Evolution and functional biology of neuropeptide signalling: from genomes to behaviour

Since arthropods have been proposed to share a common crustacean-like ancestor, it is perhaps unsurprising that many neuropeptides, neural networks and signalling pathways involved in the most fundamentally important process in arthropod life history, namely moulting, seem to be common to both insects and crustaceans. Nevertheless, despite the conservation of core mechanisms and networks involved in the act of ecdysis itself- the ‘ecdysis cascade’, that involve hormones such a crustacean cardioactive peptide and bursicon, a somewhat bewildering plethora of other neuropeptides and their cognate receptors exist. These have been revealed by transcriptome mining of insects and de novo assembly of crustacean transcriptomes, including our experimental model, the green shore crab, Carcinus maenas. Many of the peptides and receptors proposed to be involved in ecdysis have undergone neofunctionalisation or gain/loss during evolution. In particular, we still know rather little regarding the identity, distribution and function of crustacean neuropeptide receptors. Here, Professor Webster reviews the current research, highlighting the neuropeptides and receptor signalling that have been identified as involved in crustacean ecdysis, and possible novel roles for neuropeptides first identified in insects.

Sleep is among the most persistent and perplexing mysteries in biology. However, despite the fact that we devote a third of our lives to sleep, the evolutionary conservation of sleep-like states and the pervasiveness of sleep disorders, mechanisms that regulate sleep remain poorly understood. Sleep has primarily been studied using mammals, where electrophysiology can be used to define sleep and wake states. However, progress has been challenging, due in part to the complexity of mammalian brains and the poor amenability of mammals for large-scale screens. To overcome these limitations, invertebrate sleep models have been established and used to identify neuropeptidergic systems that regulate sleep-like behaviours. However, the utility of these models for understanding mammalian sleep may ultimately be limited because invertebrate brains bear little anatomical or molecular resemblance to mammalian brains, and many of the genes identified in invertebrates lack obvious vertebrate orthologues. Conversely, most neuropeptides implicated in regulating mammalian sleep lack clear invertebrate orthologues. As an alternative approach, Dr Prober’s lab is using the zebrafish, a diurnal vertebrate that combines advantages of mammalian and invertebrate models, to explore genetic and neuronal mechanisms that regulate sleep. Dr Prober will describe our use of genetic screens and candidate-gene based approaches to identify neuropeptidergic systems that regulate sleep in zebrafish, including systems that appear to be conserved with invertebrates.

Insects are exposed to a continuous multiple stressors across a range of environments. Desiccation tolerance and survival is dependent on fluid homeostasis by fluid transporting epithelia including the Malpighian tubules. Fluid transport by insect Malpighian tubules is modulated by diuretic neuropeptides which have also recently been shown to affect desiccation and starvation tolerance in the insect genetic model Drosophila melanogaster.  

However, in addition to this, a novel role for Malpighian tubules in cold tolerance has been recently demonstrated, which occurs via homeostatic control of water and ion balance and is modulated by the capa neuropeptide.  Altogether, this current evidence suggests that at least three diuretic neuropeptides (capa, DH44, kinin) perform functions in environmental stress tolerance via the Malpighian tubules.

Thus, Malpighian tubules, and diuretic neuropeptides, have much wider implications for physiology and behaviour beyond osmoregulation. 

The immune system and nervous system interact in ways that are only just beginning to be elucidated. Emerging data point to the inflammatory response being a contributor to depression and schizophrenia. Interleukin-17 (IL-17) is a major pro-inflammatory cytokine: it mediates responses to pathogens or tissue damage, and drives autoimmune diseases. Little is known about its role in the nervous system. Dr de Bono will describe experiments showing that IL-17 has neuromodulator-like properties in Caenorhabditis elegans. IL-17 can act directly on neurons to alter their response properties and contribution to behaviour. Using unbiased genetic screens, the group delineates an IL-17 signalling pathway. They show that IL-17 receptors and downstream signalling components are expressed widely in the nervous system. Dr de Bono will show how IL-17 signalling changes the properties of a tonic circuit that enables C. elegans to presistently escape 21% oxygen (O2). In this animal 21% O2 serves as a toxin of surface exposure and reprograms global animal state. IL-17 alters the properties of the RMG hub interneurons, which are connected to sensory neurons that detect O2, pheromones, noxious cues and mechanical cues. Disrupting IL-17 signalling reduces RMG responsiveness to input from O2 and pheromone sensors, and renders sustained escape from 21% O2 transient and contingent on additional stimuli. Conversely, overactivating IL-17 receptors abnormally heightens responses to 21% O2 in RMG neurons and whole animals. IL-17 deficiency can be bypassed by optogenetic stimulation of RMG. Inducing IL-17 expression in adults can rescue mutant defects within 6 h. These findings reveal a non-immunological role of IL-17 modulating circuit function and behaviour.

