The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho

Abstract

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. The receptor for MAG or signals that elicit morphological changes in neurons remained to be established. Here we show that the neurotrophin receptor p75 (p75(NTR)) is the signal transducing element for MAG. Adult dorsal root ganglion neurons or postnatal cerebellar neurons from mice carrying a mutation in the p75(NTR) gene are insensitive to MAG with regard to neurite outgrowth. MAG activates small GTPase RhoA, leading to retarded outgrowth when p75(NTR)) is present. Colocalization of p75(NTR) and MAG binding is seen in neurons. Ganglioside GT1b, which is one of the binding partners of MAG, specifically associates with p75(NTR). Thus, p75(NTR) and GT1b may form a receptor complex for MAG to transmit the inhibitory signals in neurons.

Figure 1.

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.

Figure 1.

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.

Figure 2.

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.

Figure 2.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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).