M1 Muscarinic Acetylcholine Receptor Signaling and Regulation ofAmyloid Precursor Protein Processing Público

Davis, Albert Augustus (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/wd375w627?locale=es
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Abstract

ii ABSTRACT Alzheimer's disease (AD), a progressive neurological disorder characterized by memory loss, cognitive decline, and behavioral disturbances, is the leading cause of dementia among the elderly and affects nearly half of the population over the age of 85. Although AD is a complex disease and remains incompletely understood, multiple lines of evidence point to important roles of the neurotoxic amyloid beta peptide (A) and the amyloid precursor protein (APP) from which A is derived. Activation of several neurotransmitter receptors, including muscarinic acetylcholine receptors (mAChRs), has been shown to reduce A production, but less is known about the specific mAChR subtypes that regulate APP processing in neurons. Using primary neuron cultures from wildtype and M1 mAChR-deficient mice, we demonstrate that the M1 mAChR subtype is essential for cholinergic regulation of non- amyloidogenic APP processing. In wildtype neuron cultures, treatment with the muscarinic agonist carbachol stimulated non-amyloidogenic APP processing. In M1 knockout neurons, these responses were either abolished or reversed by carbachol treatment, and M1 overexpression restored the wildtype phenotype. In vivo experiments in APP-transgenic mice demonstrate that the loss of M1 receptors accelerates amyloid pathology. Complementary experiments in the neuronotypic PC12 cell line using recently developed, highly selective allosteric M1 agonists support this finding and provide proof of principle that M1-selective drugs can regulate APP processing and are therefore good candidates for evaluation in more complex model systems.

iii We also investigated the activation and regulatory mechanisms of two structurally distinct allosteric M1 agonists. We show that allosteric agonists potently activate multiple signal transduction pathways linked to the M1 receptor but are significantly impaired in their ability to induce recruitment of arrestin-3, a protein involved in regulation of G-protein coupled receptor signaling. Consistent with their lack of arrestin recruitment, both allosteric agonists showed blunted responses in measurements of receptor desensitization, internalization, and downregulation. These results will both strengthen the understanding of basic receptor biology and help to shape the development of a new generation of drugs for the treatment of AD and other devastating neurological and neuropsychiatric diseases.

Table of Contents

v TABLE OF CONTENTS Abstract................................................................................................iv

Acknowledgements................................................................................vii

Table of Contents..................................................................................viii List of Figures.......................................................................................xii Chapter I: INTRODUCTION....................................................................1 Alzheimer's Disease: Background and Significance....................................1 Initial Report and Prevalence of Disease........................................1 Advances in Understanding the Pathophysiology of AD.....................4 AD Genetics........................................................................11 APP, Proteolytic Processing, and the Amyloid Cascade Hypothesis......13 The Cholinergic System...................................................................21 Historical Perspective............................................................21 Functional Neuroanatomy of the Cholinergic System........................23 Muscarinic Acetylcholine Receptors: Expression and Function in the CNS..........................................................................26 Cholinergic Involvement in AD.................................................31 Proposed Research.........................................................................34 Chapter II: MATERIALS AND METHODS................................................36 Primary Neuron Culture...................................................................36 Cell Culture.................................................................................37 APPSwedish/Indiana x M1KO mice...........................................................37 Antibodies, plasmids, and chemicals....................................................39 Ectodomain shedding assays..............................................................40 Western blotting............................................................................40

vi ELISA measurement of A40 and A42 peptides....................................40 Tissue Collection...........................................................................41 Histochemical Amyloid Plaque Analysis...............................................41 Sequential amyloid extraction............................................................42 Measurement of ERK 1/2 phosphorylation.............................................42 Intracellular calcium mobilization assays...............................................43 Binding assays..............................................................................44 Immunocytochemistry.....................................................................44 Arrestin recruitment........................................................................45 Statistical Analysis.........................................................................45 Chapter III: APP PROCESSING IN PRIMARY NEURON CULTURES FROM WILDTYPE AND MUSCARNIC RECEPTOR SUBTYPE KNOCKOUT MICE.................................................................................................46 Introduction.................................................................................46 Results.......................................................................................47 Discussion...................................................................................53 Chapter IV: IMPACT OF M1 MUSCARINIC RECEPTOR DELETION ON PATHOLOGY IN A MOUSE MODEL OF AMYLOIDOSIS...........................55 Introduction.................................................................................55 Results.......................................................................................58 Discussion...................................................................................61 Chapter V: REGULATION OF APP PROCESSING BY M1-SELECTIVE AGONISTS..........................................................................................64

vii Introduction.................................................................................64 Results.......................................................................................66 Effects of TBPB on APP processing...........................................66 Effects of BQCA on APP processing ..........................................69 Discussion...................................................................................69 Chapter VI: REGULATED SHEDDING OF THE LR11/SORLA ECTODOMAIN BY THE M1 MUSCARINIC RECEPTOR...................................................74 Introduction.................................................................................74 Results.......................................................................................75 M1 activation promotes shedding of the LR11 ectodomain.................75 M1-mediated LR11 shedding is dependent on a metalloprotease..........75 Discussion...................................................................................78 Chapter VII: ALLOSTERIC AGONISTS DIFFERENTIALLY REGULATE M1 MUSCARINIC RECEPTOR SIGNALING AND HOMEOSTASIS MECHANISMS....................................................................................81 Introduction.................................................................................81 Results.......................................................................................82 Activation of the M1 mAChR by orthosteric and allosteric agonists......82 Allosteric M1 agonists fail to recruit Arrestin.................................87 M1 receptors exposed to allosteric agonists remain on the cell surface...90 Allosteric agonists do not induce M1 downregulation.......................94 M1 receptors exposed to allosteric agonists remain functionally sensitive.............................................................................96

viii Discussion...................................................................................98 Chapter VIII: SUMMARY AND FUTURE DIRECTIONS.............................103 REFERENCES....................................................................................113

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