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 a CTNND2-/- mouse model of autism

摘要:

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。

Abstract:

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 glutamate 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 prefrontal 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 neurotransmitter glutamate and the inhibitory neurotransmitter γ-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 expression of Homer1b/c protein in the CTNND2^-/- mouse model of autism，and there is a reduction in the formation of Homer1b/c complexes with IP3R，Shank3，and mGluR5 proteins. It is speculated that low-expression Homer1b/c may be a key target for abnormal synaptic development in autism.