Inhibition promotes long-term potentiation at cerebellar excitatory synapses.

The ability of the cerebellar cortex to learn from experience ensures the accuracy of movements and reflex adaptation, processes which require long-term plasticity at granule cell (GC) to Purkinje neuron (PN) excitatory synapses. PNs also receive GABAergic inhibitory inputs via GCs activation of interneurons; despite the involvement of inhibition in motor learning, its role in long-term plasticity is poorly characterized. Here we reveal a functional coupling between ionotropic GABAA receptors and low threshold CaV3 calcium channels in PNs that sustains calcium influx and promotes long-term potentiation (LTP) at GC to PN synapses. High frequency stimulation induces LTP at GC to PN synapses and CaV3-mediated calcium influx provided that inhibition is intact; LTP is mGluR1, intracellular calcium store and CaV3 dependent. LTP is impaired in CaV3.1 knockout mice but it is nevertheless recovered by strengthening inhibitory transmission onto PNs; promoting a stronger hyperpolarization via GABAA receptor activation leads to an enhanced availability of an alternative Purkinje-expressed CaV3 isoform compensating for the lack of CaV3.1 and restoring LTP. Accordingly, a stronger hyperpolarization also restores CaV3-mediated calcium influx in PNs from CaV3.1 knockout mice. We conclude that by favoring CaV3 channels availability inhibition promotes LTP at cerebellar excitatory synapses.

[Botulinum neurotoxin]. / La neurotoxine botulinique.

Botulinum toxin is a multi-molecular complex comprised of a neuro-active moiety (i.e. botulinum neurotoxin) and several associated non-toxic proteins. The toxin dissociates rapidly at plasmatic pH, thereby releasing neurotoxin. Nerve terminals only take up the neurotoxin. In the peripheral nerve system, the neurotoxin mainly blocks acetylcholine release. When acting at the neuromuscular junctions, this results in paralysis of the muscle fibers. The duration of the neurotoxin action is mainly determined by the life-time of neurotoxin molecules inside the nerve terminals. Inhibition of cholinergic transmission induces rapid atrophy of the muscle fibres, and, sometimes, sprouting from poisoned nerve terminals. These effects, as well as the acetylcholine release blockade are entirely reversible. When injected in the periphery, a direct action of botulinum neurotoxin in the central nervous system remains unlikely despite its retrograde ascent demonstrated in animal models. However, indirect effects are numerous. The constituting proteins of the toxin complex can lead to immunisation against the non-toxic associated proteins and neurotoxin. Only the antibodies directed against neurotoxin are potentially neutralizing.

[A reminder of the structure and function of the skeletal neuromuscular junction]. / Rappels structuraux et fonctionnels de la jonction neuromusculaire squelettique.

The skeletal neuromuscular junction has been considered as a model of chemical synapses due to its relatively simple organization. It is made up of three cellular partners including the motoneuron nerve terminals, the peri-synaptic Schwann cells and a specialized region of skeletal muscle fibers. It has been extensively studied revealing its ultrastructural complexity involving many molecular actors. The neuromuscular junction is a highly specialized structure, optimized for the rapid transmission of information from the presynaptic nerve terminal to the post-synaptic muscle fiber. This rapid transmission requires a very close apposition of plasmic membranes of pre- and post-synaptic partners, and a strict structural and molecular arrangement on both sides of the narrow synaptic cleft separating nerve terminal and muscle membranes. In this short review, we summarize the knowledge regarding pre- and post-synaptic ultrastructural specializations and give an overview of some functional aspects of neuromuscular transmission, including the quantal acetylcholine release process, which will help to better understand the pharmacological actions of botulinum toxins in esthetic and corrective dermatology.

[Mechanisms of action of botulinum toxins and neurotoxins]. / Mécanismes d'action des toxines et neurotoxines botuliques.

