Neurotrophins regulate Schwann cell migration by activating divergent signaling pathways dependent on Rho GTPases

Abstract

Neurotrophins are recognized widely as essential factors in the developing nervous system. Previously, we demonstrated that neurotrophin 3 activation of TrkC inhibits Schwann cell myelination and enhances the migration of primary Schwann cells through the signaling pathway regulated by the Rho GTPases Rac1 and Cdc42. Here, we show that neurotrophins activate divergent signaling pathways to promote or inhibit Schwann cell migration. Endogenous brain-derived neurotrophic factor acting through p75(NTR) inhibits Schwann cell migration dramatically by Src kinase-dependent activation of the guanine-nucleotide exchange factor Vav2 and RhoA. Together, these results suggest that neurotrophins and their receptors differentially regulate Schwann cell migration through the signaling pathways that depend on Rho GTPases.

Fig. 1.

Schwann cell migration is modulated by neurotrophins. The migration of primary Schwann cells was measured by using Boyden chambers (A–C) and on fasciculated DRG axons (D). Filters were coated with DRG axonal membranes (A–C) and various extracellular substrates (A). After incubation with conditioned media from DRG neurons (A), conditioned media containing TrkB-Fc or TrkC-Fc (B), or media containing BDNF or NT3 (C), migration was assayed. (D) Schwann cell reaggregates on fasciculated DRG axons were incubated with BDNF or NT3, and the distance of migration was measured. Axons were stained for neurofilament (green) and the Schwann cells were stained with S100β (red). Scale bar indicates 50 μm. Data were evaluated by using Student's t test. *, P < 0.002; **, P < 0.01; ***, P < 0.02.

Fig. 2.

BDNF inhibits migration through p75^NTR. After incubation with (+) or without (–) BDNF, migration of primary Schwann cells isolated from sciatic nerves of rats (A and B), p75^NTR+/+ or p75^NTR–/– mice (C), and Cos-7 cells transfected with (D and E) or without (E) p75^NTR was assayed by using collagen-coated Boyden chambers. (B) Schwann cells were transfected with control or p75^NTR siRNA. To confirm the effects of siRNAs, lysates from transfected cells were immunoblotted with anti-p75^NTR or β-actin antibody.

Fig. 3.

Involvement of the RhoA signaling pathway in the BDNF inhibition of migration. Schwann cells (A and B), and Cos-7 cells (E and F) transfected with p75^NTR, were pretreated with C3 exoenzyme (A and E) or Y-27632 (B and F). After incubation with BDNF, migration was assayed by using collagen-coated Boyden chambers (A, B, E, and F). After stimulation with BDNF (0–45 min), RhoA activity was measured by pull-down assay using GST-mDia1-Rho-binding domain in the lysates from Schwann cells (C) and Cos-7 cells (G) transfected with RhoA and p75^NTR. The total RhoA in the cell lysates is also shown (C and G). RhoA activity was measured in Schwann cells (D) transfected with control or p75^NTR siRNA, and Cos-7 cells (H) were transfected with RhoA ± p75^NTR.

Fig. 4.

BDNF inhibits migration through Src kinase. Schwann cells (A) and Cos-7 cells (D) transfected with p75^NTR were pretreated with PP1 or PP3. After incubation with BDNF, migration was assayed by using collagen-coated Boyden chambers (A and D). Src activity was determined by immunoblotting with an antiphosphorylated Src antibody in the cell lysates of Schwann cells (B), and Cos-7 cells (E) were transfected with c-Src and p75^NTR. The total Src in the cell lysates is shown also (B and E). Schwann cells (C) and Cos-7 cells (F) transfected with RhoA and p75^NTR were pretreated with PP1 or PP3, and RhoA activity was then measured.

Fig. 5.

Migration is regulated by Src kinase-dependent Vav2 activation. (A) Schwann cells were transfected with control or Vav2 siRNA. (D) Cos-7 cells were transfected with or without Vav2ΔCat and p75^NTR. After incubation with BDNF, migration was assayed by using collagen-coated Boyden chambers (A and D). To confirm the effects of siRNAs, lysates from transfected cells were immunoblotted with anti-Vav2, RhoA, or β-actin antibody (A). Active Vav2 was detected by the pull-down assay using GST-RhoAG15A from the lysates of Schwann cells (B), and Cos-7 cells (E) were transfected with Vav2 and p75^NTR. The total Vav2 in the cell lysates is shown also (B and E). Schwann cells were transfected with a control or the Vav2 siRNA (C); Cos-7 cells were transfected with or without Vav2ΔCat, RhoA, and p75^NTR (F); and RhoA activity was then measured.