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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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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.
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Figure 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.

Mentions: We next addressed the receptor signaling mechanisms driving this effect in activated microglia. It is known that resting microglia are attracted to ATP or acute injury at least partly due to Rac GTPase-driven actin polymerization downstream of the Gi-coupled P2Y12 receptor3,4,9,10. However, P2Y12 is rapidly downregulated upon microglial activation5,13 (Supplementary Fig. 2), suggesting that a different receptor system may mediate chemotactic injury responses by activated microglia. While the signaling mechanisms driving microglial retraction are unknown, other cell types undergo retraction due to remodeling of the actin cytoskeleton, which often involves G12/13-coupled activation of Rho GTPase and Rho kinase (ROCK)14,15. Staining microglial actin cytoskeleton suggested that actin remodeling participates in microglial repulsion (Supplementary Fig. 3a–b). However, inhibition of either Rho GTPase or ROCK did not attenuate microglial repulsion (Fig. 4), indicating that an alternative mechanism may drive this response. Interestingly, Gs-coupled signaling can suppress motility in select cell types16,17. This pathway involves activation of adenylate cyclase, which produces cyclic adenosine monophosphate (cAMP) and thereby activates protein kinase A (PKA). We therefore tested whether ATP regulates microglial motility via Gs-coupled signaling. Indeed, inhibition of Gαs or factors downstream of Gαs, including adenylate cyclase or PKA, attenuated ATP-induced retraction and slowed motility in activated microglia (Fig. 4a–e and Supplementary Fig. 3c–d). Conversely, stimulating adenylate cyclase with forskolin induced dose-dependent retraction in both control and activated microglia (Fig. 4f). Moreover, constitutive activation of Gαs with cholera toxin blunted membrane extension in unstimulated cells and induced microglial cell rounding within 24 hours (data not shown). These results indicate that Gs signaling is necessary and sufficient for microglial repulsion.


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

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

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: We next addressed the receptor signaling mechanisms driving this effect in activated microglia. It is known that resting microglia are attracted to ATP or acute injury at least partly due to Rac GTPase-driven actin polymerization downstream of the Gi-coupled P2Y12 receptor3,4,9,10. However, P2Y12 is rapidly downregulated upon microglial activation5,13 (Supplementary Fig. 2), suggesting that a different receptor system may mediate chemotactic injury responses by activated microglia. While the signaling mechanisms driving microglial retraction are unknown, other cell types undergo retraction due to remodeling of the actin cytoskeleton, which often involves G12/13-coupled activation of Rho GTPase and Rho kinase (ROCK)14,15. Staining microglial actin cytoskeleton suggested that actin remodeling participates in microglial repulsion (Supplementary Fig. 3a–b). However, inhibition of either Rho GTPase or ROCK did not attenuate microglial repulsion (Fig. 4), indicating that an alternative mechanism may drive this response. Interestingly, Gs-coupled signaling can suppress motility in select cell types16,17. This pathway involves activation of adenylate cyclase, which produces cyclic adenosine monophosphate (cAMP) and thereby activates protein kinase A (PKA). We therefore tested whether ATP regulates microglial motility via Gs-coupled signaling. Indeed, inhibition of Gαs or factors downstream of Gαs, including adenylate cyclase or PKA, attenuated ATP-induced retraction and slowed motility in activated microglia (Fig. 4a–e and Supplementary Fig. 3c–d). Conversely, stimulating adenylate cyclase with forskolin induced dose-dependent retraction in both control and activated microglia (Fig. 4f). Moreover, constitutive activation of Gαs with cholera toxin blunted membrane extension in unstimulated cells and induced microglial cell rounding within 24 hours (data not shown). These results indicate that Gs signaling is necessary and sufficient for microglial repulsion.

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