Robert E. Sheridan

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 neurotransmitter release from peripheral cholinergic nerve terminals by cleaving different 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 factors responsible for persistence of BoNT action. Extensor digitorum longus (EDL) muscles from rats were injected locally with 5 mouse LD(50) units of BoNT/A or 20 mouse LD(50) units of BoNT/E; these doses were selected to produce total paralysis of EDL muscles within 48 hr. Additional groups of rats were injected sequentially with either BoNT/A followed 48 h later by BoNT/E or 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 monitor 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 control at these time points. Preparations injected sequentially with BoNT/A followed by BoNT/E or with BoNT/E followed by BoNT/A exhibited slow recovery times resembling those recorded in the presence of BoNT/A alone. Pronounced atrophy of the EDL muscle was observed in rats injected with BoNT/A or in those receiving serotype combinations in either sequence, whereas no loss of muscle mass was observed in animals treated with BoNT/E alone. Data suggesting 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. Evidence that BoNT/E was able to cleave SNAP-25 at its characteristic site during sequential neurotoxin exposure was demonstrated by western blot analysis of cultured primary cortical neurons. Since the sequential exposure studies 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 intracellular stability of catalytically active BoNT/A light chain.

Reduced acetylcholine receptor density, morphological remodeling, and butyrylcholinesterase activity can sustain muscle function in acetylcholinesterase knockout mice†

Nerve‐evoked contractions were studied in vitro in phrenic nerve–hemidiaphragm preparations from strain 129X1 acetylcholinesterase knockout (AChE−/−) mice and their wild‐type littermates (AChE+/+). The AChE−/− mice fail to express AChE but have normal levels of butyrylcholinesterase (BChE) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE−/− mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild‐type animals. In AChE−/− preparations, repetitive stimulation at frequencies of 20 and 50 Hz and at 200 and 400 Hz produced decremental muscle tensions; however, stimulation at 70 and 100 Hz resulted in little or no loss of tension during trains. Muscles from AChE+/+ mice maintained tension at all frequencies examined but exhibited tetanic fade after exposure to the selective AChE inhibitor 1,5‐bis(4‐allyldimethyl‐ammoniumphenyl)pentane‐3‐one (BW 284C51). The ability of diaphragm muscles from AChE−/− mice to maintain tension at 70 and 100 Hz suggests a partial compensation for impairment of acetylcholine (ACh) hydrolysis. Three mechanisms—including a reliance on BChE activity for termination of ACh action, downregulation of nicotinic acetylcholine receptors (nAChRs), and morphological remodeling of the endplate region—were identified. Studies of neuromuscular transmission in this model system provide an excellent opportunity to evaluate the role of AChE without complications arising from use of inhibitors. Muscle Nerve 30: 317–327, 2004

A study of zinc-dependent metalloendopeptidase inhibitors as pharmacological antagonists in botulinum neurotoxin poisoning

Zinc-dependent metalloprotease inhibitors phosphoramidon, captopril and a peptide hydroxamate were studied as potential pretreatment compounds by examining their ability to delay the onset or to prolong the time to 50% block of nerve-elicited muscle twitch tension in the mouse phrenic-nerve diaphragm (in vitro at 36 degrees C) after botulinum neurotoxin serotypes A and B (BoNT-A, BoNT-B). Addition of BoNT-A or BoNT-B (1 x 10(-10) M) produced 50% block of the twitch response at 56 +/- 9 min and 76 +/- 4 min, respectively. Preincubation (45 min) of muscles with phosphoramidon (0.2 mM) prolonged the time to 50% block by 15 min in BoNT-B-poisoned muscles with no effect on the time-course of paralysis in BoNT-A exposed muscles. When the same quantities of BoNT-A or BoNT-B (equivalent to 1 x 10(-10) M bath concentration) were preincubated for 2 hr with phosphoramidon (equivalent to 0.2 mM final bath concentration), and the incubation mixture was added to the muscle chamber, the times to 50% block were prolonged by 38 min and 18 min for BoNT-B and BoNT-A, respectively. Preincubation of diaphragms with captopril (up to 10 mM) or peptide hydroxamate (75 microM) failed to antagonize BoNT-A or BoNT-B-induced neuromuscular block. Among the three metalloprotease inhibitors examined here, only phosphoramidon showed a significant protection against both serotypes of BoNT. A search for better inhibitor compounds specifically tailored to match the active site on BoNT molecule deserves attention.

