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; C.B. Zachary

Disclosures

The British Journal of Dermatology. 2018;178(5):1011-1019. 

In This Article

The Structure and Function of Botulinum Neurotoxins

The Structure of Botulinum Neurotoxins

In nature, botulinum neurotoxin (BoNT) is produced by Clostridium botulinum bacteria. The distinct toxin subtypes, designated A through G, have different structures and mechanisms of action. The A, B and E subtypes cause botulism in humans owing to their action inside the axon terminal, leading to paralysis of the respiratory muscles and death resulting from respiratory failure. Pure botulinum toxin A was first synthesized as an inactive 150–kDa protein complexed with varying amounts of nontoxic companion proteins. Botulinum neurotoxin is activated when the polypeptide chain is proteolytically cleaved into the 100 kDa heavy chain and the 50 kDa light chain. The nontoxic BoNT–associated proteins include three haemagglutinin (HA) proteins and one nontoxic non–HA protein. In nature, it is believed that these associated proteins protect the inherently fragile BoNTs from the hostile environment of the gastrointestinal tract and help BoNTs pass through the intestinal epithelial barrier before they are released into general circulation (reviewed by Peng Chen et al.).[1]

Mechanism of Action of Botulinum Neurotoxin

With regard to muscle contraction under normal conditions, depolarization of the axon terminal results in acetylcholine release from the cytosol of cholinergic neurons into the synaptic cleft with subsequent muscular contraction. Various BoNT subtypes will inhibit this acetylcholine release, blocking the induction of muscular contraction. This blockade of acetylcholine release is referred to as 'chemical denervation'. For exocrine tissue, glandular secretion is blocked.

Acetylcholine release is performed by proteins from the soluble N–ethylmaleimide–sensitive factor attachment receptor (SNARE), which mediates synaptic vesicle docking/fusion with the inner surface of axonal plasma membrane at the release sites. However, the inhibition of acetylcholine exocytosis is reversible by natural SNARE protein complex turnover.

The toxic mechanism of action of BoNT comprises several distinct steps. The internalization of BoNT is achieved by endocytosis after the toxin's heavy chain has attached to cell–surface structures specifically found on cholinergic nerve terminals, such as ganglioside moieties, a vesicular protein (SV2) for BoNT/A and synaptotagmin for BoNT/B (reviewed in Peng Chen et al. and Giordano et al.).[1,2] BoNT/A can also attach to some other cell–surface proteins, such as E–cadherin,[3,4] fibroblast growth factor receptor (FGFR)3[5] and vanilloid receptors.[6] The light chain, which has zinc metalloprotease activity at its N–terminal, is released from the endocytotic vesicles upon acidification, and then reaches the cytosol wherein it cleaves one or two SNARE proteins, such as synaptosomal–associated protein (SNAP)–25 (BoNT/A, C and E), syntaxin (BoNT/C) and vesicle–associated membrane protein also known as synaptobrevin II (BoNT/B, F and G) (Figure 1). However, the BoNT receptors and intracellular targets are not unique for neurotransmission, as several of these receptors and targets have been found in both neuronal and non–neuronal cells.

Figure 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 cleft, resulting in 'chemical denervation'. Within the presynaptic terminal, ACh release is performed by the soluble N–ethylmaleimide–sensitive factor attachment receptor (SNARE), which mediates docking/fusion of the synaptic vesicle with the inner surface of axonal plasma membrane at the release sites. BoNT is internalized by endocytosis after the toxin's heavy chain (Hc) has attached to ganglioside moieties, a vesicular protein (SV2) or synaptotagmin (Syt) present on the surface of cholinergic nerve terminals and some other cell–surface proteins, such as E–cadherin, fibroblast growth factor receptor (FGFR)3 and vanilloid receptors. The toxin's light chain (Lc), which has zinc metalloprotease activity at its N–terminal, is then released from the endocytotic vesicle upon acidification, and reaches the cytosol wherein it cleaves one or two SNARE proteins, such as synaptosomal–associated protein (SNAP) 25 (BoNT/A, C and E), syntaxin (BoNT/C) and vesicle–associated membrane protein (VAMP) also known as synaptobrevin II (BoNT/B, F and G).

The Non–neuronal Cell Types Targeted by Botulinum Neurotoxin

Based on published data, several types of non–neuronal cells may be directly affected by BoNT in human skin and other tissues that produce a biological effect. The cells expressing one or more of the BoNT/A–binding proteins SV2, FGFR3 or vanilloid receptors, and/or BoNT/A cleavage target SNAP–25, include epidermal keratinocytes,[7,8] mesenchymal stem cells from subcutaneous adipose,[9] nasal mucosal cells,[10] urothelial cells,[11] intestinal epithelial cells,[12,13] prostate epithelial cells,[14] alveolar epithelial cells,[15] T47D, MDA–MB–231 and MDA–MB–453 breast cell lines,[16] neutrophils[17] and macrophages.[18] Importantly, it has been reported that, in addition to SNAP–25, BoNT/A can also cleave SNAP–23,[11,19,20] which is ubiquitously expressed in human tissues.[21] BoNT/A can inhibit SV2 expression in breast cancer cell lines.[16] Moreover, as will be discussed in detail below, BoNT/A can elicit specific biological effects in dermal fibroblasts, mast cells, sebocytes and vascular endothelial cells.

Differences in Botulinum Neurotoxin Binding to Neuronal vs. Non–neuronal Cells

There is growing evidence regarding the differences in how BoNT binds to and acts on neuronal vs. non–neuronal cells.[22] For example, the BoNT/A heavy chain enters neuronal cells mainly via a clathrin–dependent pathway, in contrast to intestinal cells where it follows a Cdc42–dependent pathway.[12] HA, one of the nontoxic components of BoNT large protein complexes, disrupts the intercellular epithelial barrier by directly binding E–cadherin.[3] It has been demonstrated that binding of the HA complex sequesters E–cadherin in the monomeric state, compromising the E–cadherin–mediated intercellular barrier and facilitating paracellular absorption of BoNT/A and BoNT/B.[4,23] In contrast, BoNT/C HA disrupts the barrier function by affecting cell morphology and viability in a ganglioside GM3–dependent manner.[24]

Differences in Botulinum Neurotoxin Action on Neuronal vs. Non–neuronal Cells

BoNT/A exhibits differential effects on gene expression in neuronal and non–neuronal cells. Microarray analysis of gene–expression changes upon exposure to BoNT/A revealed that in human HT–29 colon carcinoma cells, 167 genes were upregulated while 60 genes were downregulated, whereas in SH–SY5Y neuroblastoma cells, about 223 genes were upregulated and 18 genes were downregulated.[25] Modulation of genes and pathways involved in neuroinflammatory ubiquitin–proteasome degradation, phosphatidylinositol, calcium signalling in SH–SY5Y cells and genes relevant to focal adhesion, cell adhesion molecules, adherens– and gap–junction–related pathways in HT–29 cells suggested that affected genes play a distinct role in the biological effects of BoNT neuronal and non–neuronal cells.[25] The global transcriptional profiling of the murine alveolar macrophage cell line RAW264.7 revealed that altered genes were mainly involved in signal transduction, immunity and defence, protein metabolism and modification, neuronal activities, intracellular protein trafficking and muscle contraction.[18]

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