title: Neurotransmitter vesicle release from human model neurons (NT2) is sensitive to botulinum toxin A.
abstract: Botulinum neurotoxins (BoNTs) internalize into nerve terminals and block the release of neurotransmitters into the synapse. BoNTs are widely used as a therapeutic agent for treatment of movement disorders and recently gained more attention as a biological weapon. Consequently, there is strong interest to develop a cell-based assay platform to screen the toxicity and bioactivity of the BoNTs. In this study, we present an in vitro screening assay for BoNT/A based on differentiated human embryonal carcinoma stem (NT2) cells. The human NT2 cells fully differentiated into mature neurons that display immunoreactivity to cytoskeletal markers (βIII-tubulin and MAP2) and presynaptic proteins (synapsin and synaptotagmin I). We showed that the human NT2 cells undergo a process of exo-endocytotic synaptic vesicle recycling upon depolarization with high K(+) buffer. By employing an antibody directed against light chain of BoNT/A, we detected internalized toxin as a punctate staining along the neurites of the NT2 neurons. Using well-established methods of synaptic vesicle exocytosis assay (luminal synaptotagmin I and FM1-43 imaging) we show that pre-incubation with BoNT/A resulted in a blockade of vesicle release from human NT2 neurons in a dose-dependent manner. Moreover, this blocking effect of BoNT/A was abolished by pre-adsorbing the toxin with neutralizing antibody. In a proof of principle, we demonstrate that our cell culture assay for vesicle release is sensitive to BoNT/A and the activity of BoNT/A can be blocked by specific neutralizing antibodies. Overall our data suggest that human NT2 neurons are suitable for large scale screening of botulinum bioactivity.
authors.affiliation: Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Hannover, Germany.
authors.forename: Million Adane
authors.initials: MA
authors.lastname: Tegenge
authors.forename: Helge
authors.initials: H
authors.lastname: Böhnel
authors.forename: Frank
authors.initials: F
authors.lastname: Gessler
authors.forename: Gerd
authors.initials: G
authors.lastname: Bicker
mesh_terms: D057134:Antibodies, Neutralizing / Q000494:pharmacology; D019274:Botulinum Toxins, Type A / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism; D013572:Synaptic Vesicles / Q000187:drug effects* / Q000378:metabolism*; D018243:Teratocarcinoma / Q000473:pathology
chemical_list: D057134:Antibodies, Neutralizing; D018377:Neurotransmitter Agents; D019274:Botulinum Toxins, Type A