Peptide ligands targeting FGF receptors promote recovery from dorsal root crush injury via AKT/mTOR signaling

Abstract

Background: Fibroblast growth factor receptors (FGFRs) are key targets for nerve regeneration and repair. The therapeutic effect of exogenous recombinant FGFs in vivo is limited due to their high molecular weight. Small peptides with low molecular weight, easy diffusion, low immunogenicity, and nontoxic metabolite formation are potential candidates. The present study aimed to develop a novel low-molecular-weight peptide agonist of FGFR to promote nerve injury repair. Methods: Phage display technology was employed to screen peptide ligands targeting FGFR2. The peptide ligand affinity for FGFRs was detected by isothermal titration calorimetry. Structural biology-based computer virtual analysis was used to characterize the interaction between the peptide ligand and FGFR2. The peptide ligand effect on axon growth, regeneration, and behavioral recovery of sensory neurons was determined in the primary culture of sensory neurons and dorsal root ganglia (DRG) explants in vitro and a rat spinal dorsal root injury (DRI) model in vivo. The peptide ligand binding to other membrane receptors was characterized by surface plasmon resonance (SPR) and liquid chromatography-mass spectrometry (LC-MS)/MS. Intracellular signaling pathways primarily affected by the peptide ligand were characterized by phosphoproteomics, and related pathways were verified using specific inhibitors. Results: We identified a novel FGFR-targeting small peptide, CH02, with seven amino acid residues. CH02 activated FGFR signaling through high-affinity binding with the extracellular segment of FGFRs and also had an affinity for several receptor tyrosine kinase (RTK) family members, including VEGFR2. In sensory neurons cultured in vitro, CH02 maintained the survival of neurons and promoted axon growth. Simultaneously, CH02 robustly enhanced nerve regeneration and sensory-motor behavioral recovery after DRI in rats. CH02-induced activation of FGFR signaling promoted nerve regeneration primarily via AKT and ERK signaling downstream of FGFRs. Activation of mTOR downstream of AKT signaling augmented axon growth potential in response to CH02. Conclusion: Our study revealed the significant therapeutic effect of CH02 on strengthening nerve regeneration and suggested a strategy for treating peripheral and central nervous system injuries.

Keywords: Dorsal root crush injury; Dorsal root ganglia; Fibroblast growth factor receptor; Nerve regeneration; Peptides.

Figure 1

Screening of FGFR2-targeted small peptides using a phage display peptide library. (A) Illustration of the library screening steps. The phage library was incubated on a plate precoated with a recombinant FGFR2 extracellular domain protein, the unbound phage was washed, and the specifically bound phage was eluted. The eluted phages were amplified and used as input for the next round of biopanning. After three rounds, individual clones were sequenced to identify the phage peptides. (B) After each screening round, phage recovery (%) was assessed by phage titer. Bars represent multiple changes in the phage recovery in each round compared to the recovery in the first round. (C) Cell viability assays of FGFR2-overexpressing HEK293 cells incubated with CH01, CH02, or CH03 peptides at a concentration gradient for 48 h. (D-G) ITC analysis of FGFR1c, FGFR2c, FGFR3c, or FGFR4 binding to the CH02 peptide. Top panels show the raw data of the heat pulses resulting from FGFR1c, FGFR2c, FGFR3c, or FGFR4 titration. Bottom panels show the integrated heat pulses, normalized per mole of injectant, as a function of the molar ratio (CH02 peptide concentration/recombinant protein concentration). These binding curves were best fitted to a single-site binding model. (H) Western blot analysis of phosphorylated FGFR expression in cultured human umbilical vein endothelial cells (HUVECs) treated with CH02 in a concentration gradient. (I) Quantification of p-FGFR expression levels shown in (H). n = 3 independent experiments. The relative protein expression level was quantified after normalization to FGFR2 (***p < 0.001 by one-way ANOVA with Dunnett's test, compared to the control group). Values are means ± SEM.

