©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Neurotransmitter Release by Synthetic Proline-rich Peptides Shows That the N-terminal Domain of Vesicle-associated Membrane Protein/Synaptobrevin Is Critical for Neuro-exocytosis (*)

Fabrice Cornille (1), Florence Deloye (2), Marie-Claude Fournié-Zaluski (1), Bernard P. Roques (1)(§), Bernard Poulain (2)

From the (1)Département de Pharmacochimie Moléculaire et Structurale INSERM U266-CNRS URA D1500, Faculté des Sciences Pharmaceutiques et Biologiques, Faculté de Pharmacie-Université René Descartes, 4, avenue de l'observatoire, F-75270 Paris Cedex 06, France and the (2)Laboratoire de Neurobiologie Cellulaire et Moléculaire CNRS, 1, avenue de la terrasse, F-91198 Gif sur Yvette Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tetanus toxin and clostridial neurotoxins type B, D, F, and G inhibit intracellular Ca-dependent neurotransmitter release via the specific proteolytic cleavage of vesicle-associated membrane protein (VAMP)/synaptobrevin, a highly conserved 19-kDa integral protein of the small synaptic vesicle membrane. This results in the release of the larger part of the cytosolic domain of this synaptic protein into the cytoplasm. Microinjection of synthetic peptides corresponding to this fragment into identified presynaptic neurons of Aplysia californica led to a potent, long lasting, and dose-dependent inhibition (50% at 10 µM) of acetylcholine release, probably by hindering endogenous VAMP/synaptobrevin from interacting with synaptic proteins involved in exocytosis. Structure activity studies showed that this effect is confined to the N-terminal domain of VAMP/synaptobrevin isoform II and is related to the presence of a proline-rich motif (PGGPXGXPP or PAAPXGXPP). At higher concentrations, the inhibitory effect was lower and only transient, suggesting that the N-terminal proline-rich domain of VAMP/synaptobrevin plays opposing roles in neurotransmitter release very likely by interacting with different synaptic proteins. This probably occurs by disruption of the recently reported in vitro VAMP-synaptophysin interaction that involves the N-terminal domain of VAMP II and was proposed to hinder synaptophysin-related formation of a fusion pore. The observed recovery of neurotransmitter release following injection of high concentration of N-terminal fragments of VAMP II brings a strong in vivo support to this hypothesis. The minimum active peptide GPGGPQGGMQPPREQS could be used for rationally designing potent synthetic blockers of neurotransmission.


INTRODUCTION

Neurotransmitter release occurs via a Ca-regulated exocytosis of the content of synaptic vesicles. Over the past year, a remarkable convergence of data has given insights into the exocytotic machinery, and several proteins involved in vesicle docking and/or membrane fusion processes have been discovered(1, 2) . Among them, three proteins are specific intracellular substrates of proteolytic neurotoxins, namely tetanus and botulinum neurotoxins(3, 4) . More precisely, tetanus toxin (TeNT)()and botulinum neurotoxins (BoNT)/B, /D, /F, and /G cleave vesicle-associated membrane protein (VAMP)/synaptobrevin, an integral protein of the small synaptic vesicle membrane, whereas the target of BoNT/A and /E is 25-kDa synaptosomal-associated protein (SNAP-25), a protein associated with the plasma membrane. For its part BoNT/C cleaves syntaxin, an integral plasma membrane protein (for review, see Refs. 3 and 4). Because the cleavage of these three synaptic proteins, in vitro (see references quoted above), correlates with the inhibition of neurotransmitter release induced by TeNT and BoNTs, in vivo(5, 6, 7, 8) , it has been inferred that the cleavage of either VAMP, SNAP-25, or syntaxin prevents synaptic transmission. Collectively, these findings point out a key role for these synaptic proteins in neurotransmitter release, but their respective roles remain unclear. The three proteins associate tightly together and with synaptotagmin (9, 10) and N-ethylmaleimide-sensitive fusion protein to form a 20 S complex thought to be involved in the fusion of vesicles with membranes (11). Therefore VAMP, SNAP-25, and syntaxin have been postulated to be involved in either the docking or the fusion of synaptic vesicles with the plasma membrane at active zones of nerve terminals(2, 12, 13) .

