Persistence of botulinum neurotoxin A demonstrated by sequential administration of serotypes A and E in rat EDL muscle

Botulinum neurotoxin serotypes A (BoNT/A) and E (BoNT/E) inhibit neurotrans mitter release from peripheral cholinergic nerve terminals by cleaving diff erent sites on SNAP-25, a protein involved in synaptic vesicle docking and exocytosis. Since recovery from BoNT/A is protracted, but reversal of BoNT/ E intoxication is relatively rapid, it was of interest to determine whether sequential exposure to BoNT/A and BoNT/E could provide insight into the fa ctors responsible for persistence of BoNT action. Extensor digitorum longus (EDL) muscles from rats were injected locally with 5 mouse LD50 units of B oNT/A or 20 mouse LD50 units of BoNT/E; these doses were selected to produc e total paralysis of EDL muscles within 48 hr. Additional groups of rats we re injected sequentially with either BoNT/A followed 48 h later by BoNT/E o r with BoNT/E followed 48 h later by BoNT/A. Muscle tensions were elicited in situ in response to supramaximal stimulation of the peroneal nerve to mo nitor recovery from BoNT intoxication. Tensions returned to 53% and 94% of control, respectively, 7 and 15 days after injection of BoNT/E. In contrast , tensions in muscles injected with BoNT/A returned to only 2% and 12% of c ontrol at these time points. Preparations injected sequentially with BoNT/A followed by BoNT/E or with BoNT/E followed by BoNT/A exhibited slow recove ry times resembling those recorded in the presence of BoNT/A alone. Pronoun ced atrophy of the EDL muscle was observed in rats injected with BoNT/A or in those receiving serotype combinations in either sequence, whereas no los s of muscle mass was observed in animals treated with BoNT/E alone. Data su ggesting that BoNT/E can enter BoNT/A-treated preparations was obtained by findings that 3,4-diaminopyridine, which readily reversed muscle paralysis after BoNT/A exposure, lost this ability within 1 h of BoNT/E addition. Evi dence that BoNT/E was able to cleave SNAP-25 at its characteristic site dur ing sequential neurotoxin exposure was demonstrated by western blot analysi s of cultured primary cortical neurons. Since the sequential exposure studi es indicate that recovery from BoNT intoxication is lengthened by exposure to serotype A, but not shortened by exposure to serotype E, the duration of BoNT/A intoxication appears to be determined predominantly by the intracel lular stability of catalytically active BoNT/A light chain. Published by El sevier Science Ltd.