P2Y2 nucleotide receptor-mediated responses in brain cells

Abstract

Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimer's disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the G(q) protein-coupled P2Y(2) nucleotide receptor subtype (P2Y(2)R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y(2)R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y(2)R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y(2)R activation increases astrocyte migration. P2Y(2)R activation by UTP increases the expression in astrocytes of alpha(V)beta(3/5) integrins that bind directly to the P2Y(2)R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y(2)R, an interaction required for G(o) and G(12) protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y(2)R expression is increased by stimulation with interleukin-1beta (IL-1beta), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric beta-amyloid peptide (Abeta(1-42)), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y(2)Rs in neurons. Data with rPCNs suggest that P2Y(2)R upregulation by IL-1beta and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1beta-upregulated P2Y(2)Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y(2)Rs in glial cells can promote neuroprotective responses, suggesting that P2Y(2)Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.

Fig. 1

P2Y₂R structure and domains: the P2Y₂R is a seven pass transmembrane G protein-coupled extracellular nucleotide receptor. It is activated equipotently by ATP and UTP and has been shown to be upregulated in response to stress or injury in various cell types. Highlighted features include the consensus RGD integrin-binding domain (in pink), positively-charged amino acid residues known to be involved in ATP/UTP binding (in orange), two consensus PXXP SH3 domain binding sites (in yellow), the FLNa binding site, the intracellular loops that regulate Gq protein binding, and two glycosylation sites

Fig. 2

P2Y₂ receptor-mediated signal transduction: activation of the P2Y₂ receptor (P2Y₂R) is coupled to several intracellular signal transduction pathways including: a Gq❮-dependent activation of phospholipase C (PLC) that generates inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG), second messengers for intracellular calcium mobilization and protein kinase C activation, respectively; b Src-mediated transactivation of growth factor receptor phosphorylation that stimulates mitogen-activated protein kinase cascades to regulate gene transcription; c association with and activation of αvβ3/5 integrins that stimulates Rho kinase leading to cofilin phosphorylation; and d activation of metalloproteases (i.e., ADAM10/17) to stimulate the non-amyloidogenic processing of amyloid precursor protein (APP). Other abbreviations: AA arachidonic acid, PGE2 prostaglandin E2, VCAM-1 vascular cell adhesion molecule-1

Fig. 3

Effect of oligomeric Aβ42 on ATP release from primary cultured rat cortical astrocytes: the cells were incubated for 15 min at 37°C with HEPES buffer supplemented with 200 μM AOPCP, an inhibitor of 5′-nucleotidases, to retard ATP breakdown. Cells were washed 3 times using the same buffer and then incubated for different time periods at 37°C with or without oligomeric Aβ. Supernatants were collected and released ATP was measured with the ATP Bioluminescence Assay kit HSII (Roche). The ATP levels were calculated based on an ATP standard curve. The results are expressed as nmoles of ATP released per well of 12-well plate and are presented as means±S.E.M.; n=3. White bars are basal levels at each time point (without oligomeric Aβ) and black bars are stimulated ATP release (with oligomeric Aβ). **p<0.01

Fig. 4

P2Y₂Rs in astrocytes and microvessels: chronic inflammation caused by oxidative stress in brain is mediated by P2Y₂Rs via cytokine-like actions of nucleotides in astrocytes and microvessels through transactivation of integrins and growth factor receptors. Nucleotides are released from oxidatively-stressed brain cells activating P2Y₂Rs in astrocytes and microvessels. Activation of endogenously expressed P2Y₂Rs in glial cells leads to integrin-mediated cell migration (via the P2Y₂R RGD domain), which has been shown to be necessary for cell migration. Nucleotideinduced and integrin-dependent activation of Rho and Rac promotes glial cell migration, and P2Y₂R-induced transactivation of growth factor receptors increases cell proliferation and pro-inflammatory gene expression

Fig. 5

P2Y₂Rs in neurons: nucleotides released from oxidatively stressed brain cells activate P2Y₂Rs on neurons. P2Y₂R activation induces release of cytokines, which upregulate the expression of the P2Y₂R. Additionally, extracellular nucleotides activate matrix metal-loproteases to increase production of the non-amyloidogenic APP fragment, sAPP-α. Activation of the P2Y₂R also promotes binding of FLNa to the C-terminal domain of the receptor and phosphorylation of cofilin