Molecular Neurobiology

, Volume 46, Issue 1, pp 96–113

P2 Receptors for Extracellular Nucleotides in the Central Nervous System: Role of P2X7 and P2Y2 Receptor Interactions in Neuroinflammation

Authors

    • Department of BiochemistryUniversity of Missouri
    • Interdisciplinary Neuroscience ProgramUniversity of Missouri
    • Christopher S. Bond Life Sciences CenterUniversity of Missouri
  • Jean M. Camden
    • Department of BiochemistryUniversity of Missouri
    • Christopher S. Bond Life Sciences CenterUniversity of Missouri
  • Troy S. Peterson
    • Interdisciplinary Neuroscience ProgramUniversity of Missouri
    • Christopher S. Bond Life Sciences CenterUniversity of Missouri
  • Deepa Ajit
    • Department of BiochemistryUniversity of Missouri
    • Christopher S. Bond Life Sciences CenterUniversity of Missouri
  • Lucas T. Woods
    • Department of BiochemistryUniversity of Missouri
    • Christopher S. Bond Life Sciences CenterUniversity of Missouri
  • Laurie Erb
    • Department of BiochemistryUniversity of Missouri
    • Christopher S. Bond Life Sciences CenterUniversity of Missouri
Article

DOI: 10.1007/s12035-012-8263-z

Cite this article as:
Weisman, G.A., Camden, J.M., Peterson, T.S. et al. Mol Neurobiol (2012) 46: 96. doi:10.1007/s12035-012-8263-z

Abstract

Extracellular nucleotides induce cellular responses in the central nervous system (CNS) through the activation of ionotropic P2X and metabotropic P2Y nucleotide receptors. Activation of these receptors regulates a wide range of physiological and pathological processes. In this review, we present an overview of the current literature regarding P2X and P2Y receptors in the CNS with a focus on the contribution of P2X7 and P2Y2 receptor-mediated responses to neuroinflammatory and neuroprotective mechanisms.

Keywords

P2Y2 receptorP2X7 receptorNeuroprotectionNeuroinflammation

Introduction

Extracellular nucleotides, such as adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP), are released from cells under a variety of physiological and pathological conditions whereupon they activate P2 nucleotide receptors on the surface of neighboring cells [1, 2]. P2 receptors are a diverse family of plasma membrane proteins that can be segregated into two subtypes: the P2X receptors that are ATP-selective cation channels and the P2Y receptors for ATP, UTP, or their metabolites that are coupled to heterotrimeric G proteins [3, 4]. To date, genes for seven P2X receptors and eight P2Y receptors have been cloned and their protein products have been extensively characterized in a variety of cell and tissue types [5, 6]. In the central nervous system (CNS), multiple P2X and P2Y receptor subtypes are expressed in neurons, glial cells, oligodendrocytes, macrophages, and endothelium where they regulate physiological responses, including neurotransmission, pain perception, phagocytosis, and maintenance of the blood–brain barrier [79]. Pathophysiological responses are also regulated by P2X and P2Y receptors, including the propagation of inflammation due to the release of nucleotide agonists from damaged or diseased cells [1012]. This review describes the contributions of both P2X and P2Y receptors to cell specific functions in the CNS and focuses on the dual roles of the ionotropic P2X7 receptor (P2X7R) for ATP and the G protein-coupled P2Y2 receptor (P2Y2R) for ATP and UTP in the regulation of proinflammatory responses in the brain. Recent studies have found that the release of extracellular ATP from stressed or damaged cells of the CNS can activate microglial cell P2X7Rs, which increases cytokine release, e.g., interleukin-1β (IL-1β), and the phagocytic activity of microglial cells [13]. Additionally, IL-1β has been shown to upregulate P2Y2R expression in neurons to promote neuroprotective responses [14]. These findings are reviewed in this paper and suggest that both P2X7 and P2Y2 receptors are promising targets for the treatment of neurodegenerative and other inflammatory diseases.

P2X Receptors in the Central Nervous System

P2X receptors (P2XRs) are ligand-gated, nonselective cation channels activated by extracellular ATP. Seven pharmacologically distinct P2XR subtypes have been identified, i.e., P2X1–P2X7, and shown to be activated by ATP and its analogues [15, 16]. P2XRs range from 379 to 595 amino acids in length and, as shown in Fig. 1, consist of two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini [17]. P2XRs share ~50 % sequence homology with one third of the extracellular loop conserved, suggesting an ATP binding site [18]. P2X1 and P2X3 receptors rapidly desensitize (within milliseconds), whereas P2X2, P2X5, P2X6, and P2X7 receptors desensitize slowly (within seconds) upon activation by ATP [19]. Depending on the subtype, P2XR subunits interact in a variety of homo- or heteromeric forms to regulate a wide range of cellular responses in the CNS under physiological and pathological conditions [20, 21]. The distribution of P2XR subtypes in the CNS is dependent upon species, brain region, and cell type [2230]. The P2X2R, P2X4R, and P2X6R appear to be abundantly expressed throughout the brain, whereas the remaining subtypes are expressed in distinct regions [22, 24, 31]. In neurons, activation of P2XRs by extracellular ATP has been reported to have both presynaptic and postsynaptic effects including the modulation of transmembrane currents and neurotransmitter release [3237]. All types of glia, e.g., astrocytes, oligodendrocytes, Schwann cells, and microglial cells express P2XRs where they can regulate inflammatory, neurodegenerative, and neuroprotective responses [37]. Most notably, the P2X7R has gained recognition for its possible role in neurodegenerative disorders [38, 39]. The function of the CNS is dependent on neuronal–glial interactions, and the complexity of P2XR signaling in both cell types adds to the difficulty in interpreting ATP-mediated events in vivo. The cell and tissue distribution and functional relevance of homomeric and heteromeric P2X receptors in the nervous system are summarized in Table 1.
https://static-content.springer.com/image/art%3A10.1007%2Fs12035-012-8263-z/MediaObjects/12035_2012_8263_Fig1_HTML.gif
Fig. 1

