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.
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 Gq protein-coupled P2Y2 nucleotide receptor subtype (P2Y2R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y2R 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 P2Y2R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y2R activation increases astrocyte migration. P2Y2R activation by UTP increases the expression in astrocytes of αVβ3/5 integrins that bind directly to the P2Y2R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y2R, an interaction required for Go and G12 protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y2R expression is increased by stimulation with interleukin-1β (IL-1β), 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 β-amyloid peptide (Aβ1-42), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y2Rs in neurons. Data with rPCNs suggest that P2Y2R upregulation by IL-1β and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1β-upregulated P2Y2Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y2Rs in glial cells can promote neuroprotective responses, suggesting that P2Y2Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.
Amyloid β-protein (Aβ) deposits in brains of Alzheimer’s disease (AD) patients generate proinflammatory cytokines and chemokines that recruit microglial cells to phagocytose Aβ. Nucleotides released from apoptotic cells activate P2Y2 receptors (P2Y2Rs) in macrophages to promote clearance of dead cells. In this study, we investigated the role of P2Y2Rs in the phagocytosis and clearance of Aβ. Treatment of mouse primary microglial cells with fibrillar (fAβ1–42) and oligomeric (oAβ1–42)Aβ1–42 aggregation solutions caused a rapid release of ATP (maximum after 10 min). Furthermore, fAβ1–42 and oAβ1–42 treatment for 24 h caused an increase in P2Y2R gene expression. Treatment with fAβ1–42 and oAβ1–42 aggregation solutions increased the motility of neighboring microglial cells, a response inhibited by pre-treatment with apyrase, an enzyme that hydrolyzes nucleotides. The P2Y2R agonists ATP and UTP caused significant uptake of Aβ1–42 by microglial cells within 30 min, which reached a maximum within 1 h, but did not increase Aβ1–42 uptake by primary microglial cells isolated from P2Y2R−/− mice. Inhibitors of αv integrins, Src and Rac decreased UTP-induced Aβ1–42 uptake, suggesting that these previously identified components of the P2Y2R signaling pathway play a role in Aβ phagocytosis by microglial cells. Finally, we found that UTP treatment enhances Aβ1–42 degradation by microglial cells, but not in cells isolated from P2Y2R−/− mice. Taken together, our findings suggest that P2Y2Rs can activate microglial cells to enhance Aβ clearance and highlight the P2Y2R as a therapeutic target in AD.
J. Neurochem. (2010) 114, 1630–1638. Abstract Alzheimer’s disease (AD) is characterized by progressive neurodegeneration associated with the aggregation and deposition of β‐amyloid (Aβ40 and Aβ42) peptide in senile plaques. Recent studies suggest that copper may play an important role in AD pathology. Copper concentrations are elevated in amyloid plaques and copper binds with high affinity to the Aβ peptide and promotes Aβ oligomerization and neurotoxicity. Despite this connection between copper and AD, it is unknown whether the expression of proteins involved in regulating copper homeostasis is altered in this disorder. In this study, we demonstrate that the copper transporting P‐type ATPase, ATP7A, is highly expressed in activated microglial cells that are specifically clustered around amyloid plaques in the TgCRND8 mouse model of AD. Using a cultured microglial cell line, ATP7A expression was found to be increased by the pro‐inflammatory cytokine interferon‐gamma, but not by TNF‐α or IL‐1β. Interferon‐gamma also elicited marked changes in copper homeostasis, including copper‐dependent trafficking of ATP7A from the Golgi to cytoplasmic vesicles, increased copper uptake and elevated expression of the CTR1 copper importer. These findings suggest that pro‐inflammatory conditions associated with AD cause marked changes in microglial copper trafficking, which may underlie the changes in copper homeostasis in AD. It is concluded that copper sequestration by microglia may provide a neuroprotective mechanism in AD.
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