Abstract
The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 mum glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 mum) on CGNs markedly reduced glutamate-induced apoptosis in dose- and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 mum bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-beta-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-d-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca(2+) imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca(2+) increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [(3)H]MK-801 with an IC(50) value of 0.763 mum in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.
Highlights
The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs)
AChE inhibitors, such as tacrine and huperize A, have been shown to attenuate the apoptosis of cortical neurons induced by H2O2 or -amyloid peptide (10, 32)
Because bis(7)-tacrine is a novel, selective, and potent AChE inhibitor (1, 2), did bis(7)-tacrine prevent glutamate-induced apoptosis by inhibition of AChE? other AChE inhibitors hardly showed any neuroprotective properties on neuronal apoptosis induced by glutamate in CGNs, E2020 at 50 M possessed similar effects on inhibiting the activity of AChE in CGNs with bis(7)-tacrine at 1 M (Fig. 3, A–C)
Summary
Vol 280, No 18, Issue of May 6, pp. 18179 –18188, 2005 Printed in U.S.A. Novel Dimeric Acetylcholinesterase Inhibitor Bis(7)-tacrine, but Not Donepezil, Prevents Glutamate-induced Neuronal Apoptosis by Blocking N-Methyl-D-aspartate Receptors*. The current studies were undertaken to evaluate the ability of bis(7)-tacrine to prevent glutamate-induced apoptosis in CGNs and to systematically delineate whether this agent affected the key steps of glutamate-induced apoptosis from ERK and p38 pathways to intracellular Ca2ϩ to NMDA receptors. It appears that bis(7)-tacrine prevents glutamate-induced apoptosis in neurons by directly blocking NMDA receptors at the MK-801-binding site yet independent of the inhibition on AChE and cholinergic transmission
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