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The entry of tetanus and botulinum neurotoxins into neurons
Full text linkTetanus 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
Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry
Full text linkCerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure culture of CGN from postnatal rats/mice and an easy procedure for their intoxication with CNT. We also illustrate handy methods to evaluate CNT activity and their inhibition
Challenges in searching for therapeutics against Botulinum Neurotoxins
No full textBotulinum neurotoxins (BoNTs) are the most potent toxins known. BoNTs are responsible for botulism, a deadly neuroparalytic syndrome caused by the inactivation of neurotransmitter release at peripheral nerve terminals. Thanks to their specificity and potency, BoNTs are both considered potential bio-weapons and therapeutics of choice for a variety of medical syndromes. Several variants of BoNTs have been identified with individual biological properties and little antigenic relation. This expands greatly the potential of BoNTs as therapeutics but poses a major safety problem, increasing the need for finding appropriate antidotes. Areas covered: The authors describe the multi-step molecular mechanism through which BoNTs enter nerve terminals and discuss the many levels at which the toxins can be inhibited. They review the outcomes of the different strategies adopted to limit neurotoxicity and counter intoxication. Potential new targets arising from the last discoveries of the mechanism of action and the approaches to promote neuromuscular junction recovery are also discussed. Expert opinion: Current drug discovery efforts have mainly focused on BoNT type A and addressed primarily light chain proteolytic activity. Development of pan-BoNT inhibitors acting independently of BoNT immunological properties and targeting a common step of the intoxication process should be encouraged
An update on the mechanism of action of tetanus and botulinum neurotoxins.
Full text linkTetanus and botulinum neurotoxins, produced by anaerobic bacteria of the genus Clostridium, are the most toxic proteins
known and are the sole responsible for the pathogenesis of tetanus and botulism. They enter peripheral cholinergic
nerve terminals and cleave proteins of the neuroexocytosis apparatus causing a persistent, but reversible, inhibition of
neurotransmitter release. Botulinum neurotoxins are used in the therapy of many human syndromes caused by hyperactive
cholinergic nerve terminals. Here we focus on the many advances that were recently made on the understanding of
their molecular mechanism of action and on their use in human therapy
Impact of ageing and disuse on neuromuscular junction and mitochondrial function and morphology: Current evidence and controversies
Full text linkInactivity and ageing can have a detrimental impact on skeletal muscle and the neuromuscular junction (NMJ). Decreased physical activity results in muscle atrophy, impaired mitochondrial function, and NMJ instability. Ageing is associated with a progressive decrease in muscle mass, deterioration of mitochondrial function in the motor axon terminals and in myofibres, NMJ instability and loss of motor units. Focusing on the impact of inactivity and ageing, this review examines the consequences on NMJ stability and the role of mitochondrial dysfunction, delving into their complex relationship with ageing and disuse. Evidence suggests that mitochondrial dysfunction can be a pathogenic driver for NMJ alterations, with studies revealing the role of mitochondrial defects in motor neuron degeneration and NMJ instability. Two perspectives behind NMJ instability are discussed: one is that mitochondrial dysfunction in skeletal muscle triggers NMJ deterioration, the other envisages dysfunction of motor terminal mitochondria as a primary contributor to NMJ instability. While evidence from these studies supports both perspectives on the relationship between NMJ dysfunction and mitochondrial impairment, gaps persist in the understanding of how mitochondrial dysfunction can cause NMJ deterioration. Further research, both in humans and in animal models, is essential for unravelling the mechanisms and potential interventions for age- and inactivity-related neuromuscular and mitochondrial alterations
Double anchorage to the membrane and intact inter-chain disulfide bond are required for the low pH induced entry of tetanus and botulinum neurotoxins into neurons
No full textTetanus and botulinum neurotoxins are di-chain proteins that cause paralysis by inhibiting neuroexocytosis. These neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, whose 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 was made accessible to investigation by making it to occur at the surface of neurons. The neurotoxin, bound to the plasma membrane in the cold, was exposed to a warm 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 and botulinum neurotoxin B, C and D is similar and take places in the same slightly acidic range, which comprises that present inside synaptic vesicles. Based on these and previous findings, we propose a stepwise sequence of molecular events that lead from toxin binding to membrane insertion
Novel Botulinum Neurotoxins: Exploring Underneath the Iceberg Tip
Full text linkBotulinum neurotoxins (BoNTs), the etiological agents of botulism, are the deadliest toxins known to humans. Yet, thanks to their biological and toxicological features, BoNTs have become sophisticated tools to study neuronal physiology and valuable therapeutics for an increasing number of human disorders. BoNTs are produced by multiple bacteria of the genus Clostridium and, on the basis of their different immunological properties, were classified as seven distinct types of toxin. BoNT classification remained stagnant for the last 50 years until, via bioinformatics and high-throughput sequencing techniques, dozens of BoNT variants, novel serotypes as well as BoNT-like toxins within non-clostridial species have been discovered. Here, we discuss how the now “booming field” of botulinum neurotoxin may shed light on their evolutionary origin and open exciting avenues for future therapeutic applications
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