Phosphorylation Of Spastin Promotes The Trafficking Of AMPARs Subunit GluA2 To Mediate Dendritic Spine Plasticity

Objective:Hereditary spastic paraparesis(HSP)caused by mutations in the SPAST(SPG4)gene are autosomal-dominant inherited disorders characterized by weakness of lower extremities,spasticity and hyperreflexia.Some cases with cognitive decline have been reported and cognitive function is closely related to the expression of AMPA receptors in spines.This study aimed to investigate the effect of phosphorylation of spastin on the trafficking of AMPA receptors subunit GluA2 and developing of dendritic spines.Methods:1.Phosphorylation sites of spastin were confirmed by mass spectrometry(S210,S233,T271,S562)and the single mutant plasmids and the quadruple mutants plasmids were constructed in which serine and threonine were replaced by an alanine or a glutamine residue.2.Spastin mutants were transfected into cultured hippocampal neurons,the surface GluA2 and the development of dendritic spines were observed by immunocytochemistry and the effect on AMPA receptor?mediated m EPSCs in cultured hippocampal neurons was examined by whole-cell patch clamp.3.Spastin mutants were overexpressed into COS1 cell and the microtubule severing activity was observed by immunocytochemistry.4.GST Pull-down,Co-IP and hippocampal neurons co-localization experiments were used to identify the interaction between spastin and GluA2.Results:1.Phosphorylation of spastin promotes the maturation of dendritic spines by promoting the trafficking of GluA2 and increasing the amplitude and frequency of m EPSC.Immunofluorescence showed that mature dendritic spines in cultured hippocampal neurons overexpressing spastin(Qm D)was increased to that of control(P < 0.05)and immature dendritic spines overexpressing spastin(Qm A)was increased to that of control((P<0.05).The surface GluA2 was increased in spastin(Qm D)mutants compared with wild-type and other mutants(P<0.05),however the surface GluA2 was decreased in spastin(Qm A)mutants(P<0.05).The result of whole-cell patch clamp showed that m EPSC frequency and amplitude were increased in spastin(Qm D)overexpressed cells compared to that in controls,which were decreased in spastin(Qm A)overexpressed cells(P<0.05).2.Spastin interacts with AMPARs subunit GluA2 in vivo and in vitro and this binding is regulated by the phosphorylation of spastin.GST Pull-down,Co-IP and hippocampal neurons co-localization experiments showed that spastin interact with GluA2 in vitro and in vivo.The relationship between the level of spastin phosphorylation and the interaction was further clarified.Compared with the wild-type group,the binding of phosphorylated spastin(Qm D)to GluA2 was enhanced,while the binding of non-phosphorylated spastin(Qm A)to GluA2 was decreased.3.The non-phosphorylated spastin promoted its microtubule severing activity.Immunofluorescence showed that the fluorescence intensity of microtubule in COS1 cells overexpressing spastin(Qm D)or spastin(Qm A)was reduced to that of control(P<0.05),while overexpressing spastin(Qm A)resulted in a lower fluorescence intensity of microtubule in COS1 cells compared to wild-type(P<0.05).4.Phosphorylation of spastin S210 site promotes the membrane of GluA2 and increasing the amplitude and frequency of m EPSC.Immunofluorescence showed that the expression of surface GluA2 increased in the HEK293 cell strain stably expressing GluA2 after overexpression of spastin(Qm D)or spastin(S210D)(P<0.05),while the surface GluA2 was decreased after overexpression of spastin(Qm A)or spastin(S210A)(P<0.05).The result of whole-cell patch clamp showed that m EPSC frequency and amplitude were increased in spastin(S210D)overexpressed cells compared to that in controls,which were decreased in spastin(S210A)overexpressed cells(P<0.05).5.Phosphorylation of spastin which losses the activity of severing microtubules also promotes AMPARs GluA2 subunit trafficking to surface.Immunofluorescence showed that the fluorescence intensity of microtubule in COS1 cells overexpressing spastin was significantly reduced to that of control(P < 0.05),while the fluorescence intensity of microtubule in COS1 cells overexpressing spastin(K353A)and spastin(R464C)was comparable to that of control(P>0.05).The surface GluA2 in neurons overexpressing spastin(K353A,S210D)is increased to that of control(P < 0.05),while overexpressing spastin(K353A,S210A)is decreased(P<0.05).The result of whole-cell patch clamp showed that m EPSC frequency and amplitude were increased in spastin(K353A,S210D)overexpressed cells compared to that in controls,which were decreased in spastin(K353A,S210A)overexpressed cells(P<0.05).Conclusions:1.Phosphorylation of spastin promotes the maturation of dendritic spines by promoting the membrane of GluA2.2.Spastin interacts with AMPARs subunit GluA2 in vivo and in vitro and this binding is regulated by the phosphorylation of spastin.3.phosphorylation of spastin which losses the activity of severing microtubules also promotes AMPARs GluA2 subunit trafficking to surface and the phosphorylation of spastin S210 mediates GluA2 trafficking independent of microtubule dynamics.