Identification and characterization of the mode of action of neurotoxic phycotoxins with biological risk - Development of new sensitive detection systems. – AquaNeuroTox

This project has benefitted from the complementary scientific and technical expertise of the two participating teams (those of CNRS and CEA) in Gif-sur-Yvette. The originality of the approach used is based on a unique combination of know-how and techniques, within the consortium, combining in vitro and in vivo functional assessment approaches, through the use of conventional electrophysiology at cell and integrated levels, to toxin-receptor binding studies using selective radioligands (according to the principle of displacement of the binding between a toxin and a receptor by a ligand), to spectrofluorometry techniques, and to expression of receptors (human nicotinic and muscarinic) in heterologous systems (HEK-293 and CHO cultured cell models, Xenopus oocytes) or micro-transplantation of purified Torpedo membranes allowing the incorporation of nicotinic receptors in their native environment (with associated proteins) in Xenopus oocyte. These techniques were applied to the various biological models that we use which are complementary to each other.

The main results obtained in this project are: Confirmation of the presence of anatoxin-a in some isolates obtained when collecting biofilms of freshwater cyanobacteria. Lack of evidence for the presence of the stxA gene encoding saxitoxin in the Tarn river. Determination of selectivity profiles and mechanisms of action of some cyclic imines interacting with nicotinic and muscarinic receptors. Characterization of ionic mechanism involved in the increased in nodal volume of myelinated axons during activation of sodium channels by ciguatoxin-1B. Development of a neurotoxin detection test targeting sodium channels and nicotinic receptors using new non-radioactive tracers and spectrofluorometry. Design and production of strips, a new portable, sensitive and low cost test to detect the neurotoxins, which are present intentionally or accidentally in the environment, acting on nicotinic acetylcholine receptors.

1. To pursue the identification of cyanobacteria present in the waters of the Tarn river on the samples already available and on new samples that will be collected soon. In particular, where the biomass has been processed, the different cyanobacteria isolates will be cultured. The DNA will be extracted from each sample and analyzed by PCR and sequencing using primers specific for gene of anatoxin-a, cylindrospermopsin and saxitoxin.
2. To obtain structural data on the interaction of various shellfish toxins with nicotinic receptors to understand the origin of their selectivity of interaction (collaborations P. Marchot / Y. Bourne, CNRS Marseille).
3. Pharmacological characterization of new toxins (prorocentrolides, ptériatoxines ...) obtained by collaboration.
4. Development of new RBA detection systems targeting toxins active on sodium channels. Synthesis and labeling (biotynylation, fluorescent) of conotoxins with broad interaction with these ion channels and development of the detection system on cell lines expressing different NaV channel subtypes.
5. Optimization of the microwell RBA assays for nicotinic toxins (with new fluorescent toxin) and adaptation to ground by developing a new strip system.

From the start of this research project 7 articles have been already published in international peer-review journals, 2 articles of vulgarization in the framework of bioterrorism, and 1 article is going to be submitted. In addition, 5 book chapters (by invitation) have been published. A patent application PCT / EP2015 / 081060 with the European Patent Office was filed on 22.12.2015 by the CNRS in The Hague. Title of the invention: «Device for Detecting neurotoxins and process for manufacture thereof«.

1. Une Déclaration d’invention (DI07822-01) a été déposée le 18.03/2015 au CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE-CNRS : « NEUROTORP – Rapid Lateral Flow Tests Strips » Inventeurs : ARAOZ R., MOLGO J., SERVENT D., MOURIER G. et KESSLER P.

2. STIVALA C.E., BENOIT E., ARÁOZ R., SERVENT D., NOVIKOV A., MOLGÓ J., ZAKARIAN A. (2015). Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins. Nat. Prod. Rep., 32: 411-435

3. BOURNE Y., SULZENBACHER G., RADIC Z., ARÁOZ R., REYNAUD M., BENOIT E., ZAKARIAN A., SERVENT D., MOLGÓ J., TAYLOR P., MARCHOT P. (2015). Marine macrocyclic imines, pinnatoxins A and G: structural determinants and functional properties to distinguish …. Structure, 23: 1106-1115

4. ARAOZ R., OUANOUNOU G., IORGA B.I., GOUDET A., ALILI A., AMAR M., BENOIT E., MOLGÓ J., SERVENT D. (2015). The neurotoxic effect of 13,19-didesmethyl and 13-desmethyl spirolide C phycotoxins is mainly mediated by …. TOXICOL. SCI., 147: 156-167

5. MATTEI C., MOLGÓ J., BENOIT E. (2014). Involvement of both sodium influx and potassium efflux in ciguatoxin-induced nodal swelling of frog myelinated axons. Neuropharmacology, 85: 417-426

6. RUBIO F., KAMP L., CARPINO J., FALTIN E., LOFTIN K., MOLGÓ J., ARÁOZ R. (2014). Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors. Toxicon, 91: 45-56

Marine phytoplankton (dinoflagellates/diatoms) and freshwater cyanobacteria produce potent neurotoxins that target essential components of neurotransmission vital to life, such as voltage-gated sodium channels, nicotinic acetylcholine receptors (nAChR) and cholinesterases. In marine environments, Harmful Algal Blooms (HABs) can total hundreds of millions of dollars losses annually for aquaculture industries, and represent a threat to public health. Since shellfish can accumulate bioactive molecules through filter-feeding on toxic phytoplankton. Human poisoning typically results from consumption of contaminated shellfish giving rise to multiple syndromes such as paralytic (saxitoxin), amnesic (domoic acid), neurotoxic (brevetoxins), diarrheic (okadaic acid and dinophysistoxins) and azaspiracid shellfish poisoning. In continental waters, acute exposure to harmful cyanobacterial blooms containing anatoxin-a- or saxitoxin-producing-cyanobacteria, has been shown to be fatal for wild, stock and domestic animals as well as for humans. We recently determined the mechanism of action of gymnodimine-A, 13-desmethyl spirolide-C, and pinnatoxin-A characterizing these spirocyclic imines as the first family of highly potent phycotoxins targeting nAChRs. It is necessary to develop functional methods for Seafood Industry and freshwater supplies to detect cholinergic toxins. Therefore, we have developed a sensitive microplate-Receptor Binding Assay (microplate-RBA) using Torpedo membranes enriched in nAChRs for the detection of gymnodimines, spirolides, pinnatoxins and anatoxin-a in contaminated seafood and water samples. Furthermore, toxic producing microorganisms can be isolated from natural environments and could be easily cultured constituting a potential danger for bioterrorism purposes when aquaculture/ freshwater supplies are targeted. The objectives of this Research Program are to: i) collect and identify new cyanobacterial toxic agents, ii) determine the pharmacology of spirolides that have not been previously studied (20 methyl spirolide G) and pinnatoxins congeners, and prorocentrolides, possessing a cyclic imine group and all the newly identified toxins, iii) develop novel toxin-receptor systems for the detection of toxins targeting voltage gated sodium channels and other targets, iv) adapt the microplate-RBA for field application purposes by developing microsystems based on the ability of Torpedo membranes to coat plastic surfaces and nanofluidics for toxin-receptor interactions.