Local Tetanus Begins with a Vesicle-Associated Membrane Protein Cleavage-Associated Neuromuscular Junction Paralysis around the Site of Tetanus Neurotoxin Release.

Local tetanus develops when limited amounts of tetanus neurotoxin (TeNT) are released by Clostridium tetani generated from spores inside a necrotic wound. Within days, a spastic paralysis restricted to the muscles of the affected anatomical area develops. This paralysis follows the retrograde transport of TeNT inside the axons of spinal cord motoneurons and its uptake by inhibitory interneurons with cleavage of a vesicle-associated membrane protein required for neurotransmitter release. Consequently, incontrollable excitation of motoneurons causes contractures of innervated muscles and leads to local spastic paralysis. Here, the initial events occurring close to the site of TeNT release were investigated in a mouse model of local tetanus. A peripheral flaccid paralysis was found to occur, before or overlapping, the spastic paralysis. At variance from the confined TeNT proteolytic activity at the periphery, central vesicle-associated membrane protein cleavage can be detected within inhibitory interneurons controlling motor neuron efferents innervating muscle groups distant from the site of TeNT release. These results indicate that TeNT does have peripheral activity in tetanus and explains why the spastic paralysis observed in local tetanus, although confined to single limbs, generally affects multiple muscles. The initial TeNT neuroparalytic activity can be detected by measuring the compound muscle action potential, providing a very early diagnosis and therapy, and thus preventing the ensuing life-threatening generalized tetanus.

Three players in the 'toxic affair' between botulinum neurotoxin type A and neurons.

Joensuu and colleagues have recently shown that botulinum neurotoxin (BoNT) type A exploits a heterotrimeric complex in the presynaptic membrane to bind to and enter neurons using a Trojan horse-like mechanism. Similar processes may be relevant to the neuronal entry of different botulinum toxin serotypes and other neuropathogens.

Facial neuromuscular junctions and brainstem nuclei are the target of tetanus neurotoxin in cephalic tetanus.

Cephalic tetanus (CT) is a severe form of tetanus that follows head wounds and the intoxication of cranial nerves by tetanus neurotoxin (TeNT). Hallmarks of CT are cerebral palsy, which anticipates the spastic paralysis of tetanus, and rapid evolution of cardiorespiratory deficit even without generalized tetanus. How TeNT causes this unexpected flaccid paralysis, and how the canonical spasticity then rapidly evolves into cardiorespiratory defects, remain unresolved aspects of CT pathophysiology. Using electrophysiology and immunohistochemistry, we demonstrate that TeNT cleaves its substrate vesicle-associated membrane protein within facial neuromuscular junctions and causes a botulism-like paralysis overshadowing tetanus spasticity. Meanwhile, TeNT spreads among brainstem neuronal nuclei and, as shown by an assay measuring the ventilation ability of CT mice, harms essential functions like respiration. A partial axotomy of the facial nerve revealed a potentially new ability of TeNT to undergo intra-brainstem diffusion, which allows the toxin to spread to brainstem nuclei devoid of direct peripheral efferents. This mechanism is likely to be involved in the transition from local to generalized tetanus. Overall, the present findings suggest that patients with idiopathic facial nerve palsy should be immediately considered for CT and treated with antisera to block the potential progression to a life-threatening form of tetanus.

Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody.

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.

Toxicology and pharmacology of botulinum and tetanus neurotoxins: an update.

Tetanus and botulinum neurotoxins cause the neuroparalytic syndromes of tetanus and botulism, respectively, by delivering inside different types of neurons, metalloproteases specifically cleaving the SNARE proteins that are essential for the release of neurotransmitters. Research on their mechanism of action is intensively carried out in order to devise improved therapies based on antibodies and chemical drugs. Recently, major results have been obtained with human monoclonal antibodies and with single chain antibodies that have allowed one to neutralize the metalloprotease activity of botulinum neurotoxin type A1 inside neurons. In addition, a method has been devised to induce a rapid molecular evolution of the metalloprotease domain of botulinum neurotoxin followed by selection driven to re-target the metalloprotease activity versus novel targets with respect to the SNARE proteins. At the same time, an intense and wide spectrum clinical research on novel therapeutics based on botulinum neurotoxins is carried out, which are also reviewed here.

