Combinatorial Preconditioning of Rat Brain Cultures with Subprotective Ethanol and Resveratrol Concentrations Promotes Synergistic Neuroprotection

Preconditioning brain cultures with moderate concentrations of ethanol (EtOH) or trans-resveratrol (RES), key red wine constituents, can prevent amyloid-β (Aβ) neurotoxicity. Past studies have indicated that moderate EtOH activates synaptic N-methyl-D-aspartate receptors (NMDAR) that, in part, signal via protein kinase C (PKC) to increase protective antioxidant proteins such as peroxiredoxin-2 (Prx2). RES preconditioning also is reported to involve NMDAR and PKC. However, although moderate, the EtOH and RES concentrations used have been noticeably above circulating levels from two glasses of wine, a daily intake linked to reduced risk of cognitive decline among older social drinkers. Given their mechanistic parallels, we speculated that subprotective EtOH and RES concentrations in a combinatorial preconditioning paradigm might elicit synergistic neuroprotection. To examine this notion, rat cerebellar cultures were pretreated with 10 mM EtOH (circulating concentration after ~ 2 drinks), 5 μM RES, EtOH + RES combinatorially, or media alone (controls). After 3 days, media were removed, and fresh media aliquots containing Aβ25–35 (25 μM) were added. Assessing apoptosis 24 h later with Hoescht 33342, neurodegeneration did not differ from controls in cultures separately preconditioned with 10 mM EtOH or 5 μM RES. However, apoptosis was prevented in combinatorially preconditioned cultures. Also, immunoblotting revealed elevated Prx2 levels due to combinatorial pretreatment that correlated with subsequent neuroprotection, whereas Prx2 was unchanged in separately pretreated cultures. Although the protective mechanisms require clarification, synergistically upregulated NMDAR-PKC-Prx2 (and other antioxidant proteins) is a reasonable component. These findings imply that EtOH + RES antioxidant synergy could be involved in neurobenefits attributed to low-moderate wine consumption.