Regulation of microglial chemotactic responses duringneuroinflammation Public

Abstract

Regulation of microglial chemotactic responses during neuroinflammation By Anna G. Orr

Cell motility drives a variety of biological processes, including inflammation, development, and tumor metastasis. In the brain, microglia are phagocytic immune cells that actively survey brain tissue and scavenge sites of injury using elaborate motile processes. Motility of these processes is guided by local release of chemoattractants. Recent studies have shown that acute brain injury induces microglial process extension toward sites of tissue damage, which is thought to serve neuroprotective functions by allowing microglial scavenging of necrotic debris. This chemoattractive response is guided by the release of nucleotides from damaged cells, and involves microglial purinergic receptors. Thus, process motility enables microglial monitoring of healthy tissue and clean up of injured tissue. In contrast, prolonged brain damage is accompanied by proinflammatory, or activated, microglia with highly retracted processes. While microglial process retraction has been documented for decades and serves as a hallmark of brain trauma and neurodegeneration, its causes and consequences remain unknown. Using novel four-dimensional confocal imaging methods, we discovered that ATP triggers process retraction, slowed process motility, and repulsive migration in activated microglia. Our results indicate that while ATP is a factor that attracts naïve microglia, it instead repels activated microglia. Moreover, we found that repulsion from ATP is mediated by Gs-coupled signaling downstream of an upregulated adenosine A2A receptor. In light of evidence that the Gi-coupled P2Y12 receptor that mediates chemoattraction to ATP is downregulated upon microglial activation, we propose that a switch from P2Y12 to A2A receptor signaling drives the shift in microglial chemotaxis from attraction to repulsion. Our results further suggest that A2A stimulation inhibits phagocytosis by activated microglia and prevents scavenging during injury. Lastly, we present evidence that the A2A receptor is upregulated during neuroinflammation and induces microglial process retraction in vivo. Thus, our investigations have revealed an unexpected chemotactic switch central to CNS inflammation and suggest that other cell motility-driven biological processes may be similarly regulated.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1: Introduction

1.1. Inflammation

1.2. Resting microglia

a. Origin b. Characterization

1.3. Activated microglia a. Characterization b. Triggers of activation c. Intracellular signaling 1.4. Cell chemotaxis

1.5. Microglial morphology and process motility 1.6. Microglial scavenging 1.7. Purinergic signaling a. P1 receptors b. P2 receptors c. Adenine nucleotides in the CNS d. Physiological roles of purines in immune function e. The effects of purines on microglia

1.8. Microglial chemotactic response to injury

1.9. Summary CHAPTER 2: Materials and Methods 2.1. Animals and reagents 2.2. Cell cultures

2.3. Four-dimentional confocal imaging

a. Image acquisition

b. Image processing and analysis

c. Parameter optimization for tracking regions of interest

d. Iontophoresis

2.4. Immunohistochemistry

2.5. Reverse transcription-polymerase chain reaction (RT-PCR)

2.6. In vitro assays

a. Calcium imaging

b. Cyclic adenosine monophosphate (cAMP) analysis

c. Phagocytosis

d. Actin filament staining

2.7. Statistics

CHAPTER 3: The Effects of ATP on Microglial Motility

CHAPTER 4: The Intracellular Mechanisms Mediating Microglial Motile

Responses to ATP

CHAPTER 5: Downstream and In Vivo Effects of ATP Response Reversal in Microglia

CHAPTER 6: Conclusions and Implications

REFERENCES

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Mot-clé	motility ATP chemorepulsion GPCR neuroinflammation purinergic A2A Microglia chemotaxis cAMP
Committee Chair / Thesis Advisor	Traynelis, Stephen, Emory University
Committee Members	Hall, Randy A, Emory University Gross, Robert, Emory University Tsirka, Stella, SUNY-Stony Brook Dingledine, Raymond J, Emory University

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