Latrotoxin-Induced Neuromuscular Junction Degeneration Reveals Urocortin 2 as a Critical Contributor to Motor Axon Terminal Regeneration.

We used α-Latrotoxin (α-LTx), the main neurotoxic component of the black widow spider venom, which causes degeneration of the neuromuscular junction (NMJ) followed by a rapid and complete regeneration, as a molecular tool to identify by RNA transcriptomics factors contributing to the structural and functional recovery of the NMJ. We found that Urocortin 2 (UCN2), a neuropeptide involved in the stress response, is rapidly expressed at the NMJ after acute damage and that inhibition of CRHR2, the specific receptor of UCN2, delays neuromuscular transmission rescue. Experiments in neuronal cultures show that CRHR2 localises at the axonal tips of growing spinal motor neurons and that its expression inversely correlates with synaptic maturation. Moreover, exogenous UCN2 enhances the growth of axonal sprouts in cultured neurons in a CRHR2-dependent manner, pointing to a role of the UCN2-CRHR2 axis in the regulation of axonal growth and synaptogenesis. Consistently, exogenous administration of UCN2 strongly accelerates the regrowth of motor axon terminals degenerated by α-LTx, thereby contributing to the functional recovery of neuromuscular transmission after damage. Taken together, our results posit a novel role for UCN2 and CRHR2 as a signalling axis involved in NMJ regeneration.

Novel Small Molecule Inhibitors That Prevent the Neuroparalysis of Tetanus Neurotoxin.

Tetanus neurotoxin (TeNT) is a protein exotoxin produced by Clostridium tetani that causes the deadly spastic neuroparalysis of tetanus. It consists of a metalloprotease light chain and of a heavy chain linked via a disulphide bond. TeNT binds to the neuromuscular junction (NMJ) and it is retro-axonally transported into vesicular compartments to the spinal cord, where it is released and taken up by inhibitory interneuron. Therein, the catalytic subunit is translocated into the cytoplasm where it cleaves its target protein VAMP-1/2 with consequent blockage of the release of inhibitory neurotransmitters. Vaccination with formaldehyde inactivated TeNT prevents the disease, but tetanus is still present in countries where vaccination coverage is partial. Here, we show that small molecule inhibitors interfering with TeNT trafficking or with the reduction of the interchain disulphide bond block the activity of the toxin in neuronal cultures and attenuate tetanus symptoms in vivo. These findings are relevant for the development of therapeutics against tetanus based on the inhibition of toxin molecules that are being retro-transported to or are already within the spinal cord and are, thus, not accessible to anti-TeNT immunoglobulins.

Paper-based electrochemical peptide sensor for on-site detection of botulinum neurotoxin serotype A and C.

Botulinum neurotoxins (BoNTs) produced by soil bacterium Clostridium botulinum are cause of botulism and listed as biohazard agents, thus rapid screening assays are needed for taking the correct countermeasures in a timely fashion. The gold standard method relies on the mouse lethality assay with a lengthy analysis time, i.e., 2-5 days, hindering the prompt management of food safety and medical diagnosis. Herein, we propose the first paper-based antibody-free sensor for reliable and rapid detection of BoNT/A and BoNT/C, exploiting their cleavage capability toward a synthetic peptide able to mimic the natural substrate SNAP-25. The peptide is labelled with the electroactive molecule methylene blue and immobilized on the paper-based electrode modified with gold nanoparticles. Because BoNT/A and BoNT/C can cleave the peptide with the removal of methylene blue from electrode surface, the presence of these neurotoxins in the sample leads to a signal decrease proportional to BoNT amount. The biosensor developed with the selected peptide and combined with smartphone assisted potentiostat is able to detect both BoNT/A and BoNT/C with a linearity up to 1 nM and a detection limit equal to 10 pM. The applicability of this biosensor was evaluated with spiked samples of orange juice, obtaining recovery values equal to 104 ± 6% and 98 ± 9% for 1 nM and 0.5 nM of BoNT/A, respectively.

Tetanus and tetanus neurotoxin: From peripheral uptake to central nervous tissue targets.

