HIF-1α-ATP13A2信号通路对帕金森病多巴胺能神经元损伤的保护作用和机制研究

As one of the newly discovered gene related to Parkinson’s disease, the loss-of-function mutations of ATP13A2 could result in lysosomal dysfunction and the subsequent accumulation of alpha-synuclein, Enhanced expression of ATP13A2 can reverse the effects. In addition, the promoter of ATP13A2 contains HRE regulatory elements, hypoxia or over-expression of HIF-1 can significantly increase the levels of ATP13A2. In our previous study, we found that MPP+ can increase the mRNA and protein level of HIF-1α but the changes did not rescue the apoptosis of SH-SY5Y cells. And also, our previous studies have demonstrated that Orexin-A have the neuroprotection effects on MPP+ induced dopaminergic neuronal injury by inducting the expression of HIF-1α. On the basis of the core sequence of HRE which contains CpG island, we speculated that MPP+ has an additional effect on the methylation of the HRE element of ATP13A2 promoter which prevented the link with HIF-1α and the protective effects of Orexin-A was associated with the demethylation and the enhanced level of HIF-1α. Therefore, we hypothesized that HIF-1α-ATP13A2 pathway might play an important role in the neuroprotection of dopaminergic neuronal damage in PD, and up-regulating the expression of ATP13A2 might provide a novel therapeutic target for the treatment of PD. Our project would set up PD model in vivo and in vitro, explore the effect of Orexin-A on the promoter activity of ATP13A2 and its role in the HIF-1α-ATP13A2 pathway, and further evaluate the possibility of up-regulation of ATP13A2 in the neuroprotective treatment in PD, our study would be of great clinical significance.

帕金森病相关基因ATP13A2失功能突变可致溶酶体功能障碍及α-共核蛋白聚集，其启动子区域存在缺氧反应原件(HRE)，低氧或过表达HIF-1α可增加其活性。我们在MPP+造模中发现，MPP+作用时间延长能增加HIF-1αmRNA及蛋白水平，但未减少细胞凋亡；Orexin-A通过诱导HIF-1α对多巴胺能神经元起保护作用；HRE核心序列存在CpG岛，推测MPP+导致HRE核心序列甲基化阻碍了HIF-1α与HRE的结合而抑制了ATP13A2 的转录活性，Orexin-A的神经保护作用可能与其对该位点去甲基化及上调HIF-1α有关。OrexinA可能调控HIF-1α-ATP13A2信号通路保护多巴胺能神经元损伤，上调ATP13A2表达可能成为帕金森病治疗新靶点。本课题拟探讨Orexin-A对ATP13A2启动子活性和HIF-1α-ATP13A2通路的表达调控机制，阐明其对帕金森病的神经保护作用。

ATP13A2基因突变可致溶酶体功能障碍，在α-syn异常积聚中发挥关键作用。该基因启动子区域存在缺氧反应原件，低氧或过表达HIF-1α可增加其活性。既往研究法发现ATP13A2通过激活NLRP3炎症小体调控胶质细胞介导的神经炎症，证实了溶酶体与神经炎症的直接联系。此外，文献报道PD患者中肠道α-syn聚集相关的肠道通透性增加以及细菌和炎性产物的失调可能是引起局部炎症并促进α-syn在ENS聚集的主要原因。.研究内容及取得重要成果：1、FG-4592可促进HIF-1α表达，减少神经毒素诱导的神经元损伤。2、腹腔注射丁苯酞，利用高效液相色谱、免疫组化技术分析神经元数量、神经纤维密度及小胶质细胞激活。结果发现丁苯酞可显著改善PD模型小鼠的运动功能障碍；同时抑制小胶质细胞激活保护多巴胺能神经元。3、灌胃漆黄素，通过矿场实验、爬杆实验、悬挂实验检测小鼠行为学指标；通过蛋白免疫、尼氏染色及TUNEL检测等技术手段检测纹状体TH蛋白表达及神经元凋亡。通过高通量测序检测小鼠粪便中细菌16S rRNA V3和V4 序列的基因多样性。我们发现漆黄素可改善PD小鼠行为学损伤，增加TH阳性神经元数量。测序结果提示漆黄素可改变PD模型小鼠肠道微生物群的数量、分布和多样性。.该项目通过建立PD体内外模型，运用分子生物学等手段，证实丁苯酞通过抑制神经炎症发挥神经保护作用；还发现漆黄素改善肠道菌群失调保护多巴胺能神经元，改善模型动物的运动功能，为寻找延缓PD神经退变进程的干预靶点提供理论支撑，具有重要的科学和临床意义。本项目研究成果在Aging Dis、Hum Mol Genet等期刊杂志发表论文15篇。