title: Botulinum neurotoxin type A-cleaved SNAP25 is confined to primary motor neurons and localized on the plasma membrane following intramuscular toxin injection.
abstract: The mechanism of action of botulinum neurotoxin type A (BoNT/A) is well characterized, but some published evidence suggests the potential for neuronal retrograde transport and cell-to-cell transfer (transcytosis) under certain experimental conditions. The present study evaluated the potential for these processes using a highly selective antibody for the BoNT/A-cleaved substrate (SNAP25197) combined with 3-dimensional imaging. SNAP25197was characterized in a rat motor neuron (MN) pathway following toxin intramuscular injections at various doses to determine whether SNAP25197is confined to MNs or also found in neighboring cells or nerve fibers within spinal cord (SC). Results demonstrated that SNAP25197immuno-reactive staining was colocalized with biomarkers for MNs, but not with markers for neighboring neurons, nerve fibers or glial cells. Additionally, a high dose of BoNT/A, but not a lower dose, resulted in sporadic SNAP25197signal in distal muscles and associated SC regions without evidence for transcytosis, suggesting that the staining was due to systemic spread of the toxin. Despite this spread, functional effects were not detected in the distal muscles. Therefore, under the present experimental conditions, our results suggest that BoNT/A is confined to MNs and any evidence of distal activity is due to limited systemic spread of the toxin at higher doses and not through transcytosis within SC. Lastly, at higher doses of BoNT/A, SNAP25197was expressed throughout MNs and colocalized with synaptic markers on the plasma membrane at 6 days post-treatment. These data support previous studies suggesting that SNAP25197may be incorporated into SNARE-protein complexes within the affected MNs.
authors.affiliation: Department of Biological Sciences, Allergan plc, Irvine, CA 92612, United States.
authors.forename: Brian B
authors.initials: BB
authors.lastname: Cai
authors.affiliation: Department of Biological Sciences, Allergan plc, Irvine, CA 92612, United States.
authors.forename: Joseph
authors.initials: J
authors.lastname: Francis
authors.affiliation: Department of Biological Sciences, Allergan plc, Irvine, CA 92612, United States; Department of Neurology, University of California, Irvine, CA, 92697, United States.
authors.forename: Mitchell F
authors.initials: MF
authors.lastname: Brin
authors.affiliation: Department of Biological Sciences, Allergan plc, Irvine, CA 92612, United States. Electronic address: broide_ron@allergan.com.
authors.forename: Ron S
authors.initials: RS
authors.lastname: Broide
mesh_terms: D000818:Animals; D019274:Botulinum Toxins, Type A / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D007839:Functional Laterality; D008297:Male; D018613:Microscopy, Confocal; D009046:Motor Neurons / Q000187:drug effects* / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000166:cytology* / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009434:Neural Pathways / Q000000981:diagnostic imaging; D009465:Neuromuscular Agents / Q000494:pharmacology*; D009469:Neuromuscular Junction / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D050494:Vesicular Acetylcholine Transport Proteins / Q000378:metabolism; D050598:Vesicular Glutamate Transport Protein 1 / Q000378:metabolism
chemical_list: D009419:Nerve Tissue Proteins; D009465:Neuromuscular Agents; D011978:Receptors, Nicotinic; C493466:Slc17a7 protein, rat; C493460:Slc18a3 protein, mouse; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D050494:Vesicular Acetylcholine Transport Proteins; D050598:Vesicular Glutamate Transport Protein 1; D019274:Botulinum Toxins, Type A
keywords: SNAP25; botulinum neurotoxin; motor neuron; neuromuscular junction; onabotulinumtoxinA; spinal cord