The effects of MAG on neurons are dependent on p75^NTR. (A) Dissociated DRG neurons were incubated for 24 h with or without MAG–Fc and then immunostained with monoclonal antibody (TuJ1) recognizing the neuron-specific β–tubulin III protein. p75(+/+), wild type; p75(−/−), mice carrying a mutation in the p75^NTR gene. (B) Mean length of the longest neurite per neuron. Data are mean ± SEM. An asterisk indicates statistical significance (*p < 0.01, Student's t test). (C) Mean length of the longest neurite per neuron. Dissociated cerebellar neurons were incubated for 24 h with or without MAG–Fc.

MAG activates RhoA through a p75^NTR-dependent mechanism. (A) The effect of C3 transferase on MAG-treated DRG neurons from wild-type mice. Mean length of the longest neurite per neuron. Data are mean ± SEM. Asterisks indicate statistical significance (*p < 0.01, Student's t test). (B) Binding of MAG–Fc to 293 cells was visualized by incubation with an FITC-tagged anti–human IgG. (C) Affinity precipitation of RhoA in transfected 293 cells. MAG–Fc (25 μg/ml) elicits activation of RhoA only when 293 cells express p75^NTR.

Affinity precipitation of RhoA in postnatal cerebellar neurons. (A) RhoA activity was increased after the addition of MAG–Fc (25 μg/ml). RhoA activity is indicated by the amount of RBD-bound RhoA normalized to the amount of RhoA in the lysates. Values represent RhoA activity relative to the cells at time 0. Results are means ± SE from three experiments. Asterisks indicate statistical significance (*p < 0.01, Student's t test). (B) NGF rapidly inhibits RhoA activity (∼10 min). (C) Dose response. (D) The activation was lost in the neurons from mice carrying a mutation in the p75^NTR gene.

Colocalization of p75^NTR and MAG binding. (A) DRG neurons were stained with the anti-p75^NTR antibody and an Alexa fluor™ 568–conjugated secondary antibody. Binding of MAG–Fc was visualized by incubation with the FITC-tagged anti–human IgG. Confocal microscopy was performed on a ZEISS LSM-510 laser scanning microscope. Representative single optical sections for p75^NTR (left), MAG binding (middle), and overlay images (right) are shown. Close association of these markers on the neurites was seen in almost all of the neurons with p75^NTR immunoreactivity. (B) Binding of MAG–Fc to DRG neurons from mice carrying a mutation in the p75^NTR gene.

Association of MAG, p75^NTR, and GT1b. (A) Coprecipitation of p75^NTR and MAG–Fc using lysates prepared from P9 cerebellum. In the MAG–Fc precipitates, the anti-p75^NTR antibody revealed the presence of a protein corresponding to p75^NTR. (B) Coprecipitation of recombinant p75^NTR and GT1b. Association was examined by Western blot analysis of the precipitates produced with protein A sepharose and Fc-fused protein of p75^NTR. The anti-GT1b antibody revealed the presence of a 100-kD protein (left), which was shown to be p75^NTR by the anti-p75^NTR antibody (right). (C) Coprecipitation of recombinant p75^NTR and other gangliosides. (D) Coimmunoprecipitation of p75^NTR and GT1b using lysates prepared from P9 cerebellum. In the GT1b immunoprecipitates, the anti-p75^NTR antibody revealed the presence of a protein corresponding to p75^NTR. The bottom bands correspond to the Ig of the antibodies used. (E) Coimmunoprecipitation of p75^NTR and GT1b using transfected 293 cells. In the p75^NTR immunoprecipitates, the anti-GT1b antibody revealed the presence of a protein (left), which was shown to be p75^NTR by the anti-p75^NTR antibody (right).