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.

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.

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.

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.

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.

Melatonin promotes regeneration of injured motor axons via MT1 receptors.

Melatonin is an ancient multi-tasking molecule produced by the pineal gland and by several extrapineal tissues. A variety of activities has been ascribed to this hormone in different physiological and pathological contexts, but little is known about its role in peripheral neuroregeneration. Here, we have exploited two different types of injury to test the capability of melatonin to stimulate regeneration of motor axons: (a) the acute and reversible presynaptic degeneration induced by the spider neurotoxin α-Latrotoxin and (b) the compression/transection of the sciatic nerve. We found that in both cases melatonin administration accelerates the process of nerve repair. This pro-regenerative action is MT1 -mediated, and at least in part due to a sustained activation of the ERK1/2 pathway. These findings reveal a receptor-mediated, pro-regenerative action of melatonin in vivo that holds important clinical implications, as it posits melatonin as a safe candidate molecule for the treatment of a number of peripheral neurodegenerative conditions.

Hydrogen peroxide is a neuronal alarmin that triggers specific RNAs, local translation of Annexin A2, and cytoskeletal remodeling in Schwann cells.

Schwann cells are key players in neuro-regeneration: They sense "alarm" signals released by degenerating nerve terminals and differentiate toward a proregenerative phenotype, with phagocytosis of nerve debris and nerve guidance. At the murine neuromuscular junction, hydrogen peroxide (H2O2) is a key signal of Schwann cells' activation in response to a variety of nerve injuries. Here we report that Schwann cells exposed to low doses of H2O2 rewire the expression of several RNAs at both transcriptional and translational levels. Among the genes positively regulated at both levels, we identified an enriched cluster involved in cytoskeleton remodeling and cell migration, with the Annexin (Anxa) proteins being the most represented family. We show that both Annexin A2 (Anxa2) transcript and protein accumulate at the tips of long pseudopods that Schwann cells extend upon H2O2 exposure. Interestingly, Schwann cells reply to this signal and to nerve injury by locally translating Anxa2 in pseudopods, and undergo an extensive cytoskeleton remodeling. Our results show that, similarly to neurons, Schwann cells take advantage of local protein synthesis to change shape and move toward damaged axonal terminals to facilitate axonal regeneration.

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.

Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology.

The study of botulinum neurotoxins (BoNT) is rapidly progressing in many aspects. Novel BoNTs are being discovered owing to next generation sequencing, but their biologic and pharmacological properties remain largely unknown. The molecular structure of the large protein complexes that the toxin forms with accessory proteins, which are included in some BoNT type A1 and B1 pharmacological preparations, have been determined. By far the largest effort has been dedicated to the testing and validation of BoNTs as therapeutic agents in an ever increasing number of applications, including pain therapy. BoNT type A1 has been also exploited in a variety of cosmetic treatments, alone or in combination with other agents, and this specific market has reached the size of the one dedicated to the treatment of medical syndromes. The pharmacological properties and mode of action of BoNTs have shed light on general principles of neuronal transport and protein-protein interactions and are stimulating basic science studies. Moreover, the wide array of BoNTs discovered and to be discovered and the production of recombinant BoNTs endowed with specific properties suggest novel uses in therapeutics with increasing disease/symptom specifity. These recent developments are reviewed here to provide an updated picture of the biologic mechanism of action of BoNTs, of their increasing use in pharmacology and in cosmetics, and of their toxicology.

Hsp90 is involved in the entry of clostridial neurotoxins into the cytosol of nerve terminals.

Botulinum and tetanus neurotoxins are the most toxic substances known and form the growing family of clostridial neurotoxins. They 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 their substrates, the three SNARE proteins, are localised. L translocation is accompanied by unfolding, and it has to be reduced and reacquire the native fold to exert its neurotoxicity. The Thioredoxin reductase-Thioredoxin system is responsible for the reduction, but it is unknown whether the refolding of L is spontaneous or aided by host chaperones. Here we report that geldanamycin, a specific inhibitor of heat shock protein 90, hampers the refolding of L after membrane translocation and completely prevents the cleavage of SNAREs. We also found that geldanamycin strongly synergises with PX-12, an inhibitor of thioredoxin, suggesting that the processes of L chain refolding and interchain disulfide reduction are strictly coupled. Indeed we found that the heat shock protein 90 and the Thioredoxin reductase-Thioredoxin system physically interact on synaptic vesicle where they orchestrate a chaperone-redox machinery which is exploited by clostridial neurotoxins to deliver their catalytic part into the cytosol.

Mitochondrial alarmins released by degenerating motor axon terminals activate perisynaptic Schwann cells.

