WTAP介导的m6A修饰通过NLRP3-Caspase-1轴调控神经元焦亡在创伤性脑损伤后神经损伤中的机制研究

Traumatic brain injury (TBI) is considered as a vital global health priorities. Previous studies have suggested that N6-adenylate methylation (m6A) modification regulates nervous system diseases through epigenetic modification, but the signal-regulation of m6A modification post-TBI remains unclear. Our pre-experimental results showed that m6A level and its regulatory unit were unbalance in the cerebral cortex of TBI-mice, and m6A levels of NLRP3 and caspase-1 were up-regulated. WTAP knockdown down-regulated m6A levels of NLRP3 and caspase-1, and improved LPS-induced neuronal pyroptosis and injury. Combined with recent reported references, we hypothesized that "WTAP mediated m6A modification could regulate the NLRP3-Caspase-1 axis, affect the pyroptosis of neurons, and participate in the nerve injury post-TBI". Basis on the pre-experiment, through MeRIP-qPCR, miCLIP-seq, RNA-seq, small animal MRI, electrophysiology detection, and other experimental methods, we will further study the molecular regulation mechanism of WTAP/m6A signal-mediated neuronal pyroptosis post-TBI, which is in the hope of a breakthrough on revealing the TBI pathological mechanism, and simultaneously provides new ideas and new drug targets of the effective treatment and prognosis of TBI.

创伤性脑损伤（TBI）被认为是重要的全球卫生优先事项。既往研究指出，N6-腺苷酸甲基化（m6A）修饰通过表观遗传调控神经系统疾病，但TBI后m6A修饰的信号调控尚不清楚。预实验结果显示，TBI小鼠大脑皮层m6A水平及其调控单元失衡，NLRP3和Caspase-1 m6A水平上调；WTAP敲低可下调NLRP3和Caspase-1 m6A水平，改善LPS诱导的神经元焦亡和损伤。结合文献报道，我们提出如下假说“WTAP介导m6A修饰可调控NLRP3-Caspase-1轴，影响神经元焦亡，参与TBI后神经损伤”。在预实验的基础上，本项目将通过MeRIP-qPCR、miCLIP-seq、RNA-seq、小动物磁共振成像、电生理检测等方法，研究WTAP3/m6A信号介导TBI后神经元焦亡的分子调控机制，以期在揭示TBI病理机制上有所突破的同时，为其有效治疗和预后提供新思路和药物作用新靶点。

创伤性脑损伤（TBI）被认为是重要的全球卫生优先事项。既往研究指出，N6-腺苷酸甲基化（m6A）修饰通过表观遗传调控神经系统疾病，但TBI后m6A修饰的信号调控尚不清楚。我们前期研究表明，调控NLRP3-Caspase-1轴可改善TBI后神经元焦亡和预后，但其确切机制仍需进一步研究。预实验结果显示，TBI小鼠大脑皮层m6A水平及其调控单元失衡，NLRP3和Caspase-1 m6A水平上调；WTAP敲低可下调NLRP3和Caspase-1 m6A水平，改善LPS诱导的神经元焦亡和损伤。结合文献报道，我们提出如下假说“WTAP介导m6A修饰可调控NLRP3-Caspase-1轴，影响神经元焦亡，参与TBI后神经损伤”。在预实验的基础上，本项目将通过MeRIP-qPCR、小动物磁共振成像、荧光素酶报告系统等方法，基于TBI小鼠组织样本探究WTAP/m6A与NLRP3-Caspase-1轴的相关性，在皮层神经元中阐明WTAP/m6A/NLRP3/Caspase-1信号介导神经元焦亡的分子调控机制，并通过αCaMKⅡCre/+WTAPflox/flox-和αCaMKⅡ+/+WTAPflox/flox-TBI小鼠模型进行体内验证。 .本项目自2020年1月至2022年12月，按照原定实验内容和项目计划书执行，完成课题目标。另外，课题验证了YTHDF1参与WTAP介导m6A修饰调控TBI后NLRP3炎性小体：作为重要的m6A识别蛋白，YTHDF1可有效识别NLRP3的m6A修饰，介导NLRP3蛋白翻译但并未影响其mRNA水平，从而调控NLRP3炎性小体活性。本项目已初步阐明WTAP通过m6A调控NLRP3-Caspase-1轴在TBI后神经元焦亡和神经功能障碍中的作用及分子机制，可深入了解TBI后RNA表观调控、m6A修饰作用机制，将为TBI病生理机制及康复治疗提供新的思路和潜在靶点。此外，我们将继续关注TBI后RNA表观调控、m6A修饰作用机制，并通过TBI患者脑组织样品和脑脊液，进一步揭示m6A修饰与TBI预后的相关性，为TBI的临床治疗治疗、预后提供更完善的理论和数据支撑。