Action Of Neurotoxins Research Articles

Respiratory Muscle Injury Following Acute Monocled Cobra (Naja kaouthia) Envenoming: Histopathological Study in Rat Diaphragm

Clinical symptoms of monocled cobra (Naja kaouthia) envenoming include the paralysis of extraocular muscles, local tissue necrosis and death through respiratory failure. These neurotoxic outcomes are mainly due to the inhibitory action of postsynaptic neurotoxins to nicotinic acetylcholine receptors. However, injuries involving respiratory muscles have rarely been investigated. In this study, we determined the effect of N. kaouthia envenoming on morphological changes in the rat diaphragm. The efficacy of cobra monovalent antivenom in neutralising the histopathological effects of N. kaouthia venom was also evaluated. The intramuscular (i.m.) administration of N. kaouthia venom (2 mg/kg) caused skeletal muscle fibre atrophy and ruptures of myofibrils shown via a light microscope study. Transmission electron microscopy (TEM) revealed the zig-zagging of the Z-band, mitochondrial damages and degeneration of the synaptic fold of the neuromuscular junction following experimental cobra envenoming for 4 h. Intravenous administration of cobra antivenom at manufacturer-recommended doses diminished histopathological changes in the diaphragm following the administration of cobra venom. The expression of NF-kB and MuRF1 in the experimentally N. kaouthia-envenomed diaphragm indicated inflammation and tissue atrophy in the immunofluorescence analysis, respectively. In this study, we found that there were respiratory muscle injuries following N. kaouthia envenoming. The early administration of monovalent N. kaouthia antivenom is capable of neutralising neurotoxic outcomes following cobra envenoming.

Activity of botulinum neurotoxin X and its structure when shielded by a non-toxic non-hemagglutinin protein

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex and the crystal structure of the isolated NTNH protein. Unexpectedly, the BoNT/X complex is stable and protease-resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents very weak ganglioside binding and exposed hydrophobic surfaces.

Botulinum Toxin Accessory Proteins: Are They Just an Accessory?

Botulinum neurotoxins produced by Clostridium botulinum consist of a complex of a core neurotoxin protein and one or more nontoxin accessory proteins. The accessory proteins are generally thought to protect the neurotoxin from the gastric environment in botulism poisoning, dissociating away upon absorption. Other than their questionable immunogenicity, they are rarely mentioned in botulinum toxin therapy. To review evidence that accessory proteins potentially play a role in neurotoxin activity. Evidence suggests that the accessory proteins do not dissociate from the neurotoxin complex and enhance neurotoxin activity. Complexed type A botulinum toxin has dramatically higher endopeptidase activity than noncomplexed neurotoxin. A primary accessory protein, hemagglutinin-33, exhibits this same effect on both type A and type E core neurotoxin proteins, the latter not natively having this accessory protein. A clinical study using an objective computer assessment assay has shown a correlation between type A complex size and glabellar strain reduction, which reflects increasing clinical efficacy. Finally, a systematic review found no correlation between type A complex size and neutralizing antibody formation. Accessory proteins may play a role in the efficacy of botulinum toxin and could remain complexed to the neurotoxin for longer than previously reported.

An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the γ-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying α-neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

Structure and mechanism of action of botulinum and tetanus neurotoxins: A review

Botulinum neurotoxins and tetanus neurotoxins are the strongest known toxins that cause neuroparalytic syndromes in botulism and tetanus. This review aimed to systematize scientific data on the structures and mechanism of actions of botulinum and tetanus neurotoxins. Botulinum and tetanus neurotoxins are proteins containing functional domains responsible for receptor binding, transmembrane translocation, and proteolytic cleavage of proteins required for exocytosis of synaptic vesicles and release of neurotransmitters into the synaptic cleft. The main stages of the botulinum neurotoxins and tetanus neurotoxin action include binding to the presynaptic membrane, internalization of bound toxin into the cytosol via endocytosis, translocation of the L-chain into the cytosol via the HN domain, disruption of the interchain disulfide bond with the release of the L-chain to express its catalytic activity (as a metalloprotease) in the cytosol, and selective cleavage of one or more soluble N-ethylmaleimide-sensitive factor attachment receptor complex proteins with subsequent blockade of neurotransmitter release.

Marco Capogna, a pioneering neuroscientist and true European

Marco Capogna, a pioneering neuroscientist and true European

Human and animal botulism surveillance in France from 2008 to 2019.

