AFGF Promotes Neurite Growth By Regulating GSK3?-CRMP2 Signaling Pathway In Alzheimer's Disease

Objective:In this study,candidate proteins interacting with GSK3?were detected by co-immunoprecipitation and LTQ-Orbitrap proteomics technology.The mechanism of aFGF_14-154 and Tat-aFGF_14-154 antagonizing the neurotoxicity of A?_1-42 was studied by targeting GSK3?,which provided a theoretical basis for the development of new drugs in the therapy of Alzheimer's disease.Methods:The effect of different concentrations of aFGF_14-154 and Tat-aFGF_14-154 on the expression of GSK3?and its phosphorylation was detected in N2a-APP cells.MTT assay and Western blotting were used to explore the concentrations of BIO.Immunofluorescence was used to observe the neuroprotective effects of aFGF_14-154,Tat-aFGF_14-154 and BIO on rat primary cortical neurons injured by A?_1-42.N2a cells overexpressing APP were used as an AD model?N2a-APP?in vitro.Proteins interacting with GSK3?were captured by co-IP and LC-MS/MS?LTQ-Orbitrap?protein spectrometry at the optimal concentration of aFGF_14-154 and Tat-aFGF_14-154 in N2a-APP cells.Proteins interacting with GSK3?were identified by mass spectrometry.Protein interacting with GSK3?were analysised by bioinformatics,the interaction between screened proteins and GSK3?were verified by co-IP and Western blotting.The expression of screened proteins influenced by aFGF_14-154,Tat-aFGF_14-154 and GSK3?inhibitors were analysised by Western blotting.The rat primary cortical neurons treated with A?_1-42 were established as another AD model in vitro.Western blotting was used to analysis the expression of CRMP2and their phosphorylation by the regulation of aFGF_14-154 and Tat-aFGF_14-154 at different processing times;The effects of aFGF_14-154 and Tat-aFGF_14-154 on antagonizing the neurotoxicity of A?_1-42 was analysised by MTT assay,Western blotting,scanning electron microscopy and immunofluorescence techniques after knockdown of CRMP2 by siRNA.Result:?1?aFGF_14-154 and Tat-aFGF_14-154 repaired the activity of cortical neurons injured by A?_1-42 and promoted the regeneration of neuron;The effect of neuroprotection of aFGF was declined after using GSK3 inhibition.?2?aFGF_14-154 and Tat-aFGF_14-154 increased the phosphorylation of GSK3??Ser9?in N2a-APP cells,and total levels of GSK3?remained unchanged;The proteins?MAP1B,eEF2,CRMP2,CRAM5,PP2A B,TDP43?related to the development and formation of neuron,axons and dendrites were identified by co-IP with MS/MS;The interaction between CRMP2,CRAM5 and GSK3?were verified by co-IP and western blotting.GSK3?inhibition AR down-regulated significantly the expression of p-GSK3?and CRMP5,and almost inhibited completely the phosphorylation of CRMP2.?3?aFGF_14-154 up-regulated the phosphorylation of GSK3??Ser9?and inhibited phosphorylation of CRMP2?Thr514?,while GSK3?and CRMP2 total protein levels remained unchanged in N2a-APP cells;Both aFGF_14-154 and Tat-aFGF_14-154 could promote the phosphorylation of GSK3??Ser9?and decrease the phosphorylation of CRMP2?Thr514?after 24 h,while the total protein levels of GSK3?and CRMP2remained unchanged in the rat primary cortical neurons treated with A?_1-42.?4?In cortical neuron injured by A?_1-42,the effects of aFGF_14-154 and Tat-aFGF_14-154 on enhancing cell viability was blocked after the knockdown of CRMP2 by siRNA,and their effects on up-regulating the expression of synaptic-related proteins SYN and PSD95 was reversed.The cell morphology also showed that the soma and neurite restored to the state of A?_1-42 injuried again,including soma shrinkaged and flattenedand,the number of surface neurite reduced,neurite shorten and interrupted.The length of neurite characterized by neuron specific microtubule associated protein2?MAP2?was significantly reduced,the network structure formed by neurite disappeared.Conclution:In the AD cell model,aFGF_14-154 and Tat-aFGF_14-154 reduced the phosphorylation of CRMP2?Thr514?by promoting the phosphorylation of GSK3??Ser9?.aFGF_14-154and Tat-aFGF_14-154 inhibited the activity of GSK3?and promoted the regeneration of neurons injured by A?_1-42.aFGF_14-154 and Tat-aFGF_14-154 play an important role in the protection of neuron.