Donepezil suppresses intracellular Ca²⁺ mobilization through the PI3K pathway in rodent microglia

Microglia are resident innate immune cells which release many factors including proinflammatory cytokines or nitric oxide (NO) when they are activated in response to immunological stimuli. Pathophysiology of Alzheimer's disease (AD) is related to the inflammatory responses mediated by microglia. Intracellular Ca²⁺ signaling is important for microglial functions such as release of NO and cytokines. In addition, alteration of intracellular Ca²⁺ signaling underlies the pathophysiology of AD, while it remains unclear how donepezil, an acetylcholinesterase inhibitor, affects intracellular Ca²⁺ mobilization in microglial cells.

We examined whether pretreatment with donepezil affects the intracellular Ca²⁺ mobilization using fura-2 imaging and tested the effects of donepezil on phagocytic activity by phagocytosis assay in rodent microglial cells.

In this study, we observed that pretreatment with donepezil suppressed the TNFα-induced sustained intracellular Ca²⁺ elevation in both rat HAPI and mouse primary microglial cells. On the other hand, pretreatment with donepezil did not suppress the mRNA expression of both TNFR1 and TNFR2 in rodent microglia we used. Pretreatment with acetylcholine but not donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the nicotinic α7 receptors. In addition, sigma 1 receptors were not involved in the donepezil-induced suppression of the TNFα-mediated intracellular Ca²⁺ elevation. Pretreatment with donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the PI3K pathway in rodent microglial cells. Using DAF-2 imaging, we also found that pretreatment with donepezil suppressed the production of NO induced by TNFα treatment and the PI3K pathway could be important for the donepezil-induced suppression of NO production in rodent microglial cells. Finally, phagocytosis assay showed that pretreatment with donepezil promoted phagocytic activity of rodent microglial cells through the PI3K but not MAPK/ERK pathway.

These suggest that donepezil could directly modulate the microglial function through the PI3K pathway in the rodent brain, which might be important to understand the effect of donepezil in the brain.