银杏叶制剂纯化物经自噬信号通路调控异常磷酸化Tau蛋白缓解AD的机制研究

Alzheimer’s disease (AD) is characterized by intracellular neurofibrillary tangles (NFTs), which are primarily composed of hyperphosphorylated tau protein (p-Tau). Misfolded protein can be degraded by autophagy pathway, and this pathway is strongly activated in the AD brain. We have recently observed that to enhance neuronal autophagy by activating microglial inflammation at a low level increases degradation of p-tau in neurons. In additioin, we have also observed that Ginkgo Biloba Extract (EGb761) effectively prevents AD symptoms in APP mice by enhancing autophagy signaling pathway. However, it still remains unknown that 1) which components could activate autophagy. 2)whether p-tau is degraded in this process. 3) its underlying mechanism of activating autophagy. Thus, we hypothesized that major components of EGb761 could activate neuronal autophagy and reduce cerebral p-tau load in neuron. To test this hypothesis, we treat Tau transgenic mice with different components of EGb761 for several months. The improvement of neuronal function, neuronal autophagy, p-tau were evaluated by immunofluorescence staining, differential protein extraction. In addition, we investigate the key molecules in autophagy signaling by siRNA interference and autophagy inhibitor. In summary, the overall aim of this project is to clarify the effect and underlying mechanism of EGb761 components in degrading p-tau through autophagy signaling pathway, which will provide new theoretical basis and approach in prevention of AD.

异常磷酸化Tau蛋白（p-tau）构成神经元纤维缠结（NFTs），是阿尔茨海默病（AD）的主要病理表现；自噬能有效降解胞浆内的异常聚集蛋白，且在AD脑中激活。我们证明了适度小胶质细胞炎症激活能促进神经元自噬，进而参与p-tau的调控；我们同时发现银杏叶制剂（EGb761）能激活自噬信号通路，缓解APP模型鼠的AD症状，但其中哪些成分激活了该通路及其调控机制，能否降解p-tau尚不清楚。为此，我们提出假说：EGb761的某些纯化物能通过激活自噬清除神经元内的p-tau。为了验证该假说，我们采用免疫荧光染色、梯度蛋白萃取等方法，在体探讨EGb761纯化物对神经自噬及功能，p-tau的影响；另外，通过Atg5 siRNA干扰、自噬抑制剂等工具，体外分析纯化物经自噬途径调控p-tau可能的分子机制，为确立自噬信号通路作为防治AD新靶点提供更充分的科学依据。

阿尔茨海默病（AD）是最常见的老年痴呆。其病理特征是脑实质的细胞内神经元纤维缠结（NFT）和细胞外淀粉样蛋白（Aβ）沉积。NFT主要由高磷酸化tau蛋白（p-tau）组成。有证据表明，寡聚态Aβ能诱导Tau过度磷酸化和聚集，并促使Tau的病理进展从颞叶内侧皮质辐射至整个高级皮层。减少Tau的表达可能防止由Aβ引起的神经元死亡。本项目系统阐述了银杏叶提取纯化物EGb761改善 Tau 转基因小鼠认知的可靠证据，同时阐明银杏叶提取纯化物EGb761对Tau转基因小鼠脑内神经元存活、神经突触保护的影响。深入探究了银杏叶提取纯化物EGb761及其不同组分通过自噬信号通路介导对p-tau调控的机制，并明确了银杏叶提取纯化物EGb761及其相应单体成分通过自噬上游信号分子的磷酸化改变来介导 p-tau 的自噬性降解，揭示其中的相关分子机制，为后续寻找银杏叶制剂纯化物可能影响的靶基因及分子调控奠定了理论基础。