The actions of a red tide toxin from Ptychodiscus brevis on single sodium channels in mammalian neuroblastoma cells

The actions of brevetoxin (PbTX‐3) were studied on single, voltage‐dependent sodium channels and whole‐cell currents from the neuroblastoma × glioma cell line NG108‐15. Purified PbTX‐3 shifted the activation of sodium channels to membrane potentials negative to normal. PbTX‐3 did not alter the single‐channel mean open lifetime, suggesting that the toxin does not change the rate of sodium channel inactivation from the open state. There was also no change in single‐channel conductance. These results indicate that brevetoxin increases sodium current at rest by shifting the voltage dependence of channel activation and that the resulting depolarization is limited by channel inactivation.

Structural features of aminoquinolines necessary for antagonist activity against botulinum neurotoxin.

Certain aminoquinoline antimalarial compounds, such as chloroquine, antagonize the paralytic actions of botulinum neurotoxins (BoNT). These studies have been extended to determine the critical structural groups necessary for synthetic aminoquinolines to have antagonist activity against BoNT. Isolated mouse hemidiaphragms were maintained at 36 degrees C and indirectly stimulated; the resulting isometric twitch tensions were recorded as a measure of synaptic function. The muscles were exposed to the test compounds before being treated with a challenge concentration of BoNT (typically 0.2 nM of serotype A). The time to onset of 50% muscle paralysis due to BoNT was used to assess quantitatively the efficacy of the test compounds, which were then ranked on the basis of the concentrations necessary to delay paralysis by a specified time increment. Of the compounds tested, those having a 7-chloro-4-aminoquinoline configuration, similar to chloroquine (or the structurally similar 6-chloro-9-amino acridine group in quinacrine), were most effective. Truncation of the alkyl-amino-alkyl group from chloroquine and conversion of the 4-amino nitrogen to a primary amine did not significantly alter its effectiveness as a BoNT antagonist. However, the 6-chloro- or 8-chloro- isomers of chloroquine were essentially ineffective. These results suggest that aminoquinolines antagonize the paralytic actions of BoNT through interaction with a selective, stereospecific site that is not well correlated with antimalarial activity.

Interactions between heavy metal chelators and botulinum neurotoxins at the mouse neuromuscular junction

Exposure of isolated mouse hemidiaphragms to botulinum neurotoxins, 0.1 nM BoNT-A or BoNT-B, at 36 degrees C reduced nerve-elicited peak isometric twitch tension to 50% of control values at 55 min (BoNT-A) to 68 min (BoNT-B) after application. Either coincubation of BoNT with the heavy metal chelator TPEN, preincubation with TPEN followed by BoNT, or application of TPEN after BoNT but before neuromuscular block, delayed the onset of muscle failure in a dose-dependent manner by up to five-fold. TPEN doses between 2 and 10 microM were required to antagonize significantly the muscle block produced by BoNT, and the delay in onset was maximal between 10 and 50 microM TPEN. Treatment of muscles with a Zn(2+)-TPEN coordination complex, rather than TPEN alone, eliminated any beneficial effects of TPEN on BoNT intoxication, indicating that these effects were mediated by chelation of Zn2+. Other metal chelators that were not as membrane permeant as TPEN were ineffective in delaying BoNT paralysis, suggesting that TPEN acts by chelating intraterminal Zn2+. In the absence of BoNT, TPEN caused a dose-dependent increase in nerve-elicited twitch tension with a half-maximal concentration at 8 microM. There was no corresponding change in twitches from direct electrical stimulation of the muscle. After BoNT (A or B serotype) had reduced the muscle twitch by 20 to 70%, however, subsequent application of TPEN rapidly depressed nerve-elicited twitches. The shift from potentiation to depression after BoNT treatment suggests that presynaptic vesicle mobilization and/or release involve Zn(2+)-dependent enzymes and that BoNTs interact with these enzyme pathways.

