Elsevier

Journal of Physiology-Paris

Volume 96, Issues 1–2, January–March 2002, Pages 105-113
Journal of Physiology-Paris

Botulinum neurotoxins: from paralysis to recovery of functional neuromuscular transmission

https://doi.org/10.1016/S0928-4257(01)00086-9Get rights and content

Abstract

The neuromuscular junction is one of the most accessible mammalian synapses which offers a useful model to study long-term synaptic modifications occurring throughout life. It is also the natural target of botulinum neurotoxins (BoNTs) causing a selective blockade of the regulated exocytosis of acetylcholine thereby triggering a profound albeit transitory muscular paralysis. The scope of this review is to describe the principal steps implicated in botulinum toxin intoxication from the early events leading to a paralysis to the cellular response implementing an impressive synaptic remodelling culminating in the functional recovery of neuromuscular transmission. BoNT/A treatment promotes extensive sprouting emanating from intoxicated motor nerve terminals and the distal portion of motor axons. The current view is that sprouts have the ability to form functional synapses as they display a number of key proteins required for exocytosis: SNAP-25, VAMP/synaptobrevin, syntaxin-I, synaptotagmin-II, synaptophysin, and voltage-activated Na+, Ca2+ and Ca2+-activated K+ channels. Exo-endocytosis was demonstrated (using the styryl dye FM1-43) to occur only in the sprouts in vivo, at the time of functional recovery emphasising the direct role of nerve terminal outgrowth in implementing the restoration of functional neurotransmitter release (at a time when nerve stimulation again elicited muscle contraction). Interestingly, sprouts are only transitory since a second distinct phase of the rehabilitation process occurs with a return of synaptic activity to the original nerve terminals. This is accompanied by the elimination of the dispensable sprouts. The growth or elimination of these nerve processes appears to be strongly correlated with the level of synaptic activity at the parent terminal. The BoNT/A-induced extension and later removal of “functional” sprouts indicate their fundamental importance in the rehabilitation of paralysed endplates, a finding with ramifications for the vital process of nerve regeneration.

Introduction

BoNTs serotypes A-G, because of their unique potency and specificity for blocking transmission at the vertebrate neuromuscular junction (NMJ) are useful tools for studying transmitter release mechanisms, and long-term trophic interactions between nerve terminals, Schwann cells and muscle fibres. During the last decade, BoNTs have been sequenced, the target of their zinc-endopeptidase activity identified, and the crystal three-dimensional structure [41] and their complex with the substrates determined [33], [67], [75]. The discovery of their metalloprotease activity targeting key components of the exocytotic machinery has initiated a number of studies aimed at unravelling the intricate molecular mechanisms involved in the fusion of synaptic vesicles to the nerve terminal membrane. Since BoNTs give rise to a profound, albeit transient, paralysis when injected into skeletal muscle, a number of studies have been performed to investigate how the motoneuron and its synaptic cellular partners react to such a blockade of pre-synaptic regulated exocytosis and implement a cascade of events ultimately leading to the recovery of functional neuromuscular transmission. Motor nerve terminals are very stable throughout adulthood [64] but can undergo remarkable plasticity changes when challenged with BoNTs. The goal of this review is to summarise the molecular events involved in the blockade of neurotransmission and to examine the synaptic remodelling and recovery process taking place at BoNT-treated neuromuscular junctions.

Section snippets

Binding, internalisation and proteolytic activity of BoNTs at the nerve terminals

The neuronal acceptor(s) of BoNTs is/are still under investigation, but the characteristic of their selective binding to glycolipid and internalisation process have been described. They interact in vitro and in vivo with polysialogangliosides [31], [55], [56], in particular with members of the G1b series [7], [40], [47]. Gangliosides knockout mice [70] show a reduced sensitivity to the toxins [39] and fumonisin B1, an inhibitor of the synthesis of a wide range of glycolipids including

Blockade of quantal acetylcholine release

BoNTs drastically reduce both nerve-evoked and spontaneous quantal acetylcholine (ACh) release. The paralysis results from the inability of endplate potentials (EPPs) evoked by nerve stimuli to reach the appropriate membrane potential level to trigger an action potential in the muscle fibre [13], [30], [35], [50], [53], [60], [67], [72], [82]. Spontaneous quantal ACh release recorded as miniature endplate potentials (MEPPs) is also greatly reduced after exposure to BoNT/A [30], [50], [53], [60]

In vivo remodelling of the skeletal neuromuscular junction

A remarkable demonstration of synaptic plasticity is the ability of axons and nerve terminals of the skeletal neuromuscular system to sprout new processes and form synapses in response to the paralysis induced by BoNTs or to partial denervation. This requires changes in neuronal gene expression, de novo protein synthesis, and remodelling of synaptic contacts. Motoneurons have the ability to “sprout” new processes from their nerve terminals or nodes of Ranvier. Although many fundamental

Conclusions

The detailed analysis of BoNTs' mechanism of action has markedly improved our understanding of neurotransmitter release processes and the intricate molecular mechanisms involved in the fusion of synaptic vesicles to the nerve terminal membrane. In addition, several mechanisms are now being explored and will certainly yield new insights into synaptic vesicle trafficking within motoneurons.

Considerable evidence indicates that BoNTs promote synaptic remodelling at the NMJ. The sensor controlling

Acknowledgements

The authors' studies on botulinal neurotoxins were supported by research grants from The European Commission Biotechnology Program (grant BC104CT965119 to F.A.M.), Imperial Cancer Research Fund (to G.S.), the Association Française contre les Myopathies and the Direction des Systèmes de Forces et de la Prospective (grant DSP/STTC 01 34 029 to J. M.). The authors wish to thank the Franco-British Partnership Program Alliance (grant 00156SE) for facilitating the exchange between our laboratories.

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