ReviewPathophysiological roles of extracellular nucleotides in glial cells: differential expression of purinergic receptors in resting and activated microglia
Introduction
While the brain has been considered for a long time a privileged system from an immunological standpoint, it is instead needful of protection from external insults [40]. Microglial cells, which react to almost any kind of pathological conditions, often preceeding the reaction of the other cell types in the brain [19], are thought to play a major role in the immune response which occurs in the CNS. Upon activation, microglial cells acquire features of cytotoxic and phagocytic cells, therefore taking part in the remodeling of the nervous tissue following pathological insults. Several recent studies have focused on the extracellular mediators that trigger microglial reaction to injury. Among various substances, including growth factors, cytokines, chemoattractants, and neurotransmitters [25], extracellular ATP has been indicated as a key messenger in microglial activation. ATP can either derive from degenerating and dying cells [15] or be released from astrocytes even in the absence of cell damage [37], [43]. High levels of extracellular purines—accumulated at the site of lesion—directly induce P2-mediated calcium responses in microglial cells. On the other hand, the activation of P2 receptors on microglia at sites far away from damaged cells could proceed through indirect propagation of ATP-mediated calcium signal among astrocytes (reviewed in [30], [37], [43]). Interestingly, ATP-mediated intercellular signaling between astrocytes and microglia has been suggested to play a role in pathological rather than in physiological signaling events [30].
ATP activates specific ligand-gated P2X and G-protein-coupled P2Y receptors [1], [24]. The P2X receptor family includes seven distinct mammalian members, from P2X1 to P2X7, whose activation induces intracellular calcium responses by promoting calcium entry from the extracellular space. Eight members have been so far included in the P2Y receptor family, namely, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and the recently cloned P2Y13 and P2Y14. Very recently, a receptor responding to both AMP and adenosine provisionally classified as P2Y15 has been also reported [21]. However, this receptor is not a genuine purinergic receptor. From a phylogenetic and structural point of view, two distinct P2Y receptor subgroups with relatively high structural divergence have been identified, one encompassing the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors and the other one including the P2Y12, P2Y13, and P2Y14 receptors [2]. Receptors in the first subgroup seem to mainly couple to Gq proteins leading to activation of phospholipase-C (PLC), inositol-phosphate formation, and release of calcium from intracellular stores, whereas receptors in the second subgroup are mainly coupled to Gi and inhibition of intracellular cAMP levels [2], [27].
These receptors play important pathophysiological roles in glial cells, as also suggested by their broad expression in cells of both the astrocytic and microglial lineages (see also below). Interestingly, astrocytes concomitantly express several distinct P2X and P2Y receptors, but under resting conditions, only some of these receptors are functional (see, for example, [17]). This suggests that P2 receptors may be differentially expressed and recruited by extracellular nucleotides depending upon specific pathophysiological conditions. Regarding microglial cells, functional ionotropic and metabotropic P2 receptors have been reported both in culture [14], [31], [47] and in situ [4], [18], [22]. However, a conclusive definition of the P2 receptor profile in these cells has been hampered by the lack of subtype-specific pharmacological tools and by the heterogeneity of the species and preparations used [20]. Among P2X family members, there is presently conclusive immunohistochemical and functional evidence only for the P2X4 [42] and P2X7 receptors [8], [11], [14], [28]. Both P2X4 and P2X7 subtypes play a role in microglial response to injury. P2X7 is capable of a conformational change that results in larger pore diameter following prolonged exposure to ATP, associated with sustained calcium influx and interleukin-1β release [12], [15], [35], [44]. P2X4 has been recently shown to mediate tactile allodynia after nerve injury [42]. Concerning the P2Y family, molecular and/or functional evidence has clearly demonstrated the presence of only P2Y1, P2Y2/4 [4], and P2Y12 receptors [36]. While the physiological functions triggered by P2Y1 and P2Y2/4 receptor activation in microglia are still unknown, P2Y12 signaling has been recently suggested to mediate chemotaxis and migration of microglial cells towards damaged neurons [36]. Interestingly, pharmacological screening with subtype-specific purinergic ligands indicated that the purinoreceptor profile can change in activated as compared to resting microglia [26], [32], [45], with a general reduction in P2 receptor responsiveness being reported in cells activated with bacterial lipopolysaccharide (LPS) [28]. However, concerning the P2X7 subtype, which is associated to interleukin-1β release and is believed to be the most important receptor in microglial activation in CNS pathology, conflicting results have been reported. While calcium measurement studies indicated a general reduced function of P2 purinoreceptors in reactive cells (reviewed in [22]), immunohistochemical studies indicated a strong increase in the P2X7 receptor protein in hyperactive microglia [11], suggesting that a combination of different techniques should be utilized to fully characterize the changes of these receptors in these cells.
In the present work, the expression of all known P2 receptors was evaluated in the murine microglial N9 cell line at both mRNA and, when possible, at protein levels. Moreover, given the importance of intracellular calcium increases in short-term communication within different types of brain cells, the function of the expressed receptors was evaluated by screening responses to the available P2 subtype-specific agonists by single cell calcium measurements, both in N9 and in primary microglial cells. The analysis was carried out both in resting condition and after exposure to the activating flogogenous stimulus LPS.
Section snippets
Cell cultures
The N9 murine microglial cell line (generously provided by Prof. Paola Ricciardi-Castagnoli, Milan, Italy) was generated by infecting embryonic brain cultures with the 3RV retrovirus carrying an activated v-myc oncogene [34]. N9 cells were maintained in IMDM medium (Invitrogen, Milan, Italy), additioned with 5% fetal calf serum (Euroclone, Milan, Italy), 100 IU/ml penicillin, 10 mg/ml streptomycin, 2 mM l-glutamine, and 50 nM βME (Invitrogen, Milan, Italy) at 37 °C and 5% CO2. For calcium
Co-expression of multiple P2X and P2Y receptor subtypes in murine microglial cells
RT-PCR analysis performed in N9 murine microglial cells with primers specifically designed for the various P2X receptors cloned from this species (Table 1) revealed bands corresponding to all known P2X receptors (Fig. 1A). Western blot analysis performed on N9 homogenates revealed the presence of P2X1, P2X2, P2X3, P2X4, P2X6, and P2X7 receptors (Fig. 1B). With the exception of P2X2, and P2X6, P2X receptors were all identified by a major protein band of the predicted molecular weight, whose
N9 cells as an experimental model to study the purinergic regulation of brain microglia
The N9 cells have been extensively used as a microglial cellular model because they share with their in situ counterpart several features, including intense phagocytosis and active cytokine secretion [49]. N9 cells also appear to express a fairly complete endowment of membrane receptors, as they show functional responses to several agonists, whose activation is coupled to increases of intracellular calcium or sodium, including nicotine, acetylcholine, GABA, NAD+, glutamate, and ATP (F. Bianco
Acknowledgments
Authors are grateful to Dr. John Villiger (The Medicines Company, Auckland New Zealand) for kindly providing AR-C69931MX. This work was partially supported by The Italian Ministry of Education (MIUR, Progetto F.I.R.B. No. RBAUO19ZEN “Ruolo dell' ATP extracellulare e dei recettori purinergici centrali e periferici in processi fisiologici e patologici” to MPA).
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These authors equally contributed to the work.