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M Muscarinic Acetylcholine Receptor Modulation of Dopamine Receptor · PDF file 2017. 3. 28. · M 4 Muscarinic Acetylcholine Receptor Modulation of Dopamine Receptor Functions Nae-Yng

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  • 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

  • Table of Contents

    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

  • Table of Contents

    iii

    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

  • Table of Contents

    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

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