CRMP5 Interacts With Spastin To Release Microtubule Dynamics And Regulate Neurite Branching

Objective: The establishment of neural network is needed to pro vide material basis for the growth and development of neurite.Neurite growth is mainly controlled by cytoskeleton dynamics,which mainly depend on the polymerization and depolymerization of microtubules and microfilaments.The mechanisms of microtubules and microfilaments communication remain largely unknown.The microtubule severing protein Spastin and the microtubule-binding protein CRMP5 are involved in neurite outgrowth.We have demonstrated that Spastin interacts with CRMP5 and dtermined the binding domain.The main purpose of this study is to further clarify how Spastin cooperates with CRMP5 to regulate neurite growth and maturation by mediating cytoskeleton dynamics.Materials and methods: By using Spastin and C RMPs antibodies for immunofluorescence staining,we tried to reveal the colocalization between Spastin and CRMP5.SpartinM87 V and each truncated slice were respectively transfected into Hela cells and neurons,the change of microtubule in Hela cells and the development of hippocampal neuron neurites were observed by Laser Scanning Confocal Microscope.Similarly CRMP5 and each truncated isoforms were transfected into neurons to determine the effect on neurite.Furthermore,SpastinM87 V and CRMP5 were simultaneously overexpressed in Hela cells and neurons,and we also designed Spastin si RNA fragments to confirm the role between Spastin and CRMP5 in nurite development.In addition,SpastinM87 V and each truncated isoforms were overexpressed by calcium phosphate transfection,m EPSCs of these transfected neurons were recorded by using whole-cell recording to determine whether or not SpastinM87 V truncated isoforms affect the frequency and amplitude of m EPSC.Moreover,we overexpressed Spastin and CRMP5 simultaneously and recorded m EPSCs to analyze the variation among different groups.Results: 1)Immunocytochemistry showed that Spastin and CRMP5 colocalized in cell bodies of Hela,also in soma and neurites of neurons.2)Spastin^M87V,Spastin N190,Spastin?AAA,Spastin?N1 and Spastin?N2 were respectively transfected into Hela cells,the results revealed that microtubules were severed into small fragments by SpastinM87 Vand Spastin?N1.3)Spastin^M87V and each truncated isoforms were transfected into neurons by ca lcium phosphate transfection,the results showed that: SpastinM87 V,Spastin?N1 and Spastin?N2 could si-gnificantly promote the development of hippocampal neurons axons and dendrites.4)Whole-cell patch-clamp analysi-s results revealed that the frequency and amplitude of m EPSC in SpastinM87 V,Spastin ? N1 and Spastin ? N2 were significantly diferent from GFP group.5)The results of western-blot showed that Spastin si RNA can specifically interfere with the expressi-on of Spastin.Simultaneously,the immunostaning in cultured neurons demonstrated that si-Spastin can effectively inhibit the expression of endogenous Spastin and inhibit the development of neurites.6)CRMP5 and the truncated isoforms were transfected into neurons by calcium phosphate,the results showed that: CRMP5 and C RMP5?471 could significantly promote the development of hippocampal neuron.7)Spastin^M87V and CRMP5 were si-multaneously overexpressed in Hela cells,and CRMP5 overexpression did not affect the efficiency of microtubule severing by Spastin M87 V.8)Spastin^M87V and CRMP5 were si-multaneously overexpressed,overexpressed CRMP5 could enhance the effect of Spastin M87 V on neurites developments.9)In the neurons,the knockdown of Spastin or CRMP5 dramatically impaired neurite growth,and the knockdown of Spastin and CRMP5 simultaneously resulted in further impairment.Moreover,overexpression of CRMP5/ Spastin could rescued the inhibitory effect of si-Spastin/ si-CRMP5 on neurite growth.10)Whole-cell patch-clamp analysis results revealed that the frequency and a mplitude of m EPSC in Spastin^M87V+CRMP5,Spastin^M87V+m C herry and CRMP5+GFPwere significantly different from GFP group.Conclusions: 1)Spastin and C RMP5 colocalizes with each other in vivo.2)MTBD domain and AAA domain of Spastin play pivotal roles in severing microtubules.3)Overexpression of CRMP5 do not affect the efficiency of microtubule severing by Spastin M87 V.4)Spastin^M87V,Spastin ? N1 and Spastin ? N2 can significantly promote the development of neurites.5)CRMP5 and CRMP5?471 can significantly promote the neurite development of,and its interaction domain with Spastin is between 472-564 amino acid.6)Spastin and CRMP5 function collaboratively to regulate the development of neurites.7)Spastin and C RMP5 function collaboratively to regulate synaptic transmission.