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ADP and adenosine
Parkinsonism and Related Disorders 15S3 (2009) S195–S199
Contents lists available at ScienceDirect
Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Differential regulation of microglial motility by ATP/ADP and adenosine Stefka Gyonevaa, ∗ , Anna G. Orrb , Stephen F. Traynelisa a Department b Gladstone
of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA Institute of Neurological Disease, University of California San Francisco, San Francisco, CA, USA
article info
summary
Keywords: Microglia ATP, adenosine A2A receptor Chemotaxis Neurodegeneration
Microglia are motile immune-competent cells of the central nervous system. They assume a highly branched morphology and monitor the brain parenchyma under physiological conditions. In the presence of injury, microglia retract their branching processes, migrate to the site of injury, and help clear cellular debris by phagocytosis. This response appears to be mediated in part by ATP released at the site of injury. Here, we review the evidence for the involvement of ATP and the purinergic P2Y12 receptor in microglial process extension and chemoattraction to injury. We subsequently discuss recent findings regarding a switch of this chemotactic response to ATP in activated, or proinflammatory, microglia. Specifically, in LPS-activated microglia, ATP induces process retraction and repulsive migration, effects opposite to those seen in unstimulated cells. These repulsive effects of ATP are mediated by the Gs -coupled adenosine A2A receptor and depend on the breakdown of ATP to adenosine. Thus, ATP-induced repulsion by activated microglia involves upregulation of the adenosine A2A receptor and coincident downregulation of the P2Y12 receptor. The roles of the A2A receptor in brain pathologies such as Parkinson’s disease and ischemia are also examined. We propose that the effects of A2A receptor antagonists on brain injury may be in part due to the inactivation of A2A on activated microglia. © 2009 Elsevier Ltd. All rights reserved.
1. The response of microglia to injury Microglia are resident immune cells of the central nervous system (CNS) that are capable of phagocytosis, antigen presentation, and the expression of numerous immune-related factors [1,2]. Under normal conditions, microglia exhibit small cell bodies and highly ramified processes. However, microglia retract their processes and adopt a rounded, amoeboid morphology during various brain pathologies [3]. This transition from a ramified to an amoeboid morphology appears to proceed through a set of defined steps [3]. Moreover, retraction of microglial processes strongly correlates with the functional transformation of microglia into an activated, or proinflammatory, phenotype. Thus, retraction of microglial processes is commonly viewed as a hallmark of inflammation in the brain. The long-held view that microglia are inactive, or dormant, during normal brain function has recently been challenged by observations that microglial processes are highly motile in the normal rodent brain [4]. By utilizing in vivo two-photon microscopy, several groups have recently shown that, under normal conditions, microglia constantly move their processes and appear to sample the extracellular environment of the brain [5–7]. These studies also showed that microglia quickly respond to acute insults, * Corresponding author. Stefka Gyoneva, Department of Pharmacology, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA. Tel.: +1 404 727 1375. E-mail address: [email protected] (S. Gyoneva). 1353-8020/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
such as focal disruptions in the microvasculature of the brain. Specifically, microglia rapidly extend their processes toward the site of insult and envelop the injured area [5]. Observations like these confirmed that microglia likely play important roles in normal brain physiology and also act as sensors for brain injury [8]. Indeed, multiple studies have demonstrated the ability of microglia to sense and rapidly respond to brain injury both in vitro and in vivo (Table 1). Time-lapse imaging of acute and organotypic brain slices and in vivo two-photon imaging have revealed that microglia migrate to sites of injury and clear damaged cells and cellular debris by phagocytosis [3,6,9,10,12]. Chemoattraction, or directional process extension and/or cell migration, is an important functional feature of microglial cells that can be regulated by numerous factors. Consistent with their role in the immune response in the CNS, microglia migrate toward several known chemotactic factors, such as complement proteins, transforming growth factor b (TGFb), monocyte chemoattractant protein-1 (MCP-1), and others [14–16]. Moreover, microglia may also migrate towards neurotrophic factors, such as NGF and EGF [17,18], suggesting a possible role for microglia in brain development. Pathological protein aggregates, such as amyloid-b (Ab), have also been shown to induce microglial migration [19– 22]. Interestingly, while several reports show that the migration induced by these chemoattractants is mediated by Gai -coupled receptors [16,19,21,23], none of these studies have linked Gs coupled signaling to microglial migration.