Structural features of P2X and P2Y receptors. Based on structure and function, P2 nucleotide receptors can be divided into two classes. The P2X receptors are nonselective ligand-gated cation channels featuring two transmembrane domains and a large extracellular loop. P2X receptors interact in a wide variety of homo- and heteromeric forms depending on tissue-specific expression and receptor subtype (i.e., P2X1-7) and they are activated by extracellular ATP. The P2X7R has received much attention due to its capacity for intracellular signaling via a large C-terminal tail and its participation in inflammatory processes. The P2YRs are classical G protein-coupled receptors featuring an extracellular N-terminus, seven transmembrane domains, and an intracellular C-terminus that is structurally diverse between P2Y receptor subtypes. The Gq-coupled P2Y1,2,4,6,11 and the Gi-coupled P2Y12,13,14 receptors are activated by adenine and uridine tri- and dinucleotides with pharmacologically distinct efficacies and potencies. The P2Y2R subtype has been shown to associate with integrins via an extracellular RGD domain and to transactivate growth factor receptors via the binding of Src to Src-homology-3 (SH3) domains located within the C-terminus. Asterisk only present in the P2Y2 receptor

Table 1

P2X receptors in the nervous system

Receptor subtype

Expression

Function

Cell type

Region

Homomeric

P2X1

Neurons, astrocytes, microglia

Cerebral cortex, superior cervical ganglia

Neurogenic smooth muscle contraction, platelet activation, neuron and glial responses

P2X2

Neurons, astrocytes

Cerebral cortex, cerebellum, hippocampus, striatum, habenula, substantia nigra, dorsal root ganglia, mesenteric ganglia

Nociceptive transmission, hyperalgesia, allodynia, pre- and postnatal neurogenesis

P2X3

Neurons

Dorsal root ganglia, spinal cord

Enhance glutamate and substance P release, neuropathic pain sensation

P2X4

Neurons, astrocytes, microglia

Cerebellum, hippocampus, brainstem, spinal cord

Release of brain-derived neurotrophic factor, induce neuropathic pain, prostaglandin E2 release, synaptic strengthening, hypersensitivity to sensory stimuli

P2X5

Neurons

Cerebral cortex, cerebellum, hippocampus, hypothalamus, thalamus, olfactory bulb, globus pallidum, midbrain, and hindbrain

Interconnection of cortical areas, postsynaptic purinergic transmission

P2X6

Neurons, astrocytes

Cerebellum, hippocampus, purkinje neurons, pyramidal neurons, sensory ganglia

Rarely forms functional homomeric receptors

P2X7

Neurons, astrocytes, microglia

Cerebral cortex, hippocampus, brainstem, nucleus accumbens, spinal cord

Release of proinflammatory cytokines, apoptosis, membrane pore formation, glutamate release, ATP release, induction of synaptic plasticity

Heteromeric

P2X1/P2X2

Neurons

Superior cervical ganglia

ATP-mediated physiological responses

P2X1/P2X5

Astrocytes

Cerebral cortex

ATP-evoked membrane currents

P2X2/P2X3

Neurons

Dorsal root ganglia

Similar to P2X3, but with reduced desensitization

P2X2/P2X6

Neurons

Dogiel type II neurons in myenteric plexus

ATP-mediated physiological responses

The expression and function of P2X receptor subtypes in the nervous system are summarized. This table highlights the information presented in this review article and is not considered to be comprehensive

P2X1Rs have been shown to cause contraction of neurogenic smooth muscle [40, 41], platelet activation [42, 43], and neuronal [33, 44] and glial cell responses [45]. Among the P2XRs, the P2X1R has the highest affinity for ATP (EC50 ~1 μM) [46]. The P2X1R is often observed in a heteromeric complex with the P2X2R and P2X5R resulting in biophysical and pharmacological properties distinct from those observed when each of these receptor subtypes is expressed separately [4752]. In superior cervical ganglia neurons, the P2X1R contributes to ATP-mediated responses by forming a heteromeric unit with the P2X2R [44], whereas ATP-evoked biphasic membrane currents in mouse cortical astrocytes are regulated by P2X1R/P2X5R heteromeric channels [45].

P2X2Rs are widely expressed in the CNS with predominant expression in the cerebral cortex, cerebellum, striatum, hippocampus, habenula, substantia nigra, dorsal ganglia neurons, mesenteric ganglia neurons, and glial cells [23, 24, 5357]. The P2X2R is distinguished from other members of the P2XR family, since multiple splice variants exist in different mammalian species with diverse functional properties [58]. Many studies have shown that P2X2Rs play a role in nociceptive transmission, hyperalgesia, and allodynia, particularly when present as functional heterotrimers with P2X3Rs [56, 59, 60]. The pharmacological properties of P2X2R/P2X3R are similar to the P2X3R, but the desensitization rate of the P2X3R is reduced by its interaction with the P2X2R [61]. P2X2Rs and P2X2R/P2X3R have been implicated in pain processing [62]; however, with chronic pain, their functions are altered by the action of other P2XRs, especially those expressed in immune cells, such as microglia [63]. In addition to interactions with P2X2Rs, P2X3Rs also form homotrimeric receptors that are prominently expressed in primary sensory neurons where they enhance the release of glutamate and substance P [32, 6466], which contribute to both acute and chronic pain sensation [60]. In vivo studies using P2X2R−/−, P2X3R−/−, and P2X2R−/−/P2X3R−/− mice have contributed significantly to our understanding of neuropathic and inflammatory pain sensation and have led to the development of therapeutic antagonists to these receptors [67, 68]. The P2X2R also has been suggested to play a role in pre- and postnatal neurogenesis [69].

