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

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 (SNAP25) combined with 3-dimensional imaging. SNAP25 was characterized in a rat motor neuron (MN) pathway following toxin intramuscular injections at various doses to determine whether SNAP25 is confined to MNs or also found in neighboring cells or nerve fibers within spinal cord (SC). Results demonstrated that SNAP25 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 SNAP25 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, SNAP25 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 SNAP25 may be incorporated into SNARE-protein complexes within the affected MNs.