Abstract
Clostridium botulinum neurotoxins (BoNTs) are the most poisonous naturally occurring protein toxins known to mankind and are the causative agents of the severe and potentially life-threatening disease botulism. They are also known for their application as cosmetics and as unique bio-pharmaceuticals to treat an increasing number of neurological and non-neurological disorders. Currently, the potency of biologically active BoNT for therapeutic use is mainly monitored by the murine LD50-assay, an ethically disputable test causing suffering and death of a considerable number of mice. The aim of this study was to establish an in vitro assay as an alternative to the widely used in vivo mouse bioassay. We report a novel BoNT detection assay using mouse embryonic stem cell-derived neurons (mESN) cultured on multi-electrode arrays (MEAs). After 21 days in culture, the mESN formed a neuronal network showing spontaneous bursting activity based on functional synapses and express the necessary target proteins for BoNTs. Treating cultures for 6 h with 16.6 pM of BoNT serotype A and incubation with 1.66 pM BoNT/A or 33 Units/ml of Botox® for 24 h lead to a significant reduction of both spontaneous network bursts and average spike rate. This data suggests that mESN cultured on MEAs pose a novel, biologically relevant model that can be used to detect and quantify functional BoNT effects, thus accelerating BoNT research while decreasing animal use.
Highlights
The botulinum neurotoxin (BoNT) are produced and secreted by the bacteria Clostridium botulinum, C. butyricum, and C. baratii and are amongst the most lethal substances known, with an estimated human lethal dose of 0.1–2 ng/kg if applied intravenously (Arnon et al, 2001; Simpson, 2004)
To elicit embryoid body (EB) formation and induce differentiation of the mouse embryonic stem cell (mESC), 1–2 × 106 cells were plated on day 0 in a 10 cm tissue culture dish (TPP, Trasadingen, Switzerland) and the medium was changed to a differentiation medium termed DMNK+ consisting of a 1:1 mixture of DMEM-F12/Glutamax and Neurobasal medium, supplemented with penicillin-streptomycin-glutamine to a final concentration of 1000 Units/ml penicillin, 1000 μg/ml streptomycin, and 0.292 mg/ml L-glutamine, 2-mercaptoethanol (0.1 mM), and 15% KnockOutTM Serum Replacement
Neuronal Cultures Derived from mESCs Form Synapses and Express the Proteins Necessary for BoNT/A Intoxication
Summary
The BoNTs are produced and secreted by the bacteria Clostridium botulinum, C. butyricum, and C. baratii and are amongst the most lethal substances known, with an estimated human lethal dose of 0.1–2 ng/kg if applied intravenously (Arnon et al, 2001; Simpson, 2004). The California Department of Public Health published data on the finding of a C. botulinum strain IBCA10-7060 that produced BoNT/B and another BoNT that could not be neutralized by any of the provided monovalent polyclonal botulinum antitoxins raised against BoNTs A–G (Barash and Arnon, 2014). The novel BoNT was thereupon described as a newly defined serotype H. This has been questioned by several groups and additional studies were recommended to confirm its identity (Johnson, 2014; Rossetto et al, 2014). Recent published data has shown that BoNT/H (alternatively termed BoNT/FA) has a chimeric structure containing regions similar to the structures of BoNT/A1, BoNT/F1, and BoNT/F5 (Kalb et al, 2015; Maslanka et al, 2016; Pellett et al, 2016)
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