The P2X4R is expressed throughout the central and peripheral nervous systems [29, 44, 7072]. The P2X4R is upregulated in activated microglial cells after spinal cord or peripheral nerve injury where it appears to mediate the release of brain-derived neurotrophic factor and induce neuropathic pain [73]. Recent studies provide evidence that the functional expression of P2X4Rs in tissue-resident macrophages regulates inflammation-dependent prostaglandin E2 release [74]. Activation of homomeric P2X4Rs in hippocampal neurons has been suggested to contribute to synaptic strengthening and hypersensitivity to sensory stimuli [72, 75]. In addition, hippocampal synaptic transmission and long-term potentiation were abolished in P2X4R−/− mice [76]. A unique characteristic of the P2X4R is its modulation by trace metals; copper inhibits whereas zinc and cobalt potentiate P2X4R activity [58]. P2X4R activity has been shown to be modulated by the allosteric effector ivermectin [58]. Heteromeric assembly of the P2X4R with the P2X1, P2X6, and P2X7 receptor subtypes has been described [7779], although the functional relevance of these complexes in vivo is currently unknown.

The expression of the P2X5R subtype in the mouse CNS is most abundant in the olfactory bulb, cerebral cortex, globus pallidum, hippocampus, thalamus, hypothalamus, cerebellar cortex, and mid- and hindbrain nuclei [80]. Although in vitro data have demonstrated ATP-evoked currents coupled to P2X5R activation, little is known about the physiological relevance of P2X5Rs in the CNS. Guo et al. speculate that P2X5R expression in the molecular layer of the cerebral cortex could play a role in interconnection of local cortical areas and P2X5R expression in the olfactory bulb suggests a role in fast excitatory postsynaptic purinergic transmission [81]. In vitro studies have shown that activation of the homomeric P2X5R results in small, nondesensitizing currents, whereas activation of frequently observed heteromeric P2X5/P2X1 receptors results in slowly desensitizing ATP-evoked currents [48, 50]. A P2X5R−/− mouse has not yet been developed; however, it will be critical for evaluating the role of the P2X5R in vivo. Interestingly, a recent study indicates that most humans express only a nonfunctional isoform of the P2X5R [82].

In the CNS, the P2X6R is expressed in Purkinje cells in the cerebellum, pyramidal cells in the hippocampus, and sensory ganglia [22, 29, 8385]. The ability of the P2X6R to form functional homomeric receptors is very low due to inefficient glycosylation of the N-terminus [86, 87]. P2X6Rs readily form functional heteromers with P2X2 and P2X4 receptors, where activation of one subtype potentiates the activity of the other [88]. In the myenteric plexus, the P2X6R is expressed in Dogiel type II neurons where it likely regulates physiological responses to ATP as a heteromeric complex with P2X2Rs [89].

Among the P2X receptor subtypes, the P2X7 receptor has gained prominent recognition as a regulator of inflammatory responses [90]. P2X7Rs are expressed in many types of cells, notably in immune cells where activation by ATP increases the release of proinflammatory cytokines and apoptotic cell death [91, 92]. The P2X7R was first cloned from rat brain [93] and, subsequently, has been found to be expressed in microglia, neurons, and astrocytes [39, 92, 94, 95]. The P2X7R requires high concentrations of ATP (>0.1 mM) for activation, although the photoaffinity ligand BzATP is a more potent agonist [96, 97]. Stimulation of the P2X7R regulates the gating of nonselective cation channels, mitochondrial and plasma membrane depolarization, the formation of plasma membrane pores, plasma membrane blebbing, and the production of reactive oxygen species (ROS), responses ultimately leading to cell death [10, 90, 97103]. P2X7R activity is dramatically potentiated by decreasing the divalent cation concentration, indicating that ATP4− may be the active ligand [104106]. P2X7Rs have been shown to mediate the release of neurotransmitters, e.g., glutamate, GABA, and ATP, and may be required for the induction of synaptic plasticity [38, 107, 108]. It also has been shown that P2X7R activation induces hypoxia- and caspase-dependent neuronal cell death [109, 110]. Activation of P2X7Rs in glial cells results in the release of the proinflammatory cytokines TNFα, IL-1β, and leukotrienes, thereby triggering or potentiating neuroinflammation [111114], as described below. The P2X7R is upregulated in damaged nerves [115, 116] and in nerves obtained from neuropathic pain patients [117]. In a mouse model of neuropathic pain, hypersensitivity to pain stimuli was completely absent upon deletion of the P2X7R [117]. The P2X7R is also upregulated in microglia around β-amyloid plaques in a mouse model of Alzheimer’s disease (AD) where it mediates superoxide production [118]. Enhanced expression of P2X7Rs also was observed in microglia derived from postmortem AD brains compared with glia obtained from nondemented brains [119]. Furthermore, studies with a mouse model of Huntington’s disease suggest that P2X7Rs may play a role in disease pathogenesis [120]. Therefore, the P2X7R receptor could represent a therapeutic target for treating neurodegenerative diseases.

