甲羟戊酸途径的干预对5XFAD小鼠认知功能的影响及机制研究

In previous studies, we found that the levels of farnesylpyrophosphate/FPP and geranylgernylpyrophophate/GGPP, intermediates of mevalonate/MVA pathway, were significantly elevated in 5XFAD mice. The purpose of this study is to determine whether mevalonate pathway inhibition improves the cognitive function in 5XFAD mice, through improving oxidative stress reaction. To investigate the underlying mechanisms, we carry out the following studies. 5XFAD mice and C57 mice in vivo or neuron in vitro were treated with chemical inhibitors or shRNA of FPPS, GGPPS, FT, GGT, in order to reduce the levels of FPP and GGPP, and to weaken the activity of small G protein. Then the cognitive and memory function of mice were determined by Y maze and step down test. The morphological changes and apoptosis rate of brain and neuron were observed. The production of ROS, other oxidative stress indices and Aβ were observed. The LTP electrophysiology of mice hippocampal slices were detected. FPP or GGPP were co-incubated with cultured neuronal cells. Whether the co-incubation can reverse the influence of inhibition was observed. The purpose of our studies is to investigate whether the small G proteins in downstream of MVA pathway plays a role of “bridge” between MVA pathway and oxidative stress reaction in mechanism of AD.

本项目拟在前期研究发现5XFAD小鼠脑组织的甲羟戊酸（MVA）途径中间产物FPP、GGPP生成增多的基础上，进一步观察MVA途径的干预是否可以改善5XFAD小鼠脑组织的氧化应激从而改善其认知功能。拟开展的研究包括：（1）在体状态（5XFAD/C57小鼠）和离体状态（神经元细胞培养）下抑制FPPS、GGPPS等酶的活性及表达从而降低FPP、GGPP水平，抑制FT、GGT等酶的活性及表达从而削弱小G蛋白的活化程度，观察抑制干预前后小鼠认知、记忆功能的改变、脑组织/神经元细胞形态学和凋亡率的变化、脑组织/神经元细胞ROS等氧化应激指标的改变、脑组织/神经元细胞Aβ含量的变化、小鼠海马脑片LTP电生理的变化；（2）观察离体状态下FPP或GGPP共孵育培养神经元细胞能否逆转抑制干预所产生的影响。旨在明确MVA途径下游相关的小G蛋白是否在AD发病机制中扮演MVA途径与氧化应激反应的“桥梁角色”的作用。

本项目以5XFAD小鼠为研究对象，从在体和离体状态水平研究了MVA途径干预对5XFAD小鼠认知功能和脑组织氧化应激反应的影响。法尼基焦磷酸合成酶/FPPS抑制剂阿仑膦酸钠干预对5XFAD小鼠认知功能以及小鼠脑组织Aβ、胆固醇含量均未产生显著影响，尽管干预组较对照组小鼠脑组织氧化应激损伤有一定程度改善。细胞水平阿仑膦酸钠对氧化应激损伤的抑制作用可部分被GGPP削弱。腺相关病毒载体Y4095/4096干预GGT基因改善了5XFAD小鼠的认知功能，小鼠脑组织Aβ含量降低，活性氧自由基含量降低，氧化应激损伤减少，Rac-1活性降低。5XFAD小鼠海马神经元细胞Y4095/4096共孵育降低了Aβ水平、减轻了氧化应激损伤、减弱了Rac-1活性，上述作用可被GGPP部分性逆转。.以5XFAD小鼠为研究对象的甲羟戊酸途径干预实验结果显示，GGT阻断干预能改善5XFAD小鼠的认知功能并减轻脑组织氧化应激损伤，提示GGT介导的代谢反应可能在AD发病机制中起着重要作用，GGPP下游的小G蛋白Rac-1可能是介导甲羟戊酸途径与氧化应激反应之间的“桥梁角色”。