急性缺血性脑卒中小鼠皮层神经元自噬底物蛋白p62对神经元焦亡的调控机制研究

Acute cerebral ischemia will always do harm to the homeostasis of autophagy. The previous studies are mostly concentrated on the upstream signaling pathway of autophagy. However, its downstream effector or the relationship between autophagy and inflammation is not well documented. In our latest study, we found firstly that the accumulation of p62 induced by acute ischemia-reperfusion decreased neuronal pyroptosis via the de-activation of caspase-1 and reduction of Interleukin-1β production. Using middle cerebral artery occlusion(MCAO)/reperfusion model of mice and oxygen-glucose deprivation (OGD) cellular model, we will carry out the following studies with morphological, electrophysiological, biochemical and bioinformatics methods: (1) to find out the role of p62 in the acute ischemia or ischemia/reperfusion by targeted invention to the protein expression of p62. (2) to address the regulatory mechanism of the binding action between p62 and related inflammatory protein, and the involved pathological significance. (3) to further explore the mechanism of p62 in neuronal pyroptosis after its binding with functional proteins. (4) to identify signaling transduction pathways, the underlying molecular key proteins, and protein-protein interaction network involved in the paradigm of autopahgic dysfunction-p62-neuronal pyroptosis induced by acute cerebral ischemia. This project will focus on the crosstalk between autophagic dysfunction and neuronal inflammation to explore a new strategy on ischemic injuries, which will shed light on a new treatment against clinical ischemic stroke.

急性脑缺血可致自噬失调造成神经元损伤，由于自噬是细胞正常生理活动所需，调节自噬程度减轻缺血损伤具有极大实践难度，因此寻找自噬下游效应分子并探讨其作用机制就显得尤为重要。本课题前期工作中，我们发现缺血再灌导致自噬底物p62聚集，通过降低caspase-1激活及IL-1β产生，减少神经元焦亡。本课题拟利用在体小鼠大脑中动脉阻塞及离体皮层神经元氧糖剥夺模型，利用形态学、电生理学、蛋白组学和生物信息学等技术，开展如下研究： ①针对单纯缺血及缺血再灌时神经元p62的不同水平进行靶向干预，明确其病理意义；②探讨p62与炎性功能蛋白结合/解聚的调控机制及病理意义；③探讨p62调节神经元焦亡的分子机制；④探索急性脑缺血致自噬失调-p62-焦亡的信号转导调控系统、蛋白互作网络及关键节点蛋白。本课题聚焦p62探讨神经元自噬与炎性反应间的对话，寻找高效的脑缺血保护策略，所获成果将为临床防治缺血性脑卒中提供新思路

急性脑缺血可致自噬失调造成神经元损伤，由于自噬是细胞正常生理活动所需，调节自噬程度减轻缺血损伤具有极大难度，因此寻找自噬下游效应分子并探讨其作用机制就显得尤为重要。本团队应用在体大脑中动脉阻塞及原代培养神经元氧糖剥夺模型，发现（1）胞浆去乙酰化酶Sirt1与TRAF6存在相互作用，对早期炎性反应进行调节，缺血导致Sirt1蛋白水平降低，伴随TRAF6蛋白水平增加，TRAF6活性增强。Sirt1水平降低同时伴随ROS的大量产生，随后出现TRAF6激活，因此sirt1失能是炎性反应的起始因子。TRAF6介导的信号通路是焦亡的重要调节信号通路当TRAF6信号通路受到抑制，焦亡受到抑制，说明早期的炎性反应是TRAF6信号通路介导的。如果Sirt1激活可以降低梗死体积，提示Sirt1-TRAF6信号通路介导的炎性反应对伸进缺血损伤至关重要。急性缺血后急性氧糖剥夺后，原代培养神经元内出现LAMP1蛋白的长时程增加。研究了缺血应激对自噬和突触结构的影响。（2）急性缺血早期自噬上调，后期溶酶体数量增加，体积增大，提示溶酶体储积障碍。通过自噬抑制剂3-MA预处理或自噬相关基因Atg7敲减来抑制自噬可部分逆转这些变化；而通过自噬激活剂雷帕霉素RAPA处理神经元可模拟这些变化，表明溶酶体储积障碍与早期自噬有关。并且，再灌注时MTOR依赖性的溶酶体生物发生仅恢复到基础水平，不足以清除短暂自噬上调后的未降解物，上述原因造成的溶酶体储积障碍可导致自噬-溶酶体系统功能障碍。进一步的研究表明，突触微结构受损并伴有溶酶体储积可能是由于突触蛋白动态翻新受损所致。