Interleukin‐1β enhances nucleotide‐induced and α‐secretase‐dependent amyloid precursor protein processing in rat primary cortical neurons via up‐regulation of the P2Y₂ receptor

Abstract

The heterologous expression and activation of the human P2Y2 nucleotide receptor (P2Y2R) in human 1321N1 astrocytoma cells stimulates α‐secretase‐dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non‐amyloidogenic protein secreted amyloid precursor protein (sAPPα). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y2R‐mediated production of sAPPα occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y2R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) up‐regulated both P2Y2R mRNA expression and receptor activity by four‐fold. The up‐regulation of the P2Y2R was abrogated by pre‐incubation with Bay 11‐7085, an IκB‐α phosphorylation inhibitor, which suggests that P2Y2R mRNA transcript levels are regulated through nuclear factor‐κ‐B (NFκB) signaling. Furthermore, the P2Y2R agonist Uridine‐5′‐triphosphate (UTP) enhanced the release of sAPPα in rPCNs treated with IL‐1β or transfected with P2Y2R cDNA. UTP‐induced release of sAPPα from rPCNs was completely inhibited by pre‐treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI‐2) or the phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal‐regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y2R‐mediated release of sAPPα from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal‐regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro‐inflammatory cytokines associated with neurodegenerative diseases, such as IL‐1β, can enhance non‐amyloidogenic APP processing through up‐regulation of the P2Y2R in neurons.

Roles of extracellular nucleotides and P2 receptors in ectodomain shedding

“…Similarly, UTP induced sAPPα shedding via ADAM10 and ADAM17 in rat primary cortical neurons treated with interleukin (IL)-1β (to up-regulate P2Y2) or following transfection with P2Y2 [18]. This study also demonstrated a role for phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) in this pathway.…”

Neuroprotective roles of the P2Y2 receptor

“…Among the G protein-coupled P2YRs, the G q -coupled P2Y 2 R subtype is expressed in neurons and glial cells [13,15,48,[66][67][68]. Our studies using in situ hybridization and reverse transcriptase polymerase chain reaction with rodent brain slices have shown high levels of P2Y 2 R expression in the hippocampus and cerebellum [20], and P2Y 2 R expression can be significantly upregulated in mouse cortical neurons by the proinflammatory cytokine interleukin-1β (IL-1β) [48].…”

“…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]. Neurons express mRNAs for P2X3, 5-7 and P2Y 1,2,4,6,12,13 receptors [12,[47][48][49]. Multiple subtypes of P2Rs are expressed in monocytes (P2X1,4,7 and P2Y 1,2,4,6,11,12,13 receptors) [50] and human endothelial cells (P2X4 and P2Y 1,2,4,6,11 receptors) [51].…”

“…Our studies using in situ hybridization and reverse transcriptase polymerase chain reaction with rodent brain slices have shown high levels of P2Y 2 R expression in the hippocampus and cerebellum [20], and P2Y 2 R expression can be significantly upregulated in mouse cortical neurons by the proinflammatory cytokine interleukin-1β (IL-1β) [48]. P2YRs have been shown to be coupled either directly or indirectly to G q , G i , G o , and G 12 protein activation and downstream signaling pathways associated with alterations in PLC or adenylyl cyclase activities [1,20,[69][70][71].…”

“…For example, activation of both astrocytic P2YRs and P2X7Rs occurs in brain lesions during the functional remodeling that accompanies astrogliosis and neuroinflammation [72]. Interactions between P2X7R and P2Y 2 R signaling pathways mediate glial cell-dependent neuroprotective responses [15,75,76] in which P2X7R activation in microglial cells leads to the release of nucleotides and cytokines, including IL-1β [75], that enhance the functional expression of P2Y 2 Rs in neurons [48]. P2Y 1 R activation in astrocytes of hippocampal cultures also provides neuroprotection from oxidative stress by increasing IL-6 release [77].…”