PRNP的m6A甲基化调控小胶质细胞外泌体对糖尿病脑病认知障碍的影响及机制研究

The cognitive impairment such as learning and cognitive decline often occurs following the occurrence of diabetic encephalopathy, which have caused serious harm to diabetic patients. Therefore, it is critical to explore the molecular mechanism of diabetic encephalopathy. In our previous study, we found that the m6A modification of prion protein gene (PRNP) was significantly higher in diabetic rats than in normal rats by detecting m6A-seq in microglia. Further investigations showed that the ability of glial cells from diabetic rats to secrete exosomes was significantly higher than that from normal rats, and the expression of Tau protein was also significantly higher in the exosomes of diabetic rats than in that of normal rats. In addition, it has been reported that Tau protein can lead to neuronal dysfunction and brain cognition-related diseases, and that PRNP is involved in the regulation of secretion of exosomes. Therefore, we hypothesized that in diabetic patients, m6A methylation of microglial PRNP regulated microglia-derived exosomes to carry Tau protein which affected the function of neurons, thereby affecting the cognitive function of diabetic encephalopathy. This study validated the above hypothesis at molecular, cellular and in vivo levels, which is of great theoretical and practical significance to elucidate the regulatory mechanism of the occurrence of diabetic encephalopathy and its clinical treatment.

糖尿病脑病的发生能导致学习、认知能力下降等认知障碍疾病，给糖尿病人造成了严重危害，因此探究糖尿病脑病发生的分子机制非常关键。我们在前期研究中，通过对小胶质细胞m6A-seq检测发现，PRNP的m6A修饰在糖尿病大鼠中比正常大鼠显著升高；进一步检测发现，糖尿病大鼠来源的胶质细胞分泌外泌体的能力比正常大鼠来源的胶质细胞明显增强，且这些外泌体中，Tau蛋白的表达水平也显著升高。Tau蛋白则会导致神经细胞功能异常和脑认知相关疾病，且PRNP参与与外泌体的分泌调控。因此，我们推测：在糖尿病患者中，小胶质细胞PRNP的m6A甲基化调控小胶质细胞外泌体携带Tau 蛋白，而Tau蛋白会影响神经细胞的功能，从而影响糖尿病脑病认知功能。本项目将在从分子、细胞、体内等水平来验证以上猜测，这对阐明糖尿病脑病发生的调控机制及其临床治疗具有重要的理论和实际意义。

糖尿病脑病的发生能导致学习、认知能力下降等认知障碍疾病，给糖尿病人造成了严重危害，因此探究糖尿病脑病发生的分子机制非常关键。针对糖尿病认知障碍患者海马区转录组测序数据进行生物信息学分析发现，糖尿病认知障碍患者海马区存在大量异常表达基因，其中RPL37，EIF3D，FAM96B，HIST1H1C，HIST1H2BM，OSTA与糖尿病脑病最相关。这些核心基因广泛的参与了脑细胞RNA代谢、氧化应激、神经元发育等过程，且存在m6A修饰位点。在2型糖尿病认知障碍小鼠海马区，神经元数目明显减少、排列紊乱，m6A修饰关键蛋白mettl3、mettl14表达下降。在体外高糖环境中培养HT-22细胞，发现高糖导致HT-22细胞活力下降、m6A修饰水平显著降低、METTL3、METTL14表达水平降低，YTHDF1、YTHDF2的蛋白表达水平升高。敲除mettl14导致神经元细胞无法存活，提示mettl14的正常表达对神经元的生存是关键的。. 上述研究结果为进一步建立mettl14低表达的神经元细胞系、动物模型提供了实验依据，为探索mettl14调控的m6A修饰对糖尿病认知障碍个体认知水平、神经元细胞生物功能、基因表达差异、神经元源性外泌体差异以及基于外泌体的神经元细胞间通讯功能的影响奠定了实验基础。为将mettl14作为糖尿病认知障碍治疗靶点提供了理论支持。