FTO介导BDNF mRNA m6A修饰在微波辐射致海马 NSCs和成熟神经元参与的突触可塑性异常中的作用研究

The mechanism of learning and memory impairment caused by microwave radiation remains undetermined. The development of adult hippocampal NSCs and synaptic plasticity of mature neurons play important roles in learning and memory. The studies showed that BDNF had significant effects on the synaptic plasticity and the regulation of NSCs development in hippocampal neurons induced by microwave radiation. FTO can affect the expression of BDNF by the demethylation m6A modification of BDNF mRNA. Therefore, we speculated that the mRNA demethylation effect of FTO should be a potential mechanism of abnormal development of NSCs and plasticity of mature neurons, which were involved in the abnormal expression of BDNF after microwave radiation. In this study, the regulation of FTO on BDNF expression as a target, we aimed to explore the mechanism of abnormalities of NSCs development and mature neurons plasticity induced by microwave radiation. Firstly, we established the animal and cell models, and evaluated the effects of microwave radiation on FTO, BDNF and mRNA m6A modification. Secondly, in cell models, we planed to assess the effects of microwave radiation on FTO upstream transcription factor and the intermediate link between FTO demethylation effect and BDNF mRNA.Then the RNA interference or inhibitors of the key moleculars would be applied, to further uncover the mechanism of BDNF mRNA m6A demethylation on the synaptic plasticity abnormalities of NSCs and mature neurons mediated by FTO after microwave radiation. In conclusion, the purpose of this study was to clarify the mechanism of learning and memory impairment, provide a new target for prevention and treatment of brain injury by microwave radiation.

微波辐射致学习记忆障碍的机制尚未阐明。海马NSCs发育及神经元突触可塑性在学习记忆中扮演重要角色，BDNF在微波辐射致神经元突触可塑性异常及NSCs发育中发挥关键作用，FTO可通过BDNFmRNA m6A修饰影响BDNF表达。故我们推测FTO的mRNA去甲基化作用是微波辐射后BDNF表达改变引起NSCs发育及成熟神经元突触可塑性异常的潜在机制。本项目拟从FTO调控BDNF表达着手，探讨微波辐射致NSCs发育和成熟神经元突触可塑性异常的机制。首先建立突触可塑性异常的动物和细胞模型，研究微波辐射对FTO、BDNF及mRNA m6A修饰的影响，在细胞水平进一步探讨辐射对FTO转录因子的影响和FTO对BDNFmRNA去甲基化作用的中间环节及相关信号通路，进而对关键分子进行干扰，研究微波辐射通过FTO介导BDNFmRNA m6A修饰调控突触可塑性的机制，为阐明微波辐射致学习记忆障碍的机制奠定基础。

一定剂量微波辐射可引起海马突触可塑性异常，海马齿状回颗粒下区神经干细胞可分化为神经元并参与海马神经环路。脑源性神经营养因子是促进NSCs增殖、分化、迁移，调节突触可塑性的中心环节。FTO可能通过调节mRNA m6A修饰调控BDNF蛋白表达。本研究在建立微波辐射致NSCs介导的突触可塑性异常的动物和细胞模型基础上，在动物水平，通过对BDNF和FTO的蛋白表达和总mRNA m6A修饰水平检测，初步明确微波辐射对BDNF、FTO以及总mRNA m6A修饰水平的影响；在细胞水平，检测BDNF和FTO蛋白表达以及总mRNA m6A修饰水平和去甲基化酶活性的改变，进一步通过MeRIP-seq明确微波辐射致BDNF及其相关信号通路关键分子mRNA m6A修饰水平的影响并验证其蛋白表达改变，以初步阐明微波辐射致大鼠海马神经元突触可塑性异常的转录组表观遗传学机制；给予FTO抑制剂，明确其在介导微波辐射致BDNF表达改变和神经元可塑性异常的作用，并对其上游转录因子进行表达检测，明确微波辐射引起FTO表达改变的上游调控因子。结果表明，在动物水平：大鼠空间学习记忆能力和事件记忆障碍，海马DG区神经元突触结构异常，表现为突触后致密物分布异常，NSCs增殖、分化能力减弱引起的苔藓纤维出芽生长抑制；微波辐射后BDNF的mRNA和蛋白表达异常是NSCs增殖分化减弱介导的大鼠海马突触可塑性异常的重要原因；FTO上调导致总mRNA m6A修饰水平降低可能是BDNF表达异常的机制；在细胞水平，成功建立了NSCs与原代海马神经元共培养细胞模型，为微波辐射致NSCs介导的海马突触可塑性异常的分子机制研究提供更好的体外实验模型；发现TrkB和MAPK mRNA m6A修饰水平及其蛋白表达改变可能是微波辐射致BDNF下游相关信号通路异常并诱导NSCs和成熟神经元可塑性改变的关键转录组表观遗传学机制；FTO和其他m6A去甲基化酶共同参与微波辐射致大鼠海马NSCs和成熟神经元可塑性异常调控；Foxa2可能是微波辐射影响FTO表达的上游关键转录因子。