Among the three synaptic proteins cleaved by clostridial neurotoxins, VAMP appears to be of special interest because it is the target of five of these neurotoxins (3, 4) and is the only putative vesicular SNAP receptor so far identified(10) . VAMP homologs are highly conserved in eucaryotes, from yeast to human(14, 15) . In all species, the cytoplasmic domain of VAMP consists of a N-terminal proline-rich region followed by a large and highly conserved hydrophilic core (Fig. 1). VAMP is anchored in the synaptic vesicle membrane by a short lipophilic domain. Its C-terminal region is intravesicular and of variable size. The first Met residue is probably removed during maturation, and, most likely, the N-terminal end is capped by an acetyl group(11, 16) . VAMP is hydrolyzed at distinct sites by TeNT and BoNT/B, /D, /F, and /G (see Fig. 1and, for review, see Refs. 3 and 4). The enzymatic cleavage of VAMP by these neurotoxins results in the release of a large N-terminal cytosolic fragment whose size varies with the neurotoxin type. Because they are released in the cytoplasm, these soluble fragments may compete with endogenous VAMP in its function, thus leading to an alteration of neurotransmission. Our aims were (i) to test whether a synthetic fragment corresponding to that released by one of these toxins (for instance TeNT) can perturb neurotransmission when intracellularly applied and (ii) if so, to identify the putative VAMP domains underlying this action. The best model for this purpose is dissected ganglia from the central nervous system of Aplysia californica as some of their largest neurons make identified synapses (17) and afford an easy access to intracellular spaces(5, 18, 19) . In addition, neurotransmitter release can be easily quantified using conventional electrophysiological techniques (for details, see Ref. 18).


Figure 1: A, alignment of the isoform II of the human VAMP (41) and of Aplysia VAMP (21). The sequence of the C-terminal intravesicular tail of the Aplysia isoform is not reported. B, schematic structure of VAMP. a and b, cytosolic domain comprising a variable N-terminal portion (a) and a highly conserved region (b); c membrane-anchoring domain; and d, C-terminal tail protruding in the lumen of synaptic vesicles. C, the sites of cleavage of Aplysia VAMP by clostridial neurotoxins are indicated according to Yamasaki et al. (21, 40) and Niemann et al. (4). N-terminal sequences of various VAMPS are as follows: 1, Di Antonio et al. (41); 2, Südhof et al. (14); 3, Yamasaki et al. (21); 4, Hunt et al. (27); 5, Trimble et al. (42); 6, Elferink et al. (43); 7, Archer et al. (39); and 8, Mac Mahon et al. (28).




MATERIALS AND METHODS

Peptide Syntheses

Assembly of the protected peptide chain was carried out using the stepwise solid-phase method of Merrifield (22) on an Applied Biosystems 431A peptide synthesizer, with dicyclohexylcarbodiimide/hydroxybenzotriazole as coupling reagents and N-methylpyrrolidone as solvent. Fluoren-9-ylmethoxycarbonyl was employed as temporary protecting groups for amino acid amines, and deprotection was achieved by treatment with piperidine. The syntheses were run on a 0.1-mmol scale (114 mg of Wang resin loaded at 0.88 mmol of hydroxyl group/g of dry resin) except for the large peptides A-syb 1-83, ac-A-syb 2-66, A-syb 20-83, H-syb II 1-93, and H-syb II 24-93 for which successive removal of peptidyl-resin at different steps of the synthesis led us to finish on a 25-µmol scale. Peptides with proline in the last or penultimate position were synthesized with dichlorotrityl resin, which is known to prevent diketopiperazine formation during synthesis. The peptide resin was treated for 2 h with 40 ml of trifluoroacetic acid in the presence of phenol (3 g), 1,2-ethanedithiol (1 ml), thioanisole (2 ml), and HO (2 ml) in order to cleave the peptide from the resin and to remove the protecting groups of the amino acid side chains. Purifications of the peptides were carried out by reverse phase HPLC on a Vydac C4 column (250 10 mm) with a linear gradient of 10-60% B in 90 min (A, 0.1% trifluoroacetic acid in HO; B, 0.09% trifluoroacetic acid, 70% acetonitrile in HO; flow 1.5 ml/min; detection wavelength, 214 nm). The purified peptides were then lyophilized and kept as a powder at -80 °C. Their purity was checked by amino acid analysis, capillary electrophoresis, and electrospray mass spectrometry.

