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

The accumulation of amyloid β protein (Aβ) 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. In the present study, we investigated effects of S-2474 on Aβ-induced cell death in primary cultures of rat cortical neurons. 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 Aβ(25 – 35)- and Aβ(1 – 40)-induced cell death. S-2474 ameliorated Aβ-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA completely. Prior to cell death, Aβ(25 – 35) generated prostaglandin D2 (PGD2) and free radicals from neurons. PGD2 is a product of cyclo-oxygenase (COX), and caused neuronal cell death. S-2474 significantly inhibited the Aβ(25 – 35)-induced generation of PGD2 and free radicals. 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. In conclusion, S-2474 exhibits protective effects against neurotoxicity of Aβ. 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. Keywords: Alzheimer's disease, amyloid β protein, apoptosis, nonsteroidal anti-inflammatory drug, S-2474, cyclo-oxygenase-2, free radical, neuroprotection Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed in drug therapy for inflammatory diseases such as Alzheimer's disease (AD) (McGeer et al., 1996; Stewart et al., 1997). Currently available NSAIDs have dual inhibitory activities against cyclo-oxygenase (COX) and 5-lipoxygenase (5-LO) (Vane & Botting, 1998; Hidaka et al., 1984; Ikuta et al., 1987). COX is classified into two distinct isoforms, a constitutive form, COX-1, and a mitogen-inducible form, COX-2. By COX and 5-LO, arachidonic acid (AA) is metabolized to prostaglandins (PGs) and leukotrienes (LTs), respectively. S-2474 is a novel NSAID containing the γ-sultam skeleton and the di-tert-butylphenol antioxidant moiety (Inagaki et al., 2000). This compound very strongly suppresses the COX-2 pathway (IC50=0.2 – 3.6 nM), and inhibits production of the multiple inflammatory mediators such as LTs, interleukin (IL)-1, IL-6, IL-8 and NO (IC50=0.5 – 30 μM) (Matsumoto et al., 1997). On the other hand, it does not affect the COX-1 pathway (IC50>10 μM) (Inagaki et al., 2000). COX-1 is responsible for the synthesis of cytoprotective prostaglandins (PGs) in the gastrointestinal tract and its inhibition of COX-1 leads to adverse effects, i.e. gastrointestinal ulceration (Miller, 1983). Oral S-2474 exerts anti-inflammatory and analgestic action without side effects, gastrointestinal ulceration (Jyoyama et al., 1997). Thus, S-2474 is thought to offer anti-inflammatory therapeutic effects via inhibition of COX-2, 5-LO, IL production and radical generation. Alzheimer's disease (AD) is characterized clinically by progressive dementia and pathologically by cortical atrophy, neuronal loss, neurofibrillary tangles, senile plaques, and deposits of amyloid β protein (Aβ) in the various region of brain such as cerebral cortex and hippocampus (Selkoe, 1991). Aggregated deposits of Aβ are generally assumed to have a causative role in neurodegeneration and development of AD (Selkoe, 1994; Cummings & Cotman, 1995). Aβ is a 39 to 43-amino-acid hydrophobic peptide, and causes neuronal cell death in primary cultures (Pike et al., 1991; Ueda et al., 1994). Aβ-induced neuronal cell death is typified by several characteristic features of apoptosis, such as formation of cell surface blebs, chromatin condensation, and DNA fragmentation (Forloni et al., 1993; Ueda et al., 1996). Therefore, neuroprotective compounds against Aβ toxicity are drug candidates for the clinical management of AD. Aβ causes peroxidation of plasma membrane (Behl et al., 1994; Ueda et al., 1997a). Aβ neurotoxicity is attenuated by a number of antioxidants, e.g., vitamin E (Behl et al., 1994; Ueda et al., 1997a). Aβ-induced lipid peroxidation impairs plasma membrane ion-motive ATPases, leading to depolarization and the activation of L-type voltage-dependent calcium channels (Weiss et al., 1994; Ueda et al., 1997a). Subsequently, intracellular Ca2+ levels ([Ca2+]i) are elevated (Mattson et al., 1992; Ueda et al., 1997b). The resulting increase in [Ca2+]i activates phospholipase A2 (PLA2), which releases AA from the membrane. Cytosolic PLA2 immunoreactivity is increased in AD brain (Stephenson et al., 1996). However, it has not yet been clearly demonstrated how AA metabolites are involved in Aβ neurotoxicity and AD. A clinical trial of AD patients with a COX inhibitor, indomethacin, indicated a beneficial effect (Rogers et al., 1993; McGeer, 2000). In the brain, both COX-1 and COX-2 are expressed (Yasojima et al., 1999). COX-2 is up-regulated in AD brain and in Aβ-treated SH-SY5Y neuroblastoma cells (Pasinetti & Aisen, 1998), suggesting the involvement of COX-2 in AD. Among PGs, the formation of PGD2 is significantly increased in AD brain (Iwamoto et al., 1989). On the other hand, 5-LO inhibitors protect hippocampal neurons against Aβ-toxicity (Goodman et al., 1994), suggesting the association of LTs with AD. Recently, NSAIDs are reported to reduce apoptotic neuronal cell death via activation of peroxysome proliferator-activated receptor γ (PPAR γ) in rat cerebella granule cells. (Heneka et al., 2000). However, an endogenous ligand for PPAR γ, 15-deoxy-Δ12,14-prostaglandin J2 (15d-Δ12,14-PGJ2) induces apoptosis in rat cortical neurons (Rohn et al., 2001). Thus, it is not yet understood how NSAIDs exhibit clinically beneficial effects on AD. Aβ-induced neuronal cell death is established as in vitro model of AD (Pike et al., 1991). Using this model, we found neuroprotective effects of S-2474 against Aβ neurotoxicity in the primary culture of rat cortical neurons. In the present study, we examined how S-2474 exhibits neuroprotective effects in this model.