Neuritogenic Actions of Botulinum Neurotoxin A on Cultured Motor Neurons

• Published in: The Journal of pharmacology and experimental therapeutics 2009-07, Vol.330 (1), p.352-358
• Main Authors: Coffield, Julie A., Yan, Xiuzhen
• Format: Article
• Language: English
• Subjects: Animals; Botulinum Toxins, Type A - pharmacology; Cells, Cultured; Cytoplasmic Vesicles - microbiology; Cytoplasmic Vesicles - physiology; Cytoplasmic Vesicles - secretion; Embryonic Stem Cells - cytology; Embryonic Stem Cells - microbiology; Embryonic Stem Cells - physiology; Female; Intracellular Fluid - microbiology; Intracellular Fluid - physiology; Mice; Motor Neurons - cytology; Motor Neurons - microbiology; Motor Neurons - physiology; Neurites - microbiology; Neurites - physiology; Neurogenesis - physiology; Pregnancy; Signal Transduction - physiology; Toxicology
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.108.147744

Botulinum neurotoxins (BoNTs) are extremely potent neuromuscular poisons that act through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein cleavage to inhibit neurotransmitter release. The ability of BoNT serotype A (BoNT/A) to eliminate localized transmitter release at extremely low doses is well characterized. In the current study, we investigated the less understood characteristic of BoNT/A to induce nerve outgrowth, sometimes referred to as sprouting. This phenomenon is generally considered a secondary response to the paralytic actions of BoNT/A, and other potential factors that may initiate this sprouting have not been investigated. Alternatively, we hypothesized that BoNT/A induces sprouting through presynaptic receptor activation that is independent of its known intracellular actions on the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) synaptosomal associated protein of 25 kDa (SNAP-25). To test this, the effects of BoNT/A application on neurite outgrowth were examined using primary cultures enriched with motor neurons isolated from embryonic mouse spinal cord. In this system, BoNT/A potently stimulated neuritogenesis at concentrations as low as 0.01 nM. The neuritogenic effects of BoNT/A exposure were concentration dependent and antagonized by Triticum vulgaris lectin, a known competitive antagonist of BoNT. Similar results were observed with the isolated BoNT/A binding domain, revealing that neuritogenesis could be initiated solely by the binding actions of BoNT/A. In addition, the presence or absence of SNAP-25 cleavage by BoNT/A was not a determinant factor in BoNT/A-induced neuritogenesis. Collectively, these results suggest that binding of BoNT/A to the motor neuronal membrane activates neuritogenesis through as yet undetermined intracellular pathway(s), independent of its known action on vesicular release.