Tetanus is a deadly but preventable disease caused by a protein neurotoxin produced by Clostridium tetani. Spores of C. tetani may contaminate a necrotic wound and germinate into a vegetative bacterium that releases a toxin, termed tetanus neurotoxin (TeNT). TeNT enters the general circulation, binds to peripheral motor neurons and sensory neurons, and is transported retroaxonally to the spinal cord. It then enters inhibitory interneurons and blocks the release of glycine or GABA causing a spastic paralysis. This review attempts to correlate the metalloprotease activity of TeNT and its trafficking and localization into the vertebrate body to the nature and sequence of appearance of the symptoms of tetanus.

Genome Sequence of the Fish Brain Bacterium Clostridium tarantellae.

Eubacterium tarantellae was originally cultivated from the brain of fish affected by twirling movements. Here, we present the draft genome sequence of E. tarantellae DSM 3997, which consists of 3,982,316 bp. Most protein-coding genes in this strain are similar to genes of Clostridium bacteria, supporting the renaming of E. tarantellae as Clostridium tarantellae.

Clinical duration of action of different botulinum toxin types in humans.

The Botulinum NeuroToxin (BoNT) comprises several serotypes with distinct properties, mechanisms of action, sensitivity and duration of effect in different species. The serotype A (BoNT/A) is the prevalent neurotoxin applied in human's disease. In this paper we present an overview of the current knowledge regarding the duration of effect and the neuromuscular sprouting of different BoNT serotypes in humans. Then, we report the original results of a study in healthy subjects treated with BoNT/A, B, C and F using different neurophysiological techniques. Twelve healthy volunteers (7 men, 5 women) are treated with BoNT/A, B, C and F or placebo in Abductor digiti minimi (ADM) muscle of the hand. Before and after injections, an extensive neurophysiological study is performed with the CMAP amplitude variation, Multi-Motor Unit Action Potentials (MUAPs) analysis, the Turns/Amplitude ratio of interference pattern (IP) and determination of jitter and Fiber Density (FD) at single-fiber electromyography (SFEMG), at week 2 (w2), 4 (w4), 6 (w6) and 8 (w8). A maximal neuromuscular block is obtained at w2 for all the serotypes. Afterwards, the CMAP trend appear similar for BoNT/A, B, and C while, BoNT/F shows a faster recover. Multi-MUAPs analysis and IP detect mild changes at w2 for all serotypes, except for BoNT/F that shows a greater change since w4. SFEMG have minimal changes in FD while, Jitter increase at w2 with a slower decrease over the time for all BoNTs. In conclusion, BoNT/F has earlier sprouting and complete recovery at w8. Other serotypes present a slower and similar profile. The EMG appear useful to study the functional recovery in humans, and these results should provide new evidence for assessing different serotypes. These findings improve our knowledge regarding the methods to evaluate duration of effects and dose equivalents in different serotypes, that in the future could change the clinicians strategy for disease-tailored BoNT therapies.

Tables of Toxicity of Botulinum and Tetanus Neurotoxins.

Tetanus and botulinum neurotoxins are the most poisonous substances known, so much so as to be considered for a possible terrorist use. At the same time, botulinum neurotoxin type A1 is successfully used to treat a variety of human syndromes characterized by hyperactive cholinergic nerve terminals. The extreme toxicity of these neurotoxins is due to their neurospecificity and to their metalloprotease activity, which results in the deadly paralysis of tetanus and botulism. Recently, many novel botulinum neurotoxins and some botulinum-like toxins have been discovered. This large number of toxins differs in terms of toxicity and biological activity, providing a potential goldmine for novel therapeutics and for new molecular tools to dissect vesicular trafficking, fusion, and exocytosis. The scattered data on toxicity present in the literature require a systematic organization to be usable by scientists and clinicians. We have assembled here the data available in the literature on the toxicity of these toxins in different animal species. The internal comparison of these data provides insights on the biological activity of these toxins.

The role of the single interchains disulfide bond in tetanus and botulinum neurotoxins and the development of antitetanus and antibotulism drugs.

A large number of bacterial toxins consist of active and cell binding protomers linked by an interchain disulfide bridge. The largest family of such disulfide-bridged exotoxins is that of the clostridial neurotoxins that consist of two chains and comprise the tetanus neurotoxins causing tetanus and the botulinum neurotoxins causing botulism. Reduction of the interchain disulfide abolishes toxicity, and we discuss the experiments that revealed the role of this structural element in neuronal intoxication. The redox couple thioredoxin reductase-thioredoxin (TrxR-Trx) was identified as the responsible for reduction of this disulfide occurring on the cytosolic surface of synaptic vesicles. We then discuss the very relevant finding that drugs that inhibit TrxR-Trx also prevent botulism. On this basis, we propose that ebselen and PX-12, two TrxR-Trx specific drugs previously used in clinical trials in humans, satisfy all the requirements for clinical tests aiming at evaluating their capacity to effectively counteract human and animal botulism arising from intestinal toxaemias such as infant botulism.

