Molecular targets of botulinum toxin at the mammalian neuromuscular junction.
The molecular targets of botulinum neurotoxins (BoNTs) are SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins necessary for neurotransmitter release. BoNT are powerful therapeutic agents in the treatment of numerous neurological disorders. The goals of this study were to (1) assess toxin diffusion by measuring substrate cleavage in adjacent and distant muscles, and (2) characterize the clinical course using SNARE protein chemistry. A small volume of BoNT/A was injected unilaterally into the mouse gastrocnemius muscle. Motor impairment was limited to the toxin-treated limb. No systemic illness or deaths occurred. At five time points, a subset of mice were killed, and muscles from both hindlimbs, and the diaphragm, were collected. Protein samples were examined for changes in SNAP-25 (synaptosomal-associated protein of Mr = 25 kDa) using immunochemistry. SNAP-25 cleavage product was noted in the toxin-treated limb as early as 1 day postinjection and continued through day 28. Onset and peak levels of substrate cleavage corresponded to the onset and peak clinical response. Cleavage was observed in adjacent and distant muscles, demonstrating that substrate cleavage is a sensitive indicator of toxin diffusion. Significant increases in full-length SNAP-25 and vesicle-associated membrane protein II were evident early in the impaired limb and continued through day 28. The increased SNARE protein most likely originates from nerve terminal sprouts.
Whelchel
Dorothy D
DD
Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, 30602, USA.
Brehmer
Tonia M
TM
Brooks
Paula M
PM
Darragh
Neely
N
Coffield
Julie A
JA
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001905:Botulinum Toxins / Q000494:pharmacology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009043:Motor Activity / Q000187:drug effects; D009132:Muscles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009469:Neuromuscular Junction / Q000166:cytology / Q000187:drug effects*; D009498:Neurotoxins / Q000494:pharmacology*; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D001905:Botulinum Toxins