Regular articleDynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration are dictated by the sites of SNAP-25 truncation
Introduction
In the peripheral and central nervous systems, neuronal plasticity plays pivotal roles in recovery from injury and in learning/memory, respectively. Yet, the intrinsic neuronal determinants for regulation of this fundamental process remain poorly defined. A unique strategy for investigating these in motor nerves entails elimination of regulated neuroexocytosis with botulinum neurotoxin (BoNT) while leaving the viability of the nerve endings unaltered. There are seven serotypes (A–G) of BoNT—all Zn2+-dependent, highly specific proteases—that inhibit the Ca2+-dependent exocytotic release of transmitters by cleaving the SNARE proteins, SNAP-25 Blasi et al 1993, Foran et al 1996, Schiavo et al 1993a, synaptobrevin Schiavo et al 1993a, Schiavo et al 1993b, or syntaxin (Schiavo et al., 1995). Interestingly, both BoNT/A and E cleave SNAP-25 within the C-terminus at Gln197–Arg and Arg180–Ile, respectively, removing 9 and 26 residues but strikingly inducing paralysis of very different durations (Eleopra et al., 1998). Neuroparalysis produced by BoNT/A elicits nerve sprouting (reviewed in Meunier et al., 2002) and the newly created synapses are responsible for the initial synaptic transmission at the onset of recovery from muscular paralysis (de Paiva et al., 1999). Early studies have suggested that the lifetime of these outgrowths depends on muscle activity since direct and chronic electrical stimulation of BoNT/A-injected muscle greatly reduces sprouting (Brown et al., 1981). However, it was recently observed that the return of synaptic activity to endings originally poisoned with BoNT/A coincides with retraction of the neurite outgrowths (de Paiva et al., 1999). Careful in vivo imaging of exo-endocytosis activity, using a styryl dye (FM1-43, known to fluoresce only in a lipidic environment), revealed that the level of synaptic activity was lower at the nascent sprouts than in normal, untreated, parent nerve endings. Based on the collective findings, it was proposed that resumption of a threshold activity at the toxin A-poisoned original terminal may signal sprout elimination and suppression of synapse plasticity.
Herein, the latter hypothesis (de Paiva et al., 1999) was evaluated by monitoring motor terminal remodeling employing three BoNT serotypes, alone or in combination, to perturb transmitter release and, thereby, manipulate the extent and time course of sprouting. Arresting transmitter release for different durations, afforded by the various BoNTs, revealed that neuromuscular transmission switches over days from mature stable end plates to nascent extrajunctional synapses, if neuroparalysis persists for more than 3 days. Reversion of this process occurs upon eventual recovery of endo-/exocytosis at the parent terminals. Thus, blockade of regulated, but not constitutive, exocytosis with these toxins provides an elegant means of investigating synaptic plasticity. The use of BoNT/E also revealed that residues 181–197 of BoNT/A-cleaved SNAP-25 (SNAP-25A) contribute to its persistence within the nerve ending and ability to retard replenishment with intact functional SNARE; this unveils a basis for the unique long-lasting effectiveness of BoNT/A in the clinical treatment of dystonias (Brin, 1997), including spasticity.
Section snippets
Nerve sprouting is triggered by inhibition of regulated synaptic vesicle recycling for more than 3 days
As a prerequisite to using BoNT/A, E, or F (Table 1) to block transmitter release for different periods in the hind leg of mice, we first established the dose of each which caused a maximal loss of toe spread reflex (TSR; score = 5), due to blocking the compounded response of all muscles producing this flexion, without other obvious symptoms of botulism. This was termed the effective dose (ED); the corresponding doses for each of the neurotoxins used are given in Table 2. BoNT/A induced a
Remodeling of nerve terminals following exposure to BoNT is determined by the period of neuroparalysis—a process reflective of the adaptiveness provided by synaptic plasticity
The data herein establish, for the first time, the contributions which various parameters make to the appearance, maintenance, and eventual retraction of sprouts—a cascade triggered by abolishing transmitter release with BoNT/A or F. Initiation of the remodeling process was found to require the synapse to be devoid of activity for more than 3 days because recovery from paralysis started within 2 days of administering BoNT/E and it failed to induce nerve outgrowths; consistently, the onset of
Comparison of recovery times from neuromuscular paralysis induced by BoNT/A, E, or F in mice
Tyler’s ordinary mice (20 g) were lightly anesthetized with halothane (4%) and injected (0.1 ml) intramuscularly into the dorsal surface of the right hind leg with an ED (Table 2) of either BoNT/A–hemagglutinin complex [BOTOX (List Biologics Ltd., CA)], or electrophoretically pure BoNT/E supplied by Dr. B.R. DasGupta or BoNT/F complex, dual combinations (BoNT/A/E or A/F), and sequential injections of various amounts of BoNT/E, following a 3-day prior injection of 0.5 ED of BoNT/A (Table 2). It
Acknowledgements
This research was funded by a BBSRC studentship (to G.L.) and, in part, by Allergan Inc. The authors thank Drs. G. Lawrence, N. Mohammed, and G. O’Sullivan for their input. Dr. P.G. Foran, of this laboratory, is thanked for advice and the provision of some background information.
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- 1
Contributed equally to this work.
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Present address: Department of Physiology and Pharmacology, The University of Queensland, 4072 St. Lucia, Australia.