Neuronal Sensitivity to Tetanus Toxin Requires Gangliosides

Abstract

Tetanus toxin produces spastic paralysis in situ by blocking inhibitory neurotransmitter release in the spinal cord. Although di-and trisialogangliosides bind tetanus toxin, their role as productive toxin receptors remains unclear. We examined toxin binding and action in spinal cord cell cultures grown in the presence of fumonisin B 1 , an inhibitor of ganglioside synthesis. Mouse spinal cord neurons grown for 3 weeks in culture in 20 M fumonisin B 1 develop dendrites, axons, and synaptic terminals similar to untreated neurons, even though thin layer chromatography shows a greater than 90% inhibition of ganglioside synthesis. Absence of tetanus and cholera toxin binding by toxin-horseradish peroxidase conjugates or immunofluorescence further indicates loss of mono-and polysialogangliosides. In contrast to control cultures, tetanus toxin added to fumonisin B 1 -treated cultures does not block potassiumstimulated glycine release, inhibit activity-dependent uptake of FM1-43, or abolish immunoreactivity for vesicle-associated membrane protein, the toxin substrate. Supplementing fumonisin B 1 -treated cultures with mixed brain gangliosides completely restores the ability of tetanus toxin to bind to the neuronal surface and to block neurotransmitter release. These data demonstrate that fumonisin B 1 protects against toxin-induced synaptic blockade and that gangliosides are a necessary component of the receptor mechanism for tetanus toxin.Tetanus toxin (TeNT) 1 blocks the release of inhibitory neurotransmitters in the spinal cord leading to hyperactivity of motor neurons and consequent spastic paralysis (for review, see Ref. 1). The toxin is synthesized as a single polypeptide (M r ϭ 150,000), released by bacterial lysis, and cleaved to form an active dichain toxin with the heavy and light chains linked through one disulfide bond. The carboxyl-terminal half of the heavy chain constitutes the receptor binding domain, the amino-terminal half of the heavy chain is responsible for membrane translocation of the toxin, and the light chain contains the catalytic domain (for review, see Ref.2). Upon entering the synaptic cytosol, the toxin arrests neurotransmitter release by its action as a zinc endopeptidase, which cleaves vesicle-associated membrane protein (VAMP), a synaptic vesicle integral membrane protein believed to be critical for neuroexocytosis (3, 4). Whereas TeNT has been shown to bind to the surface of neurons and to act intracellularly (for reviews, see Refs. 2 and 5-7), its cell surface receptor(s) and mechanism of delivery to the cytosol are not well understood.Since the demonstration of the affinity of TeNT for gangliosides present on the neuronal surface (8), there have been a number of studies (for review, see Ref. 6) characterizing the binding of TeNT to gangliosides in various in vitro and in vivo preparations. Although it is difficult to assign an affinity to this binding (7), there is general agreement that TeNT shows the highest affinity for GT1b and GD1b polysialogangliosides, although not nearly as ...

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

“…The requirement for gangliosides as a component of the neuronal receptor(s) for TeNT is well established (9,10,29). Although numerous structural and biochemical studies have described the interactions of TeNT with gangliosides in vitro, few studies have addressed ganglioside function in vivo.…”

“…This suggests the simultaneous binding of 2 mol of GT1b to TeNT via the R and W pockets (9,14). Attempts to deplete endogenous gangliosides with PPMP in PC12 cells, reload exogenous gangliosides, and then measure VAMP cleavage were not successful because of PPMP toxicity to PC12 cells during extended incubations (data not shown).…”

“…Although the molecular basis for TeNT entry remains undetermined, an unambiguous role for gangliosides has been demonstrated (5)(6)(7)(8)(9). Current models implicate a dual receptor mechanism for the binding of the clostridial neurotoxins to neurons, which includes a ganglioside-binding component (10).…”

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

“…Complex gangliosides, a class of glycosphingolipids, which are particularly abundant in the outer leaflet of nerve cell membranes, were recognized to function as receptors for clostridial neurotoxins (CNTs) (5)(6)(7)(8). Their important role was recently pinpointed by inhibiting their biosynthesis (9)(10)(11). However, the existence of two classes of binding sites distinguished by different affinities and the discovery of protease-sensitive binding to neurons (12,13) resulted in a double-receptor concept.…”

Electrospray mass spectrometry of neuac oligomers associated with the c fragment of the tetanus toxin

“…oth botulinum and tetanus neurotoxins interact with motor neurons, blocking acetylcholine release and causing muscle paralysis and death (for an excellent review, see Niemann, 1991, [1]). The first step required for toxin entrance into neurons is the recognition of gangliosides at the cell surface by the receptor-binding domain of the heavy chain [2,3]. Following the binding of the toxin to the cell surface, an endosome forms around the toxin, and the translocation domain of the toxin transfers the catalytic domain, a zinc protease, into the cell.…”