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Table 1 Summary of studies examining the roles of ATP/ADP and adenosine on microglial motility Finding
Approach
Reference(s)
Migration to injury, dead cell phagocytosis
Time-lapse video microscopy of acute slices, 3D imaging of organotypic slices
[9,10]
ATP/ADP act as chemoattractants
In vitro migration assays
[11]
Migration to ATP released by injured neurons
Time-lapse confocal microscopy of acute slices
[12]
Resting microglia are highly dynamic
In vivo time-lapse two-photon microscopy
[5,6]
Migration to injury/ATP in vivo
In vivo time-lapse two-photon microscopy
[5–7]
P2Y12 receptor mediates migration to ATP
P2Y12 -knock out microglia, confocal and in vivo two-photon microscopy
[7]
A2A receptor upregulation in activation, migration away from ATP
Confocal imaging of microglia inside Matrigel™
[13]
In addition to the classic chemokines, brain injury also induces microglial chemotaxis in a process that involves ATP/ADP release from injured tissue. These nucleotides can act as potent chemoattractants for microglia in various in vitro migration assays [11]. As with many other chemoattractants, signaling through the Gi -coupled receptors is required for migration toward ATP/ADP [11]. Kurpius et al. [12] and Davalos et al. [6] confirmed the role of ATP as a microglial chemoattractant to the site of injury in slice preparations and in vivo, respectively. In addition, localized ATP application or ATP release from dying cells induced microglial process extension toward the source of ATP. On the other hand, addition of apyrase, an enzyme that breaks down ATP to AMP, or interfering with P2Y receptor signaling with the non-selective P2Y antagonists reactive blue 2 or PPADS prevented microglial chemoattraction towards ATP [6,12]. Using a genetic approach, Haynes et al. [7] identified the Gi -coupled P2Y12 ATP/ADP receptor as a critical mediator of microglial chemoattraction to ATP and to brain injury. Specifically, while the lack of P2Y12 did not have an effect on baseline motility of microglial processes in vivo, the lack of P2Y12 prevented microglial process extension and chemotaxis in response to localized ATP application in vitro and in hippocampal slices. Moreover, microglia lacking P2Y12 exhibited delayed process extension toward acute brain injury in vivo. Interestingly, while P2Y12 is abundant in ramified microglia, its expression is quickly downregulated in microglia activated by brain injury [7]. Process extension and process retraction are two distinct, yet tightly linked, functional responses in microglia [3]. While process extension has been hypothesized to play an important role in active surveillance of brain parenchyma, process retraction is apparent in activated microglia present during neurodegeneration and neuroinflammation. Moreover, Gi -coupled signaling, and in particular P2Y12 receptor signaling, is essential for microglial response to pathological insults, and the downregulation of P2Y12 is associated with activated microglia that lack extensive branching [7]. These observations raise new questions regarding the chemotactic activity of activated microglia. Specifically, do activated microglia with diminished P2Y12 levels fail to sense ATP released during acute brain injury? How would activated microglia respond to an insult in the absence of P2Y12 receptor-mediated signaling? If activated microglia continue to exhibit an ATP response despite P2Y12 downregulation, is this response mediated by another purinergic receptor? To address these questions, we studied the effects of ATP on the chemotaxis and morphology of activated microglia. 2. The response of activated microglia to ATP Most previous studies evaluating the effects of ATP on microglial activity have examined cells derived from healthy brains, in which microglial P2Y12 is highly expressed. However, in our studies, we examined the effects of ATP on microglia pretreated with lipopolysaccharide (LPS), a well-known trigger of microglial
activation and microglial downregulation of P2Y12 [7,13]. In contrast to the established role of ATP as a chemoattractant for microglia, we found that local ATP application onto LPS-activated microglia caused chemorepulsive migration away from ATP. In addition, while bath-applied ATP induced process extension in untreated microglia, ATP induced rapid process retraction in LPS-activated microglia (Fig. 1). This reversal in microglial chemotactic and morphological response to ATP was not specific to microglia activated with LPS, as pretreatment with other toll-like receptor (TLR) agonists, such as lipotechoic acid (LTA) or CpG oligodeoxynucleotides, or with the proinflammatory cytokine tumor necrosis factor-a (TNF-a), had the same effect on responses to ATP. 3. Functional effects of Gs signaling in microglia Considering that the Gi -coupled P2Y12 receptor mediates process extension toward ATP/ADP in resting microglia, we tested the possibility that Gs -coupled signaling might mediate process retraction in response to ATP in activated microglia. Indeed, activating adenylate cyclase directly with forskolin caused process retraction in both LPS-treated and untreated microglia. As additional support for the involvement of the Gs pathway in microglial motility, blocking the pathway at the level of Gas , adenylate cyclase, or PKA reduced process retraction in response to ATP. Taken together, these observations suggest that, in addition to Gi -coupled signaling, the Gs -coupled pathway may also control microglial process motility. Because ATP triggered a motile response in cells that exhibited minimal P2Y12 receptor expression and because this motile response was mediated by Gs -coupled signaling, we hypothesized that ATP activates a Gs -coupled purinergic receptor to mediate motile responses in activated microglia. We identified this receptor to be the Gs -coupled adenosine A2A receptor. To further support the involvement of adenosine receptors, which are activated by adenosine but not by ATP, we provided evidence that ATP responses in activated microglia are dependent on the breakdown of ATP to adenosine. Moreover, while the A2A receptor mRNA was undetectable in microglia prior to LPS exposure, A2A was rapidly upregulated following microglial activation with either LPS, LTA, CpG, or TNF-a. This is consistent with recent findings showing that LPS can upregulate the A2A receptor in primary human macrophages as well as mouse and rhesus monkey microglia [24–26]. We also provided evidence that P2Y12 is simultaneously downregulated during microglial activation, supporting previous studies [7]. Similar effects of LPS on A2A and P2Y12 receptor expression were observed in acutely isolated human microglia, suggesting that the switch in microglial response to ATP from chemoattraction to chemorepulsion may be relevant for neuroinflammation in humans. Additionally, A2A receptor activation may play a similar role in microglia in vivo, since administration of an A2A -selective antagonist reversed LPS-induced process retraction as seen in fixed brain slices from mice.