P2Y Receptors in the Central Nervous System

P2Y receptors (P2YRs) are classical heterotrimeric G protein-coupled seven-pass transmembrane receptors, as shown in Fig. 1. The extracellular N-terminus contains several potential glycosylation sites, and the C-terminus contains consensus phosphorylation sites for protein kinases [121124]. The intracellular loops and C-terminus have structural diversity among P2YR subtypes, thereby influencing the degree of coupling with Gq/11, Gs, and Gi proteins [125]. The length of human P2YRs varies from 328 (P2Y6R) to 377 (P2Y2R) amino acids, and the composition reveals two structurally distinct subgroups within the P2YR family, the Gq-coupled P2Y1, P2Y2, P2Y4, P2Y6,and P2Y11 receptors and the Gi-coupled P2Y12, P2Y13, and P2Y14 receptors [125, 126]. The degree of sequence homology among members of the human P2YR family ranges from 20 to 50 %, suggesting a relatively high functional diversity [127]. It has been demonstrated that positively charged amino acids within transmembrane domains of P2YRs contribute to agonist binding [122, 128, 129]. P2YRs, whose agonists are adenine and/or uridine nucleotides, are expressed in many cell types comprising the CNS and have been shown to regulate neurotransmission, inflammation, cell growth, and apoptosis [130133]. P2Y1, P2Y12, P2Y13, and P2Y14 receptors are activated by adenine nucleotides only, whereas the P2Y2R and rodent P2Y4R can be activated by either adenine or uridine nucleotides and the human P2Y4 and P2Y6 receptors are selective for uridine nucleotides [125]. The P2Y14R subtype is activated by uridine 5′-diphosphate (UDP)-glucose [6, 84, 125]. All eight P2Y receptor subtypes are expressed in primary rat astrocytes or astrocytoma cells [125, 134136], although the expression patterns vary with age [137, 138]. Rodent neurons express P2Y1,2,4,6,12,13 receptors [14, 139, 140]. P2YRs are expressed at postsynaptic terminals where P2Y1, P2Y2, and P2Y4 receptors are neuromodulators that have inhibitory roles in synaptic transmission [34, 141143]. The cell and tissue distribution, agonist specificities, and functional relevance of P2Y receptors in the nervous system are summarized in Table 2.
Table 2

P2Y receptors in the nervous system

Receptor subtype

Agonist

Expression

Function

Cell type

Region

P2Y1

ADP, ATP

Neurons, astrocytes, microglia, oligodendrocytes

Cerebral cortex, cerebellum, hippocampus, midbrain, caudate nucleus, putamen, globus pallidus, habenula, subthalamic nucleus, dorsal root ganglia, dorsal horn

Synaptic transmission modulation, provides neuroprotection by stimulating IL-6 release from astrocytes, brain development and repair, sensory reception

P2Y2

ATP, UTP

Neurons, astrocytes, microglia

Cerebral cortex, cerebellum, hippocampus, nucleus accumbens, spinal cord

Promote neurite outgrowth, stimulate α-secretase-dependent processing of amyloid precursor protein, increase phagocytosis of Aβ peptide, regulate intracellular calcium waves, stimulate proliferation, modulate pain sensation, increase cell motility

P2Y4

ATP, UTP

Neurons, astrocytes, microglia

Cerebral cortex, hippocampus

Synaptic transmission modulation, regulation of blood–brain barrier function, blood flow, metabolic trafficking, water homeostasis

P2Y6

UDP, UTP

Neurons, astrocytes, microglia

Cerebral cortex, cerebellum, hippocampus, amygdala, cingulate gyrus, putamen, nucleus accumbens, superior cervical ganglia, dorsal root ganglia

Stimulate phagocytic activity, neuroinflammatory responses

P2Y11

ATP, ADP

Neurons

Cerebellum, hippocampus, parahippocampal gyrus, putamen, striatum, nucleus accumbens

Neuroinflammatory responses

P2Y12

ADP

Neurons, astrocytes, microglia, oligodendrocytes

Cerebral cortex, cerebellum, hippocampus, nucleus accumbens

Regulation of migration and chemotaxis

P2Y13

ADP

Neurons, astrocytes

Brainstem

Modulation of synaptic transmission, modulates expression of cell survival genes

P2Y14

UDP-glucose

Astrocytes, microglia

Cerebral cortex, cerebellum

Modulation of immune system’s anti-tumor response

The agonists, expression, and function of P2Y receptor subtypes in the nervous system are summarized. This table highlights the information presented in this review article and is not considered to be comprehensive

The P2Y1R has a widespread distribution in mammalian brain, including the cerebral cortex, hippocampus, caudate nucleus, putamen, globus pallidus, habenula, subthalamic nucleus, midbrain, and cerebellum, as demonstrated in autoradiographic and immunohistochemical studies [144146]. The P2Y1R is intensely expressed in Purkinje cells, in deep layers of the cerebral cortex, and in areas of the hippocampus sensitive to ischemia [146]. P2Y1R immunoreactivity has also been observed in oligodendrocytes and astrocytes in brain white matter tracts and optic nerves [134, 147]. P2Y1Rs have been suggested to play important roles in glial cell functions [146]. P2Y1R activation in astrocytes of hippocampal cultures has been suggested to provide neuroprotection from oxidative stress by increasing IL-6 release [148]. P2Y1Rs are also expressed in microglial cells [116, 134, 144, 149, 150], rat neuroprogenitor cells [151], and various sensory neurons such as dorsal root ganglia and dorsal horn neurons [152154]. Studies have suggested potential roles for P2Y1Rs in brain development and repair [151] and sensory reception [153, 155].

