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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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ATP induces process retraction and slowed motility in activated microgliaMicroglial 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
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Figure 3: ATP induces process retraction and slowed motility in activated microgliaMicroglial 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

Mentions: To better understand the intracellular mechanisms driving these opposing chemotactic responses by microglia, we performed time-lapse three-dimensional imaging during bath application of agonist. This method enabled us to quantify changes in microglial cell structure as well as motile responses by individual cell processes without interference from cell migration, since no agonist gradient exists in the recording chamber under these conditions (Fig. 3a–f and Supplementary Fig. 1). In control microglia, we observed that global ATP exposure triggered a rapid and reversible increase in microglial surface area-to-volume ratio (SA:V, Fig. 3g and Supplementary Video 2), reflecting cell process extension. Moreover, tracking individual cell processes revealed that ATP also significantly increased the velocity of process movement, which we interpret as increased microglial process motility (Fig. 3h). These four-dimensional analyses in control microglia support previous findings that ATP serves as a chemoattractant for resting cells4,5,9,10.


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

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

ATP induces process retraction and slowed motility in activated microgliaMicroglial 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
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Figure 3: ATP induces process retraction and slowed motility in activated microgliaMicroglial 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
Mentions: To better understand the intracellular mechanisms driving these opposing chemotactic responses by microglia, we performed time-lapse three-dimensional imaging during bath application of agonist. This method enabled us to quantify changes in microglial cell structure as well as motile responses by individual cell processes without interference from cell migration, since no agonist gradient exists in the recording chamber under these conditions (Fig. 3a–f and Supplementary Fig. 1). In control microglia, we observed that global ATP exposure triggered a rapid and reversible increase in microglial surface area-to-volume ratio (SA:V, Fig. 3g and Supplementary Video 2), reflecting cell process extension. Moreover, tracking individual cell processes revealed that ATP also significantly increased the velocity of process movement, which we interpret as increased microglial process motility (Fig. 3h). These four-dimensional analyses in control microglia support previous findings that ATP serves as a chemoattractant for resting cells4,5,9,10.

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