Effect Of KLF4 In ASD-like Behaviors And Its Potential Mechanisms

Background:Autism(ASD)refers to a dysfunctional disease with persistent social interaction impairments and repetitive stereotyped behavioral patterns which start from infant stage ASD is a neurodevelopmental disorder.The pathogenesis of ASD is complicated,which can be attributed to genetic factors,environmental factors,and their interactions.Evidence suggests that local neural network hyperfunction caused by abnormal integration of neural networks,abnormal activation of glial cells,and imbalance of excitatory/inhibitory ratio of synaptic transmission is strongly associated with the development of ASD.In particular,synaptic transmission dysfunction is a key neurophysiological change.The VPA model is a widely used pharmacological model for ASD-related basic and preclinical research for ASD pathogenesis and evaluation of drugs.VPA,as a non-selective deacetylation inhibitor(HDACi),can affect neurological development during gestation and early development,possibly by affecting the expression of key genes related to synaptic function,causing abnormal integration of neural networks and leading to local neural network hyperfunction.However,the mechanism by which the VPA model causes ASD-like behavioral phenotypes in the offspring is not fully understood,especially based on different brain regions in the regulation of ASD-like behavioral phenotypes still needs to be further investigated.KLF4(Kruppel-like factor 4)is a eukaryotic zinc finger protein transcription factor,which is a member of the Kruppel-like transcription factor protein family.It has been reported that KLF4 together with three transcription factors,including Sox2,Oct4 and c-Myc,can induce the transformation of mouse embryonic or adult fibroblasts into pluripotent stem cells(Ips)under vitro embryonic stem cell culture conditions.This suggests that they play an important regulatory role in embryonic development and cell proliferation and differentiation.KLF4 can promote dendritic spinogenesis and affect axonal regeneration by binding to the phosphorylation site of stat3 and inhibiting stat3-mediated downstream transcriptional activity,suggesting that stat3 is a key downstream target factor for KLF4 to play a regulatory role in the nervous system.These results suggest that KLF4 may be involved in the neurophysiological regulation of neurodevelopmental diseases.However,there are no reports on the relationship between KLF4 and ASD,especially on its regulatory role in prenatal VPA exposure-induced offspring ASD models.Based these results,this present study aimed to investigate the role of KLF4 in prenatal VPA exposure-induced ASD models and Effect of KLF4 in ASD-like behaviors and its potential mechanisms.Objective:To validate the face validity of VPA model and to explore the possible neurophysiological mechanisms of social impairment in the VPA model by studying the social activity-induced neuronal activity changes in the medial prefrontal cortex(mPFC)and hippocampal(HP);to explore the regulatory role of KLF4 in VPA model and the effect of KLF4 in mPFC on ASD-like behaviors and the possible molecular mechanisms.Based on the above studies,we aim to provide a complement to the mechanisms of ASD-like behaviors,especially the social impairments,and to provide a possible molecular target for study of ASD pathogenesis and screening of intervention drugs.Methods:Construct VPA models by giving a single 500 mg/kg dose of VPA or saline intraperitoneally injection to pregnant rats at embryonic day 12.5(E12.5),and the open field test,marble burying test and three-chamber test were performed on the VPA model until P21,respectively.We compared the c-Fos(a protein marker for neuronal activation)expression in different subsites of mPFC and HP after social activities by immunohistochemical staining to explore the possible neurophysiological mechanisms underlying the development of social impairment in the VPA model.The expression of KLF4,Sox2,Oct4 and c-Myc were test by immunoblotting in P21 offspring rats.The neuronal protein marker(Neun),astrocyte protein marker(GFAP)and microglia protein marker(Iba1)in mPFC were test by immunofluorescence staining at P1,P7,P21 and P35.Animal models of mPFC with low and high KLF4 expression were constructed by brain stereotaxic targeting of mPFC injected with lentivirus-mediated interference/overexpression KLF4 vector,the ASD-like behavioral phenotypes of mice were explored by open field test,nest building test,marble burying test and three chamber test.The expression of KLF4 and pstat3/stat3 were verified by immunoblotting;the expression of Neun,GFAP and Iba1 were investigated by immunoblotting and immunofluorescence staining,and the changes in the expression of excitatory and inhibitory synapse-associated proteins were measured by immunoblotting assays.Results:1.Social activity stimulated significantly less IL cFos expression in mPFC if VPA models than SAL group,while no significant difference in c-Fos expression was observed when comparing the CA1 CA3 and DG nuclei of HP in VPA and SAL group,suggesting that prenatal VPA exposure causes social impairment by disrupting the ability of mPFC neurons to respond to social stimulation and signals.2.KLF4 expression was significantly increased in the P21 offspring of VPA models,but there was no such trend in HP,Stria and Amyg,nor in the expression of Sox2,c-Myc.P21 was the key time point for the increase of Iba1 and GFAP expression in the mPFC of VPA model.VPA group showed higher GFAP expression levels than SAL group at P35,while there was no such trend for Iba1.It suggests that KLF4 of mPFC may be involved in the development of prenatal VPA exposure-induced ASD in the offspring by affecting glial cell expression and activation.3.Abnormal mPFC KLF4 expression levels can induce social impairments in mice.mPFC KLF4 overexpression inhibited the socialization-induced increase in IL c-Fos expression.mPFC KLF4 overexpression upregulated the expression of Iba1 and GFAP,and interference with mPFC KLF4 expression downregulated the expression of Iba1 and GFAP.mPFC KLF4 expression levels were correlated with the expression of Iba1 and GFAP.mPFC KLF4 overexpression inhibited the expression of Glu A1 and upregulated the expression levels of GAD65 and GABAA ?1.Conclusions;1.Prenatal exposure to VPA causes social impairment by disrupting the responsiveness of mPFC neurons to social stimulation and related signals.2.mPFC KLF4 may be involved in the development of ASD in offspring induced by VPA prenatal exposure by affecting glial cell expression and activation.3.mPFC KLF4 can regulate social behavior.mPFC.KLF4 overexpression attenuates the ability of IL neurons to respond to social stimulation and related signals.mPFC KLF4 upregulates Iba1 and GFAP,inhibits Glu A1 expression,upregulates GAD65 and GABAA ?1 expression levels,and thus disrupts excitatory/inhibitory transmission.