Blockade of acetylcholine release by botulinum neurotoxin type A at the neuromuscular junction induces the formation of an extensive network of nerve-terminal sprouts. By repeated in vivo imaging of N-(3-triethyl ammonium propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide uptake into identified nerve endings of the mouse sternomastoid muscle after a single intramuscular injection of the toxin, inhibition of stimulated uptake of the dye at the terminals was detected within a few days, together with an increase in staining of the newly formed sprouts. After 28 days, when nerve stimulation again elicited muscle contraction, regulated vesicle recycling occurred only in the sprouts [shown to contain certain soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs) and to abut acetylcholine receptors] and not at the parent terminals. Therefore, only these sprouts could be responsible for nerve-muscle transmission at this time. However, a second, distinct phase of the rehabilitation process followed with a return of vesicle turnover to the original terminals, accompanied by an elimination of the by then superf luous sprouts. This extension and later removal of ''functional'' sprouts indicate their fundamental importance in the repair of paralyzed endplates, a finding with ramifications for the vital process of nerve regeneration.The ability of nerve endings at the neuromuscular junction to sprout after the potent blockade of neurotransmission by botulinum neurotoxin type A (BoTx͞A) is a striking example of synaptic plasticity (1-4). Although significant progress already has been made in identifying the factors triggering such outgrowth (see ref. 5), as well as that induced on, for example, partial denervation by nerve crush (1, 6), the significance of endplate remodeling in the intricate series of interactions between presynaptic and postsynaptic cells during recovery of transmission at paralyzed synapses has yet to be shown. The selective blockade of the regulated exocytosis of acetylcholine (ACh) by BoTx͞A after its intracellular proteolytic cleavage of synaptosomal-associated protein with a molecular mass of 25 kDa ref. 7 and 8) causes an unique and long-term eradication of synaptic activity, while, advantageously, avoiding removal of the nerve endings (9, 10). Despite the extent of the paralysis induced by this toxin, recovery of neurotransmission does occur eventually, as manifested in animal experiments (2, 11-13) and in the clinical treatment of dystonias involving involuntary movement of certain skeletal muscles (see ref. 14). Although BoTx͞A is known to promote nerve sprouting, it has remained unclear what the precise contribution, if any, these newly formed outgrowths make to the recovery from this initially dramatic, but ultimately temporary, paralysis.Limitations encountered in earlier studies on this fundamentally important question originated from the need to employ conventional histological techniques in vitro to excised tissues. These limitations have been overcome in our stu...

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Specific proteolysis by the tetanus toxin light chain of a vesicle-associated membrane protein (VAMP) involved in exocytosis is thought to underlie its intracellular blockade of neurotransmitter release. To substantiate this mechanism, recombinant light chain was expressed as a maltose binding protein-light chain fusion product in Escherichia coli. After purification of affinity chromatography and cleavage with factor Xa, the resultant light chain was isolated and its identity confirmed by Western blotting and N-terminal sequencing. It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-residue synthetic polypeptide spanning the cleavage site of the substrate. The importance of Glu234 in the catalytic activity of the light chain, possibly analogous to Glu143 of thermolysin, was examined using site-directed mutagenesis. Changing Glu234 to Ala abolished the protease activity of the light chain, but its ability to bind the polypeptide substrate was retained. Each recombinant light chain could be reconstituted with the heavy chain of tetanus toxin, yielding the same level of disulfide-linked species as the two native chains. Whereas the toxin formed with wild-type light chain exhibited appreciable neuromuscular paralysis activity and mouse lethality, the equivalent dichain material containing the Ala234 mutant lacked neurotoxicity in both the in vitro and in vivo assays. Thus, these results demonstrate directly, for the first time, that the lethality of tetanus toxin and its inhibition of exocytosis in intact neurons are attributable largely, if not exclusively, to endoprotease activity.

Although botulinum neurotoxin (BoNT) types A and B and tetanus toxin (TeTx) are specific inhibitors of transmitter release whose light chains contain a zinc-binding motif characteristic of metalloendoproteases, only the latter two proteolyse synaptobrevin. Chelation of zinc or its readdition at high concentration hindered blockade of neuromuscular transmission by BoNT/A and B, indicating that type A also acts via a zinc-dependent mechanism. Such treatments prevented proteolysis of synaptobrevin II in rat brain synaptic vesicles by BoNT/B and TeTx but only the activity of the latter was antagonised appreciably by ASQFETS, a peptide spanning their cleavage site. The toxin's neuroparalytic activities were attenuated by phosphoramidon or captopril, inhibitors of certain zinc requiring proteases. However, these agents were ineffective in reducing the toxins' degradation of synaptobrevin except that a high concentration of captopril partially blocked the activity of TeTx but not BoNT/B, as also found for these drugs when tested on synaptosomal noradrenaline release. These various criteria establish that a zinc-dependent protease activity underlies the neurotoxicity of BoNT/A, a finding confirmed at motor nerve endings for type B and TeTx. Moreover, the low potencies of captopril and phosphoramidon in counteracting the toxins' effects necessitate the design of improved inhibitors for possible use in the clinical treatment of tetanus or botulism.