Antagonism of neuronal prostaglandin E(2) receptor subtype 1 mitigates amyloid β neurotoxicity in vitro

Abstract

Multiple lines of evidence indicate that regional brain eicosanoid signaling is important in initiation and progression of neurodegenerative conditions that have neuroinflammatory pathologic component, such as AD. We hypothesized that PGE(2) receptor subtype 1 (EP1) signaling (linked to intracellular Ca(2+) release) regulates Aβ peptide neurotoxicity and tested this in two complementary in vitro models: a human neuroblastoma cell line (MC65) producing Aβ(1-40) through conditional expression of the APP C-terminal portion, and murine primary cortical neuron cultures exposed to Aβ(1-42). In MC65 cells, EP1 receptor antagonist SC-51089 reduced Aβ neurotoxicity ~50 % without altering high molecular weight Aβ immunoreactive species formation. Inositol-3-phosphate receptor antagonist 2-aminoethoxy-diphenyl borate offered similar protection. SC-51089 largely protected the neuron cultures from synthetic Aβ(1-42) neurotoxicity. Nimodipine, a Ca(2+) channel blocker, was completely neuroprotective in both models. Based on these data, we conclude that suppressing neuronal EP1 signaling may represent a promising therapeutic approach to ameliorate Aβ peptide neurotoxicity.

Figure 1. Inhibiting innate immunity as a therapeutic strategy for neurodegenerative diseases

Figure 2

A. MC65 cells were plated on Day 0 in medium with (Tet+) or without (Tet-) tetracycline. Cell viability was assessed with the MTT assay and data presented as % (SEM) of Tet+ cultures on Day 1. Tet+ cultures continued to expand over the next 2 days before reaching a plateau. In contrast, Tet- cultures showed delayed but complete toxicity that developed over Days 1 to 3. B. Cultures of MC65 cells were incubated with (Tet+) or without (Tet-) tetracylcline (1 μg/ml) in the presence of the reagents for 48 h and then harvested in RIPA buffer, proteins separated by SDS-PAGE, and then probed with antibody 6E10 that recognizes an epitope in amino acids 1–16 of Aβ peptides. All Tet+ cultures failed to display any detectable 6E10-immunorective bands. Cultures exposed to drug vehicle (Veh) show the expected multiple species of 6E10-immunoreactive bands. Incubation with α-tocopherol (VitE, 100 μM) suppressed HMW aggregates of 6E10-immunoreative species, as previously described. In contrast, incubation with nimodipine (Nim, 25 μM) or SC-51089 (SC, 50 μM) did not block the formation of HMW species. Western blot for β-actin is loading control.

Figure 3

A. MC65 cells were seeded in 96-well plate in mediumwith (Tet+) or without (Tet-) tetracycline in the presence of the inhibitors at Day 0. The cells were cultured for an additional 72 h and then cell viability was assessed with an MTT assay with data presented as % (SEM) of Tet+ cultures with vehicle. Inhibitors were SC-51089 (SC, 20 μM), nimodipine (Nim, 25 μM), or 2-aminoethoxy-diphenyl borate (2APB, 20 μM). Two-way ANOVA had P<0.0001 for Tet+ vs. Tet-, for agents, and for interaction between these two terms. Bonferroni-corrected post-tests Tet- with Vehicle, SC, or 2APB. B. Murine primary cerebral cortical neurons were incubated with fresh medium (Med, clear bar) or medium that contained 20 μMAβ1–42 (filled bars) and the inhibitors for 48 h. Cell viability was assessed with an MTT assay. SC-51089 protected neurons from Aβ1–42 toxicity at all concentrations used (P<0.001 compared to vehicle for all concentrations of SC-51089) with the greatest protection provided at 50 μM; however, protection by SC-51089 was partial and significantly less than nimodipine (P<0.01 for SC-51089 at 10 or 25 μM, and P<0.05 for SC-51089 at 50 μM). One-way ANOVA had P<0.0001 with Bonferroni-corrected multiple paired comparisons having *P<0.001 compared to Med and ⁺P<0.001 compared to Veh