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b Amyloid protein (Ab) and acetylcholinesterase (AChE) have been shown to be closely implicated in the pathogenesis of Alzheimer's disease. In the current study, we investigated the effects of bis(7)-tacrine, a novel dimeric AChE inhibitor, on Ab-induced neurotoxicity in primary cortical neurons. Bis(7)-tacrine, but not other AChE inhibitors, elicited a marked reduction of both fibrillar and soluble oligomeric forms of Ab-induced apoptosis as evidenced by chromatin condensation and DNA specific fragmentation. Both nicotinic and muscarinic receptor antagonists failed to block the effects of bis(7)-tacrine. Instead, nimodipine, a blocker of L-type voltage-dependent Ca 2+ channels (VDCCs), attenuated Ab neurotoxicity, whereas N-, P/Q-or R-type VDCCs blockers and ionotropic glutamate receptor antagonists did not.Fluorescence Ca 2+ imaging assay revealed that, similar to nimodipine, bis(7)-tacrine reversed Ab-triggered intracellular Ca 2+ increase, which was mainly contributed by the extracellular Ca 2+ instead of endoplasmic reticulum and mitochondria Ca 2+ . Concurrently, using whole cell patch-clamping technique, it was found that bis (7)-tacrine significantly reduced the augmentation of high voltage-activated inward calcium currents induced by Ab. These results suggest that bis(7)-tacrine attenuates Ab-induced neuronal apoptosis by regulating L-type VDCCs, offers a novel modality as to how the agent exerts neuroprotective effects.

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The excessive activation of the N-methyl-D-aspartate receptor (NMDAR)/nitric oxide (NO) pathway has been proposed to be involved in the neuropathology of various neurodegenerative disorders. In this study, NO was found to mediate glutamateinduced excitotoxicity in primary cultured neurons. Compared with the NO synthase (NOS) inhibitor, N G -monomethyl-L-arginine (L-NMMA), and the NMDAR antagonist memantine, bis(7)-tacrine was found to be more potent in reducing NO-mediated excitotoxicity and the release of NO caused by glutamate. Moreover, like L-NMMA but not like 5H-dibenzo [a,d]cyclohepten-5,10-imine (MK-801) and memantine, bis(7)-tacrine showed greater neuroprotection and inhibition on NO release when neurons were pretreated for a prolonged time between 0 and 24 h and remained quite potent even when neurons were post-treated 1 h after the glutamate challenge. Bis(7)-tacrine was additionally found to be as moderately potent as memantine in competing with [ 3 H]MK-801, inhibiting NMDA-evoked currents and reducing glutamate-triggered calcium influx, which eventually reduced neuronal NOS activity. More importantly, at neuroprotective concentrations, bis(7)-tacrine substantially reversed the overactivation of neuronal NOS caused by glutamate without interfering with the basal activity of NOS. Furthermore, in vitro pattern analysis demonstrated that bis(7)-tacrine competitively inhibited both purified neuronal and inducible NOS with IC 50 values at 2.9 and 9.3 M but not endothelial NOS. This result was further supported by molecular docking simulations that showed hydrophobic interactions between bis(7)-tacrine and three NOS isozymes. Taken together, these results strongly suggest that the substantial neuroprotection against glutamate by bis(7)-tacrine might be mediated synergistically through the moderate blockade of NMDAR and selective inhibition of neuronal NOS.The precise mechanisms leading to the pathogenesis of chronic and acute neurodegenerative disorders have not yet been fully elucidated. However, increasing evidence has shown that these diseases may share a final common pathway to neuronal injury as a result of the excitotoxicity caused by the overstimulation of glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype (Yuan and Yankner, 2000;