S. Gyoneva et al. / Parkinsonism and Related Disorders 15S3 (2009) S195–S199
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Fig. 1. ATP application causes process extension in untreated microglia, but process retraction in LPS-treated microglia. Images show two different cells at different time points after the beginning of ATP application.
4. Significance of A2A receptor upregulation in activated microglia The chemoattractive effect of ATP release on microglia is wellestablished. However, we showed that upregulation of the adenosine A2A receptor upon microglial activation switches the chemotactic response of microglia from chemoattraction toward ATP to chemorepulsion away from ATP. We propose that this effect is a consequence of the rapid ATP breakdown to adenosine by extracellular and membrane-associated nucleotidases (e.g. CD39 and CD73) and subsequent activation of upregulated A2A receptors. This adenosine-induced effect on activated microglia represents a novel feature of the microglial response and raises many important questions. Specifically, are the effects of microglial A2A expression following inflammation and subsequent activation by adenosine important determinants of microglial response to injury? If so, which features of microglial function are impacted by A2A activation, and what are the consequences for tissue injury and recovery? Does activation of A2A on microglia during inflammation have additional downstream effects on microglial function beyond chemotaxis? To assess the effects of A2A signaling on microglial function, one could use pharmacological or genetic disruption of A2A signaling to investigate the effects on downstream targets. For example, Makranz et al. [27] and Bryn et al. [28] have shown that elevated intracellular cAMP inhibited myelin phagocytosis by macrophages. Similarly, we reported that A2A agonists decreased phagocytosis of fluorescein-labeled E. coli particles by LPS-activated microglia, perhaps through a similar cAMP-dependent mechanism. The pattern of cytokine secretion, another major function of microglia, changes after microglial activation with LPS [29]. However, the role of A2A receptor stimulation in microglial cytokine secretion is only beginning to be appreciated [25].
5. Involvement of the A2A receptor in brain pathology The A2A receptor has garnered considerable attention in the context of brain injury and chronic neurodegeneration in a wide range of neuropathological conditions [30–33]. However, a central question that remains unanswered is what fraction of the effects attributed to A2A receptors in the context of disease are related to the changes observed in microglia following neuroinflammation. We suggest that A2A upregulation in microglia and the consequent switch to process retraction in response to ATP/adenosine may contribute in part to the characteristic morphological transformation that occurs in microglia during inflammation. Moreover, we suggest that A2A receptors on activated microglia may play a key role in certain neuropathological contexts. One condition in which the A2A receptor has been implicated to play a role is Parkinson’s disease (PD). Indeed, both epidemiological and experimental data support the involvement of the A2A receptor in PD. For instance, consumption of caffeine, a non-selective adenosine receptor antagonist is correlated with lower incidence of PD [34,35]. Blocking the A2A receptor with caffeine or selective A2A antagonists attenuates the depletion of dopamine observed in the MPTP and 6-OHDA models of PD without inducing negative motor symptoms [36–41]. As a result, the A2A receptor has been proposed as a therapeutic target in the treatment of PD [31]. Based on our recent findings in microglia, we speculate that the effects of A2A receptor blockade on neurodegeneration during PD may partly involve the inhibition of A2A receptors expressed by activated microglia. Blockade of A2A receptor signaling may be neuroprotective in models of cerebral ischemia [32,42–44]. Interestingly, the expression or activity of ecto-5 -nucleotidase, an enzyme that carries out the final step of ATP conversion to adenosine, is increased after ischemia [45]. Thus, enhanced activity of this
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Fig. 2. Two purinergic signaling pathways that control microglial motility. ATP released at the site of brain injury, and its breakdown product ADP, activate Gi -coupled P2Y12 receptors on resting, highly ramified microglia. P2Y12 receptor activation causes microglia to extend their processes and migrate towards the ATP/ADP source. As microglia become activated, P2Y12 receptors are downregulated while the Gs -coupled A2A receptors are upregulated. Adenosine (formed from the breakdown of ATP) activates A2A receptors and causes microglia to retract their processes and adopt an amoeboid phenotype; in vitro results also suggest that adenosine might repel microglia away from acute sites of injury, or perhaps counteract the chemoattractive effects of other chemotactic factors released from the injured site. Modified from ref. [13].
