The internalization of a short acyl chain analogue of ganglioside GM1 in polarized neurons

Abstract

In order to study the endocytosis of membrane lipids during the development of neuronal polarity, we examined the internalization of a short acyl chain fluorescent derivative of ganglioside GM1, N-(6-(4-nitrobenz-2-oxa-1,3-diazole-7-yl)-aminohexanoyl)-GM1 (C6-NBD-GM1), in hippocampal neurons cultured at low density. C6-NBD-GM1 was internalized by temperature- and energy-dependent mechanisms, and after short times of incubation, accumulated in endosomes in the axon, cell body and dendrites of neurons maintained for up to 4–5 days in culture. C6-NBD-GM1 was subsequently transported in a retrograde direction to a pool of recycling endosomes in the cell body, with little transport to lysosomes, as indicated by the lack of degradation of C6-NBD-GM1 even after long times, and the re-appearance of intact C6-NBD-GM1 at the cell surface after recycling; similarly, little degradation of C6-NBD-GM1 was detected in N18TG-2 neuroblastoma cells. In hippocampal neurons maintained for longer than 6 days in culture, there was little internalization of C6-NBD-GM1 along the length of axons, but the amount of endocytosis from dendrites was similar to that observed in younger neurons. These results demonstrate that gangliosides turnover rapidly in dendritic membranes at all stages of neuronal development, whereas ganglioside turnover in axons is much less rapid, at least in mature, polarized neurons.