OnabotulinumtoxinA (onabotA), a Botulinum neurotoxin type A (BoNT/A), is an effective treatment for chronic migraine, but its direct mechanism of action on human sensory neurons has not been fully elucidated. While rodent studies on dorsal root ganglion (DRG) and trigeminal ganglion (TG) show that BoNT/A inhibits neurotransmission, including calcitonin gene-related peptide (CGRP/CALCA) release, by cleaving synaptosomal-associated protein 25 (SNAP-25), only one previous study has assessed its effect on human DRG neurons. The objective of this study was to understand the mechanism of action of BoNT/A in cultured human sensory neurons and assess, using RNA sequencing, the transcriptomic consequences of BoNT/A treatment. Using DRGs obtained from organ donors, the expression of key targets, including synaptic vesicle glycoprotein 2C (SV2C), SNAP25, & CALCA, was validated by mining existing transcriptomic datasets as well as immunohistochemistry. Cultured dissociated human DRG neurons treated with BoNT/A were used to examine cleavage of SNAP25, release of CGRP, and transcriptomic changes after BoNT/A treatment. SV2C was found to be widely expressed in human DRG neurons in a pattern that completely overlapped with CGRP expression. Consistent with this finding, BoNT/A disrupted soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes in human DRG neurons as demonstrated by SNAP-25 cleavage in most somatosensory neurons and a reduction in capsaicin-evoked CGRP release, indicating impaired vesicle fusion. Moreover, bulk RNA sequencing experiments revealed downregulated expression of a large subset of genes responsible for neurotransmitter and neuropeptide release from neurons, suggesting a novel mechanism through which BoNT/A regulates neurotransmission. These results provide new insight into the molecular mechanisms by which BoNT/A may exert its pain-relieving effects in humans.

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A’s protease domain (LC/A) could expand its therapeutic applications; however, LC/A’s extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A’s active site could guide the design of improved BoNT proteases and inhibitors.

To evaluate the effect of a cell-penetrating peptide (CPP) in a prototype botulinum neurotoxin type A (BoNT/A) formulation using toxin adsorption, in vivo potency, and duration analyses.

Botulinum neurotoxins (BoNTs), produced by Clostridia and other bacteria, are the most potent toxins known. Their cleavage of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins in neurons prevents the release of neurotransmitters, thus resulting in the muscle paralysis that is characteristic of botulism. This mechanism of action has been exploited for a variety of therapeutic and cosmetic applications of BoNTs. This chapter provides an overview of the native BoNTs, including the classical serotypes and their clinical use, mosaic BoNTs, and novel BoNTs that have been recently identified in clostridial and non-clostridial strains. In addition, the modular structure of native BoNTs, which are composed of a light chain and a heavy chain, is amenable to a multitude of novel fusions and mutations using molecular biology techniques. These novel recombinant BoNTs have been used or are being developed to further characterize the biology of toxins, to assist in vaccine production, to serve as delivery vehicles to neurons, and to be utilized as novel therapeutics for both neuronal and non-neuronal cells.

Differences in botulinum neurotoxin manufacturing, formulation, and potency evaluation can impact dose and biological activity, which ultimately affect duration of action. The potency of different labeled vials of incobotulinumtoxinA (Xeomin®; 50 U, 100 U, or 200 U vials; incobotA) versus onabotulinumtoxinA (BOTOX®; 100 U vial; onabotA) were compared on a unit-to-unit basis to assess biological activity using in vitro (light-chain activity high-performance liquid chromatography (LCA-HPLC) and cell-based potency assay (CBPA)) and in vivo (rat compound muscle action potential (cMAP) and mouse digit abduction score (DAS)) assays. Using LCA-HPLC, incobotA units displayed approximately 54% of the protease activity of label-stated equivalent onabotA units. Lower potency, reflected by higher EC50, ID50, and ED50 values (pooled mean ± SEM), was displayed by incobotA compared to onabotA in the CBPA (EC50: incobotA 7.6 ± 0.7 U/mL; onabotA 5.9 ± 0.5 U/mL), cMAP (ID50: incobotA 0.078 ± 0.005 U/rat; onabotA 0.053 ± 0.004 U/rat), and DAS (ED50: incobotA 14.2 ± 0.5 U/kg; onabotA 8.7 ± 0.3 U/kg) assays. Lastly, in the DAS assay, onabotA had a longer duration of action compared to incobotA when dosed at label-stated equivalent units. In summary, onabotA consistently displayed greater biological activity than incobotA in two in vitro and two in vivo assays. Differences in the assay results do not support dose interchangeability between the two products.

