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

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. (Journal of Biomolecular Screening 2007:370-377)

Phenotypic effect of botulinum toxin A onCaenorhabditis elegans

“…BoTX/A cleaves SNAP-25 present in neurons of affected animals including mammals and chicken (Stahl et al 2007). However, exocytosis of Xenopus is significantly inhibited by BoTX/A, despite of relatively low sequence preservation in SNAP-25, near the cleavage site for BoTX/A (Yao et al 1999;Kanno et al 2009; Figure 6).…”

“…Therefore, a variety of assay system has been developed to determine the activity of the toxin, utilizing model systems including chick neurons (Stahl et al 2007). However, it is unknown whether BoTX/A has a physiological effect on Caenorhabditis elegans.…”

Progress in Cell Based Assays for Botulinum Neurotoxin Detection

“…Research on primary cells has helped immensely in our understanding of the biology and characteristics of BoNTs, including SNARE protein cleavage, inhibition of neurotransmitter release, duration of action, and cell entry mechanisms., A variety of primary cells derived from rat, mouse, or chicken embryos have been used (Keller et al 2004;Pellett et al 2007Pellett et al , 2010Sheridan et al 2005;Stahl et al 2007;Bigalke et al 1985). Among these are cortical neurons Stahl et al 2007;Saadi et al 2012), spinal cord neurons (Keller et al 2004;Pellett et al 2007Pellett et al , 2010Bigalke et al 1985), hippocampal neurons (Verderio et al 2007;Dong et al 2007), and dorsal root ganglion cells (Welch et al 2000;Stahl et al 2007;Duggan et al 2002).…”

“…Spinal cord cells or motor-neuron-enriched spinal cord cells would be expected to be the most relevant and sensitive BoNT cell model based on the neuromuscular symptomatology of botulism. However other primary cells such as cortical neurons, hippocampal cells, and dorsal root ganglion cells also seem to be exquisitely sensitive to BoNTs and have been used as study models Welch et al 2000;Stahl et al 2007;Saadi et al 2012;Verderio et al 2007;Dong et al 2007;Duggan et al 2002).…”

Current Methods for Detecting the Presence of Botulinum Neurotoxins in Food and Other Biological Samples

“…A number of different neuronal and nonneuronal derived cell lines have been generated for use in BoNT assays. These include: rat spinal cord cells (Pellett et al, 2007); chick embryo neuronal cells (Stahl et al, 2007); neuroblastoma cells N2A (Eubanks et al, 2007); and BE(2)-M17 cells (Hale et al, 2011). The read-out for most of the cell-based assays for detection of BoNT/A is the cleavage of SNAP-25.…”