Mechanism Of Spastin In Regulating AMPA Receptor Subunit GluA1 Internalization

ObjectiveThe study focused on how the microtubule-severing protein Spastin regulate the internalization of the GluA1 subunit of the AMPA receptor.Materials and methodsFirst,we constructed the various expression vectors of Spastin and its truncated fragments to identify the interaction and the binding site between Spastin and GluA1 by using pull down and Co-IP.Secondly,the colocalization of dendrite and dendritic spine was observed in hippocampal nruron with Spastin and GluA1 by Laser Scanning Confocal Microscope.Thirdly,Spastin was overexpressed in GluA1-293 T stable cell lines and hippocampal neurons to observe the changes of Total GluA1 and Surface GluA1 by immunofluorescence,membrane protein extraction and patch clamp.At last,the eukaryotic expression mutants of the Spastin domains were constructed and overexpressed in stable cell lines and neurons to clarify the specific effects of the Spastin domains with GluA1.Results1)The interaction between Spastin and GluA1 was confirmed by pull down and Co-IP experiments,and the site was located in the MIT domain of Spastin and the Cterminal of GluA1;2)Overexpression of Spastin WT in GluA1-HEK293 T stable cell lines and neuros would inhibit the expression of GluA1 on the cell membrane;3)The microtubule were severed into small flagments by Spastin WT and Spastin△MIT,instead of the control group,Spastin △MTBD,Spastin △AAA or Spastin C413Y;4)Overexpression of Spastin WT and its domain mutants in stable cell lines and neuros,we found that the function of microtubule and MIT domain together promote GluA1 internalization.Conclusions1)Spastin interacted with GluA1,the interactional site was located in the MIT domain of Spastin and the C-terminal of GluA1;2)Spastin WT promote the internalization of GluA1;3)The microtubule severing and MIT domain of Spastin together promote the internalization of GluA1.