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. 2009 Jul;12(7):872-8.
doi: 10.1038/nn.2341. Epub 2009 Jun 14.

Adenosine A(2A) receptor mediates microglial process retraction

Anna G Orr  1 , Adam L Orr, Xiao-Jiang Li, Robert E Gross, Stephen F Traynelis
Affiliations

Adenosine A(2A) receptor mediates microglial process retraction

Anna G Orr et al. Nat Neurosci. 2009 Jul.

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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Figures

Fig. 1
Fig. 1. Untreated microglia exhibit low expression of activation markers
RT-PCR analysis in cultured mouse microglia indicates that control microglia (C) exhibit low mRNA levels for known proinflammatory factors, as indicated. Upon LPS treatment (100 ng/ml, 24 h), microglia show robust upregulation of these factors (n = 3). Abbreviations: iNOS: inducible nitric oxide synthase, IL-1β interleukin-1β, COX-2: cyclooxygenase-2, TNF-α: tumor necrosis factor-α, MMP-9: matrix metalloproteinase-9, MCP-1: monocyte chemoattractant protein-1.
Fig. 2
Fig. 2. ATP induces migratory repulsion in activated microglia
(a) Three-dimensional reconstruction of a microglial cell shown before and after 25 minutes of local ATP exposure (0.5 mM, yellow dot marks starting position of cell nucleus, yellow line shows migratory path, dashed arrow indicates net displacement of cell nucleus). Scale: 10 µm2 per grid square. (b) Vector displacement of microglia during ATP ejection for ≤ 1 hour, as plotted after X-axis alignment to pipette tip location. (c) Microglia exhibit enhanced migration toward ATP source (n = 5, p < 0.05 compared to baseline). Negative values were assigned for net migration away from pipette. (d–f) LPS-activated microglia migrate away from ATP (LPS: 100 ng/ml; n = 6, p < 0.01 compared to baseline).
Fig. 3
Fig. 3. ATP induces process retraction and slowed motility in activated microglia
Microglial three-dimensional volume (a, b), surface (c, d), and tracked process movement (e, f) during baseline and ATP exposure (Scale: 10 µm2 per grid square). (g) ATP increases process ramification in control microglia (Con, n = 6, p < 0.01 compared to baseline), but causes retraction in activated microglia (n = 5–10; LPS: p < 0.01; LTA: 10 µg/ml, p < 0.01; CpG: 10 µM, p < 0.001; TNF-α: 20 ng/ml, p < 0.001; compared to baseline). (h) ATP increases motility in control microglia, but decreases motility in activated microglia (n = 6–8, compared to baseline, pre-ATP baseline was set to 100% track speed). (i) Chemotactic reversal requires > 12 hours (n = 4–8, compared to baseline). (j) Process retraction is blocked by NF-κB inhibitors (1 µM QNZ, 20 µM SN50, n = 6–10, p < 0.05 compared to responses in LPS-activated cells). (k) NF-κB inhibition prevents ATP-induced decline in process motility (n = 4–5, compared to responses in LPS-activated cells; pre-ATP baseline was set to 100% track speed). (l) C5a (20 nM) increases ramification in LPS-activated microglia (n = 8, p < 0.05 compared to baseline). All graphs show mean + s.e.m; *p < 0.05, #p < 0.01, ##p < 0.001
Fig. 4
Fig. 4. Gs-coupled signaling mediates microglial repulsion from ATP
(a–c) Microglial process retraction was attenuated by inhibition of Gαs with NF449 (50 µM, n = 8), inhibition of adenylate cyclase (AC) with ddAdo (50 µM, n = 6), or inhibition of PKA with H89 (50 µM, n = 8). (d) Summary of inhibitor effects on retraction in LPS-activated microglia (Con) is shown. Inhibition of Rho with C3 exoenzyme (20 µg/ml, n = 5) or ROCK with Y27632 (10 µM, n = 9) had no effect. (e) Process motility decline in activated microglia was attenuated with Gαs, AC, or PKA inhibitors, but not with Rho or ROCK inhibitors. For graphs a–e: values were compared to responses in LPS-treated cells. (f) Adenylate cyclase activation with forskolin triggered retraction in both control and activated microglia (values were compared to baseline). All graphs show mean + s.e.m. *p < 0.05, #p < 0.01, ##p < 0.001.
Fig. 5
Fig. 5. Adenosine A2A receptor upregulation mediates process retraction and reverses chemotaxis in activated microglia
(a) A2A receptor mRNA is upregulated upon microglial activation with LPS (n = 4), LTA (n = 3), TNF-α (n = 3), CpG (n = 2), or amyloid-β (Aβ, n = 3, 1 µM), while P2Y12 is downregulated. (b) A2A agonist triggered retraction in LPS-treated microglia (CGS: 20 µM, n = 6, p < 0.01 compared to baseline), but not in untreated cells (Con: n = 3). (c) The A2A antagonist SCH-58261 (SCH: 5 µM) and adenosine deaminase (ADA: 5 U/ml) inhibited ATP-induced retraction in LPS-activated microglia (ATP: n = 5; ATP + SCH: n = 7, p < 0.05; ATP + ADA: n = 5, p < 0.05 compared to responses in ATP-treated cells). All graphs show mean + s.e.m.
Fig. 6
Fig. 6. Human microglia exhibit a similar shift in purinergic receptor expression and chemotactic response to ATP
(a) Activated human microglia upregulate A2A mRNA and downregulate P2Y12 mRNA (n = 4). (b) Untreated human microglia migrate toward ATP (Control: n = 6, 0.5 mM), while LPS-activated microglia migrate away from ATP (LPS: n = 5).
Fig. 7
Fig. 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).
Fig. 8
Fig. 8. A2A stimulation inhibits uptake by LPS-treated microglia
Treatment with indicated agonists (50 µM) for 20 minutes following microglial exposure to LPS (100 ng/ml, 24 h) led to a decline in microglial uptake of fluorescein-labeled E. coli bioparticles, which were applied for 2 hours along with agonists. CGS-21680 (CGS). n ≥ 7, *p < 0.05. Graph shows mean + s.e.m.
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