Figure 2

Molecular docking and MDS analysis of CH02 interactions with FGFR2 receptors. (A) Binding pattern between CH02 and the FGFR2 extracellular segment. The surface model represents the FGFR2 extracellular domain, the cartoon model shows a partial enlargement of the D3 area, and the stick model shows the CH02 peptide. (B) Two-dimensional binding mode shows noncovalent interaction between CH02 and FGFR2. Each balloon displays different residues, and non-covalent bonds are shown as dashed lines in different colors. (C) MDS results of two CH02-FGFR2 docking complexes (CH02-FGFR2:CH02-FGFR2) renamed Complex-2. The remaining components (FGFR2:FGFR2) after removing the CH02 ligand from Complex-2 were considered the control group and called Complex-1. (a) Complex-1. (b) Average structure in the last 50 ns of MDS from (a). (c) Complex- 2. (d) Average structure in the last 50 ns of MDS from (c). (D) Average structure of the interaction between CH02 and FGFR2 in Complex-2. CH02 is shown as a blue stick model, and partial residues of FGFR2 that interacted with CH02 are shown as purple stick models. (E) Types of noncovalent interactions between CH02 and FGFR2 in Complex-2. Each balloon represents different residues, and dashed lines in different colors show noncovalent bonds. (F) Number of hydrogen bonds formed by different residues of CH02 and FGFR2 in the simulation process for Complex 2.

Figure 3

Effect of the CH02 peptide on axon growth and neuron survival. (A) Representative images of the CH02 peptide effect on axon growth in adult primary DRG neurons. DRG neurons were obtained from adult Sprague-Dawley rats. After 12-h of culture, neurons were stimulated with CH02 or bFGF for 48 h. Axonal outgrowth was assessed by immunocytochemistry using an antibody against β-tubulin, an axonal marker. Images were acquired from random neurons by fluorescence microscopy. Scale bars = 50 μm. (B) Quantification of the longest axon length of each neuron randomly selected in three independent experiments from (A), using Image-Pro Plus 6.0 software (n = 48 for each condition; *p < 0.05, ****p < 0.0001 by one-way ANOVA with Dunnett's test; ns, not significant; mean ± SD). (C) Representative images of the CH02 effect on axon growth in ex vivo cultured adult DRG explants. The axon is shown in fluorescence (β3-tubulin) images. Scale bars = 100 μm. (D) Quantification of axon growth of DRG explants from (C) using the Neurite-J software (n=4 for each condition; *control vs. CH02; #control vs. bFGF; *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01 by two-way ANOVA with Dunnett's test; ns, not significant; mean ± SEM). (E) Cell cytotoxicity assay of primary DRG neurons induced by H₂O₂. LDH released from damaged cell membranes was detected by an LDH assay kit. The results are expressed as the mean ± SEM (**p < 0.01 by one-way ANOVA with Dunnett's test; ns, not significant, compared with the control group). (F) Representative TUNEL staining of apoptotic neurons induced by H₂O₂ in the control, CH02, and FGF2 groups. Scale bars = 50 μm. Apoptotic neurons were detected by a TUNEL apoptosis assay kit. TUNEL (green); NeuN, neuronal marker (red); DAPI (blue). (G) Quantification of TUNEL-positive neurons from (F) (n = 3 for each condition; **p < 0.01 by one-way ANOVA with Dunnett's test; ns, not significant, compared with the control group). DRG neurons were obtained from adult Sprague-Dawley rats. After 12-h of culture, neurons were pretreated with CH02 or bFGF for 24 h before H₂O₂ (500 μM) treatment for 12 h.

Figure 4

CH02 peptide treatment after dorsal root crush injury improves axon regeneration and sensory function recovery. (A) Procedure of the dorsal root crush model in adult Sprague-Dawley rats. Connective tissue and muscles were removed to expose the right spinal segments from the fourth cervical (C4) to the second thoracic (T2) levels. After a C5 to T1 dorsal laminectomy and dura matter opening, the right C5 to T1 dorsal roots were fully exposed. Each root was crushed three times (5s per crush) using no. 7 forceps between the dorsal root ganglia and DREZ. (B, C) Effect of CH02 treatment on sensory behavioral recovery in rats after dorsal root crush injury. The functional sensory recovery of rats was assessed using the von Frey test (B) and plantar test (C) at the indicated time points. Behavioral results showed that, compared to the PBS group, the recovery of pressure and thermal sensation in the CH02 and FGF2 groups was significantly improved (n = 5 for each condition; *sham vs. PBS; #sham vs. bFGF; *p < 0.05, **p < 0.01, ****p < 0.0001, ##p < 0.01, ####p < 0.0001 by two-way ANOVA with Dunnett's test; mean ± SEM). (D-H) Effect of CH02 treatment on axon densities in the dorsal horn of the spinal cord. (D) Experimental timeline: adult Sprague-Dawley rats were subjected to concurrent C5 -T1 dorsal root crush injury, followed by daily treatment with the CH02 peptide (20 μM, 400 μL) via subcutaneous injection near the injury site. After 25 days, the rats were euthanized for spinal dorsal horn tissue collection. (E, F) Representative fluorescence images of immunostaining for CGRP (E) or IB4+ (F), and laminin in spinal dorsal horn tissues collected from Sprague-Dawley rats 25 days after dorsal root injury. Laminin (green); CGRP and IB4+ (red); DAPI (blue). Scale bar = 50 μm. (G, H) Quantification of immunofluorescence intensity for CGRP (G) or IB4+ (H) from (E, F) (n = 4 animals per group; *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired t-test; mean ± SEM). (I-K) CH02 peptide treatment enhanced axon regeneration across the DREZ. (I) Experimental timeline: Sprague-Dawley rats were traced with 10% biotinylated dextran amine (BDA) (0.8 μL) 4 weeks after injury. After 7 days of BDA labeling, the rats were euthanized to harvest spinal cord tissues with attached dorsal roots. (J) Representative images of BDA-labeled regenerated axons observed in transverse spinal cord tissue sections containing the dorsal root and DREZ. Transganglionic tracing with BDA. BDA (green); Laminin (red); DAPI (blue). Scale bars = 20 μm. (K) Quantification of axonal regeneration across the DREZ from (J) (n = 4 animals per group; *p < 0.05 by unpaired t-test; mean ± SEM).

