ATM调控Notch信号通路维持神经干细胞静息的分子机制研究

The quiescence of neural stem cells has deep relationships with brain homeostasis, regeneration and aging, but the mechanisms under which are known little to us. Due to the scarity of number of stem cell and the lack of powerful identification and purification technique, the study of neural stem cell quiescence is still limit. ATM is a very important protein kinase that responds to DNA damage, it plays an impotant role in the maintenance of genomic integrity, and whether ATM could maintain the neural stem cells quiescence is unknown. Our preliminary data showed that, in the ATM knockout mice, ATM deficiency reduces NSC quiescence, thus leading to increased activation, proliferation and eventual exhaustion of neural stem cells. Furthermore, based on next-generation, high-throughput RNA sequencing, we find that loss of ATM inactivate Notch signaling pathway through bioinformatics analysis. These results suggest that ATM may play an essential role in maintaining the quiescent state of NSCs through mediate Notch signaling pathway. In this project, we will clearly elucidate the funtion and mechanism of ATM in maintaining the quiescence of NSCs through regulating Notch signaling pathway by the means of in vitro culture system for quiescent NSCs and ATM knockout mice at levels of whole animal, cell and molecular models. A new theoretical basis will be established for the application of NSCs in the regenerative medicine.

神经干细胞(NSCs)的静息与脑功能稳态、再生、衰老等密切相关，但我们对其调控机制知之甚少。目前关于NSCs静息的相关研究很少，主要受限于其数量的稀少和分离纯化鉴定技术的缺乏。ATM是DNA损伤应答的关键蛋白，在维持基因组稳定性中起到关键作用，ATM能否维持NSCs的静息状态更是未见报道。我们前期实验结果显示，在ATM基因敲除小鼠中，ATM缺失破坏了NSCs的静息状态，导致NSCs瞬时大量扩增，继而出现NSCs库的耗竭。进一步在二代转录组测序的基础上，我们通过生物信息学分析发现ATM的缺失导致Notch信号通路下调。提示ATM可能通过调控Notch信号通路维系NSCs的静息。本课题拟应用ATM基因敲除小鼠和已建立的体外成体NSCs静息培养体系，从分子、细胞、动物水平系统阐明ATM通过调控Notch信号通路对维持神经干细胞静息状态的作用机制，为神经干细胞在再生医学中的应用奠定新的理论基础。

神经干细胞(NSCs)的静息与脑功能稳态、再生、衰老等密切相关，但我们对其调控机制知之甚少。ATM是DNA损伤应答的关键蛋白，在维持基因组稳定性中起到关键作用，ATM能否维持NSCs的静息状态尚无报道。本研究发现1月龄的ATM-/-鼠，在SVZ区神经前体细胞出现异常高的增殖水平，但是在3月龄的ATM-/-鼠，SVZ区神经前体细胞的增殖能力大大降低。体外实验发现ATM的缺失导致细胞周期蛋白ccnb2, cdk1,ccne1,ccnb1的表达水平上调。进一步在二代转录组测序的基础上，我们通过生物信息学分析发现ATM的缺失导致Notch信号通路下调，并通过体内免疫荧光染色、QRT-PCR、Western blot、双荧光素酶报告基因等实验对Notch信号通路中的关键基因进行验证。在静息培养体系中沉默ATM基因，Notch信号通路相关基因（Hes5,NICD）被抑制。在静息培养体系中加入γ-分泌酶抑制剂DAPT抑制 Notch 信号通路后，发现静息培养体系中ki67阳性细胞的比例明显增加。在静息培养体系中沉默ATM后，通过加入Dll1重组蛋白或通过过表达Hes5后，静息培养体系中ki67阳性细胞的比例明显降低。上述结果证实ATM能通过调控Notch信号通路来维持神经干细胞的静息状态。