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

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.