Galantamine potentiates the neuroprotective effect of memantine against NMDA-induced excitotoxicity

Abstract

The combination of memantine, an N-methyl-d-aspartate (NMDA) receptor antagonist, with an acetylcholinesterase inhibitor (AChEI) is the current standard of care in Alzheimer's disease (AD). Galantamine, an AChEI currently marketed for the treatment of AD, exerts memory-enhancing and neuroprotective effects via activation of nicotinic acetylcholine receptors (nAChRs). Here, we investigated the neuroprotective properties of galantamine in primary cultures of rat cortical neurons when given alone or in combination with memantine. In agreement with previous findings, we found that memantine was fully effective in reversing NMDA toxicity at concentrations of 2.5 and 5 μmol/L. Galantamine also completely reversed NMDA toxicity at a concentration of 5 μmol/L. The α7 and α4β2 nAChR antagonists, methyllycaconitine, and dihydro-β-erythroidine blocked the neuroprotective effect of galantamine, demonstrating the involvement of nAChRs. The combination of memantine with galantamine produced synergistic actions, such that full neuroprotective efficacy, was obtained at inactive concentrations of memantine (0.1 μmol/L) and galantamine (1 μmol/L). A similar potentiation was also observed when memantine was replaced with ifenprodil, suggesting a possible involvement of the NR2B subunit of the NMDA receptor. In summary, our study reports for the first time at a cellular level that memantine and galantamine interact on the same excitotoxic cascade and that the combination of these two drugs can result in a remarkable neuroprotective effect.

Keywords: Alzheimer's disease; NMDA neurotoxicity; NR2B; drug combination; polypharmacology; primary cortical neurons.

Figure 1

Protective effects afforded by individual treatments with memantine, ifenprodil, and galantamine against NMDA neurotoxicity in cultured rat cortical neurons. Memantine (A, B), ifenprodil (C, D), and galantamine (E, F) caused a significant and dose-dependent decrease in the toxicity of cortical neuron cultures when administered simultaneously to 100 μmol/L NMDA. Treatment duration was 3 h. Neurotoxicity was assessed using the MTT (A, C, E) or LDH (B, D, F) assays; n = 4 (A, B, C, D); n = 5 (E, F). NMDA, N-methyl-d-aspartate; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase.

Figure 2

Protective effects afforded by combined treatments with memantine, ifenprodil, and galantamine against NMDA neurotoxicity in cultured rat cortical neurons. Treatment with galantamine (1 μmol/L)/memantine (0.1 μmol/L) or galantamine (1 μmol/L)/ifenprodil (0.1 μmol/L) combinations yielded significant neuroprotection against the toxicity of NMDA. Treatment duration was 3 h. Neurotoxicity was assessed using the LDH assay (A) or the MTT assay (B). ++: P < 0.01, +++: P < 0.001 compared with NMDA + Mem 0.1 μmol/L or IF 0.1 μmol/L; xxx: P < 0.001 compared with NMDA + Gal 1 μmol/L; ###: compared with NMDA, n = 4. NMDA, N-methyl-d-aspartate; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; IF, ifenprodil.

Figure 3

Blockade of α7 or α4β2 nAChRs decreases galantamine neuroprotection against NMDA toxicity, and activation of α7 nAChR with memantine or ifenprodil shows neuroprotective effect. Exposure of neuronal cultures to different concentrations of (A) methyllycaconitine (MCC) or (B) dihydro-β-erythroidine (DHBE) together with 5 μmol/L of galantamine and 100 μmol/L of NMDA resulted in a dose-dependent decrease in the neuroprotective effect of galantamine. Treatment duration was 3 h. Neurotoxicity was assessed using the LDH (white bars) or MTT (black bars) assays. ###, +++: P < 0.001 compared with NMDA + Gal 5 μmol/L, n = 3. (C) Administration of 0.1 and 1 μmol/L of AR-R17779 in combination with nonactive doses of memantine (0.1 μmol/L) or ifenprodil (0.1 μmol/L) prevents the neurotoxic effect of NMDA exposure in primary cultures of rat cortical neurons. Treatment duration was 3 h. Neurotoxicity was assessed using the MTT (black bars) or LDH (white bars) assays. ###: P < 0.001 compared with NMDA; *P < 0.05, ***P < 0.001 compared with NMDA + Mem 0.1 μmol/L + Gal 1 μmol/L or NMDA + IF 0.1 μmol/L + Gal 1 μmol/L, n = 3. NMDA, N-methyl-d-aspartate; nAChR, nicotinic acetylcholine receptor; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; IF, ifenprodil.

Figure 4

Combined administration of α7 and α4β2 nAChR antagonists abolishes the neuroprotective effect of memantine or ifenprodil plus galantamine. The neuroprotective effect of (A) memantine/galantamine and (B) ifenprodil/galantamine combinations was partially lost through treatment with either methyllycaconitine (MCC) or dihydro-β-erythroidine (DHBE). Simultaneous exposure to both nAChR antagonists completely blocks the protection of the NMDA antagonist/galantamine combination against 100 μmol/L of NMDA. Treatment duration was 3 h. Neurotoxicity was assessed using the MTT (black bars) or LDH (white bars) assays. ###: P < 0.001 compared with NMDA; **P < 0.01, ***P < 0.001 compared with NMDA + Mem 0.1 μmol/L + Gal 1 μmol/L or NMDA + IF 0.1 μmol/L + Gal 1 μmol/L, n = 3. NMDA, N-methyl-d-aspartate; nAChR, nicotinic acetylcholine receptor; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; IF, ifenprodil.