Spastin Phosphorylation And SUMO Ylation Together Regulate The Trafficking Of AMPA Receptor GluA2 Subunits

Objective:To investigate that how the phosphorylation and SUMOylation modifications of Spastin regulate the morphology and function of dendritic spines in hippocampal neurons and the trafficking of AMPA receptor GluA2 subunits.Methods : Construction of SUMO-deficient mutant Spastin-K427 R prokaryotic and eukaryotic expression vector plasmids;first,identify the SUMOylation sites by Co-IP assay,overexpress in COS-1 cells to identify the target protein microtubule cutting function;Over-expression of Spastin-K427 R in hippocampal neurons to detect the neuronal dendritic spines morphology and synaptic function changes by confocal imaging and whole-cell patch-clamp,and identify changes of GluA2 on neuronal membranes by immunocytofluorescence staining;finally,GST Pull-down and Co-IP experiments were used to clarify the interaction between Spastin-K427 R and GluA2 and its binding sites.Similarly,we construcedt the dephosphorylated Spastin-210A-K427 R,pseudo-phosphorylated Spastin-210D-K427 R prokaryotic and eukaryotic expression vector plasmids;firstly,GST Pull-down and Co-IP experiments were used to identify the interaction between phosphorylated Spastin-K427 R and GluA2;secondly,over-expression of phosphorylated mutants in primary cultured hippocampal neurons,and the effect of Spastin-K427 R on the change of the number of GluA2 on the neuron membrane after phosphorylation modification was clarified by immunocytofluorescence staining;finally,we confirmed that Spastin-K427 R altered the synaptic function of neurons after phosphorylation modification through whole-cell patch clamp detection.Result:1.Overexpression of Spastin-K427 R in COS-1 cells lines.The results of cellular immunofluorescence showed that the de-Sumoylation Spastin mutant lost its microtubule severing function.Overexpression of the de-SUMO mutant Spastin-K427 R in cultured primary hippocampal neurons.Confocal images showed dendritic spines and whole cell patch clamp experiments to detect AMPAR-mediated mEPSC.The results showed that the density of dendritic spines and the proportion of mature dendritic spines increased,and the amplitude and frequency of mEPSC increased significantly(P <0.05).The immunocytofluorescence labelled the GluA2 subunits on neuronal membrane.After overexpression of the de-SUMO mutant,the GluA2 fluorescence intensity on the cell membrane increased significantly(P <0.05).2.Through GST Pull-down and Co-IP experiments,we found that Spastin and GluA2 interacted directly in vitro and in vivo.The combination of Spastin-K427 R and GluA2 was significantly increased(P <0.05).The MIT domain of Spastin interacted with the C-terminus of GluA2 subunits.3.GST Pull-down and Co-IP experiments found that phosphorylation modification increased the direct interaction between Spastin-K427 R and GluA2 in vitro and in vivo,and the dephosphorylation modification decreased the interaction.Spastin-210A-K427 R and Spastin-210D-K427 R were overexpressed in primary hippocampal neurons.AMPAR-mediated m EPSC and immunocytofluorescence staining of GluA2 subunits on the neuronal membranes were detected by whole-cell patch clamp experiments.The results indicated that phosphorylation modification can promote the membrane trafficking of GluA2 increased by Spastin-K427 R,while dephosphorylation modifications had the opposite effect.Conclusion:1.Spastin 427-site de-SUMO mutant losing its microtubule severing function promotes the maturation of the morphology and function of dendritic spines,and enhances its interaction with AMPA receptor GluA2 subunits.Spastin binds to the C-terminus of GluA2 through its MIT domain and the binding complex together trafficking to the neuronal membrane.2.The phosphorylation modification at 210-site further enhances the binding between the de-SUMO mutant of Spastin and GluA2,and increases the membrane trafficking and the function of GluA2 subunits.