Kelvin K.W. Kan

Bis(7)-tacrine attenuates β amyloid-induced neuronal apoptosis by regulating L-type calcium channels

β Amyloid protein (Aβ) and acetylcholinesterase (AChE) have been shown to be closely implicated in the pathogenesis of Alzheimers disease. In the current study, we investigated the effects of bis(7)‐tacrine, a novel dimeric AChE inhibitor, on Aβ‐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 Aβ‐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 Ca2+ channels (VDCCs), attenuated Aβ neurotoxicity, whereas N‐, P/Q‐ or R‐type VDCCs blockers and ionotropic glutamate receptor antagonists did not. Fluorescence Ca2+ imaging assay revealed that, similar to nimodipine, bis(7)‐tacrine reversed Aβ‐triggered intracellular Ca2+ increase, which was mainly contributed by the extracellular Ca2+ instead of endoplasmic reticulum and mitochondria Ca2+. 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 Aβ. These results suggest that bis(7)‐tacrine attenuates Aβ‐induced neuronal apoptosis by regulating L‐type VDCCs, offers a novel modality as to how the agent exerts neuroprotective effects.

Synergistic neuroprotection by bis(7)-tacrine via concurrent blockade of N-methyl-D-aspartate receptors and neuronal nitric-oxide synthase

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 glutamate-induced excitotoxicity in primary cultured neurons. Compared with the NO synthase (NOS) inhibitor, NG-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 [3H]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 IC50 values at 2.9 and 9.3 μM 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.

Alkylene tether-length dependent γ-aminobutyric acid type A receptor competitive antagonism by tacrine dimers

Bis(7)-tacrine was previously demonstrated as an antagonist of gamma-aminobutyric acid type A (GABA(A)) receptors. In this study, the effects of a series of alkylene-linked tacrine dimers on GABA(A) receptors were examined. In radioligand binding assay, the analogues differed in binding affinity for GABA(A) receptors, and potency monotonically increased as the tether was shortened from nine to two methylenes. Bis(2)-tacrine, the shortest tacrine dimer, could displace [(3)H]muscimol from rat brain membranes with an IC(50) of 0.48 microM, which was 11, 13 and 525 times more potent than the GABA(A) receptor antagonist (+)-bicuculline, bis(7)-tacrine and tacrine, respectively. In whole-cell patch-clamp recordings, these dimeric tacrine analogues competitively antagonized GABA-induced inward current with a rank order of potency of bis(2)-tacrine>bicuculline>bis(7)-tacrine>bis(9)-tacrine>tacrine, and the potency of bis(2)-tacrine was 11, 18 and 487 times higher than that of (+)-bicuculline, bis(7)-tacrine and tacrine, respectively. Bis(2)-tacrine shifted the GABA concentration-response curve to the right in a parallel manner, and the inhibition was voltage-independent between -80 and +20 mV. It can be concluded that the shorter the alkylene linkage in tacrine dimers the stronger the binding affinity and higher the antagonistic effect on the GABA(A) receptor will be.