小胶质细胞caspase-1/GSDMD焦亡信号通路激活参与AD发病机理研究

Microglia dysfunction plays a role in the pathogenesis of Alzheimer's disease (AD), and its mechanism is still unknown. Cell is pyroptosis by the activated-caspase-1 cleaved GSDMD protein, and releases the inflammatory mediators. In our previous study, we found that Aβ, the key risk factor of AD, activated caspase-1 and cleaved GSDMD. Microglia but not neurons or astrocytes were pyroptosis, and released IL-1β. In APP/PS1 transgenic mice, caspase-1 was activated and cleaved GSDMD, which induced microglia but not neurons or astrocytes pyroptosis. Accordingly, we suggest a hypothesis that aging activated caspase-1/GSDMD pathway and caused microglia pyroptosis, which impaired the clearance of Aβ and other metabolites, leading inflammatory mediators release and spine plasticity impairment, speeding up the course of AD. In the present proposal, we will interfere caspase-1/GSDMD pathway and investigate the pyroptosis of microglia by western blot, immunofluorescence and electrophysiology, and analyze the cognitive functions by behavioral tests. The successful fulfillment of this proposal will shred novel insight into the form and mechanism of AD, and provide preliminary basis for exploring novel therapeutic target for AD.

小胶质细胞功能异常参与老年痴呆（AD）发病，其机制不明。激活的caspase-1剪切GSDMD蛋白使细胞发生焦亡，释放炎性介质。申请者发现：AD关键发病因子Aβ激活caspase-1并剪切GSDMD，且仅引起神经细胞中小胶质细胞焦亡，炎性介质释放显著增加；APP/PS1小鼠中观察到同样现象。为此，我们推测：在AD进程中，老化等因素激活caspase-1/GSDMD通路，导致小胶质细胞焦亡，进而引起Aβ及其他异物清除能力丧失，释放的炎性介质进一步损害神经元突触可塑性，加剧AD发病。本项目拟在细胞和AD动物模型中，采用药物及基因干预caspase-1/GSDMD焦亡途径，运用行为学、免疫蛋白印迹、免疫荧光、电生理等方法检测小胶质细胞焦亡水平及动物认知功能，探讨抑制caspase-1/GSDMD焦亡信号通路对AD模型的影响及机制。该研究结果将丰富AD发病机制，并为AD治疗提供潜在治疗靶点。

小胶质细胞功能异常参与老年痴呆（AD）发病，其机制不明。激活的caspase-1剪切GSDMD 蛋白使细胞发生焦亡，释放炎性介质。本项目以GSDMD为中心，从细胞水平和动物水平研究 AD 模型的小胶质细胞焦亡情况及对认知功能的影响；通过敲除GSDMD基因及使用GSDMD-CT质粒转染，观察Aβ42干预的小胶质细胞的细胞焦亡水平及炎症。构建APP/PS1/GSDMD-/-基因鼠，观察敲除GSDMD基因的AD模型鼠的小胶质细胞和其它细胞的焦亡水平、以及学习、记忆功能的情况。研究结果显示Aβ诱发BV2小胶质细胞焦亡，表现为细胞存活率下降、NLRP3、caspase-1及其剪切片段、GSDMD-NT表达增高，这些现象在GSDMD-CT转染的细胞中得到逆转。而在GSDMD-/-的原代小胶质细胞培养中，Aβ不能诱发细胞的焦亡。与正常野生型小鼠相比，6月龄的APP/PS1小鼠表现出焦虑情绪和认知功能障碍，而相同月龄的APP/PS1/GSDMD-/-完全逆转了APP/PS1小鼠的焦虑情绪和认知功能障碍。本项目发现GSDMD-CT对Aβ引起的小胶质细胞焦亡具有保护作用，敲除GSDMD对APP/PS1老年痴呆小鼠模型的认知功能具有很强的保护作用。该项目为老年痴呆的预防和治疗提供了重要的靶点。