The contribution of the P2Y2R subtype to CNS functions is becoming better understood [9, 156, 157] and appears to be most relevant under pathophysiological conditions, such as inflammation and bacterial infection [9, 158]. The mammalian P2Y2R is equipotently activated by ATP or UTP [7, 125] and is upregulated in many cell and animal models of inflammation or injury [14, 159163], including acute and chronic stages of spinal cord injury [164], brain ischemia, mechanical injury to the nucleus accumbens, and brain trauma [116, 165], suggesting that P2Y2R upregulation represents a cellular response to tissue damage and inflammation. P2Y2R expression under proinflammatory conditions is regulated by NF-κB binding to the P2Y2R promoter [166], consistent with the established role of NF-κB activation in the induction of inflammation [167]. In addition to the typical Gq-coupled activation of the PLC/IP3/PKC pathway, the P2Y2R has a variety of structural motifs that enable it to activate integrin and growth factor receptor signaling cascades, as shown in Fig. 2. For example, P2Y2R activation by ATP or UTP has been shown to induce phosphorylation of growth factor receptors which increases the activities of the MAP kinases ERK1/2 and the related adhesion focal tyrosine kinase (RAFTK) via a pathway dependent upon Src and Shc/Grb2 [168170]. However, other studies show that P2Y2R-mediated epidermal growth factor receptor (EGFR) phosphorylation is Src-independent, but requires the release of growth factors via P2Y2R-dependent activation of the matrix metalloproteases ADAM10 and ADAM17 [171]. These differences appear to be due to cell type, e.g., endothelial vs. epithelial. The P2Y2R is unique among GPCRs in that it contains the consensus integrin-binding motif Arg-Gly-Asp (RGD) in a putative extracellular domain that enables the association of the P2Y2R with αvβ3/5 integrins allowing activation of heterotrimeric Go and G12 proteins that regulate the activities of the small GTPases Rho and Rac [172174], regulators of actin polymerization and cytoskeletal rearrangements required for cell migration [175, 176]. In addition, the P2Y2R contains SH3-binding motifs in its C-terminal domain that can interact with the actin-binding protein filamin A (FLNa), another known regulator of cytoskeletal rearrangements, suggesting that the P2Y2R may regulate migration of cells by both RGD-dependent interactions with integrins and C-terminal-dependent association with actin-binding proteins [177]. Moreover, the P2Y2R has been reported to regulate migration of some cell types via transactivation of growth factor receptors [173, 178], suggesting that a complex array of signaling events uniquely coupled to P2Y2R activation may be required to optimize the cytoskeletal rearrangements whereby the P2Y2R regulates cell-specific functions, e.g., neurite outgrowth, phagocytosis, synapse formation, and chemokinesis.
https://static-content.springer.com/image/art%3A10.1007%2Fs12035-012-8263-z/MediaObjects/12035_2012_8263_Fig2_HTML.gif
Fig. 2

P2Y2R signaling pathways. The P2Y2R modulates a variety of cellular processes through classical G protein-coupled receptor pathways and unique receptor motifs. Activation of the P2Y2R by ATP or UTP stimulates the Gq-dependent activation of PLC leading to the generation of IP3 and DAG. IP3 triggers a release of Ca2+ from intracellular stores leading to an increase in [Ca2+]i and the activation of Ca2+-dependent proteins, whereas DAG serves to activate PKC leading to activation of a variety of downstream proteins including RAFTK (also known as Pyk2) and the MAP kinases ERK1/2. The Src-homology-3 (SH3) domains in the C-terminus allow the P2Y2R to stimulate Src-dependent transactivation of growth factor receptors and their downstream signaling molecules Shc and Grb2 leading to ERK1/2 and p38 activation of cell proliferation and neurite outgrowth. The P2Y2R also has been shown to upregulate VCAM-1 through a pathway involving VEGFR-2. Furthermore, P2Y2R activation has been shown to stimulate the PI3K/Akt pathway to inhibit apoptosis in neurons, a response that was dependent on Src activation. SH3 domains also allow the P2Y2R to interact with the actin cytoskeleton via the actin-binding protein filamin A (FLNa). Alternatively, the P2Y2R can access the actin cytoskeleton through an extracellular RGD domain that interacts with αvβ3/5 integrins to enable activation of Go and G12 proteins allowing the P2Y2R to stimulate cell migration, phagocytosis, neurite outgrowth, and diapedesis by activating the cytoskeletal regulators Rac and Rho. Lastly, the P2Y2R is able to activate the matrix metalloproteases ADAM10 and ADAM17 to induce non-amyloidogenic APP processing and shedding of growth factors

P2Y2R expression in rat primary cortical neurons is upregulated in response to IL-1β [14], a cytokine whose levels are elevated in the brains of AD patients [179, 180]. Subsequent activation of these upregulated P2Y2Rs in neurons promotes neurite outgrowth [181] and generates the non-amyloidogenic soluble APPα peptide, rather than neurotoxic Aβ1-42 peptide aggregates associated with AD [14]. In mouse primary microglial cells, the P2Y2R is upregulated in the presence of Aβ1-42 and when activated can increase the phagocytosis and degradation of neurotoxic forms of Aβ [9, 182, 183]. In astrocytic cells, the P2Y2R has been suggested to contribute to synaptic transmission through the regulation of intracellular calcium waves [184] and upregulates anti-apoptotic protein expression to promote cell survival [185]. Thus, P2Y2R upregulation in response to proinflammatory conditions likely serves a neuroprotective role in the CNS that requires contributions from both glial and neuronal P2Y2Rs, as described in more detail below.

