BJD REVIEW ARTICLE British Journal of Dermatology The non-neuronal and nonmuscular effects of botulinum toxin: an opportunity for a deadly molecule to treat disease in the skin and beyond* S.A. Grando iD and C.B. Zachary Department of Dermatology, University of California, Irvine, Irvine, CA, U.S.A. Linked Comment: Ozog. Br J Dermatol 2018; 178:999. Summary Correspondence There is growing evidence that botulinum neurotoxins (BoNTs) exhibit biological Sergei A. Grando. effects on various human cell types with a host of associated clinical implications. E-mail: [email protected] This review aims to provide an update on the non-neuronal and nonmuscular Accepted for publication effects of botulinum toxin. We critically analysed recent reports on the structure 16 October 2017 and function of cellular signalling systems subserving biological effects of BoNTs. The BoNT receptors and intracellular targets are not unique for neurotransmission. Funding sources They have been found in both neuronal and non-neuronal cells, but there are None. differences in how BoNT binds to, and acts on, neuronal vs. non-neuronal cells. The non-neuronal cells that express one or more BoNT/A-binding proteins, and/or Conflicts of interest None to declare. cleavage target synaptosomal-associated protein 25, include: epidermal ker- atinocytes; mesenchymal stem cells from subcutaneous adipose; nasal mucosal cells; *Plain language summary available online urothelial cells; intestinal, prostate and alveolar epithelial cells; breast cell lines; neutrophils; and macrophages. Serotype BoNT/A can also elicit specific biological DOI 10.1111/bjd.16080 effects in dermal fibroblasts, sebocytes and vascular endothelial cells. Nontraditional applications of BoNT have been reported for the treatment of the following derma- tological conditions: hyperhidrosis, Hailey–Hailey disease, Darier disease, inversed psoriasis, aquagenic palmoplantar keratoderma, pachyonychia congenita, multiple eccrine hydrocystomas, eccrine angiomatous hamartoma, eccrine sweat gland naevi, congenital eccrine naevus, Raynaud phenomenon and cutaneous leiomy- omas. Experimental studies have demonstrated the ability of BoNT/A to protect skin flaps, facilitate wound healing, decrease thickness of hypertrophic scars, produce an anti-ageing effect, improve a mouse model of psoriasiform dermatitis, and have also revealed extracutaneous effects of BoNT arising from its anti-inflam- matory and anticancer properties. BoNTs have a much wider range of applications than originally understood, and the individual cellular responses to the cholinergic impacts of BoNTs could provide fertile ground for future studies. What’s already known about this topic? • Botulinum neurotoxins (BoNTs) have been used therapeutically in different medi- cal conditions, predominantly in relation to muscle relaxation. What does this study add? • Recent research indicates that serotype BoNT/A can also elicit specific biological effects in skin cells, leading to profound clinical changes in dermatological conditions. • BoNTs appear to have a wider range of applications than originally understood. © 2017 British Association of Dermatologists British Journal of Dermatology (2018) 178, pp1011–1019 1011 1012 Applications of botulinum toxins in dermatology, S.A. Grando and C.B. Zachary (Fig. 1). However, the BoNT receptors and intracellular targets The structure and function of botulinum are not unique for neurotransmission, as several of these neurotoxins receptors and targets have been found in both neuronal and non-neuronal cells. The structure of botulinum neurotoxins In nature, botulinum neurotoxin (BoNT) is produced by The non-neuronal cell types targeted by botulinum Clostridium botulinum bacteria. The distinct toxin subtypes, desig- neurotoxin nated A through G, have different structures and mechanisms of action. The A, B and E subtypes cause botulism in humans Based on published data, several types of non-neuronal cells owing to their action inside the axon terminal, leading to may be directly affected by BoNT in human skin and other paralysis of the respiratory muscles and death resulting from tissues that produce a biological effect. The cells expressing respiratory failure. Pure botulinum toxin A was first synthe- one or more of the BoNT/A-binding proteins SV2, FGFR3 or sized as an inactive 150-kDa protein complexed with varying vanilloid receptors, and/or BoNT/A cleavage target SNAP-25, 7,8 amounts of nontoxic companion proteins. Botulinum neuro- include epidermal keratinocytes, mesenchymal stem cells 9 10 toxin is activated when the polypeptide chain is proteolytically from subcutaneous adipose, nasal mucosal cells, urothelial 11 12,13 14 cleaved into the 100 kDa heavy chain and the 50 kDa light cells, intestinal epithelial