Synergistic Neuroprotection by Bis(7)-tacrine via Concurrent Blockade of N-Methyl-d-aspartate Receptors and Neuronal Nitric-Oxide Synthase

Abstract

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;

Novel Anti-Alzheimer's Dimer Bis(7)-Cognitin: Cellular and Molecular Mechanisms of Neuroprotection Through Multiple Targets

“…35 Pretreatment of cells with bis(7)-Cognitin or tacrine attenuated hydrogen peroxide (H 2 O 2 )-induced cell toxicity in pheochromocytoma (PC12) cells, and bis(7)-Cognitin demonstrated higher potency than tacrine in improving redox desequilibrium. 36 Recently, we have also found that bis(7)-Cognitin prevents glutamate-induced apoptosis in cerebellar granule neurons (CGNs) 22,37 and attenuates ␤-amyloid-induced neuronal apoptosis in rat cortical neurons, 38 and it reverses the inhibition of long-term potentiation (LTP) by A␤ in hippocampal slices. In the in vivo study, bis(7)-Cognitin reverses the occlusion of bilateral common carotid arteries-induced injuries in brain and latency delay in Morris water maze, and reduces the infarction size and neurological scoring caused by middle cerebral arty occlusion in rats.…”

“…22,40 The neuroprotective properties of bis(7)-Cognitin on glutamate-induced excitotoxicity were investigated in primary cultured CGNs. 37 Exposure of CGNs to 75 M glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL (FIG.…”

“…To date, three novel series of tacrine/HA analogs have been developed by linking tacrine, or the fragment of HA to itself, or the fragment of HA to tacrine. 21,22 These dimeric drugs are much easier to produce and have been shown to be more potent than either tacrine or HA alone, and represent a combination of the best of the Western and Chinese palliative drugs for AD. In this review, we demonstrate that the novel promising anti-Alzheimer's candidate bis(7)-tacrine (FIG.…”

The anti‐cancer agent SU4312 unexpectedly protects against MPP+‐induced neurotoxicity via selective and direct inhibition of neuronal NOS

“…The survival of neurons in the presence of SU4312 and/or MPP + was assayed by determining the activity of mitochondrial dehydrogenases with 3(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay (Li et al, 2007). The assay was performed according to the specifications of the manufacturer (MTT kit I; Roche Applied Science, Pen-zberg, Germany).…”

“…For the in vitro NOS activity assay (Li et al, 2007), purified recombinant human nNOS, endothelial NOS (eNOS) and inducible NOS (iNOS) were bought from Alexis Biochemicals (Lausen, Switzerland). NOS activity was determined by monitoring the conversion of L-[ 3 H]arginine to [ 3 H]citrulline following the instructions provided in the kit (Merck, Darmstadt, Germany).…”