Tetanus toxin association with developing neuronal cell cultures. Kinetic parameters and evidence for ganglioside-mediated internalization.

Rat cerebral neurons maintained in monolayer culture accumulate 125I-labeled tetanus toxin. Accumulation is receptor-mediated; i.e. it can be prevented by including unlabeled tetanus toxin, gangliosides, or tetanus antitoxin in the incubation medium but not by including tetanus toxoid, high concentrations of serum, or thyrotropin. Accumulation is time-dependent, reaching a plateau after approximately 3 h when 60% of the added toxin is associated with the cells. It is better at 0 degrees C than at ambient temperature and is significantly higher when 0.25 M sucrose replaces physiological salts in a medium containing 5% serum. Unlabeled tetanus toxin, tetanus antitoxin, and tetanus toxoid do not release the accumulated 125I-labeled tetanus toxin to any significant degree; however, gangliosides (50 micrograms/ml) can release 30% of the accumulated 125I-labeled toxin. Treatment of cells with Triton X-100, under conditions where over 90% of the lipids and 70% of the gangliosides are removed, extracts only 15% of the cell-associated 125I-labeled toxin. Evidence is presented that over 50% of the accumulated toxin is internalized in a cellular compartment which is not in immediate equilibrium with the extracellular environment and which is associated with detergent-insoluble cellular constituents. The tetanus toxin accumulated in this compartment has the same gel electrophoretic pattern as the native toxin and is bioactive. The role of gangliosides as potential shuttle vehicles for tetanus toxin internalization is discussed as are the implications of these data to in vitro studies of the pathogenesis of tetanus-induced neurotoxicity.