Brevetoxin depresses synaptic transmission in guinea pig hippocampal slices

Extracellular recordings were obtained from area CA1 of guinea pig hippocampal slices. PbTx-3, a brevetoxin fraction isolated from the red tide dinoflagellate Ptychodiscus brevis, was applied by bath perfusion. The toxin produced a concentration-dependent depression of the orthodromically evoked population spike with an EC50 of 37.5 nM. Brevetoxin concentrations below 10 nM were without effect, and concentrations above 100 nM led to total inhibition of evoked responses. PbTx-3 did not produce spontaneous synchronous discharges but did induce afterdischarges following evoked responses in about 50% of the slices tested, particularly at concentrations between 10 nM and 100 nM. Orthodromically evoked responses were more sensitive to PbTx-3 than were those elicited by antidromic stimulation. High-Ca2+ solution, 4-aminopyridine, and tetraethylammonium failed to antagonize either orthodromic or antidromic effects of the toxin. Although the precise mechanism by which PbTx-3 depresses evoked responses is not certain, depolarization of the presynaptic nerve terminals leading to failure of transmitter release could explain the toxins actions. This is the first report of the effects of brevetoxin applied directly to central nervous system tissue.

Anomalous enhancement of botulinum toxin type A neurotoxicity in the presence of antitoxin

The neutralization of botulinum toxin serotype A with polyclonal equine antitoxin was studied in isolated mouse hemidiaphragms and compared to the same action in live mice. The biological activity of the toxin in the isolated muscle could be markedly reduced with excess antitoxin, estimated as 3:1 molar ratios of IgG Ab:toxin or better. Toxin neutralization in vivo required higher ratios of Ab:toxin, ranging from 30:1 at high toxin doses and increasing to 100:1 at 10xLD50 toxin. At equimolar Ab to toxin ratios in the isolated muscle, the biological activity of the toxin underwent a statistically significant increase. This paradoxical effect of the polyclonal antisera was serotype selective and independent of the presence or absence of hemagglutinin in the toxin. The enhancement of toxin activity was subsequently localized to occupancy of one of four epitopes on the toxin using monoclonal antibodies to mimic the effect of the antitoxin. The enhancement of toxin activity suggests that botulinum toxin may undergo a conformational change upon binding antibodies to certain domains. This phenomenon could contribute to the observed concentration dependent changes in neutralization efficacy with antitoxin in vivo.

Differential actions of brevetoxin on phrenic nerve and diaphragm muscle in the rat

The mechanism of inhibition of skeletal muscle function by brevetoxin (PbTX-3) was examined in vitro in the rat phrenic nerve-diaphragm preparation. PbTX-3 in low concentrations (< 0.06 microM) preferentially blocked conduction in the phrenic nerve without altering the resting membrane potential of the muscle fibers. Endplate potential failure occurred in an all-or-none fashion in the presence of PbTX-3 (> 0.06 microM). An increase in the frequency of miniature endplate potentials resulting from nerve terminal depolarization was observed only after endplate potential failure. Higher concentrations of toxin (> 0.3 microM) depressed directly-elicited muscle twitches and produced significant muscle membrane depolarization. Tetrodotoxin was effective in reversing membrane depolarization and alterations in MEPP frequency caused by PbTX-3. These findings suggest that diaphragmatic failure in PbTX-3 is primarily caused by a block of impulse conduction in the phrenic nerve due to a higher sensitivity of nerve than muscle membrane to the toxin.

The effects of pumiliotoxin-B on sodium currents in guinea pig hippocampal neurons

The actions of pumiliotoxin-B, extracted from the skin of the frog Dendrobates pumilio, were examined on hippocampal slices and on acutely dissociated hippocampal neurons from the adult guinea pig. Application of 0.5-1 microM pumiliotoxin-B to hippocampal slices caused spontaneous, repetitive field discharges in the CA3 subfield. In whole-cell patch-clamp recordings of isolated CA1 and CA3 neurons, 1-2 microM pumiliotoxin-B shifted the midpoint of Na+ current activation by -11.4 +/- 1.1 mV. This shift was not dependent upon prior activation of the sodium channel. Pumiliotoxin-B did not block macroscopic Na+ inactivation but did reduce the apparent voltage-dependence of inactivation such that currents decayed faster at membrane potentials more negative than -30 mV. Single-channel recordings of sodium currents from excised membrane patches indicated that pumiliotoxin-B had little or no effect on channel closings due to entry into inactivated state(s) but did increase the rate of channel closings due to reversal of channel opening. The increase in the channel closing rate was consistent with a +8.7 mV shift in voltage sensitivity. Negative shifts in activation and positive shifts in closing rates implied a negative shift in the voltage-dependence of channel opening, suggesting that pumiliotoxin-B increases the rate of Na+ channel opening and closing in cells at rest, which could result in spontaneous activity in the neurons.