The human P2Y4R is preferentially activated by uridine nucleotides, whereas the rat and mouse P2Y4Rs are stimulated equipotently by ATP and UTP [186188]. P2Y4R mRNA is highly expressed in human brain [189]. Single cell RT-PCR demonstrated the expression of P2Y4Rs in rat hippocampal pyramidal neurons [34]. The expression of P2Y4Rs in astrocytes and microglial cells has been extensively documented [134, 137, 150, 186]. P2Y4Rs, as well as P2Y2Rs, are strongly expressed in glial endfeet in proximity to blood vessel walls [190] where their activation by ATP has been postulated to regulate blood–brain barrier function, blood flow, metabolic trafficking, and water homeostasis [190, 191].

The P2Y6R is activated by UDP and to a lesser extent UTP [192]. In 18 areas of the human brain, the level of P2Y6R mRNA expression was highest in the amygdala, cingulate gyrus, nucleus accumbens, and putamen [189]. Single cell RT-PCR revealed P2Y6R mRNA in 2 of 12 pyramidal neurons of rat hippocampus [34]. In addition, P2Y6R mRNA has been demonstrated in superior cervical ganglion [44, 193] and dorsal root ganglion neurons [152, 153]. Functional studies have revealed the presence of P2Y6R activity in cerebellar and cortical astrocytes [134, 194]. P2Y6R activation has been shown to increase phagocytotic activity of microglia, postulated to occur in vivo in response to UTP released from damaged cells [195, 196]. Consistent with this hypothesis, injury has been shown to induce increased P2Y6R expression in astroglial cells [165]. In microglial cells stimulated overnight with bacterial lipopolysaccharide, P2Y6R-mediated increases in the intracellular calcium concentration were observed, suggesting a role for the P2Y6R in neuroinflammation [150].

The P2Y11R can couple to multiple G proteins to regulate the activity of two second messenger systems: adenylate cyclase-mediated cAMP production and PLC-dependent production of IP3 and DAG that modulate calcium release from intracellular storage sites and protein kinase C activation, respectively [197]. The P2Y11R is activated by ATP or ADP, but not by uridine nucleotides [197]. P2Y11R mRNA expression is prominent in nucleus accumbens, parahippocampal gyrus, putamen, and striatum [84]. The P2Y11R has been localized to single rat hippocampal pyramidal neurons and to Purkinje cells in adult rat cerebellum [34, 198]. Inhibition of the P2Y11R has been shown to delay ATP-induced neutrophil apoptosis, suggesting a role for the P2Y11R in the regulation of neuroinflammatory responses [199].

The P2Y12R is widely distributed in the brain with a pattern consistent with expression in astrocytes [200, 201]. RT-PCR has demonstrated the presence of P2Y12R mRNA in single rat hippocampal pyramidal neurons [34]. Cortical and cerebellar astrocytes and astrocytes in the rat nucleus accumbens also express P2Y12Rs [134, 165, 202]. P2Y12Rs have been suggested to regulate the migration of microglial cells towards damaged neurons [203]. P2Y12R expression in microglia is robust in the “resting” state, but dramatically reduced in activated microglia, and P2Y12R−/− mice have significantly diminished directional branch extension toward sites of cortical damage in vivo [204]. In contrast, a recent study concludes that the expression of the P2Y12R in the CNS is restricted to oligodendrocytes [205]. It also has been suggested P2Y12Rs contribute to the migration and adhesion of glial cell processes to axons during pre-myelination [205].

The P2Y13R is activated by ADP [206] and 2-methylthio ADP is a potent synthetic agonist [207], similar to the P2Y12R; however, ATP and ATP analogues are inactive at the P2Y13R [208]. P2Y13R expression has been localized to brainstem astrocytes and glutamatergic neurons [145, 189, 209]. P2Y13Rs, along with P2Y1 and P2Y12 receptors, have been shown to regulate Na+ and Cl-dependent synaptic glycinergic neurotransmitter transporters to increase transport of glycine from the synaptic cleft, thereby maintaining quantal glycine levels in inhibitory synaptic vesicles [209, 210]. The P2Y13R can also activate the glycogen synthase kinase-3 (GSK-3)-dependent phosphatidylinositoI 3-kinase (PI3K)/Akt survival pathway to increase translocation of the GSK-3 substrate β-catenin to the nucleus, where it modulates expression of cell survival genes [211].

The P2Y14R is expressed in astrocytes [189], and RT-PCR and single cell Ca2+ imaging has documented the functional expression of P2Y14Rs in rat cortical and cerebellar astrocytes [134, 202]. Agonists of the P2Y14R include UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine, but not adenine or uridine nucleotides [212214]. UDP-glucose has been shown to be released from a variety of cell lines, and UDP-glucose levels can exceed those of ATP under various conditions [215]. Functionally, P2Y14Rs in primary microglial cells from rat brain have been shown to modulate the calcium response to bacterial lipopolysaccharide [150]. P2Y14Rs expressed in immature dendritic cells have been suggested to play a role in the immune system’s anti-tumor response [143, 216].