An acute and highly reproducible motor axon terminal degeneration followed by complete regeneration is induced by some animal presynaptic neurotoxins, representing an appropriate and controlled system to dissect the molecular mechanisms underlying degeneration and regeneration of peripheral nerve terminals. We have previously shown that nerve terminals exposed to spider or snake presynaptic neurotoxins degenerate as a result of calcium overload and mitochondrial failure. Here we show that toxin-treated primary neurons release signaling molecules derived from mitochondria: hydrogen peroxide, mitochondrial DNA, and cytochrome c. These molecules activate isolated primary Schwann cells, Schwann cells cocultured with neurons and at neuromuscular junction in vivo through the MAPK pathway. We propose that this inter- and intracellular signaling is involved in triggering the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases.

On the translocation of botulinum and tetanus neurotoxins across the membrane of acidic intracellular compartments.

Tetanus and botulinum neurotoxins are produced by anaerobic bacteria of the genus Clostridium and are the most poisonous toxins known, with 50% mouse lethal dose comprised within the range of 0.1-few nanograms per Kg, depending on the individual toxin. Botulinum neurotoxins are similarly toxic to humans and can therefore be considered for potential use in bioterrorism. At the same time, their neurospecificity and reversibility of action make them excellent therapeutics for a growing and heterogeneous number of human diseases that are characterized by a hyperactivity of peripheral nerve terminals. The complete crystallographic structure is available for some botulinum toxins, and reveals that they consist of four domains functionally related to the four steps of their mechanism of neuron intoxication: 1) binding to specific receptors of the presynaptic membrane; 2) internalization via endocytic vesicles; 3) translocation across the membrane of endocytic vesicles into the neuronal cytosol; 4) catalytic activity of the enzymatic moiety directed towards the SNARE proteins. Despite the many advances in understanding the structure-mechanism relationship of tetanus and botulinum neurotoxins, the molecular events involved in the translocation step have been only partially elucidated. Here we will review recent advances that have provided relevant insights on the process and discuss possible models that can be experimentally tested. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

Animal models for studying motor axon terminal paralysis and recovery.

An extraordinary property of the peripheral nervous system is that nerve terminals can regenerate after damage caused by different physical, chemical, or biological pathogens. Regeneration is the result of a complex and ill-known interplay among the nerve, the glia, the muscle, the basal lamina and, in some cases, the immune system. This phenomenon has been studied using different injury models mainly in rodents, particularly in mice, where a lesion can be produced in a chosen anatomical area. These approaches differ significantly among them for the nature of the lesion and the final outcomes. We have reviewed here the most common experimental models employed to induce motor axon injury, the relative advantages and drawbacks, and the principal read-outs used to monitor the regenerative process. Recently introduced tools for inducing reversible damage to the motor axon terminal that overcome some of the drawbacks of the more classical approaches are also discussed. Animal models have provided precious information about the cellular components involved in the regenerative process and on its electrophysiological features. Methods and tools made available recently allow one to identify and study molecules that are involved in the crosstalk among the components of the endplate. The time-course of the intercellular signaling and of the intracellular pathways activated will draw a picture of the entire process of regeneration as seen from a privileged anatomical site of observation. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Identification and characterization of Clostridium botulinum group III field strains by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Animal botulism is primarily due to botulinum neurotoxin (BoNT) types C, D or their chimeric variants C/D or D/C, produced by Clostridium botulinum group III, which appears to include the genetically indistinguishable Clostridium haemolyticum and Clostridium novyi. In the present study, we used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS) to identify and characterize 81 BoNT-producing Clostridia isolated in 47 episodes of animal botulism. The instrument's default database, containing no entries for Clostridium botulinum, permitted reliable identification of 26 strains at the genus level. Although supplementation of the database with reference strains enhanced the instrument's ability to identify the neurotoxic strains at the genus level, resolution was not sufficient to recognize field strains at species level. Characterization by MALDI TOF confirmed the well-documented phenotypic and genetic differences between Clostridium botulinum strains of serotypes normally implicated in human botulism (A, B, E, F) and other Clostridium species able to produce BoNTs type C and D. The chimeric and non-chimeric field strains grouped separately. In particular, very low similarity was found between two non-chimeric type C field strains isolated in the same outbreak and the other field strains. This difference was comparable with the differences among the various Clostridia species included in the study. Characterization by MALDI TOF confirmed that BoNT-producing Clostridia isolated from animals are closely related and indistinguishable at the species level from Clostridium haemolyticum and Clostridium novyi reference strains. On the contrary, there seem to be substantial differences among chimeric and some non-chimeric type C strains.

An animal model of Miller Fisher syndrome: Mitochondrial hydrogen peroxide is produced by the autoimmune attack of nerve terminals and activates Schwann cells.

