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Adenosine A(2A) receptor mediates microglial process retraction.

Orr AG, Orr AL, Li XJ, Gross RE, Traynelis SF - Nat. Neurosci. (2009)

Bottom Line: Thus, A(2A) receptor stimulation by adenosine, a breakdown product of extracellular ATP, caused activated microglia to assume their characteristic amoeboid morphology during brain inflammation.Our results indicate that purine nucleotides provide an opportunity for context-dependent shifts in receptor signaling.Thus, we reveal an unexpected chemotactic switch that generates a hallmark feature of CNS inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, USA. anna.orr@gladstone.ucsf.edu

ABSTRACT
Cell motility drives many biological processes, including immune responses and embryonic development. In the brain, microglia are immune cells that survey and scavenge brain tissue using elaborate and motile cell processes. The motility of these processes is guided by the local release of chemoattractants. However, most microglial processes retract during prolonged brain injury or disease. This hallmark of brain inflammation remains unexplained. We identified a molecular pathway in mouse and human microglia that converted ATP-driven process extension into process retraction during inflammation. This chemotactic reversal was driven by upregulation of the A(2A) adenosine receptor coincident with P2Y(12) downregulation. Thus, A(2A) receptor stimulation by adenosine, a breakdown product of extracellular ATP, caused activated microglia to assume their characteristic amoeboid morphology during brain inflammation. Our results indicate that purine nucleotides provide an opportunity for context-dependent shifts in receptor signaling. Thus, we reveal an unexpected chemotactic switch that generates a hallmark feature of CNS inflammation.

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A2A receptor upregulation and involvement in microglial retraction in vivo(a) Fixed cortical tissue sections from BAC-transgenic mice expressing eGFP upstream of BAC A2A coding sequence were immunostained for eGFP. LPS-treated animals (LPS: 2 mg/kg) exhibit A2A upregulation after 48 hours, as evidenced by increased eGFP expression. Scale bar: 50 µm. Inset: Constitutive A2A expression within striatal neurons served as a positive control. (b) Fixed cortical tissue sections from Cx3cr1-eGFP transgenic mice. Intracortical blockade of the A2A receptor with the antagonist SCH-58261 (SCH: 1 mM) triggered microglial process ramification in LPS-exposed animals (n = 4; scale bar: 50 µm).
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Figure 7: A2A receptor upregulation and involvement in microglial retraction in vivo(a) Fixed cortical tissue sections from BAC-transgenic mice expressing eGFP upstream of BAC A2A coding sequence were immunostained for eGFP. LPS-treated animals (LPS: 2 mg/kg) exhibit A2A upregulation after 48 hours, as evidenced by increased eGFP expression. Scale bar: 50 µm. Inset: Constitutive A2A expression within striatal neurons served as a positive control. (b) Fixed cortical tissue sections from Cx3cr1-eGFP transgenic mice. Intracortical blockade of the A2A receptor with the antagonist SCH-58261 (SCH: 1 mM) triggered microglial process ramification in LPS-exposed animals (n = 4; scale bar: 50 µm).

Mentions: We next investigated whether the A2A receptor plays a role in the retracted morphology assumed by activated microglia during neuroinflammation in vivo. For this, we utilized a well-established animal model of neuroinflammation involving systemic LPS exposure7. Using transgenic Adora-eGFP mice that express eGFP under control of the A2A promoter, we first confirmed that A2A receptor upregulation takes place in vivo following LPS treatment (Fig. 7a). Next, we utilized transgenic Cx3Cr1-eGFP mice that exhibit microglia-specific eGFP labeling3–5. We note that although eGFP is localized exclusively in microglial cells among CNS-resident cellular elements, the fluorescent reporter does not differentiate between resident microglia and infiltrating mononuclear phagocytes, which may exhibit microglial morphology. Upon LPS exposure, these animals displayed characteristic retracted microglia throughout the brain, a hallmark of activated microglia and induction of CNS inflammation (Supplementary Fig. 5). In these LPS-treated animals, we observed that intracortical injection of the A2A-specific antagonist SCH-58261 resulted in microglial process re-extension within 30 minutes, an effect not seen in animals injected with vehicle alone (Fig. 7b and Supplementary Fig. 5). These observations are consistent with our in vitro data and suggest that activated microglia may assume an amoeboid phenotype in vivo due to A2A receptor stimulation by purine nucleotides released in the brain.


