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Animal presynaptic neurotoxins provide a relevant novel model of moto axon terminal degeneration followed by regeneration
Abstracts / Toxicon 116 (2016) 72e86
to opioid receptors, suggesting that the opioid-like activity of the peptide could result from the release of endogenous opioids. This was recently confirmed by the demonstration that crotalphine-induced antinociception involves activation of peripheral CB2 receptors, which stimulates dynorphin A release from peripheral skin tissues. Dynorphin A, in turn, induces antinociception by acting on k-opioid receptors expressed on primary afferent neurons. Initial studies using peritoneal macrophage cultures incubated with fluorescein-labelled crotalphine showed that the peptide internalizes on these cells. Using antibody anti-CB2 cannabinoid receptors, co-localization of labelled crotalphine and cannabinoid CB2 receptors was detected, indicating that internalization of crotalphine on macrophages is mediated by cannabinoid CB2 receptors. The release of dynorphin A (A-13) in the macrophage culture medium was also detected. In contrast, the presence of internalized crotalphine was not observed in DRG neurons. In co-cultures of macrophages and DRG neurons, internalization of k opioidreceptors was detected. These results indicate that internalization of these receptors is an indirect effect, mediated by the release of endogenous opioids. Taken together, these results contribute to the understanding of the mechanisms involved in crotalphine induced antinociception and corroborate the existence of a close connection between the opioid and cannabinoid systems in pain control. ~o de Amparo a Pesquisa do Estado de Sa ~o Paulo Financial support: Fundaça (FAPESP, Brazil, Grant Nos. 2009/14203-5, 2010/12917-8 and 2013/074671), Instituto Nacional de Ciencia e Tecnologia em Toxinologia (INCTTOX PROGRAM) of Conselho Nacional de Desenvolvimento Científico e Tecgico (CNPq)/FAPESP (Grant No. 2008/57898-0), and Fundaça ~o do nolo Desenvolvimento Administrativo (FUNDAP). CROTOXIN: NEW TARGETS AND NEW FUNCTIONS G. Faure. Unit e R ecepteurs e Canaux, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris, France Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus exhibits PLA2 activity and presynaptic neurotoxicity. Previously, we solved the crystal structure of this heterodimeric CACB complex at 1.35 Å resolution (Faure et al., 2011). The three-dimensional structure revealed the binding interface between the acidic (CA) and basic (CB) PLA2 subunits of crotoxin and key residues involved in the stability, toxicity and enzymatic activity of this potent b-neurotoxin. More recently, using surface plasmon resonance and other biochemical and biophysical techniques, we identified two novel specific targets of rattlesnake PLA2: (1) the cystic fibrosis transmembrane conductance regulator (CFTR) and (2) bacterial pentameric proton-gated ion channel (GLIC). We investigated the possible physiological relevance of CB-CFTR and CB-GLIC interactions, and diverse electrophysiological studies enabled us to discover new functions of crotoxin. The talk will focus on the description of these new targets and new functions of crotoxin. Faure G., Xu H., Saul, F.A., 2011. Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric b-neurotoxin. J. Mol. Biol. 412, 176e191. STUDY OF THE INVOLVEMENT OF ITCH MEDIATORS IN THE NEUROPEPTIDE RELEASE INDUCED BY PACIFIC-CIGUATOXIN-2 IN A CO-CULTURE MODEL OF SENSORY NEURONS AND KERATINOCYTES K. L’herondelle a, *, R. Philippe b, O. Mignen b, I. Vetter c, R.J. Lewis c, L. Misery a, d, R. Le Garrec a. a Laboratoire de Neurosciences de Brest, Universit e de Bretagne Occidentale, 22 avenue Camille Desmoulins, 29200 e Bretagne Occidentale, 46 rue Brest, France; b INSERM U1078, Universit Felix Le Dantec, 29218 Brest, France; c Institute for Molecular Bioscience, epartement de The University of Queensland, St Lucia, Australia; d D Dermatologie, Centre Hospitalier R egional Universitaire de Brest, 2 avenue Foch, 29609 Brest, France
* Corresponding author.
