Bis(12)-hupyridone (B12-hup) is a novel dimeric acetylcholinesterase (AChE) inhibitor modified from huperzine A, an unique anti-Alzheimer's agent first isolated from Chinese medicinal plant. In this study, we investigated the neuroprotection of B12-hup against glutamate-induced excitotoxicity in primary cultured rat cerebellar granule neurons (CGNs). Exposure of CGNs to glutamate induces apoptosis, as assessed by MTT and LDH assays, FDA staining, Hoechst 33342 staining and DNA fragmentation. B12-hup, but not other currently used anti-Alzheimer's agents, could effectively attenuate glutamate-induced apoptosis in dose- and time-dependent manners. Meanwhile, MK-801 could also protect the CGNs from the glutamate-induced apoptosis, suggesting that glutamate-induced apoptosis is largely media...[ Read more ]

Bis(12)-hupyridone (B12-hup) is a novel dimeric acetylcholinesterase (AChE) inhibitor modified from huperzine A, an unique anti-Alzheimer's agent first isolated from Chinese medicinal plant. In this study, we investigated the neuroprotection of B12-hup against glutamate-induced excitotoxicity in primary cultured rat cerebellar granule neurons (CGNs). Exposure of CGNs to glutamate induces apoptosis, as assessed by MTT and LDH assays, FDA staining, Hoechst 33342 staining and DNA fragmentation. B12-hup, but not other currently used anti-Alzheimer's agents, could effectively attenuate glutamate-induced apoptosis in dose- and time-dependent manners. Meanwhile, MK-801 could also protect the CGNs from the glutamate-induced apoptosis, suggesting that glutamate-induced apoptosis is largely mediated by N-methyl-D-aspartate (NMDA) receptor. Using the receptor-ligand binding assay, it is found bis(12)-hupyridone displaces the binding of [³H] MK-801 on NMDA receptor with K_i of 586 nM. In addition, B12-hup inhibits the formation of NO and cGMP triggered by glutamate, but is not able to protect the sodium nitroprusside (SNP)-induced death in CGNs. On the other hand, after the exposure to glutamate, the dephosphorylations of protein kinase B (PKB or AKT) and glycogen synthase kinase 3β (GSK3β) were found to rapidly increase. B12-hup inhibited this dephosphorylation and apoptosis in CGNs to a similar extent as the GSK3β inhibitors. Furthermore, the neuroprotective effect of B12-hup might possibly involve the modulation of the cleavage of Cdk5/p35 to Cdk5/p25, which has been first activated by glutamate treatment. More interestingly, we also detect that Cdk5/p35 and Cdk5/p25 might regulate the phosphorylation and dephosphorylation of GSK3β respectively. In conclusion, the ensemble of results indicates that B12-hup protects CGNs against glutamate-induced apoptosis at least in part by possibly regulating the AKT/GSK3β and Cdk5/p25 pathways.