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Axonal elongation triggered by stimulus-induced local translation of a polarity complex protein.

Hengst U, Deglincerti A, Kim HJ, Jeon NL, Jaffrey SR - Nat. Cell Biol. (2009)

Bottom Line: We found that the PAR complex, like local translation, is required for stimulated, but not basal, outgrowth.Selective ablation of Par3 mRNA from axons abolishes the outgrowth-promoting effect of NGF.These results identify a new role for local translation and the PAR complex in axonal outgrowth.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Weill Medical College, Cornell University, NY 10065, USA.

ABSTRACT
During development, axon growth rates are precisely regulated to provide temporal control over pathfinding. The precise temporal regulation of axonal growth is a key step in the formation of functional synapses and the proper patterning of the nervous system. The rate of axonal elongation is increased by factors such as netrin-1 and nerve growth factor (NGF), which stimulate axon outgrowth using incompletely defined pathways. To clarify the mechanism of netrin-1- and NGF-stimulated axon growth, we explored the role of local protein translation. We found that intra-axonal protein translation is required for stimulated, but not basal, axon outgrowth. To identify the mechanism of translation-dependent outgrowth, we examined the PAR complex, a cytoskeleton regulator. We found that the PAR complex, like local translation, is required for stimulated, but not basal, outgrowth. Par3 mRNA is localized to developing axons, and NGF and netrin-1 trigger its local translation. Selective ablation of Par3 mRNA from axons abolishes the outgrowth-promoting effect of NGF. These results identify a new role for local translation and the PAR complex in axonal outgrowth.

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Proteins constituting the polarity complex are localized to growth cones of DRG and DSC neurons after axon formation(a, b) Immunolabelling of DRG (b) and DSC (c) explants at 5 DIV for polarity complex proteins shows localization of PAR3, PAR6 and aPKCζ to growth cones. (c, d) Dissociated DRG neurons were transfected with siRNAs targeting PAR3 or PAR6 mRNA or a non-tageting control siRNA and immunolabelling against PAR3 (c) or PAR6 (d) was performed 48 h later. (e, f) Quantification of the average staining intensity for PAR3 and PAR6 normalized to tau-1 staining reveals that transfection with targeting siRNAs causes a significant decrease in staining intensities demonstrating the specificity of the PAR3 and PAR6 signal observed in axons and growth cones. t-test, PAR3, *P=0.0104; PAR6, ***P=0.005. n = 13 to 21 axons per condition. Scale bar, 20 µm.
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Figure 2: Proteins constituting the polarity complex are localized to growth cones of DRG and DSC neurons after axon formation(a, b) Immunolabelling of DRG (b) and DSC (c) explants at 5 DIV for polarity complex proteins shows localization of PAR3, PAR6 and aPKCζ to growth cones. (c, d) Dissociated DRG neurons were transfected with siRNAs targeting PAR3 or PAR6 mRNA or a non-tageting control siRNA and immunolabelling against PAR3 (c) or PAR6 (d) was performed 48 h later. (e, f) Quantification of the average staining intensity for PAR3 and PAR6 normalized to tau-1 staining reveals that transfection with targeting siRNAs causes a significant decrease in staining intensities demonstrating the specificity of the PAR3 and PAR6 signal observed in axons and growth cones. t-test, PAR3, *P=0.0104; PAR6, ***P=0.005. n = 13 to 21 axons per condition. Scale bar, 20 µm.

Mentions: Consistent with prior studies16, we find PAR3, PAR6, and aPKCζ localized to the tips of the presumptive axon in embryonic day 18 (E18) cultured rat hippocampal neurons at day in vitro 1 (DIV1) (Supplementary Information, Fig. S2a). At time points at which axon specification has been completed, we find each of the PAR complex proteins still present in growth cones and axons of DIV5 DRG and DSC neurons and DIV9 hippocampal neurons (Fig. 2a,b and Supplementary Information, Fig. S2b). The specificity of the immunofluorescence staining for PAR3 and PAR6 was established by the lack of staining in dissociated DRG neurons transfected with PAR3 or PAR6 siRNA (Fig. 2c–f). Together, these data indicate the PAR complex is present in axons at the same time that they are responsive to NGF and netrin-1.


