How botulinum and tetanus neurotoxins block neurotransmitter release* Yann Humeau a , Frédéric Doussau a **, Nancy J. Grant b , Bernard Poulain a *** a Laboratoire de Neurobiologie Cellulaire, UPR 9009 du CNRS, Centre de Neurochimie, 5, rue Blaise-Pascal, 67084 Strasbourg cedex, France b Biologie de la Communication Cellulaire, INSERM U-338, Centre de Neurochimie, 5, rue Blaise-Pascal, 67084 Strasbourg cedex, France (Received 17 January 2000; accepted 17 March 2000) Abstract — Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M r ≈50) disulfide linked to a heavy chain (M r ≈100). By inhibiting neurotransmitter release at distinct synapses, these toxins cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin), syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS botulinum neurotoxins / tetanus toxin / Clostridium / quantal release / neurotransmission / membrane fusion / synaptic vesicle / SNARE complex / VAMP / synaptobrevin / SNAP-25 / syntaxin 1. Introduction: key milestones over 2000 years Tetanus (from the greek tetanos = to contract) was first referred to more than 20 centuries ago when Greek physicians recognized this condition as a neurological disease. Botulism, or sausage poisoning (from the latin botulus = sausage), has been well documented since the 18th century. The infectious nature of tetanus and the bacterial etiology and toxicopathological origin of botu- lism were recognized at the end of the 19th century, notably by Carle and Rattone [1] and Van Ermengen [2] (for historical considerations see [3–6]). Major progress in understanding this disease was the discovery by Burgen and collaborators in 1949 that botulinum toxin blocks neurotransmitter release [7]. However, it was not until 30 years later that Simpson proposed a cellular mechanism of action involving three sequential steps, namely binding, internalization and intraneuronal action [8]. Nearly one century after their initial discovery, the amino acid se- quences of tetanus toxin (1986) and one of the botulinum toxins (1990) were determined in Niemann’s laboratory [9, 10]. Thereafter, the intracellular mechanism of tetanus toxin action was quickly deciphered in Montecucco’s laboratory in 1992 [11, 12]. Over the next 3 years, other clostridial neurotoxins were also demonstrated to be zinc-endopeptidases that selectively cleave the SNAREs (soluble NSF associated protein receptors) proteins (VAMP (vesicle associated membrane protein), SNAP-25 (synaptosome associated protein of 25 kDa) and syntaxin) implicated in neurotransmitter exocytosis (reviewed in [13, 14]). Finally, the tridimensional structure of botuli- num neurotoxin type A has now been resolved [15]. This article attempts to summarize what we know about the molecular actions of clostridial neurotoxins and how differences between toxins account for their distinct ef- fects on neurotransmission. To simplify the text, data has been included in a series of tables in which the reader will find relevant references. However, only a fraction of the vast literature on this subject has been cited and we apologize for any essential omissions. 2. Clostridial neurotoxins Seven distinct serotypes of botulinum toxins (BoNTs, A-G) have been identified through the examination of botulism outbreaks in man (BoNT/A, /B, /E and /F), birds (BoNT/C), cattle (BoNT/D), or isolated from soils (BoNT/G). The various BoNTs are produced by distinct strains of Clostridium botulinum that display heteroge * This paper is dedicated to the memory of Heiner Niemann. ** Present address: Departement of Neurobiology, Duke Univer- sity Medical Center, 27710 Durham, NC, USA *** Correspondence and reprints: poulain@neurochem.u-strasbg.fr Biochimie 82 (2000) 427-446 © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400002169/FLA