M4 Muscarinic Acetylcholine Receptor Modulation
of Dopamine Receptor Functions
Nae-Yng Amy Chen
BPharmSc (Hons)
A thesis submitted for the degree of Doctor of Philosophy
at Monash University in 2016
Drug Discovery Biology
Monash Institute of Pharmaceutical Sciences
Faculty of Pharmacy and Pharmaceutical Sciences
Monash University
Parkville, Victoria, Australia
Copyright notice
© Nae-Yng Amy Chen (2016). Except as provided in the Copyright Act 1968, this
thesis may not be reproduced in any form without the written permission of the
author.
I CERTIFY THAT I HAVE MADE ALL REASONABLE EFFORTS TO SECURE
COPYRIGHT PERMISSIONS FOR THIRD-PARTY CONTENT INCLUDED IN
THIS THESIS AND HAVE NOT KNOWINGLY ADDED COPYRIGHT CONTENT
TO MY WORK WITHOUT THE OWNER'S PERMISSION.
Table of Contents
i
Table of Contents
List of Figures .................................................................................................................... viii
List of Tables ....................................................................................................................... xii
List of Abbreviations ......................................................................................................... xiii
Abstract ............................................................................................................................... xvi
Publications ..................................................................................................................... xviii
Declaration.......................................................................................................................... xix
Acknowledgements ............................................................................................................. xx
Chapter 1: General Introduction ........................................................................................... 1
1.1 G Protein-Coupled Receptors ................................................................................. 2
1.1.1 GPCR Classification ........................................................................................ 2
1.1.2 GPCR Signalling ............................................................................................. 3
1.1.3 GPCR Allostery ............................................................................................... 6
1.2 Schizophrenia ............................................................................................................ 11
1.2.1 GPCRs in Schizophrenia ............................................................................... 13
1.2.2 Muscarinic Acetylcholine Hypothesis of Schizophrenia............................... 16
1.3 Muscarinic Acetylcholine Receptors ......................................................................... 19
1.3.1 M1 mAChR .................................................................................................... 19
1.3.2 M2 mAChR .................................................................................................... 20
1.3.3 M3 mAChR .................................................................................................... 21
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ii
1.3.4 M4 mAChR .................................................................................................... 21
1.3.5 M5 mAChR .................................................................................................... 23
1.4 Challenges in Translational Research ................................................................... 25
1.5 Prepulse Inhibition and Locomotor Activity ........................................................ 28
1.5.1 Prepulse Inhibition of the Startle Reflex ...................................................... 28
1.5.2 Locomotor Activity ...................................................................................... 31
1.6 Scope of Thesis ..................................................................................................... 33
Chapter 2: Detection and Quantification of Allosteric Modulation of Endogenous M4
Muscarinic Acetylcholine Receptor Using Impedance-Based Label-Free Technology in a
Neuronal Cell Line .............................................................................................................. 35
Chapter 3: Determination of Signalling Cross-Talk between M4 Muscarinic Acetylcholine
and Dopamine Receptors Endogenously Expressed in a Neuronal Cell Line ..................... 45
3.1 Introduction ........................................................................................................... 46
3.2 Materials and Methods .......................................................................................... 49
3.2.1 Materials ........................................................................................................ 49
3.2.2 Cell Culture ................................................................................................... 49
3.2.3 cAMP Bioluminescence Resonance Energy Transfer Biosensor Assay ....... 50
3.2.4 ERK1/2 Phosphorylation Assay .................................................................... 50
3.2.5 Data Analysis ................................................................................................. 52
3.3 Results ................................................................................................................... 53
3.3.1 NG108-15 Cells Endogenously Express D2-like, but Not D1-like, Dopamine
Receptors ..................................................................................................................... 53
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3.3.2 Interaction Studies Reveal a Lack of Signalling Cross-Talk between M4
Muscarinic Acetylcholine and D2-like Dopamine Receptor Ligands ......................... 58
3.4 Discussion ............................................................................................................. 66
Chapter 4: Studying the Effect of Positive Allosteric Modulation of M4 Muscarinic
Acetylcholine Receptors on Psychosis-like Behaviours Induced by a D1 Dopamine
Receptor-selective Agonist in Mice .................................................................................... 70
4.1 Introduction ........................................................................................................... 71
4.2 Material and Methods ........................................................................................... 75
4.2.1 Materials ........................................................................................................ 75
4.2.2 Cell Culture ................................................................................................... 75
4.2.3 Preparation of Cell Membranes ..................................................................... 76
4.2.4 Radioligand Binding Assays in Membrane Preparations .............................. 76
4.2.5 ERK1/2 Phosphorylation Assays................................................................... 77
4.2.6 Animals .......................................................................................................... 78
4.2.7 Drugs ............................................................................................................. 79
4.2.8 Prepulse Inhibition of the Acoustic Startle Response (PPI) .......................... 80
4.2.9 Locomotor Activity (LMA) ........................................................................... 82
4.2.10 Assessment of Compound Exposure in Brain and Plasma ............................ 82
4.2.11 Data and Statistical Analysis ......................................................................... 85
4.3 Results ................................................................................................................... 90
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iv
4.3.1 Potentiation of ACh Function at M4 mAChRs by a Next Generation M4
Muscarinic Receptor Positive Allosteric Modulator, ML253, is Subject to Species
Variability .................................................................................................................... 90
4.3.2 In Vitro and In Vivo Characterisation of R(+)-6-Br-APB, a Selective D1
Dopamine Receptor Agonist ....................................................................................... 95
4.3.3 Drug Vehicles do not Affect Prepulse Inhibition and Locomotor Activity
compared to Saline and Water for Injection in Mice................................................. 102
4.3.4 Assessment of Compound Exposure in Plasma and Brain .......................... 104
4.3.5 Treatments of LY2033298 alone or with Donepezil, an Acetylcholinesterase
Inhibitor, Showed a Trend to Reverse Disruption of Prepulse Inhibition Induced by
R(+)-6-Br-APB .......................................................................................................... 106
4.3.6 Combined Treatment of LY2033298 and Donepezil Reversed
Hyperlocomotor Activity Induced by R(+)-6