Adenosine A(2A) receptor mediates microglial process retraction.

Orr AG, Orr AL, Li XJ, Gross RE, Traynelis SF - Nat. Neurosci. (2009)

A2A receptor upregulation and involvement in microglial retraction in vivo(a) Fixed cortical tissue sections from BAC-transgenic mice expressing eGFP upstream of BAC A2A coding sequence were immunostained for eGFP. LPS-treated animals (LPS: 2 mg/kg) exhibit A2A upregulation after 48 hours, as evidenced by increased eGFP expression. Scale bar: 50 µm. Inset: Constitutive A2A expression within striatal neurons served as a positive control. (b) Fixed cortical tissue sections from Cx3cr1-eGFP transgenic mice. Intracortical blockade of the A2A receptor with the antagonist SCH-58261 (SCH: 1 mM) triggered microglial process ramification in LPS-exposed animals (n = 4; scale bar: 50 µm).
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Figure 7: A2A receptor upregulation and involvement in microglial retraction in vivo(a) Fixed cortical tissue sections from BAC-transgenic mice expressing eGFP upstream of BAC A2A coding sequence were immunostained for eGFP. LPS-treated animals (LPS: 2 mg/kg) exhibit A2A upregulation after 48 hours, as evidenced by increased eGFP expression. Scale bar: 50 µm. Inset: Constitutive A2A expression within striatal neurons served as a positive control. (b) Fixed cortical tissue sections from Cx3cr1-eGFP transgenic mice. Intracortical blockade of the A2A receptor with the antagonist SCH-58261 (SCH: 1 mM) triggered microglial process ramification in LPS-exposed animals (n = 4; scale bar: 50 µm).
Mentions: We next investigated whether the A2A receptor plays a role in the retracted morphology assumed by activated microglia during neuroinflammation in vivo. For this, we utilized a well-established animal model of neuroinflammation involving systemic LPS exposure7. Using transgenic Adora-eGFP mice that express eGFP under control of the A2A promoter, we first confirmed that A2A receptor upregulation takes place in vivo following LPS treatment (Fig. 7a). Next, we utilized transgenic Cx3Cr1-eGFP mice that exhibit microglia-specific eGFP labeling3–5. We note that although eGFP is localized exclusively in microglial cells among CNS-resident cellular elements, the fluorescent reporter does not differentiate between resident microglia and infiltrating mononuclear phagocytes, which may exhibit microglial morphology. Upon LPS exposure, these animals displayed characteristic retracted microglia throughout the brain, a hallmark of activated microglia and induction of CNS inflammation (Supplementary Fig. 5). In these LPS-treated animals, we observed that intracortical injection of the A2A-specific antagonist SCH-58261 resulted in microglial process re-extension within 30 minutes, an effect not seen in animals injected with vehicle alone (Fig. 7b and Supplementary Fig. 5). These observations are consistent with our in vitro data and suggest that activated microglia may assume an amoeboid phenotype in vivo due to A2A receptor stimulation by purine nucleotides released in the brain.

Bottom Line: Thus, A(2A) receptor stimulation by adenosine, a breakdown product of extracellular ATP, caused activated microglia to assume their characteristic amoeboid morphology during brain inflammation.Our results indicate that purine nucleotides provide an opportunity for context-dependent shifts in receptor signaling.Thus, we reveal an unexpected chemotactic switch that generates a hallmark feature of CNS inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, USA. anna.orr@gladstone.ucsf.edu

ABSTRACT
Cell motility drives many biological processes, including immune responses and embryonic development. In the brain, microglia are immune cells that survey and scavenge brain tissue using elaborate and motile cell processes. The motility of these processes is guided by the local release of chemoattractants. However, most microglial processes retract during prolonged brain injury or disease. This hallmark of brain inflammation remains unexplained. We identified a molecular pathway in mouse and human microglia that converted ATP-driven process extension into process retraction during inflammation. This chemotactic reversal was driven by upregulation of the A(2A) adenosine receptor coincident with P2Y(12) downregulation. Thus, A(2A) receptor stimulation by adenosine, a breakdown product of extracellular ATP, caused activated microglia to assume their characteristic amoeboid morphology during brain inflammation. Our results indicate that purine nucleotides provide an opportunity for context-dependent shifts in receptor signaling. Thus, we reveal an unexpected chemotactic switch that generates a hallmark feature of CNS inflammation.

Show MeSH
Related in: MedlinePlus