Microglial motility under pro-inflammatory conditions in situ: Relevance to neurodegeneration Open Access

Gyoneva, Stefka Ivanova (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/qb98mf94h?locale=en
Published

Abstract

Microglia are the resident immune cells of the central nervous system. In the healthy brain, microglia continuously survey the parenchyma to maintain homeostasis. When a disturbance is detected, microglial processes migrate toward the site of injury and surround it to help with tissue repair in a process that depends on ATP release and activation of P2Y12 receptors on microglia. However, in prolonged injury and in many neurodegenerative conditions microglia assume an amoeboid "activated" phenotype commonly associated with release of pro-inflammatory factors. Microglial activation with lipopolysaccharide (LPS) in vitro results in P2Y12 receptor downregulation, concurrent adenosine A2A receptor upregulation, and ATP- and adenosine-induced process retraction (rather than extension). The work presented here examines the effects of A2A receptor activation under pro-inflammatory conditions in acute brain slices and in vivo with emphasis on the response of microglia to tissue damage. Time-lapse confocal microscopy showed that activated microglia in slices from LPS-treated CX3CR1GFP/+ mice, which have microglia-specific GFP expression, displayed reduced process displacement in the direction of mechanically induced tissue injury. Similar results were seen in slices prepared from the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Consistent with the in situ observations, microglia in LPS-treated CX3CR1GFP/+ mice imaged with in vivo two-photon microscopy showed a delayed response to a laser-induced localized tissue damage. The involvement of A2A receptors in modulating the delayed response to damage was confirmed with the use of the selective adenosine A2A receptor antagonist preladenant. Finally, the effects of norepinephrine receptor activation in microglia were characterized to determine if the purinergic (ATP/adenosine) receptor system is unique among neurotransmitters in modulating microglial motility. Application of norepinephrine to both resting and LPS-activated primary microglia in vitro resulted in process retraction. Interestingly, simultaneous activation of adrenergic and purinergic receptors prevented ATP-induced motility responses in microglia in vitro, suggesting that norepinephrine might interfere with the ability of microglia to respond to tissue damage in vivo. The differential regulation of microglial motility under resting and neuroinflammatory conditions by various G protein-coupled receptors can have implications on microglial ability to effectively respond to tissue disturbances such as the extensive neuronal death in neurodegenerative conditions.

Table of Contents

1. CHAPTER 1: Background

1

1. Abstract

1

2. Introduction to microglia

1

a. Basic properties

1

b. Origin and turnover

3

c. Microglial phenotypes

5

3. Microglia in the healthy brain

8

a. Tissue surveillance

8

b. Synapse surveillance

9

c. Microglia promote neurogenesis

10

4. Microglia in disease states

11

a. Inflammation in Parkinson's disease

12

b. Inflammation in Alzheimer's disease

16

5. Expression of G protein-coupled receptors (GPCRs) on microglia

20

6. Modulation of microglial motility

22

a. Regulation of microglial motility by purinergic signaling

22

b. Regulation of microglial motility by adrenergic signaling

27

7. Dissertation outline

27

CHAPTER 2: Methods

29

1. Reagents and animals

29

2. Primary microglia culture

30

3. Reverse transcriptase PCR (RT-PCR)

30

4. Real time quantitative PCR (qPCR)

32

5. cAMP assay

35

6. Preparation of acute brain slices

35

7. Immunohistochemistry

37

8. Confocal imaging

39

a. Confocal imaging of isolated cells

39

b. Confocal imaging of acute brain slices

40

9. Two-photon imaging

42

10. Image analysis

44

a. 3D cell reconstructions

44

b. Analysis of process length in acute brain slices

45

c. Analysis of response to mechanical damage in slices

48

d. Analysis of in vivo two-photon imaging data

48

11. Iontophoresis

51

12. Live-cell calcium imaging

53

13. Statistical analysis

53

CHAPTER 3: Modulation of microglial motility by purinergic receptors in vitro

55

1. Abstract

55

2. Introduction

55

3. Results

57

a. Changes in purinergic receptor expression in primary microglia

57

b. Differential modulation of microglial process motility by ATP and adenosine receptors

63

c. Involvement of other adenosine receptors in regulating microglial process motility

71

4. Discussion

73

a. Adenosine A2A receptor expression in the brain

76

b. Modulation of microglial motility by adenosine receptors

77

c. Modulation of non-motility microglial functions by A2A receptors

78

CHAPTER 4: Microglial responses to tissue damage in acute brain slices

80

1. Abstract

80

2. Introduction

80

3. Results

82

a. Microglia in acute brain slices respond to localized mechanical damage

82

b. Activated microglia have a reduced capacity to respond to tissue damage

87

c. MPTP treatment impairs microglial response to tissue damage

89

d. Antagonism of adenosine A2A receptors restores microglial responses to tissue injury

91

4. Discussion

96

a. Microglial motility in acute brain slices

96

b. Activated microglia in Parkinson's disease

99

c. Possibility of differential motility patterns of microglia in PD

99

d. Modulation of microglial motility by A2A receptors in tissues

100

CHAPTER 5: Purinergic control of microglial motility in vivo

102

1. Abstract

102

2. Introduction

102

3. Results

104

a. Characterization of activated microglia in vivo

105

b. Response of activated microglia to tissue damage in vivo

105

c. Effects of A2A receptor antagonists on microglial motility in vivo

111

4. Discussion

117

a. Motility of activated microglia in vivo

117

b. Mechanism underlying delayed response to tissue damage in vivo

120

c. Neuroprotective strategies and microglial motility

121

CHAPTER 6: Modulation of microglial motility by adrenergic receptors

123

1. Abstract

123

2. Introduction

123

3. Results

125

a. Noradrenergic receptor expression in microglia is dependent on their activation status

125

b. NE modulates microglial process motility in tissues

126

c. Norepinephrine modulates microglial process dynamics in vitro

131

d. Mechanisms underlying NE control of microglial process motility

134

e. Adrenergic receptor activation interferes with ATP response in resting microglia

136

4. Discussion

141

a. Adrenergic receptor expression in microglia

142

b. Differential modulation of microglial process dynamics by NE and ATP

143

c. Mechanism underlying the interaction between ATP and NE signaling in microglia

146

CHAPTER 7: Discussion and conclusions

148

1. Summary

148

2. Mechanisms of microglial activation

149

a. Mechanisms initiated in the brain

150

b. Systemic inflammation and neurodegeneration

152

3. Microglial motility in neurodegeneration

154

a. Receptor switching in microglia

154

b. Implications of impaired microglial motility under pro-inflammatory conditions

157

c. Neuroprotection by A2A antagonists

162

d. Involvement of other GPCRs in modulating microglial motility

164

4. Functional implications for the interaction between ATP and other neurotransmitters in microglia

166

5. Conclusion

168

CHAPTER 8: References

170

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