基于齿状回神经元树突棘形态探讨ROCK2敲减改善APP/PS1小鼠认知功能的作用机制

Knockdown of ROCK2 improves cognitive function in APP/PS1 mice by protecting morphology of dendritic spines in dentate gyrus

目的:观察海马区敲减Rho相关激酶2(ROCK2)对阿尔茨海默病模型APPswe/PS1dE9(APP/PS1)转基因小鼠的干预效果,并探索其对海马齿状回区神经元树突棘形态的影响及潜在机制。方法:选用雄性8月龄的C57BL/6及APP/PS1小鼠,随机分为4组,分别为野生型(WT)组(8只)、APP/PS1小鼠生理盐水(NS)组(8只)、APP/PS1小鼠敲减对照(shRNA)组(12只)和APP/PS1小鼠ROCK2敲减(shROCK2)组(12只)。通过腺相关病毒共转染shRNA的方法,下调APP/PS1转基因小鼠双侧海马区神经元ROCK2蛋白表达,实现海马区局部神经元ROCK2敲减。利用水迷宫和Y迷宫实验检测小鼠的认知功能;免疫荧光染色和Western blot检测AD相关病理改变;激光共聚焦和Imairs软件观察并分析海马齿状回区神经元的树突棘形态变化;Western blot检测海马组织中突触相关蛋白的表达水平。结果:水迷宫实验中,与WT组的小鼠相比,NS和shRNA组小鼠到达平台的时间、到达平台的距离及第一次进入平台所在象限的时间均显著增加(P<0.05或P<0.01);敲减ROCK2可以显著降低APP/PS1小鼠到达平台的时间和距离及第一次进入平台象限的时间(P<0.05)。Y迷宫实验中,敲减ROCK2使APP/PS1小鼠在新异臂的时间占总时间的比例和自发交替率显著增加(P<0.01或P<0.05)。敲减ROCK2显著降低了APP/PS1小鼠脑组织中β-淀粉样蛋白和磷酸化τ蛋白水平(P<0.05或P<0.01)。shROCK2组小鼠海马齿状回神经元树突棘的长度和树突棘颈的平均长度较shRNA组显著增加(P<0.01),颈的平均直径显著减小(P<0.05)。与WT组相比,NC组及shRNA组小鼠海马区突触结合蛋白1(Syt1)、囊泡谷氨酸转运体1(VGLUT1)及突触后致密蛋白95(PSD95)的含量显著下调(P<0.01或P<0.05),shROCK2小鼠海马区的Syt1、VGLUT1和PSD95蛋白表达显著高于shRNA小鼠(P<0.05或P<0.01)。结论:海马齿状回区神经元ROCK2敲减可以显著改善APP/PS1小鼠的认知功能,调控海马齿状回区树突棘的形态,并有效抑制APP/PS1小鼠海马区兴奋性突触数量的减少。

AIM:To observe the therapeutic effect of hippocampal Rho-associated kinase 2(ROCK2) knockdown on the cognitive impairment in APPswe/PS1dE9(APP/PS1) transgenic mice, and to explore its underlying mechanisms. METHODS:Male 8-month-old C57BL/6 and APP/PS1 mice were selected and randomly divided into 4 groups:C57BL/6 control(wild-type, WT) group(n=8), APP/PS1 mice treated with saline(normal saline, NS) group(n=8), APP/PS1 mice treated with control short hairpin RNA(shRNA) group(n=12), and APP/PS1 mice treated with ROCK2shRNA(shROCK2) group(n=12). To achieve knockdown of ROCK2 in the hippocampal neurons, adeno-associated viral vectors co-expressing shRNA were used to specifically target ROCK2 in the hippocampal neurons of APP/PS1 transgenic mice. The Morris water maze test and Y-maze test were used to assess the cognitive function of the mice. Typical pathological changes associated with Alzheimer disease were detected by immunofluorescence and Western blot. Confocal microscopy and the software Imairs were used to analyze the dendritic spines of dentate gyrus(DG) neurons in shRNA and shROCK2 mice. The expression levels of synaptic proteins in the hippocampus were detected by Western blot. RESULTS:The time of latency to target, mean distance to the target, and latency of the first entrance to the southwest zone were increased in mice in the NS and shRNA groups when compared to the WT mice(P<0. 05 or P<0. 01), while the shROCK2 mice exhibited a significantly shorter time and distance to the platform and a shorter time of the first entrance to the SW zone when compared to the shRNA mice(P<0. 05). In the Y-maze test, knockdown of ROCK2 markedly increased the percentage of time spent in the novel arm and the spontaneous alternation rate of APP/PS1 mice(P<0. 05 or P<0. 01). Down-regulation of ROCK2 significantly inhibited the expression of amyloid β-protein and phosphorylated Tau protein in the APP/PS1 mice(P<0. 05 or P<0. 01). Compared with the shRNA mice, the shROCK2 mice exhibited an increased dendritic spine length, increased dendritic spine neck length, and decreased diameter of the dendritic spine neck in the DG of the hippocampus(P<0. 05 or P<0. 01). The expression levels of synaptotagmin 1(Syt1), vesicular glutamate transporter 1(VGLUT1), and postsynaptic density protein 95(PSD95) in the hippocampus of mice were significantly down-regulated in the NC and shRNA groups when compared with the WT group(P<0. 05 or P<0. 01), and the knockdown of ROCK2 markedly increased the levels of Syt1, VGLUT1, and PSD95 in the hippocampus of the APP/PS1 mice(P<0. 05 or P<0. 01). CONCLUSION:Knockdown of ROCK2 improves the cognitive function of APP/PS1 mice, regulates the morphology of dendritic spines in the DG, and effectively inhibits the reduction of the number of excitatory synapses in the hippocampus of APP/PS1 mice.