Article Figures & Data
Figures
- Figure 1.
MAG inhibits NgR1-deficient postnatal CGNs. A, P7 CGNs isolated from wild-type (NgR1+/+; n = 5) and NgR1-deficient (NgR1−/−; n = 5) mice were cultured on CHO control or CHO-MAG feeder layers and stained with TuJ1. Scale bar, 50 μm. B, Quantification of neurite lengths on CHO (gray bars) and CHO-MAG cells (black bars) revealed strong inhibition of both wild-type and NgR1−/− neurons on CHO-MAG cells (p < 0.05). MAG inhibition is fully reversed in the presence of Y27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide] (20 μm). The number of neurites quantified for each condition is indicated in parentheses. Results are presented as mean ± SEM from five independent experiments, and the percentile of neurite outgrowth compared with CHO controls is shown. No significant difference (n.s.) in neurite length is observed between wild-type and NgR1-deficient neurons cultured on CHO-MAG cells. Wild-type and NgR1−/− CGNs show robust and very similar growth on control CHO feeder cells [Kruskal–Wallis one-way ANOVA (post hoc Dunn's test)]. C, Western blot analysis for NgR1, NgR2, and p75NTR normalized to actin in P7 brain lysates of wild-type and NgR1 mutants. In wild-type brains, NgR1 protein is detected at an apparent molecular weight of 65 kDa. No NgR1 protein is detected in brain lysates of NgR1−/− mice. Loss of NgR1 does not result in altered expression levels of NgR2 or p75 protein.
- Figure 2.
NgR1 is dispensable for OMgp-mediated inhibition of neurite extension. A, Postnatal day 25 DRG neurons of wild-type (NgR1+/+; n = 3) and NgR1 mutants (NgR1−/−; n = 3) were either cultured on control substrate (PDL/laminin) or plates also containing recombinant Nogo-66 or OMgp. Neurons were labeled by TuJ1 immunofluorescence. Scale bar, 100 μm. B, Quantification of neurite lengths of wild-type (black bars) and NgR1−/− (gray bars) neurons. Compared with control substrate, Nogo-66 and OMgp substrates significantly inhibit growth of DRG neurons from wild-type mice (Nogo-66, p = 0.006; OMgp, p = 0.003) and NgR1−/− mice (Nogo-66, p = 0.001; OMgp, p = 0.0009). No significant (n.s.) difference in neurite outgrowth between wild-type and NgR1 mutant DRGs was observed. Results are presented as mean ± SEM from three independent experiments, and the number of neurites quantified for each condition is indicated in parentheses. Statistical analyses were done using Student's t test.
- Figure 3.
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 PC12NgR1 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)].
- Figure 4.
MAG- and OMgp-induced growth cone collapse is attenuated in NgR1-null neurons. A, Growth cones of postnatal DRG explants of wild-type (NgR1+/+) and NgR1 mutant (NgR1−/−) mice were treated with no inhibitor, AP-Sema3A (10 nm), MAG-Fc (100 nm), or OMgp-Fc (6 nm) for 30 min and stained with rhodamine-conjugated phalloidin. Scale bar, 10 μm. B, Quantification of collapsed growth cones. Results are presented as mean ± SEM from 6–16 independent experiments with NgR1+/+ (black bars) and NgR1−/− (gray bars). For MAG-Fc, n = 6 NgR1+/+ and n = 6 NgR1−/−; for OMgp-Fc, n = 9 NgR1+/+ and n = 10 NgR1−/−; for Sema3A, n = 15 NgR1+/+ and n = 16 NgR1−/−; and for no inhibitor, n = 15 NgR1+/+ and n = 16 NgR1−/−. The number of growth cones quantified for each condition is indicated in parentheses. *p < 0.001, significant difference between NgR1+/+ and NgR1−/− DRG neurons treated with MAG-Fc. **p < 0.001, significant difference between NgR1+/+ and NgR1−/− DRG neurons treated with OMgp-Fc [Kruskal–Wallis one-way ANOVA (post hoc Dunn's test)].
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental figure legends
- supplemental material - Figure 1
- supplemental material - Figure 2
