Taurine enhances mouse cochlear neural stem cells proliferation and differentiation to sprial gangli through activating sonic hedgehog signaling pathway

Abstract

To investigate the molecular mechanism underlying taurine-stimulated proliferation and differentiation of cochlear neural stem cells (NSCs) and potential involvement of Sonic Hedgehog (Shh) pathway. The NSCs were characterized with immunofluorescence stained with nestin antibody. Cell viability was determined by MTT assay. The relative proliferation was measured by BrdU incorporation assay. The morphologic index was measured under light microscope. The relative protein level was determined by immunoblotting. Here we presented our findings that taurine stimulated proliferation and neurite outgrowth of NSCs, which was completely abolished by Shh inhibitor cyclopamine. In addition, cyclopamine antagonized taurine's effect on glutamatergic and GABAergic neuron population via suppressing expressions of Ptc-1, Smo and Gli-1. Our data supported the critical role of Shh pathway underlying the protective effect of taurine on auditory neural system.

Keywords: Cochlear neural stem cells; Sonic Hedgehog; Taurine; cyclopamine; differentiation.

FIGURE 1.

Cyclopamine inhibited the effect of taurine on the viability and proliferation of mouse cochlea NSCs in vitro. (A) Chemical structure of taurine. (B) The identification of NSCs, revealed by immunostaining against nestin (green), and cell nuclei were counterstained with DAPI (blue). (C) Representative photomicrographs of NSCs to three groups of the control (0 mM, Ctrl), taurine treatment (10 mM, Tau), taurine (10 mM) combined with cyclopamine (2.5 mM) treatment (Tau+ Cyc). Cells were immunostained by tubulin (red) and nuclei were counterstained with DAPI (blue). (D, E) Relative cell viability and proliferation of the NSCs to the control following taurine treatment, OR taurine combined with cyclopamine treatment, measured by MTT assay (D) and BrdU incorporation assay (E). (F) Representative photomicrographs of BrdU labeled cells with their nuclei counterstained by the nuclear fluorescent stain DAPI in three experimental groups. Scale bar: 50 μm. Histogram bars represent mean ± SEM (*P < 0.05, **P < 0.01). n = 8 each group. Cyc, cyclopamine. Tau, taurine.

FIGURE 2.

Cyclopamine inhibited the effect of taurine on the neurite outgrowth of mouse cochlea NSCs. (A) Representative images of the NSCs culture at DIV14 after taurine and cyclopamine treatment compared to control. Cells were stained by tuj-1. Scale bar, 20 μm. (B-D) Statistical analysis of the number of primary dendrites per cell (B), number of dendritic end tips number (C) and average length of neurite (D) in experimental groups on DIV14. (E-F) Western blot analysis of GAP-43 expression and the relative optical densities in the experimental groups. Histogram bars represent mean ± SEM (*P < 0.05, **P < 0.01). Cyc, cyclopamine. Tau, taurine.

FIGURE 3.

Cyclopamine inhibited the effect of taurine-increased glutamatergic neuron population and taurine-reduced GABAergic neuron population in vitro. (A, C) Control, taurine alone or taurine combined with cyclopamine treated cultures in vitro, glutamatergic neurons (A) and GABAergic neurons (C) were indicated by positive staining by VGLUT1 (green) (A) and GAT1 (green) (C). Part of the cell population was nestin-positive (red). Nuclei were stained DAPI (blue). (B, D) Percentage of glutamatergic neurons (B) and GABAergic neurons (D) in culture relative to the control after taurine alone or taurine combined with cyclopamine treatment. Scale bar, 100 μm. Histogram bars represent mean ± SEM (*P < 0.05, **P < 0.01). n = 6 each group. Cyc, cyclopamine. Tau, taurine.

FIGURE 4.

Cyclopamine inhibited the effect of taurine on VGLUT1 and GAT1 proteins expression in NSCs in vitro. (A, C) VGLUT1 and GAT1 protein levels, as detected by western blotting. (B, D) Quantitative analysis of VGLUT1 and GAT1 protein levels. GAPDH was used as a loading control. Histogram bars represent mean ± SEM (*P < 0.05, **P < 0.01). n = 6 each group. Cyc, cyclopamine. Tau, taurine.

FIGURE 5.

Cyclopamine inhibited the effect of taurine on the expression of Shh, Ptc-1, Smo, Gli-1 proteins in NSCs in vitro. (A) Shh, Ptc-1, Smo, and Gli-1 protein levels, as detected by western blotting. (B) Quantitative analysis of Shh, Ptc-1, Smo, and Gli-1 levels. Histogram bars represent mean ± SEM (*P < 0.05, **P < 0.01). n = 5 each group. Cyc, cyclopamine. Tau, taurine.