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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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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.
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fig8: 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.

Mentions: To obtain an independent confirmation of these results, we applied the RNA interference techniques using short interfering double stranded RNA oligomers (siRNAs) (Elbashir et al., 2001). mDia1-specific siRNA was designed and its efficiency in knocking down mDia1 protein was tested in NIH3T3 cells by Western blot analysis (Fig. 8 A). Loss of mDia1 immunoreactivity was specifically obtained in cells expressing a cotransfection marker enhanced GFP (EGFP; unpublished data). Using identical transfection procedures, EGFP-positive cerebellar granule neurons were screened to identify the neurons, which have taken up the mDia1 siRNA, and axon lengths were measured in these neurons. A significant reduction was observed both in axon numbers and in axon length (Fig. 8, B and C), compared with scramble siRNA-treated neurons, in keeping with the DN approach.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172896&req=5

fig8: 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.
Mentions: To obtain an independent confirmation of these results, we applied the RNA interference techniques using short interfering double stranded RNA oligomers (siRNAs) (Elbashir et al., 2001). mDia1-specific siRNA was designed and its efficiency in knocking down mDia1 protein was tested in NIH3T3 cells by Western blot analysis (Fig. 8 A). Loss of mDia1 immunoreactivity was specifically obtained in cells expressing a cotransfection marker enhanced GFP (EGFP; unpublished data). Using identical transfection procedures, EGFP-positive cerebellar granule neurons were screened to identify the neurons, which have taken up the mDia1 siRNA, and axon lengths were measured in these neurons. A significant reduction was observed both in axon numbers and in axon length (Fig. 8, B and C), compared with scramble siRNA-treated neurons, in keeping with the DN approach.

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