肾脑蛋白介导的突触可塑性下降在慢性脑低灌注认知功能障碍中的作用及机制

Role and mechanisms of synaptic plasticity decrease mediated by KIBRA in cognitive dysfunction by chronic cerebral hypoperfusion

目的探讨肾脑蛋白(kidney brain protein, KIBRA)下调在慢性脑低灌注所致认知功能障碍中的作用和机制。方法 90只雄性SPF Sprague Dawley (SD)大鼠, 按照随机数字表法分为四组:假手术组(n=15)、慢性脑低灌注组(2VO组, n=25)、慢性脑低灌注脑立体定位注射AAV-KIBRA组(2VO+AAV-KIBRA组, n=25)、慢性脑低灌注脑立体定位AAV-vector组(2VO+AAV-vector组, n=25)。采用双侧结扎颈总动脉方法建立慢性脑低灌注模型, 脑立体定位注射2 μL AAV-KIBRA或AAV-vector, 观察30 d。利用Morris水迷宫、离体电生理、p21激活的蛋白激酶3(p21-activated kinase 3, PAK3)酶活性检测、蛋白印迹、免疫共沉淀和Golgi染色方法, 分别检测大鼠的空间学习记忆能力、长时程增强(long-term potentiation, LTP)、KIBRA水平表达、PAK3酶活性的变化, 并观察树突棘的分布。用SPSS 16.0统计软件分析实验数据, 采用单因素方差分析(one-way ANOVA)比较各组间差异, 采用LSD检验进行两组间数据差异显著性比较, 方差不齐则采用Welch检验。结果大鼠慢性脑低灌注1月后, 取大鼠海马进行匀浆和蛋白印迹检测KIBRA水平, 发现2VO组KIBRA水平较假手术组明显下降[(73.49±4.12)%](P<0.01), 海马CA1区注射AAV-KIBRA显著上调KIBRA水平[(91.91±7.01)%](P<0.01)。Morris水迷宫检测发现:2VO组大鼠在7 d的平台位置学习训练中的潜伏期[第3~7天学习结果:(48.18±2.82)s、(43.45±2.27)s、(32.27±2.22)s、(26.55±2.37)s、(17.18±2.67)s]显著长于假手术组[(41.67±2.74)s、(32.58±2.57)s、(22.50±2.94)s、(16.91±2.39)s、(8.75±1.52)s](均P<0.05), 2VO+AAV-KIBRA组在7 d的平台位置学习潜伏期[第3~7天分别为:(43.83±2.95)s、(35.25±2.15)s、(26.58±2.03)s、(19.92±2.17)s、(17.75±1.35)s]显著短于2VO组(均P< 0.01)。撤除平台的Morris水迷宫检测, 短期记忆结果显示2VO组大鼠达到平台区域的潜伏期显著长于假手术组, 而2VO+AAV-KIBRA组大鼠到达平台潜伏期显著短于2VO组(P<0.01)。同时2VO大鼠的平台区域滞留时间和穿梭次数均少于假手术组(P<0.01), 而2VO+AAV-KIBRA组的平台区域滞留时间和穿梭次数则显著多于2VO组(P<0.01)。离体脑片电生理记录显示:高频刺激后, 2VO组相对兴奋性突触后场电位(1.43±7.43)显著低于假手术组(2.21±6.54), 而2VO+AAV-KIBRA组相对兴奋性突触后场电位(1.90±8.15)高于2VO组(P<0.01)。大鼠海马组织进行免疫共沉淀发现沉淀KIBRA时, 蛋白免疫印迹实验可以检测到PAK3。PAK3酶活性检测结果显示:2VO海马组织PAK3的相对酶活性(0.64±0.04)显著低于假手术组(1.02±0.07), 而2VO+AAV-KIBRA组PAK3的相对酶活性(0.86±0.03)显著高于2VO组。Golgi染色显示:2VO海马神经元树突棘密度[(6.85±0.43)个/10 μm]显著低于假手术组[(11.83±0.58)个/10 μm], 而2VO+AAV-KIBRA组神经元树突棘密度[(10.22±0.39)个/10 μm]显著高于2VO组。结论慢性脑低灌注后KIBRA下降在认知功能障碍中发挥重要作用, 与突触功能可塑性下降有关, KIBRA下调通过调控PAK3活性而参与树突的结构可塑性。因此, KIBRA可能是防治慢性脑低灌注认知功能的重要靶点。

