Inhibition of the Rho signaling pathway improves neurite outgrowth and neuronal differentiation of mouse neural stem cells

Abstract

Neurons in the adult mammalian CNS do not spontaneously regenerate axons after injury due to CNS myelin and other inhibitory factors. Previous studies have showed that inhibition of the Rho-ROCK pathway promotes axonal outgrowth in primary neurons or in spinal cord injury models. Furthermore, RhoA inhibitor C3 transferase has a potential effect to induce neural differentiation in primary cultured neurons and cell lines. As stem cells and stem cell-derived neural progenitor cells have emerged as a regenerative medicine for stroke, Parkinson's disease and other neurological disorders, strategies that can promote axonal outgrowth and neuronal differentiation appear to have promising benefits in the cell-based therapy. Currently, how changes in the Rho-ROCK pathway may affect the neurite outgrowth and neuronal differentiation of stem cells has been poorly understood. The present investigation examined the effects of RhoA inhibition on neurite outgrowth and neuronal differentiation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of the mouse. Our results show that inhibition of RhoA leads to neurite outgrowth of NSCs not only on normal culture substrate, poly-D-lysine (PDL), but also on myelin substrate. Moreover, inhibition of RhoA improves neuronal differentiation of NSCs and up-regulates biomarkers of neuronal gene expression. These results support that the Rho signaling pathway plays an important role in neurite development and neuronal differentiation of NSCs.

Keywords: Neural stem cells; Rho signaling pathway; myelin; neurite outgrowth; neuronal differentiation.

Figure 1

Cell culture and characterization of NSCs from mouse SVZ. A. Cells isolated from mouse brain SVZ region formed neurospheres after 7 days in culture. B. Many cells in neurospheres were neural progenitor marker Nestin (red) positive. DAPI (blue) staining shows all cells. C. 12 days after neuronal differentiation with the RA protocol, many cells became positive to the neuronal marker MAP-2 (red) or astrocyte marker GFAP (green). Scale bar = 100 μm. D. Voltage-gated Na⁺ currents were recorded in neuron-like cells 12 days after differentiation. The inward current was triggered by voltage steps from -70 mV to +50 mV in 10 mV increments in the presence of K⁺ channel blockers. Individual current traces are superimposed in the figure. Bath application of the selective Na⁺ channel blocker tetrodotoxin (TTX, 500 nM) completely blocked the voltage-gated inward current. E. Voltage-gated outward K⁺ currents were recorded in the presence of TTX. Individual current traces evoked by depolarizing voltage steps are superimposed in the figure. The outward currents were inhibited by the K⁺ channel blocker 3 mM TEA. Traces are representative for recordings from more than 15 cells.

Figure 2

Effect of C3 phosphotase on cell viability of NSCs. Cell viability after C3 phosphotase treatment was measured using MTT assay. A. NSCs of passage 2, 3 and 4 (P2, P3 and P4) were exposed to different concentration (0.5, 1.0 and 2.0 μg/ml) of C3 phosphotase for 4 hrs, MTT assay was performed 24 hrs later. No change in cell viability was detected in all tests. B. NSCs were subjected to different duration of 1.0 μg/ml C3 treatment and MTT assay was performed 24 hr later. Prolonged exposure to C3 phosphotase for 24 hrs resulted in about 20% reduction in cell viability. N ≥ 3 independent assays per group. P> 0.05 for all comparisons between experimental groups.

Figure 3

Regulation of MAPK pathway molecules in C3-treated NCSs. Western blot was applied to measure the protein levels of signaling molecules in the MAPK pathway. A. Representative immunoblots with antibodies against MAPK, pMAPK, Akt, pAkt, ROCK II and RhoA in cells exposed to 4-hr C3 (1.0 μg/ml) treatment. B. Quantification of Western blot bands for MAPK/pMAPK , Akt/pAkt , ROCK II and RhoA in C3 (1.0 μg/ml) treated cells. *. p<0.05 compared with control group. N=4 assays.

Figure 4

Neuronal differentiation of NSCs after C3 pretreatment. C3 treatment promoted neuronal differentiation of NSCs. A and B. MAP-2 (red) and GFAP (green) fluorescent images of control cells (A) and C3 phosphotase treated cells (B). C3 (1.0 μg/ml, 4 hrs) pretreatment was performed before the 12-16 day neuronal differentiation using the RA protocol. C. Percentage quantification of NeuN-positive and GFAP-positive cells 12-16 days into neuronal differentiation. *. p<0.05 compared with control; n ≥3 assays. C3 pretreatment significantly increased NeuN-positive cells compared to control cells. There is a trend of reduction in GFAP-positive cells in C3-treated group. D and E. Gene expression changes after 1.0 μg/ml C3 treatment detected using RT-PCR. Quantified analysis is shown in E. MAP-2 and NF-L expression was significantly increased in C3-treated cells. *. p<0.05 vs. control group; n=3 assays.

Figure 5

C3 transferase promotes neurite outgrowth of NSCs. NSC cultures was pre-treated with C3 for 4 hrs for its effect on neurite development A. Phase contrast images were taken 5 days after plating from NSCs pre-treated with different C3 concentrations. Scale bar: 200 μm. B. Quantification of neurite length of NSCs at different time points. *. p<0.05 compared with time matched controls. N≥5 independent assays.

Figure 6

C3 transferase promotes neurite outgrowth of NSCs on myelin substrate. NSCs were planted on myelin substrate for the effect of C3 pre-treatment on neurite development. A. Phase contrast photos were taken 2 days after planting from NSC cultures treated with C3 (1.0 μg/ml). B. Quantification of neurite length of NSCs under different conditions and at different time after neuronal differentiation. *. p<0.05 compared with poly-D-lysine coated controls, #. p<0.05 compared with myelin control. Scale Bar=200 μm.

Figure 7

Y-27632 promotes neurite outgrowth of NSCs on myelin substrate. The effect of ROCK inhibitor Y-27632 on neurite outgrowth of NSCs was compared on poly-D-lysine (PDL) coated and myelin coated culture dishes. Y-27632 (10 uM, 4 hrs) was applied before the RA induction. A. Images of NSCs cultures on PDL or myelin base under control and Y-27632 treated condition. B. Quantification of neurite length of NSCs and comparisons between different culture conditions. *. p<0.05 compared with PDL control group, #. p<0.05 compared with myelin control. N≥5 assays.