Transynaptic Action of Botulinum Neurotoxin Type A at Central Cholinergic Boutons.

Botulinum neurotoxin Type A (BoNT/A) is an effective treatment for several movement disorders, including spasticity and dystonia. BoNT/A acts by cleaving synaptosomal-associated protein of 25 kDa (SNAP-25) at the neuromuscular junction, thus blocking synaptic transmission and weakening overactive muscles. However, not all the therapeutic benefits of the neurotoxin are explained by peripheral neuroparalysis, suggesting an action of BoNT/A on central circuits. Currently, the specific targets of BoNT/A central activity remain unclear. Here, we show that catalytically active BoNT/A is transported to the facial nucleus (FN) after injection into the nasolabial musculature of rats and mice. BoNT/A-mediated cleavage of SNAP-25 in the FN is prevented by intracerebroventricular delivery of antitoxin antibodies, demonstrating that BoNT/A physically leaves the motoneurons to enter second-order neurons. Analysis of intoxicated terminals within the FN shows that BoNT/A is transcytosed preferentially into cholinergic synapses. The cholinergic boutons containing cleaved SNAP-25 are associated with a larger size, suggesting impaired neuroexocytosis. Together, the present findings indicate a previously unrecognized source of reduced motoneuron drive after BoNT/A via blockade of central, excitatory cholinergic inputs. These data highlight the ability of BoNT/A to selectively target and modulate specific central circuits, with consequent impact on its therapeutic effectiveness in movement disorders.SIGNIFICANCE STATEMENT Botulinum neurotoxins are among the most potent toxins known. Despite this, their specific and reversible action prompted their use in clinical practice to treat several neuromuscular pathologies (dystonia, spasticity, muscle spasms) characterized by hyperexcitability of peripheral nerve terminals or even in nonpathological applications (i.e., cosmetic use). Substantial experimental and clinical evidence indicates that not all botulinum neurotoxin Type A (BoNT/A) effects can be explained solely by the local action (i.e., silencing of the neuromuscular junction). In particular, there are cases in which the clinical benefit exceeds the duration of peripheral neurotransmission blockade. In this study, we demonstrate that BoNT/A is transported to facial motoneurons, released, and internalized preferentially into cholinergic terminals impinging onto the motoneurons. Our data demonstrate a direct central action of BoNT/A.

Primary resistance of human patients to botulinum neurotoxins A and B.

Botulinum neurotoxin serotypes A and B are successfully used to treat a variety of human diseases characterized by hyperactive peripheral nerve terminals. However, a number of patients are primary resistant to these pharmaceuticals, without having antitoxin-neutralizing antibodies. A straightforward explanation of this phenomenon posits that mutations of the toxin sites of interaction with their receptors or protein substrates prevent their neuroparalytic action. After a careful investigation of available human genomic databases, we conclude that it is very unlikely that humans are resistant to these two therapeutic neurotoxins because of mutations that would affect their binding or intracellular proteolytic actions.

Variability in venom composition of European viper subspecies limits the cross-effectiveness of antivenoms.

Medically relevant cases of snakebite in Europe are predominately caused by European vipers of the genus Vipera. Systemic envenoming by European vipers can cause severe pathology in humans and different clinical manifestations are associated with different members of this genus. The most representative vipers in Europe are V. aspis and V. berus and neurological symptoms have been reported in humans envenomed by the former but not by the latter species. In this study we determined the toxicological profile of V. aspis and V. berus venoms in vivo in mice and we tested the effectiveness of two antivenoms, commonly used as antidotes, in counteracting the specific activities of the two venoms. We found that V. aspis, but not V. berus, is neurotoxic and that this effect is due to the degeneration of peripheral nerve terminals at the NMJ and is not neutralized by the two tested antisera. Differently, V. berus causes a haemorrhagic effect, which is efficiently contrasted by the same antivenoms. These results indicate that the effectiveness of different antisera is strongly influenced by the variable composition of the venoms and reinforce the arguments supporting the use polyvalent antivenoms.

