The tyrosine phosphatases LAR and PTPRδ act as receptors of the nidogen-tetanus toxin complex.

Tetanus neurotoxin (TeNT) causes spastic paralysis by inhibiting neurotransmission in spinal inhibitory interneurons. TeNT binds to the neuromuscular junction, leading to its internalisation into motor neurons and subsequent transcytosis into interneurons. While the extracellular matrix proteins nidogens are essential for TeNT binding, the molecular composition of its receptor complex remains unclear. Here, we show that the receptor-type protein tyrosine phosphatases LAR and PTPRδ interact with the nidogen-TeNT complex, enabling its neuronal uptake. Binding of LAR and PTPRδ to the toxin complex is mediated by their immunoglobulin and fibronectin III domains, which we harnessed to inhibit TeNT entry into motor neurons and protect mice from TeNT-induced paralysis. This function of LAR is independent of its role in regulating TrkB receptor activity, which augments axonal transport of TeNT. These findings reveal a multi-subunit receptor complex for TeNT and demonstrate a novel trafficking route for extracellular matrix proteins. Our study offers potential new avenues for developing therapeutics to prevent tetanus and dissecting the mechanisms controlling the targeting of physiological ligands to long-distance axonal transport in the nervous system.

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.

Sulfation at Glycopolymer Side Chains Switches Activity at the Macrophage Mannose Receptor (CD206) In Vitro and In Vivo.

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO4-3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO4-3-Gal-glycopolymer-CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO4-3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies.

Models and methods to study Schwann cells.

Schwann cells (SCs) are fundamental components of the peripheral nervous system (PNS) of all vertebrates and play essential roles in development, maintenance, function, and regeneration of peripheral nerves. There are distinct populations of SCs including: (1) myelinating SCs that ensheath axons by a specialized plasma membrane, called myelin, which enhances the conduction of electric impulses; (2) non-myelinating SCs, including Remak SCs, which wrap bundles of multiple axons of small caliber, and perysinaptic SCs (PSCs), associated with motor axon terminals at the neuromuscular junction (NMJ). All types of SCs contribute to PNS regeneration through striking morphological and functional changes in response to nerve injury, are affected in peripheral neuropathies and show abnormalities and a diminished plasticity during aging. Therefore, methodological approaches to study and manipulate SCs in physiological and pathophysiological conditions are crucial to expand the present knowledge on SC biology and to devise new therapeutic strategies to counteract neurodegenerative conditions and age-derived denervation. We present here an updated overview of traditional and emerging methodologies for the study of SCs for scientists approaching this research field.

Mannosylated Polycations Target CD206+ Antigen-Presenting Cells and Mediate T-Cell-Specific Activation in Cancer Vaccination.

Immunotherapy is deemed one of the most powerful therapeutic approaches to treat cancer. However, limited response and tumor specificity are still major challenges to address. Herein, mannosylated polycations targeting mannose receptor- are developed as vectors for plasmid DNA (pDNA)-based vaccines to improve selective delivery of genetic material to antigen-presenting cells and enhance immune cell activation. Three diblock glycopolycations (M15A12, M29A25, and M58A45) and two triblock copolymers (M29A29B9 and M62A52B32) are generated by using mannose (M), agmatine (A), and butyl (B) derivatives to target CD206, complex nucleic acids, and favor the endosomal escape, respectively. All glycopolycations efficiently complex pDNA at N/P ratios <5, protecting the pDNA from degradation in a physiological milieu. M58A45 and M62A52B32 complexed with plasmid encoding for antigenic ovalbumin (pOVA) trigger the immune activation of cultured dendritic cells, which present the SIINFEKL antigenic peptide via specific major histocompatibility complex-I. Importantly, administration of M58A45/pOVA elicits SIINFEKL-specific T-cell response in C56BL/6 mice bearing the melanoma tumor model B16-OVA, well in line with a reduction in tumor growth. These results qualify mannosylation as an efficient strategy to target immune cells in cancer vaccination and emphasize the potential of these glycopolycations as effective delivery vehicles for nucleic acids.

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.

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.

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.

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.

Postnatal Development and Distribution of Sympathetic Innervation in Mouse Skeletal Muscle.

Vertebrate neuromuscular junctions (NMJs) have been conceived as tripartite synapses composed of motor neuron, Schwann cell, and muscle fiber. Recent work has shown the presence of sympathetic neurons in the immediate vicinity of NMJs and experimental and clinical findings suggest that this plays an eminent role in adult NMJ biology. The present study examined the postnatal development and distribution of sympathetic innervation in different muscles using immunofluorescence, confocal microscopy, and Western blot. This demonstrates the proximity of sympathetic neurons in diaphragm, extensor digitorum longus, tibialis anterior, soleus, and levator auris longus muscles. In extensor digitorum longus muscle, sympathetic innervation of NMJs was quantified from perinatal to adult stage and found to increase up to two months of age. In diaphragm muscle, an extensive network of sympathetic neurons was prominent along the characteristic central synapse band. In summary, these data demonstrate that an elaborate sympathetic innervation is present in several mouse skeletal muscles and that this is often next to NMJs. Although the presence of sympathetic neurons at the perisynaptic region of NMJs increased during postnatal development, many synapses were already close to sympathetic neurons at birth. Potential implications of these findings for treatment of neuromuscular diseases are discussed.

