Deciphering neuropeptide signalling via mass spectrometry-based peptidomic approaches: from discovery to function
Professor Lingjun Li, University of Wisconsin-Madison, USA
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.
Regulation of synaptic transmission by peptides in cortex and striatum
Professor Bernardo L. Sabatini, Harvard Medical School, USA
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.
Structural biology of opioid receptors
Dr Sébastien Granier, INSERM-Institut de Génomique Fonctionnelle Montpellier, France
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.
Alternative receptors for neuropeptides - ion channels!
Professor Stefan Gründer, Institute of Physiology, RWTH Aachen University, Germany
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.
The development of Functional Genomics Platforms for nematode pathogens: informing the biology of neuropeptides and their receptors
Dr Angela Mousley, Queen's University Belfast, UK
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.
Discussion of day 2 and closing remarks
Professor Maurice Elphick, Queen Mary University of London, UK