Electrophysiological Measurements of Acetylcholine Release in A. californica

Experiments were performed at 22 °C on identified cholinergic synapses in the buccal ganglion of Aplysia. In this ganglion, two cholinergic presynaptic neurons, B4 and B5, make well known chloride-dependent inhibitory synapses with the same postsynaptic cells as B3, B6, and several others(17) . Both presynaptic cells (125-180 µM in diameter) were impaled with two glass microelectrodes (3 M KCl Ag/AgCl, 4-10 megaohms). Alternately, once a minute, a presynaptic neuron was depolarized to elicit an action potential. The ensuing postsynaptic response was recorded in the postsynaptic neuron as a current change using the conventional two-electrode voltage-clamp technique. Subsequently, by taking into account its reversal potential, i.e. the null potential for Cl, the postsynaptic response was expressed as membrane conductance, a value that is directly proportional to the amount of acetylcholine released per impulse (for details, see Refs. 18 and 19).

Injection of Peptides

Buccal ganglia were continuously superfused (30 ml/h, experimental chamber volume of 1 ml) with a physiological medium (artificial sea water) containing 460 mM NaCl, 10 mM KCl, 11 mM CaCl, 25 mM MgCl, 28 mM MgSO, and 10 mM Tris-HCl, pH 7.8. Before intracellular administration, peptides dissolved in 50 mM Tris-HCl, pH 7.8, 100 mM NaCl were mixed together with a solution of dye (10% by volume; fast green FCF, Sigma). Peptides were air pressure-injected (1% by volume of the cell body) into one of the two presynaptic neurons under visual and electrophysiological monitoring. For this purpose, a third electrode was implanted into the cell body and was removed after injection.


RESULTS

The VAMP Fragment Released by TeNT Inhibits Neurotransmission

Intracellular injection of TeNT (Fig. 2a) into identified presynaptic neurons of the buccal ganglion of Aplysia produces a fast and complete blockade of neurotransmitter release(20) . This results from the cleavage of Aplysia VAMP at the Gln-Phe bond(21, 22) . Given that the N-terminal fragment is released into the cytosol, an Aplysia VAMP acetyl 2-66 peptide (ac-A-syb 2-66) was synthesized by solid-phase peptide synthesis (23) using the Fmoc (Fluoren-9-ylmethoxycarbonyl)/t-butyl strategy(22) . All of the peptides used in this study were synthesized by this method. In all cases, the peptidyl-resin was cleaved using trifluoroacetic acid in the presence of scavengers, and the crude peptide was purified by reverse phase HPLC. The purity of each peptide was checked by amino acid analysis, capillary electrophoresis, and electrospray mass spectrometry and was estimated to be higher than 95%. The synthetic ac-A-syb 2-66 peptide was microinjected as reported previously (18, 19) into presynaptic neurons of Aplysia at a final intrasomatic concentration of 10 µM. This resulted in an almost 50% inhibition of acetylcholine release as shown by the depression of the amplitude of the postsynaptic responses evoked by presynaptic action potentials (Fig. 2b). The inhibition of acetylcholine release was maximal within 1 h following injection, and it remained for several hours (>6 h, not shown). Importantly, no alteration of the presynaptic action potential was detected. This suggested that the inhibition of neurotransmission was not the consequence of a change in nerve conduction properties. In addition, neither the time to peak nor the decay time of the postsynaptic responses were modified (not shown). This indicated that the kinetics of the release process remained unchanged.


Figure 2: The fragment of VAMP released by TeNT induces a 50% inhibition of neurotransmitter release in Aplysia. Neurotransmitter release was evoked at identified cholinergic synapses in the buccal ganglion of Aplysia and monitored (percent) following the microinjection of TeNT (A) or of various fragments of VAMP into the cell body of a presynaptic (either B4 or B5) neuron (B-D). The release of neurotransmitter was quantified via the measurement of the amplitude of postsynaptic responses recorded as described elsewhere (18). The peptides were synthesized according to Cornille et al. (23); they span various domains of either Aplysia of human isoform II VAMPs (A-syb and H-syb II, respectively). The first and last amino acids are indicated as numbers according to the published sequences (21, 42). Note that the ac-A-syb 2-66 peptide corresponds to the fragment of VAMP released in Aplysia neurons by either TeNT or BoNT/B. The final intracellular concentration of each peptide is indicated. Each of the panels reports a typical experiment from a series of 3-5. The average inhibition value obtained for an intraneuronal injection of 10 µM of the following peptides is given: A-syb 2-66 (45 ± 3%), A-syb 1-83 (39 ± 6%), H-syb-II 1-93(44 ± 4%).



