Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis.

Abstract

Abstract. Using pharmacological (Simpson, L. L., 1980, J. Pharmacol. Exp. Ther. 212:16-21) and autoradiographic techniques (Black, J. D., and J. O. Dolly, 1986, J. Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT-induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with L~5I-BoNT. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target.

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

“…In line with previous studies using radioiodinated BoNT/A to label murine motor nerve terminals (27,57,58), gold-tagged BoNT/A-Hc bound predominantly to the membrane of hippocampal neurons at the level of nerve terminals and was internalized in response to stimulation. In the presynaptic nerve terminals, BoNT/A-Hc was detected predominantly in the lumen of synaptic vesicles, with a smaller proportion present in clathrin-coated structures and early endosomes.…”

Drug Insight: biological effects of botulinum toxin A in the lower urinary tract

“…[13][14][15][16] Studies on cultured hippocampal neurons from the rat or mouse phrenic nerve of the hemidiaphragm have shown that BTX-A recognizes and enters the ganglioside during neurotransmitter exocytosis when more-active receptors are exposed, exploiting the secretory vesicle recycling pathway. 17,18 Thus, endocytosis of BTX-A is enhanced by synaptic activity.…”

Development of vaccines for prevention of botulism

“…There is evidence suggesting that polysialogangliosides [13] or synaptotagmin [14,15] could act as receptors for the toxins but the data supporting a specific receptor remain equivocal [16]. After binding, the toxin is internalized into an endosome through receptor-mediated endocyctosis [17,18]. The amino-terminal half of the heavy chain (H N ) is believed to participate in the translocation mechanism of the light chain across the endosomal membrane [4,[19][20][21][22].…”