enzyme may increase A2A receptor activation in microglia by accelerating the formation of adenosine. Since A2A receptor activation also increases COX2 and iNOS/nitric oxide levels [46,47], a possible mechanism of A2A -induced brain damage might involve the generation of inflammatory mediators. However, the actions of adenosine receptors inferred from the studies mentioned above are complex, and perhaps interrelated with other adenosine receptor family members. For instance, activation of the Gi -coupled A1 adenosine receptor is neuroprotective [32,42,48]. Interestingly, Pedata et al. [32] have proposed that A2A receptors may antagonize the actions of A1 receptors, suggesting that the neuroprotective effects of A2A blockade are mediated by increased A1 receptor signaling. The interactions between receptors within the adenosine receptor family might be even more complicated. Notably, van der Putten et al. [25] have recently found that, in addition to increased A2A receptor signaling, activated microglia showed a decline in A3 receptor signaling. This dynamic adenosine receptor expression in activated microglia is proposed to mediate the potent suppression of cytokine release upon adenosine exposure. The study suggests that blockade of A2A receptors may increase pro-inflammatory cytokine release by activated microglia, thereby enhancing CNS inflammation and secondary tissue damage, a possible problem for the development of A2A antagonists for therapeutic purposes. Clearly, in order to predict the effects of therapeutic interventions that target adenosine receptors in the brain, more studies are needed to further examine how adenosine receptor signaling in microglia may change in different pathophysiological contexts and how adenosine receptors may modulate the various facets of microglial function in these contexts. 6. Conclusions The motile activity of microglia, the resident immune cells of the CNS, is controlled by two opposing purinergic signaling pathways: the Gi -coupled P2Y12 receptor and the Gs -coupled A2A receptor (Fig. 2). Although these pathways clearly control microglial cell shape, process motion, and migration, the effects of these pathways on cytokine secretion, phagocytosis, and other microglial
functions require further investigation. However, the extensive chemotactic effects observed in microglia thus far, together with the involvement of the A2A receptor in brain pathologies and cytokine release, make a compelling case for further studies of microglial A2A receptor function in the brain. Acknowledgements The experiments and writing of the manuscript were supported in part by the US National Institutes of Health (NIH) Pharmacological Sciences Training Grant (T32 GM008602, S.G.), NIH National Research Service Award fellowship (NS054515, A.G.O.), a pilot grant from NIH (ES016731, S.T.) and the National Parkinson’s Foundation (S.T.). Conflict of interests The authors declare no conflict of interest. References 1. Ulvestad E, Williams K, Bjerkvig R, Tiekotter K, Antel J, Matre R. Human microglial cells have phenotypic and functional characteristics in common with both macrophages and dendritic antigen-presenting cells. J Leukocyte Biol 1994;56:732–40. 2. Streit WJ. Microglia as neuroprotective, immunocompetent cells of the CNS. Glia 2002;40:133–9. 3. Stence N, Waite M, Dailey ME. Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices. Glia 2001;33:256–66. 4. Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends Neurosci 1996;19(8):312–8. 5. Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 2005;308:1314–8. 6. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 2005;8(6):752–8. 7. Haynes S, Hollopeter G, Yang G, Kurpius D, Dailey ME, Gan W-B, et al. The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci 2006;9(12):1512–9. 8. Hanisch U-K, Kettenman H. Microglia: active sensors and versatile effector cells in the normal and pathologic brain. Nat Neurosci 2007;10(11):1387–94. 9. Brockhaus J, Moller T, Kettenman H. Phagocytozing ameboid microglial cells studied in a mouse corpus callosum slice preparation. Glia 1996;16:81–90. 10. Petersen MA, Dailey ME. Diverse microglial motility behaviors during clearance of dead cells in hippocampal slices. Glia 2004;46:195–206.
