Understanding spatiotemporal GPCR signalling in astrocytes using designer receptors and microfluidic technology

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

Astrocytes are multifunctional CNS glial cells, regulating neurotransmitter uptake and metabolic partitioning, actively signalling to neurons and vasculature via gliotransmission and undergoing phenotypic changes (reactive gliosis) in response to injury. The extensive astrocyte G protein coupled receptor (GPCR) repertoire has been implicated in neuron-astrocyte communication, metabolic control and the transition to a reactive state. Targeting astrocytic GPCRs is therefore a feasible approach to inhibit gliosis and other forms of astrocyte dysfunction during pathogenesis of many neurodegenerative diseases. However GPCR signalling is increasingly complex, involving ordered recruitment of G protein, arrestin and other effectors to determine particular functional outcomes. Crucially these mechanisms depend on temporal organisation conferred by kinetics of the stimulus, its nature (e.g. orthosteric versus allosteric ligand) and the specific signalling properties of the receptor subtype targeted. Thus we need approaches to isolate astrocytic signalling in response to particular ligand-receptor combinations - from a backdrop of many receptors for a particular neurotransmitter. Second, we should study such behaviours following delivery of agonists and other ligands in a precisely controlled manner - for example to reflect of the kinetics of physiological stimuli rather than traditional equilibrium measurements.

This project will make use of two novel technologies to address these aims. It will first employ designer receptors (DREADDs) based on the Gq coupled muscarinic receptor family (MR), selectively activated by a synthetic ligand (clozapine-N-oxide), rather than the endogenous neurotransmitter acetylcholine. The student will characterise the pharmacology of these receptors to MR agonists, antagonists and allosteric modulators using model cell lines (transfected CHO cells) and routine signalling assays. In addition, using novel MR fluorescent ligands, ligand binding kinetics will be investigated by establishment of TR-FRET competition association methods. These studies will be complemented by single cell calcium imaging studies in DREADD transfected astrocytes to relate the kinetic properties of the ligand (e.g. its dissociation rate) to the resultant calcium response profile. In parallel, the student will develop our in house microfluidics technology to deliver controlled patterns of DREADD ligand stimulus to transfected CHO and astrocyte cell cultures - monitoring the calcium response profile or fluorescent ligand binding as the concentration of agonist and competing drug (antagonist, allosteric modulator) is varied in real time with microsecond precision. These twin strands will combine to provide an increased understanding of how the dynamics of ligand-GPCR interaction drive spatiotemporal signalling in both model cell systems and astrocytes. It will also demonstrate the central role of kinetic properties of muscarinic ligands, under consideration as CNS therapeutics, as determinants of their behaviour in a dynamic neurotransmitter environment.

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M008770/1 01/10/2015 30/09/2023
1644276 Studentship BB/M008770/1 01/10/2015 30/09/2019 Jessica Carpenter
 
Description I have now characterised the designer receptor (DREADD) of muscarinic M1. DREADD receptors have been altered to stop the normal ligand binding (ACh) and rather a bioloigcally inert substance (CNO) now bindings. This allows for a system in which there is less non-specific binding and when the cell reacts to CNO, we know that the signalling must only be through the M1 pathway. I have also tested allosteric modulators on this system. Allosteric modulators are drugs which do not bind at the site of the receptor as ACh or CNO would, but they bind the receptor and this alters how ligands signal here. We have found that there is increased M1 signalling with the drug BQCA. We are now going to test this using fluorescent ligands and use competition binding to answer the kinetics of these drugs binding the M1 receptor. I am testing the clozapine derivatives on non-transfected astrocytes as a control in both the calcium imaging assay and stellation.
Exploitation Route All together these results have answered novel questions of receptor-ligand interactions within a temporal and spatial manner and in future work I will delineate how this effects are encoded by cells, and the link to diseases especially using the astrocytes. Ligand occupancy is a hot topic at the moment and there is interest in how this results in different therapeutic outcomes which will have a large societal impact. In vitro studies like mine, can be built on in the drug discovery process and will give novel tools to the science world with DREADD receptors, allosteric modulators and the microfluidic system to carry on with this such research in a highly specific and targeted manner.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://molpharm.aspetjournals.org/content/molpharm/83/2/521.full.pdf?with-ds=yes
 
Description My research focuses on looking at the spatiotemporal patterns of ligand-receptor binding and how these aspects alter cell signalling pathways. There is a strong pharmacological link to this project, which means that current and future therapeutics (and the process in which these are made) will benefit from increased knowledge of this area. Improved understanding of this area means that patients may be able to benefit from drugs which have improved design properties, therefore addressing an illness more effectively with less side effects. The links to health and disease from the cellular based project I am undertaking means that there is certainly scope for a large social impact in the future. New cellular tools (such as designer-receptors/optogenetic receptors) will also encourage the use of the three Rs of animal work that is encouraged by the government and ethics groups (as discussed in Anticipated Outputs) due to more specific ligand-receptor binding with fewer side-effects expected. I am also currently using a more novel tool (microfluidic setup) to investigate the kinetics of cell signalling. Once fully characterised a microfluidic setup will have wide-ranging effects on kinetic/cell signalling scientists and how they can add a drug to cells, and expand the questions which are answered within labs, especially involving the non-equilibrium phase of ligand-receptor interactions which is a hot topic at the moment.
First Year Of Impact 2016
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic,Policy & public services

 
Description UNICAS@Graduate School Inter-disciplinary Sandpit Programme Funding Application
Amount £5,000 (GBP)
Organisation University of Nottingham 
Department University of Nottingham Interdisciplinary Centre for Analytical Science (UNICAS)
Sector Academic/University
Country United Kingdom
Start 04/2018 
End 06/2018