Botulism is a human and animal neurological disease caused by the action of bacterial neurotoxins (botulinum toxins) produced by bacteria from the genus Clostridium. This disease induces flaccid paralysis that can result in respiratory paralysis and heart failure. Due to its serious potential impact on public health, botulism is a closely monitored notifiable disease in France through a case-based passive surveillance system. In humans, this disease is rare, with an average of 10 outbreaks reported each year, mainly due to the consumption of contaminated foods. Type B and to a lesser extend type A are responsible for the majority of cases of foodborne botulism. Each year, an average of 30 outbreaks are recorded on poultry farms, about 20 cases in wild birds and about 10 outbreaks in cattle, involving a large number of animals. Mosaic forms C/D and D/C in birds and cattle, respectively, are the predominant types in animals in France. Types C and D have also been observed to a lesser extent in animals. With the exception of botulinum toxin E, which was exceptionally detected throughout the period in wild birds, the types of botulism found in animal outbreaks are different from those identified in human outbreaks over the last ten years in France and no human botulism outbreaks investigated have been linked to animal botulism. In line with the One Health concept, we present the first integrative approach to the routine surveillance of botulism in humans and animals in France.

Pharmacological Dissection of the Crosstalk between NaV and CaV Channels in GH3b6 Cells.

Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.

Organophosphates modulate tissue transglutaminase activity in differentiated C6 neural cells.

The organophosphate compounds chlorpyrifos (O, O-diethyl O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate, CPF) and phenyl saligenin phosphate (PSP) have been widely implicated in developmental neurotoxicity and neurodegeneration. However, the underlying mechanism remains unclear. Transglutaminase (TG)2 is a calcium ion (Ca2+)-dependent enzyme with an important role in neuronal cell outgrowth and differentiation and in neurotoxin activity and is modulated by organophosphates. We studied TG2 activity modulation by CPO and PSP during differentiation in C6 glioma cells. We studied the effects of CPO or PSP treatment with or without the TG2 inhibitor Z-DON and identified potential TG2 protein substrates via mass spectrometry. PSP and CPO did not affect cell viability but affected TG2 activity in differentiating cells. Our results indicate that the organophosphate-induced amine incorporation activity of TG2 may have a direct effect on neuronal outgrowth, differentiation, and cell survival by modifying several essential microtubule proteins, including tubulin. Inhibiting TG2 reduced neurite length but not cell survival. TG2 inhibitors can protect against organophosphate-induced neuropathy and could be used for developing novel therapeutic strategies for treating brain cancer and neurodegenerative disorders.

Sublethal toxicity of organophosphate pesticides on development organ and survival rate of silver rasbora (Rasbora argyrotaenia)

The use of pesticides in agriculture only functions as much as 90%. The rest has polluted the surrounding environment, especially fish. Organophosphates are active neurotoxins. The organophosphate does not require any other conversion to inhibit the acetylcholinesterase enzyme. Organophosphates have the ability inhibit the action of the acetylcholinesterase (AChE) enzyme which results in disruption of acetylcholine when it provides stimulation impulses from pre-synapse to post-synapse (neurotransmitter), this results in impaired muscle work. So that the work of the undirected muscles that can cause symptoms of poisoning that affect the work of the whole body. This study aims to know the effect of sublethal toxicity of organophosphate pesticides on development organ and the survival rate of silver rasbora larvae, and to determine the exposure of organophosphate pesticides that can affect development organ and the survival rate of larvae. The research method was an experiment with a Completely Randomized Design as an experimental design. The treatments used were different organophosphate concentrations, that is 0, 0.5, 1, 1.5, and 2 ppm. Each treatment was repeated four times. The main parameters observed were the development organ of larvae, egg yolk absorption rate, abnormality larvae. The results showed that organophosphate pesticide exposure of 0.5 - 2 ppm could affect the development of the organ, survival rate, and abnormality silver rasbora larvae. Exposure to organophosphate pesticides can also increase the absorption rate of egg yolks in larvae. Exposure dosage to organophosphate pesticides is directly proportional to the percentage of larva abnormalities and inversely proportional to the value of survival rate larva.

Recombinant L-HN Fusion Antigen Derived from the L and HN Domains of Botulinum Neurotoxin B Stimulates a Protective Antibody Response Against Active Neurotoxin.

Botulinum neurotoxin (BoNT) is a neurotoxin produced by Clostridium botulinum in an anaerobic environment. BoNT is the most toxic protein among bacteria, animals, plants, and chemical substances reported to date. BoNTs are 150kDa proteins composed of three major functional domains: catalytic (L domain, 50kDa), translocation (HN domain, 50kDa), and receptor-binding (Hc domain, 50kDa) domains. Most studies have focused on the use of the Hc domain as an antigen because it is capable of generating robust protective immunity and contains some functional neutralizing epitopes. In the present study, we produced and characterized a recombinant L-HN fusion fragment of the parent BoNT/B (BL-HN) composed of L and HN domains with a deletion in the Hc domain (BHc). When the BL-HN protein was expressed in E. coli, it retained its stable structure and antigenicity. As a vaccine antigen, the recombinant BL-HN protein was found to induce sufficient protection against native BoNT/B in a mouse model. The BL-HN subunit vaccine could also induce a strong humoral immune response and generate sufficient neutralizing antibodies in immunized mice. Therefore, BL-HN may retain the native neurotoxin structure and critical epitopes responsible for inducing serum neutralizing antibodies. Studies of the dose-dependent immunoprotective effects further confirmed that the BL-HN antigen could provide potent protective immunity. This finding suggests that BL-HN can play an important role in immune protection against BoNT/B. Therefore, the BL-HN fusion fragment provides an excellent platform for the design of recombinant botulinum vaccines and neutralizing antibodies.

