Ganglioside-mediated enhancement of the cytoskeletal organization and activity in neuro-2a neuroblastoma cells

Abstract

Our previous studies 21,22 have demonstrated that a mixture of bovine brain gangliosides (BBG) applied to Neuro-2a neuroblastoma cells markedly increased the degree and rate of neurite formation. In the present study, the cytoskeletal basis for BBG-mediated neurite outgrowth was investigated by comparing cells grown in the presence or absence of BBG (250 μ/ml). After 24–48 h, neurite morphology and the distribution of cytoskeletal components were analyzed with correlative whole-cell transmission electron microscopy, thin-section transmission electron microscopy and scanning electron microscopy. BBG treatment enhanced markedly the organization of the microfilamentous system, and had a less pronounced effect on the number and organization of microtubules. The most prominent changes in microfilament organization were in the distal segment of the neurite and the growth cone. BBG-treated cells had a complex cytoskeleton consisting of numerous bundles of microfilaments. These filament bundles were distributed into the secondary and tertiary neuritic branches. Cells grown in serum-depleted medium to stimulate neurite outgrowth, lacked these bundles of microfilaments, suggesting that the formation of microfilament bundles was not required for non-BBG-mediated neuritogenesis. The role that the cytoskeletal components play in BBG-induced neurite outgrowth was examined following disruption of microtubules or microfilaments with Colcemid and cytochalasin D, respectively. Simultaneous treatment of cells with BBG and Colcemid (0.25 μg/ml) at the time of plating resulted in cells with numerous spine-like projections which did not extend neurites. In contrast, the simultaneous treatment of cells with BBG and cytochalasin D (2 μg/ml) at the time of plating resulted in cells devoid of spines, but exhibiting anomalous neurite outgrowth consisting of many long, thin, unbranched neurites. These neurites lacked characteristic flattened growth cones and had a tendency to grow in a circular fashion. These results demonstrate that neurite outgrowth under microfilament-limiting conditions results in reduced neuritic branching while growth under microtubule-limiting conditions allows initiation, but prevents significant elongation. The different neuritic growth patterns induced by serum deprivation, ganglioside treatment or the various cytoskeletal disruptive agents reflect changes in the organization of microtubules and microfilaments. Our studies suggest that the organizational state and activity of these cytoskeletal elements determine neurite morphology. Microfilaments appear to be the primary determinants in ganglioside-mediated growth.