Marco Pirazzini

Botulinum neurotoxins: genetic, structural and mechanistic insights

Botulinum neurotoxins (BoNTs) are produced by anaerobic bacteria of the genus Clostridium and cause a persistent paralysis of peripheral nerve terminals, which is known as botulism. Neurotoxigenic clostridia belong to six phylogenetically distinct groups and produce more than 40 different BoNT types, which inactivate neurotransmitter release owing to their metalloprotease activity. In this Review, we discuss recent studies that have improved our understanding of the genetics and structure of BoNT complexes. We also describe recent insights into the mechanisms of BoNT entry into the general circulation, neuronal binding, membrane translocation and neuroparalysis.

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.

Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.

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

Tetanus 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.

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.

Botulinum Neurotoxin Type A is Internalized and Translocated from Small Synaptic Vesicles at the Neuromuscular Junction

Botulinum neurotoxin type A (BoNT/A) is the most frequent cause of human botulism and, at the same time, is largely used in human therapy. Some evidence indicates that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been directly 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 microscope, 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 synaptic vesicle protein 2, 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 trans-membrane pH gradient, we show that the neurotoxin translocation 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 no more than two neurotoxin molecules.

The thioredoxin reductase-thioredoxin system is involved in the entry of tetanus and botulinum neurotoxins in the cytosol of nerve terminals.

Tetanus and botulinum neurotoxins cause paralysis by cleaving SNARE proteins within the cytosol of nerve terminals. They are endocytosed inside acidic vesicles and the pH gradient across the membrane drives the translocation of their metalloprotease L domain in the cytosol. This domain is linked to the rest of the molecule by a single interchain disulfide bridge that has to be reduced on the cytosolic side of the membrane to free its enzymatic activity. 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.

Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain

Botulinum neurotoxins translocate their enzymatic domain across vesicular membranes. The molecular triggers of this process are unknown. Here, we tested the possibility that this is elicited by protonation of conserved surface carboxylates. Glutamate‐48, glutamate‐653 and aspartate‐877 were identified as possible candidates and changed into amide. This triple mutant showed increased neurotoxicity due to faster cytosolic delivery of the enzymatic domain; membrane translocation could take place at less acidic pH. Thus, neutralisation of specific negative surface charges facilitates membrane contact permitting a faster initiation of the toxin membrane insertion.

Re-Assembled Botulinum Neurotoxin Inhibits CNS Functions without Systemic Toxicity

The therapeutic potential of botulinum neurotoxin type A (BoNT/A) has recently been widely recognized. BoNT/A acts to silence synaptic transmission via specific proteolytic cleavage of an essential neuronal protein, SNAP25. The advantages of BoNT/A-mediated synaptic silencing include very long duration, high potency and localized action. However, there is a fear of possible side-effects of BoNT/A due to its diffusible nature which may lead to neuromuscular blockade away from the injection site. We recently developed a “protein-stapling” technology which allows re-assembly of BoNT/A from two separate fragments. This technology allowed, for the first time, safe production of this popular neuronal silencing agent. Here we evaluated the re-assembled toxin in several CNS assays and assessed its systemic effects in an animal model. Our results show that the re-assembled toxin is potent in inhibiting CNS function at 1 nM concentration but surprisingly does not exhibit systemic toxicity after intraperitoneal injection even at 200 ng/kg dose. This shows that the re-assembled toxin represents a uniquely safe tool for neuroscience research and future medical applications.