Several bacteria of the Clostridium genus (C. botulinum) produce 150 kDa di-chainal protein toxins referred as botulinum neurotoxins or BoNTs. They associate with non-toxic companion proteins and form a complex termed botulinum toxin. BoNTs specifically inhibit vesicular neurotransmitter release. The cellular action of BoNTs can be depicted according to a multi-step model : The toxin's heavy chain mediates binding to specific receptors comprised of a ganglioside moiety and a vesicular protein (SV2 for BoNT type A, synaptotagmin for BoNT type B), followed by endocytotic internalisation of the BoNT/receptor complex. Vesicle recycling induces BoNT internalisation. Upon acidification of vesicles, the light chain of the neurotoxin is translocated into the cytosol. Here, this zinc-endopeptidase cleaves one or two among three synaptic proteins (VAMP-synapto-brevin, SNAP25, and syntaxin). As the three protein targets of BoNT play major role in fusion of synaptic vesicles at the release sites, their cleavage is followed by blockade of neurotransmitter exocytosis. Importantly, as the BoNT receptors and intracellular targets are present in all nerve terminals, the BoNTs are not specific for cholinergic transmission. Duration of their inhibitory action is mainly determined by the the life-time of the toxin's light chain in the cytosol. Sprouting of new nerve-endings, which are retracted when the poisoned nerve terminals have recovered full functionality, may lead to anticipated recovery of the poisoned nerve terminals.

Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells.

Clostridium perfringens epsilon toxin (ET) is a potent pore-forming cytotoxin causing fatal enterotoxemia in livestock. ET accumulates in brain and kidney, particularly in the renal distal-collecting ducts. ET binds and oligomerizes in detergent-resistant membranes (DRMs) microdomains and causes cell death. However, the causal linkage between membrane permeabilization and cell death is not clear. Here, we show that ET binds and forms 220-kDa insoluble complexes in plasma membrane DRMs of renal mpkCCD(cl4) collecting duct cells. Phosphatidylinositol-specific phospholipase C did not impair binding or the formation of ET complexes, suggesting that the receptor for ET is not GPI anchored. ET induced a dose-dependent fall in the transepithelial resistance and potential in confluent cells grown on filters, transiently stimulated Na+ absorption, and induced an inward ionic current and a sustained rise in [Ca2+]i. ET also induced rapid depletion of cellular ATP, and stimulated the AMP-activated protein kinase, a metabolic-sensing Ser/Thr kinase. ET also induced mitochondrial membrane permeabilization and mitochondrial-nuclear translocation of apoptosis-inducing factor, a potent caspase-independent cell death effector. Finally, ET induced cell necrosis characterized by a marked reduction in nucleus size without DNA fragmentation. DRM disruption by methyl-beta-cyclodextrin impaired ET oligomerization, and significantly reduced the influx of Na+ and [Ca2+]i, but did not impair ATP depletion and cell death caused by the toxin. These findings indicate that ET causes rapid necrosis of renal collecting duct cells and establish that ATP depletion-mediated cell death is not strictly correlated with the plasma membrane permeabilization and ion diffusion caused by the toxin.

[Mode of action of botulinum neurotoxin: pathological, cellular and molecular aspect]. / Le mode d'action des neurotoxines botuliques: aspects pathologiques, cellulaires et moléculaires.

Several bacteria of the Clostridium genus (C. botulinum) produce 150 kDa di-chainal protein toxins referred as botulinum neurotoxins or BoNTs. They associate with non-toxic companion proteins and form a complex termed botulinum toxin or BoTx. The latter is used in clinic for therapeutic purpose. BoNTs affect cholinergic nerve terminals in periphery where they block acetylcholine release, thereby causing dysautonomia and motorparalysis (i.e. botulism). The cellular action of BoNTs can be depicted according to a three steps model: binding, internalisation and intraneuronal action. The toxins heavy chain mediates binding to specific receptors followed by endocytotic internalisation of BoNT/receptor complex. BoNT receptors may comprise gangliosides and synaptic vesicle-associated proteins as synaptotagmins. Vesicle recycling induces BoNT internalisation. Upon acidification of vesicles, the light chain of the neurotoxin is translocated into the cytosol. Here, this zinc-endopeptidase cleaves one or two among three synaptic proteins (VAMP-synaptobrevin, SNAP25, and syntaxin). As the three protein targets of BoNT play major role in fusion of synaptic vesicles at the release sites, their cleavage is followed by blockage of neurotransmitter exocytosis. The duration of the paralytic effect of the BoNTs is determined by 1) the turnover of their protein target; 2) the time-life of the toxin light chain in the cytosol, and 3) the sprouting of new nerve-endings that are retracted when the poisoned nerve terminal had recovered its full functionality.