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Contents lists available at ScienceDirect
Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Differential regulation of microglial motility by ATP/ADP and adenosine Stefka Gyonevaa, ∗ , Anna G. Orrb , Stephen F. Traynelisa a Department b Gladstone
of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA Institute of Neurological Disease, University of California San Francisco, San Francisco, CA, USA
article info
summary
Keywords: Microglia ATP, adenosine A2A receptor Chemotaxis Neurodegeneration
Microglia are motile immune-competent cells of the central nervous system. They assume a highly branched morphology and monitor the brain parenchyma under physiological conditions. In the presence of injury, microglia retract their branching processes, migrate to the site of injury, and help clear cellular debris by phagocytosis. This response appears to be mediated in part by ATP released at the site of injury. Here, we review the evidence for the involvement of ATP and the purinergic P2Y12 receptor in microglial process extension and chemoattraction to injury. We subsequently discuss recent findings regarding a switch of this chemotactic response to ATP in activated, or proinflammatory, microglia. Specifically, in LPS-activated microglia, ATP induces process retraction and repulsive migration, effects opposite to those seen in unstimulated cells. These repulsive effects of ATP are mediated by the Gs -coupled adenosine A2A receptor and depend on the breakdown of ATP to adenosine. Thus, ATP-induced repulsion by activated microglia involves upregulation of the adenosine A2A receptor and coincident downregulation of the P2Y12 receptor. The roles of the A2A receptor in brain pathologies such as Parkinson’s disease and ischemia are also examined. We propose that the effects of A2A receptor antagonists on brain injury may be in part due to the inactivation of A2A on activated microglia. © 2009 Elsevier Ltd. All rights reserved.
1. The response of microglia to injury Microglia are resident immune cells of the central nervous system (CNS) that are capable of phagocytosis, antigen presentation, and the expression of numerous immune-related factors [1,2]. Under normal conditions, microglia exhibit small cell bodies and highly ramified processes. However, microglia retract their processes and adopt a rounded, amoeboid morphology during various brain pathologies [3]. This transition from a ramified to an amoeboid morphology appears to proceed through a set of defined steps [3]. Moreover, retraction of microglial processes strongly correlates with the functional transformation of microglia into an activated, or proinflammatory, phenotype. Thus, retraction of microglial processes is commonly viewed as a hallmark of inflammation in the brain. The long-held view that microglia are inactive, or dormant, during normal brain function has recently been challenged by observations that microglial processes are highly motile in the normal rodent brain [4]. By utilizing in vivo two-photon microscopy, several groups have recently shown that, under normal conditions, microglia constantly move their processes and appear to sample the extracellular environment of the brain [5–7]. These studies also showed that microglia quickly respond to acute insults, * Corresponding author. Stefka Gyoneva, Department of Pharmacology, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA. Tel.: +1 404 727 1375. E-mail address: [email protected] (S. Gyoneva). 1353-8020/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
such as focal disruptions in the microvasculature of the brain. Specifically, microglia rapidly extend their processes toward the site of insult and envelop the injured area [5]. Observations like these confirmed that microglia likely play important roles in normal brain physiology and also act as sensors for brain injury [8]. Indeed, multiple studies have demonstrated the ability of microglia to sense and rapidly respond to brain injury both in vitro and in vivo (Table 1). Time-lapse imaging of acute and organotypic brain slices and in vivo two-photon imaging have revealed that microglia migrate to sites of injury and clear damaged cells and cellular debris by phagocytosis [3,6,9,10,12]. Chemoattraction, or directional process extension and/or cell migration, is an important functional feature of microglial cells that can be regulated by numerous factors. Consistent with their role in the immune response in the CNS, microglia migrate toward several known chemotactic factors, such as complement proteins, transforming growth factor b (TGFb), monocyte chemoattractant protein-1 (MCP-1), and others [14–16]. Moreover, microglia may also migrate towards neurotrophic factors, such as NGF and EGF [17,18], suggesting a possible role for microglia in brain development. Pathological protein aggregates, such as amyloid-b (Ab), have also been shown to induce microglial migration [19– 22]. Interestingly, while several reports show that the migration induced by these chemoattractants is mediated by Gai -coupled receptors [16,19,21,23], none of these studies have linked Gs coupled signaling to microglial migration.
