P2Y2 Nucleotide Receptors Enhance α-Secretase-dependent Amyloid Precursor Protein Processing

Abstract

The amyloid precursor protein (APP) is proteolytically processed by ␤-and ␥-secretases to release amyloid ␤, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid ␤ domain by ␣-secretase releasing the non-amyloidogenic product sAPP␣, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate ␣-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G proteincoupled P2Y 2 receptor (P2Y 2 R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPP␣ in a time-and dose-dependent manner. P2Y 2 Rmediated sAPP␣ release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y 2 R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPP␣ release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPP␣ release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y 2 Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-ArgVal-Lys-Arg-chloromethylketone also diminished UTPinduced sAPP␣ release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y 2 R-activated sAPP␣ release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPP␣ release. These results may contribute to an understanding of the non-amyloidogenic processing of APP.P2 nucleotide receptors modulate a wide range of physiological responses following their activation by extracellular nucleotides (1, 2). The G protein-coupled P2Y 2 receptor (P2Y 2 R) 1 subtype is fully activated by equivalent concentrations of ATP or UTP (3-5) and is up-regulated in salivary gland models of stress and disease (6 -8) as well as in blood vessels after balloon angioplasty and in collared carotid arteries, where it induces intimal hyperplasia and inflammation by increasing smooth muscle cell proliferation and leukocyte infiltration (9, 10). Moreover, nucleotides are released from damaged cells of all tissues and from excited neurons, aggregating platelets, and contracting smooth muscle under physiological conditions (2, 11).The diversity of cellular responses mediated by P2Y 2 Rs is due in part to unique structural features that enable these receptors to stimulate a variety of signal transduction pathways. In addition to the classical stimulation of G␣ q -dependent phospholipase C (12, 13), the P2Y 2 R c...

Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925

“…1) [9]. Furthermore, a neuroprotective role of the P2Y 2 receptor was postulated via the induction of α-secretase-dependent amyloid precursor protein processing in astrocytoma cells [10].…”

Neuroprotective roles of the P2Y2 receptor

“…The P2R agonist ATP is a neuro-and gliotransmitter released by exocytosis from neurons and by diffusion through hemichannels, pannexins, and voltage-gated channels in various cell types [12,17,[34][35][36][37]. P2Rs (both P2X ligandgated cation channels and P2Y G protein-coupled receptors) [1,4,6] are expressed in neuroglia (astrocytes, oligodendrocytes) and microglia of the CNS [14], where they regulate differentiation, nociceptive transmission, cytokine release, apoptosis, and metalloprotease-dependent degradation of amyloid precursor protein (APP) [16,27,31,[38][39][40]. Among cell types that comprise the CNS, mRNAs for P2X1-7 and P2Y 1,2,4,6,11,12,13,14 receptor subtypes have been identified in primary rat astrocytes [4,[41][42][43][44], and their expression patterns can vary with the age of the animal [45,46].…”

“…Activation of P2Y 2 Rs expressed in human astrocytoma cells or rat primary cortical neurons (rPCNs) stimulates α-secretases, i.e., the MMPs adamalysin 10/17 (ADAM10/17), that mediate the proteolytic processing of APP to generate the non-amyloidogenic soluble APPα (sAPPα) peptide [38,48]. P2Y 2 R-mediated α-secretase activity is dependent on activation of the PI3-kinase/Akt pathway and partially dependent on activation of PKC and ERK1/2.…”

“…These interactions lead to Src-dependent activation of MMPs and consequent release of the HB-EGF ligand to enable autocrine activation of the EGFR [119]. Since G q -coupled P2Y 2 R activation can induce Src-dependent activation of the EGFR [21,22,88,89], activation of the MMPs α-secretases [38], and integrin-dependent increases in cell motility [24,95], it is intriguing to speculate that arrestins and receptor internalization play a role in these processes. Signaling pathways known to be coupled to P2Y 2 R activation are shown in Fig.…”

“…The C terminus of the P2Y 2 R also has been shown to interact with the actin-binding protein filamin A (FLNa). P2Y 2 R activation can stimulate the activity of matrix metalloproteases (e.g., ADAM10 and ADAM17) leading to the α-secretase-dependent processing of APP to the non-amyloidogenic peptide sAPPα [38,48] and release of growth factors (e.g., NRG1) [94] cells as a result of caspase 3/7 activation, also have been shown to induce cell migration of phagocytic cells [143]. In an in vivo model of cell migration, supernatants from apoptotic cells produced a three-fold greater recruitment of monocytes and macrophages than supernatants from control cells and depletion of nucleotides in the apoptotic cell supernatants diminished cell migration [143].…”