Figure 5

Role of FGFR signaling in response to the CH02 peptide on promoting nerve regeneration. (A) Western blot analysis of phosphorylated FGFR expression in C5-T1 DRGs subjected to dorsal root crush injury, followed by CH02 treatment (20 μM). DRGs were collected after 3 days. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were used as the loading control. (B) Western blot analysis of phosphorylated FGFR expression in ex vivo cultured DRG explants of adult Sprague-Dawley rats in response to CH02 treatment at different induction times. (C) Western blot analysis of DRG explants treated with DMSO (control), CH02 (20 μM), CH02+BGJ398, or BGJ398 (2 μM) for 4 h to examine phospho-FGFR levels after 6 days in ex vivo culture. Tubulin antibodies were used as the loading control. (D-F) Quantification of p-FGFR expression levels in panels A, B, and C. n = 3 independent experiments. Relative protein expression levels were quantified after normalization to FGFR2 or FGFR1. Values are means ± SEM. (G) Representative images of the CH02 effect on axonal growth in ex vivo DRG explants cultured for 1 day and treated with DMSO (control), CH02 (20 μM), CH02+BGJ398, or BGJ398 (2 μM) for 4 days. DRG explants were fixed in 4% paraformaldehyde and immunostained for NF200 antibodies. Scale bar = 100 μm. (H) Quantification of axon growth in DRG explants (G) using Neurite-J software (n = 5 for each condition; *control vs. CH02; #control vs. BGJ398; **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ##p < 0.01 by two-way ANOVA with Dunnett's test; mean ± SEM). (I) Representative images of the CH02 effect on axonal growth in primary cultured DRG neurons treated with DMSO (control), CH02 (20 μM), CH02+BGJ398, orBGJ398 (2 μM) for 48 h after 12- h of culture. Subsequently, DRG neurons were fixed in 4% paraformaldehyde and immunostained with β-tubulin antibodies. Images of random neurons were acquired by fluorescence microscopy. Scale bar = 25 μm. (J) Quantification of the longest axon length of each neuron randomly selected from (I) using Image-Pro Plus 6.0 software (n = 100 for each condition; *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA with Dunnett's; ns, not significant; mean ± SD).