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S. Gyoneva et al. / Parkinsonism and Related Disorders 15S3 (2009) S195–S199
Table 1 Summary of studies examining the roles of ATP/ADP and adenosine on microglial motility Finding
Approach
Reference(s)
Migration to injury, dead cell phagocytosis
Time-lapse video microscopy of acute slices, 3D imaging of organotypic slices
[9,10]
ATP/ADP act as chemoattractants
In vitro migration assays
[11]
Migration to ATP released by injured neurons
Time-lapse confocal microscopy of acute slices
[12]
Resting microglia are highly dynamic
In vivo time-lapse two-photon microscopy
[5,6]
Migration to injury/ATP in vivo
In vivo time-lapse two-photon microscopy
[5–7]
P2Y12 receptor mediates migration to ATP
P2Y12 -knock out microglia, confocal and in vivo two-photon microscopy
[7]
A2A receptor upregulation in activation, migration away from ATP
Confocal imaging of microglia inside Matrigel™
[13]
In addition to the classic chemokines, brain injury also induces microglial chemotaxis in a process that involves ATP/ADP release from injured tissue. These nucleotides can act as potent chemoattractants for microglia in various in vitro migration assays [11]. As with many other chemoattractants, signaling through the Gi -coupled receptors is required for migration toward ATP/ADP [11]. Kurpius et al. [12] and Davalos et al. [6] confirmed the role of ATP as a microglial chemoattractant to the site of injury in slice preparations and in vivo, respectively. In addition, localized ATP application or ATP release from dying cells induced microglial process extension toward the source of ATP. On the other hand, addition of apyrase, an enzyme that breaks down ATP to AMP, or interfering with P2Y receptor signaling with the non-selective P2Y antagonists reactive blue 2 or PPADS prevented microglial chemoattraction towards ATP [6,12]. Using a genetic approach, Haynes et al. [7] identified the Gi -coupled P2Y12 ATP/ADP receptor as a critical mediator of microglial chemoattraction to ATP and to brain injury. Specifically, while the lack of P2Y12 did not have an effect on baseline motility of microglial processes in vivo, the lack of P2Y12 prevented microglial process extension and chemotaxis in response to localized ATP application in vitro and in hippocampal slices. Moreover, microglia lacking P2Y12 exhibited delayed process extension toward acute brain injury in vivo. Interestingly, while P2Y12 is abundant in ramified microglia, its expression is quickly downregulated in microglia activated by brain injury [7]. Process extension and process retraction are two distinct, yet tightly linked, functional responses in microglia [3]. While process extension has been hypothesized to play an important role in active surveillance of brain parenchyma, process retraction is apparent in activated microglia present during neurodegeneration and neuroinflammation. Moreover, Gi -coupled signaling, and in particular P2Y12 receptor signaling, is essential for microglial response to pathological insults, and the downregulation of P2Y12 is associated with activated microglia that lack extensive branching [7]. These observations raise new questions regarding the chemotactic activity of activated microglia. Specifically, do activated microglia with diminished P2Y12 levels fail to sense ATP released during acute brain injury? How would activated microglia respond to an insult in the absence of P2Y12 receptor-mediated signaling? If activated microglia continue to exhibit an ATP response despite P2Y12 downregulation, is this response mediated by another purinergic receptor? To address these questions, we studied the effects of ATP on the chemotaxis and morphology of activated microglia. 2. The response of activated microglia to ATP Most previous studies evaluating the effects of ATP on microglial activity have examined cells derived from healthy brains, in which microglial P2Y12 is highly expressed. However, in our studies, we examined the effects of ATP on microglia pretreated with lipopolysaccharide (LPS), a well-known trigger of microglial
activation and microglial downregulation of P2Y12 [7,13]. In contrast to the established role of ATP as a chemoattractant for microglia, we found that local ATP application onto LPS-activated microglia caused chemorepulsive migration away from ATP. In addition, while bath-applied ATP induced process extension in untreated microglia, ATP induced rapid process retraction in LPS-activated microglia (Fig. 1). This reversal in microglial chemotactic and morphological response to ATP was not specific to microglia activated with LPS, as pretreatment with other toll-like receptor (TLR) agonists, such as lipotechoic acid (LTA) or CpG oligodeoxynucleotides, or with the proinflammatory cytokine tumor necrosis factor-a (TNF-a), had the same effect on responses to ATP. 3. Functional effects of Gs signaling in microglia Considering that the Gi -coupled P2Y12 receptor mediates process extension toward ATP/ADP in resting microglia, we tested the possibility that Gs -coupled signaling might mediate process retraction in response to ATP in activated microglia. Indeed, activating adenylate cyclase directly with forskolin caused process retraction in both LPS-treated and untreated microglia. As additional support for the involvement of the Gs pathway in microglial motility, blocking the pathway at the level of Gas , adenylate cyclase, or PKA reduced process retraction in response to ATP. Taken together, these observations suggest that, in addition to Gi -coupled signaling, the Gs -coupled pathway may also control microglial process motility. Because ATP triggered a motile response in cells that exhibited minimal P2Y12 receptor expression and because this motile response was mediated by Gs -coupled signaling, we hypothesized that ATP activates a Gs -coupled purinergic receptor to mediate motile responses in activated microglia. We identified this receptor to be the Gs -coupled adenosine A2A receptor. To further support the involvement of adenosine receptors, which are activated by adenosine but not by ATP, we provided evidence that ATP responses in activated microglia are dependent on the breakdown of ATP to adenosine. Moreover, while the A2A receptor mRNA was undetectable in microglia prior to LPS exposure, A2A was rapidly upregulated following microglial activation with either LPS, LTA, CpG, or TNF-a. This is consistent with recent findings showing that LPS can upregulate the A2A receptor in primary human macrophages as well as mouse and rhesus monkey microglia [24–26]. We also provided evidence that P2Y12 is simultaneously downregulated during microglial activation, supporting previous studies [7]. Similar effects of LPS on A2A and P2Y12 receptor expression were observed in acutely isolated human microglia, suggesting that the switch in microglial response to ATP from chemoattraction to chemorepulsion may be relevant for neuroinflammation in humans. Additionally, A2A receptor activation may play a similar role in microglia in vivo, since administration of an A2A -selective antagonist reversed LPS-induced process retraction as seen in fixed brain slices from mice.