Neuroinflammatory P2X7Rs Regulate Neuroprotective P2Y2R Expression

P2X7R activation contributes to neuroinflammation by promoting mitochondrial and plasma membrane depolarization, the formation of plasma membrane pores, plasma membrane blebbing, and the production of ROS [10, 90, 98103]. In addition, P2X7R activation promotes neuroinflammation by causing the release of proinflammatory cytokines, such as IL-1β and TNF-α [90, 217, 218], and activation of MAP kinases and NF-κB, resulting in upregulation of proinflammatory gene products, including COX-2, chemokines, and cell adhesion molecules [90, 219223] and the P2Y2R [166]. Importantly, P2X7R-mediated pore formation initially increases ATP release through P2X7R interactions with a pannexin hemi-channel in cells [91]. P2X7R-mediated IL-1β and ATP release is a mechanism whereby the P2X7R regulates functional P2Y2R expression in neurons and provides agonist for the activation of the upregulated P2Y2R and other P2 receptors [9]. ATP release also can occur from activated microglia and astrocytes in response to oxidative stress [9], following neuronal excitation [224, 225], via volume-activated anion channels [225], or upon exposure of cells to fibrillar or oligomeric forms of amyloidogenic Aβ1-42 peptides [157, 183, 226, 227]. Thus, P2X7 and P2Y2 receptors may represent promising targets to control inflammatory responses associated with neurodegenerative diseases. Indeed, mice deficient in the P2X7R (P2X7R−/− mice) exhibit decreased inflammatory responses [117, 228230], including a reduction in pulmonary fibrosis in a mouse model of lung inflammation [230] and the absence of pain hypersensitivity in mouse models of chronic inflammation and neuropathic pain [117]. Phase I and II clinical trials for selective P2X7R antagonists are presently underway for the treatment of rheumatoid arthritis and other inflammatory diseases [231, 232].

Upregulation of the P2Y2R in response to P2X7R activation appears to promote neuroprotective responses. The ability of the P2Y2R to stimulate neuroprotective responses depends upon the coupling of the receptor to intracellular signaling pathways that are distinct among the P2YR family (see Fig. 2). These responses associated with P2Y2R upregulation include the outgrowth and stabilization of dendritic spines [9, 176, 233], which requires RGD-dependent P2Y2R/αv integrin interaction to stimulate Rac and Rho and induce cytoskeletal rearrangements [173, 174] and upregulation of neurofilament M and neurofilaments that promote neurite outgrowth [181]. P2Y2Rs also require Src to co-localize with the tyrosine receptor kinase A in the presence of nerve growth factor, a pathway that regulates neurite outgrowth and cell division via the activation of p38 and ERK1/2 MAP kinases [234, 235]. In neural progenitor cells isolated from the subventricular zone of adult mouse brain, P2Y2R activation was shown to induce proliferative responses such as the transient activation of the EGFR, the MAP kinases ERK1/2, and the transcription factor CREB [236]. Other studies indicate that the P2Y2R mediates the activation of PI3-kinase/Akt and MAP kinases to inhibit apoptosis of PC12 pheochromocytoma cells and dorsal root ganglion neurons [235, 237]. P2Y2R upregulation by IL-1β and subsequent activation in primary cortical neurons increases amyloid precursor protein (APP) processing via activation of matrix metalloproteases (i.e., α-secretases), a neuroprotective response that produces a non-amyloidogenic soluble APP peptide (i.e., sAPPα) rather than neurotoxic amyloidogenic Aβ peptide [14]. IL-1β is known to stimulate neuronal synthesis of APP and increase the release of neurotoxic Aβ, which further enhances IL-1β production [238]. We postulate that upregulation of the P2Y2R induced by IL-1β in vivo counteracts the potential neurotoxic effects of IL-1β-dependent elevations in APP levels by promoting generation of non-toxic sAPPα instead of Aβ. Thus, P2Y2R upregulation in the CNS may delay the progression of neurodegeneration associated with reactive gliosis and chronic inflammation in AD and other neurological disorders.

Glial cells, including astrocytes and microglia, play important neuroprotective roles. Astrocytes contribute to the maintenance of the blood–brain barrier (BBB) [239241], which prevents invasion of pathogenic, neurotoxic substances into the brain from the circulation [242, 243]. Astrocytes also release neurotrophic factors that regulate neuronal survival and sprouting and supply energy substrates to neurons [244]. Astrocytes have been shown to release ATP under a variety of pathological conditions [245247] and ATP levels are elevated sufficiently by inflammation in vivo to activate P2 nucleotide receptors [245]. P2Y2Rs are upregulated in reactive astrocytes of the rat cortex and nucleus accumbens in response to mechanical injury [165] and have been suggested to enhance astrocyte survival [185, 248]. In addition, interactions between the P2Y2R and integrins have been demonstrated to regulate the migration of astrocytes [173, 174, 249]. It also has been shown that P2X7R activation increases the expression of P2Y2Rs in rat astrocytes [250] likely via P2X7R-mediated IL-1β release [112, 226, 242, 251253].