Animals have to make decisions many times per day. These decisions include decisions about where and how to find optimal food sources while avoiding toxic foods or predators. The decisions and behaviours, however, need to be flexible to meet individual needs and particular circumstances. For instance, metabolic states largely influence an animal’s interest in food in general, but also for specific nutrients. How gravid females identify foods to match their specific dietary needs remains elusive. Among these needs are pungent smelling polyamines, such as putrescine and spermidine, which are essential for cell proliferation, reproduction, and embryonic development in all animals. Using Drosophila, the group have identified a behavioural, neuronal, and genetic mechanism that adapts the senses of smell and taste, the major modalities for food quality, to the physiological needs of a gravid female. The group shows that female flies seek fermenting fruit, which are rich in polyamines, through specific olfactory and gustatory ionotropic receptors (IRs) and bitter taste neurons. Polyamine attraction is enhanced in a sex-specific manner in gravid females through a G-protein coupled receptor and its neuropeptide ligands that act directly in the polyamine-detecting chemosensory neurons. Together, these data show that neuropeptide-mediated modulation of chemosensory neurons increases pregnant females’ preference for important nutrients to ensure optimal conditions for her growing progeny. 

All nervous systems employ a large number of amines, amino acids and neuropeptides as neurotransmitters and neuromodulators. Comprehensive characterisation of all signalling molecules in a nervous system with chemical, spatial and temporal information is often critical to deciphering the functionality of a neural circuit yet it presents a daunting challenge. The group has chosen to work with a simpler and well-defined crustacean nervous system to both facilitate technology development and address fundamental neuroscience problems related to neuromodulation and network plasticity. In this talk, Professor Li will present the progress on the development of a multi-faceted mass spectrometry-based analytical platform to probe neuronal signalling with enhanced sensitivity and selectivity. By combining chemical labelling, micro-scale separation, and tandem mass spectrometry sequencing techniques, the group has discovered a large number of novel neuropeptides in crustacean nervous systems. Moreover, both mass spectrometric imaging technology and in vivo microdialysis sampling tools have been developed and implemented to follow neuropeptide spatial distribution, dynamic secretion, and sequential degradation with unprecedented details. Furthermore, isotopic and novel tandem mass tagging reagents based on dimethylated amino acids have been developed and employed to produce differential display of neuropeptidomes under different physiological conditions. Examples of neuropeptide regulation of feeding behaviour, environmental stress, and neural network development will be highlighted. Collectively, these combined studies will help elucidate the functional roles that neuropeptides play in regulating neural plasticity as well as the functional consequences of neuropeptide diversity and multiplicity.

Neurons within the mouse brain are often categorised by the patterns of expression of neuropeptides and their receptors. Here the group described the function of neuropeptides in regulating synapse and circuit function in two parts of the mouse brain. First, within striatum Enkephalin is produced by neurons of the indirect pathway and detected by neurons of multiple classes that express mu and delta opioid receptors. The group finds that Enk, acting primarily via delta receptors, inhibits GABAergic synapses that connect the indirect and direct pathway striatal projection neurons to facilitate the latter pro-kinetic pathway. This effect is limited to synapses found within the ‘patch/striosome’ sub-compartment of the striatum. Second, the group discovers an intracortical system by which a peptide made by cortical pyramidal cells acts on cortical Vip interneurons to disinhibit cortical activity and promote cortical plasticity. In summary, this work demonstrates multiple pathways by which peptide based signalling regulates the efficacy of traditional fast-neurotransmitter based intra-cellular signalling.

Opioid receptors (OR), members of the G protein-coupled receptor (GPCR) superfamily, constitute the major and the most effective target for the treatment of pain. µ-opioid receptors (µOR) are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone.

