端粒参与神经元中毒应激机制 ──TRF2介导端粒和SIRT3亚端粒长距离染色质互动，保护线粒体功能

The occurrence of toxic encephalopathy might be linked to the neuron damage，which is non regenerative or post-mitotic cells. However, the mechanism of neuronal toxic stress is unknown. We have found that telomeric specific protein TRF2 (telomeric repetitive factor 2) protects neuron mitochondria during toxic stress. Previous work showed that: 1) TRF2 is highly expressed in the central nervous system; 2) TRF2 regulates the expression of SIRT3, a very important mitochondria protein highly activated and expressed in neurons and is involved in mitochondrial metabolism. .In this project, we asked the question how telomeres and TRF2 play a role in protecting against toxic stress damaging post mitotic cells including neuron? .We will try to answer the question by focusing on three tasks: 1) we will establish toxic stress models by using rotenone or Antimycin A. If we observe the decrease of TRF2 level and the increase of mitochondria injury simultaneously in neurons under toxic stress, it will suggest that TRF2 is involved in the protection of mitochondria function and energy dynamic in neuron toxic stress. The mitochondria function will be analyzed by electron microscopy, respiratory chain enzyme complexes I-V and MitoSOX™ MitoTrackerR Red CMXRos, etc.2) We will then use 3C method (chromatin conformation capture) and 3D-FISH to study TRF2 induced long distance chromatin interaction between telomeres and SIRT3 subtelomeric region. We will also use CRISP-CAS9 to knockout the ITS (interstitial telomeric sequence) at SIRT3 subtelomeric region to study the transcriptional regulation of TRF2 in SIRT3 expression. 3）We will use TRF2 knockout mouse model and transgenic overexpression of TRF2 mouse model, combining with optogenetics technique, lightsheet microscopy and Morris Water Maze behavior study to show that TRF2-SIRT3 axis protects mitochondria metabolism, neuron function, brain morphology and brain cognitive capability. All these studies will confirm our hypothesis that the TRF2-SIRT3 axis is a crucial, self-protective and unique pathway in the non-generative tissues like brain composed of post-mitotic cells, including neuron, to act against environmental pollution and poisoning injury compared to those generative tissues.

中毒性脑病可能与神经元损伤且不可再生有关，但机制不详。我们发现端粒蛋白TRF2保护神经元线粒体，抑制中毒应激。前期工作发现，TRF2在神经组织高表达，调控线粒体代谢重要蛋白SIRT3表达。本项目研究：⑴运用鱼藤酮等建立中毒应激模型结合电镜、线粒体酶和呼吸链复合物测定，研究中毒应激时神经元TRF2表达下降，线粒体损伤加重。⑵染色质构象捕获和3D-FISH技术证实TRF2在端粒和亚端粒区SIRT3基因形成长距离染色质互动环，结合CAS9技术敲除SIRT3亚端粒序列，共同证明TRF2作为转录增强子调控SIRT3表达的机制，参与线粒体能量代谢。⑶在敲除和过表达TRF2的小鼠模型，用光遗传学和Lightsheet体内证实TRF2-端粒-SIRT3环维护神经元线粒体稳定，保护脑功能。该机制是非分裂细胞独有的抵御中毒损伤的自我保护通路，为筛选以端粒为靶点的保护神经元线粒体和治疗中毒应激的药物提供新思路。

本项目按计划执行，汇集缺氧中毒应激、氧化损伤、线粒体功能障碍、端粒和神经系统生物学知识解决以下基本问题：端粒在中枢神经组织，包括分裂细胞和非分裂细胞（神经元）的保护功能，特别是保护神经元中毒应激中的作用机制。在原定研究计划的基础上，不仅阐明了TRF2-SIRT3信号通路，发挥端粒外作用，保护线粒体，抑制神经损伤和神经递质异常释放，维护神经功能，还发现了新的调控通路：TRF2下调不仅激活了DNA损伤通路ATM的磷酸化，还转录下调了PP2A磷酸酶的神经特异性调节亚基PPP2R2C的表达，参与调控ATM磷酸化，促进DNA损伤的持续作用，超额完成了原定计划。相关内容已发表标注该基金的SCI通讯作者论文三篇，共计发表标注该基金文章5篇，待发表文章一篇，拟投稿SCI的中科院I区杂志。共同参与三项中国专利申请。