TIGAR在脑缺血复灌过程中经非酶途径激活线粒体自噬的保护作用及机制研究

Cerebral ischemia induces mitophagy in neurons. Mitophagy protects against ischemic neuronal injury by clearance of damaged mitochondria. Ischemia-reperfusion-induced oxidative stress may activate mitophagy. In hypoxic neurons, glucose is metabolized by pentose phosphate pathway (PPP) to NADPH which diminishes oxidative stress. Our preliminary experiments, however, unexpectedly find that TP53-induced glycolysis and apoptosis regulator (TIGAR), which promotes PPP metabolism, activates rather than inhibits mitophagy in primary neurons treated with oxygen-glucose deprivation and reperfusion. Furthermore, TIGAR-induced mitophagy protects against ischemic neuronal injury. In addition, our preliminary data indicate that the TIGAR recruitment to mitochondria, but not the phosphatase activity of TIGAR, is required for mitophagy activation in ischemic neurons. It was reported that TIGAR binds to UB2L3, an ubiquitin-conjugating enzyme. We therefore hypothesized that TIGAR translocated to mitochondria may recruit UB2L3, which further passing ubiquitin to the E3 ubiquitin ligase Parkin and thus activates mitophagy in ischemic neurons. By employing neuron-specific knockout and overexpressed TIGAR mutant mice, the present investigation aims to identify that TIGAR-induced mitophagy protects against cerebral ischemia-reperfusional injury. We further aimed to explore the mechanisms by which TIGAR activates mitophagy in an enzyme-independent manner. This investigation may provide novel drug targets for neuroprotection by interrupting mitophagy in cerebral ischemia.

脑缺血复灌诱发神经元线粒体自噬，并清除损伤线粒体，发挥神经保护作用。复灌诱发的氧化应激可能启动线粒体自噬。神经元低氧时，葡萄糖经由戊糖磷酸途径代谢减少氧化应激，并可能由此抑制自噬。申请人的预实验意外发现，p53诱导的糖酵解及凋亡调节因子TIGAR促进戊糖磷酸代谢，却激活缺血复灌神经元的线粒体自噬、保护神经元。我们还发现，TIGAR促线粒体自噬作用可能与其磷酸酶活性无关，而与其向线粒体转位有关。TIGAR结合E2泛素结合酶UBE2L3，推测TIGAR可能于线粒体上招募UBE2L3，进而向Parkin提供泛素，介导线粒体自噬。因此，本研究拟利用选择性TIGAR基因敲除等模式动物，结合药理学、分子生物学等手段，首先明确脑缺血复灌过程中TIGAR经促进线粒体自噬的保护作用，并试图阐明TIGAR经非酶途径诱导线粒体自噬的机制。本研究有望为干预脑缺血过程中的线粒体自噬进而保护神经提供新的药物靶点。

氧化应激损伤是脑缺血复灌过程中的主要细胞损伤机制。神经元可通过TIGAR经戊糖磷酸通路将葡萄糖代谢为还原性物质NADPH。长时程缺血后葡萄糖的可获得性显著降低，但神经元仍保留一定的抗氧化能力，其机制尚不清楚。本项目探索了不同时程脑缺血后复灌所导致的氧化应激损伤，率先发现了长时程脑缺血后，TIGAR通过非戊糖磷酸途径发挥抗氧化作用。进一步研究发现，TIGAR的上述抗氧化作用与其激活细胞自噬密切相关，但激活的自噬并非通过清除线粒体、过氧化物酶体等氧化物产生的细胞器发挥作用，而是通过激活Nrf2进而上调抗氧化酶表达，最终发挥减少氧化应激、保护缺血大脑的作用。本项目的发现为进一步理解在长时程缺血后葡萄糖减少情况下细胞如何实现氧化还原平衡提供了新的视角，提出调节TIGAR-Nrf2途径可能是一种潜在的抗脑缺血复灌损伤药物干预新策略。本项目成果标注发表论文5篇，培养研究生6人。