Hsp90 and Thioredoxin-Thioredoxin Reductase enable the catalytic activity of Clostridial neurotoxins inside nerve terminals.

Botulinum (BoNTs) and tetanus (TeNT) neurotoxins are the most toxic substances known and form the growing family of Clostridial neurotoxins (CNT), the etiologic agents of botulism and tetanus. CNT are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the membrane translocation of L into the cytosol, where its substrates, the three SNARE proteins, are localized. L translocation is accompanied by unfolding and, once delivered on the cytosolic side of the endosome membrane, it has to be reduced and reacquire the native fold to be active. The Thioredoxin-Thioredoxin Reductase system (Trx-TrxR) specifically reduces the interchain disulfide bond while the cytosolic chaperone protein Hsp90 mediates L refolding. Both steps are essential for CNT activity and their inhibition efficiently blocks the neurotoxicity in cultured neurons and mice. Trx and its reductase physically interact with Hsp90 and are loosely bound to the cytosolic side of synaptic vesicles, the organelle exploited by CNT to enter nerve terminals and wherefrom L is translocated into the cytosol. Therefore, Trx, TrxR and Hsp90 orchestrate a chaperone-redox molecular machinery that enables the catalytic activity of the L inside nerve terminals. Given the fundamental role of L reduction and refolding, this machinery represents a rational target for the development of mechanism-based antitoxins.

High Conservation of Tetanus and Botulinum Neurotoxins Cleavage Sites on Human SNARE Proteins Suggests That These Pathogens Exerted Little or No Evolutionary Pressure on Humans.

The Genome Aggregation Database presently contains >120,000 human genomes. We searched in this database for the presence of mutations at the sites of tetanus (TeNT) and botulinum neurotoxins (BoNTs) cleavages of the three SNARE proteins: VAMP, SNAP-25 and Syntaxin. These mutations could account for some of the BoNT/A resistant patients. At the same time, this approach was aimed at testing the possibility that TeNT and BoNT may have acted as selective agents in the development of resistance to tetanus or botulism. We found that mutations of the SNARE proteins are very rare and concentrated outside the SNARE motif required for the formation of the SNARE complex involved in neuroexocytosis. No changes were found at the BoNT cleavage sites of VAMP and syntaxins and only one very rare mutation was found in the essential C-terminus region of SNAP-25, where Arg198 was replaced with a Cys residue. This is the P1' cleavage site for BoNT/A and the P1 cleavage site for BoNT/C. We found that the Arg198Cys mutation renders SNAP-25 resistant to BoNT/A. Nonetheless, its low frequency (1.8 × 10-5) indicates that mutations of SNAP-25 at the BoNT/A cleavage site are unlikely to account for the existence of BoNT/A resistant patients. More in general, the present findings indicate that tetanus and botulinum neurotoxins have not acted as selective agents during human evolution as it appears to have been the case for tetanus in rats and chicken.

Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission.

Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absence of neuromuscular damage. Two triple mutants of BoNT/C, namely BoNT/C S51T/R52N/N53P (BoNT/C α-51) and BoNT/C L200W/M221W/I226W (BoNT/C α-3W), were recently reported to selectively cleave syntaxin and have been used here to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to the effect of BoNT/C in vivo. Although BoNT/C α-51 and BoNT/C α-3W toxins cleave syntaxin with similar efficiency, we unexpectedly found also cleavage of SNAP-25, although to a lesser extent than wild type BoNT/C. Interestingly, the BoNT/C mutants exhibit reduced lethality compared to wild type toxin, a result that correlated with their residual activity against SNAP-25. In spite of this, a local injection of BoNT/C α-51 persistently impairs neuromuscular junction activity. This is due to an initial phase in which SNAP-25 cleavage causes a complete blockade of neurotransmission, and to a second phase of incomplete impairment ascribable to syntaxin cleavage. Together, these results indicate that neuroparalysis of BoNT/C at the neuromuscular junction is due to SNAP-25 cleavage, while the proteolysis of syntaxin provides a substantial, but incomplete, neuromuscular impairment. In light of this evidence, we discuss a possible clinical use of BoNT/C α-51 as a botulinum neurotoxin endowed with a wide safety margin and a long lasting effect.