Axonal elongation triggered by stimulus-induced local translation of a polarity complex protein.

Hengst U, Deglincerti A, Kim HJ, Jeon NL, Jaffrey SR - Nat. Cell Biol. (2009)

Proteins constituting the polarity complex are localized to growth cones of DRG and DSC neurons after axon formation(a, b) Immunolabelling of DRG (b) and DSC (c) explants at 5 DIV for polarity complex proteins shows localization of PAR3, PAR6 and aPKCζ to growth cones. (c, d) Dissociated DRG neurons were transfected with siRNAs targeting PAR3 or PAR6 mRNA or a non-tageting control siRNA and immunolabelling against PAR3 (c) or PAR6 (d) was performed 48 h later. (e, f) Quantification of the average staining intensity for PAR3 and PAR6 normalized to tau-1 staining reveals that transfection with targeting siRNAs causes a significant decrease in staining intensities demonstrating the specificity of the PAR3 and PAR6 signal observed in axons and growth cones. t-test, PAR3, *P=0.0104; PAR6, ***P=0.005. n = 13 to 21 axons per condition. Scale bar, 20 µm.
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Related In: Results  -  Collection

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Figure 2: Proteins constituting the polarity complex are localized to growth cones of DRG and DSC neurons after axon formation(a, b) Immunolabelling of DRG (b) and DSC (c) explants at 5 DIV for polarity complex proteins shows localization of PAR3, PAR6 and aPKCζ to growth cones. (c, d) Dissociated DRG neurons were transfected with siRNAs targeting PAR3 or PAR6 mRNA or a non-tageting control siRNA and immunolabelling against PAR3 (c) or PAR6 (d) was performed 48 h later. (e, f) Quantification of the average staining intensity for PAR3 and PAR6 normalized to tau-1 staining reveals that transfection with targeting siRNAs causes a significant decrease in staining intensities demonstrating the specificity of the PAR3 and PAR6 signal observed in axons and growth cones. t-test, PAR3, *P=0.0104; PAR6, ***P=0.005. n = 13 to 21 axons per condition. Scale bar, 20 µm.
Mentions: Consistent with prior studies16, we find PAR3, PAR6, and aPKCζ localized to the tips of the presumptive axon in embryonic day 18 (E18) cultured rat hippocampal neurons at day in vitro 1 (DIV1) (Supplementary Information, Fig. S2a). At time points at which axon specification has been completed, we find each of the PAR complex proteins still present in growth cones and axons of DIV5 DRG and DSC neurons and DIV9 hippocampal neurons (Fig. 2a,b and Supplementary Information, Fig. S2b). The specificity of the immunofluorescence staining for PAR3 and PAR6 was established by the lack of staining in dissociated DRG neurons transfected with PAR3 or PAR6 siRNA (Fig. 2c–f). Together, these data indicate the PAR complex is present in axons at the same time that they are responsive to NGF and netrin-1.

Bottom Line: We found that the PAR complex, like local translation, is required for stimulated, but not basal, outgrowth.Selective ablation of Par3 mRNA from axons abolishes the outgrowth-promoting effect of NGF.These results identify a new role for local translation and the PAR complex in axonal outgrowth.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Weill Medical College, Cornell University, NY 10065, USA.

ABSTRACT
During development, axon growth rates are precisely regulated to provide temporal control over pathfinding. The precise temporal regulation of axonal growth is a key step in the formation of functional synapses and the proper patterning of the nervous system. The rate of axonal elongation is increased by factors such as netrin-1 and nerve growth factor (NGF), which stimulate axon outgrowth using incompletely defined pathways. To clarify the mechanism of netrin-1- and NGF-stimulated axon growth, we explored the role of local protein translation. We found that intra-axonal protein translation is required for stimulated, but not basal, axon outgrowth. To identify the mechanism of translation-dependent outgrowth, we examined the PAR complex, a cytoskeleton regulator. We found that the PAR complex, like local translation, is required for stimulated, but not basal, outgrowth. Par3 mRNA is localized to developing axons, and NGF and netrin-1 trigger its local translation. Selective ablation of Par3 mRNA from axons abolishes the outgrowth-promoting effect of NGF. These results identify a new role for local translation and the PAR complex in axonal outgrowth.

Show MeSH