Dephosphorylation Of Spastin By PTEN Inhibits Trafficking Of AMPA Receptor Subunit GluA1 To Regulate Dendritic Spine Plasticity

Objective: To investigate the effect of PTEN on the regulation of AMPA receptor GluA1 subunit transport by phosphorylation of spastin.Materials and methods: First,hippocampal neurons were transfected with spastin and its Ser210 mutant plasmids constructed by point mutation,which to observed the changes of dendritic growth and surface GluA1 by immunofluorescence and patch clamp technology.Next,we constructed prokaryotic and eukaryotic expression vectors of PTEN and its functional domains,and confirmed the interaction between PTEN and spastin,as well as the possible binding domains with GST pull-down and Co-IP.Then,spastin and PTEN were coexpressed in HEK293 cells,meanwhile,we also transfected spastin and si PTEN in HEK293 cells,and levels of spastin S210 D was shown by Western blot.Afterward,we transfected si PTEN and GFP-PTEN in hippocampal neurons individually.Similarly,the influence of PTEN on dendritic growth of neurons and levels of surface GluA1 were observed by immunofluorescence and patch clamp technology.Finally,hippocampal neurons were transfected with PTEN and si Spastin,either individually or together,and the differences of m EPSC were observed using patch clamp technology.Results: 1.Spastin and its Ser210 mutant plasmid were transfected into hippocampal neurons,Sholl analysis results showed that the dephosphorylation of spastin inhibited the length of dendrites and the number of branches of hippocampal neurons,patch clamp statistics showed that the frequency and amplitude of m EPSC decreased,and immunofluorescence results showed that level of surface of GluA1 decreased,while the phosphorylation of spastin promoted the growth of dendritic and level of surface GluA1(P<0.05).2.We constructed PTEN and its function domains prokaryotic and eukaryotic expression vectors successfully,purified protein for GST pull-down,and transfected into HEK293 cells to collect the protein for Co-IP.The interaction between PTEN and spastin was demonstrated,and the MIT domain of spastin binds the C-tail and PDZ domain of PTEN.Western blot analysis showed that PTEN expression resulted in a reduction of P-Spastin(S210),while PTEN knockdown increased level of P-Spastin(S210)(P<0.05).But there was no difference in levels of total spastin.3.Overexpressed PTEN in hippocampal neurons and sholl analysis showed that the length of dendrites and the number of branches decreased,the frequency and amplitude of m EPSC decreased according to the statistical analysis of patch clamp,and the level of surface GluA1 decreased by immunofluorescence analysis(P<0.05).Knockdown PTEN in hippocampal neurons,the length of dendrites and the number of branches increased,the frequency and amplitude of m EPSC increased,and the level of surface GluA1 increased(P<0.05).4.Overexpress of PTEN and knockdown spastin in hippocampal neurons.Compared with the group overexpressed PTEN,patch clamp analysis showed increase in m EPSC frequency and amplitude(P<0.05).But compared with the group knockdown PTEN,there was no significant difference in m EPSC frequency and amplitude(P>0.05).Conclusions:1.The phosphorylation of spastin Ser210 promoted the growth of dendrites and the expression of surface GluA1,while the dephosphorylation of spastin Ser210 inhibited the growth of dendrites and the expression of surface GluA1.2.C-terminal of PTEN interactes with MIT domain of spastin.3.PTEN dephosphorylates spastin to inhibit trafficking of AMPA receptor subunit GluA1 to regulate dendritic spine plasticity.