The injection of a C-terminally extended peptide encompassing the whole cytoplasmic domain of Aplysia VAMP (A-syb 1-83) at the same intracellular concentration as ac-A-syb 2-66 did not lead to a greater inhibition (Fig. 2c). Because instead of an acetyl group, the first Met was present in this peptide; this result suggested that, most likely, the processing of the Met and the capping of Ser are not essential for the intracellular activity of the peptide. These data suggested also that TeNT might induce inhibition of neurotransmitter release, at least partially, by generating an inhibitory peptide. However, since a total inhibition of release was observed upon intraneuronal injection of this toxin (Fig. 2a), it was important to test whether a total blockade of transmission could be obtained following microinjection of higher concentrations of the released cytosolic fragment of VAMP. However, the intracellular concentration of the ac-A-syb 2-66 and A-syb 1-83 peptides could not be increased due to solubility problems in the injection micropipette (note that to reach an intracellular concentration of 100 µM requires an intrapipette concentration of 10 mM). This problem was solved later by the identification of a smaller domain with similar inhibitory properties but with increased solubility.

The Inhibitory Domain of VAMP Is Confined in its N-terminal Portion

The cytosolic region of VAMP has a variable N-terminal domain and a central region that is conserved among various species (Fig. 1C). Thus, it was postulated that a comparison of the abilities of various VAMPs to interfere with neurotransmission might help in the identification of the inhibitory domain. A peptide corresponding to the whole cytosolic domain of the human II isoform was synthesized (H-syb II 1-93); it differed mainly from the A-syb 1-83 peptide by its first 26 amino acids and several punctual changes in the conserved domain (Fig. 1). Intraneuronal injection of H-syb II 1-93 (10 µM final) induced a 45% inhibition of acetylcholine release (Fig. 2d) similar, on a molar ratio, to that obtained with either A-syb 2-66 or A-syb 1-83 (Fig. 2b). Since Aplysia and human II VAMPs differ mainly in their N-terminal portion, the highly conserved region (H-syb II 24-93 and A-syb 20-83) was suspected to contain the protein domain responsible for this inhibition. Therefore, the corresponding peptides were synthesized and intraneuronally applied, each at 10 µM; no alteration in the neurotransmission was detected (Fig. 3c). Contrastingly, intracellular application of peptides corresponding to the variable N-terminal proline-rich part of the two isoforms tested A-syb 1-19 and H-syb II 1-26 () blocked neurotransmitter release to the same extent and over a similar time scale (Fig. 3b) as the longer peptides (A-syb 2-66, A-syb 1-83, and H-syb II 1-93) ( Fig. 2and 4).


Figure 3: The proline-rich domain of VAMP is responsible for the inhibition of neurotransmitter release in Aplysia neurons. a, schematic representation of synaptobrevin. PPPP represents the proline-rich domain of synaptobrevin. b and c show the same kind of experiments as in Fig. 2. The final intracellular concentration of each peptide is indicated. Each of the panels reports a typical experiment from a series of 3-5. The average inhibition value obtained for an intraneuronal injection of 10 µM of the peptide A-syb 1-19 is 45 ± 6%.