A role for phospholipase D1 in neurotransmitter release.

Phosphatidic acid produced by phospholipase D (PLD) as a result of signaling activity is thought to play a role in membrane vesicle trafficking, either as an intracellular messenger or as a cone-shaped lipid that promotes membrane fusion. We recently described that, in neuroendocrine cells, plasma membrane-associated PLD1 operates at a stage of Ca(2+)-dependent exocytosis subsequent to cytoskeletal-mediated recruitment of secretory granules to exocytotic sites. We show here that PLD1 also plays a crucial role in neurotransmitter release. Using purified rat brain synaptosomes subjected to hypotonic lysis and centrifugation, we found that PLD1 is associated with the particulate fraction containing the plasma membrane. Immunostaining of rat cerebellar granule cells confirmed localization of PLD1 at the neuronal plasma membrane in zones specialized for neurotransmitter release (axonal neurites, varicosities, and growth cone-like structures). To determine the potential involvement of PLD1 in neurotransmitter release, we microinjected catalytically inactive PLD1(K898R) into Aplysia neurons and analyzed its effects on evoked acetylcholine (ACh) release. PLD1(K898R) produced a fast and potent dose-dependent inhibition of ACh release. By analyzing paired-pulse facilitation and postsynaptic responses evoked by high-frequency stimulations, we found that the exocytotic inhibition caused by PLD1(K898R) was not the result of an alteration in stimulus-secretion coupling or in vesicular trafficking. Analysis of the fluctuations in amplitude of the postsynaptic responses revealed that the PLD1(K898R) blocked ACh release by reducing the number of active presynaptic-releasing sites. Our results provide evidence that PLD1 plays a major role in neurotransmission, most likely by controlling the fusogenic status of presynaptic release sites.

High sensitivity of mouse neuronal cells to tetanus toxin requires a GPI-anchored protein.

Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis. Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP. Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin.

Synapsin controls both reserve and releasable synaptic vesicle pools during neuronal activity and short-term plasticity in Aplysia.

Neurotransmitter release is a highly efficient secretory process exhibiting resistance to fatigue and plasticity attributable to the existence of distinct pools of synaptic vesicles (SVs), namely a readily releasable pool and a reserve pool from which vesicles can be recruited after activity. Synaptic vesicles in the reserve pool are thought to be reversibly tethered to the actin-based cytoskeleton by the synapsins, a family of synaptic vesicle-associated phosphoproteins that have been shown to play a role in the formation, maintenance, and regulation of the reserve pool of synaptic vesicles and to operate during the post-docking step of the release process. In this paper, we have investigated the physiological effects of manipulating synapsin levels in identified cholinergic synapses of Aplysia californica. When endogenous synapsin was neutralized by the injection of specific anti-synapsin antibodies, the amount of neurotransmitter released per impulse was unaffected, but marked changes in the secretory response to high-frequency stimulation were observed, including the disappearance of post-tetanic potentiation (PTP) that was substituted by post-tetanic depression (PTD), and increased rate and extent of synaptic depression. Opposite changes on post-tetanic potentiation were observed when synapsin levels were increased by injecting exogenous synapsin I. Our data demonstrate that the presence of synapsin-dependent reserve vesicles allows the nerve terminal to release neurotransmitter at rates exceeding the synaptic vesicle recycling capacity and to dynamically change the efficiency of release in response to conditioning stimuli (e.g., post-tetanic potentiation). Moreover, synapsin-dependent regulation of the fusion competence of synaptic vesicles appears to be crucial for sustaining neurotransmitter release during short periods at rates faster than the replenishment kinetics and maintaining synchronization of quanta in evoked release.

How botulinum and tetanus neurotoxins block neurotransmitter release.

Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M(r) approximately 50) disulfide linked to a heavy chain (M(r) approximately 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.

