Neuroprotection by donepezil against glutamate excitotoxicity involves stimulation of alpha7 nicotinic receptors and internalization of NMDA receptors.
OBJECTIVE
Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer's disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil.


METHODS
Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca(2+) concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD.


RESULTS
Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepezil. In addition, glutamate-mediated Ca(2+) entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of alpha7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca(2+) entry. LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca(2+) entry induced by donepezil.


CONCLUSIONS
Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of alpha7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system.
Shen
H
H
Department of Neuroscience for Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Kihara
T
T
Hongo
H
H
Wu
X
X
Kem
W R
WR
Shimohama
S
S
Akaike
A
A
Niidome
T
T
Sugimoto
H
H
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D000077265:Donepezil; D018698:Glutamic Acid / Q000494:pharmacology*; D007189:Indans / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D064569:alpha7 Nicotinic Acetylcholine Receptor; D019061:src-Family Kinases / Q000378:metabolism
C579165:Chrna7 protein, rat; D007189:Indans; D018696:Neuroprotective Agents; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D018698:Glutamic Acid; D000077265:Donepezil; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D002118:Calcium