Dual Action of the N-terminal Proline-rich Domain of VAMP

Because the more soluble A-syb 1-19 and H-syb II 1-26 fragments retained entirely the inhibitory properties of the ac-A-syb 2-66 peptide or H-Syb II 1-93, it was possible to perform dose-dependent experiments in order to test whether a complete inhibition could be reached (Fig. 4). Upon intraneuronal injection of either A-syb 1-19 or H-syb II 1-26 peptides at 1 µM, the observed inhibition was around 20% and reached about 50% at 10 µM. However at a final concentration of 100 µM, no gain in inhibition was observed, but, instead, the extent of inhibition was reduced, and the depression of neurotransmitter release was only transient (Fig. 4, a-c and d-f). This suggested that the N-terminal domain of VAMP altered the release process via two counteracting effects. In the 1-10 µM range, the inhibitory effect predominated, whereas in the 10-100 µM range, an opposite so-called facilitatory effect superimposed and allowed neurotransmission to recover to near control levels. This was confirmed in a series of experiments where a presynaptic neuron was submitted to two injections. When the A-syb 1-19 peptide was first injected at a final concentration of 5 µM, the second injection of 5 µM (2 h later) produced a further inhibition (Fig. 5a). Contrastingly when the neuron was injected twice with 10 µM of A-syb 1-19 peptide, the second application produced an increase in neurotransmission (Fig. 5b). Because the amino acid sequence of the A-syb 1-19 and H-syb II 1-26 peptides are different (), it was possible that they produced similar effects via interactions with different intraneuronal targets. However this appeared unlikely because coinjection of 10 µM of each peptide (Fig. 5c) led to the same weak and transient effect observed with 20 µM of either peptide (not shown).


Figure 4: The proline-rich domain of VAMP from Aplysia (1-19) and human isoform II (1-26) have the same dual effect on neurotransmitter release. Inhibition in the 1-10 µM range and this inhibition is lowered and transient at the 10-100 µM range. The same kind of experiments as in Figs. 2 and 3 are shown. Each of the panels reports a typical experiment from a series of 3-5. The average inhibition values obtained for an intraneuronal injection of the following peptides are as follows: at 1 µM, A-syb 1-19 (20 ± 12%), H-syb-II 1-26 (18 ± 12%); at 10 µM, A-syb 1-19 (45 ± 6%), H-syb-II 1-26 (44 ± 4%).




Figure 5: Double injection of A-syb 1-19 at 5 and 10 µM concentrations (a and b). Coinjection of 10 µM A-syb 1-19 and A-syb 1-26 is shown in c. Injection of 20 nM TeNT following a 50% inhibition of acetylcholine release induced by injection of 10 µM A-syb 1-19 is shown in d. a-c, the 50% inhibition reached by injection of 10 µM of A-syb 1-19 peptide can be reversed by a second injection of 10 µM of the same peptide or can be total by the addition of 20 nM of tetanus toxin. d, the A-syb 1-19 and H-syb II 1-26 peptides are acting on the same receptor targets. The coinjection of 10 µM of each of these peptides is similar to the injection of 20 µM of one of them (not shown). The same kind of experiments as in Figs. 2-4 are shown.



In addition, an intraneuronal injection of TeNT (20 nM), 60 min after injection of 10 µM of A-syb 1-19 peptide giving a half-inhibition of neurotransmitter release led to a complete inhibition of neurotransmission within 2 h (Fig. 5d). This demonstrated that endogenous vesicular synaptobrevin molecules remained accessible to the toxin and able to interact with protein targets involved in the neurotransmitter release process.

A Motif PXPXGXPP Is Responsible for the Dual Effect Observed in Aplysia Neurons

In order to examine whether the ability of the VAMP N-terminal fragments to alter neurotransmission was due to the presence of a common motif, the minimum active domain of the A-syb 1-19 peptide was first determined. Various analogues of this peptide, shortened at either their N and/or C terminus, were synthesized and injected into presynaptic neurons of Aplysia. The minimal active peptide corresponded to the Gly-Ser sequence of Aplysia synaptobrevin (A-syb 4-19) (). When intracellularly applied at 10 µM, it exhibited a 36% inhibitory activity (), and at 100 µM, it induced the typical transient action observed with A-syb 1-19 or H-syb II 1-26 (not shown). Furthermore, both Aplysia and human VAMP N-terminal domains share the striking feature of being mainly composed of amino acids with no, or short, lateral chains (Gly or Ala) and of several proline residues. However these N-terminal domains differ by their length and by the positions of these residues in the amino acid sequences. In order to test the specificity of the responses observed, the synthetic 1-28 fragment of human synaptobrevin isoform I (H-syb I 1-28) () was used. Indeed this peptide shares about the same amino acid content as H-syb II 1-26 but differs from it by the relative positions of the proline moieties. Injection of H-syb I 1-28 (at either 10 or 100 µM) into presynaptic neurons did not modify neurotransmission (). These observations suggested the existence of a common motif in A-syb 4-19 and H-syb II 1-26, but not in H-syb I 1-28, is responsible for the intracellular activity of the two former peptides. This motif is suggested to be PXPXGXPP by comparison of the sequences of the two active peptides ().