Microglia have important immunoregulatory functions in the CNS. Injury or other insults to the CNS trigger transformation of quiescent microglia into activated phenotypes, i.e., phagocytic macrophages [254, 255]. Activated microglia have neuroprotective functions [255259], although sustained activation can be neurotoxic [256, 260262]. Microglial cell activation by proinflammatory cytokines has been shown to increase cell motility and proliferation [263], responses associated with reactive gliosis in neurodegenerative diseases. Adenine and uridine nucleotides have been shown to increase the motility of microglial cells [195, 204, 264] via activation of P2Y2 and P2Y12 receptors [204, 265] and ATP release can significantly increase microglial process extension towards a site of injury [266]. The endogenous expression of P2Y2Rs has been reported in mouse microglia [267, 268] where they have been shown to regulate responses associated with reactive gliosis [7, 9, 165, 185, 248, 249]. For example, the P2Y2R agonists UTP and ATP released from apoptotic cells have been shown to induce migration of phagocytic cells [269], which presumably serves to enhance the clearance of cellular debris. Microglial cells exposed to Aβ also have been shown to release ATP [226, 270]. Studies using peritoneal macrophages in mice have shown that stimulation of P2Y2 and P2Y12 receptors induces the formation of lamellipodia in membrane protrusions which is required for cell motility [271]. Co-activation of P2Y2 and P2Y6 receptors in human monocytes enhances migration, a response shown to involve toll-like receptor-induced IL-8 release [272, 273]. We have found that P2Y2R activation increases mouse microglial cell migration and phagocytic activity, such as the uptake of neurotoxic oligomeric Aβ1-42, responses that are absent in microglia from P2Y2R−/− mice [183]. Both activated astrocytes and microglia internalize and degrade Aβ [274278], a pathway that reduces Aβ toxicity in neurons that is postulated to play a role in the progression of AD. We speculate that P2Y2Rs in glial cells contribute to the phagocytosis and degradation of neurotoxic forms of Aβ in vivo under conditions where elevated levels of ATP release and IL-1β generation occur [279, 280].

Recent studies suggest that peripheral leukocytes and hematopoietic cells that differentiate into microglia have important functions in the CNS [281], particularly in response to tissue injury [282]. P2Y2Rs in endothelial cells that form the BBB may also regulate the migration of leukocytes across the BBB towards sites of injury or disease in the brain. Activation of the endothelial P2Y2R has been shown to enhance the diapedesis of neutrophils towards the chemoattractant lipopolysaccharide of gram-negative bacteria [273] through a mechanism involving Rho kinase activation, suggesting that P2Y2R associations with integrins may be involved [173, 174]. Microglia derived from bone marrow have been shown to phagocytose Aβ deposits in the brain of AD mice to a greater extent than resident brain microglia [283]. Thus, diapedesis of microglia across the BBB, in addition to neurite outgrowth, non-amyloidogenic APP processing, and phagocytosis of neurotoxic forms of Aβ, may comprise a neuroprotective phenotype linked to P2Y2R activation in several cell types that comprise the brain (i.e., neurons, glial cells, and endothelium). The neuroprotective pathways by which P2X7R-mediated upregulation and activation of P2Y2Rs are suggested to contribute to neuroprotection in the brain are shown in Fig. 3.
https://static-content.springer.com/image/art%3A10.1007%2Fs12035-012-8263-z/MediaObjects/12035_2012_8263_Fig3_HTML.gif
Fig. 3

P2X7R-mediated neuroinflammation stimulates P2Y2R-mediated neuroprotective responses. (1) ATP released under neuroinflammatory conditions can (2) activate the P2X7R to stimulate (3) the release of proinflammatory cytokines, including interleukin-1β (IL-1β), and further ATP release via interaction of the P2X7R with pannexin hemi-channels. In response to the proinflammatory environment, quiescent microglia take on an (4a) activated phenotype and P2Y2R activation by extracellular ATP increases (5a) cell motility. In addition, (4b) IL-1β upregulates P2Y2R expression in neurons and glia through NF-κB activation. (6) ATP release (from Aβ exposure, cytokine exposure, oxidative stress, etc.) provides agonist for the (5b) P2Y2R to stimulate non-amyloidogenic APP processing and neurite outgrowth through P2Y2R interactions with matrix metalloproteases and the actin cytoskeleton, respectively. Another neuroprotective response to (7) P2Y2R activation in microglial cells is increased phagocytosis and degradation of neurotoxic oligomeric Aβ1-42

Conclusion

This review summarizes data indicating that seven ionotropic P2X and eight G protein-coupled P2Y receptors for extracellular nucleotides are expressed in cell types comprising the CNS and these P2X and P2Y receptor subtypes have been shown to regulate diverse physiological and pathological responses under a variety of conditions. Recent studies indicate that activation of the P2X7R subtype during inflammation causes upregulation and activation of P2Y2Rs to promote neuroprotective responses. These findings suggest that ATP released from injured or stressed cells in the CNS can activate P2X7Rs in microglial cells to increase the release of proinflammatory cytokines, such as IL-1β, that increase the expression of the P2Y2R, particularly in neurons. Other studies indicate that both P2X7R and P2Y2R activation can increase phagocytosis of neurotoxic forms of Aβ and that activation of the P2Y2R increases non-amyloidogenic APP processing, neuroprotective responses that are postulated to delay the onset or retard the progression of neurodegenerative diseases, such as Alzheimer’s disease. In addition, P2Y2R activation in neurons has been shown to increase neurite outgrowth. The P2Y2R contains multiple motifs that enable its activation to directly couple to integrin and growth factor receptor signaling pathways that play a role in cell proliferation and differentiation and cytoskeletal rearrangements that are critical for tissue repair. Thus, the studies described in this review suggest that the P2X7R and P2Y2R are promising targets for the treatment of neurodegenerative diseases.

Copyright information

© Springer Science+Business Media, LLC 2012