Botulinum neurotoxins A, B, C, E, and F preferentially enter cultured human motor neurons compared to other cultured human neuronal populations

Abstract

Human‐induced pluripotent stem cell (hiPSC)‐derived neurons can be exquisitely sensitive to botulinum neurotoxins (BoNTs), exceeding sensitivity of the traditionally used mouse bioassay. In this report, four defined hiPSC‐derived neuronal populations including primarily GABAergic, glutamatergic, dopaminergic, and motor neurons were examined for BoNT/A, B, C, D, E, and F sensitivity. The data indicate that sensitivity varies markedly for the BoNTs tested. Motor neurons are significantly more sensitive than other neuron types for all BoNTs except BoNT/D. Examination of SNARE protein levels and BoNT‐specific cell surface protein receptors reveals few differences between the cell types except greater expression levels of the receptor protein SV2C and synapsin‐IIa in motor neurons. This indicates that differential toxicity of BoNTs for motor neurons compared to other neuronal cell types involves multiple mechanisms.

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

“…Historically, these assays have used primary neuronal cultures derived from rodents or chickens, as well as continuous cell lines derived from cancer cells. In the past decade, an explosion of advancements in tissue culture techniques and genetics, materials, and derivation and culture of stem cell-derived neurons have enabled the refinement and optimization of several NCB assays that can reliably and quantitatively detect BoNTs with greater sensitivity and less variation than the mouse bioassay [96,98,[102][103][104][105][106][107][108][109][110].…”

“…Thus, when comparing a rodent and a human cell model for sensitivity to a specific BoNT, greater sensitivity in the human cell model does not necessarily imply greater sensitivity in vivo in humans, and mechanistic studies demonstrating human specificity would be required for that conclusion. In fact, a recent study has shown that BoNT sensitivity of various hiPSC derived neuronal cell models varies significantly for the same BoNT with motor-neurons being the most sensitive, and that the sensitivity of the same model varied significantly for various BoNT serotypes [110].…”

“…Early studies using radiolabeled BoNT/A or/B exposure of in vitro vertebrate tissue samples indicated a preferential selectivity for cholinergic motor nerve ending and minimal binding to non-neuronal cells, but also binding to in vitro exposed brain section regions [121,122]. Cell-based assays have shown large variability in the sensitivity of various cell models, with primary neuronal cell models and stem cell-derived neurons being the most sensitive, and hiPSC-derived motor neurons being more sensitive for most BoNTs than other hiPSC-derived neuronal classes [89,110]. However, further studies examining the molecular mechanisms controlling potential neuronal cell selectivity by BoNTs are required to confirm the preferential uptake of BoNTs by cholinergic neurons, as the results of in vivo studies could be due to pharmacodynamic properties of the toxins, and the results of the NCB studies could be due to extracellular or cell model-specific factors other than their cholinergic properties.…”

Neuronal selectivity of botulinum neurotoxins

“…BoNT/A1, BoNT/B1, BoNT/C, and BoNT/E are more potent in motor-neurons than in GABAergic, DOPAergic, and glutamatergic neurons (Pellett et al, 2019). Thereby, motorneurons are more sensitive than the other neuronal cell types to BoNTs.…”

“…Thereby, motorneurons are more sensitive than the other neuronal cell types to BoNTs. This greater sensitivity of motor-neurons can result from multiple factors (expression of the intracellular target, expression of the protein and ganglioside receptors, glucosylation of cell surface proteins, cell maturity and differentiation, and/or synaptic activity) (Pellett et al, 2019).…”