cells, prostate epithelial cells, 15 chain. The nontoxic BoNT-associated proteins include three alveolar epithelial cells, T47D, MDA-MB-231 and MDA-MB- 16 17 18 haemagglutinin (HA) proteins and one nontoxic non-HA pro- 453 breast cell lines, neutrophils and macrophages. tein. In nature, it is believed that these associated proteins pro- Importantly, it has been reported that, in addition to SNAP- 11,19,20 tect the inherently fragile BoNTs from the hostile environment 25, BoNT/A can also cleave SNAP-23, which is ubiqui- 21 of the gastrointestinal tract and help BoNTs pass through the tously expressed in human tissues. BoNT/A can inhibit SV2 16 intestinal epithelial barrier before they are released into gen- expression in breast cancer cell lines. Moreover, as will be eral circulation (reviewed by Peng Chen et al.).1 discussed in detail below, BoNT/A can elicit specific biological effects in dermal fibroblasts, mast cells, sebocytes and vascular Mechanism of action of botulinum neurotoxin endothelial cells. With regard to muscle contraction under normal conditions, Differences in botulinum neurotoxin binding to neuronal depolarization of the axon terminal results in acetylcholine vs. non-neuronal cells release from the cytosol of cholinergic neurons into the synap- tic cleft with subsequent muscular contraction. Various BoNT There is growing evidence regarding the differences in how subtypes will inhibit this acetylcholine release, blocking the BoNT binds to and acts on neuronal vs. non-neuronal cells.22 induction of muscular contraction. This blockade of acetyl- For example, the BoNT/A heavy chain enters neuronal cells choline release is referred to as ‘chemical denervation’. For mainly via a clathrin-dependent pathway, in contrast to exocrine tissue, glandular secretion is blocked. intestinal cells where it follows a Cdc42-dependent pathway.12 Acetylcholine release is performed by proteins from the sol- HA, one of the nontoxic components of BoNT large protein uble N-ethylmaleimide-sensitive factor attachment receptor complexes, disrupts the intercellular epithelial barrier by (SNARE), which mediates synaptic vesicle docking/fusion directly binding E-cadherin.3 It has been demonstrated that with the inner surface of axonal plasma membrane at the binding of the HA complex sequesters E-cadherin in the release sites. However, the inhibition of acetylcholine exocyto- monomeric state, compromising the E-cadherin-mediated sis is reversible by natural SNARE protein complex turnover. intercellular barrier and facilitating paracellular absorption of The toxic mechanism of action of BoNT comprises several BoNT/A and BoNT/B.4,23 In contrast, BoNT/C HA disrupts distinct steps. The internalization of BoNT is achieved by the barrier function by affecting cell morphology and viability endocytosis after the toxin’s heavy chain has attached to cell- in a ganglioside GM3-dependent manner.24 surface structures specifically found on cholinergic nerve ter- minals, such as ganglioside moieties, a vesicular protein (SV2) Differences in botulinum neurotoxin action on neuronal for BoNT/A and synaptotagmin for BoNT/B (reviewed in vs. non-neuronal cells Peng Chen et al. and Giordano et al.).1,2 BoNT/A can also attach to some other cell-surface proteins, such as E-cad- BoNT/A exhibits differential effects on gene expression in 3,4 5 herin, fibroblast growth factor receptor (FGFR)3 and vanil- neuronal and non-neuronal cells. Microarray analysis of gene- 6 loid receptors. The light chain, which has zinc expression changes upon exposure to BoNT/A revealed that in metalloprotease activity at its N-terminal, is released from the human HT-29 colon carcinoma cells, 167 genes were upregu- endocytotic vesicles upon acidification, and then reaches the lated while 60 genes were downregulated, whereas in cytosol wherein it cleaves one or two SNARE proteins, such as SH-SY5Y neuroblastoma cells, about 223 genes were upregu- synaptosomal-associated protein (SNAP)-25 (BoNT/A, C and lated and 18 genes were downregulated.25 Modulation of E), syntaxin (BoNT/C) and vesicle-associated membrane pro- genes and pathways involved in neuroinflammatory ubiquitin- tein also known as synaptobrevin II (BoNT/B, F and G) proteasome degradation, phosphatidylinositol, calcium British Journal of Dermatology (2018) 178, pp1011–1019 © 2017 British Association of Dermatologists Applications of botulinum toxins in dermatology, S.A. Grando and C.B. Zachary 1013 G=ganglioside P=protein Syt and SV2 isoforms *=other proteins, e.g. E-cadherin, FGFR3, vanilloid receptor Fig 1. Molecular mechanisms of botulinum neurotoxins (BoNT) action (modified from Peng Chen et al.).1 BoNT subtypes inhibit the release of acetylcholine (ACh) from the synaptic vesicle of the nerve ending into the synaptic