The neuromuscular junction is a tripartite synapse composed of the presynaptic nerve terminal, the muscle and perisynaptic Schwann cells. Its functionality is essential for the execution of body movements and is compromised in a number of disorders, including Miller Fisher syndrome, a variant of Guillain-Barré syndrome: this autoimmune peripheral neuropathy is triggered by autoantibodies specific for the polysialogangliosides GQ1b and GT1a present in motor axon terminals, including those innervating ocular muscles, and in sensory neurons. Their binding to the presynaptic membrane activates the complement cascade, leading to a nerve degeneration that resembles that caused by some animal presynaptic neurotoxins. Here we have studied the intra- and inter-cellular signaling triggered by the binding and complement activation of a mouse monoclonal anti-GQ1b/GT1a antibody to primary cultures of spinal cord motor neurons and cerebellar granular neurons. We found that a membrane attack complex is rapidly assembled following antibody binding, leading to calcium accumulation, which affects mitochondrial functionality. Consequently, using fluorescent probes specific for mitochondrial hydrogen peroxide, we found that this reactive oxygen species is rapidly produced by mitochondria of damaged neurons, and that it triggers the activation of the MAP kinase pathway in Schwann cells. These results throw light on the molecular and cellular pathogenesis of Miller Fisher syndrome, and may well be relevant to other pathologies of the motor axon terminals, including some subtypes of the Guillain Barré syndrome.

The adjuvant MF59 induces ATP release from muscle that potentiates response to vaccination.

Vaccines are the most effective agents to control infections. In addition to the pathogen antigens, vaccines contain adjuvants that are used to enhance protective immune responses. However, the molecular mechanism of action of most adjuvants is ill-known, and a better understanding of adjuvanticity is needed to develop improved adjuvants based on molecular targets that further enhance vaccine efficacy. This is particularly important for tuberculosis, malaria, AIDS, and other diseases for which protective vaccines do not exist. Release of endogenous danger signals has been linked to adjuvanticity; however, the role of extracellular ATP during vaccination has never been explored. Here, we tested whether ATP release is involved in the immune boosting effect of four common adjuvants: aluminum hydroxide, calcium phosphate, incomplete Freund's adjuvant, and the oil-in-water emulsion MF59. We found that intramuscular injection is always associated with a weak transient release of ATP, which was greatly enhanced by the presence of MF59 but not by all other adjuvants tested. Local injection of apyrase, an ATP-hydrolyzing enzyme, inhibited cell recruitment in the muscle induced by MF59 but not by alum or incomplete Freund's adjuvant. In addition, apyrase strongly inhibited influenza-specific T-cell responses and hemagglutination inhibition titers in response to an MF59-adjuvanted trivalent influenza vaccine. These data demonstrate that a transient ATP release is required for innate and adaptive immune responses induced by MF59 and link extracellular ATP with an enhanced response to vaccination.

Evidence for a radial SNARE super-complex mediating neurotransmitter release at the Drosophila neuromuscular junction.

The SNARE proteins VAMP/synaptobrevin, SNAP-25 and syntaxin are core components of the apparatus that mediates neurotransmitter release. They form a heterotrimeric complex, and an undetermined number of SNARE complexes assemble to form a super-complex. Here, we present a radial model of this nanomachine. Experiments performed with botulinum neurotoxins led to the identification of one arginine residue in SNAP-25 and one aspartate residue in syntaxin (R206 and D253 in Drosophila melanogaster). These residues are highly conserved and predicted to play a major role in the protein-protein interactions between SNARE complexes by forming an ionic couple. Accordingly, we generated transgenic Drosophila lines expressing SNAREs mutated in these residues and performed an electrophysiological analysis of their neuromuscular junctions. Our results indicate that SNAP-25-R206 and syntaxin-D253 play a major role in neuroexocytosis and support a radial assembly of several SNARE complexes interacting via the ionic couple formed by these two residues.

The apoptogenic toxin AIP56 is a metalloprotease A-B toxin that cleaves NF-κb P65.

AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39)-Glu(40) peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

The blockade of the neurotransmitter release apparatus by botulinum neurotoxins.

The high toxicity of the seven serotypes of botulinum neurotoxins (BoNT/A to G), together with their specificity and reversibility, includes them in the list A of potential bioterrorism weapons and, at the same time, among the therapeutics of choice for a variety of human syndromes. They invade nerve terminals and cleave specifically the three proteins which form the heterotrimeric SNAP REceptors (SNARE) complex that mediates neurotransmitter release. The BoNT-induced cleavage of the SNARE proteins explains by itself the paralysing activity of the BoNTs because the truncated proteins cannot form the SNARE complex. However, in the case of BoNT/A, the most widely used toxin in therapy, additional factors come into play as it only removes a few residues from the synaptosomal associate protein of 25 kDa C-terminus and this results in a long duration of action. To explain these facts and other experimental data, we present here a model for the assembly of the neuroexocytosis apparatus in which Synaptotagmin and Complexin first assist the zippering of the SNARE complex, and then stabilize and clamp an octameric radial assembly of the SNARE complexes.