Control of axon elongation via an SDF-1alpha/Rho/mDia pathway in cultured cerebellar granule neurons

Abstract

Rho-GTPase has been implicated in axon outgrowth. However, not all of the critical steps controlled by Rho have been well characterized. Using cultured cerebellar granule neurons, we show here that stromal cell-derived factor (SDF)-1alpha, a neural chemokine, is a physiological ligand that can turn on two distinct Rho-dependent pathways with opposite consequences. A low concentration of the ligand stimulated a Rho-dependent pathway that mediated facilitation of axon elongation. In contrast, Rho/ROCK activation achieved by a higher concentration of SDF-1alpha caused repression of axon formation and induced no more increase in axon length. However, even at this higher concentration a Rho-dependent axon elongating activity could be recovered upon removal of ROCK activity using Y-27632. SDF-1alpha-induced axon elongating activity under ROCK inhibition was replicated by the dominant-active form of the mammalian homologue of the Drosophila gene Diaphanous (mDia)1 and counteracted by its dominant-negative form. Furthermore, RNAi knockdown of mDia1 abolished SDF-1alpha-induced axon elongation. Together, our results support a critical role for an SDF-1alpha/Rho/mDia1 pathway in mediating axon elongation.

Figure 1.

SDF-1α facilitates axon elongation via a Rho-dependent pathway. (A) Facilitation of axon elongation by 12-h exposure to SDF-1α (top). SDF-1α–induced facilitation is blocked by C3 treatment (bottom), indicating the existence of a Rho-dependent mode of neurite extension. β-tubulin immunostaining was employed to completely trace the entire length of axons. (B) Axon elongating activity induced by SDF-1α reveals a bell-shaped response curve, whereas axon numbers are reduced at only higher concentrations. SDF-1α effect on axon length and number are both blocked by C3 treatment. n ≈ 42–209. (C) 1- or 12-h exposure to SDF-1α (100 or 500 ng/ml) leads to a substantial increase in the amount of GTP-bound Rho (RBD pulldown) but not that of GTP-bound Rac (unpublished data) in cerebellar granule cells. **P < 0.01; ***P < 0.001. Bar, 5 μm.

Figure 2.

Distinct C3 dose effectiveness on axon elongation and axon number in cultured cerebellar granule neurons. Axon elongation reveals a bell-shaped C3 responsiveness consistent with the presence of two opposing effects downstream of Rho. However, the C3 effect on axon number is saturated at doses over 10 μg/ml. n ≈115–424; ***P < 0.001.

Figure 3.

SDF-1α–induced axon elongation is antagonized by ROCK. 12-h exposure to high concentration (500 ng/ml) of SDF-1α represses axonogenesis (A, top, and B, right). Addition of 50 μM Y-27632, a specific ROCK inhibitor, blocked this SDF-1α–induced inhibition of axonogenesis (A, bottom, and B, right). Under the same condition, SDF-1α significantly facilitated axon elongation in ROCK-inhibited neurons (B, left), while having little effect on axon numbers (B, right). n ≈ 82–74. ***Compared with untreated cells or ###compared with Y-27632-treated cells; P < 0.001. Bars, 5 μm.

Figure 4.

A putative role for mDia1 in facilitated process outgrowth. (A) Domain structure of wild-type and dominant mutant constructs of mDia1. (B) Coupling of elevated mDia1 activity (by overexpression of mDia1-ΔN3) with lower ROCK activity (in the presence of Y-27632) is sufficient to induce elongated axon-like processes in Swiss3T3 cells. Note the high amount of F-actin stained with phalloidin in the thin processes (arrowheads) of the GFP-mDia1-ΔN3–expressing cells. Bars, 50 μm.

Figure 5.

