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, 10 (9), 1117-1128

Trimebutine, a Small Molecule Mimetic Agonist of Adhesion Molecule L1, Contributes to Functional Recovery After Spinal Cord Injury in Mice

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Trimebutine, a Small Molecule Mimetic Agonist of Adhesion Molecule L1, Contributes to Functional Recovery After Spinal Cord Injury in Mice

Junping Xu et al. Dis Model Mech.

Abstract

Curing spinal cord injury (SCI) in mammals is a daunting task because of the lack of permissive mechanisms and strong inhibitory responses at and around the lesion. The neural cell adhesion molecule L1CAM (L1) has been shown to favor axonal regrowth and enhance neuronal survival and synaptic plasticity but delivery of full-length L1 or its extracellular domain could encounter difficulties in translation to therapy in humans. We have, therefore, identified several small organic compounds that bind to L1 and stimulate neuronal survival, neuronal migration and neurite outgrowth in an L1-dependent manner. Here, we assessed the functions of two L1 mimetics, trimebutine and honokiol, in regeneration following SCI in young adult mice. Using the Basso Mouse Scale (BMS) score, we found that ground locomotion in trimebutine-treated mice recovered better than honokiol-treated or vehicle-receiving mice. Enhanced hindlimb locomotor functions in the trimebutine group were observed at 6 weeks after SCI. Immunohistology of the spinal cords rostral and caudal to the lesion site showed reduced areas and intensities of glial fibrillary acidic protein immunoreactivity in both trimebutine and honokiol groups, whereas increased regrowth of axons was observed only in the trimebutine-treated group. Both L1- and L1 mimetic-mediated intracellular signaling cascades in the spinal cord lesion sites were activated by trimebutine and honokiol, with trimebutine being more effective than honokiol. These observations suggest that trimebutine and, to a lesser extent under the present experimental conditions, honokiol have a potential for therapy in regeneration of mammalian spinal cord injuries.

Keywords: Honokiol; L1CAM mimetics; Mouse; Small organic compounds; Spinal cord injury; Trimebutine.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Effects of trimebutine or honokiol on functional recovery from spinal cord injury. (A-H) Time course and degree of recovery of locomotor functions after SCI in mice treated with vehicle control (VC), trimebutine (TMB) or honokiol (HNK). Shown are mean±s.e.m. values for BMS scores (A), foot-stepping angles (C), rump-height indices (E), and recovery indices (RI) after injury as measured by BMS scores (B), foot-stepping angles (D), and rump-height (F) at 1, 2, 3, 4, 5 and 6 weeks after SCI. Group mean values and individual values of overall recovery indices are shown in G and H, respectively. *P<0.05, **P<0.01 versus the vehicle control; one-way ANOVA with Tukey's post hoc test; n=6 mice/group.
Fig. 2.
Fig. 2.
Lesion areas at 6 weeks after spinal cord injury in mice treated with trimebutine or honokiol. (A) Representative images of H&E-stained longitudinal sections at 6 weeks after SCI in vehicle control (VC), trimebutine (TMB) or honokiol (HNK) groups. (B) Quantitative analysis of lesion area at 6 weeks after SCI in vehicle control, trimebutine or honokiol groups. **P<0.01 versus the vehicle control; one-way ANOVA with Tukey's post hoc test; n=3 mice/group.
Fig. 3.
Fig. 3.
GFAP, βIII-tubulin and NF200 immunoreactivity at 6 weeks after SCI in mice treated with trimebutine or honokiol. (A-I) Representative micrographs of GFAP (A,E,F), βIII-tubulin (C,E) and NF200 (F) staining, and quantitative analysis of intensity of GFAP (B,G), βIII-tubulin (D,H) and NF200 (I) immunoreactivity. *P<0.05, **P<0.01 versus the vehicle control; one-way ANOVA with Tukey's post hoc test; n=3 mice/group. HNK, honokiol; TMB, trimebutine; VC, vehicle control.
Fig. 4.
Fig. 4.
L1, pCK2α, mTOR and PTEN immunoreactivity at 6 weeks after SCI in mice treated with trimebutine or honokiol. (A-D) Representative images and quantitative analysis of L1 (A), pCK2α (B), mTOR (C) and PTEN (D) immunohistochemical staining. *P<0.05, **P<0.01 versus the vehicle control; one-way ANOVA with Tukey's post hoc test; n=3 mice/group. HNK, honokiol; TMB, trimebutine; VC, vehicle control.
Fig. 5.
Fig. 5.
Analysis of L1 and pCK2α immunofluorescence intensities rostral, central and caudal to the lesion site of the injured spinal cord at 6 weeks post-SCI in mice treated with trimebutine or honokiol. (A,C) Representative images of L1 (A) and pCK2α (C) rostral (1 mm to the lesion center), central and caudal (1 mm to the lesion center) to the lesion site. (B,D) Quantitative analysis of L1 (B) and pCK2α (D) immunoreactivities. *P<0.05, **P<0.01 versus the vehicle control; one-way ANOVA with Tukey's post hoc test; n=3 mice/group. HNK, honokiol; TMB, trimebutine; VC, vehicle control.
Fig. 6.
Fig. 6.
Activation of L1-mediated intracellular signaling cascades at 6 weeks after SCI in mice treated with trimebutine or honokiol. Western blot analysis of L1 (A), phosphorylation levels of Akt1 (B), Erk1/2 (C), CK2α (D) and mTOR (E), protein levels of PTEN (F), p53 (G) and GFAP (H), and Bcl-2/Bax ratio (I) of mice treated with vehicle control (VC), trimebutine (TMB) or honokiol (HNK) at 5 mm rostral to the lesion center, the lesion center, and 5 mm caudal to the lesion center. *P<0.05, **P<0.01 versus the vehicle control; one-way ANOVA with Tukey's post hoc test; n=4 mice/group.
Fig. 7.
Fig. 7.
Activation of L1-mediated intracellular signaling cascades in cultured mouse cerebellar granule cells treated with trimebutine. (A-E) Protein levels of L1 (A), phosphorylation levels of Akt1 (B) and Erk1/2 (C), protein levels of mTOR (D), and the ratio of Bcl-2/Bax levels (E) in cultured mouse cerebellar granule cells treated with trimebutine (TMB) at concentrations of 0, 5, 10 and 20 nM. *P<0.05, **P<0.01, ***P<0.001 versus the vehicle control; one-way ANOVA with Tukey's post hoc test; three independent experiments. Blots in C-E are from the same gel and therefore have the same β-actin loading controls.
Fig. 8.
Fig. 8.
Activation of L1-mediated intracellular signaling cascades in cultured mouse cerebellar granule cells treated with honokiol. (A-E) L1 levels (A), phosphorylation levels of Akt1 (B) and Erk1/2 (C), protein levels of mTOR (D), and the ratio of Bcl-2/Bax levels (E) in cultured mouse cerebellar granule cells treated with honokiol (HNK) at concentrations of 0, 50, 100 and 200 nM. *P<0.05, **P<0.01, ***P<0.001 versus the vehicle control; one-way ANOVA with Tukey's post hoc test; three independent experiments. Blots in B-E are from the same gel and therefore have the same β-actin loading controls.

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