Ablation of S1P3 receptor protects mouse soleus from age-related drop in muscle mass, force, and regenerative capacity.

We investigated the effects of S1P3 deficiency on the age-related atrophy, decline in force, and regenerative capacity of soleus muscle from 23-mo-old male (old) mice. Compared with muscle from 5-mo-old (adult) mice, soleus mass and muscle fiber cross-sectional area (CSA) in old wild-type mice were reduced by ~26% and 24%, respectively. By contrast, the mass and fiber CSA of soleus muscle in old S1P3-null mice were comparable to those of adult muscle. Moreover, in soleus muscle of wild-type mice, twitch and tetanic tensions diminished from adulthood to old age. A slowing of contractile properties was also observed in soleus from old wild-type mice. In S1P3-null mice, neither force nor the contractile properties of soleus changed during aging. We also evaluated the regenerative capacity of soleus in old S1P3-null mice by stimulating muscle regeneration through myotoxic injury. After 10 days of regeneration, the mean fiber CSA of soleus in old wild-type mice was significantly smaller (-28%) compared with that of regenerated muscle in adult mice. On the contrary, the mean fiber CSA of regenerated soleus in old S1P3-null mice was similar to that of muscle in adult mice. We conclude that in the absence of S1P3, soleus muscle is protected from the decrease in muscle mass and force, and the attenuation of regenerative capacity, all of which are typical characteristics of aging.

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.

Beyond neuromuscular activity: botulinum toxin type A exerts direct central action on spinal control of movement.

Overt muscle activity and impaired spinal locomotor control hampering coordinated movement is a hallmark of spasticity and movement disorders like dystonia. While botulinum toxin A (BoNT-A) standard therapy alleviates mentioned symptoms presumably due to its peripheral neuromuscular actions alone, the aim of present study was to examine for the first time the toxin's trans-synaptic activity within central circuits that govern the skilled movement. The rat hindlimb motor pools were targeted by BoNT-A intrasciatic bilateral injection (2 U per nerve), while its trans-synaptic action on premotor inputs was blocked by intrathecal BoNT-A-neutralising antitoxin (5 i.u.). Effects of BoNT-A on coordinated and high intensity motor tasks (rotarod, beamwalk swimming), and localised muscle weakness (digit abduction, gait ability) were followed until their substantial recovery by day 56 post BoNT-A. Later, (day 62-77) the BoNT-A effects were examined in unilateral calf muscle spasm evoked by tetanus toxin (TeNT, 1.5 ng). In comparison to peripheral effect alone, combined peripheral and central trans-synaptic BoNT-A action induced a more prominent and longer impairment of different motor tasks, as well as the localised muscle weakness. After near-complete recovery of motor functions, the BoNT-A maintained the ability to reduce the experimental calf spasm evoked by tetanus toxin (TeNT 1.5 ng, day 62) without altering the monosynaptic reflex excitability. These results indicate that, in addition to muscle terminals, BoNT-A-mediated control of hyperactive muscle activity in movement disorders and spasticity may involve the spinal premotor inputs and central circuits participating in the skilled locomotor performance.

Time course and temperature dependence of the membrane translocation of tetanus and botulinum neurotoxins C and D in neurons.

Tetanus and botulinum neurotoxins act inside nerve terminals and, therefore, they have to translocate across a membrane to reach their targets. This translocation is driven by a pH gradient, acidic on the cis side and neutral on the cytosol. Recently, a protocol to induce translocation from the plasma membrane was established. Here, we have used this approach to study the temperature dependence and time course of the entry of the L chain of tetanus neurotoxin and of botulinum neurotoxins type C and D across the plasma membrane of cerebellar granular neurons. The time course of translocation of the L chain varies for the three neurotoxins, but it remains in the range of minutes at 37 °C, whilst it takes much longer at 20 °C. BoNT/C does not enter neurons at 20 °C. Translocation also depends on the dimension of the pH gradient. These data are discussed with respect to the contribution of the membrane translocation step to the total time to paralysis and to the low toxicity of these neurotoxins in cold-blood vertebrates.

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.

Introduction to the Special Issue "Skeletal Muscle Atrophy: Mechanisms at a Cellular Level".

Skeletal muscle is the most abundant tissue in the body and requires high levels of energy to function properly. Skeletal muscle allows voluntary movement and body posture, which require different types of fiber, innervation, energy, and metabolism. Here, we summarize the contribution received at the time of publication of this Introductory Issue for the Special Issue dedicated to "Skeletal Muscle Atrophy: Mechanisms at a Cellular Level". The Special Issue is divided into three sections. The first is dedicated to skeletal muscle pathophysiology, the second to disease mechanisms, and the third to therapeutic development.