High expression of mDia1 in cerebellar EGL during early postnatal development. (A) Western blot analysis of mDia1 expression in cerebellar lysates at P1, P2, P9, or in adult (A). (B) mDia1-like immunoreactivity is highly concentrated at and beneath the EGL at P1. TO-PRO nuclear stain indicates the locations of the cells. Bar, 50 μm. (C) mDia1 is highly expressed at the neck of a nascent process (top, arrowhead) or in the growth cones (bottom, arrowheads) in cerebellar granule cells in culture at 6 h (top) and 12 h (bottom) in vitro. mDia1 heavily colocalized with F-actin (phalloidin) and microtubules (tubulin) structures. Bars, 5 μm.

Figure 6.

DA mDia1 facilitates axon elongation. Morphology of cerebellar granule cells overexpressing GFP (A), GFP-mDia1-ΔN3 alone (B), or GFP-mDia1-ΔN3 in the presence of Y-27632 (C). When ROCK activity was reduced, expression of GFP-mDia1-ΔN3 resulted in a significantly enhanced elongation (D, left) of axons (n ≈ 65–157) compared with EGFP-expressing controls (A). Overexpression of GFP-mDia1-ΔN3 alone successfully induced an axon, which, however, exhibited a significantly altered shape (enlarged width, premature stop), presumably due to an increased actin stability in the presence of intact ROCK activity (B). Basal ROCK activity, in the context of excessive mDia1 activity, might cause a prominent increase in actin polymerization, while also sustaining a tonic level of actomyosin contractility, thereby negatively acting on axon elongation. *P < 0.05; ***P < 0.001. Bars, 5 μm.

Figure 7.

A DN mDia1 mutant interferes with SDF-1α–dependent axon elongation. (A) Coexpression of the GFP-mDia1-ΔN3(HindIII) mutant abolished the effect of FLAG-mDia1-ΔN3 expression on axon length (left). n ≈ 33–80. (B and C) The effect of GFP-mDia1-ΔN3(HindIII) overexpression was examined on SDF-1α–facilitated axon elongation in the presence of Y-27632. A potent inhibition on both SDF-1α–dependent axon elongation (B and C, left) and axon initiation (B and C, right) was detected. n ≈ 35–145. Bars, 5 μm.

Figure 8.

mDia1 knockdown by RNAi using siRNA completely abolishes SDF-1α–dependent axon elongation. (A) Significant reduction of mDia1 protein is achieved in NIH3T3 within 24 h by RNAi using siRNA. (B and C) mDia1 knockdown by RNAi annihilates both SDF-1α–dependent axon elongation (C, left) and axon initiation (C, right) back to baseline levels, thereby confirming the DN experiments. n ≈ 26–140. ***P < 0.001. Bars, 5 μm.

Figure 9.

Expression of the morphological effects of mDia1 is mediated, at least in part, by Rac activity. (A) Co-expression of GFP-N17Rac, a DN mutant of Rac, suppresses expression of the facilitatory effects of FLAG-mDia1-ΔN3 on either axon length (left) in the presence of Y-27632 (50 μM) back to baseline levels. n ≈ 45–137. (B) Inhibition of ROCK activity in the presence of high concentration of SDF-1α (500 ng/ml) increases the GTP-bound form of Rac. ***P < 0.001.

Figure 10.

Proposed model for the role of mDia in SDF-1α– dependent axon outgrowth in cultured cerebellar granule neurons. Usually Rho and Rac antagonistically controls axon outgrowth. During the very early stages in the primary culture, ROCK action predominates to favor suppression of precocious outgrowth of axons from the cerebellar granule cells. However, as these neurons express a high amount of mDia1 gradual decline in ROCK activity facilitates expression of Rho-dependent mDia1 activity and a subsequent recruitment of a signaling complex, which in concert with the Rac-dependent signaling cascade may help the transition from inhibition to stimulation of axon outgrowth and elongation. Thus, the SDF-1α/Rho/mDia1 pathway may play a critical role in defining and modulating the balance between the Rho- and Rac-based signaling pathways during axon outgrowth.