Differential regulation of microglial motility by ATP/ADP and adenosine

Abstract

Microglia are very active 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 G_s-coupled adenosine A_2A receptor and depend on the breakdown of ATP to adenosine. Thus, ATP-induced repulsion by activated microglia involves upregulation of the adenosine A_2A receptor and coincident downregulation of the P2Y₁₂ receptor. The roles of the A_2A receptor in brain pathologies such as Parkinson’s disease and ischemia are also examined. We propose that the effects of A_2A receptor antagonists on brain injury may be in part due to the inactivation of A_2A on activated microglia.

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 idea 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, 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, 9-11].

Table 1.

Summary of studies examining the roles of ATP/ADP and adenosine on microglial motility

Finding	Aprroach	Reference
Migration to injury, dead cell phagocytosis	Time-lapse video microscopy of acute slices, 3D imaging of organotypic slices	[9, 11]
ATP/ADP act as chemoattractants	In vitro migration assays	[22]
Migration to ATP released by injured neurons	Time-lapse confocal microscopy of acute slices	[10]
Resting microglia are highly dynamic	In vivo time-lapse two-photon microscopy	[5]
Migration to injury/ATP in vivo	In vivo time-lapse two-photon microscopy	[6]
P2Y12 receptor mediates migration to ATP	P2Y12-knock out migroglia, confocal and in vivo two-photon microscopy	[7]
A2A receptor upregulation in activation, migration away from ATP	Confocal imaging of microglia inside Matrigel	[23]

Chemoattraction, or direction 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 β (TGFβ), monocyte chemoattractant protein-1 (MCP-1), and others [6, 12-14]. Moreover, microglia may also migrate towards neurotrophic factors, such as NGF and EGF [15, 16], suggesting a possible role for microglia in brain development. Pathological protein aggregates, such as amyloid-β (Aβ), have also been shown to induce microglial migration [17-20]. Interestingly, while several reports show that the migration induced by these chemoattractants is mediated by Gα_i-coupled receptors [14, 17, 19, 21], none of these studies have linked G_s-coupled signaling to microglial migration.

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 [22]. As with many other chemoattractants, signaling through the G_i-coupled receptors is required for migration toward ATP/ADP [22]. Kurpius et al. [10] and Davalos et al. [6] confirmed the role for 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, 10]. Using a genetic approach, Haynes et al. [7] identified the G_i-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 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, G_i-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 P2Y 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, 23]. 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 (Figure 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-α (TNF-α), had the same effect on responses to ATP.

Figure 1.

ATP application causes process extension in untreated microglia, but process retraction in LPS-treated microglia. Images show a single cell at different time points after the beginning of ATP application.

3. Functional Effects of G_s Signaling in Microglia

Considering that the G_i-coupled P2Y₁₂ receptor mediates process extension toward ATP/ADP in resting microglia, we tested the possibility that G_s-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 G_s pathway in microglial motility, blocking the pathway at the level of Gα_s, adenylate cyclase, or PKA reduced process retraction in response to ATP. Taken together, these observations suggest that, in addition to G_i-coupled signaling, the G_s-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 G_s-coupled signaling, we hypothesized that ATP activates a G_s-coupled purinergic receptor to mediate motile responses in activated microglia. We identified this receptor to be the G_s-coupled adenosine A_2A 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 A_2A receptor mRNA was undetectable in microglia prior to LPS exposure, A_2A was rapidly upregulated following microglial activation with either LPS, LTA, CpG, or TNF-α. This is consistent with recent findings showing that LPS can upregulate the A_2A receptor in primary human macrophages as well as mouse and rhesus monkey microglia [24, 25, 26]. We also provided evidence that P2Y₁₂ is simultaneously downregulated during microglial activation, supporting previous studies [7]. Similar effects of LPS on A_2A 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, A_2A receptor activation may play a similar role in microglia in vivo, since administration of an A_2A-selective antagonist reversed LPS-induced process retraction as seen in fixed brain slices from mice.

4. Significance of A_2A Receptor Upregulation in Activated Microglia

The chemoattractive effect of ATP release on microglia is well-established. However, we showed that upregulation of the adenosine A_2A 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 A_2A 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 A_2A 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 A_2A activation, and what are the consequences for tissue injury and recovery? Does activation of A_2A on microglia during inflammation have additional downstream effects on microglial function beyond chemotaxis?

To assess the effects of A_2A signaling on microglial function, one could use pharmacological or genetic disruption of A_2A 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 A_2A 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 A_2A receptor stimulation in microglial cytokine secretion is only beginning to be appreciated [25].

5. Involvement of the A_2A Receptor in Brain Pathology

The A_2A 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 A_2A receptors in the context of disease are related to the changes observed in microglia following neuroinflammation. We suggest that A_2A 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 A_2A receptors on activated microglia may play a key role in certain neuropathological contexts.

One condition in which the A_2A receptor has been implicated to play a role is Parkinson’s disease (PD). Indeed, both epidemiological and experimental data support the involvement of the A_2A 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 A_2A receptor with caffeine or selective A_2A antagonists attenuated the depletion of dopamine observed in the MPTP and 6-OHDA models of PD without inducing negative motor symptoms in several animal PD models [36-41]. As a result, the A_2A 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 A_2A receptor blockade on neurodegeneration during PD may partly involve the inhibition of A_2A receptors expressed by activated microglia.

Blockade of A_2A receptor signaling may be neuroprotective in models of cerebral ischemia [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 enzyme may increase A_2A receptor activation in microglia by accelerating the formation of adenosine. Since A_2A receptor activation also increases COX2 and iNOS/nitric oxide levels [46, 47], a possible mechanism of A_2A-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 G_i-coupled A₁ adenosine receptor is neuroprotective [32, 42, 48]. Interestingly, Pedata et al. [32] have proposed that A_2A receptors may antagonize the actions of A₁ receptors, suggesting that the neuroprotective effects of A_2A 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 A_2A 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 A_2A 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 A_2A 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 G_i-coupled P2Y₁₂ receptor and the G_s-coupled A_2A receptor (Figure 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 A_2A receptor in brain pathologies and cytokine release, make a compelling case for further studies of microglial A_2A receptor function in the brain.

Figure 2.

Two purinergic signaling pathways that control microglial motility. ATP released at the site of brain injury, and its breakdown product ADP, activate G_i-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 G_s-coupled A_2A receptors are upregulated. Adenosine (formed from the breakdown of ATP) activates A_2A 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.