A novel catalytically inactive construct of botulinum neurotoxin A (BoNT/A) directly inhibits visceral sensory signaling

Botulinum neurotoxin A (BoNT/A) is a potent neurotoxin that silences cholinergic neurotransmission through the cleavage of the synaptic protein SNAP-25. Previous studies have shown that, in addition to its paralytic effects, BoNT/A can inhibit sensory nerve activity. The aim of this study was to identify how BoNT/A inhibits afferent signalling from the bladder. To investigate the role of SNAP-25 cleavage in the previously reported BoNT/A-dependent inhibition of sensory signalling, we developed a recombinant form of BoNT/A with an inactive light chain, rBoNT/A (0), unable to paralyse muscle. We also developed recombinant light chain (LC)-domain-only proteins to better understand the entry mechanisms, as the heavy chain (HC) of the protein is responsible for the internalisation of the light chain. We found that, despite a lack of catalytic activity, rBoNT/A (0) potently inhibited the afferent responses to bladder distension to a greater degree than catalytically active rBoNT/A. This was also clear from the testing of the LC-only proteins, as the inactive rLC/A (0) protein inhibited afferent responses significantly more than the active rLC/A protein. Immunohistochemistry for cleaved SNAP-25 was negative, and purinergic and nitrergic antagonists partially and totally reversed the sensory inhibition, respectively. These data suggest that the BoNT/A inhibition of sensory nerve activity in this assay is not due to the classical well-characterised ‘double-receptor’ mechanism of BoNT/A, is independent of SNAP25 cleavage and involves nitrergic and purinergic signalling mechanisms

The Isolated Mouse Jejunal Afferent Nerve Assay as a Tool to Assess the Effect of Botulinum Neurotoxins in Visceral Nociception

For the past two decades, botulinum neurotoxin A (BoNT/A) has been described as a strong candidate in the treatment of pain. With the production of modified toxins and the potential new applications at the visceral level, there is a real need for tools allowing the assessment of these compounds. In this study, we evaluated the jejunal mesenteric afferent nerve assay to investigate BoNT/A effects on visceral nociception. This ex vivo model allowed the continuous recording of neuronal activity in response to various stimuli. BoNT/A was applied intraluminally during three successive distensions, and the jejunum was distended every 15 min for 3 h. Finally, samples were exposed to external capsaicin. BoNT/A intoxication was validated at the molecular level with the presence of cleaved synaptosomal-associated protein of 25 (SNAP25) in nerve terminals in the mucosa and musculosa layers 3 h after treatment. BoNT/A had a progressive inhibitory effect on multiunit discharge frequency induced by jejunal distension, with a significant decrease from 1 h after application without change in jejunal compliance. The capsaicin-induced discharge was also affected by the toxin. This assay allowed the description of an inhibitory effect of BoNT/A on afferent nerve activity in response to distension and capsaicin, suggesting BoNT/A could alleviate visceral nociception

BoNT/A in the Urinary Bladder—More to the Story than Silencing of Cholinergic Nerves

Botulinum neurotoxin (BoNT/A) is an FDA and NICE approved second-line treatment for overactive bladder (OAB) in patients either not responsive or intolerant to anti-cholinergic drugs. BoNT/A acts to weaken muscle contraction by blocking release of the neurotransmitter acetyl choline (ACh) at neuromuscular junctions. However, this biological activity does not easily explain all the observed effects in clinical and non-clinical studies. There are also conflicting reports of expression of the BoNT/A protein receptor, SV2, and intracellular target protein, SNAP-25, in the urothelium and bladder. This review presents the current evidence of BoNT/A’s effect on bladder sensation, potential mechanisms by which it might exert these effects and discusses recent advances in understanding the action of BoNT in bladder tissue

Botulinum toxin intoxication requires retrograde transport and membrane translocation at the ER

Abstract Botulinum neurotoxin A (BoNT/A) is a highly potent proteolytic toxin specific for neurons with numerous clinical and cosmetic uses. After uptake at the synapse, the protein is proposed to translocate from synaptic vesicles to cytosol. Surprisingly, we found that after intoxication proteolysis of a fluorescent reporter occurs in the neuron soma first and then centrifugally in neurites. To investigate the molecular mechanisms at play, we use a genome-wide siRNA screen in genetically engineered neurons and identify over three hundred genes. An organelle-specific split-mNG complementation indicates BoNT/A traffic from the synapse to the soma-localised Golgi in a retromer dependent fashion. The toxin then moves to the ER and appears to require the Sec61 complex for retro-translocation to the cytosol. Our study identifies genes and trafficking processes hijacked by BoNT/A, revealing an unexpected complex route for efficient intoxication

The Expanding Therapeutic Utility of Botulinum Neurotoxins

Botulinum neurotoxin (BoNT) is a major therapeutic agent that is licensed in neurological indications, such as dystonia and spasticity. The BoNT family, which is produced in nature by clostridial bacteria, comprises several pharmacologically distinct proteins with distinct properties. In this review, we present an overview of the current therapeutic landscape and explore the diversity of BoNT proteins as future therapeutics. In recent years, novel indications have emerged in the fields of pain, migraine, overactive bladder, osteoarthritis, and wound healing. The study of biological effects distal to the injection site could provide future opportunities for disease-tailored BoNT therapies. However, there are some challenges in the pharmaceutical development of BoNTs, such as liquid and slow-release BoNT formulations; and, transdermal, transurothelial, and transepithelial delivery. Innovative approaches in the areas of formulation and delivery, together with highly sensitive analytical tools, will be key for the success of next generation BoNT clinical products

