Microglial motility under pro-inflammatory conditions in situ: Relevance to neurodegeneration Pubblico
Gyoneva, Stefka Ivanova (2013)
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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