Effect Of Cul4b Deficiency On Neuronal Development

CUL4B,a member of Cullin family,is a component of Cullin 4B-Ring ubiquitin ligase complex(CRL4B complex).The CRL4B complex recognizes and targets various substrates for ubiquitination,by which it regulates a variety of physiological and developmental processes.Previously we found that mutation in CUL4B gene led to X-linked mental retardation.The clinical features of the patients include mental retardation,short stature,impaired speech,and increased monocyte counts in peripheral blood.Mice lacking of Cul4b in nervous system(Cul4bNestin-Cre)exhibited learning and memory defects,suggestting that CUL4B plays an important role in the development of nervous system.However,the mechanisms underlying mental retardation caused by mutations in CUL4B gene remain to be clarified.In this study,we examined the effect of depletion of CUL4B on the length and complexity of neuron dendrites,the density of neuron synapses,and the expression of nervous system development-related proteins.Part 1.Loss of Cul4b led to the abnormal dendrite morphology and reduced synapse densityTo investigate the effect of CUL4B deletion on the dendritic growth and branching,as well as the density of synapses,we crossed Cul4b floxed mice with Nestin-Cre transgenic mice to obtain nervous systerm specific Cul4b knockout mice(Cul4bNeslin-Cre mice)and littermate control mice,and isolated neurons from cerebral cortex of E18.5 Cul4bNestin-Cre mice and littermate control mice for in vitro culture.Immunostainning with neuron-specific markers(Neuro-ChromTM Pan Neuronal Maker)in DIV1,DIV2 and DIV4 neurons showed that the growth of neurites of Cul4b-deficient neurons was slightly slower than that of control neurons.After 14 days in vitro(DIV14)culture,the neurons were immunostained with dendritic specific marker MAP-2,and the total length of dendrites was analyzed using Image J software.We found that the total dendrite length of Cul4b-deficient neurons was significantly shorter than that of control neurons.Then Sholl analysis software was used to analyze the branches of dendrites.The results showed that the branches of dendrities of Cwl4b-deficient neurons was significantly reduced when compared with control neurons,suggesting that CUL4B may affect the dendritic morphology of neurons.In order to determine whether the synaptic density of neurons was altered by loss of Cul4b,we used immunofluorescence assays to detect the expression of four synapse-associated proteins Synapsin1,PSD95,GAD67 and Gephyrin,and quantified the positive synapses within 100?m dendrite from soma.The densities of synapses with Synapsin1,PSD95,GAD67 and Gephyrin,were all reduced in Cul4b-deficient neurons,and the colocalization of Synapsin1 with PSD95,and the colocalization of GAD67 with Gephyrin were also reduced in Cul4b-deficient neurons,suggesting that CUL4B may affect synapses formation.The above results suggest that mental retardation in CUL4B mutants may be attributed to the abnormal dendritic morphology and decreased synaptic density of neurons.Part 2.Loss of Cul4b resulted in the reduced NEFM expression in forebrain of newborn miceWe previously found that the expressions of some nervous system development-related proteins were changed after knockout of Cul4b gene,part of which were associated with synapse formation or synaptic vesicles.We extracted proteins from forebrains of nervous system specific Cul4b knockout mice(Cul4bNestin-Cre mice)and littermate control mice at newborn(PO),developmental stages(2 weeks and 1 month)and adulthood(2 months),and determined the expression levels of NEFM,NEFL,SNAP-25,Synaptotagmin-1,STXBP1,VTI1B and Drebrin by Western blot assay.The results showed that compared with the control mice,the expression of NEFM in the forebrain tissue of PO Cul4b knockout mice was significantly reduced,while there was no significant change in the expression of other nervous system development-related proteins.Interestingly,the reduced NEFM expression in Cul4b knockout mice was only observed in the forebrain tissue of neonatal mice,but not in the hippocampus,cortex and corpus callosum in developmental and adult mice,suggesting that the effect of CUL4B deletion on NEFM expression only occurs in mouse embryonic developmental stage.We then verified the above results in newborn neuron-specific Cul4b knockout mice(Cul4bNse-Cre mice),and the expression of NEFM in Cul4b-deficient primary cultured neurons was also reduced.In addition,we extracted RNA from forebrain of neonatal(PO day)Cul4bNestin-Cre mice and littermate control mice,and detected mRNA expression of NEFM by real-time quantitative PCR.We found that the mRNA level of NEFM was not decreased after Cul4b deletion,suggesting that CRL4B regulates NEFM expression may be at the post-transcriptional level.