Homer1b/c“支架”在CTNND2-/-自闭症模型鼠中的作用研究

Role of scaffolding protein Homer protein homolog 1b and 1c in aCTNND2-/- mouse model of autism

目的:观察CTNND2-/-自闭症模型鼠前额叶皮层中支架蛋白荷马1b/c(homer protein homolog 1b and 1c,Homer1b/c)与三磷酸肌醇受体(inositol 1,4,5-trisphosphate receptor,IP3R)、代谢型谷氨酸受体5(metabotropic glutamate receptor 5,mGluR5)和香克3蛋白(sh3 and multiple ankyrin repeat domains 3,Shank3)蛋白相关复合体的变化;前额叶皮层中常见氨基酸的变化,发现自闭症模型鼠中可能参与疾病发生的关键靶点。方法:蛋白免疫印迹法(Western blot,WB)法检测CTNND2-/-自闭症模型鼠前额叶皮层中支架蛋白Homer1b/c、突触后密度蛋白-95(postsynaptic density protein 95,PSD-95)、突触素蛋白(synaptophysin,SYP)、囊泡谷氨酸转运体1(vesicular glutamate transporter 1,vGluT1)的表达的变化;通过免疫荧光(immunofluorescence,IF)观察Homer1b/c与IP3R、mGluR5和Shank3蛋白的表达与共定位;免疫共沉淀(co-immunoprecipitation,CO-IP)技术分别观察Homer1b/c与IP3R、mGluR5或Shank3结合的变化;使用液相色谱(liquid chromatography,LC)观察前额叶皮层中常见氨基酸的变化。结果:与对照组相比,CTNND2-/-模型鼠前额叶皮层中Homer1b/c(P=0.003)、PSD-95(P=0.003)以及SYP蛋白(P=0.046)的表达均明显降低;同时,Homer1b/c与IP3R、mGluR5、Shank3蛋白相互之间结合减少;前额叶皮层中常见的兴奋性神经递质谷氨酸(glutamate,Glu)和抑制性神经递质γ-氨基丁酸(γ-aminobutyric acid,GABA)的表达无明显变化(P=0.366,P=0.355),组氨酸(histidine,His)(P=0.036),酪氨酸(tyrosine,Tyr)(P=0.030)表达则明显增高。结论:CTNND2-/-自闭症模型鼠中Homer1b/c蛋白低表达,同时Homer1b/c与IP3R、Shank3、mGluR5蛋白复合物的形成减少,并推测低表达的Homer1b/c可能是导致自闭症神经元突触异常发育的关键靶点。

Objective:To observe the changes in scaffolding protein Homer protein homolog 1b and 1c(Homer1b/c),inositol 1,4,5-trisphosphate receptor(IP3R),metabotropic glutamate receptor 5(mGluR5),sh3 and multiple ankyrin repeat domains 3(Shank3)protein-related complexes,and common amino acids in the prefrontal cortex of CTNND2-/-mice with autism,and to investigate the key targets that may be involved in the development of the disease in mice with autism.Methods:Western blot(WB)was used to measure the changes in the protein expression levels of Homer1b/c,postsynaptic density-95(PSD-95),synaptophysin(SYP),and vesicular glu-tamate transporter 1(vGluT1)in the prefrontal cortex of CTNND2-/-mice with autism;immunofluorescent(IF)staining was used to investigate the expression and colocalization of Homer1b/c with IP3R,mGluR5,or Shank3 proteins;co-immunoprecipitation(CO-IP)was used to observe the binding of Homer1b/c to IP3R,mGluR5 or Shank3;liquid chromatography(LC)was used to analyze the changes in common amino acids in the prefrontal cortex of mice.Results:Compared with the control group,the CTNND2-/-model mice had significant reductions in the expression of Homer1b/c(P=0.003),PSD-95(P=0.003),and SYP(P=0.046)in the prefron-tal cortex and a significant reduction in the binding of Homer1b/c to IP3R,mGluR5,and Shank3 proteins,and there were no significant changes in the expression levels of the common excitatory neu-rotransmitter glutamate and the inhibitory neurotransmitterγ-aminobutyric acid in the prefrontal cortex(P=0.366 and 0.355),while there were significant increases in the expression levels of histidine(P=0.036)and tyrosine(P=0.030).Conclusion:There is low ex-pression of Homer1b/c protein in the CTNND2-/-mouse model of autism,and there is a reduction in the formation of Homer1b/c com-plexes with IP3R,Shank3,and mGluR5 proteins.It is speculated that low-expression Homer1b/c may be a key target for abnormal syn-aptic development in autism.