73
Ciguatera Fish Poisoning (CFP), which is called “La Gratte” (the itch) in the Pacific islands, is caused by eating fish contaminated with ciguatoxins (CTXs). The most characteristic clinical signs are cutaneous sensory disorders such as cold-induced dysesthesia, paresthesia and an intense pruritus. CFP-associated itch, which can persist for several weeks, is poorly or not relieved by antihistamine drugs. At the cellular and molecular levels, it is recognized that CTXs bind to voltage-gated sodium channels (VGSCs) leading to Na+ entry and increased excitability with various cellular, including secretory, consequences. Recently, it was shown that the cold allodynia induced by CTX in mice is VGSC-dependent and involves TRPA1, and that sensory neurons which respond to CTX express Calcitonin GeneRelated Peptide (CGRP). However, the molecular basis of the mechanisms underlying CTX-induced itch remain unclear. Pruritus is the unpleasant sensation that leads to the need to scratch. It can seriously impair quality of life, especially when it is chronic. Whatever its origin is, its pathophysiology is complex and still incompletely known, despite recent advances. Whereas the best-known pruritogen in humans is histamine, many types of itch do not respond to anti-H1 drugs. Other substances released from skin cells, such as substance P (SP) and CGRP, have been shown to mediate or facilitate pruritus. Furthermore, substantial evidence indicate the involvement of emerging receptors expressed by various skin cells, including some TRP (Transient Receptor Potential) channels and some Protease-Activated Receptors (PARs). The purpose of this work was to study the release of the neuropeptides substance P (SP) and CGRP in a coculture of human keratinocytes and rat DRG neurons exposed to PacificCiguatoxin-2 (P-CTX-2). Neuropeptides were quantified in the culture supernatant by ELISA. For the first time, we showed that low nanomolar concentrations of P-CTX-2 cause a large release of SP and CGRP in this model. In addition, calcium imaging experiments on cultured keratinocytes alone showed an increase of intracellular Ca2+. Pharmacological tools were used to study the involvement of some TRP channels and of PAR-2 receptor in these effects of the P-CTX-2. Up to now, CFP-associated pruritus has never been the subject of any study. This work is not only an original way to better understand the pathophysiology of itch but it will also supplement the knowledge of the molecular and cellular mechanisms of CFP. The long-term aim of this study is to identify potential targets for treating sensory disorders of ciguatera. ANIMAL PRESYNAPTIC NEUROTOXINS PROVIDE A RELEVANT NOVEL MODEL OF MOTO AXON TERMINAL DEGENERATION FOLLOWED BY REGENERATION C. Montecucco a, b, *, E. Duregotti a, S. Negro a, M. Scorzeto a, I. Zornetta a, B.C. Dickinson c, C.J. Chang c, M. Rigoni a,b. a Department of Biomedical Sciences, University of Padua, Padua 35131, Italy; b CNR Institute of Neuroscience, Padua 35131, Italy; c Department of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA Several animal presynaptic neurotoxins cause an acute motor axon terminal degeneration with a botulism-like flaccid paralysis. This is followed by complete regeneration within few days in mice and 4-6 weeks in humans. Mice injected with these neurotoxins in different muscles provide an appropriate and controlled system to dissect the molecular mechanisms underlying degeneration and regeneration of peripheral nerve terminals. We have previously shown that the toxin induced neurodegeneration is caused by calcium overload and mitochondrial failure. Here we show that toxin-treated primary neurons and neuromuscular junctions release danger signaling molecules derived from mitochondria: hydrogen peroxide, mito-DNA and cytochrome c. These molecules activate isolated primary Schwann cells, Schwann cells co-cultured with neurons and at neuromuscular junction in vivo, through the ERK1/2 pathway. We propose that these inter- and intra-cellular signaling events are involved in triggering the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases.
