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

Arakawa Y, Bito H, Furuyashiki T, Tsuji T, Takemoto-Kimura S, Kimura K, Nozaki K, Hashimoto N, Narumiya S - J. Cell Biol. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Pharmacology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8315, Japan.

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.

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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.
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fig1: 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.

Mentions: We tested whether SDF-1α was able to trigger any axon growth and whether this morphological change correlated, at least in part, with an alteration in either Rho or Rac activity in cultured cerebellar granule cells, where axonogenesis is well known to precede dendritogenesis. A 12-h exposure to SDF-1α at a concentration of 100 ng/ml induced a significant increase in the mean length of first appearing process compared with control (Fig. 1, A and B ; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200210149/DC1), while having no effect on axon number (Fig. 1, A and B). This effect was abolished in the presence of C3 exoenzyme, a Rho inhibitor (Fig. 1, A and B). SDF-1α promoted a bell-shaped response in axon elongation with a peak effect at 100 ng/ml (Fig. 1, A and B). However, at a larger concentration this axon outgrowth effect was abolished and indistinguishable from the control (Fig. 1 B). In contrast, SDF-1α treatment significantly reduced axon number at concentrations over 250 ng/ml. Presence of C3 exoenzyme completely flattened either response (Fig. 1 B). Together, this indicated that SDF-1α may promote axon elongation via Rho, at least at lower concentrations, whereas it inhibited initiation of axon at higher concentrations. The reduction in SDF-1α–dependent axon growth with the maximal concentration of SDF-1α was unlikely to be caused by receptor desensitization or inactivation, since we confirmed that axon numbers were now negatively affected at the same dose. Furthermore, either 1- or 12-h exposure to either 100 or 500 ng/ml SDF-1α was accompanied with a similarly strong elevation in the amount of GTP-bound form of Rho (Fig. 1 C), whereas no apparent increase was discerned for GTP-bound Rac (unpublished data) as determined by pull-down assays using either a GST-fused Rho-binding domain (RBD) of Rhotekin or a GST-fused Cdc42/Rac interactive binding (CRIB) domain of p21-associated kinase (PAK) were performed (Ren et al., 1999; Tsuji et al., 2002). These results indicate the possibility that stimulation of Rho pathway with a physiological ligand such as SDF-1α may mediate axon elongation.


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

Arakawa Y, Bito H, Furuyashiki T, Tsuji T, Takemoto-Kimura S, Kimura K, Nozaki K, Hashimoto N, Narumiya S - J. Cell Biol. (2003)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172896&req=5

fig1: 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.
Mentions: We tested whether SDF-1α was able to trigger any axon growth and whether this morphological change correlated, at least in part, with an alteration in either Rho or Rac activity in cultured cerebellar granule cells, where axonogenesis is well known to precede dendritogenesis. A 12-h exposure to SDF-1α at a concentration of 100 ng/ml induced a significant increase in the mean length of first appearing process compared with control (Fig. 1, A and B ; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200210149/DC1), while having no effect on axon number (Fig. 1, A and B). This effect was abolished in the presence of C3 exoenzyme, a Rho inhibitor (Fig. 1, A and B). SDF-1α promoted a bell-shaped response in axon elongation with a peak effect at 100 ng/ml (Fig. 1, A and B). However, at a larger concentration this axon outgrowth effect was abolished and indistinguishable from the control (Fig. 1 B). In contrast, SDF-1α treatment significantly reduced axon number at concentrations over 250 ng/ml. Presence of C3 exoenzyme completely flattened either response (Fig. 1 B). Together, this indicated that SDF-1α may promote axon elongation via Rho, at least at lower concentrations, whereas it inhibited initiation of axon at higher concentrations. The reduction in SDF-1α–dependent axon growth with the maximal concentration of SDF-1α was unlikely to be caused by receptor desensitization or inactivation, since we confirmed that axon numbers were now negatively affected at the same dose. Furthermore, either 1- or 12-h exposure to either 100 or 500 ng/ml SDF-1α was accompanied with a similarly strong elevation in the amount of GTP-bound form of Rho (Fig. 1 C), whereas no apparent increase was discerned for GTP-bound Rac (unpublished data) as determined by pull-down assays using either a GST-fused Rho-binding domain (RBD) of Rhotekin or a GST-fused Cdc42/Rac interactive binding (CRIB) domain of p21-associated kinase (PAK) were performed (Ren et al., 1999; Tsuji et al., 2002). These results indicate the possibility that stimulation of Rho pathway with a physiological ligand such as SDF-1α may mediate axon elongation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Pharmacology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8315, Japan.

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.

Show MeSH
Related in: MedlinePlus