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Fig. 1. ATP application causes process extension in untreated microglia, but process retraction in LPS-treated microglia. Images show two different cells at different time points after the beginning of ATP application.
4. Significance of A2A receptor upregulation in activated microglia The chemoattractive effect of ATP release on microglia is wellestablished. However, we showed that upregulation of the adenosine A2A receptor upon microglial activation switches the chemotactic response of microglia from chemoattraction toward ATP to chemorepulsion away from ATP. We propose that this effect is a consequence of the rapid ATP breakdown to adenosine by extracellular and membrane-associated nucleotidases (e.g. CD39 and CD73) and subsequent activation of upregulated A2A receptors. This adenosine-induced effect on activated microglia represents a novel feature of the microglial response and raises many important questions. Specifically, are the effects of microglial A2A expression following inflammation and subsequent activation by adenosine important determinants of microglial response to injury? If so, which features of microglial function are impacted by A2A activation, and what are the consequences for tissue injury and recovery? Does activation of A2A on microglia during inflammation have additional downstream effects on microglial function beyond chemotaxis? To assess the effects of A2A signaling on microglial function, one could use pharmacological or genetic disruption of A2A signaling to investigate the effects on downstream targets. For example, Makranz et al. [27] and Bryn et al. [28] have shown that elevated intracellular cAMP inhibited myelin phagocytosis by macrophages. Similarly, we reported that A2A agonists decreased phagocytosis of fluorescein-labeled E. coli particles by LPS-activated microglia, perhaps through a similar cAMP-dependent mechanism. The pattern of cytokine secretion, another major function of microglia, changes after microglial activation with LPS [29]. However, the role of A2A receptor stimulation in microglial cytokine secretion is only beginning to be appreciated [25].
5. Involvement of the A2A receptor in brain pathology The A2A receptor has garnered considerable attention in the context of brain injury and chronic neurodegeneration in a wide range of neuropathological conditions [30–33]. However, a central question that remains unanswered is what fraction of the effects attributed to A2A receptors in the context of disease are related to the changes observed in microglia following neuroinflammation. We suggest that A2A upregulation in microglia and the consequent switch to process retraction in response to ATP/adenosine may contribute in part to the characteristic morphological transformation that occurs in microglia during inflammation. Moreover, we suggest that A2A receptors on activated microglia may play a key role in certain neuropathological contexts. One condition in which the A2A receptor has been implicated to play a role is Parkinson’s disease (PD). Indeed, both epidemiological and experimental data support the involvement of the A2A receptor in PD. For instance, consumption of caffeine, a non-selective adenosine receptor antagonist is correlated with lower incidence of PD [34,35]. Blocking the A2A receptor with caffeine or selective A2A antagonists attenuates the depletion of dopamine observed in the MPTP and 6-OHDA models of PD without inducing negative motor symptoms [36–41]. As a result, the A2A receptor has been proposed as a therapeutic target in the treatment of PD [31]. Based on our recent findings in microglia, we speculate that the effects of A2A receptor blockade on neurodegeneration during PD may partly involve the inhibition of A2A receptors expressed by activated microglia. Blockade of A2A receptor signaling may be neuroprotective in models of cerebral ischemia [32,42–44]. Interestingly, the expression or activity of ecto-5 -nucleotidase, an enzyme that carries out the final step of ATP conversion to adenosine, is increased after ischemia [45]. Thus, enhanced activity of this
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Fig. 2. Two purinergic signaling pathways that control microglial motility. ATP released at the site of brain injury, and its breakdown product ADP, activate Gi -coupled P2Y12 receptors on resting, highly ramified microglia. P2Y12 receptor activation causes microglia to extend their processes and migrate towards the ATP/ADP source. As microglia become activated, P2Y12 receptors are downregulated while the Gs -coupled A2A receptors are upregulated. Adenosine (formed from the breakdown of ATP) activates A2A receptors and causes microglia to retract their processes and adopt an amoeboid phenotype; in vitro results also suggest that adenosine might repel microglia away from acute sites of injury, or perhaps counteract the chemoattractive effects of other chemotactic factors released from the injured site. Modified from ref. [13].
