的重要性还鲜有研究。我们发现TRF2作为关键的端粒保护蛋白，在人类骨骼肌中的水平随年龄增长会下降。在培养的人肌管细胞中，TRF2下调并不触发端粒功能障碍，而是抑制线粒体sirtuin3基因表达，导致线粒体呼吸功能障碍和活性氧水平升高。重要的是，恢复TRF2受损的肌管细胞中的SIRT3水平完全补救线粒体功能。靶向敲除小鼠骨骼肌中的Terf2基因导致线粒体功能障碍和SIRT3下调，导致肌肉早衰。这些结果揭示了TRF2-SIRT3轴控制肌肉线粒体功能。本研究揭示了在长寿的有丝分裂后细胞衰老中端粒的作用。.我们还从上述研究的Chip-Seq结果中挖掘出TRF2参与复制衰老的新功能，即TRF2不仅特异性结合于端粒，还结合在端粒外如sirtuin3基因附近，以及结合近着丝粒。端粒在解旋酶RTEL1 的共同作用下维护近着丝粒的稳定性，并且发现TRF2对于复制叉通过近着丝粒、确保全基因组异染色质稳定性很重要。.我们还提供强有力的证据证明TRF2作用蛋白RAP1保护极短端粒。RAP1可以防止53BP1 (p53结合蛋白1)在端粒上的聚集，并在衰老细胞中保护端粒使其避免发生融合，但在活跃分裂的细胞中则没有该功能。.本项目严格按计划完成任务。发表标注本项目的SCI文章共计4篇（均为通讯作者）。本课题的其他主要研究成果正在投稿国际影响力的杂志上（SCI>10）论著1篇，准备投稿1篇，拟投稿于国际顶级杂志。申请国家专利一项。培养博士后1名，并获得博士后科学基金(面上)一等奖，博士研究生5名(已毕业2名，在读3名)，参与培养博士研究生2名(在读)及硕士研究生3名(已毕业2名，在读1名)。