虫草素靶向PDK2调控MG代谢重编程介导AD神经元保护作用的机制研究

Microglial metabolic reprogramming is related to its mediated neuro-immune microenvironment and participates in the pathogenesis and development of AD. It was found that cordycepin improved the learning and memory impairment in APP/PS1 mice by inhibiting Aβ deposition and neuronal apoptosis, which was associated with the induction of MG-M2 polarization and maintenance of mitochondrial homeostasis, accompanied by the down-regulation of PDK2 expression; cordycepin could increase LPS+Aβ-induced OCR in BV2, and promote MG-M2 polarization and neuronal survival in AD. Informatics predicted that cordycepin could combine with PDK2 at the sites of Ala251, Glu254 and Asp282, suggesting that PDK2-mediated metabolic reprogramming was involved in the protective effect of cordycepin-induced MG-M2 polarization in AD, but it needs to be confirmed. In this project, taking the microglial phenotype switch mediated by metabolic reprogramming as the breakthrough point, to investigate the correlation between metabolic reprogramming and cordycepin-induced MG-M2 polarization, to confirm that PDK2 was regulated by cordycepin-induced metabolic reprogramming and MG-M2 polarization, to clear the binding mode and key sites between cordycepin and PDK2, to elucidate the molecular mechanism by which cordycepin targeting PDK2-mediated microglial metabolic reprogramming and MG-M2 polarization to protect neurons in AD, and this study will provide new targets and novel strategies for AD therapy and the development of anti-AD drugs.

MG代谢重编程与其介导的神经免疫微环境相关，参与AD发生发展。申请者发现：虫草素通过抑制Aβ沉积和神经元凋亡改善APP/PS1小鼠学习记忆障碍与诱导MG-M2极化及维持线粒体稳态相关，伴有PDK2表达下调；虫草素可提高LPS+Aβ诱导的BV2的OCR，促进MG-M2极化和AD神经元存活。信息学预测发现虫草素可与PDK2关键位点Ala251、Glu254和Asp282结合，提示PDK2介导代谢重编程参与虫草素诱导MG-M2极化发挥AD神经元保护作用，但尚需研究证实。本项目以代谢重编程介导的MG表型转化为切入点，考察代谢重编程与虫草素诱导MG-M2极化的相关性，明确PDK2在虫草素调控代谢重编程和MG-M2极化中的作用，确认虫草素与PDK2关键位点结合的作用方式，阐明虫草素靶向PDK2调控MG代谢重编程介导MG-M2极化发挥AD神经元保护作用的分子机制，为AD治疗和新药研发提供新靶点和新策略。

小胶质细胞（MG）介导的神经炎性微环境，特别是其M1/M2表型转化和线粒体能量代谢重编程，对阿尔茨海默病（AD）进程中神经元的最终命运归属发挥决定性作用，与AD发生发展密切相关，而线粒体代谢重编程在MG-M1/M2表型转化中的作用有待阐明。本研究聚焦于探究线粒体重编程与MG-M1/M2表型转化的联动效应在AD中的作用及虫草素的抗AD作用及机制，首次发现有氧代谢水平降低介导的线粒体能量代谢失衡可诱导MG-M1极化，是引发AD神经炎性微环境导致神经元损伤的关键环节。而虫草素具有改善APP/PS1小鼠学习记忆功能的抗AD作用，与其促进MG-M2极化，恢复线粒体稳态，进而增加AD神经元存活有关。具体来说，我们应用线粒体分离联合LC-MS/MS技术明确虫草素可分布于线粒体，阐明虫草素与丙酮酸脱氢酶激酶同工酶2（PDK2）靶向结合上调有氧代谢途径中的OCR水平和葡萄糖消耗，诱导IL-1β等M1表型相关炎性因子表达明显降低，而Arg-1等M2表型相关因子表达增加，从而促进神经元存活。本研究为揭示代谢重编程和MG-M1/M2表型转化的联动效应与AD神经保护作用的相关性提供了新思路，也为研发虫草素作为新型治疗AD的候选药物奠定了实验基础。