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Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling.

Joset A, Dodd DA, Halegoua S, Schwab ME - J. Cell Biol. (2010)

Bottom Line: Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system.Pincher-mediated macroendocytosis results in the formation of NogoDelta20-containing signalosomes that direct RhoA activation and growth cone collapse.Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain Research Institute, University of Zurich, Zurich, Switzerland. hatkic@hifo.uzh.ch

ABSTRACT
Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system. The Nogo-A-specific fragment NogoDelta20 induces growth cone collapse, and inhibits neurite outgrowth and cell spreading by activating RhoA. Here, we show that NogoDelta20 is internalized into neuronal cells by a Pincher- and rac-dependent, but clathrin- and dynamin-independent, mechanism. Pincher-mediated macroendocytosis results in the formation of NogoDelta20-containing signalosomes that direct RhoA activation and growth cone collapse. In compartmentalized chamber cultures, NogoDelta20 is endocytosed into neurites and retrogradely transported to the cell bodies of dorsal root ganglion neurons, triggering RhoA activation en route and decreasing phosphorylated cAMP response element binding levels in cell bodies. Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.

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Internalization of NogoΔ20 occurs by macroendocytosis and is Pincher and Rac1 dependent. (A) PC12 cells were transfected with HA-tagged dn PincherG68E (red). 24 h after transfection, cells were incubated with 300 nM NogoΔ20-T7 (green) for 30 min at 37°C. NogoΔ20 remained on the cell surface; no internalized NogoΔ20 vesicles are seen. (B) Transferrin (green) uptake is unaffected by overexpression of dn PincherG68E (red). (C and D) PC12 cells were transfected with T7-tagged wt Rac1 (C, red) or dn Rac1N17 (D, red) before incubation with 300 nM of Fc-tagged NogoΔ20 (green). After 30 min at 37°C, NogoΔ20 appeared in a large number of endosomal vesicles and cisterns inside the wt Rac1 cells (arrow), but the internalized proportion was reduced in the dn RacN17 transfected cells. Bar, 5 µm. (E) Quantification of NogoΔ20 and transferrin colocalization with dn PincherG68E. Values are given as the mean of three independent experiments ± SEM. (F) Quantification of internalization revealed that the uptake of NogoΔ20 was reduced by 84% upon overexpression of dn PincherG68E and by 73% upon overexpression of dn Rac1N17. Values are given as the mean from three independent experiments ± SEM (*, P < 0.05; ***, P < 0.001; Student’s t test).
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fig3: Internalization of NogoΔ20 occurs by macroendocytosis and is Pincher and Rac1 dependent. (A) PC12 cells were transfected with HA-tagged dn PincherG68E (red). 24 h after transfection, cells were incubated with 300 nM NogoΔ20-T7 (green) for 30 min at 37°C. NogoΔ20 remained on the cell surface; no internalized NogoΔ20 vesicles are seen. (B) Transferrin (green) uptake is unaffected by overexpression of dn PincherG68E (red). (C and D) PC12 cells were transfected with T7-tagged wt Rac1 (C, red) or dn Rac1N17 (D, red) before incubation with 300 nM of Fc-tagged NogoΔ20 (green). After 30 min at 37°C, NogoΔ20 appeared in a large number of endosomal vesicles and cisterns inside the wt Rac1 cells (arrow), but the internalized proportion was reduced in the dn RacN17 transfected cells. Bar, 5 µm. (E) Quantification of NogoΔ20 and transferrin colocalization with dn PincherG68E. Values are given as the mean of three independent experiments ± SEM. (F) Quantification of internalization revealed that the uptake of NogoΔ20 was reduced by 84% upon overexpression of dn PincherG68E and by 73% upon overexpression of dn Rac1N17. Values are given as the mean from three independent experiments ± SEM (*, P < 0.05; ***, P < 0.001; Student’s t test).

Mentions: The pinocytic chaperon protein Pincher belongs to the family of Eps15 homology (EH) domain–containing proteins (EHDs/RME-1), which have been implicated in clathrin-independent endocytosis (Shao et al., 2002; Valdez et al., 2007) and recycling from endosomes (Grant et al., 2001; Caplan et al., 2002). Overexpression of a dn form of Pincher (PincherG68E) has been shown to prevent NGF-induced internalization of TrkA (Shao et al., 2002). To assess a possible role of Pincher for NogoΔ20-T7 endocytosis, we overexpressed dn HA-PincherG68E in PC12 cells. In agreement with previous observations (Shao et al., 2002), HA-PincherG68E was associated with the plasma membrane in PC12 cells. Interestingly, the expression of dn Pincher dramatically reduced the endocytosis of NogoΔ20 (15.31 ± 3.68% vs. 99.46 ± 5.49% in control, n = 47 cells; ***, P < 0.001; Fig. 3, A and F). Confocal analysis revealed that NogoΔ20-T7 localization remained restricted to the plasma membrane and that 73.37 ± 2.91% of NogoΔ20-T7 colocalized with PincherG68E (Fig. 3, B and E). In contrast, transferrin was internalized and appeared in a punctate pattern in the cytoplasm of the PC12 cells, which is consistent with previous results showing that PincherG68E does not interfere with clathrin-mediated endocytosis (Shao et al., 2002). Accordingly, we observed only 9.74 ± 2.02% of transferrin overlap with PincherG68E (Fig. 3, B and E). These results indicate that Pincher function is essential for NogoΔ20 endocytosis.


Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling.

