Effects of S-2474, a novel nonsteroidal anti-inflammatory drug, on amyloid beta protein-induced neuronal cell death

Abstract

1. The accumulation of amyloid beta protein (Abeta) in the brain is a characteristic feature of Alzheimer's disease (AD). Clinical trials of AD patients with nonsteroidal anti-inflammatory drugs (NSAIDs) indicate a clinical benefit. NSAIDs are presumed to act by suppressing inhibiting chronic inflammation in the brain of AD patients. 2. In the present study, we investigated effects of S-2474 on Abeta-induced cell death in primary cultures of rat cortical neurons. 3. S-2474 is a novel NSAID, which inhibits cyclo-oxygenase-2 (COX-2) and contains the di-tert-butylphenol antioxidant moiety. S-2474 significantly prevented neurons from Abeta(25 - 35)- and Abeta(1 - 40)-induced cell death. S-2474 ameliorated Abeta-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA completely. 4. Prior to cell death, Abeta(25 - 35) generated prostaglandin D(2) (PGD(2)) and free radicals from neurons. PGD(2) is a product of cyclo-oxygenase (COX), and caused neuronal cell death. 5. S-2474 significantly inhibited the Abeta(25 - 35)-induced generation of PGD(2) and free radicals. 6. The present cortical cultures contained little non-neuronal cells, indicating that S-2474 affected neuronal survival directly, but not indirectly via non-neuronal cells. Both an inhibitory effect of COX-2 and an antioxidant effect might contribute to the neuroprotective effects of S-2474. 7. In conclusion, S-2474 exhibits protective effects against neurotoxicity of Abeta. Furthermore, the present study suggests that S-2474 may possess therapeutic potential for AD via ameliorating degeneration in neurons as well as suppressing chronic inflammation in non-neuronal cells.

Figure 1

Effects of S-2474 on Aβ(25 – 35)-induced neuronal cell death. Cortical neurons were treated with S-2474 at the indicated concentrations in the presence (open circles) or absence (closed circles) of 10 μM Aβ(25 – 35). MTT reducing activity was determined 48 h after Aβ(25 – 35) treatment. Data are expressed as mean±s.e.mean values (n=4). **P<0.01, compared with Aβ(25 – 35) control by ANOVA followed by Dunnett's test. Vehicle control is treated with culture medium containing 1% deionized water and 0.1% DMSO. Aβ control is treated with culture medium containing 10 μM Aβ (25 – 35) and 0.1% DMSO.

Figure 2

Morphologic changes in cortical neurons by Aβ(25 – 35). Immunocytochemical analysis for anti-MAP2 (A), anti-GFAP (B) or OX-42 (C) was performed on the present cortical cultures. Magnification, ×95. Cortical neurons were treated with vehicle control (D), 10 μM Aβ(25 – 35) (E), or 10 μM Aβ(25 – 35)+10 μM S-2474 (F). Neurons were examined by phase-contrast microscopy 48 h later. Magnification, ×95. Vehicle control is treated with culture medium containing 1% deionized water and 0.1% DMSO. Aβ control is treated with culture medium containing 10 μM Aβ (25 – 35) and 0.1% DMSO.

Figure 3

Apoptotic features of cortical neurons induced by Aβ(25 – 35). Cortical neurons were treated with vehicle control (A and D), 10 μM Aβ(25 – 35) control (B and E) or 10 μM Aβ(25 – 35)+10 μM S-2474 (C and F). Neurons were stained with 1 μM Hoechst 33258 for 10 min 48 h later (A, B, and C). Neurons were fixed with 4% paraformaldehyde, washed twice with PBS, and stained by the TUNEL technique 48 h later (D, E, and F). Magnification, ×95. Vehicle control is treated with culture medium containing 1% deionized water and 0.1% DMSO. Aβ control is treated with culture medium containing 10 μM Aβ (25 – 35) and 0.1% DMSO.

Figure 4

Aβ(25 – 35)-induced neuronal cell death and generation of eicosanoids from Neurons. Cortical neurons were treated with 10 μM Aβ(25 – 35). MTT-reducing activity (A) and eicosanoids (B) were measured at the indicated time points after Aβ treatment. PGD₂ (circles), PG E₂ (triangles) and LBT₄ (squares) were measured with their RIA kits. The control level of PGD₂, PGE₂ or LTB₄ was 73±3, 198±20 or 752±38 pg/ml, respectively. Data are expressed as mean±s.e.mean values (n=4). **P<0.01, compared with vehicle control by ANOVA followed by Dunnett's test. Vehicle control is treated with culture medium containing 1% deionized water and 0.1% DMSO.

Figure 5

Effect of S-2474 on Aβ(25 – 35)-induced generation of PGD₂ from Neurons. (A) PGD₂-induced neuronal cell death: Cortical neurons were treated with 10 μM PGD₂. MTT-reducing activity was determined at the indicated times after the PG treatment. *P<0.05, **P<0.01, compared with vehicle control by ANOVA followed by Dunnett's test. Vehicle control is treated with culture medium containing 0.1% ethanol. (B) Effect of S-2474 on Aβ(25 – 35)-induced generation of PGD₂ from Neurons: Cortical neurons were treated with S-2474 at the indicated concentration in the presence of 10 μM Aβ(25 – 35). Generation of PGD₂ was determined 29 h later. Data are expressed as mean±s.e.mean values (n=4). **P<0.01, compared with Aβ control by ANOVA followed by Dunnett's test. Aβ control is treated with culture medium containing 10 μM Aβ (25 – 35) and 0.1% DMSO.

Figure 6

Involvement of free radicals in Aβ(25 – 35) neurotoxicity. (A) Level of free radicals measured by DCFDA assay: Cortical neurons were treated with 10 μM Aβ(25 – 35) in the presence or absence of S-2474 or 100 μM vitamin E, and the DCFDA assay was performed 24 h later. Data are expressed as mean±s.e.mean (n=4), per cent of control cultures, which were incubated for the same amount of time as experimental cultures (n=4). **P<0.01, compared with vehicle control by ANOVA followed by Dunnett's test. ##P<0.01, compared with Aβ control by ANOVA followed by Dunnett's test. Vehicle control is treated with culture medium containing 1% deionized water and 0.1% DMSO. Aβ control is treated with culture medium containing 10 μM Aβ (25 – 35) and 0.1% DMSO. (B) Effect of S-2474 on Aβ(25 – 35)-induced neuronal cell death. Cortical neurons were treated with S-2474 at the indicated concentration or 100 μM vitamin E in the presence of 10 μM Aβ(25 – 35). Forty-eight hours later, cell viability was measured according to the morphological criteria. Data are expressed as mean±s.e.mean values (n=4). **P<0.01, compared with vehicle control by ANOVA followed by Dunnett's test. #P<0.05, ##P<0.01, compared with Aβ control by ANOVA followed by Dunnett's test. Vehicle control is treated with culture medium containing 1% deionized water and 0.1% DMSO. Aβ control is treated with culture medium containing 10 μM Aβ (25 – 35) and 0.1% DMSO.