AMPK/TFEB乙酰化调控自噬及其在AD样病变中的作用和机制研究

Autophagy-lysosome dysfunction is a major early pathological feature of Alzheimer's disease (AD). Transcription factor EB (TFEB)is an important regulator of autophagosome formation and clearance, but the regulatory network that governs TFEB activity is not yet fully defined. In our preliminary experiments, we found that the abnormal aggregation of Aβ in 8-month-old APP/PS1 AD mice was closely related to the autophagosome clearance disorder; Overexpression of AMPK reduced the level of TFEB acetylation and Aβ in N2a-APP cells, suggesting that TFEB acetylation regulation is a key regulator of AD autophagy. Therefore, we hypothesized that AMPK activity decreased-the level of TFEB acetylation increased-TFEB transcriptional regulation activity decreased, resulting in abnormal expression of autophagic protein, dysfunction of autophagy-lysosome, Aβ clearance weakened. This study focuses on clarifying acetylation regulates the activity of TFEB；the role of TFEB acetylation in the regulation of autophagy and its mechanism; AMPK affects autophagy-lysosome function and Aβ clearance by regulating the level of TFEB acetylation. LC-MS/MS, lentivirus expression system and brain stereotaxic localization technology will be used in animal and cell levels. The study will reveal the regulation mechanism of AMPK/TFEB on autophagy and its role in the pathogenesis of AD, and provide a new target for the prevention and treatment of AD.

自噬溶酶体功能障碍是AD的主要早期病变特征之一。TFEB是自噬体形成和清除的重要调节因子，但其活性调节机制并不清楚。我们预实验发现，8月龄APP/PS1 AD模型鼠中Aβ异常聚集与自噬体清除障碍密切相关；在N2a-APP细胞中过表达AMPK，可降低TFEB乙酰化水平和Aβ的含量，提示TFEB乙酰化调节是AD自噬障碍的关键调控开关。由此，提出课题假设：AMPK活性降低-TFEB乙酰化水平增高-TFEB转录调节活性降低，引起自噬蛋白表达异常，自噬溶酶体功能降低，Aβ清除减弱。本项目重点阐明乙酰化对TFEB活性的影响；TFEB乙酰化在调控自噬中的作用和机制；AMPK通过调节TFEB乙酰化的水平影响Aβ的清除。研究将运用LC-MS/MS、慢病毒表达系统及脑立体定位技术进行动物整体和细胞水平的研究。研究将揭示AMPK/TFEB对自噬功能的调控机制及其在AD发病中的作用，为AD防治策略提供新靶点。

清除体内异常聚集的Aβ和Tau蛋白是治疗AD的关键，Aβ和Tau蛋白大量沉积与自噬功能的降低存在显著相关性。TFEB可调节自噬体的形成、自噬体和溶酶体的融合以及溶酶体的生成和功能的正常发挥，AMPK在啮齿类动物和人类大脑的不同脑区（除下丘脑）广泛表达，随着年龄的增加，AMPK 活性降低；小鼠海马CA3区下调AMPK，可导致小鼠的空间学习记忆能力下降。但AMPK能否通过TFEB调控Tau蛋白和Aβ的降解目前尚不清楚。本研究实验结果显示：①在APP/PS1小鼠和散发型AD样动物模型小鼠的海马中，AMPK的活性降低和Tau蛋白乙酰化水平增加；激活AMPK可降低上述模型中Tau蛋白的乙酰化水平和改善认知功能，其机制与Sirt1相关。②APP/PS1小鼠海马区自噬体清除障碍，TFEB表达水平降低和Aβ沉积；激活AMPK后TFEB的表达水平和入核均增加，改善自噬功能和降低Aβ的含量；细胞水平上进一步验证，AMPK通过TFEB促进Aβ降解。③在AD样动物模型中，TFEB过表达可改善认知功能和AD样病理变化；乙酰化影响TFEB的活性，AD动物模型中TFEB的乙酰化水平增加，降低TFEB的乙酰化水平可改善认知功能和AD样病理变化。本研究证实 AMPK和TFEB在 AD 发生发展中的作用，为靶向调节AMPK和TFEB作为 AD 治疗新策略提供理论和实验依据。