Vol. 288, Issue 1, 6-13, January 1999

ONO-1603, a Potential Antidementia Drug, Delays Age-Induced Apoptosis and Suppresses Overexpression of Glyceraldehyde-3-Phosphate Dehydrogenase in Cultured Central Nervous System Neurons

Nobuo Katsube, Katsuyoshi Sunaga, Hideki Aishita, De-Maw Chuang and Ryoichi Ishitani

Minase Research Institute, Ono Pharmaceutical Company, Ltd., Osaka, Japan (N.K., H.A.); Group on Cellular Neurobiology, Josai University, Sakado, Saitama, Japan (K.S., R.I.); and Section on Molecular Neurobiology, Biological Psychiatry Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland (D.-M.C.)

Primary cultures of rat cerebral cortical cells and cerebellar granule cells die by an apoptotic mechanism after more than 2 weeks in cultures in the absence of medium change and glucose supplement, a process termed age-induced apoptosis of cultured neurons. Our preliminary study has shown that age-induced apoptosis of cerebellar granule cells is protected by pretreatment with tetrahydroaminoacridine (THA), an antidementia drug. In this study, we systematically compared the neuroprotective effects of THA with those of (S)-1-[N-(4-chlorobenzyl)succinamoyl]pyrrolidine-2-carbaldehyde (ONO-1603), a novel prolyl endopeptidase inhibitor and potential antidementia drug. Both ONO-1603 and THA effectively delay age-induced apoptosis of cerebral and cerebellar neurons, as demonstrated morphologically with toluidine blue and fluorescein diacetate/propidium iodide staining or biochemically by DNA laddering analysis on agarose gels. ONO-1603 is about 300 times more potent than THA, with a maximal protective effect at 0.03 and 10 µM, respectively. ONO-1603 shows a wide protective range of 0.03 to 1 µM in contrast to a narrow effective range of 3 to 10 µM for THA. Moreover, ONO-1603 is nontoxic to neurons, even at the high concentration of 100 µM, whereas THA elicits severe neurotoxicity at a dose of 30 µM. Both ONO-1603 and THA robustly suppress overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) mRNA and accumulation of GAPDH protein in a particulate fraction of cultured neurons undergoing age-induced apoptosis. Because we documented that GAPDH overexpression participates in neuronal apoptosis induced by various insults, we conclude that the neuroprotective actions of ONO-1603 and THA appear to be mediated by suppression of this protein overexpression.