ReviewBotulinum neurotoxin type A: Actions beyond SNAP-25?
Introduction
Botulinum toxin type A (BoNT/A) is a protein derived from Gram (−) anaerobic bacterium Clostridium botulinum. BoNT/A and other botulinum toxin serotypes (B-G) cause a neuroparalytic disease called botulism in both animals and humans. At the same time in small doses it is widely used for treatment of pathological muscle contractions and autonomic hyperactivity. So far, BoNT/A has been registered for treatment of facial wrinkles, different movement disorders (strabism, blepharospasm, hemifacial spasm, cervical dystonia, upper limb spasticity), and autonomic disorders (primary axillar hyperhidrosis and neurogenic detrusor overactivity). Chronic migraine is the only pain disorder with approved BoNT/A use (Chen, 2012). In addition, there many other clinical conditions with reported BoNT/A efficacy. In small doses BoNT/A is safe and usually does not induce adverse distant effects, however, it is contraindicated in disorders with pronounced muscle weakness such as myasthenia gravis, Lambert–Eaton syndrome and lower motoneuron disease (Wheeler and Smith, 2013). BoNT/A enters neurons by endocytosis into synaptic vesicles, mediated by toxin’s heavy chain (HC) interaction with membrane protein acceptors (SV2 and FGF3) and gangliosides (Dong et al., 2006, Jacky et al., 2013, Rossetto et al., 2014). After the BoNT/A light chain (LC) is translocated into the synapse cytosol, it enzymatically cleaves a peptide bond on synaptosomal-associated protein of 25 kDa (SNAP-25), a synaptic protein localized on the inner side of plasma membrane (Blasi et al., 1993). SNAP-25 and two other proteins: syntaxin, and vesicle-associated membrane protein (VAMP)/Synaptobrevin forms heterotrimeric soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) complex, which mediates the fusion of vesicular and synaptic membrane. BoNT/A-mediated SNAP-25 protein cleavage prevents the membrane fusion and neurotransmitter exocytosis (Rossetto et al., 2014). In the periphery BoNT/A paralyses the neuromuscular junctions and autonomic synapses, while in central neurons it inhibits the excitatory neurotransmission (Verderio et al., 2004, Kato et al., 2013). Apart from synaptic neurotransmitter release, BoNT/A may prevent other SNARE-dependent physiological functions in both neuronal and non-neuronal cells (reviewed by Matak and Lacković, 2014).
Up to now, SNAP-25 is the only accepted BoNT/A target molecule, and it is widely believed that the therapeutic and toxic actions of BoNT/A are exclusively mediated by prevention of synaptic neurotransmitter release induced by SNAP-25 cleavage (Wheeler and Smith, 2013, Rossetto et al., 2014). Only recently it is being investigated if some of the BoNT/A actions might be mediated by other SNARE-dependent physiological functions, such as the involvement of membrane trafficking of receptors in the antinociceptive action of BoNT/A (Shimizu et al., 2012). However, in addition to BoNT/A actions mediated by SNAP-25, several observations suggest the actions apparently unassociated with SNAP-25 cleavage, linked to: (1) arachidonic acid pathway, (2) neuritogenesis, (3) cell cycle and apoptosis, and possibly (5) gene expression. In present review we summarize these observations and evaluate their significance.
Section snippets
BoNT/A and arachidonic acid-mediated neuroexocytosis
Arachidonic acid (AA) is formed from membrane phospholipids by different phospholipase A2 (PLA2) isoenzymes. In addition to its involvement in the synthesis of eicosanoids, AA promotes neurotransmitter release. BoNT/A effects on AA-mediated neuroexocytosis have been initially explored in a model of neuronal growth factor-differentiated pheochromocytoma-12 (PC-12) cell line (Ray et al., 1993). BoNT/A applied to the cell culture reduced the K+-stimulated acetylcholine and arachidonic acid
BoNT/A and neuritogenesis
A well known property of BoNT/A action is the induction of transient sprouting at the motor nerve terminals in the proximity of neuromuscular junctions (NMJ). Due to the similar time course of sprouting and paralysis, it has been assumed that BoNT/A effects on neuritogenic outgrowth at the NMJ represent a secondary response to synaptic paralysis mediated by SNAP-25 cleavage (Morbiato et al., 2007, Harrison et al., 2011, Jiang et al., 2014).
The apparent association of sprouting activity of
Effects on cell cycle and apoptosis
Karsenty et al. (2009) studied the effect of commercially available BoNT/A in human prostate cancer cell lines and prostate cancer xenografts in mice. BoNT/A dose-dependently reduced the mitotic index and increased the apopototic index in LNCaP cell lines at low BoNT/A concentrations (0.25–1 U/ml). In xenografts, BoNT/A reduced the tumor size and serum PSA levels (Karsenty et al., 2009). Proietti et al. (2012) performed similar experiments in LNCaP and PC-3 prostatic cancer cell lines, and at
In vitro and in vivo effects on gene expression
In line with possibility that the BoNT/A molecule may induce a more complex host response within the interacting cells, two studies employed a microarray analysis of gene expression in toxin-exposed cell cultures (Thirunavukkarasusx et al., 2011, Scherf et al., 2014). To mimic the food-borne toxin exposure, Thirunavukkarasusx et al. (2011) employed human intestine epithelial cell line (HT-29) (lacking SNAP 25) and human neuroblastoma cell line (SH-SY5Y). In HT-29 cells, 167 genes were
Relevance of BoNT/A interaction with targets beyond SNAP-25
Molecular interactions leading to SNAP-25-unrelated events may be related to either known of unknown BoNT/A binding abilities (Fig. 1). BoNT/A actions on neuritogenesis are most likely mediated via BoNT/A HC-binding molecules (SV2, FGF3, and polysialoglangliosides) which mediate its entrance into neurons (Coffield and Yan, 2009). Hypothetically, BoNT/A endopeptidase might cleave additional SNARE or non-SNARE proteins: BoNT/A at high concentration may cleave a non-neuronal SNAP-25 protein
Conclusion
The effects of BoNT/A described in the present review point to the existence of additional toxin actions unrelated to SNAP-25, occurring at pharmacologically relevant BoNT/A doses/concentrations. Proapoptotic and antitumor activity might be the most important novel BoNT/A action. Therefore, identification of additional mechanisms and target molecules might elucidate other pharmacological or toxic effects of BoNT/A.
Acknowledgements
Authors’ work is funded by Croatian Ministry of Science, Education and Sports (Project no. 101-1010003-0001), and Deutscher Academischer Austauch Dienst (DAAD). The authors declare no conflict of interest.
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