Botulinal neurotoxins: revival of an old killer

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Botulinal neurotoxins (BoNTs) produced by anaerobic bacteria of the genus Clostridium are the most toxic proteins known, with mouse LD50 values in the range of 1–5 ng/kg. They are responsible for the pathophysiology of botulism. BoNTs are metalloproteinases that enter peripheral cholinergic nerve terminals, where they cleave one or two of the three core proteins of the neuroexocytosis apparatus and elicit persistent but reversible inhibition of neurotransmitter release. Their specificity of action has made them useful therapeutic agents for many human syndromes caused by hyperactivity of cholinergic nerve terminals. Their range of clinical applications is continuously growing, and BoNT/A is being used extensively as a pharmaco-cosmetic.

Introduction

Seven biochemically and serologically distinct botulinum neurotoxins (designated BoNT/A to/G) have been characterized from anaerobic spore-forming bacteria of the genus Clostridium. The bacterial spores are widely present in the environment and are resistant to various physical and chemical agents. Therefore, they can contaminate food and, under suitably anaerobic conditions, germinate and yield the vegetative bacterial cells that produce one or more of the toxins. Ingestion of BoNT-poisoned food causes an intoxication known as botulism, which was first described at the beginning of the 17th century and associated to a protein neurotoxin at the end of that same century [1, 2, 3]. More recently, infant botulism was described as a syndrome caused by the colonization of the intestinal tract of infants by neurotoxigenic strains of C. botulinum, which subsequently results in infant intoxication [2, 3]. Another form of the disease is wound botulism, which results from the production of BoNTs by C. botulinum growing in anaerobic wounds [2, 3].

This brief overview highlights the structural organization and mode of action of BoNTs on neuroexocytosis. The review also focuses on the therapeutic uses and current limitations of BoNTs, as well as the ongoing developments in their use.

Section snippets

Botulism

The signs and symptoms of botulism are essentially the same for all forms of the disease, and are a consequence of sustained blockade of acetylcholine (ACh) release at somatic and autonomic nerve terminals. The facial and throat muscles are the first skeletal muscles that become weak and progressively paralyzed, causing diplopia, ptosis, dysphagia and facial paralysis. The paralysis progressively descends to affect muscles of the trunk, including the respiratory and visceral muscles (if death

Structure of botulinal neurotoxins

Before their release via bacterial autolysis, BoNTs are produced as progenitor toxins; that is, as variously sized complexes composed of inactive single polypeptide toxin chains (Mr ∼150 kDa) and other non-toxic accessory proteins. The accessory proteins protect BoNTs during their passage through the stomach, and they dissociate from the progenitor toxins when the complexes reach the small intestine — the main site at which the BoNTs are absorbed into the bloodstream and subsequently reach their

Botulinal neurotoxin-elicited inhibition of neuroexocytosis

The BoNTs are the most potent toxins known. The mouse LD50 values for the BoNTs range from 1 ng/kg to 5 ng/kg, and similar or lower values have been estimated for humans [16]. Their toxicity is usually expressed in units: 1 unit is 1 mouse LD50 for a 20 g Swiss strain mouse, and the LD50 of BoNT/A for an adult human (∼ 70 kg) has been estimated to be ∼ 3000 units. This potency derives from their high neurospecificity and from their catalytic activity, which results in the blockade of essential

Therapeutic uses of botulinal neurotoxins

BoNT/A, under the trade names of BOTOX (Allergan) and Dysport (Ipsen), has become a multi-million dollar drug used in the treatment of many human syndromes and as a ‘pharmaco-cosmetic’ [18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33]; BoNT/B is also commercially available (Neurobloc, Elan Pharmaceuticals). The increasing use and commercial success of BoNTs is due to their high specificity for peripheral cholinergic nerve terminals and to the remarkable fact that, when proper

Current limitations of the therapeutic use of botulinal neurotoxins

As mentioned above, the effect of BoNTs is reversible; therefore, to sustain the desired therapeutic effect, treatments usually have to be repeated. Thus, as BoNTs are antigenic, an immune response leading to the formation of neutralizing antibodies can occur [47, 48]. This possibility is rare because most treatments require only minute amounts of BoNT/A; however, it could become a problem when larger amounts of toxin are needed to treat powerful muscles of body regions rich in lymph nodes

Conclusions

In conclusion, BoNTs are zinc metalloendoproteases that exhibit extraordinary specificities for proteins involved in the neurotransmitter release process, and their toxicities make them responsible for animal and human botulism. However, these extremely poisonous molecules have become useful therapeutic agents in a number of expanding applications in human medicine. Ongoing efforts to elucidate BoNTs’ mechanisms of action for reducing pain and other pathological conditions will provide an

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We apologize to our colleagues whose papers could not be cited owing to a restriction on space. We thank Dr Arnold S. Kreger for helpful suggestions during the copyediting of the manuscript. The authors’ studies of BoNTs were supported by research grants from Telethon-Italia Grant GP0272/01 (to CM) and from the Direction des Systèmes de Forces et de la Prospective (Grant # 026065093 to JM).

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