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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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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.
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fig6: 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.

Mentions: To ask directly whether mDia1 activity facilitated, at least in part, axon formation and elongation in cerebellar granule neurons, we tested whether expression of the DA-mDia1 mutant was sufficient to replicate the SDF-1α–induced axon elongating activity in the absence of ROCK pathway which was blocked with Y-27632. Transfection was performed immediately after trituration and during initial plating of the neurons so that expression of exogenous protein was initiated ∼6 h after plating (Bito et al., 2000, and this study). Neurons were fixed using PFA at 12 h after transfection to examine the effect of mDia1 activity on axon initiation and elongation. Indeed, mDia1-ΔN3–expressing cerebellar granule cells revealed a similar number of axons as GFP-expressing control cells; however, the axons were significantly longer compared with the mock-transfected neurons in the presence of Y-27632 (Fig. 6 , A–D), consistent with the idea that mDia1 may work downstream of Rho to facilitate axon growth.


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)

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.
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Related In: Results  -  Collection

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fig6: 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.
Mentions: To ask directly whether mDia1 activity facilitated, at least in part, axon formation and elongation in cerebellar granule neurons, we tested whether expression of the DA-mDia1 mutant was sufficient to replicate the SDF-1α–induced axon elongating activity in the absence of ROCK pathway which was blocked with Y-27632. Transfection was performed immediately after trituration and during initial plating of the neurons so that expression of exogenous protein was initiated ∼6 h after plating (Bito et al., 2000, and this study). Neurons were fixed using PFA at 12 h after transfection to examine the effect of mDia1 activity on axon initiation and elongation. Indeed, mDia1-ΔN3–expressing cerebellar granule cells revealed a similar number of axons as GFP-expressing control cells; however, the axons were significantly longer compared with the mock-transfected neurons in the presence of Y-27632 (Fig. 6 , A–D), consistent with the idea that mDia1 may work downstream of Rho to facilitate axon growth.

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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