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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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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.
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fig5: 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.

Mentions: We also found that expression of mDia1 coincided with early axonogenesis in postnatal cerebellum in mice (Fig. 5, A and B) . Immunohistochemical analysis showed that mDia1 was especially abundant in postnatal day 1 (P1) cerebellum at and beneath the external granule cell layer where the earliest events in axonogenesis occurred (Fig. 5 B). In round cerebellar granule cells, mDia1 protein was already colocalized with F-actin and tubulin at spots where an axon was likely to initiate (Fig. 5 C). After axon outgrowth started, mDia1 was heavily enriched at the base of early initiating process and within its growth cones (Fig. 5 C, arrowheads) in close spatial vicinity with actin filaments and microtubules (Figs. 5 C).


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)

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.
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Related In: Results  -  Collection

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fig5: 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.
Mentions: We also found that expression of mDia1 coincided with early axonogenesis in postnatal cerebellum in mice (Fig. 5, A and B) . Immunohistochemical analysis showed that mDia1 was especially abundant in postnatal day 1 (P1) cerebellum at and beneath the external granule cell layer where the earliest events in axonogenesis occurred (Fig. 5 B). In round cerebellar granule cells, mDia1 protein was already colocalized with F-actin and tubulin at spots where an axon was likely to initiate (Fig. 5 C). After axon outgrowth started, mDia1 was heavily enriched at the base of early initiating process and within its growth cones (Fig. 5 C, arrowheads) in close spatial vicinity with actin filaments and microtubules (Figs. 5 C).

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