The Molecular Mechanism Of Neurite Outgrowth Regulated By Cereblon

Background and purposeMental retardation,also called intellectual disability,is a common disorder appearing before adulthood.The mutation of cereblon(CRBN),the substrate receptor of E3 ubiquitin ligase,is associated with mental retardation.A nonsense single nucleotide mutation(c.1255 C>T)in CRBN was discovered by a genetic screening for a group of patients with mild mental retardation.The mutation is called CRBN R419X,where the 419th amino acid,arginine,is replaced by a stop codon,causing a loss of the last 24 amino acids.A recent study also found that another mutant C391R(c.1171 T>C)can lead to intelligence developmental disorders.However,the molecular mechanism by which CRBN regulates mental retardation is still elusive.Through quantitative proteomics,we discovered that Nogo A was one of the most significantly downregulated proteins upon CRBN expression.Nogo A,a member of reticulon family,has inhibitory activity for axon extension.Therefore,we examine whether CRBN regulates the neurite outgrowth through Nogo A.Methods1.Overexpress CRBN and Nogo A in HEK293T cells,purify CRBN protein through immunoprecipitation,and then detect Nogo A through Western blotting.Pull down the endogenous Nogo A through immunoprecipitation and verify the interaction between CRBN and Nogo A by Western blotting;2.Treat HEK293T cells with MG132,and then pull down Nogo A to detect the ubiquitination level of Nogo A and investigate whether CRBN regulates the Nogo A protein level through the ubiquitin-proteasome pathway;3.Treat HEK293T cells with cycloheximide(CHX)to inhibit protein synthesis,detect Nogo A protein level under the regulation of CRBN in different time points to investigate whether CRBN regulates the half-time and stability of Nogo A;4.To verify the effect of CRBN on neurite outgrowth through expressing or knocking down CRBN and/or Nogo A in N2a cells and stimulating the neuronal differentiation;5.To build CRBN and its mutant stable expressed N2a cells line by lentivirus.Results1.CRBN interacts with Nogo A;2.CRBN wild type but not C391R can regulate Nogo A protein level through the ubiquitin-proteasome pathway;3.CRBN reduces the half-life of Nogo A and destablize Nogo A;4.CRBN overexpression enhances neurite outgrowth while Nogo A knockdown abolishes this effect;5.Nogo A knockdown eliminates the effect of CRBN on neurite outgrowth.ConclusionsCRBN,acting as a substrate receptor of CRL4-CRBN E3 ubiquitin ligase,regulates Nogo A protein level and stability by enhancing the Nogo A ubiquitination,thus affecting the neurite outgrowth.In this study,we first elucidated the regulatory relationship between CRBN and neurite outgrowth on the molecular level,which might provide important theoretical basis for further research and therapeutical intervention on mental retardation.