Developmental Exposure To Nonylphenol Induced Axonal Injury Through The Activation Of Wnt-Dvl-GSK-3β Cascade In Vivo And In Vitro

Objective: Nonylphenol(NP),one of the persistent organic pollutants(POPs)is ubiquitous in our diets and environment.The central nervous system(CNS)is quite sensitive to POPs during perinatal period.Recently,increasing evidence indicates that developmental NP exposure causes learning and memory impairments.Axon is a basic and essential structure for learning and memory function.Thus,we investigated whether NP exposure during developmental period induced axonal injury and its effects on the development of axon in cultured neurons.Furthermore,we explored the relationship between change of Wnt-Dvl-GSK-3β cascade and impairment of the axon caused by NP exposure.Methods: 1.Pregnant rats were randomly divided into control and NP treatment groups.NP treatment groups were given NP daily by oral gavage at 10,50,100 mg/kg/day,respectively,from gestational day 0 till postnatal day 21(PN21).The control group received the corn oil vehicle.Bielschowsky silver staining and immunofluorescence staining were performed on the progeny hippocampus to observe axonal morphological changes.Immunoblotting assay was used to detect the levels of axon associated protein CRMP-2,Wnt,Dvl,and GSK-3β.2.Primary cortical neurons were prepared from newborn Wistar rats.Neurons were treated with a final concentration(0,30,50,70 μM)of NP for 24 h.Immunofluorescence staining was used to observe the axon morphology of neurons.FM1-43 loading and destaining were assessed to evaluate the axon function.Immunoblotting assay was used to detect the levels of CRMP-2,Wnt,Dvl,and GSK-3β.3.GSK-3β inhibitor SB-216763 was used to further analyze the role of Wnt-DvlGSK-3β cascade in axon impairments caused by NP.Immunofluorescence staining was used to observe the axon morphology of neurons,and the experiments of FM1-43 evaluated neuronal axon function.Western blotting was performed to detect the levels of CRMP-2.Results: Developmental exposure to NP reduced the number of axons in offspring hippocampus on PN21 and 80.Moreover,perinatal exposure to NP decreased microtubule-associated protein CRMP-2 levels and increased p‐CRMP-2 levels.Besides,NP exposure significantly decreased the levels of p-GSK-3β,Dvl and Wnt.In cultured neurons,NP significantly decreased the average length of neurites and the length of axon.FM dye uptake and release assay showed that synaptic vesicle recycling was damaged in NP treated neurons.NP increased the levels of p‐CRMP-2 and decreased the level of CRMP-2,p-GSK-3β,Dvl and Wnt in cultured neurons,which are consistent with the results in vivo.GSK-3β inhibitor SB-216763 reversed the altered levels of CRMP-2 and p-CRMP-2 caused by NP.Meanwhile,SB-216763 pretreatment significantly increased the length of neurites and recovered axonal functions in NP treated neurons.Conclusion: 1.Developmental NP exposure impairs axon development and activates Wnt-Dvl-GSK-3β cascade in offspring hippocampus.2.NP exposure causes axonal damage in primary cultured neurons,which were ameliorated by SB-216763 via inhibiting GSK-3β.3.The activation of Wnt-Dvl-GSK-3β cascade may contribute to the axonal damage caused by developmental NP exposure.