ABSTRACT
Regulated secretion is critical for diverse biological processes ranging from immune and endocrine signaling to synaptic transmission. Botulinum and tetanus neurotoxins, which specifically proteolyze vesicle fusion proteins involved in regulated secretion, have been widely used as experimental tools to block these processes. Genetic expression of these toxins in the nervous system has been a powerful approach for disrupting neurotransmitter release within defined circuitry, but their current utility in the brain and elsewhere remains limited by lack of spatial and temporal control. Here we engineered botulinum neurotoxin B so that it can be activated with blue light. We demonstrate the utility of this approach for inducibly disrupting excitatory neurotransmission, providing a first-in-class optogenetic tool for persistent, light-triggered synaptic inhibition. In addition to blocking neurotransmitter release, this approach will have broad utility for conditionally disrupting regulated secretion of diverse bioactive molecules, including neuropeptides, neuromodulators, hormones, and immune molecules. VIDEO ABSTRACT.
Subject(s)
Botulinum Toxins/pharmacology , Optogenetics/methods , Synaptic Transmission/drug effects , Animals , Arabidopsis Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Botulinum Toxins/genetics , Botulinum Toxins/radiation effects , Caenorhabditis elegans , Cells, Cultured , Cryptochromes/genetics , Female , HEK293 Cells , Humans , Light , Male , Mice , Mice, Inbred C57BL , Rats , Rats, Sprague-Dawley , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Recombinant Proteins/radiation effects , SNARE Proteins/metabolism , Synapses/metabolism , Synapses/physiology , Vesicle-Associated Membrane Protein 2/metabolismABSTRACT
The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation.