DISCUSSION

The complete blockade of neurotransmission that follows the proteolytic removal of VAMP from the surface of synaptic vesicles by several clostridial neurotoxins and the fact that this protein was found in a fusogenic protein complex comprising the two t-SNAP receptors, syntaxin and SNAP-25, led to the implication of VAMP in the exocytotic process of small synaptic vesicles(3, 4, 10, 11, 16, 24) . Nevertheless, until now, the mechanism of action of VAMP-synaptobrevin in the neurotransmitter release process has been unclear. In addition, several attempts to characterize in vivo the role of VAMP using anti-VAMP antibodies have been unsuccessful(5, 25, 26) . Our in vivo experiments show clearly that the potent inhibition of acetylcholine release induced by the intraneuronal injection in presynaptic neurons of A. californica of N-terminal proline-rich fragments of VAMP involves the N-terminal region of this small protein in the regulated secretion of neurotransmitters. Comparison of the minimum active domain of Aplysia VAMP with the N terminus of the human VAMP II isoform suggests that the functional sequence could comprise a PGGPXGXPP or a PAAPXGXPP motif. The squid VAMP 8-91 fragment contains also two similar motifs (PGGPXPP and PGGPXGPP) in its N-terminal sequence and, as expected, this 8-91 fragment inhibits partially neurotransmitter release when injected inside the giant nerve terminal of squid(27) . This biologically active motif is not found in vertebrate VAMP I isoforms (for example the inactive H-syb I 1-28 contains a PGXPXGXPP sequence), in rat cellubrevin (28) (Fig. 1) or in a Drosophila VAMP(14) .

The neuronal effects observed with the N-terminal domain of VAMP suggest a competition of VAMP N-terminal fragments with endogenous VAMP in its interaction with proteins of the release machinery. However, the long lasting inhibition of neurotransmitter release induced by VAMP or its N-terminal fragments reached a maximum of 50%, contrasting with the complete blockade ensured by intraneuronal injection of TeNT alone (Fig. 2a) or in the presence of inhibitory peptides (Fig. 5d). This suggests that the mechanism of action of TeNT and by inference of BoNT/B, /D, /F, and /G is probably not related to toxin-induced release of inhibitory peptides but rather to a dissociation of the N-terminal part of synaptobrevin from the synaptic vesicle. Moreover, these results could be interpreted in the light of recent in vitro results. Thus, in neurosecretory cells, synaptobrevin is distributed in two main pools. One corresponds to the 20 S complex, which include the SNAPs, syntaxin, SNAP-25, and the vesicular protein synaptotagmin(10, 24) . In this complex, synaptobrevin was shown to strongly interact with syntaxin and SNAP-25 by its well conserved domain (sequence 27-96)(29, 30, 31) . In the second pool, about 40-50% of VAMP/synaptobrevin was also recently found to be associated in vitro with synaptophysin(32, 33, 34) , a glycosylated protein with four transmembrane helices and a long intracytoplasmic domain enriched in tyrosine residues, which shows the propensity to form channels in membranes(35) . This in vitro interaction was shown to be blocked to 60-65% at concentration of 0.18 mM by the 1-32 N-terminal fragment of rat VAMP II but not rat VAMP I (sequence 1-34), showing that the interaction synaptophysin-VAMP II is mediated by the N-terminal domain of VAMP II(33, 34) . The interaction between VAMP and synaptophysin was proposed to inhibit the oligomerization of this latter protein to form a pore or to interact with its synaptic targets with subsequent formation of a fusion pore(32) . Indeed synaptophysin was not found in the 20 S complex, suggesting that the secretion process could be ensured by a disruption of the cytoplasmic synaptophysin-synaptobrevin 56-kDa complex allowing reconstitution of the temporary assembly of the 20 S complex and the oligomerization of synaptophysin to transiently form transmembrane channels ensuring neurosecretion (Fig. 6A)(32) .


