| Objective: The failure of axons to regenerate is a major obstacle for visual functional recovery after optic nerve injury in central nervous system(CNS).Recent studies have identified several way to stimulate retinal ganglion cells to regenerate their axons by knocking-out neuron growth inhibitory factor,it still remain risky and difficult to knock out gene of interest in human,which make it only feasible in experimental research.Here we screen several neurotrophic factors in mice optic nerve crush model to demonstrate their abilities to promote axon regeneration.We have identified OPN promotes axon regeneration with IGF-1,which maybe useful in clinical trials.Further,we investigate the mechanism of how OPN to interact with receptors,how integrin bind to IGF-1R and influence surface axon growth.Methods:(1)AAV-f OPN was injected intravitreally with a fine glass pipette.Optic nerves were crushed with a pair of Dumont #5 forceps 2 weeks after injection.IGF-1,NGF,NT-3 or BDNF(1 μl,1 μg/μL,Peprotech)was injected into the vitreal space of the eye at 0 dpc and 7 dpc.1μl of Alexa-conjugated CTB568 or 647(Invitrogen)was injected intravitreally 2 days before transcardially perfused with 4% paraformaldehyde,CTB-labelled optic nerve axons were used to evaluate the ability of axon regeneration;The effects of different doses of IGF-1 in promoting axon regeneration were also performed in the optic nerve crush moderl.(2)With bioinformatics analysis of functional domain in OPN sequence,three AAV2/2 viruses were packaged,respectively expressing N-terminus of OPN,Signal peptides conjuncted C-terminus OPN and △143-153 OPN.Based on our optic nerve crush model,combinated administration of AAV-f OPN,AAV-n OPN,AAV-c OPN,AAV-OPN △ 143-153 with r IGF-1 were perfomed intravitreal injection,then CTB-labelled optic nerve axons were used to evaluate the ability of axon regeneration.(3)To investigate the effects of IGF-1 and OPN/IGF-1 in activating IGFR signaling,in vitro experments were performed in HEK293 cell and mouse cortical neurons by stimulated with IGF-1 or OPN/IGF-1.The expresson levels of IGFR,phosphorylated IGFR and phosphorylated S6 detected by western blot were used to evaluate the activation of IGFR signaling and AKT/m TOR signaling.(4)To understand the role of IGFR signaling in lipid raft.We administrated three diffierent treatment(blank,IGF-1 and OPN/IGF-1)in HEK2293 cells and extraction lipid raft and non-lipid raft protein respectively.The expression of IGFR was detected by western blot;To clarify whether the disruption of lipid raft affect IGF-R protein traffic in the cell,membrane lipid rafts of 293 cells were isolated after the treatment of 100 m M M-β-CD;Then to understand the role of lipid rafts in promoting axon regeneration induced by OPN/IGF-1,two groups of in vivo experiments were performed in mouse optic nerve crush model by administration of OPN/IGF-1or OPN/IGF-1/M-β-CD.(5)IGF-1,OPN,OPN/IGF-1 were respectively treated to HEK293 cells.Anti-IGF-IR immunoprecipitates from lipid raft fraction 3-5 immunoblotted with anti-β1 and β3 integrin m Abs demonstrated IGF-IR,β1 and β3 integrin complex formation;Also To clarify whether the disruption of lipid raft affect interaction between IGF-R and HEK293 cells,membrane lipid rafts of 293 cells were isolated after the treatment of 100 m M M-β-CD;To understand the role of integrin in promoting axon regeneration induced by OPN/IGF-1,two groups of in vivo experiments were performed in mouse optic nerve crush model by administration of OPN/IGF-1or Integrin-Ab/OPN/IGF-1.(6)To explorer the binding of IGFR with integrin or Caveolin-1,co-IP and western blot were perfomed.Also anti-Caveolin-1 immunoprecipitates with anti-IGFR was used detected their interaction;Then anti-Caveolin-1 immunoprecipitates with anti-P-Caveolin-1 was used to evaluated the phosphorylation of Caveolin-1 after treatment of IGF-1 and OPN/IGF-1.Results:(1)CTB labeling showed regeneration was significantly more effective in OPN/IGF-1 or OPN/BDNF infection than in control retinas,especially for OPN/IGF-1 treatment.Ciliary neurotrophic factor(CNTF)has effects on promoting the survival and axon regeneration of RGCs after optic nerve crush.OPN plus CNTF did not further stimulate regeneration signanfcantly comparing with CNTF alone.(2)OPN showed integrin bindind poteintial in STRING database analysis;Also 1-16 aa signal peptide was predicted by Signa IP platform;In vivo data revealed that N-OPN was effective at promoting regeneration.However,C-OPN failed to stimulated regeneration;Relative to wild type OPN,integrin binding sequence deletion OPN failed to promote regeneration.(3)In both HEK293 cells and mouse cortical neurons,IGF-1R was phosphorylated at tyrosine residues in response to IGF-1 stimulation.IGF-1 and OPN combination enhanced signal for phospho-IGF-1R.Simultaneously,we used the same samples to detect the phosphorylation of the main signaling proteins,Akt and S6.We found that enhanced signal of phosphorylated AKT in OPN and IGF-1 combination comparing with IGF-1(4)We found that OPN induced IGF-1R translocation into the lipid raft in HEK293 cells.The percentage of IGF-1R in lipid rafts after IGF-1 and the combination of IGF-1 and OPN treatment is not significant different after treated with M-β-CD,as well as in vivo models,which proved that OPN enhanced IGF-1induced IGFR activation in lipid raft-depedent microdomain in plasma.(5)Anti-IGF-IR immunoprecipitates from lipid raft immunoblotted with anti-β1 and β3 integrin m Abs demonstrated IGF-IR,β1 and β3 integrin complex formation.When we use intergrin-Ab to block antigen bind sites in integrin,OPN could not enhance the translocation.As well as in vivo experiments,intergrin-Ab significantly abolished the ability of OPN/IGF-1 induced axon regeneration.(6)The IGFR-1R was immobilized on beads,followed by incubation with lysate.After extensive washing,the bound caveolin was eluted from the resin.Caveolin-1 bound to the IGF-1R after IGF-1 stimulation and OPN promote the binding.OPN also increased tyrosine phosphorylated Cav-1 upon IGF-1 stimulation.Conclusion: In conclusion,this data suggest the mechanism of combination treatment of OPN/IGF-1 to promote axon regeneration could be :OPN promoted IGF-IR activation is coupled with integrin β1 plasma membrane reorganization and phosphorylation of Cav-1 protein sustained IGF-IR traveling and signaling and keep the activation of IGF-1 pathway,as well as downstream AKT/m TOR signaling axis to promote optic nerve regeneration in CNS. |
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