PMC

Ganglioside GD1a and the biosynthetic pathway for major brain gangliosides. [Ganglioside nomenclature is that of Svennerholm ().] Gangliosides are synthesized by sequential addition of sugars to ceramide by specific glycosyltransferases (). Pharmacological (P4) and genetic (GalNAcT −/−) blocks in the pathway are indicated.

Neurons from complex ganglioside knockout mice are less responsive to MAG-mediated neurite outgrowth inhibition. CGNs from GalNAcT heterozygote (A) and knockout mice (B) were grown for 48 h on substrata adsorbed with a detergent extract of myelin and then were immunostained for GAP-43. Images are presented as the reverse grayscale (black on white) for clarity. (Bar = 50 μm.) Neurite outgrowth inhibition from 25 heterozygote and 21 knockout littermates, quantified by image analysis as described in Materials and Methods (presented as mean ± SD), indicates that neurons lacking complex gangliosides are significantly less susceptible to MAG-mediated neurite outgrowth inhibition (C). *, P < 0.01.

IgG-class mouse mAbs against GD1a or GT1b attenuate MAG-mediated neurite outgrowth inhibition. CGNs were grown on PDL- (A) or MAG-adsorbed surfaces (B and C) in the absence (B) or presence (C) of 10 μg/ml of anti-GD1a monoclonal IgG. Images are presented as the reverse grayscale (black on white) for clarity. (Bar = 100 μm.) The effects of adding 10 μg/ml of the indicated antiganglioside Abs on MAG-mediated neurite outgrowth inhibition were quantified by image analysis (D), as described in Materials and Methods, and are presented as mean percent inhibition ± SD. *, P < 0.002; **, P < 0.02.

Clustering complex gangliosides on neuronal surfaces inhibits neurite outgrowth. CGNs grown on PDL-coated surfaces were treated for 48 h with 10 μg/ml of anti-GM1 mAb (A and B) or anti-GD1a mAb (C and D). Abs were added either alone (A and C) or after precomplexing for 1 h with anti-mouse IgG (5 μg/ml; B and D). Neurites were visualized by GAP-43 immunostaining, with images presented as the reverse grayscale (black on white) for clarity. (Bar = 100 μm.) Neurite outgrowth inhibition was quantified by image analysis (E) as described in Materials and Methods. *, P < 0.0002 (the small inhibitory effect of uncomplexed anti-GM1 mAb was not statistically significant, P > 0.15).

Neurite outgrowth inhibition by MAG is sialic acid-dependant. CGNs were plated on culture surfaces adsorbed with membranes of MAG-expressing CHO cells or detergent extracts of myelin for 48 h in the presence or absence of neuraminidase (Neu; 7.5 milliunits/ml) or anti-MAG Ab (513; 20 μg/ml). Neurite outgrowth was assessed by scoring the percent of cells with neurites longer than four cell bodies in multiple fields from duplicate wells per experiment. Data are normalized with respect to the appropriate control (membranes of control CHO cells or detergent-containing buffer, respectively). Data are presented as the mean ± SD of at least 3 independent experiments. *, P < 0.005.

Blocking glycosphingolipid biosynthesis reverses MAG-mediated inhibition of neurite outgrowth. Rat CGNs were treated without (control, A) or with (B) the glycosphingolipid biosynthetic inhibitor P4 (2.5 μM) for 48 h. Cell surface complex gangliosides were detected by neuraminidase treatment followed by labeling with FITC-cholera toxin B-subunit as described in Materials and Methods. Fluorescent images of the cholera B-subunit-labeled cultures (A and B) demonstrate nearly complete depletion of complex cell surface gangliosides after P4 treatment. In separate experiments, cells were cultured on MAG-containing inhibitory substrata in the absence (control, C) or presence of 2.5 μM P4 (D). After 48 h, cells were immunostained for GAP-43 and visualized by Cy3-conjugated secondary Ab to detect neurites [images are presented as the reverse grayscale (black on white) for clarity], revealing enhanced neurite outgrowth by P4-treated neurons. Neurite outgrowth inhibition, quantified as described in the legend to Fig. , is presented as the mean ± SD of at least 3 independent experiments (E). *, P < 0.005. (Bars = 50 μm.) Equivalent reversal of neurite outgrowth inhibition by P4 treatment was quantified with semiautomated image analysis (see Fig. 7).