* Corresponding author.
to opioid receptors, suggesting that the opioid-like activity of the peptide could result from the release of endogenous opioids. This was recently confirmed by the demonstration that crotalphine-induced antinociception involves activation of peripheral CB2 receptors, which stimulates dynorphin A release from peripheral skin tissues. Dynorphin A, in turn, induces antinociception by acting on k-opioid receptors expressed on primary afferent neurons. Initial studies using peritoneal macrophage cultures incubated with fluorescein-labelled crotalphine showed that the peptide internalizes on these cells. Using antibody anti-CB2 cannabinoid receptors, co-localization of labelled crotalphine and cannabinoid CB2 receptors was detected, indicating that internalization of crotalphine on macrophages is mediated by cannabinoid CB2 receptors. The release of dynorphin A (A-13) in the macrophage culture medium was also detected. In contrast, the presence of internalized crotalphine was not observed in DRG neurons. In co-cultures of macrophages and DRG neurons, internalization of k opioidreceptors was detected. These results indicate that internalization of these receptors is an indirect effect, mediated by the release of endogenous opioids. Taken together, these results contribute to the understanding of the mechanisms involved in crotalphine induced antinociception and corroborate the existence of a close connection between the opioid and cannabinoid systems in pain control. ~o de Amparo a Pesquisa do Estado de Sa ~o Paulo Financial support: Fundaça (FAPESP, Brazil, Grant Nos. 2009/14203-5, 2010/12917-8 and 2013/074671), Instituto Nacional de Ciencia e Tecnologia em Toxinologia (INCTTOX PROGRAM) of Conselho Nacional de Desenvolvimento Científico e Tecgico (CNPq)/FAPESP (Grant No. 2008/57898-0), and Fundaça ~o do nolo Desenvolvimento Administrativo (FUNDAP). CROTOXIN: NEW TARGETS AND NEW FUNCTIONS G. Faure. Unit e R ecepteurs e Canaux, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris, France Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus exhibits PLA2 activity and presynaptic neurotoxicity. Previously, we solved the crystal structure of this heterodimeric CACB complex at 1.35 Å resolution (Faure et al., 2011). The three-dimensional structure revealed the binding interface between the acidic (CA) and basic (CB) PLA2 subunits of crotoxin and key residues involved in the stability, toxicity and enzymatic activity of this potent b-neurotoxin. More recently, using surface plasmon resonance and other biochemical and biophysical techniques, we identified two novel specific targets of rattlesnake PLA2: (1) the cystic fibrosis transmembrane conductance regulator (CFTR) and (2) bacterial pentameric proton-gated ion channel (GLIC). We investigated the possible physiological relevance of CB-CFTR and CB-GLIC interactions, and diverse electrophysiological studies enabled us to discover new functions of crotoxin. The talk will focus on the description of these new targets and new functions of crotoxin. Faure G., Xu H., Saul, F.A., 2011. Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric b-neurotoxin. J. Mol. Biol. 412, 176e191. STUDY OF THE INVOLVEMENT OF ITCH MEDIATORS IN THE NEUROPEPTIDE RELEASE INDUCED BY PACIFIC-CIGUATOXIN-2 IN A CO-CULTURE MODEL OF SENSORY NEURONS AND KERATINOCYTES K. L’herondelle a, *, R. Philippe b, O. Mignen b, I. Vetter c, R.J. Lewis c, L. Misery a, d, R. Le Garrec a. a Laboratoire de Neurosciences de Brest, Universit e de Bretagne Occidentale, 22 avenue Camille Desmoulins, 29200 e Bretagne Occidentale, 46 rue Brest, France; b INSERM U1078, Universit Felix Le Dantec, 29218 Brest, France; c Institute for Molecular Bioscience, epartement de The University of Queensland, St Lucia, Australia; d D Dermatologie, Centre Hospitalier R egional Universitaire de Brest, 2 avenue Foch, 29609 Brest, France
* Corresponding author.