Duration of Symptom Relief Between Injections for AbobotulinumtoxinA (Dysport®) in Spastic Paresis and Cervical Dystonia: Comparison of Evidence From Clinical Studies.

Background: Botulinum toxin-A is a well-established treatment for adult and pediatric spastic paresis and cervical dystonia. While guidelines and approved labels indicate that treatment should not occur more frequently than every 12 weeks, studies and real-world evidence show that the timing of symptom recurrence between treatments may vary.Methods: We report retreatment criteria and response duration (retreatment intervals) from four pivotal, double-blind, placebo-controlled studies with open-label extensions involving patients treated with abobotulinumtoxinA (aboBoNTA) for upper limb (NCT01313299) or lower limb (NCT01249404) spastic paresis in adults, lower limb spastic paresis in children (NCT01249417), and cervical dystonia in adults (NCT00257660). We review results in light of recently available preclinical data.Results: In spastic paresis, 24.0–36.9% of upper limb patients treated with aboBoNTA and 20.1–32.0% of lower limb patients did not require retreatment before 16 weeks. Moreover, 72.8–93.8% of aboBoNTA-treated pediatric patients with lower limb spastic paresis did not require retreatment before 16 weeks (17.7–54.0% did not require retreatment before 28 weeks). In aboBoNTA-treated patients with cervical dystonia, 72.6–81.5% did not require retreatment before 16 weeks.Conclusion: AboBoNTA, when dosed as recommended, offers symptom relief beyond 12 weeks to many patients with spastic paresis and cervical dystonia. From recently available preclinical research, the amount of active neurotoxin administered with aboBoNTA might be a factor in explaining this long duration of response.

Transferability study of the BINACLE (binding and cleavage) assay for in vitro determination of botulinum neurotoxin activity

The muscle-relaxing effects of the botulinum neurotoxin (BoNT) serotypes A and B are widely used in clinical and aesthetic medicine. The standard method for measuring the biological activity of pharmaceutical BoNT products is a mouse bioassay. In line with the European Directive 2010/63/EU, a replacement by an animal-free method would be desirable. Whereas the existing approved in vitro methods for BoNT activity measurements are product-specific and not freely available for all users, the “binding and cleavage” (BINACLE) assay could become a widely applicable alternative. This method quantifies active BoNT molecules based on their specific receptor-binding and proteolytic properties and can be applied to all BoNT products on the European market.Here we describe the results of a transferability study, in which identical BoNT samples were tested in the BINACLE assay in four laboratories. All participants successfully performed the method and observed clear dose-response relationships. Assay variability was within an acceptable range. These data indicate that the BoNT BINACLE assay is robust and can be straightforwardly transferred between laboratories. They thus provide an appropriate basis for future studies to further substantiate the suitability of the BINACLE assay for the potency determination of BoNT products.

Anticoagulant activity of krait, coral snake, and cobra neurotoxic venoms with diverse proteomes are inhibited by carbon monoxide

A phenomena of interest is the in vitro anticoagulant effects of neurotoxins found in elapid venoms that kill by paralysis. These enzymes include phospholipase A2 (PLA2), and it has recently been demonstrated that carbon monoxide inhibits the PLA2-dependent neurotoxin contained in Mojave rattlesnake type A venom. The purpose of this investigation was to assess if the anticoagulant activity of elapid venoms containing PLA2 and/or three finger toxins could be inhibited by carbon monoxide. Venoms collected from Bungarus multicinctus, Micrurus fulvius, and five Naja species were exposed to carbon monoxide via carbon monoxide releasing molecule-2 prior to placement into human plasma. Coagulation kinetics were assessed via thrombelastography. Compared with plasma without venom addition, all venoms had significant anticoagulant effects, with a 160-fold range of concentrations having similar anticoagulant effects in a species-specific manner. Carbon monoxide significantly inhibited the anticoagulant effect of all venoms tested, but inhibition was not complete in all cases. Given that individual neurotoxin activity often depends on intact activity that includes anticoagulant action, it may be possible that carbon monoxide inhibits neurotoxicity. Future investigation is justified to assess such carbon monoxide mediated inhibition with purified neurotoxins in vitro and in vivo.

