M1 Muscarinic Acetylcholine Receptor Signaling and Regulation of Amyloid Precursor Protein Processing Open Access

Davis, Albert Augustus (2009)

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


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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.


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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

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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

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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

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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

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Discussion...................................................................................98

Chapter VIII: SUMMARY AND FUTURE DIRECTIONS.............................103

REFERENCES....................................................................................113

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