The role of gangliosides in neurite extension of neuroblastoma cells

Abstract

An assay procedure for quantification of GM1 and other gangliotetraoses was developed. Gangliosides were absorbed onto multiwell ELISA plate and treated with neuraminidase (for GM1 this step was omitted) and then with cholera toxin B subunit (cholera B) conjugated with horseradish peroxidase. Color was developed and quantified spectrophotometrically. Linearity was obtained from 0.5 to 3 picomoles. Applying this procedure to 11 neuroblastoma lines revealed a general correlation between gangliotetraoses, particularly GM1, content in these cells and their neurite forming ability, either spontaneously or induced by serum deprivation, exogenous bovine brain gangliosides (BBG) or retinoic acid. Treatment of N2A, B104 and B50 with neuraminidase induced neurite sprouting, which was blocked by cholera B and anti-GM1 antibody, suggesting the effect is due to increase of GM1 on the membrane surface. This effect was also inhibited by partial reduction of Ca{dollar}\sp{lcub}2+{rcub}{dollar} or addition of La{dollar}\sp{lcub}3+{rcub}{dollar} in the culture medium, suggesting an involvement of Ca{dollar}\sp{lcub}2+{rcub}{dollar} in GM1 modulated neuritogenesis. A modest but significant enhancement of Ca{dollar}\sp{lcub}2+{rcub}{dollar} influx was caused in these 3 lines by neuraminidase, which was also blocked by cholera B. These results suggest endogenous GM1 has crucial role in neuronal differentiation and the one of the mechanisms by which GM1 may carry out is to modulate Ca{dollar}\sp{lcub}2+{rcub}{dollar} flux.;The effect of exogenous gangliosides was also Ca{dollar}\sp{lcub}2+{rcub}{dollar} dependent. BBG-induced neurite outgrowth of N2A cells was associated with increase in both Ca{dollar}\sp{lcub}2+{rcub}{dollar} influx and efflux. Depletion, but not partial reduction, of Ca{dollar}\sp{lcub}2+{rcub}{dollar} in the culture medium inhibit BBG-induced neurite outgrowth, in contrast, depletion of Ca{dollar}\sp{lcub}2+{rcub}{dollar} did not inhibit the neuritogenesis initiated by retinoic acid or dibutyryl cyclic AMP. They did not influence Ca{dollar}\sp{lcub}2+{rcub}{dollar} flux either. These results suggest neuritogenesis induced by BBG requires Ca{dollar}\sp{lcub}2+{rcub}{dollar} and involves modulation of Ca{dollar}\sp{lcub}2+{rcub}{dollar} flux which is probably by a mechanism different from endogenous GM1.