[Analysis of synaptic neurotransmitter release mechanisms using bacterial toxins]

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.