大电导钙激活钾通道（BKCa)开放剂治疗阿尔茨海默病模型小鼠的神经生物学研究

For the treatment of Alzheimer's disease (AD) is limited, explore new therapeutic method is the hot spot of the current study. It is proposed that intracellular amyloidβ (Aβ) , before extracellular plaque formation, triggers cognitive deficits in Alzheimer's disease (AD). Our previous work found that spike width was broadened by injecting Aβ1-42 protein into mouse neocortical pyramidal cells through whole-cell patch pipettes. The effect was mimicked by charybdotoxin, a blocker of large-conductance Ca2+ activated K+ (BKCa) channels, and blocked by isopimaric acid, a BKCa channel opener. In young AD model mice, We also confirmed spike broadening by Aβ1-42. These findings suggest that the suppression of BKCa channels by intracellular Aβ1-42 is a key mechanism for early dysfunction in the AD brain. This project put forward the application of BKCa channel opener in the treatment of AD model mice, to explore new methods for the treatment of AD. We will use behavioral methods to evaluate learning and memory function of mice. We will observe the changes of synaptic plasticity in the hippocampus and neural excitability in neocortical pyramidal cells by electrophysiological techniques. We will check soluble Aβ42 concentration and the expression of homer proteins in the brain by molecular biology techniques. This study will provide important theoretical basis for new drug targets for the treatment of AD.

目前对于阿尔茨海默病（AD）的治疗手段有限，相关的药物疗效一般,探寻新的治疗方法是当前研究的热点。β-淀粉样蛋白(Aβ)是AD核心的病理机制，是最早的损害突触完整性和可塑性的因素。我们前期工作发现向大鼠的新皮层锥体细胞注射Aβ1-42使锋电位增宽，该作用可以被钾通道阻滞剂模拟，被大电导钙激活钾通道（BKCa）开放剂逆转。提示细胞内Aβ1-42抑制BKCa通道是AD脑早期功能障碍的关键机制。在年轻AD模型小鼠中也证实了细胞内Aβ抑制BKCa通道活性。本项目据此提出应用BKCa通道开放剂治疗AD模型小鼠，探索治疗AD的新方法。我们将利用行为学方法评价小鼠的学习和记忆功能；通过电生理技术观察海马区突触的可塑性及新皮层锥体细胞的兴奋性变化；通过分子生物学方法观察脑内homer1a及homer1b/c蛋白表达及可溶性Aβ42 含量变化。本研究将为寻找新的药物靶点治疗AD提供重要的理论依据。

我们前期工作发现向大鼠的新皮层锥体细胞注射Aβ42使锋电位增宽，该作用可以被钾通道阻滞剂模拟，被大电导钙激活钾通道（BKCa）开放剂逆转。提示细胞内Aβ42抑制BKCa通道是AD脑早期功能障碍的关键机制。据此提出应用BKCa通道开放剂—异海松酸（ISO）治疗AD模型小鼠，探索治疗AD的新方法。方法 应用脑立体定位仪向4月龄雄性APP/PS1小鼠和野生型（WT）小鼠左侧侧脑室植入连接Alzet微量渗透压泵的套管，持续性微量注射ISO或DMSO治疗。第3周开始在体进行行为学实验后制备海马脑片，检测海马CA1区突触可塑性及神经元的兴奋性。采用ELISA方法检测海马Aβ42含量变化。结果 APP/PS1+DMSO组的新物体识别指数与WT+DMSO组比较显著降低，APP/PS1+ISO组较APP/PS1+DMSO组的新物体识别指数显著升高（P＜0.01）。定位航行试验的逃避潜伏期，第3-5天APP/PS1+DMSO组与WT+DMSO组比较显著延长，第4-5天APP/PS1+ISO组较APP/PS1+DMSO组显著缩短（P＜0.05）。空间探索实验显示APP/PS1+DMSO组小鼠穿越平台区时间百分比和穿越平台次数较WT+DMSO组显著降低，APP/PS1+ISO组小鼠穿越平台区时间百分比和穿越平台次数较APP/PS1+DMSO组显著增加（P＜0.05）。与WT+DMSO组相比，APP/PS1+DMSO组海马LTP显著下降，APP/PS1+ISO组海马LTP与APP/PS1+DMSO组相比显著升高（P＜0.01）。与WT+DMSO组比较，APP/PS1+DMSO组海马神经元的动作电位发放频率显著增加（P＜0.01），５峰电位序列平均峰电位半宽度显著增宽（P＜0.05）；与APP/PS1+DMSO组相比，APP/PS1+ISO组海马神经元的动作电位发放频率显著降低（P＜0.01），５峰电位序列平均峰电位半宽度显著缩窄（P＜0.05）。与WT+DMSO组相比，APP/PS1+DMSO组海马Aβ42含量显著升高，APP/PS1+ISO组海马Aβ42含量与APP/PS1+DMSO组相比显著下降（P＜0.05）。结论 ISO通过易化BKCa通道，降低神经元兴奋性、清除Aβ42、提高海马突触可塑性并改善学习和记忆能力。本研究为寻找新的药物靶点治疗AD提供重要的理论依据。