神经元凋亡时GSK-3/Egr-1上调PUMA的作用及其机制

GSK-3 (Glycogen synthase kinase-3) plays a critical role in mediating neuronal apoptosis, however its downstream pro-apoptotic effectors still need to be identified. Although it has been reported that GSK-3 mediates the up-regulation of PUMA through p53, we found that GSK-3 potentiated the induction of PUMA in a p53-independent manner during neuronal apoptosis by using the p53 knock-out mice. Via further anlysis of the puma promoter, we have located a classical Egr-1 binding site (-104 to -95 bp). Inhibition of Egr-1 down regulated PUMA expression; dominant-negative Egr-1 (dn-Egr-1) suppressed the activity of puma promoter-driven luciferase reporter. These data suggest that Egr-1 transcriptionally regulates PUMA expression. In addition, GSK-3 inhibitor dramatically suppressed the mRNA and protein expression and the transcriptional activity of Egr-1. Thus, we propose that GSK-3-mediated up-regulation of PUMA is Egr-1-dependent but not p53-dependent, during neuronal apoptosis. By using adenovirus expressing shRNA, ChIP, EMSA etc., we aim to demonstrate a new mechanism of PUMA regulation and identify a GSK-3 downstream pro-apoptotic effector, which would shed a light on discoverying novel theraputic targets for neurodenerative disease.

GSK-3是促神经元凋亡的关键激酶，然而其促凋亡底物不清楚。尽管文献报道: GSK-3通过激活p53上调PUMA促凋亡，但我们用p53敲除鼠观察到：神经元凋亡时GSK-3通过一种新的、p53非依赖机制介导PUMA上调。进一步分析发现puma启动子包含一个经典的Egr-1结合位点 (-104至-95 bp)；抑制Egr-1，PUMA表达下调；负显性Egr-1抑制PUMA 启动子驱动的报告基因；提示Egr-1介导PUMA表达。进一步结果显示，GSK-3抑制剂抑制Egr-1 mRNA、蛋白及转录活性。由此，我们提出假设：神经元凋亡时，GSK-3通过Egr-1，而非p53，介导PUMA转录上调。本项目拟采用腺病毒干扰、EMSA和CHIP等方法，旨在获得GSK-3通过Egr-1介导PUMA上调的可靠证据，阐明PUMA上调新机制、确立GSK-3促凋亡新底物，为寻找神经退行性疾病治疗新靶点提供科学依据。

帕金森病的主要病理特征是黑质致密部多巴胺能神经元进行性死亡，神经炎症在其中发挥重要作用。Egr-1是一个锌指蛋白转录因子，已报道既参与神经元死亡又参与炎症反应。但Egr-1是否在PD病程中发挥作用，目前尚无人研究。通过利用神经毒素MPTP诱导的小鼠帕金森病模型，我们首次发现，Egr-1在模型极早期选择性在黑质致密部上调。原位免疫荧光结果显示，Egr-1主要在黑质AldoC阳性的原生质型星形胶质细胞核内上调。基因敲除Egr-1或使用Egr-1抑制剂Mithramycin A显著的抑制小胶质细胞和星形胶质细胞激活、炎症因子表达及多巴胺神经元的死亡。本研究说明PD模型中Egr-1的上调促进神经炎症产生及多巴胺神经元退化，并提示星形胶质细胞的Egr-1可能在炎症反应中起关键作用。