突触黏附分子NECL4对大脑皮质Ca^2+-CaMKII-CDC42信号通路的影响

Effect of synaptic adhesion molecule NECL4 on Ca^2+-CaMKII-CDC42 signal pathway in cerebral cortex

目的探究突触黏附分子NECL4对Ca^2+-CaMKII-CDC42信号通路和树突棘数量的影响。方法以Necl4全身性敲除小鼠为研究对象,对8周龄的Necl4野生(WT)及敲除(KO)小鼠脑组织进行高尔基染色,对大脑皮质第Ⅱ/Ⅲ层椎体神经元二级树突上树突棘密度进行统计。通过Western blot和RT-qPCR实验对Ca^2+-CaMKII信号通路及其下游信号分子的蛋白和mRNA表达水平进行检测,包括CDC42、Rac1、RhoA、H-Ras、ERK等信号蛋白。结果高尔基染色结果显示,Necl4敲除小鼠大脑皮质第Ⅱ/Ⅲ层椎体神经元二级树突上树突棘数量与野生型小鼠相比减少(P<0.01)。Western blot实验结果显示CaMKIIα(P<0.05)及phospho-CaMKIIα(Thr286)(P<0.05)在Necl4敲除小鼠中表达降低,CaMKIIα的下游信号分子CDC42的mRNA水平(P<0.05)和蛋白水平(P<0.05)在Necl4敲除小鼠中均表达降低。结论NECL4通过Ca^2+-CaMKII-CDC42信号通路对小鼠大脑皮质树突棘数量进行调控。

Objective To investigate the effect of synaptic adhesion molecule nectin-like molecule 4(NECL4)on Ca^2+-CaMKII-CDC42 signal pathway and the number of dendritic spines.Methods Using Necl4 knockout mice as the research animal,the brain tissues of 8-week-old Necl4 wild type(WT)and knockout(KO)mice were stained with Golgi staining,then the density of dendritic spines on the secondary dendrites in layer Ⅱ/Ⅲ of cerebral cortex neuron was examined.Western blot and RT-qPCR were used to detect the protein and mRNA expression of Ca^2+-CaMKII signal pathway and downstream signal molecules,including CDC42,Rac1,RhoA,H-Ras,ERK and other signal proteins.Results The Golgi staining showed that the number of dendritic spines on the secondary dendrites of the cerebral cortex Ⅱ/Ⅲ layer neuron in Necl4 knockout mice was less than that in wild type mice(P<0.01).The results of Western blot showed that the expression of CaMKIIα(P<0.05)and phospho-CaMKIIα(Thr286)(P<0.05)decreased in Necl4 knockout mice.The mRNA(P<0.05)and protein(P<0.05)levels of CDC42 were decreased in Necl4 knockout mice.Conclusions NECL4 regulates the number of dendritic spines in mouse cerebral cortex through Ca^2+-CaMKII-CDC42 signal pathway.