New Modified Recombinant Botulinum Neurotoxin Type F with Enhanced Potency

Botulinum neurotoxins (BoNTs) are notorious toxins and powerful agents and can be lethal, causing botulism, but they are also widely used as therapeutics, particularly to treat neuromuscular disorders. As of today, the commercial BoNT treatments available are from native A or B serotypes. Serotype F has shown efficacy in a clinical trial but has scarcely been used, most likely due to its medium duration of effect. Previously, the uniqueness of the light chain of the F7 subtype was identified and reported, showing an extended interaction with its substrates, VAMPs 1, 2 and 3, and a superior catalytic activity compared to other BoNT/F subtypes. In order to more extensively study the properties of this neurotoxin, we engineered a modified F7 chimera, mrBoNT/F7-1, in which all the regions of the neurotoxin were identical to BoNT/F7 except the activation loop, which was the activation loop from BoNT/F1. Use of the activation loop from BoNT/F1 allowed easier post-translational proteolytic activation of the recombinant protein without otherwise affecting its properties. mrBoNT/F7-1 was expressed, purified and then tested in a suite of in vitro and in vivo assays. mrBoNT/F7-1 was active and showed enhanced potency in comparison to both native and recombinant BoNT/F1. Additionally, the safety profile remained comparable to BoNT/F1 despite the increased potency. This new modified recombinant toxin F7 could be further exploited to develop unique therapeutics to address unmet medical needs

BoNT/A1 Secondary Failure for the Treatment of Neurogenic Detrusor Overactivity: An Ex Vivo Functional Study

Management of neurogenic detrusor overactivity (NDO) remains a clinical priority to improve patients’ quality of life and prevent dramatic urological complications. Intradetrusor injection of onabotulinumtoxinA (BoNT/A1, botulinum neurotoxin A1) is approved as second therapeutic line in these patients, demonstrating a good efficacy. However, a loss of its efficacy over time has been described, with no clear understanding of the underlying mechanisms. This paper aims at shedding new light on BoNT/A1 secondary failure in NDO through functional and structural analysis. Three groups of patients (either non-NDO, NDO with no toxin history or toxin secondary failure) were investigated using an ex vivo bladder strip assay. Detrusor strips were tensed in organ baths and submitted to electrical field stimulation to generate contractions. Recombinant BoNT/A1 was then added at various concentrations and contractions recorded for 4 h. Histology exploring BoNT/A1 targets, fibrosis and neuronal markers was also used. Detrusor strips from patients with BoNT/A1 secondary failure displayed a smaller sensitivity to toxin ex vivo at 3 nM compared to the other groups. Histological evaluation demonstrated the presence of cleaved Synaptosomal-Associated Protein, 25 kDa (c-SNAP25) in the detrusor from the toxin-secondary failure population, indicating some remaining in vivo sensitivity to BoNT/A1 despite the therapeutic escape. Moreover, residual c-SNAP25 did not affect parasympathetic-driven contractions observed ex vivo. This study confirms the slightly lower efficacy of BoNT/A1 in the BoNT/A1 secondary failure NDO group, suggesting that the escape from BoNT/A1 efficacy in NDO occurs at least at the parasympathetic level and could imply compensatory mechanisms for detrusor contraction

BoTest cell-free light chain activity assay.

<p>(A) BoNT/B1, rBoNT/B1 or rBoNT/B1_(S201P) (0.5 pM—1.25 nM) were incubated with 200 nM BoTest Reporter (VAMP-2 [33–94] flanked by N-terminal cyan fluorescent protein [CFP] and C-terminal yellow fluorescent protein [YFP]) for 18 hours at 30°C. Fluorescence intensity at 526 nm (YFP) and 470 nm (CFP) was determined using a BioTek Synergy HT plate reader. A diminishing 526/470 nm relative fluorescent unit (RFU) ratio indicates substrate cleavage. Data were fitted to a four-parameter equation. (B) The concentration of each toxin required for 50% maximum inhibition of 526/470 RFU ratio (pIC₅₀) was determined from the fitted curves. Data represent the mean ± s.e.m. of <i>n</i> = 6 (BoNT/B1) or <i>n</i> = 3 (rBoNT/B1 and rBoNT/B1_(S201P)) independent experiments, each performed in duplicate. (C) BoNT/B1, rBoNT/B1 or rBoNT/B1_(S201P) (0.1 pM—10 nM) were incubated with 2 μM VAMP-1 (2–96)-GFP at 37°C for 24 hr. Reactions were terminated by addition of NuPAGE LDS buffer, 1 mM DTT and separated by SDS-PAGE on 12% Bis-tris gels. Gels were stained and densitometry performed to determine the percentage substrate cleavage of each sample. Data were fitted to a four-parameter equation. (D) The concentration of each toxin required for 50% maximum substrate cleavage (pEC₅₀) was determined from the fitted curves. Data represent the mean ± s.e.m. of <i>n</i> = 3 independent experiments, each performed in triplicate.Significant differences in potency are denoted by **(P<0.01) and ***(P<0.001, one-way ANOVA, Dunnett’s <i>post-hoc</i> test).</p

Purification of rBoNT/B1 and rBoNT/B1_(S201P).

<p>(A) The wild-type molecule, rBoNT/B1 (<i>left)</i> and single mutant, rBoNT/B1_(S201P) (<i>right</i>) were purified using a four step process–an initial passage through an anionic exchange column (AEC) before capture by hydrophobic interaction chromatography (HIC), followed by an intermediate AEC purification step and final HIC polish step after proteolytic cleavage into the active di-chain. Purified samples were resolved by PAGE and visualised with SafeStain. Western blot analysis of rBoNT/B1 (B) and rBoNT/B1_(S201P) (C) confirm the presence of the parent di-chain at ~150 kDa (“oxidised”) and light and heavy chains at ~50 kDa and ~100 kDa, respectively (“reduced”).</p