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Rôles de la GTPase Rac et de la synapsine dans la (...)

- 1 Ann Readapt Med Phys. 2003 Jul ;46(6):265-75.

[Mode of action of botulinum neurotoxin : pathological, cellular and molecular aspect]

[Article in French]

Poulain B, Humeau Y.

Neurotransmission et secretion neuroendocrine, UPR 2356 du CNRS, IFR 37 des neurosciences, 5, rue Blais e-Pascal, 67084 Strasbourg cedex, France. poulain

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.

Publication Types : Review

PMID : 12928128

- 2 : J Neurosci. 2002 Sep 15 ;22(18):7968-81.

Rac GTPase plays an essential role in exocytosis by controlling the fusion competence of release sites.

Humeau Y, Popoff MR, Kojima H, Doussau F, Poulain B.

Neurotransmission et Secretion Neuroendocrine, UPR2356 du Centre National de la Recherche Scientifique, IFR-37 des Neurosciences, F-67084 Strasbourg Cedex, France.

The role of small GTPases of the Rho family in synaptic functions has been addressed by analyzing the effects of lethal toxin (LT) from Clostridium sordellii strain IP82 (LT82) on neurotransmitter release at evoked identified synapses in the buccal ganglion of Aplysia. LT82 is a large monoglucosyltranferase that uses UDP-glucose as cofactor and glucosylates Rac (a small GTPase related to Rho), and Ras, Ral, and Rap (three GTPases of the Ras family). Intraneuronal application of LT (50 nm) rapidly inhibits evoked acetylcholine (ACh) release as monitored electrophysiologically. Injection of the catalytic domain of the toxin similarly blocked ACh release, but not when key amino acids needed for glucosylation were mutated. Intraneuronal application of competitive nucleotide sugars that differentially prevent glucosylation of Rac- and Ras-related GTPases, and the use of a toxin variant that affects a different spectrum of small GTPases, established that glucosylation of Rac is responsible for the reduction in ACh release. To determine the quantal release parameters affected by Rac glucosylation, we developed a nonstationary analysis of the fluctuations in postsynaptic response amplitudes that was performed before and after the toxin had acted or during toxin action. The results indicate that neither the quantal size nor the average probability for release were affected by lethal toxin action. ACh release blockage by LT82 was only caused by a reduction in the number of functional release sites. This reveals that after docking of synaptic vesicles, vesicular Rac stimulates a membrane effector (or effectors) essential for the fusion competence of the exocytotic sites.

PMID : 12223550

- 3 : Proc Natl Acad Sci U S A. 2001 Dec 18 ;98(26):15300-5.

A role for phospholipase D1 in neurotransmitter release.

Humeau Y, Vitale N, Chasserot-Golaz S, Dupont JL, Du G, Frohman MA, Bader MF, Poulain B.

Centre National de la Recherche Scientifique, Unite Propre de Recherche 2356, Neurotransmission et Secretion Neuroendocrine, 5 Rue Blaise Pascal, IFR37, 67084 Strasbourg, France.

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.

PMID : 11752468

- 4 : Biochem Biophys Res Commun. 2001 Nov 30 ;289(2):623-9.

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

Munro P, Kojima H, Dupont JL, Bossu JL, Poulain B, Boquet P.

INSERM Unite 452, Faculte de Medecine, 28 Avenue de Valombrose, F-06107 Nice, France.

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.

PMID : 11716521

- 5 Annales-de-readaptation-et-de-medecine-physique. 2003 ; 46 (6) : 265-275 Le mode d’ action des neurotoxines botuliques : aspects pathologiques, cellulaires et moleculaires. La toxine botulique : mise au point sur les principales indications therapeutique = Mode of action of botulinum neurotoxin : pathological, cellular and molecular aspect POULAIN-B ; HUMEAU-Y AF : Neurotransmission et secretion neuroendocrine, UPR 2356 du CNRS, IFR 37 des neurosciences, 5, rue Blaise-Pascal, 67084 Strasbourg, France ; Freidrich Miecher Institute, Basel, Switzerland

Les neurotoxines botuliques (BoNTs, 7 serotypes, A-G) sont des proteines produites par des bacteries du genre Clostridium. Les BoNTs sont associees a des proteines non toxiques et forment un complexe appele toxine botulique (BoTx). C’ est ce complexe qui est utilise en clinique pour un but therapeutique. L’ intoxination avec la BoNT cause le botulisme qui comprend une dysautonomie et une paralysie qui sont provoquees par l’ inhibition de la liberation d’ acetylcholine en peripherie. Apres leur liaison a des recepteurs neuronaux comprenant des gangliosides et peut-etre la synaptotagmine, les BoNTs sont internalisees dans les terminaisons nerveuses par endocytose. L’ acidification de la vesicule de capture entraine la translocation de la chaine legere de BoNT dans le cytoplasme neuronal. La chaine legere de BoNT est une metalloprotease a zinc qui, selon le toxinotype, clivent une ou deux des proteines VAMP/synaptobrevine, SNAP25 ou syntaxine. Comme ces trois proteines interviennent dans le processus de fusion des vesicules synaptiques avec la membrane plasmique, leur clivage provoque l’ inhibition de l’ exocytose d’ acetylcholine. La duree du blocage de la neurotransmission cholinergique depend de la remanence des neurotoxines dansles neurones, de la vitesse de remplacement des proteines qu’ elles ont alterees et de la mise en place temporaire de nouvelles terminaisons nerveuses qui sont retractees apres que la plaque motrice originelle a retrouve sa pleine fonctionnalite.

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