Dysregulation Of Wnt/β-catenin Signaling Pathway And Related Molecular Mechanisms In Fragile X Syndrome

Background Fragile X syndrome(FXS)is the most common autism spectrum disorder(ASD)single gene mutation pathogenic factor,an inherited intellectual disability caused by the deficiency of fragile X mental retardation protein FMRP(the protein encoded by the Fmr1 gene).The disease is characterized by the impairment of cognitive function and social communication.The Wnt/β-catenin signaling pathway is involved in the regulation and remodeling of neural synapses,maintains synaptic morphology and function,and plays an important role in learning and memory.Studies have shown that GSK3β,a key component of Wnt signaling,is abnormally activated in FXS model mice,and inhibition of GSK3β improves cognitive and behavioral deficits in FXS mice.Proteomic and genomic analysis found that abnormal expression of Wnt signaling components and cell adhesion function were found in ASD patients or model mice,which may be related to the dysregulation of Wnt/β-catenin signaling pathway.However,the expression and function of canonical Wnt signaling central protein β-catenin in FXS is unclear,and the role and molecular mechanism of Wnt/β-catenin signaling pathway in FXS is unclear.Objective The purpose of this study was to investigate the role and molecular mechanism of Wnt/β-catenin signaling in FXS during the critical period of development.Methods WT and Fmr1 KO mice were used as FXS mice model.Western blot was used to detect the key proteins of Wnt/β-catenin signaling pathway and their phosphorylation levels in the prefrontal cortex and hippocampus.Cell fractions were separated to detect the subcellular distribution of β-catenin and Active β-catenin;the co-localization of β-catenin and N-cadherin was detected by immunofluorescence.Taking Fmr1 knockdown HT22 and N2 a cells as the research objects,the key proteins of Wnt/β-catenin signaling pathway and their phosphorylation expression levels were detected by Western blot.Taking primary cortical neurons of Fmr1 KO mice as the research object,the key proteins of Wnt/β-catenin signaling pathway and their phosphorylation expression levels were detected by Western blot;the expression of Wnt target genes and related genes of presynaptic and postsynaptic protein were detected by q RT-PCR;The co-localization of β-catenin and N-cadherin was detected by immunofluorescence;the branching and length of neuronal dendrites were detected by MAP2 staining.Results In the prefrontal cortex and hippocampus of Fmr1 KO mice,the expression of key proteins in the Wnt/β-catenin signaling pathway was abnormal,subcellular localization of β-catenin and Active β-catenin was abnormal,co-localization of β-catenin and N-cadherin was decreased;Abnormal expression of key proteins in Wnt/β-catenin signaling pathway were observed in Fmr1 knockdown HT22 and N2 a cells;In primary cortical neuron of Fmr1 KO mice,the expression of key proteins in Wnt/β-catenin signaling pathway was abnormal,the m RNA expression levels of Wnt target genes and presynaptic and postsynaptic proteins are abnormal,and the branches and length of neuronal dendrites are reduced.The activation of Wnt signaling pathway has a corrective effect.Conclusion The expressions of Wnt/β-catenin signaling pathway key proteins are abnormal,the expression and subcellular localization of β-catenin are abnormal,and the expression of β-catenin-N-cadherin complex are abnormal in Fmr1 KO mice,Wnt/β-catenin signaling pathway is dysregulated,which affects the expression of synapse-related proteins and neuron morphology.Our study will provide theoretical basis and molecular targets for the diagnosis and treatment of FXS.