Figure 6

Phosphoproteomic profiling of the intracellular signaling pathway used by CH02 for augmenting nerve regeneration. (A) GO biological process analysis of proteins corresponding to differentially expressed phosphorylated peptides. Ordinates indicate the enriched GO terms; abscissae represent the gene ratio; size of the dots indicates the number of proteins enriched in this biological process. (B) KEGG pathway analysis of proteins corresponding to differentially expressed phosphorylated peptides. Ordinates indicate the enriched pathway term; abscissae represent the gene ratio; size of the dots indicates number of proteins enriched in this pathway. (C) Immunoblot showing phosphorylated ERK1/2 and AKT expression in C5-T1 DRGs subjected to dorsal root injury following CH02 (20 μM) treatment. DRG samples were collected after 3 days. (D-E) Quantification of p-ERK1/2 and p-AKT expression levels. n = 3 independent experiments. Relative protein expression level was quantified after normalization to the corresponding total proteins. Values are means ± SEM. (F) Immunoblot showing phosphorylated ERK1/2 and AKT expression in ex vivo cultured DRG explants treated with DMSO (control), CH02 (20 μM), CH02+BGJ398, or BGJ398 (2 μM) for 4 h. (G-H) Quantification of p-ERK1/2 and p-AKT expression levels. n = 3 independent experiments. Relative protein expression level was quantified after normalization to the corresponding total proteins. Values are means ± SEM. (I) Representative images of the CH02 effect on axon growth in DRG explants treated with DMSO (control), CH02 (20 μM), CH02+AKTi, AKTi (2 μM), CH02+U0126, or U0126 (2 μM) for 4 days. The axon is shown in fluorescence (NF200) images. Scale bar = 100 μm. (J) Quantification of axon growth in DRG explants (I) using Neurite-J software (n = 4 for each condition; *control vs. CH02; #CH02 vs. CH02+AKTi, CH02+U0126; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 by two-way ANOVA with Dunnett's test; mean ± SEM). (K) Representative images showing CH02 effect on axonal growth of DRG neurons treated with DMSO (control), CH02 (20 μM), CH02+AKTi, AKTi (2 μM), CH02+U0126, or U0126 (2 μM) for 48 h after 12 h of culture. For quantification of axon length, neurons were immunostained using an antibody against β-tubulin. Images were acquired from random neurons for further analysis. Scale bar = 50 μm. (L) Quantification of the longest axon length of each neuron randomly selected from (K) using Image-Pro Plus 6.0 software (n = 40 for each condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Dunnett's test; ns, not significant; mean ± SD). (M) Representative TUNEL staining of primary cultured apoptotic neurons induced by H₂O₂ for 12 h after pretreatment with DMSO (control), CH02, CH02+AKTi, or CH02+U0126for 24 h. Scale bars = 50 μm. TUNEL (green); NeuN (red). (N) Quantification of TUNEL-positive neurons (M) using ImageJ software (n = 5 for each condition; ***p < 0.001 by one-way ANOVA with Dunnett's test; mean ± SEM).

Figure 7

CH02-stimulated activation of AKT/mTOR signaling is required for growth potential augmentation of axons. (A) Immunoblot analysis showing phosphorylated mTOR expression in C5-T1 DRGs subjected to dorsal root injury followed by CH02 treatment (20 μM). DRG samples were collected 3 days post-treatment. (B) Quantification of p-mTOR expression levels in A. n = 3 independent experiments. Relative protein expression levels were quantified after normalization to mTOR. Values are means ± SEM. (C) Immunoblot showing phosphorylated mTOR expression in DRG explants pretreated with DMSO (control), CH02 (20 μM), CH02+AKTi (2 μM) or CH02+ U0126 (2 μM) for 4 h. (D) Quantification of the p-mTOR expression from C. n = 3 independent experiments. Relative protein expression levels were quantified after normalization to mTOR. Values are means ± SEM. (E) Representative images of the CH02 effect on axon growth in DRG neurons pretreated with DMSO (control), CH02 (20 μM), CH02+Torin1, or Torin1 (1 μM) for 48 h after 12-h culture. The axon is shown in fluorescence (β3-tubulin) images. Scale bar = 50 μm. (F) Quantification of the longest axon length of each neuron from (E) (n = 62 for each condition; *Control vs. CH02, CH02+Torin1, Torin1; # CH02 vs. CH02+Torin1; **p < 0.01, ****p < 0.0001, #p < 0.05 by one-way ANOVA with Dunnett's test; ns, not significant; mean ± SD). (G) Representative images of the CH02 effect on axon growth in DRG explants treated with DMSO (control), CH02 (20 μM), CH02+Torin1, and Torin1 (1 μM) for 4 days. DRG explants were fixed in 4% paraformaldehyde and immunostained for NF200 antibodies. Scale bar = 100 μm. (H) Quantification of axon growth in DRG explants from (G) using Neurite-J software (n = 4 for each condition; *control vs. CH02, CH02+Torin1, Torin1; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Dunnett's test; mean ± SEM). (I) Schematic illustration showing putative signaling pathways involved in CH02-mediated axon regeneration in rat sensory neurons. FGFR signal activation is the primary factor by which the CH02 peptide promotes axon regeneration. Activation of AKT/mTOR signaling in response to the CH02 peptide to enhance axon regeneration primarily depends on upstream FGFR signaling. The CH02 peptide also has an affinity for VEGFR2 and MET and can simultaneously activate VEGFR2 and MET at the cellular level. The dotted line represents unverified results.