Figure 4.
The R377Q and R377W-NgR1 variants are dominant-negative disruptors of myelin signaling. A, Chick E7 retinal neurons were cultured with or without herpes simplex virus directing the expression of WT-NgR, R377Q-NgR1, or R377W-NgR1 as indicated. The cultures were exposed to 100 nm GST (glutathione S-transferase)-Nogo-66, for 30 min, and then fixed and stained with phalloidin for F-actin and with anti-NgR1 antibody. Note that the viruses drive the expression of NgR1 protein. WT-NgR1 allows growth cones to collapse in response to Nogo-66, but R-377Q-NgR1 does not. B, The fraction of collapsed E7 retinal growth cones expressing WT-NgR1 is significantly greater than that for growth cones expressing GFP after exposure to 100 nm Nogo-66 (*p < 0.05, one-way ANOVA). C, Neurite outgrowth over 16 h from E20 rat DRG was assessed on a monolayer of control or MAG-expressing CHO cells after infection of neurons with virus expressing GFP, WT-NgR, R377Q-NgR1, or R377W-NgR. Neurons are visualized with anti-βIII-tubulin immunohistology. D, Mean neurite outgrowth from neurons expressing WT-NgR1 is significantly decreased on MAG cells relative to control monolayers (*p < 0.05, one-way ANOVA). Outgrowth from neurons expressing variant NgR1 is not reduced by MAG. E, The percentage of collapsed E13 chick DRG growth cones expressing the indicated proteins is reported from cultures similar to those in A. The values with R377Q-NgR1 and R377W-NgR1 virus are significantly different from those with control GFP virus (*p < 0.05, one-way ANOVA). F, Growth cone collapse of E13 chick DRG growth cones in the presence of 100 nm Nogo-66, 100 nm MAG-Fc, 3 μg/ml myelin protein, or 10 nm Sema3A. Collapse was assessed without peptide, with 1 μm WT peptide, with 1 μm R377Q peptide, or with 1 μm R377W peptide. Note that the R377W peptide suppresses collapse by Nogo-66, MAG, and myelin but not by Sema3A. Values with the variant NgR1 peptides are significantly different from those with the WT NgR1 peptide (*p < 0.05, one-way ANOVA). G, E13 chick DRG growth cone collapse in presence of 100 nm MAG-Fc and various concentrations of R377W peptide. All data are mean ± SEM from n = 3–7 experiments.