A Rho-related GTPase is involved in Ca(2+)-dependent neurotransmitter exocytosis.

Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the actin cytoskeleton and phosphoinositide metabolism and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible implication in Ca(2+)-dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA, RhoB, Rac1, and Cdc42 are present in rat brain synaptosomes; however, only Rac1 was associated with highly purified synaptic vesicles. To determine the synaptic function of these GTPases, toxins that impair Rho-related proteins were microinjected into Aplysia neurons. We used lethal toxin from Clostridium sordellii, which inactivates Rac; toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42; and C3 exoenzyme from Clostridium botulinum and cytotoxic necrotizing factor 1 from Escherichia coli, which mainly affect Rho. Analysis of the toxin effects on evoked acetylcholine release revealed that a member of the Rho family, most likely Rac1, was implicated in the control of neurotransmitter release. Strikingly, blockage of acetylcholine release by lethal toxin and toxin B could be completely removed in <1 s by high frequency stimulation of nerve terminals. Further characterization of the inhibitory action produced by lethal toxin suggests that Rac1 protein regulates a late step in Ca(2+)-dependent neuroexocytosis.

[Analysis of synaptic neurotransmitter release mechanisms using bacterial toxins]. / Dissection des mécanismes synaptiques de la libération des neurotransmetteurs par l'utilisation de toxines bactériennes.

Several bacterial toxins are powerful and highly specific tools for studying basic mechanisms involved in cell biology. Whereas the clostridial neurotoxins are widely used by neurobiologists, many other toxins (i.e. toxins acting on small G-proteins or actin) are still overlooked. Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT), known under the generic term of clostridial neurotoxins, are characterized by their unique ability to selectively block neurotransmitter release. These proteins are formed of a light (Mr approximately 50) and a heavy (Mr approximately 100) chain which are disulfide linked. The cellular action of BoNT and TeNT involves several steps: heavy chain-mediated binding to the nerve ending membrane, endocytosis, and translocation of the light chain (their catalytic moiety) into the cytosol. The light chains each cleaves one of three, highly conserved, proteins (VAMP/synaptobrevin, syntaxin, and SNAP-25 also termed SNAREs) implicated in fusion of synaptic vesicles with plasma membrane at the release site. Hence, when these neurotoxins are applied extracellularly, they can be used as specific tools to inhibit evoked and spontaneous transmitter release from certain neurones whereas, when the membrane limiting steps are bypassed by the mean of intracellular applications, BoNTs orTeNT can be used to affect regulated secretion in various cell types. Several members of the Rho GTPase family have been involved in intracellular trafficking of synaptic vesicles and secretory organelles. As they are natural targets for several bacterial exoenzymes or cytotoxins, their role in neurotransmitter release can be probed by examining the action of these toxins on neurotransmission. Such toxins include: i) the non permeant C3 exoenzymes from C. botulinum or C. limosum which ADP-ribosylate and thereby inactivate Rho, ii) exoenzyme S from Pseudomonas aeruginosa which ADP-ribosylates different members of the Ras, Rab, Ral and Rap families, iii) toxin B from C. difficile which glucosylates Rho, Rac and CDC42, iv) lethal toxin from C. sordellii which glucosylates Rac, Ras and to a lesser extent, Rap and Ral, but not on Rho or CDC42, and v) CNF deamidases secreted by pathogenic strains of E. coli which activate Rho and, to a lesser extent, CDC42. Since these toxins or exoenzymes have no or little ability to enter into the neurones, they must be applied intraneuronally to bypass the membrane limiting steps. Injection of several of these toxins into Aplysia neurones allowed us to reveal a new role for Rac in the control of exocytosis. ADP-ribosylating enzymes, which specifically act on monomeric actin (C2 binary toxin from C. botulinum and iota toxin from C. perfringens), are potential tools to probe the role of actin filaments during secretion.

Calcium-dependent regulation of rab3 in short-term plasticity.

