Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling

Abstract

Individuals with spinal cord injury (SCI) suffer from permanent disabilities such as severe motor, sensory and autonomic dysfunction. Neural stem cell transplantation has proven to be a potential strategy to promote regeneration of the spinal cord, since NSCs can produce neurotrophic growth factors and differentiate into mature neurons to reconstruct the injured site. However, it is necessary to optimize the differentiation of NSCs before transplantation to achieve a better regenerative outcome. Inhibition of Notch signaling leads to a transition from NSCs to neurons, while the underlying mechanism remains inadequately understood. Our results demonstrate that overexpression of fucosyltransferase 9 (Fut9), which is upregulated by Wnt4, promotes neuronal differentiation by suppressing the activation of Notch signaling through disruption of furin-like enzyme activity during S1 cleavage. In an in vivo study, Fut9-modified NSCs efficiently differentiates into neurons to promote functional and histological recovery after SCI. Our research provides insight into the mechanisms of Notch signaling and a potential treatment strategy for SCI.

Keywords: Notch; Wnt; fucosyltransferase; neural stem cell transplantation; neural stem cells; neuronal differentiation.

Figure 1

Fut9 is upregulated in the process by which Wnt4 promotes neuronal differentiation in NSCs (A,B) RNA sequencing of NSCs treated with Wnt4 for 3 days. (A) Volcano plots of differentially expressed genes (DEGs) in NSCs treated with Wnt4 in comparison with the untreated group. (B) Heatmap of DEGs of NSCs treated with Wnt4 in comparison with the untreated group. (C,D) RT-qPCR and western blot analysis of Fut9 expression in NSCs stimulated with Wnt4 for 3 days. (E) Quantification of Fut9 protein expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the untreated group.

Figure 2

Wnt4 upregulates Fut9 expression through the β-catenin signaling pathway (A) GO pathway enrichment analysis of the differentially expressed genes between the NT group and Wnt4 group. (B,C) RT-qPCR and western blot analysis of Fut9 expression in NSCs treated with a specific pathway inhibitor and then stimulated with Wnt4 for 3 days. (D) Quantification of Fut9 protein expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the untreated group; #P<0.05 compared with the Wnt4 group. IWR-1: Wnt/β-catenin inhibitor.

Figure 3

Overexpression of Fut9 promotes neuronal differentiation (A,B) mRNA and protein expression levels of Fut9 in the NT, LV-Con, and LV-Fut9 groups. (C) Immunofluorescence staining for neural-differentiated markers of NSCs in the NT, LV-Con, and LV-Fut9 groups. Scale bar: 100 μm. (D,E) Quantification of dendritic length and neural-differentiated marker-positive cells of NSCs. (F,G) mRNA and protein expression levels of NF200, β3-tubulin and MAP2 in the NT, LV-Con, and LV-Fut9 groups. (H) Quantification of neural-differentiated marker expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the nontreatment group and LV-Con group.

Figure 4

Fut9 rescues the negative effect of Notch signaling to promote neuronal differentiation (A) Immunofluorescence staining for neural-differentiated markers of NSCs in the NT, LV-Con+Jag1, and LV-Fut9+Jag1 groups. Scale bar: 100 μm. (B, C) Quantification of dendritic length and neural-differentiated marker-positive cells of NSCs. (D,E) mRNA and protein expression levels of NF200, β3-tubulin and MAP2 in the NT, LV-Con+Jag1, and LV-Fut9+Jag1 groups. (F) Quantification of neural-differentiated marker expression by western blot analysis. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the LV-Con+Jag1 group.

Figure 5

Fut9 inhibits the furin enzyme activity of S1 cleavage to suppress the activation of the Notch signaling pathway (A) Western blot analysis of Notch1 and NICD expression in NSCs treated with furin. (B) Quantification of Notch1 and NICD expression by western blot analysis. (C) Furin-like enzyme activity was measured in NSCs in different groups. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the untreated group. (D,E) RT-qPCR and western blot analysis of Hes1 and Hes5 in the LV-Con, LV-Con+furin and LV-Fut9+furin groups. (F) RT-qPCR analysis of neural transcription factor expression in the LV-Con, LV-Con+furin and LV-Fut9+furin groups. Data are presented as the mean±SD from one representative experiment of three independent experiments performed in triplicate. *P<0.05 compared with the LV-Con+furin group.

Figure 6

Fut9 promotes functional recovery and tissue repair (A) Images showing hind limb climb from Sham, SCI, LV-Con and LV-Fut9 groups at the eighth week postinjury; white arrows point to the hind limb. (B) BBB scores of the different groups. (C) H&E, MRI and Nissl staining analyses of the spinal cord in different groups. Sections at 2 mm rostral and caudal to the lesion epicenter were counted for each rat. (D,F) Quantification of H&E, MRI and Nissl staining analyses in different groups. Data are presented as the mean±SD. *P<0.05 compared with the sham group; #P<0.05 compared with the SCI group. (G) Immunofluorescence staining of the spinal cord in different groups. Scale bar: 50 μm. (H) Quantification of immunofluorescence staining. Data are presented as the mean±SD from two independent experiments. **P<0.001.