Spastin Interact With CRMP5 To Regulate Neurite Outgrowth In Hippocampal Neuron

Objective: The aim of the experiment is to investigate whether spastin can interact with C RMP5 and how to combine with CRMP5,at the same time, the development of hippocampal neuron neurite can be influenced by their interac tion. Methods: At First,the prokaryotic plasmid of GST-spastin^M87V、 GST-spastin N190（residues 87-190）、GST-spastin△AAA（residues 87-305）、GST-spastin△N1（residues 191-616） and GST-spastin△N2（residues 306-616） were constructed,and the fusion protein of GST and GST-spastin^M87V、GST-spastin N190、GST-spastin △AAA、GST-spastin△N1 and GST-spastin△N2 were expressed. Then the GST pull-down assays were applied to detect whether spastin could bind to CRMP5 and how to combine with. Secondly, the prokaryotic plasmid of GST-spastin^M87V、GST-spastin N190、GST-spastin△AAA、GST-spastin△N1 and GST-spastin△N2 were constructed, Hela cells and hippocampal neuron cells were respectively transfected with GFP-spastin, the of microtubuline of Hela and the development of hippocampal neuron neurite were observed by Laser Scanning Confocal Microscope. Results: 1) The results of recombinant plasmid digested by restriction enzyme show that the sizes of target genes were consistent with the anticipation, and there were no mismatch and mutation existing in the DNA sequencing of GST-spastin^M87V、GST-spastin N190、GST-spastin△AAA、GST-spastin△N1、GST-spastin△N2; 2) The fusion protein of spastin^M87V、spastin N190、spastin△AAA、spastin△N1、spastin△N2 and GST were expressed successfully, and their molectular weight were about 84.19 k Da、37.44 k Da、50.09 k Da、69k Da,60.21 k Da and 26 k Da, which consistent with the expected size of the sμ m of target protein and GST protein size;3) In vitro,GST pull-down experiment results showed the fusion proteins of spastin^M87V、spastin△AAA and spastin△N1 can bind with C RMP5 in rat brain lysate,but the fusion proteins of spastin N190、spastin△N2 can’t bind with CRMP5; 4) The results of recombinant plasmid digested by restriction enzyme show that the sizes of target genes were consistent with the anticipation, and there were no mismatch and mutation existing in the DNA sequencing of GFP-spastin^M87V、GFP-spastin N190、GFP-spastin△AAA、GFP-spastin△N1 and GFP-spastin△N2; 5) Overexpression of spastin M87 V in Hela cells, the microtubule of Hela cells were severed into small flagments by spastin; 6) Overexpression of spastin M87 V in hippocampal neuron,we found that the nμ mber and length of dendrites and axon are increased; Conclusions: 1) Prokaryotic expression vector of GST-spastin^M87V、GST-spastin N190、GST-spastin△AAA、GST-spastin△N1、GST-spastin△N2have been constructed successfully; 2) The fusion protein of spastin^M87V、spastin N190、spastin△AAA、spastin△N1、spastin△N2 and GST were expressed successfully in vitro; 3) In vitro The result of GST pull-down was that the protein of spastin^M87V、spastin△AAA and spastin△N1 can interact with CRMP5, on the contrary,the protein of spastin N190、spastin△N2 can not interact with CRMP5. So the site of spastin combined with the C RMP5 was 191-305 amino acid; 4) Prokaryotic expression vector of GFP-spastin^M87V、GFP-spastin N190、GFP-spastin△AAA、GFP-spastin△N1 and GFP-spastin△N2 have been constructed successfully; 5) Spastin M87 V can sever microtubule of Hela cell;6) Spastin M87 V can promote the development of neurite outgrowth in hippocampal neuron.