The Rab3 proteins are monomeric GTP-binding proteins associated with secretory vesicles. In their active GTP-bound state, Rab3 proteins are involved in the regulation of hormone secretion and neurotransmitter release. This action is thought to involve specific effectors, including two Ca2+-binding proteins, Rabphilin and Rim. Rab3 acts late in the exocytotic process, in a cell domain in which the intracellular Ca2+ concentration is susceptible to rapid changes. Therefore, we examined the possible Ca2+-dependency of the regulatory action of GTP-bound Rab3 and wild-type Rab3 on neuroexocytosis at identified cholinergic synapses in Aplysia californica. The effects of recombinant GTPase-deficient Aplysia-Rab3 (apRab3-Q80L) or wild-type apRab3 were studied on evoked acetylcholine release. Intraneuronal application of apRab3-Q80L in identified neurons of the buccal ganglion of Aplysia led to inhibition of neurotransmission; wild-type apRab3 was less effective. Intracellular chelation of Ca2+ ions by EGTA greatly potentiated the inhibitory action of apRab3-Q80L. Train and paired-pulse facilitation, two Ca2+-dependent forms of short-term plasticity induced by a rise in intraterminal Ca2+ concentration, were increased after injection of apRab3-Q80L. This result suggests that the inhibition exerted by GTP-bound Rab3 on neuroexocytosis is reduced during transient augmentations of intracellular Ca2+ concentration. Therefore, a Ca2+-dependent modulation of GTP-bound Rab3 function may contribute to short-term plasticity.

[Action mechanisms of botulinum neurotoxins and tetanus neurotoxins]. / Mécanismes d'action des neurotoxines botuliques et de la neurotoxine tétanique.

Tetanus (TeNT) neurotoxin and botulinum (BoNT, serotypes A-G) neurotoxins are di-chain bacterial proteins of MW-150 kDa which are also termed as clostridial neurotoxins. They are the only causative agents of two severe neuroparalytic diseases, namely tetanus and botulism. The peripheral muscle spasms which characterise tetanus are due to a blockade of inhibitory (GABAergic and glycinergic) synapses in the central nervous system leading to a motor neurones desinhibition. In contrast, botulism symptoms are only peripheral. They are consequent to a near irreversible and highly selective inhibition of acetyl-choline release at the motor nerve endings innervating skeletal muscles. During the past decade, the cellular and molecular modes of action of clostridial neurotoxins has been near completely elucidated. After a binding step of the neurotoxins to specific membrane acceptors located only on nerve terminals, BoNTs and TeNT are internalized into neurons. Inside their target neurones, the intracellularly active moiety (their light chain) is translocated from the endosomal compartment to the cytosol. The neurotoxins' light chains are zinc-dependent (endopeptidases which are specific for one among three synaptic proteins (VAMP/synaptobrevin, syntaxin or SNAP-25) implicated in neurotransmitter exocytosis. The presence of distinct targets for BoNTs and TeNT correlates well with the observed quantal alterations of neurotransmitter release which characterize certain toxin serotypes. In addition, evidence for a second, non-proteolytic, inhibitory mechanism of action has been provided recently. Most likely, this additional blocking action involves the activation of neurone transglutaminases. Due to their specific action on key proteins of the exocytosis apparatus, clostridial neurotoxins are now widely used as molecular tools to study exocytosis.

Evidence for a functional link between Rab3 and the SNARE complex.

Rab3 is a monomeric GTP-binding protein associated with secretory vesicles which has been implicated in the control of regulated exocytosis. We have exploited Rab3 mutant proteins to investigate the function of Rab3 in the process of neurotransmitter release from Aplysia neurons. A GTPase-deficient Rab3 mutant protein was found to inhibit acetylcholine release suggesting that GTP hydrolysis by Rab3 is rate-limiting in the exocytosis process. This effect was abolished by a mutation in the effector domain, and required the association of Rab3 with membranes. In order to determine the step at which Rab3 interferes with the secretory process, tetanus and botulinum type A neurotoxins were applied to Aplysia neurons pre-injected with the GTPase-deficient Rab3 mutant protein. These neurotoxins are Zn(2+)-dependent proteases that cleave VAMP/synaptobrevin and SNAP-25, two proteins which can form a ternary complex (termed the SNARE complex) with syntaxin and have been implicated in the docking of synaptic vesicles at the plasma membrane. The onset of toxin-induced inhibition of neurotransmitter release was strongly delayed in these cells, indicating that the mutant Rab3 protein led to the accumulation of a toxin-insensitive component of release. Since tetanus and botulinum type A neurotoxins cannot attack their targets, VAMP/synaptobrevin and SNAP-25, when the latter are engaged in the SNARE complex, we propose that Rab3 modulates the activity of the fusion machinery by controlling the formation or the stability of the SNARE complex.