The recent structures of the µOR in inactive and agonist-induced active states provide snapshots of the receptor at the beginning and end of a signalling event. The group used solution-state NMR to examine the process of µOR activation. These results suggest a weak allosteric coupling between the agonist-binding pocket and G-protein-coupling interface (transmembrane TM 5 and 6). Furthermore, the analysis provides clues on the successive structural events leading to the full active conformation of µOR. 

However, much remains to be learned about the mechanisms by which different agonists can induce distinct levels of G-protein activation and/or arrestin recruitment upon activation of µOR. In this study, the group are investigating the conformational landscape of the µOR in distinct pharmacological conditions using liquid-state NMR spectroscopy and advanced pharmacology signalling characterisation. In particular, the group have developed a double labelling scheme to monitor signals from distinct methyl probes in specifically labelled µOR domains. And In combination, the group assesses the potential for biased agonism to activate different cell signalling pathways, such as G-proteins activation, β-arrestins recruitment and µOR trafficking.

The goal is to provide a mechanistic understanding of opioid receptor activation upon binding of ligands presenting distinct efficacy and/or biased signalling properties. A better knowledge of the structural basis for opioid drug efficacy may lead to new therapeutic approaches with limited side effects.

Chemical synapses use small molecule transmitters and neuropeptides for transmission. It is textbook knowledge that only small molecule neurotransmitters directly gate ion channels, mediating fast neurotransmission, whereas neuropeptides activate exclusively G-protein coupled receptors, mediating slow neurotransmission. The first exception to this rule was the FMRFamide-activated Na+ channel (FaNaC), which is directly activated by the evolutionary old neuropeptide FMRFamide. So far, FaNaC has been cloned exclusively from different molluscs, which belong to the clade of protostomes (Bilateria). A sister group to Bilateria are Cnidaria, animals with a simple body plan. The group used a model Cnidarian, the freshwater polyp Hydra, to clone and functionally characterise ion channels that are related to FaNaC and directly gated by neuropeptides, the Hydra Na+ channels (HyNaCs). 

The Hydra genome contains genes encoding 12 HyNaCs that assemble into different heterotrimeric ion channels, which are directly activated by RFamide-neuropeptides of the Hydra nervous system. HyNaCs are expressed by epitheliomuscular cells at the base of the tentacles or the foot region, suggesting that the simple nervous system of Hydra extensively uses neuropeptides for fast neurotransmission, perhaps for neuromuscular transmission. The presence of related peptide-gated channels in Cnidaria and Bilateria suggests that peptide-gated channels have a deep evolutionary origin. Recently, the group cloned another peptide-gated channel, closely related to FaNaC, from the marine annelid worm Platynereis, which is directly activated by a neuropeptide that is not a RFamide. These results suggest that peptide-gated channels may be present in many animals and activated by a range of chemically diverse neuropeptides. 

Traditional approaches to the discovery, localisation and functional characterisation of nematode neuropeptides have included immunocytochemical techniques, classical biochemical characterisation methods, PCR-based gene detection tools, and muscle-based physiology assays. The data generated highlighted the importance of neuropeptides to nematode biology, and flagged the candidature of the neuropeptidergic system as a putative anthelmintic target. More recently, these datasets have been enhanced by the omic-analyses of nematode genome, transcriptome and peptidome datasets enabling the identification and prioritisation of neuropeptides and their receptors that exhibit therapeutic appeal, but are not yet validated. Unfortunately, the development of functional biology tools in nematode parasites has not kept pace. Indeed, a key hurdle to the exploitation of putative targets is the absence of tools that allow the elucidation of target function in therapeutically-relevant nematode pathogens. Broadly, reverse genetics is being applied to probe the biology of many organisms through sophisticated methods of transgenesis, gene silencing (RNA interference), and genome editing (CRISPR/Cas9 technology). The application of these experimental tools to nematode parasites has been eagerly awaited; however their translation has either been difficult or is in early stages of development, such that their impact on novel drug discovery for the control of nematode pathogens is yet to be realised. This presentation provides an overview of Functional Genomics Platforms that are currently available for use in parasitic nematodes, and describes progress in their application to the understanding of the neuropeptidergic system.