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FRIMEDBIO-Fri prosj.st. med.,helse,biol

Development of fully functional, species-specific nicotinic acetylcholine receptor models from arthropods

Alternative title: Utvikling av funksjonelle, artsspesifikke modeller for nikotinerge acetylcholinreseptorer i artropoder

Awarded: NOK 12.0 mill.

Electrical signals are transmitted from cell to cell by means of transmitting substances that are released from one cell and activate special receptors on the next cell. A very important neurotransmitter is acetylcholine, and the corresponding receptors are called acetylcholine receptors. In an organism, there are several variants of these receptors, including some that are activated by nicotine, and thus have been named nicotinic acetylcholine receptors (nAChR). The nAChRs are composed of five different proteins, and different combinations of the same or different proteins can give rise to receptors with slightly different properties. One of the most widely used groups of insecticides in the world are the neonicotinoids. These activate the receptors in the same way as nicotine and acetylcholine, but with a longer duration of effect. This group of insecticides are of significant concern because they can pose adverse effects on beneficial insects, e.g. bees. They are directly toxic, but can in lower doses have other undesirable effects, such as impacting their ability to navigate. One of the neonicotinoids was also recently marketed against salmon lice, which is a small crustacean and a parasite on salmonid fish. It has been difficult to study the effects of neonicotinoids at the receptor level in insects and other arthropods because no one has previously been able to express a complete receptor in an experimental model system. However, our group recently managed this, by cloning the necessary genes from the salmon louse into immature frog eggs (oocytes), and then precisely measuring the properties of the receptors. It was shown that there are different combinations of proteins that form acetylcholine-sensitive receptors in the salmon louse, and that they respond differently to neonicotinoids. This project builds on these results. Although this type of receptor has significant similarities between different arthropods, they are not identical. Therefore, the receptor must be expressed based on genes for each species to be studied. The goal now is to assemble functional receptors from two insects (bees and mosquitoes), as well as the arachnids spider mites and castor bean ticks. In addition, the effect of activation with acetylcholine, nicotine and neonicotinoids will be examined. So far, more than 50 genes coding for nAChR proteins have been identified from the various arthropod species. Different protein combinations are being tested in the experimental model system to identify what gives a response after activation with acetylcholine, and new combinations have already shown activity. When all combinations from the various species have been tested, the effects of neonicotinoids will be mapped and compared. Since we have progressed a little further with the receptors from salmon lice, it is a goal to determine the exact spatial location (stoichiometry) of the separate proteins that the salmon lice receptors are composed of. This work consists of computer modelling, followed by experimental tests in the model system. We hope this work will pave the way for a simpler determination of the spatial location in the other species as well.

Nicotinic acetylcholine receptors (nAChRs) mediate chemical neurotransmission in the nervous system and at neuromuscular junctions in vertebrates. The natural transmitter is acetylcholine, but they are also the direct targets for neonicotinoids, some of the worlds largest selling insecticides. These chemicals are widely used against ectoparasites, arthropod vectors and agricultural pests. There are significant concerns regarding adverse effects of neonicotinoids on beneficial insects, and some neonicotinoids are now banned for out-door use in many countries. In-depth studies on the function of these receptors in arthropods are surprisingly scarce. Until 2020, no research groups had managed to express functional nAChRs in an ex-vivo system, using genes coding for both a- and ß-subunits from the target arthropod species. Co-expression of nAChR a-subunits from insects and ß-subunits from vertebrates (mainly chicken) had been the only feasible way of obtaining functional receptors with both subunit types in Xenopus laevis oocytes. In 2020, two groups independently overcame this hurdle, one being the applicant here. We managed to express two nAChRs from the marine arthropod Lepeophtheirus salmonis into Xenopus laevis oocytes using genes from this species only. In this project, these results will be used to attempt cloning of nAChRs from other arthropods like bees, mites, ticks, and mosquitos and test their sensitivity towards natural transmitters, antiparasitic agents, pesticides and other compounds. Thus, the present project aims to lead to a greatly improved way of obtaining nAChRs from arthropods, with a switch from arthropod-vertebrate chimeric nAChRs to arthropod-specific native nAChRs. These nAChRs will further serve as tools for fundamental research on the function of the arthropod nervous system and serve as elegant models to determine the effect of various existing and novel compounds on these receptors from parasites, pests, and non-target organisms.

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FRIMEDBIO-Fri prosj.st. med.,helse,biol

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