Figure 6: Proposed target sites of N-terminal proline-rich fragments of VAMP II injected into Aplysia neurons. A, synaptobrevin is associated by its central part with syntaxin and SNAP-25 to form the 20 S complex while synaptophysin forms oligomers, a step putatively required for a fusion pore formation and a subsequent neurotransmitter release (32). The intraneuronal injection of the VAMP peptide 1-19 may prevent the formation of the 20 S complex (target 1, whitearrow in B) at low concentration (<10 µM) inhibiting neurotransmitter release. B, synaptophysin interacts with synaptobrevin to form a 56-kDa complex, and this interaction prevents oligomerization of synaptophysin (32-34). At concentration higher than 100 µM, the intraneuronally injected N-terminal fragments of the VAMP inhibit this protein-protein interaction (target 2, whitearrow) this facilitating neurosecretion (see Fig. 4d).



Our in vivo results bring strong physiological support to these proposals, deduced from in vitro biochemical experiments. Thus, at the membrane level, the N-terminal peptides of VAMP/synaptobrevin probably inhibit the binding of endogenous synaptobrevin simply by hindering the highly conserved domain of the native peptide from reaching its site of binding to SNAP-25 and syntaxin (Fig. 6B). This proposition is relevant for the inhibition observed after injection of N-terminal fragments of VAMP II at concentration below 10 µM. It is interesting to observe that peptides corresponding to the conserved region of VAMP such as Syb 20-83 are unable to reduce neurotransmitter release (Fig. 3c), suggesting that these peptides have no accessibility to the complex formed between VAMP and SNAP-25 and syntaxin. Furthermore, in our in vivo conditions, the intraneuronally injected N-terminal fragments of synaptobrevin, disjoined from the linked vesicles, behave as pseudoagonists devoid of intrinsic activities leading to 50% inhibition of neurotransmitter release. The U-shaped dose-response curves (Fig. 4, c and f, and Fig. 5, b and c) often observed in pharmacological experiments, could be due to the interaction of the N-terminal fragments at higher concentrations (10-100 µM) with another site resulting in activation of the neurosecretory process. This interaction could occur at the level of the 56-kDa complex (Fig. 6B) with the N-terminal fragments of synaptobrevin disrupting the interaction between endogenous synaptobrevin and synaptophysin, leading to the formation of pores in the plasma membrane and neurosecretion (Fig. 6A). In vitro, the inhibition of synaptobrevin-synaptophysin by the 1-32 fragment of VAMP II occurs also in the concentration range of 0.18 mM(32) , like it was observed in our in vivo conditions. This counteracting process would account for the reversion of the inhibition of acetylcholine release (Fig. 4, c and f, and Fig. 5, b and c). It is interesting to observe that, in contrast to the present findings, full and reversible inhibition of transmitter release was observed following injections of peptide fragments of synaptotagmin or SNAPs to squid giant presynaptic terminals(36, 37) . This seems to indicate that, in this case, only one target is critically involved in the neurosecretory function of these proteins. Moreover, the reversibility of the inhibition was interpreted by a dilution of the inhibitory peptides in the axon of squid giant neuron(36, 37) . Interestingly, injection of 8-91 domain of squid synaptobrevin in the squid giant synapse was reported to cause a slow and irreversible inhibition of about 60% of neurotransmitter release(27) , a result in complete agreement with our observations, supporting a difference in the mechanism of action of these different proteins in neurosecretion and the occurrence of two binding sites for synaptobrevin.

In conclusion, in this study the N-terminal proline-rich domain of VAMP II isoforms has been identified as playing a physiological role in neurotransmitter release certainly due to its interaction with synaptophysin. Since this domain is clearly distinct from the region involved in the interaction with SNAP receptors (domain 27-96), this confirms that in vivo synaptobrevin has more than one role in neuroexocytosis.

An important observation is the potent antagonist properties of peptides as short as 16 residues (in the case of Aplysia) toward the Ca-dependent neurotransmitter release. This could facilitate the development of synthetic blockers of neurotransmission, which could be used as surrogates for BoNT/A in the therapeutic treatment of dystonia induced by motoneuronal hyperactivity (strabism, hemifacial spasm, blepharospasm, etc.)(38) .

  
Table: 4


FOOTNOTES

*
This work was supported by Direction des Etudes et Recherches Techniques (Contract 92-102) and a grant from Association Franaise contre les Myopathies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: TeNT, tetanus toxin; BoNT, botulinum neurotoxin; VAMP, vesicle-associated membrane protein; SNAP, synaptosomal-associated protein; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank D. Ficheux and L. Martin for helpful technical assistance and Dr. A. Beaumont for stylistic revision of the manuscript.


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