The entry of tetanus and botulinum neurotoxins into neurons

Tetanus and botulinum neurotoxins cause neuroparalysis by inhibiting neuroexocytosis. They are composed by two main chains: the 100 kDa heavy chain (H) mediates the neurospecific binding to target cells and chaperons the entry of the 50 kDa light chain (L). After binding on the plasma membrane, these neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, as shown here for the first time by immuno-electron microscopy. The lumenal acidic pH induces a structural change of the neurotoxin molecule that becomes capable of translocating its L chain into the cytosol, via a transmembrane protein-conducting channel made by the H chain. This is the least understood step of the intoxication process primarily because it takes place inside vesicles within the cytosol. In the present study, we describe how this passage can be made accessible to investigation by making it to occur at the plasma membrane of neurons. The neurotoxin, bound to the plasma membrane of cerebellar granular neurons in the cold, was exposed to a low pH extracellular medium and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. We found that the neurotoxin has to be bound to the membrane via at least two anchorage sites in order for a productive low-pH induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT) /B, /C and /D is similar and takes place in the same slightly acidic range, which comprises that present inside synaptic vesicles. Thanks to this reliable method we have also studied the temperature dependence and the time course of TeNT, BoNT/C and BoNT/D L chain entry across the plasma membrane. The time course of translocation of the L chain varies for the three neurotoxins, but 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. Another fundamental event along CNTs neuron intoxication process is the reduction of the interchain disulphide bond. This is a conditio sine qua non to free the catalytic part of the molecule in the cytosol of neurons. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-Thioredoxin reductase-Thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins. BoNT/A is the most frequent cause of human botulism and at the same time is largely used in human therapy. Some evidences indicate that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been formally proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscopy, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor SV2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle transmembrane pH gradient, we show that translocation of the neurotoxin into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by one or two neurotoxin molecules. Another important aspect regarding CNTs research is their employment in human therapy. Accordingly, BoNT/A is almost invariably used in the treatment of many human diseases characterized by hyperactivity of peripheral cholinergic nerve terminals. Unfortunately, some patients are or become resistant to it. This drawback can be overcome by using other botulinum toxins, and pre-clinical studies have been performed with different toxin serotypes. Botulinum neurotoxin type D has never been tested in human muscles in vivo. Here we show that BoNT/D is very effective upon injection in mice, on the mouse hemidiaphragm preparation and on different rat primary neuronal cultures. From these experiments, doses to be injected in human volunteers were determined. The effect of the injection into the human Extensor Digitorum Brevis muscle was assayed by measuring the compound muscle action potential at different times after injection. Botulinum toxin type D was found to be very uneffective in inducing human skeletal muscle paralysis. These results are interpreted in terms of recent reports on neuronal surface receptors of this neurotoxin and of the established double receptor model of binding

Snake and spider toxins induce a rapid recovery of function of botulinum neurotoxin paralysed neuromuscular junction

Botulinum neurotoxins (BoNTs) and some animal neurotoxins (-Bungarotoxin, -Btx, from elapid snakes and -Latrotoxin, -Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis. This result provides a proof of principle that, by causing the complete degeneration, reabsorption, and regeneration of a paralysed nerve terminal, one could favour the recovery of function of a biochemically- or genetically-altered motor axon terminal. These observations might be relevant to dying-back neuropathies, where pathological changes first occur at the neuromuscular junction and then progress proximally toward the cell body

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

Measuring utility with diffusion models

The diffusion model (DDM) is a prominent account of how people make decisions. Many of these decisions involve comparing two alternatives based on differences of perceived stimulus magnitudes, such as economic values. Here, we propose a consistent estimator for the parameters of a DDM in such cases. This estimator allows us to derive decision thresholds, drift rates, and subjective percepts (i.e., utilities in economic choice) directly from the experimental data. This eliminates the need to measure these values separately or to assume specific functional forms for them. Our method also allows one to predict drift rates for comparisons that did not occur in the dataset. We apply the method to two datasets, one comparing probabilities of earning a fixed reward and one comparing objects of variable reward value. Our analysis indicates that both datasets conform well to the DDM. Interestingly, we find that utilities are linear in probability and slightly convex in reward

Design and manufacture of hybrid aluminum/composite co-cured tubes with viscoelastic interface layer

Abstract The development of hybrid FRP-metal axisymmetric components is a matter of increasing interest in the automotive and aerospace industry for a lightweight design. The hybridization of the technology enables a cost reduction of components production and an increase of mechanical performances together with the ability of machining the surface of the metal tube; this technique guarantees a production improvement since the coating of the tube is no longer required and the pieces can be manufactured in one-step curing cycle. The improvement in bending and torsional stiffness, corrosion resistance and mass reduction of hybrid tubes, in comparison to the single material-built component, has been already demonstrated. A great challenge is to find a way to make hybrid tubes with external metal, avoiding delaminations and detachments that could occur during and after the curing process, due to the different coefficients of thermal expansion between FRP and metal along the axial direction of the tube. For this reason, a component manufacturing process has been studied by experimental and numerical analysis (FEM), including custom process machines and inserting a viscoelastic layer as an interface between the two tubes. Genetic algorithm method has been used to optimize the stacking sequence of a hybrid co-cured metal/composite tube to maximize the flexural stiffness, while applying a strength constraint condition

CXCL12/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axonterminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by -latrotoxin. CXCL12 acts via binding to the neuronal CXCR4 receptor. A CXCL12-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration invivo. Recombinant CXCL12 invivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons invitro. These findings indicate that the CXCL12-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage