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Novel insights into botulinum neurotoxins mechanism of action and the discovery of prophylactic inhibitors against botulism
Full text linkBotulinum neurotoxins (BoNTs), the most poisonous substances identified so far, are protein toxins that cause botulism, a severe neuroparalytic disease. They are produced by different species of neurotoxigenic Clostridia and can be grouped into seven serotypes (BoNT/A to /G). Using genomic and proteomic approach, many novel BoNTs have been recently identified and are classified as subtypes, though they cannot be completely neutralized by currently available immunological methods. However, all BoNTs have a similar molecular architecture which reflects a conserved mechanism of action. Therefore this situation can be tackled by developing inhibitors targeting the BoNT intracellular intoxication process.
The BoNTs consist of two main chains linked by a unique inter-chain disulfide bond: the heavy chain (H, 100 kDa) and the catalytic light chain (L, 50 kDa). The C-terminal part of H (HC) is responsible for the neurospecific binding and the internalization within an endocytic compartment, whilst the N-terminal part (HN) is involved in the translocation of L across the endosome membrane. L is a Zn2+ dependent metalloprotease that targets specifically the SNARE proteins, the three proteins constituting the core of neuroexocytosis. This cleavage results in a prolonged inactivation of neurotransmitter release and causes the flaccid paralysis typical of botulism. To penetrate into neurons, BoNTs exploit synaptic vesicles (SV) recycling and their lumen acidification induces the HN-mediated membrane translocation of L. It has been demonstrated that, once on the cytosolic side, the L metalloprotease remains connected to H via the interchain disulphide bridge and the reduction of this bond is necessary to release the protease in the cytosol and enable their catalytic activity.
Using a series of well characterized inhibitors of Thioredoxin Reductase (TrxR)/Thioredoxin (Trx) system, we found that this redox system is involved in the cytosolic reduction of the interchain disulphide bond of BoNTs. In neuronal cultures, these molecules prevent the metalloproteolytic activity of all toxin serotypes without significantly affecting cell viability. Moreover, such compounds are very effective in vivo, lowering the severity and the duration of paralysis caused by a local BoNT injection. More importantly, one of these drugs elicits a remarkable protection in mice systemically injected with lethal doses of different serotypes. These results entail that the reduction of the interchain disulphide bond is a strict prerequisite for the activity of BoNTs and that this class of inhibitors can prevent the neurotoxicity regardless of their different immunogenicity. Intriguingly, we also found that the TrxR/Trx system is bound to the cytosolic side of the SV membrane and that it is enriched in those SV that are docked to active zones. We speculated that this redox system may play a role in maintaining SV protein function by controlling the redox state of the different SV protein disulfides.
Another step in BoNTs mechanism of action that might offer a good template for drug design is their trafficking. Recently, an inhibitor of different pathogens that require a passage through acidic endosomes to invade cells has been identified and dubbed EGA. We tested the effect of this molecule in neurons treated with BoNTs as also their neurotoxicity is strictly dependent on the passage through an intracellular acidic compartment. We focused our investigation on BoNT/A and BoNT/B, the two serotypes mainly associated with human botulism and used in therapy, and BoNT/D, that scarcely affects humans, but frequently causes botulism in animals. We found that EGA inhibits BoNTs activity on neuronal cultures, without interfering with any of the main steps characterising their cellular mechanism of intoxication. We speculated that, rather than having a direct effect on BoNTs, this compound impinges on an intracellular target which is responsible for their trafficking. Importantly, we found that EGA is not toxic per se in vivo, and is particularly efficacious in preventing botulism induced by BoNT/B and BoNT/D. Instead, in the case of BoNT/A the lethality was not reduced, but botulism symptoms developed later. We argued that the trafficking of the different BoNT types might be differently impacted by EGA and this compound may be used as a new tool for studying different intracellular routes exploited by BoNTs.
On the basis of the present knowledge about BoNTs mechanism of action, it is clear that once the LC has been released in the cytosol, the inhibitors tested here are no longer effective. Therefore, these drugs are to be considered as prophylactics. However, if given soon after diagnosis, these compounds could reduce symptoms severity by preventing the entry into neurons of circulating BoNTs, thus reducing the severity of poisoning and shortening the period of hospitalization that is related to the high costs of intensive care. Moreover, these molecules may be administered without knowing the BoNT serotype and subtype, therefore saving the time needed for toxin characterization
Endocytic proteins: An expanding repertoire of presynaptic functions
No full textFrom a presynaptic perspective, neuronal communication mainly relies on two interdependent events: The fast Ca2+-triggered fusion of neurotransmitter-containing synaptic vesicles (SVs) and their subsequent high-fidelity reformation. To allow rapid neurotransmission, SVs have evolved into fascinating molecular nanomachines equipped with a well-defined set of proteins. However, upon exocytosis, SVs fully collapse into the presynaptic plasma membrane leading to the dispersal of their molecular components. While the canonical function of endocytic proteins at the presynapse was believed to be the retrieval of SV proteins via clathrin-mediated endocytosis, it is now evident that clathrin-independent endocytic mechanisms predominate. We will highlight in how far these mechanisms still rely on the classical endocytic machinery and discuss the emerging functions of endocytic proteins in release site clearance and SV reformation from endosomal-like vacuoles
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
Mouse-derived Synaptosomes Trypsin Cleavage Assay to Characterize Synaptic Protein Sub-localization
Full text linkNeurons communicate through neurotransmission at highly specialized junctions called synapses. Each neuron forms numerous synaptic connections, consisting of presynaptic and postsynaptic terminals. Upon the arrival of an action potential, neurotransmitters are released from the presynaptic site and diffuse across the synaptic cleft to bind specialized receptors at the postsynaptic terminal. This process is tightly regulated by several proteins at both presynaptic and postsynaptic sites. The localization, abundance, and function of these proteins are essential for productive neurotransmission and are often affected in neurological and neurodegenerative disorders. Here, we outline a method for purifying mouse synaptosomes and using limited tryptic digestion to assess the subcellular localization of synaptic proteins. During synaptosomes purification, presynaptic terminals reseal and are protected from proteolysis, while postsynaptic proteins remain susceptible to tryptic cleavage. These changes can easily be evaluated by western blot analysis. This approach offers a straightforward and reliable method to evaluate the subcellular localization of synaptic proteins based on their proteolytic sensitivity, providing valuable insights into synaptic physiology and pathology
Presynaptic endocytic factors in autophagy and neurodegeneration
No full textNeuronal signaling depends on the exocytic fusion and subsequent endocytic retrieval and reformation of neurotransmitter-containing synaptic vesicles at synapses. Recent findings have uncovered surprising roles of presynaptic endocytic proteins in the formation and transport of autophagosomes. These include functions of the membrane remodelling protein endophilin and its downstream effector, the phosphoinositide phosphatase synaptojanin, in autophagosome formation and in Parkinson's disease, the endocytic sorting adaptor CALM in protein degradation via the autophagy/lysosomal pathway in Alzheimer's disease, and the clathrin adaptor complex AP-2 in retrograde transport of signaling autophagosomes to prevent neurodegeneration. These findings reveal unanticipated connections between the machineries for synaptic neurotransmission and neuronal proteostasis and identify presynaptic endocytic proteins as potential targets to treat neurodegenerative diseases
The axonal endolysosomal and autophagic systems
No full textNeurons, because of their elaborate morphology and the long distances between distal axons and the soma as well as their longevity, pose special challenges to autophagy and to the endolysosomal system, two of the main degradative routes for turnover of defective proteins and organelles. Autophagosomes sequester cytoplasmic or organellar cargos by engulfing them into their lumen before fusion with degradative lysosomes enriched in neuronal somata and participate in retrograde signaling to the soma. Endosomes are mainly involved in the sorting, recycling, or lysosomal turnover of internalized or membrane-bound macromolecules to maintain axonal membrane homeostasis. Lysosomes and the multiple shades of lysosome-related organelles also serve non-degradative roles, for example, in nutrient signaling and in synapse formation. Recent years have begun to shed light on the distinctive organization of the autophagy and endolysosomal systems in neurons, in particular their roles in axons. We review here our current understanding of the localization, distribution, and growing list of functions of these organelles in the axon in health and disease and outline perspectives for future research. (Figure presented.)
Inhibition of botulinum neurotoxins interchain disulfide bond reduction prevents the peripheral neuroparalysis of botulism
Full text linkThe first non Clostridial botulinum-like toxin cleaves VAMP within the juxtamembrane domain
Full text linkThe genome of Weissella oryzae SG25T was recently sequenced and a botulinum neurotoxin (BoNT) like gene was identified by bioinformatics methods. The typical three-domains organization of BoNTs with a N-terminal metalloprotease domain, a translocation and a cell binding domains could be identified. The BoNT family of neurotoxins is rapidly growing, but this was the first indication of the possible expression of a BoNT toxin outside the Clostridium genus. We performed molecular modeling and dynamics simulations showing that the 50 kDa N-terminal domain folds very similarly to the metalloprotease domain of BoNT/B, whilst the binding part is different. However, neither the recombinant metalloprotease nor the binding domains showed cross-reactivity with the standard antisera that define the seven serotypes of BoNTs. We found that the purified Weissella metalloprotease cleaves VAMP at a single site untouched by the other VAMP-specific BoNTs. This site is a unique Trp-Trp peptide bond located within the juxtamembrane segment of VAMP which is essential for neurotransmitter release. Therefore, the present study identifies the first non-Clostridial BoNT-like metalloprotease that cleaves VAMP at a novel and relevant site and we propose to label it BoNT/Wo
Hsp90 is involved in the entry of clostridial neurotoxins into the cytosol of nerve terminals
Full text linkHsp90 and Thioredoxin-Thioredoxin Reductase enable the catalytic activity of Clostridial neurotoxins inside nerve terminals
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