DAO-EPAC2蛋白互作介导缺血性脑卒中突触可塑性损伤的机制探究

D型氨基酸氧化酶（DAO）是D型氨基酸主要代谢酶，以往认为DAO通过产物H2O2诱导神经损伤。申请人前期首次发现DAO抑制剂阻止脑缺血再灌注损伤，但对产物H2O2抑制作用远未达到其阻止脑损伤效应，提示DAO介导神经损伤至少部分不依赖H2O2。EPAC2是cAMP主要感应器，被活化后可增强突触传递并参与突触可塑性。申请人发现DAO和EPAC2共定位，存在直接相互作用，且DAO+/-小鼠海马EPAC2表达增加，脑缺血再灌注后损伤减弱。因此提出：DAO可能与EPAC2互作，抑制其功能，介导脑缺血再灌注损伤。申请人拟通过免疫共沉淀结合分子模拟技术探究DAO-EPAC2互作的结合位点；应用膜片钳等技术阐明两者互作调控突触可塑性的分子机制；在DAO-/-小鼠转入DAO-EPAC2结合位点突变的daomut基因，探究DAO介导脑缺血再灌注损伤的作用机理。研究有望为靶向DAO治疗缺血性脑卒中提供理论基础。

D-type amino acid oxidase (DAO) is the main enzyme responsible for D-type amino acids metabolism. DAO was previously regarded to induce neuronal damage through the increase of H2O2. We have found, for the first time, that DAO inhibitor could prevent cerebral ischemia-reperfusion-induced injury. However, the inhibition efficacy of DAO inhibitor on H2O2 was much smaller than its efficacy on the prevention of brain injury, suggesting that DAO-mediated neuronal injury was at least partially independent of H2O2 production. EPAC2 is the main sensor of cAMP. EPAC2 could enhance synaptic transmission and participate in synaptic plasticity after activated by cAMP. We have found that DAO and EPAC2 were co-localized in the neurons. Moreover, DAO and EPAC2 had direct interaction. The expression of EPAC2 in the hippocampus was increased, while the brain damage is decreased after cerebral ischemia-reperfusion in DAO+/- mice. Therefore, we hypothesized that DAO might directly interact with EPAC2, inhibit the physiological function of EPAC2, and therefore mediate cerebral ischemia-reperfusion-induced injury. We will explore the binding information of DAO-EPAC2 interaction by using immunoprecipitation and molecular simulation technology. Moreover, we will clarify the molecular mechanisms underlying the action between DAO-EPAC2 interaction and synaptic plasticity by using patch clamp. Furthermore, we will knock DAO-EPAC2 binding site mutant dao in DAO-/- mice to explore DAO-mediated neuronal damage in ischemia stroke. Our study might provide a theoretical basis for targeting DAO to treat ischemic stroke.