Evaluation of a Chemical Genetic Approach to be used in vivo to Investigate the Role of the M1 Muscarinic Acetylcholine Receptor in Learning, Memory, and Neurodegenerative Disease.
thesisposted on 01.02.2019, 10:29 by Simon M. Brooke
In this project I performed the in vitro characterisation of a chemical genetic approach, using mutant receptors and an inert synthetic ligand, to dissect the role of the M1 muscarinic acetylcholine receptor (mAChR) in learning, memory, and neurodegenerative disease. The mutant receptors being employed are designer receptors exclusively activated by designer drugs (DREADDs) and phosphorylation deficient (PD) receptors. DREADDs contain mutations within the orthosteric binding pocket; reducing the affinity and potency of the natural ligand, acetylcholine, and increasing the potency and efficacy of an inert synthetic ligand, clozapine-N-oxide (CNO). For phosphorylation deficient mutant receptors, phosphorylated serine residues were substituted for alanine residues. Using M1-DREADD and M1-DREADD-PD mutations in vivo, our ultimate aim is to study role of M1-mAChR signalling, and G-protein dependent signalling, on learning and memory. My investigations of the function and pharmacology of the M1-DREADD and M1-DREADD-PD mAChRs found the DREADD mutations significantly reduced the potency of acetylcholine, as well as the affinity of orthosteric antagonists such as N-methyl-scopolamine. These mutations also increased both the efficacy, relative to acetylcholine, and potency of CNO for the M1 mAChR. Phosphorylation-deficient mutants were found to have impaired agonist dependent internalisation when compared to wild-type and DREADD M1-mAChRs. Although reported to be an inert ligand, CNO was found to act in an antagonist mode at the mouse M1, M2, and M4 mAChRs. This antagonistic mode, as well as the potential for CNO to be metabolised into clozapine, suggests alternative DREADD ligands need to be considered to prevent off-target side effects, even though previous transgenic studies have been successful. Given the successful modification of M1-mAChR, these mutants can now be used to genetically engineer mice for in vivo studies. With these mice, behavioural studies can be performed to characterise the role played by total M1-mAChR signalling, and its G-protein-dependent and independent signalling, on learning and memory. These observations can be used to inform drug discovery efforts targeting the M1-mAChR, such as the treatment of Alzheimer’s disease.