Abstract
The botulinum neurotoxins (BoNTs) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. No pharmacologic therapies have been identified that clinically relieve botulinum poisoning. The black widow spider venom α-latrotoxin (LTX) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling. The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons (ESNs), using a novel screening assay designed around the kinetics of BoNT/A activation. Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved SNAP-25 within 48 h, whereas treatment with 60 mM K+ had no effect. Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca2+ influx and evidence of excitotoxicity, though ESNs remained viable 48 h after LTX treatment. This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity. These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery. They further demonstrate that ESNs are a novel, responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery.
Highlights
The Clostridium botulinum neurotoxins (BoNTs) are the most poisonous substances known, with human toxicities estimated to be as low as 1–2 ng/kg [1]
We reported that 0.81 pM BoNT/A treatment for 24 h results in cleavage of 50% of SNAP-25 in embryonic stem cell-derived neurons (ESNs) [28]
In comparing the percent cleaved SNAP-25 at 24 h following either 3 or 24 h intoxication, we found that roughly three-quarters of toxin internalization occurs within the first few hours
Summary
The Clostridium botulinum neurotoxins (BoNTs) are the most poisonous substances known, with human toxicities estimated to be as low as 1–2 ng/kg [1]. Inhalation or injection, BoNTs gain access to the presynaptic termini of neuromuscular junctions and target the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins VAMP-2, SNAP-25, or syntaxin-1 for proteolysis [2]. Passive immunotherapy can reduce vascular toxin load, once the toxin is sequestered within the presynaptic terminus there are currently no therapeutic approaches that restore normal synaptic activity. Interference with toxin internalization or activation involves a short therapeutic window; delays the onset of paralysis but does not prevent intoxication; and does not appear to add significant clinical value to the current post-exposure prophylaxis offered by passive immunizations [3,4]. Once the toxin is cleared from poisoned nerve termini, the synapse must be regenerated and coordinated neuromuscular control re-established [7,8,9]
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