title: Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research.
abstract: There are no pharmacological treatments to rescue botulinum neurotoxin (BoNT)-mediated paralysis of neuromuscular signaling. In part, this failure can be attributed to the lack of a cell culture model system that is neuron-based, allowing detailed elucidation of the mechanisms underlying BoNT pathogenesis, yet still compatible with modern cellular and molecular approaches. We have developed a method to derive highly enriched, glutamatergic neurons from suspension-cultured murine embryonic stem (ES) cells. Hypothesizing that ES cell-derived neurons (ESNs) might comprise a novel platform to investigate the neurotoxicology of BoNTs, we evaluated the susceptibility of ESNs to BoNT/A and BoNT/E using molecular and functional assays. ESNs express neuron-specific proteins, develop synapses and release glutamate in a calcium-dependent manner under depolarizing conditions. They express the BoNT substrate SNARE proteins SNAP25, VAMP2 and syntaxin, and treatment with BoNT/A and BoNT/E holotoxin results in proteolysis of SNAP25 within 24 h with EC50s of 0.81 and 68.6 pM, respectively. Intoxication with BoNT/A results in the functional inhibition of potassium-induced, calcium-dependent glutamate release. ESNs remain viable and susceptible to intoxication for up to 90 days after plating, enabling longitudinal screens exploring toxin-specific mechanisms underlying persistence of synaptic blockade. The evidence suggests that derived neurons are a novel, biologically relevant model system that combines the verisimilitude of primary neurons with the genetic tractability and scalable expansion of a continuous cell line, and thus should significantly accelerate BoNT research and drug discovery while dramatically decreasing animal use.
authors.affiliation: The United States Army Medical Research Institute of Chemical Defense, APG, MD 21010, USA. patrick.mcnutt@us.army.mil
authors.forename: Patrick
authors.initials: P
authors.lastname: McNutt
authors.forename: Jeremy
authors.initials: J
authors.lastname: Celver
authors.forename: Tracey
authors.initials: T
authors.lastname: Hamilton
authors.forename: Mariano
authors.initials: M
authors.lastname: Mesngon
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000633:toxicity*; D019274:Botulinum Toxins, Type A / Q000633:toxicity*; D002118:Calcium / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology*; D005089:Exocytosis / Q000187:drug effects; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology* / Q000187:drug effects*; D014176:Protein Biosynthesis; D013569:Synapses / Q000187:drug effects / Q000502:physiology; D046509:Tissue Culture Techniques
chemical_list: D018698:Glutamic Acid; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D002118:Calcium; C096326:botulinum toxin type E