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments.

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

MODELING OF PARKINSON’S DISEASE USING ENVIRONMENTAL NEUROTOXINS (REVIEW)

In recent years, there has been an increase in the prevalence of neurodegenerative diseases including Parkinson’s disease (PD). It is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to disability of patients and large financial costs of the treatment and rehabilitation. In this regard, the understanding of the environmental factors causing this disease, the development of adequate experimental models for studying its pathogenesis, and the search for strategies to prevent its development, as well as possible neuroprotective drugs, have fundamental scientific value. Although some researchers believe that genetic mutations and aging of the population are the main factors for the development of PD, a lot of studies have shown that PD may be caused by exposure to a number of toxins which enter the body from the environment. This review discusses the main toxic substances that cause the development of PD and, therefore, are used to model this disease in animals and cell cultures, as well as the mechanisms of action of neurotoxins, and the advantages and disadvantages of specific models.

The binacle (binding and cleavage) assay for in vitro determination of botulinum neurotoxin activity

The binacle (binding and cleavage) assay for in vitro determination of botulinum neurotoxin activity

A novel protein binding strategy for energy-transfer-based photoelectrochemical detection of enzymatic activity of botulinum neurotoxin A

In this work, we propose a novel energy-transfer-based photoelectrochemical (PEC) platform for probing of protein-protein interaction, which associates intimately with zinc-dependent cleavage and substrate specificities in the enzymatic activities of botulinum neurotoxin (BoNT). Specifically, by using substrate protein SNAP-25 as the energy-transfer nanoprobe, an exciton-plasmon interaction (EPI) based strategy between CdS quantum dots (QDs) and Au nanoparticles (NPs) in a PEC system is constructed with the photocurrent declining. Interestingly, the EPI effect is then interrupted by the target botulinum neurotoxin serotype A light chain (BoNT-LCA) special cleavage of the probe SNAP-25, leading to the photocurrent recovery. Therefore, the enzymatic activity of BoNT-LCA could be sensitively detected with a detection limit of 1 pg/mL. Unlike conventional DNA-programable assembly, a protein probe is used to bridge the excitons and plasmons in this work, which provides a new route for the investigation of the EPI-based bioassay.

The Effect of Neurotoxin MPTP and Neuroprotector Isatin on the Profile of Ubiquitinated Brain Mitochondrial Proteins.

Mitochondria are a crucial target for the actions of neurotoxins, causing symptoms of Parkinson’s disease in various experimental animal models, and also neuroprotectors. There is evidence that mitochondrial dysfunction induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) influences functioning of the ubiquitin-proteasomal system (UPS) responsible for selective proteolytic degradation of proteins from various intracellular compartments (including mitochondria) and neuroprotective effects of certain anti-Parkisonian agents (monoamine oxidase inhibitors) may be associated with their effects on the UPS. In this study, we have investigated the effect of the neurotoxin MPTP and neuroprotector isatin, and their combination on the profile of ubiquitinated brain mitochondrial proteins. The development of movement disorders induced by MPTP administration caused dramatic changes in the profile of ubiquitinated proteins associated with mitochondria. Pretreatment with the neuroprotector isatin decreased manifestations of MPTP-induced Parkinsonism, and had a significant impact on the profile of ubiquitinated mitochondrial proteins (including oxidative modified proteins). Administration of isatin alone to intact mice also influenced the profile of ubiquitinated mitochondrial proteins, and increased the proportion of oxidized proteins carrying the ubiquitination signature. These alterations in the ubiquitination of mitochondrial proteins observed within 2 h after administration of MPTP and isatin obviously reflect immediate short-term biological responses to these treatments.

A novel role of C-terminus in introducing a functionally flexible structure critical for the biological activity of botulinum neurotoxin

Botulinum neurotoxin (BoNT) is responsible for botulism, a clinical condition resulting in flaccid muscle paralysis and potentially death. The light chain is responsible for its intracellular toxicity through its endopeptidase activity. Available crystal structures of BoNT/A light chains (LCA) are based on various truncated versions (tLCA) of the full-length LCA (fLCA) and do not necessarily reflect the true structure of LCA in solution. The understanding of the mechanism of action, longevity of intoxication, and an improved development of endopeptidase inhibitors are dependent on first having a better insight into the structure of LCA in solution. Using an array of biophysical techniques, we report that the fLCA structure is significantly more flexible than tLCA in solution, which may be responsible for its dramatically higher enzymatic activity. This seems to be achieved by a much stronger, more rapid binding to substrate (SNAP-25) of the fLCA compared to tLCA. These results suggest that the C-terminus of LCA plays a critical role in introducing a flexible structure, which is essential for its biological function. This is the first report of such a massive structural role of the C-terminus of a protein being critical for maintaining a functional state.