虫草素诱导MG-M2表型转化上调NGF表达介导AD神经元保护作用的分子机制

Neuro-immune microenvironment mediated by microglial M1/M2 polarization is the crucial factor for the pathogenesis and development in Alzheimer’s disease. In previous studies, we found that anti-AD drugs exerted the neuroprotective effects by adjusting the balance of NGF-TrkA/p75NTR signaling. Cordycepin improved the cognitive deficits of APP/PS1 transgenic AD mice, promoted microglial M2 polarization and increased NGF expression, accompanied by the enhanced CREB phosphorylation. Bioinformatics predicted that CREB bound with NGF promoter to regulate the NGF transcription. The results above mentioned suggested that cordycepin increased NGF expression by promoting microglial M2 polarization to elicit the anti-AD effects via balancing the NGF-TrkA/p75NTR signaling in AD neurons . However, it still needs to be further studied. In this study, based on the key CRE site confirmation of CREB bound with NGF promoter to reveal that cordycepin promoted microglial M2 polarization to secrete NGF via JAK/CREB signaling. Furthermore, it was testified that the neuronal beneficial effects of the secreted NGF upregulated by cordycepin-induced microglial M2 polarization was mediated by the balance of NGF-TrkA/p75NTR signaling in AD neurons by MG/neuron co-culture. The study will provide new ideas on the interaction between neuro-immune microenvironment modulated by microglial polarization and the progression and development of AD, and find novel targets for AD therapy.

MG-M1/M2表型转化介导神经免疫微环境影响AD发生发展。申请者发现：抗AD药物调节NGF-TrkA/p75NTR通路平衡发挥神经保护作用；虫草素改善APP/PS1小鼠认知障碍与脑内M1/M2表型标志物及NGF表达相关，并伴有CREB激活；信息学预测CREB可与NGF启动子结合调控NGF表达，提示JAK/CREB通路参与虫草素诱导MG-M2表型转化上调NGF分泌与表达，并通过神经元NGF-TrkA/p75NTR通路平衡发挥AD神经元保护作用，但尚需研究证实。本项目在确认CREB调控NGF表达关键位点基础上，阐明虫草素诱导MG-M2表型转化通过JAK/CREB通路介导NGF表达的分子机制，明确虫草素诱导MG-M2表型转化上调NGF表达调控AD神经元NGF-TrkA/p75NTR通路平衡介导AD神经元保护作用。本研究为深入探讨MG表型转化介导免疫微环境与AD发生发展相关性提供新思路和新靶点。

小胶质细胞M1/M2表型转化介导神经免疫微环境改变是影响AD发生发展的重要因素，诱导MG-M2表型转化改善神经元免疫微环境以保护AD神经元，成为AD治疗新策略。课题组发现，CCS可显著改善APP/PS1转基因AD小鼠学习记忆障碍，并诱导MG-M2表型转化，上调NGF，与激活CREB相关，提示CCS可能通过激活CREB通路诱导MG-M2表型转化上调NGF发挥神经保护作用。细胞实验显示，CCS可增加静息态BV-2细胞（MG-M0）和Aβ+LPS诱导BV2细胞（MG-M0）的M2表型标志物Arg1、TGFβ、IL10、CD206表达，并降低M1表型标志物IL-1β、TNFα及iNOS水平，同时伴有NGF表达及分泌增高，提示CCS通过促进MG-M2表型转化上调NGF。进而，我们过表达CREB及给予CREB选择性抑制剂KG501干预CREB，发现过表达CREB的MG-M1细胞显著向MG-M2表型极化，同时上调NGF；再用KG501处理后，M2表型标记物水平降低，而M1表型标记物水平增高，NGF下调；提示CREB是调控CCS诱导MG-M2表型上调NGF的重要分子机制。生物信息学预测，CREB作为转录因子可与NGF启动子区6个CRE结合位点结合，我们通过构建NGF启动子区系列截短荧光素酶质粒p2000GL3、p1078GL3、p1023GL3、p816GL3、p688GL、p520GL及p283GL，并分别将其转入CREB过表达的293T细胞及BV-2细胞，进一步通过ChIP实验验证，发现CREB与NGF启动子区的关键CRE位点-1085~-1078，-291~-284直接结合，调控NGF转录。进而，采用共培养的方法，以rNGF及anti-NGF干预体系NGF后，发现共培养体系中NGF水平与神经元的存活率、细胞衰老、凋亡及突触可塑性指标GAP-43、MAP2表达密切相关；并以APP/PS1小鼠与C57 BL/6J小鼠原代神经元与CCS处理后的MG-M1共培养后发现，改善NGF-TrkA/p75NTR通路平衡是CCS诱导MG-M2表型介导NGF对AD神经元的保护作用的分子机制。综上，CCS可激活CREB通路诱导MG-M2表型转化，并通过CREB与NGF启动子区-1085~-1078和-291~-283 CRE关键位点的直接结合作用调控NGF表达，进而通过维持神经元NGF-TrkA/