Joset A, Dodd DA, Halegoua S, Schwab ME - J. Cell Biol. (2010)

Internalization of NogoΔ20 occurs by macroendocytosis and is Pincher and Rac1 dependent. (A) PC12 cells were transfected with HA-tagged dn PincherG68E (red). 24 h after transfection, cells were incubated with 300 nM NogoΔ20-T7 (green) for 30 min at 37°C. NogoΔ20 remained on the cell surface; no internalized NogoΔ20 vesicles are seen. (B) Transferrin (green) uptake is unaffected by overexpression of dn PincherG68E (red). (C and D) PC12 cells were transfected with T7-tagged wt Rac1 (C, red) or dn Rac1N17 (D, red) before incubation with 300 nM of Fc-tagged NogoΔ20 (green). After 30 min at 37°C, NogoΔ20 appeared in a large number of endosomal vesicles and cisterns inside the wt Rac1 cells (arrow), but the internalized proportion was reduced in the dn RacN17 transfected cells. Bar, 5 µm. (E) Quantification of NogoΔ20 and transferrin colocalization with dn PincherG68E. Values are given as the mean of three independent experiments ± SEM. (F) Quantification of internalization revealed that the uptake of NogoΔ20 was reduced by 84% upon overexpression of dn PincherG68E and by 73% upon overexpression of dn Rac1N17. Values are given as the mean from three independent experiments ± SEM (*, P < 0.05; ***, P < 0.001; Student’s t test).
© Copyright Policy - openaccess
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fig3: Internalization of NogoΔ20 occurs by macroendocytosis and is Pincher and Rac1 dependent. (A) PC12 cells were transfected with HA-tagged dn PincherG68E (red). 24 h after transfection, cells were incubated with 300 nM NogoΔ20-T7 (green) for 30 min at 37°C. NogoΔ20 remained on the cell surface; no internalized NogoΔ20 vesicles are seen. (B) Transferrin (green) uptake is unaffected by overexpression of dn PincherG68E (red). (C and D) PC12 cells were transfected with T7-tagged wt Rac1 (C, red) or dn Rac1N17 (D, red) before incubation with 300 nM of Fc-tagged NogoΔ20 (green). After 30 min at 37°C, NogoΔ20 appeared in a large number of endosomal vesicles and cisterns inside the wt Rac1 cells (arrow), but the internalized proportion was reduced in the dn RacN17 transfected cells. Bar, 5 µm. (E) Quantification of NogoΔ20 and transferrin colocalization with dn PincherG68E. Values are given as the mean of three independent experiments ± SEM. (F) Quantification of internalization revealed that the uptake of NogoΔ20 was reduced by 84% upon overexpression of dn PincherG68E and by 73% upon overexpression of dn Rac1N17. Values are given as the mean from three independent experiments ± SEM (*, P < 0.05; ***, P < 0.001; Student’s t test).
Mentions: The pinocytic chaperon protein Pincher belongs to the family of Eps15 homology (EH) domain–containing proteins (EHDs/RME-1), which have been implicated in clathrin-independent endocytosis (Shao et al., 2002; Valdez et al., 2007) and recycling from endosomes (Grant et al., 2001; Caplan et al., 2002). Overexpression of a dn form of Pincher (PincherG68E) has been shown to prevent NGF-induced internalization of TrkA (Shao et al., 2002). To assess a possible role of Pincher for NogoΔ20-T7 endocytosis, we overexpressed dn HA-PincherG68E in PC12 cells. In agreement with previous observations (Shao et al., 2002), HA-PincherG68E was associated with the plasma membrane in PC12 cells. Interestingly, the expression of dn Pincher dramatically reduced the endocytosis of NogoΔ20 (15.31 ± 3.68% vs. 99.46 ± 5.49% in control, n = 47 cells; ***, P < 0.001; Fig. 3, A and F). Confocal analysis revealed that NogoΔ20-T7 localization remained restricted to the plasma membrane and that 73.37 ± 2.91% of NogoΔ20-T7 colocalized with PincherG68E (Fig. 3, B and E). In contrast, transferrin was internalized and appeared in a punctate pattern in the cytoplasm of the PC12 cells, which is consistent with previous results showing that PincherG68E does not interfere with clathrin-mediated endocytosis (Shao et al., 2002). Accordingly, we observed only 9.74 ± 2.02% of transferrin overlap with PincherG68E (Fig. 3, B and E). These results indicate that Pincher function is essential for NogoΔ20 endocytosis.

Bottom Line: Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system.Pincher-mediated macroendocytosis results in the formation of NogoDelta20-containing signalosomes that direct RhoA activation and growth cone collapse.Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain Research Institute, University of Zurich, Zurich, Switzerland. hatkic@hifo.uzh.ch

ABSTRACT
Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system. The Nogo-A-specific fragment NogoDelta20 induces growth cone collapse, and inhibits neurite outgrowth and cell spreading by activating RhoA. Here, we show that NogoDelta20 is internalized into neuronal cells by a Pincher- and rac-dependent, but clathrin- and dynamin-independent, mechanism. Pincher-mediated macroendocytosis results in the formation of NogoDelta20-containing signalosomes that direct RhoA activation and growth cone collapse. In compartmentalized chamber cultures, NogoDelta20 is endocytosed into neurites and retrogradely transported to the cell bodies of dorsal root ganglion neurons, triggering RhoA activation en route and decreasing phosphorylated cAMP response element binding levels in cell bodies. Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.

Show MeSH
Related in: MedlinePlus