TAG-1 Regulates NRP1 in Schwann Cells and Participates in Regulating Nerve Regeneration in Rats with Sciatic Nerve Crush Injury

Abstract

Schwann cells (SCs) are necessary for peripheral nerve regeneration due to their plasticity and trophic supply after sciatic nerve injury (SNI). However, the multiple adaptations of SCs are still poorly understood. This study explored the effects of transient axonal glycoprotein type-1 (TAG-1) on cell migration and neuropilin1 (NRP1) expression in SCs and examined the impact of TAG-1 on nerve regeneration in rats with SNI. The expression of TAG-1 and NRP1 in SCs, RSC96 cells, was measured using co-immunoprecipitation and immunofluorescence. TAG-1 silence in RSC96 cells was established by TAG-1 siRNA transfection. The effects of TAG-1 silence on cell migration and NRP1 expression were measured in cells. Male adult Wistar rats suffered sciatic nerve crush injury and were treated with exogenous TAG-1 protein. The sciatic function was observed every week. The histological changes of sciatic nerves and expressions of S100β, NRP1, GAP43, and NCAM in the nerves were observed after injury 28 days. The TAG-1 and NRP1 were expressed in the RSC96 cells, and there was an interaction between TAG-1 and NRP1. TAG-1 silence suppressed the cell migration and NRP1 expression in cells. In rats with SNI, the TAG-1 treatment improved the sciatic function and promoted nerve regeneration by increasing the expressions of S100β, NRP1, GAP-43, and NCAM in the nerves. This study showed that TAG-1 regulated cell migration and NRP1 expression in SCs, and TAG-1 treatment might be a strategy for nerve regeneration after the SNI.

Keywords: Co-immunoprecipitation; Schwann cells; Sciatic function; Sciatic nerve injury.

Fig. 1

Expression of TAG-1 and NRP1 in the RSC96 cells. A Expression of TAG-1 and NRP1 was measured by immunofluorescence. B The specific proteins in the precipitates were measured using immunoblotting (IB) with the indicated antibodies. Three repeats of each experiment

Fig. 2

TAG-1 expression in RSC96 cells was suppressed by TAG-1 siRNA transfection. A Expression of S100β and TAG-1 was measured by immunofluorescence, scale = 50 μm. B The mean gray of TAG-1 was measured using ImageJ software. C TAG-1 expression was measured by western blotting. The expression of TAG-1 was normalized by β-actin. ^**p < 0.01, n = 3

Fig. 3

Effects of TAG-1 silence on cell migration and NCAM expression in the RSC96 cells. A Cell migration was measured by Transwell, scale = 50 μm. The numbers of transwell cells were counted using ImageJ software. B NCAM expression was measured by immunofluorescence, scale = 50 μm. The mean gray of NCAM was analyzed using ImageJ software. ^**p < 0.01, n = 3

Fig. 4

Effects of TAG-1 silence on NRP1 expression in RSC96 cells. A Expression of S100β and NRP1 was measured by immunofluorescence, scale = 50 μm. B The mean gray of NRP1 was measured using ImageJ software. C NRP1 expression was measured by western blotting. The quantitative analysis of bands was performed using ImageJ software. The expression of NRP1 was normalized by β-actin. ^**p < 0.01, n = 3

Fig. 5

Effects of TAG-1 treatment on sciatic function and histological changes of nerves in rats with sciatic nerve injury (SNI). A The sciatic functional index (SFI) was measured at different times. Compared with the sham group, ^**p < 0.01; Compared with the vehicle group, ^#p < 0.05, ^##p < 0.01, 7 rats in each group. B After injury for 28 days, the amplitude and latency of compound muscle action potentials among consecutive stimulations were recorded by electrophysiological assay. ^*p < 0.05, ^**p < 0.01. C Histological changes of nerves were measured by H&E staining. Seven rats in each group

Fig. 6

Effects of TAG-1 treatment on myelination regeneration in the rats with sciatic nerve injury (SNI). A The myelin sheets were stained by LFB staining. Black arrows: myelin sheaths; red arrows: axons. B The area of LFB staining was analyzed using ImageJ software. C The density of myelin sheaths was observed using TEM. D Numbers of myelin sheets per 100 µm². ^*p < 0.05, ^**p < 0.01, n = 7

Fig. 7

Effects of TAG-1 treatment on the S100-β expression in the rats with SNI. A The expression of S100-β and TAG-1 was stained by immunofluorescence. Scale = 20 μm. B The mean gray of S100-β and TAG-1 was analyzed by ImageJ software. ^*p < 0.05, ^**p < 0.01, n = 7

Fig. 8

Effects of TAG-1 treatment on the NRP1 expression in the rats with SNI. A The expression of S100-β and NRP1 was stained by immunofluorescence. Scale = 20 μm. B The mean gray of S100-β and NRP1 was analyzed by ImageJ software. ^*p < 0.05, ^**p < 0.01, n = 7

Fig. 9

Effects of TAG-1 treatment on the GAP-43 and NCAM expression in the rats with SNI. A The expression of GAP-43 was measured by immunohistochemistry. Scale = 50 μm. Black arrow: GAP-43 expression. The expression of GAP-43 was analyzed using ImageJ software. B The expression of NCAM was measured by immunofluorescence. The mean gray of NCAM was analyzed by ImageJ software. ^*p < 0.05, ^**p < 0.01, n = 7