Differences in the multiple step process of inhibition of neurotransmitter release induced by tetanus toxin and botulinum neurotoxins type A and B at Aplysia synapses.

In order to gain insights into the steps (binding, uptake, intracellular effect) which differ in the inhibitory actions of tetanus toxin and botulinum neurotoxins types A or B, their temperature dependencies were investigated at identified cholinergic and non-cholinergic synapses in Aplysia. Upon lowering the temperature from 22 degrees C to 10 degrees C, extracellularly applied botulinum neurotoxin type A and B appeared unable to inhibit transmitter release whilst tetanus toxin exhibited a residual activity. Binding of each toxin to the neuronal membrane appeared virtually unaltered following this temperature change. By contrast, the intracellular effects of botulinum neurotoxin type B and tetanus toxin were strongly attenuated by temperature reduction whereas the inhibitory action of botulinum neurotoxin type A was only moderately reduced. Importantly, this discrepancy relates to the known proteolytic cleavage of different synaptic proteins by these two toxin groups. Since both the binding and intracellular activity of botulinum neurotoxin type A are minimally affected at 10 degrees C, its inability to inhibit neurotransmission at this low temperature when applied extracellularly indicated attenuation of its uptake. Due to the strict temperature dependence of the intracellular action of tetanus toxin and botulinum neurotoxin type B, but not A, an examination of the effects of changes in temperature on the internalization step was facilitated by the use of heterologous mixtures of the toxins' heavy and light chains. At 10 degrees C, heavy chain from tetanus toxin but not from botulinum neurotoxin type B mediated uptake of botulinum neurotoxin type A light chain. Collectively, these results provide evidence that, at least in Aplysia, the uptake mechanism for botulinum neurotoxin types A and B differs from that of tetanus toxin.

Tetanus toxin inhibits neuroexocytosis even when its Zn(2+)-dependent protease activity is removed.

Tetanus toxin (TeTX) is a dichain protein that blocks neuroexocytosis, an action attributed previously to Zn(2+)-dependent proteolysis of synaptobrevin (Sbr) by its light chain (LC). Herein, its cleavage of Sbr in rat cerebrocortical synaptosomes was shown to be minimized by captopril, an inhibitor of certain metalloendoproteases, whereas this agent only marginally antagonized the inhibition of noradrenaline release, implicating a second action of the toxin. This hypothesis was proven by preparing three mutants (H233A, E234A, H237A) of the LC lacking the ability to cleave Sbr and reconstituting them with native heavy chain. The resultant dichains were found to block synaptosomal transmitter release, albeit with lower potency than that made from wild type LC; as expected, captopril attenuated only the inhibition caused by the protease-active wild type toxin. Moreover, these protease-inactive toxins or their LCs blocked evoked quantal release of transmitter when micro-injected inside Aplysia neurons. TeTX was known to stimulate in vitro a Ca(2+)-dependent transglutaminase (TGase) (Facchiano, F., and Luini, A. (1992) J. Biol. Chem. 267, 13267-13271), an affect found here to be reduced by an inhibitor of this enzyme, monodansylcadaverine. Accordingly, treatment of synaptosomes with the latter antagonized the inhibition of noradrenaline release by TeTX while not affecting Sbr cleavage. This drug also attenuated the inhibitory action of all the mutants. Hence, it is concluded that TeTX inhibits neurotransmitter release by proteolysis of Sbr and a protease-independent activation of a neuronal TGase.

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.

Tetanus toxin and clostridial neurotoxins type B, D, F, and G inhibit intracellular Ca(2+)-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 (approximately 50% at 10 MicroM) 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 (PGGPXGX3PP or PAAPXGX3PP). 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 synatophysin-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.