Objective To explore the role and mechanism of kidney brain protein(KIBRA)down-regulation in cognitive dysfunction caused by chronic cerebral hypoperfusion.Methods Ninety male SPF grade Sprague Dawley(SD)rats were divided into four groups according to random number table:sham operation group(n=15),chronic hypoperfusion group(2VO group,n=25),chronic hypoperfusion stereotaxic injection of AAV-KIBRA group(2VO+AAV-KIBRA group,n=25),chronic hypoperfusion stereotaxic injection of AAV-Vector group(2VO+AAV-vector group,n=25).Chronic cerebral hypoperfusion model was established by bilateral ligation of common carotid artery,and stereotactic injection of 2μL AAV-KIBRA or AAV-vector was performed for 30 days.Morris water maze,in vitro electrophysiology,p21-activated kinase 3(PAK3)activity detection,Western blot,immunoprecipitation and Golgi staining were used to detect spatial learning and memory ability,long-term potentiation(LTP),KIBRA level expression,PAK3 activity changes and the distribution of dendritic spines.SPSS 16.0 statistical software was used for statistical data.One-way ANOVA was used to compare the differences between groups.LSD test was used to compare the significance of data differences between the two groups.Welch test was used for uneven variance.Results After 1 month of chronic cerebral hypoperfusion,the level of KIBRA in the hippocampus of rats was detected by homogenate and Western blot,and it was found that the level of KIBRA in 2VO group was lower than that of sham group(73.49±4.12)%(P<0.01).AAV-KIBRA injection in hippocampal CA1 region significantly up-regulated the level of KIBRA to(91.91±7.01)%over 2VO group(P<0.01).Morris water maze test showed that the latency of the 2VO group(3rd-7th day trail data:(48.18±2.82)s,(43.45±2.27)s,(32.27±2.22)s,(26.55±2.37)s,(17.18±2.67)s)were significantly longer than those of the sham group((41.67±2.74)s,(32.58±2.57)s,(22.50±2.94)s,(16.91±2.39)s,(8.75±1.52)s)(all P<0.05),and the latencies of the 2VO+AAV-KIBRA group 3rd-7th day trail data:(43.83±2.95)s,(35.25±2.15)s,(26.58±2.03)s,(19.92±2.17)s,(17.75±1.35)s)was significantly shorter than that of the 2VO group((all P<0.01).The Morris water maze test with the platform removed showed that the latency of rats in the 2VO group to reach the platform region was significantly longer than that of the sham group,while the latency of rats in the 2VO+AAV-KIBRA group to reach the platform region was significantly shorter than that in the 2VO group(P<0.01).At the same time,the retention time and the crossing times in the platform region of 2VO group were less than those of the sham group(P<0.01),but the retention time and the crossing times in the platform region of 2VO+AAV-KIBRA group were significantly higher than those in the 2VO group(P<0.01).The electrophysiological records of the brain slices showed that the relative excitatory postsynaptic field potential of 2VO group(1.43±7.43)was significantly lower than that of sham group(2.21±6.54)after high frequency stimulation,while the relative excitatory postsynaptic field potential of 2VO+AAV-KIBRA group(1.90±8.15)was higher than that of 2VO group(P<0.01).Immunoprecipitation in rat hippocampus revealed that PAK3 could be detected by Western blot assay when KIBRA was precipitated.The results showed that the relative enzyme activity of PAK3 in 2VO hippocampal tissue(0.64±0.04)was significantly lower than that in sham group(1.02±0.07),while the relative enzyme activity of PAK3 in 2VO+AAV-KIBRA group(0.86±0.03)was significantly higher than that in 2VO group.Golgi staining showed that the density of dendritic spines in 2VO hippocampal neurons((6.85±0.43)/10μm)was significantly lower than that in sham group((11.83±0.58)/10μm),while the density of dendritic spines in 2VO+AAV-KIBRA group((10.22±0.39)/10μm)was significantly higher than that in 2VO group.Conclusion The down-regulated of KIBRA after chronic cerebral hypoperfusion plays a key role in cognitive dysfunction and is also involved in the decrease of synaptic functional plasticity.The downregulation of KIBRA is involved in the structural plasticity of dendrites through the regulation of PAK3 activity.Therefore,KIBRA may be an important target for the prevention and treatment of cognitive function of chronic cerebral hypoperfusion.