Primary Cultures of Embryonic Chicken Neurons for Sensitive Cell-Based Assay of Botulinum Neurotoxin: Implications for Therapeutic Discovery

¹ U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland.
² Target Structure-Based Drug Discovery Group, SAIC-Frederick Inc., NCI-Frederick, Frederick, Maryland.

^* To whom correspondence should be addressed. E-mail: gordon.ruthel{at}amedd.army.mil; sina.bavari@amedd.army.mil.

	Abstract

Botulinum toxin is an exceedingly potent inhibitor of neurotransmission across the neuromuscular junction, causing flaccid paralysis and death. The potential for misuse of this deadly poison as a bioweapon has added a greater urgency to the search for effective therapeutics. The development of sensitive and efficient cell-based assays for the evaluation of toxin antagonists is crucial to the rapid and successful identification of therapeutic compounds. The authors evaluated the sensitivity of primary cultures from 4 distinct regions of the embryonic chick nervous system to botulinum neurotoxin A (BoNT/A) cleavage of synaptosomal-associated protein of 25 kD (SNAP-25). Although differences in sensitivity were apparent, SNAP-25 cleavage was detectable in neuronal cells from each of the 4 regions within 3 h at BoNT/A concentrations of 1 nM or lower. Co-incubation of chick neurons with BoNT/A and toxin-neutralizing antibodies inhibited SNAP-25 cleavage, demonstrating the utility of these cultures for the assay of BoNT/A antagonists.

Key Words: botulinum neurotoxin, chick neurons, SNAP-25

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