enzyme may increase A2A receptor activation in microglia by accelerating the formation of adenosine. Since A2A receptor activation also increases COX2 and iNOS/nitric oxide levels [46,47], a possible mechanism of A2A -induced brain damage might involve the generation of inflammatory mediators. However, the actions of adenosine receptors inferred from the studies mentioned above are complex, and perhaps interrelated with other adenosine receptor family members. For instance, activation of the Gi -coupled A1 adenosine receptor is neuroprotective [32,42,48]. Interestingly, Pedata et al. [32] have proposed that A2A receptors may antagonize the actions of A1 receptors, suggesting that the neuroprotective effects of A2A blockade are mediated by increased A1 receptor signaling. The interactions between receptors within the adenosine receptor family might be even more complicated. Notably, van der Putten et al. [25] have recently found that, in addition to increased A2A receptor signaling, activated microglia showed a decline in A3 receptor signaling. This dynamic adenosine receptor expression in activated microglia is proposed to mediate the potent suppression of cytokine release upon adenosine exposure. The study suggests that blockade of A2A receptors may increase pro-inflammatory cytokine release by activated microglia, thereby enhancing CNS inflammation and secondary tissue damage, a possible problem for the development of A2A antagonists for therapeutic purposes. Clearly, in order to predict the effects of therapeutic interventions that target adenosine receptors in the brain, more studies are needed to further examine how adenosine receptor signaling in microglia may change in different pathophysiological contexts and how adenosine receptors may modulate the various facets of microglial function in these contexts. 6. Conclusions The motile activity of microglia, the resident immune cells of the CNS, is controlled by two opposing purinergic signaling pathways: the Gi -coupled P2Y12 receptor and the Gs -coupled A2A receptor (Fig. 2). Although these pathways clearly control microglial cell shape, process motion, and migration, the effects of these pathways on cytokine secretion, phagocytosis, and other microglial
functions require further investigation. However, the extensive chemotactic effects observed in microglia thus far, together with the involvement of the A2A receptor in brain pathologies and cytokine release, make a compelling case for further studies of microglial A2A receptor function in the brain. Acknowledgements The experiments and writing of the manuscript were supported in part by the US National Institutes of Health (NIH) Pharmacological Sciences Training Grant (T32 GM008602, S.G.), NIH National Research Service Award fellowship (NS054515, A.G.O.), a pilot grant from NIH (ES016731, S.T.) and the National Parkinson’s Foundation (S.T.). Conflict of interests The authors declare no conflict of interest. References 1. Ulvestad E, Williams K, Bjerkvig R, Tiekotter K, Antel J, Matre R. Human microglial cells have phenotypic and functional characteristics in common with both macrophages and dendritic antigen-presenting cells. J Leukocyte Biol 1994;56:732–40. 2. Streit WJ. Microglia as neuroprotective, immunocompetent cells of the CNS. Glia 2002;40:133–9. 3. Stence N, Waite M, Dailey ME. Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices. Glia 2001;33:256–66. 4. Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends Neurosci 1996;19(8):312–8. 5. Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 2005;308:1314–8. 6. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 2005;8(6):752–8. 7. Haynes S, Hollopeter G, Yang G, Kurpius D, Dailey ME, Gan W-B, et al. The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci 2006;9(12):1512–9. 8. Hanisch U-K, Kettenman H. Microglia: active sensors and versatile effector cells in the normal and pathologic brain. Nat Neurosci 2007;10(11):1387–94. 9. Brockhaus J, Moller T, Kettenman H. Phagocytozing ameboid microglial cells studied in a mouse corpus callosum slice preparation. Glia 1996;16:81–90. 10. Petersen MA, Dailey ME. Diverse microglial motility behaviors during clearance of dead cells in hippocampal slices. Glia 2004;46:195–206.
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