73
Ciguatera Fish Poisoning (CFP), which is called “La Gratte” (the itch) in the Pacific islands, is caused by eating fish contaminated with ciguatoxins (CTXs). The most characteristic clinical signs are cutaneous sensory disorders such as cold-induced dysesthesia, paresthesia and an intense pruritus. CFP-associated itch, which can persist for several weeks, is poorly or not relieved by antihistamine drugs. At the cellular and molecular levels, it is recognized that CTXs bind to voltage-gated sodium channels (VGSCs) leading to Na+ entry and increased excitability with various cellular, including secretory, consequences. Recently, it was shown that the cold allodynia induced by CTX in mice is VGSC-dependent and involves TRPA1, and that sensory neurons which respond to CTX express Calcitonin GeneRelated Peptide (CGRP). However, the molecular basis of the mechanisms underlying CTX-induced itch remain unclear. Pruritus is the unpleasant sensation that leads to the need to scratch. It can seriously impair quality of life, especially when it is chronic. Whatever its origin is, its pathophysiology is complex and still incompletely known, despite recent advances. Whereas the best-known pruritogen in humans is histamine, many types of itch do not respond to anti-H1 drugs. Other substances released from skin cells, such as substance P (SP) and CGRP, have been shown to mediate or facilitate pruritus. Furthermore, substantial evidence indicate the involvement of emerging receptors expressed by various skin cells, including some TRP (Transient Receptor Potential) channels and some Protease-Activated Receptors (PARs). The purpose of this work was to study the release of the neuropeptides substance P (SP) and CGRP in a coculture of human keratinocytes and rat DRG neurons exposed to PacificCiguatoxin-2 (P-CTX-2). Neuropeptides were quantified in the culture supernatant by ELISA. For the first time, we showed that low nanomolar concentrations of P-CTX-2 cause a large release of SP and CGRP in this model. In addition, calcium imaging experiments on cultured keratinocytes alone showed an increase of intracellular Ca2+. Pharmacological tools were used to study the involvement of some TRP channels and of PAR-2 receptor in these effects of the P-CTX-2. Up to now, CFP-associated pruritus has never been the subject of any study. This work is not only an original way to better understand the pathophysiology of itch but it will also supplement the knowledge of the molecular and cellular mechanisms of CFP. The long-term aim of this study is to identify potential targets for treating sensory disorders of ciguatera. ANIMAL PRESYNAPTIC NEUROTOXINS PROVIDE A RELEVANT NOVEL MODEL OF MOTO AXON TERMINAL DEGENERATION FOLLOWED BY REGENERATION C. Montecucco a, b, *, E. Duregotti a, S. Negro a, M. Scorzeto a, I. Zornetta a, B.C. Dickinson c, C.J. Chang c, M. Rigoni a,b. a Department of Biomedical Sciences, University of Padua, Padua 35131, Italy; b CNR Institute of Neuroscience, Padua 35131, Italy; c Department of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA Several animal presynaptic neurotoxins cause an acute motor axon terminal degeneration with a botulism-like flaccid paralysis. This is followed by complete regeneration within few days in mice and 4-6 weeks in humans. Mice injected with these neurotoxins in different muscles provide an appropriate and controlled system to dissect the molecular mechanisms underlying degeneration and regeneration of peripheral nerve terminals. We have previously shown that the toxin induced neurodegeneration is caused by calcium overload and mitochondrial failure. Here we show that toxin-treated primary neurons and neuromuscular junctions release danger signaling molecules derived from mitochondria: hydrogen peroxide, mito-DNA and cytochrome c. These molecules activate isolated primary Schwann cells, Schwann cells co-cultured with neurons and at neuromuscular junction in vivo, through the ERK1/2 pathway. We propose that these inter- and intra-cellular signaling events are involved in triggering the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases.
* Corresponding author.