Botulinum neurotoxin type A-cleaved SNAP25 is confined to primary motor neurons and localized on the plasma membrane following intramuscular toxin injection

Neuroscience. 2017 Jun 3;352:155-169. doi: 10.1016/j.neuroscience.2017.03.049. Epub 2017 Apr 5.

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. SNAP25197 was characterized in a rat motor neuron (MN) pathway following toxin intramuscular injections at various doses to determine whether SNAP25197 is confined to MNs or also found in neighboring cells or nerve fibers within spinal cord (SC). Results demonstrated that SNAP25197 immuno-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 SNAP25197 signal 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, SNAP25197 was expressed throughout MNs and colocalized with synaptic markers on the plasma membrane at 6 days post-treatment. These data support previous studies suggesting that SNAP25197 may be incorporated into SNARE-protein complexes within the affected MNs.

Keywords: SNAP25; botulinum neurotoxin; motor neuron; neuromuscular junction; onabotulinumtoxinA; spinal cord.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Botulinum Toxins, Type A / pharmacology*
  • Cell Membrane / drug effects*
  • Dose-Response Relationship, Drug
  • Functional Laterality
  • Male
  • Microscopy, Confocal
  • Motor Neurons / drug effects*
  • Motor Neurons / ultrastructure
  • Muscle, Skeletal / cytology*
  • Muscle, Skeletal / drug effects
  • Nerve Tissue Proteins / metabolism
  • Neural Pathways / diagnostic imaging
  • Neuromuscular Agents / pharmacology*
  • Neuromuscular Junction / drug effects
  • Neuromuscular Junction / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, Nicotinic / metabolism
  • Synaptosomal-Associated Protein 25 / drug effects
  • Synaptosomal-Associated Protein 25 / metabolism*
  • Time Factors
  • Vesicular Acetylcholine Transport Proteins / metabolism
  • Vesicular Glutamate Transport Protein 1 / metabolism

Substances

  • Nerve Tissue Proteins
  • Neuromuscular Agents
  • Receptors, Nicotinic
  • Slc17a7 protein, rat
  • Slc18a3 protein, mouse
  • Snap25 protein, mouse
  • Synaptosomal-Associated Protein 25
  • Vesicular Acetylcholine Transport Proteins
  • Vesicular Glutamate Transport Protein 1
  • Botulinum Toxins, Type A