The Nogo-66 Receptor NgR1 Is Required Only for the Acute Growth Cone-Collapsing But Not the Chronic Growth-Inhibitory Actions of Myelin Inhibitors

shRNAi knock-down of NgR1 in cortical neurons does not release MAG inhibition. A, Cortical neurons of E18.5 rats were either transfected with pEGFP alone or pEGFP and shRNAi using the Amaxa nucleofection technology. Three days after transfection, NgR1 expression in GFP⁺ neurons was analyzed by anti-NgR1 and anti-GFP double-immunofluorescence labeling. The single arrowheads indicate a transfected neuron that is GFP⁺ and NgR1⁺; the double arrowheads indicate transfected neurons with successful knock-down of NgR1. The asterisks show untransfected NgR1⁺ neurons. Scale bar, 50 μm. B, Western blot analysis for NgR1 of PC12^NgR1 cells and primary cortical neurons either transfected with pEGFP or double-transfected with pEGFP plus NgR1 shRNAi. Lysates of NgR1-shRNAi transfected cells show a decrease in total NgR1 expression. Actin immunoblotting is shown as a loading control. C, Quantification of neurite outgrowth on CHO (gray bars) and CHO-MAG (black bars) feeder cells of GFP⁺ neurons of pEGFP transfected and pEGFP plus shRNAi double-transfected cultures revealed strong and comparable inhibition. D, E18.5 cortical neurons of wild-type (NgR1^+/+) and mutant (NgR1^−/−) mice are strongly inhibited on CHO-MAG feeder cells. Results are presented as mean ± SEM from six independent experiments for shRNAi. *p < 0.05, significantly different from neurons grown on CHO feeder cells; n.s. indicates that there was no significant difference in neurite growth between control and NgR1-depleted neurons [Kruskal–Wallis one-way ANOVA (post hoc Dunn's test)].