Zfp462 Deficiency Causes Anxiety-like Behaviors In Mice

Objective:To investigate whether Zfp462 heterozygous(Zfp462 Het)mice have anxiety phenotype and further explore the possible pathogenesis of anxiety phenotype.Methods:Western blot analysis was used to determine the protein expression levels of Zfp462 in different tissues and different brain regions of mice and to detect the protein expression levels of Zfp462 and Pbxl in different developmental stages of mice.Western blot analysis was used to determine the protein expression levels of Zfp462 in brain tissues of Zfp462 knockout(Zfp462 KO)mice and control mice(Zfp462 WT).RT-PCR was used to detect the mRNA expression level of Zfp462 in Zfp462 KO mice and Zfp462 WT mice.Similarly,Western blot method was used to measure the protein expression level of Zfp462 and the anxiety-related gene Pbxl in the brain tissues of Zfp462 Het mice and Zfp462 WT mice.Q-PCR method was used to measure mRNA expression of Zfp462 and related genes Pbx1,Hoxb8 and Meis2 in the brain tissues of Zfp462 Het mice and Zfp462 WT mice.By immunofluorescence staining,the neural tube patterning and the protein level of Pbxl were detected.By CO-immunoprecipitation(Co-IP),the interaction between Zfp462 and Pbxl was detected.By Open field test,Elevated plus maze test,Light-dark box test,Marble burying test and other behavioral tests validate animal model to detect Whether the Zfp462 Het mice have anxiety phenotype.To observe whether there have excessive self-grooming behaviors in Het Zfp462 mice by self-grooming test.By the Water maze test,the Rotarod test,the Tail suspension test(TST),the Forced swimming test(FST)and other behavioral tests validate animal model to detect Whether the Zfp462 Het mice have learning and memory disorders,motor disorders and depressive-like behaviors.By measuring the body weight at different time points,Zfp462 Het mice were observed whether there were developmental disorders.To study the effect of anti-anxiety drugs on the treatment of anxiety-like behavior in Zfp462 mice,Zfp462 Het mice and Zfp462 WT mice were given daily intraperitoneal injection of IM(20mg/kg weight)and the same dose of normal saline.After three weeks treatment.the changes of behaviors and weight were detected.Results:The results of Western blot showed that the protein expression of Zfp462 in the mouse brain was higher,the cortical and hippocampal areas expressed highest in the brain.The results of Western blot and RT-PCR showed that Zfp462 KO mice did not have the protein and mRNA expression of Zfp462 compared with Zfp462 WT mice.Take mouse embryos.Zfp462 KO embryos were found dead with open eyes before embryonic day 18.5(E18.5).The results of Western blot showed that the protein expression of Zfp462 and anxiety-related gene Pbxl in Zfp462 Het mice was decreased compared with WT mice(P<0.05).Q-PCR results showed that the mRNA expression levels of Zfp462 and anxiety-related genes Pbxl.Hoxb8 in Zfp462 Het mice were decreased compared with WT mice(P<0.05).but there was no significant difference between the mRNA expression level of Meis2(P>0.05).The results of Immunofluorescence staining showed that the protein expression of Pbxl was decreased in Zfp462 Het mice.Take Zfp462 KO mice,Zfp462 Het mice and Zfp462 WT mice at E10.5 for immunofluorescence staining,their neural tubes patterning were normal.The results of Co-IP showed that there was a significant interaction between Zfp462 protein and Pbxl protein.The results of the Open field test,Elevated plus maze test,Light-dark box test,Marble burying test showed that Zfp462 Het mice have obvious anxiety-like behaviors(P<0.05).The results of the Water maze test,the Rotarod test,the Tail suspension test,the Forced swimming test(FST)and other behavioral tests validate animal model showed that Zfp462 Het mice did not have learning and memory disorders,motor disorders and depressive-like behaviors(P>0.05).By measuring the body weight,Zfp462 Het mice were observed developmental disorders compared with Zfp462 WT mice.Through the observation.Zfp462 Het mice have hair loss with the growth of age.After imipramine treatment for 3 weeks,we found that anxiety-like behaviors and excessive self-grooming behaviors in Zfp462 het mice were attenuated,but body weight have no significant change.Conclusions:Zfp462 KO caused prenatal death in mice but no neural tube defects.Zfp462 deficiency causes anxiety-like behaviors with excessive self-grooming in mice and developmental delay.In Zfp462 Het mice,the expression of Zfp462 was decreased in the anxiety related to orbital cortex,amygdala and other brain regions,the related genes Pbxl and Hoxb8 also decreased,which may be the cause of anxiety-like behaviors.Imipramine treatment can alleviated the anxiety phenotype,therefore,Zfp462 Het mice is an ideal model for the study of anxiety mechanisms and can be used for screening of anti-anxiety drugs.