Effects Of SUMOylation Modification On Plasticity Of Phosphorylated CRMP2-mediated Dendritic Spine

Objective: 1.To investigate the effects of phosphorylated and non phosphorylated CRMP2 on neurite outgrowth,dendritic spines formation and electrophysiology in hippocampal neurons.2.To investigate the effects of SUMOylation and de SUMOylation CRMP2 on dendritic spine formation and electrophysiological regulation of hippocampal neurons.3.To investigate the effects of phosphorylated CRMP2 whatever is modified by SUMOylation on plasticity of dendritic spine in hippocampal neurons.Methods: Construction of dephosphorylation CRMP2 GFP-T514 D and dephosphorylation of CRMP2 GFP-T514 A plasmid.Primary hippocampal neurons culture was made when a SD rat has been postnatal first day.Cultured hippocampal neurons to the 8-10 th days,using calcium phosphate transfection technology transfected the plasmid GFP,CRMP2,GFP-T514 D and GFP-T514 A into the primary hippocampal neurons.When hippocampal neurons was cultured continuely to the 13 th days,the collected cell were used to make cellular immunochemistry,and the photographs about neurite outgrowth were snaped with the upright fluorescence microscope,and the dendritic spines,the Glu A1 and the PSD95 potos were obtained by making use of the confocal microscope.Moreover,we also examined the electrophysiological properties of the transfected neurons by using patch clamp technique.In the same way,CRMP2,HA-SUMO1,Ubc9 and de SUMOylation CRMP2 K374 A were transferred into hippocampal neurons.Confocal laser was used to detect dendritic spine morphology.We also detected the m EPSC of this transfected neuron using patch clamp technique.Results: 1.The effect of phosphorylated CRMP2 on neurite outgrowth:CRMP2,nonphosphorylated CRMP2 T514 A promotes neurite outgrowth,but the phosphorylated CRMP2 T514 D do not play a role in neurite growth.2.The regulation of phosphorylated CRMP2 on dendritic spines: CRMP2,non-phosphorylated CRMP2 promotes the formation and maintenance of dendritic spines and enhances the amplitude and frequency of m EPSC,and accumulate the expression of PSD95 and Glu A1.But phosphorylated CRMP2 T514 D has no effect on dendritic spines.3.The regulation of SUMOylation CRMP2 on dendritic spines: SUMOylation CRMP2 reduce the amplitude of m EPSC.But the de SUMOylation of CRMP2 promotes the formation and maintenance of dendritic spines and strengthen the amplitude and frequency of m EPSC.4.The regulation of phosphorylation CRMP2 on dendritic spines does not depend on SUMOylation modification.Regardless of CRMP2 514 amino acid phosphorylation or not,can be SUMO modified,so that the amplitude of m EPSC decreased;Regardless of the phosphorylation of amino acid at position 514 of CRMP2,the amplitude and frequency of m EPSC can be increased in the SUMOylation modification.Conclusions: 1.CRMP2 and non-phosphorylated CRMP2 T514 A do promote neurite outgrowth.2.Non-phosphorylated CRMP2 promotes the formation and maturation of dendritic spines 3.De SUMOylation of CRMP2 to promote the formation and maturation of dendritic spines 4.Non-phosphorylated CRMP2 promotes the formation and maturation of dendritic spines that do not depend on SUMO modification.