Beyond the Connectome: Unravelling Neuropeptide Signalling in Parasitic Nematodes to Inform Drug Discovery Pipelines

Nematodes, or roundworms, are highly successful invertebrates. There are >25,000 species which can be either free-living or parasites of humans, animals and plants, acting as a common source of disease. Human parasites are particularly prevalent in less economically developed countries where poverty, inadequate healthcare provision, and poor living conditions are commonplace and favour nematode persistence. Many human parasitic nematodes live in the gastrointestinal system, lymphatic system, or body tissues, where they cause serious health problems to the host. Infections in children impact on their physical and intellectual development, while nematode disease in adults can result in an inability to work and provide for their families. Globally, nematode infections are also common in agricultural livestock negatively impacting animal productivity (including meat and milk production) and the subsequent economic sustainability of the UK agri-food industry.

Unfortunately, the drugs currently available to treat parasitic nematode infections (anthelmintics) no longer work effectively. Indeed, anthelmintic resistance in nematode parasites is a major local and global problem. In some areas of the world (for example, Scotland and New Zealand), sheep and cattle cannot be farmed due to nematode parasite control problems. Therefore, the development of new drugs to treat such resistant nematode parasites is urgently needed both in the UK and globally. New drugs are critical to the long-term sustainability of livestock farming for future food production.

The nematode nerve-muscle system (neuromuscular system) is a proven source of drug targets. Indeed, the majority of anthelmintic drugs used to control nematode infectionhave exerted their effects on this system. The neuromuscular system of nematodes coordinates behaviour, controlling vital processes such as movement, feeding and reproduction. If these processes can be effectively interrupted then nematode parasite survival and transmission will be significantly reduced. The neuromuscular system continues to receive attention from academics and pharmaceutical companies searching for new drug targets. Indeed the neuromuscular system in nematodes is currently underexploited as a drug target source, with many novel resistance-breaking targets awaiting discovery. In order to develop new drugs that target the neuromuscular system of parasites we need a better understanding of the nematode nervous system structure and function.

The nematode nervous system is complex and our current knowledge on its structure is based on a free-living model nematode called Caenorhabditis elegans. We have a detailed map of every single nerve cell (neuron) in C. elegans and all of the connections between them. We call this the connectome. However, we do not know anything about the communication that can occur outside of the connectome, via the fluid-filled body cavity. This is important as signalling beyond the connectome (extrasynaptic signalling via the body cavity fluid) is believed to be a key part of the communication system in nematodes, and yet we know very little about it. A more comprehensive understanding of nematode extrasynaptic signalling will enable us to better exploit the neuromuscular system as a drug target resource.

This project uses a variety of sophisticated technologies and tools to investigate the extent and significance of extrasynaptic signalling in nematodes, by exploiting the large gastrointestinal nematode parasite, Ascaris suum, as a model system. The size of Ascaris allows us to easily collect its body cavity fluid and analyse the signalling molecules it contains - this has not been done before and would be very difficult in other nematode species. The information generated from this project will help us to better understand nematode biology and will provide valuable data for drug discovery by the pharmaceutical industry.

Neural circuit synaptic connections provide the anatomical foundation for our understanding of nematode nervous system function. However, other non-synaptic routes of communication are known in invertebrates, including extrasynaptic volume transmission (EVT). Indeed, in Caenorhabditis elegans wired connections do not facilitate many of the receptor-ligand interactions that have been functionally validated, highlighting the potential for alternative communication routes via wireless mechanisms. EVT pathways in nematodes are underappreciated and would add an additional level of complexity to our current understanding of nematode communication beyond the connectome. Ascaris suum, a gastrointestinal parasite of pigs, offers a unique opportunity to probe EVT in nematodes due to its large size and experimental tractability. Significantly, nematode parasites impact global health and food security which costs the UK agri-food industry ~£80 million/year. Key issues to the sustained control of nematode pathogens include the inability to effectively control nematodiasis due to multi-drug resistance and a lack of novel therapeutic avenues in development. Neuromuscular signalling is an a priori drug target, however our ability to exploit this system would be enhanced by a better understanding of the EVT component governing neuromuscular function in nematodes. Here we will harness recent developments in omics technologies to provide the first direct experimental evidence supporting extrasynaptic (wireless) routes of neurotransmission in nematodes. Our inter-disciplinary academic and industrial alliance offers a unique opportunity to interface LC-MS/genomic/transcriptomic and nematode behaviour expertise with industrial perspectives and will determine, for the first time, the role and significance of EVT in nematode biology. The data generated will inform fundamental biology that directly underpins drug discovery programs for nematode pathogens.

COMMERCIAL PRIVATE SECTOR industries, the PUBLIC SECTOR, and POLICY MAKERS will benefit directly from this research.

Beneficiaries within the COMMERCIAL PRIVATE SECTOR include local and global pharmaceutical companies and agricultural/farming enterprises. This research to interrogate extrasynaptic volume transmission pathways in nematodes is proposed in collaboration with a world leading research-driven pharmaceutical company (Boehringer Ingelheim; BI) who provide added-value through innovation and the provision of a pathway for translation of basic biology into end-user impact. In the short-term this collaboration will engage BI personnel in cutting-edge academic research activities that will shape the BI-Parasitology division business and encourage the recruitment of additional staff as they enhance their parasite R&D portfolio. In the longer-term, the development of novel drugs, directed against targets emerging from this research, to combat parasite disease will generate significant revenue for BI that will enable substantial reinvestment. In addition, drugs emerging from BI development pipelines will positively impact local and global farming enterprises who will ultimately benefit through enhanced economic returns associated with parasite-free livestock. This is significant given that nematode parasites are estimated to cost farmers $80 million / year. Resultant productivity will drive economic success in all UK Agri-Food related businesses involved in the 'farm to fork' production process.

Within the PUBLIC SECTOR impact will be realised through multiple channels, reaching beyond global enterprises and SMEs, to benefit the individual small-scale farmer and local farming communities in both developed and less economically developed countries. Nematode parasites have a devastating impact on global agriculture where many farmers and their families rely on livestock for income and food. Further, they infect >1/4 of the world's population causing serious morbidity that reduce the quality of life and economic gain. From a 'One Health' perspective, the development of novel drugs to combat nematodiasis in livestock, will also improve the quality of life of those afflicted with human parasites. Education will form an important channel for the delivery of impact from this research activity. By extending our engagement with education providers at all levels (primary, secondary, tertiary) we will improve the reach of this research to (i) enhance scientific knowledge amongst students and teachers, (ii) inspire the next generation of STEM subject scientists, (iii) raise awareness about the importance of basic scientific research to our society, and (iv) instil an appreciation of social/ethical issues surrounding the impact of parasitism. Our research findings will form the basis of research-led teaching to QUB undergraduate and postgraduate students. Our lines of communication with the general public will facilitate a greater appreciation of the role and benefits of non-clinical research in our society. In addition to enhancing knowledge, the general public will also benefit through the availability of animal produce derived from livestock with reduced exposure to ineffective drugs. This feeds directly into consumer-led demand for organic and chemical residue-free food that is produced from animals maintained in a welfare-friendly environment. A novel, resistance-free anthelmintic will facilitate a reduction in drug use, limiting food contamination and facilitating the organic farmer who is restricted to reduced treatment regimes.

POLICY MAKERS and stakeholders will benefit through research findings that will directly inform policy development. UK-based government bodies (DEFRA/DAERA), Levy boards (EBLEX, BPEX), and other representatives of the Agri-Food industry will benefit as this project will provide an evidence-base for policy development and informs EU-directed changes in agricultural legislation.