Botulinum neurotoxin type B (BoNT/B) initiates its toxicity by binding to synaptotagmin II (SytII) and gangliosides GD1a and GT1b on the neural membrane. We synthesized two 27-residue peptides that carry the BoNT/B binding sites on mouse SytII (mSytII 37–63) or human SytII (hSytII 34–60). BoNT/B bound to these peptides, but showed substantially higher binding to mSytII peptide than to hSytII peptide. The mSytII peptide inhibited almost completely BoNT/B binding to synaptosomes (snps) and displayed a high affinity. BoNT/B bound strongly to mSytII peptide and binding was inhibited by the peptide. Binding of BoNT/B to snps was also inhibited (~80 %) by a larger excess of gangliosides GD1a or GT1b. The mSytII peptide inhibited very strongly (at least 80 %) the toxin binding to snps, while the two gangliosides were much less efficient inhibitors requiring much larger excess to achieve similar inhibition levels. Furthermore, gangliosides GD1a or GT1b inhibited BoNT/B binding to mSytII peptide at a much larger excess than the inhibition by mSytII peptide. Conversely, BoNT/B bound well to each ganglioside and binding could be inhibited by the correlate ganglioside and much less efficiently by the mSytII peptide. There was no apparent collaboration between mSytII peptide and either ganglioside. mSytII peptide displayed some protective activity in vivo in mice against a lethal BoNT/B dose. We concluded that SytII peptide and gangliosides bind independently but, with their binding sites on BoNT/B being spatially close, each can influence BoNT/B binding to the other due to regional conformational perturbations or steric interference or both. Ganglioside involvement in BoNT/B binding might help in toxin translocation and endocytosis.

Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

A surface-simulation peptide, SQMIN[GG]TTNI[G]NSIS[G]RDTH[G]NLES, (SS-peptide) was synthesized that described the spatial interrelationships of 21 residues on the surface of botulinum neurotoxin type A (BoNT/A). The glycine residues in brackets were spacers between surface segments of BoNT/A. The SS-peptide did not contain an antigenic or a synaptosome (snps)-binding site of BoNT/A and it did not bind anti-BoNT/A antibodies (Abs) or inhibit toxin binding to synaptosomes. Antibodies prepared by immunization with the free peptide or with peptide-ovalbumin (OVA) conjugate did not protect mice in vivo against a lethal dose of the toxin. Early Abs (day 52) against free SS-peptide recognized the peptide and showed a small cross-reaction with native toxin, but later Abs (day 115) exhibited a higher cross-reaction with to active toxin. Similarly, early Abs (day 52) against peptide-OVA conjugate displayed a low cross-reaction with native toxin, but the cross-reaction also increased in later bleeds (day 115). Both, the free peptide or its OVA conjugate, elicited predominantly IgG Abs that in the course of immunization were increasingly more capable of binding to a peptide conformation resembling the shape of the surface area on the native BoNT/A. The Abs were able to detect the conformational changes of the toxoid. This demonstrates that Abs could be prepared essentially against a peptide that mimics a surface area and such Abs could recognize and bind to the correlate surface area on the native protein. The area selected could, but need not, be an antigenic site when the native protein is used as an immunogen. The ability to make Abs against protein surface areas that are mimicked by surface-simulation synthesis provides versatile and valuable tools for analytical, therapeutic, clinical and diagnostic applications.