-dependent neuronal death in a similar concentration range. Molecular cloning of TRPM2 cDNA from rat brain revealed several differences in amino acid sequences within the Nudix box region as compared with those of human and mouse TRPM2. ADPR-induced current responses, H 2 O 2 -induced Ca 2+ influx, and H 2 O 2 -induced cell death were induced in human embryonic kidney cells heterologously expressing rat TRPM2. Treatment of cultured neurons with small interfering RNA against rat TRPM2, which efficiently suppressed immunoreactive TRPM2 content and the H 2 O 2 -induced Ca 2+ influx, significantly inhibited H 2 O 2 -induced neuronal death. These results suggest that TRPM2 plays a pivotal role in H 2 O 2 -induced neuronal death as redox-sensitive Ca 2+-permeable channels expressed in neurons.

Donepezil is a potent and selective acetylcholinesterase (AChE) inhibitor developed for the treatment of Alzheimer's disease. To elucidate whether donepezil shows neuroprotective action in addition to amelioration of cognitive deficits, we examined the effects of donepezil on glutamate-induced neurotoxicity using primary cultures of rat cortical neurons. A 10-min exposure of cultures to glutamate followed by a 1-h incubation with glutamatefree medium caused a marked loss of viability, as determined by Trypan blue exclusion. Glutamate neurotoxicity was prevented by 24-h pretreatment of donepezil in a concentration-dependent manner. Among AChE inhibitors examined, donepezil and certain AChE inhibitors such as tacrine and galanthamine showed potent neuroprotective action, although physostigmine did not affect glutamate neurotoxicity. Neuroprotective action of donepezil was antagonized by mecamylamine, a nicotinic acetylcholine receptor (nAChR) antagonist, but not by scopolamine, a muscarinic acetylcholine receptor antagonist. Furthermore, both dihydro-␤-erythroidine, an ␣4␤2-neuronal nAChR antagonist, and methyllycaconitine, an ␣7-nAChR antagonist, each also significantly antagonized the effect of donepezil. Next, we examined the effects of donepezil on glutamate-induced apoptosis. Exposure of 100 M glutamate to cortical neurons for 24 h induced apoptotic neuronal death and nuclear fragmentation. Donepezil for 24 h before and 24 h during glutamate exposure prevented nuclear fragmentation and glutamate-induced apoptosis. These results suggest that donepezil not only protects cortical neurons against glutamate neurotoxicity via ␣4␤2-and ␣7-nAChRs but also prevents apoptotic neuronal death.

No abstract