Whether moderate ethanol preconditioning (MEP) causes events analogous to those triggered during/by ischemic and other types of neuroprotective preconditioning has been little studied. Therefore, the objective of this project was to elucidate the possible “sensor” roles of certain cell surface receptors in MEP-mediated neuroprotection from Aβ in vitro. Specifically, Gαi protein-coupled receptors and/or glutamatergic N-methyl-D-aspartate receptors (NMDAR) have been shown to be involved in other neuroprotective preconditioning mechanisms. I hypothesize that MEP mediates neuroprotection from Aβ by the sensor activity of one or more of these cell surface receptors. The experiments employed rat cerebellar mixed cultures that show dose-related neurotoxicity in response to Aβ challenge and robust neuroprotection by MEP.
The results of these studies implicate NMDAR activation—but rule against adenosine A1 receptors or other Gαi/o protein-coupled receptors—in attaining the neuroprotected phenotype promoted by MEP. Whereas inhibition of adenosine A1 receptors with the selective antagonist, DPCPX, or of Gαi/o protein-coupled receptors with pertussis toxin failed to block MEP neuroprotection, inhibition of NMDAR with either the selective antagonist, AP5, or open channel blocker, memantine, effectively blocked it. Support for these findings was further demonstrated by the ability of preconditioning with NMDA itself to neuroprotect from Aβ-insult directly.
MEP was then shown to modulate NMDAR by increasing receptor subunit expression and enhancing synaptic receptor localization, ultimately triggering pathways favoring prosurvival and protection against the neurotoxic Aβ. Specifically, NMDAR subunits NR1, NR2B and NR2C were elevated by day 2 of MEP treatment and remained increased through day 6. MEP also increased post-synaptic scaffolding protein PSD-95 and the phosphorylation of tyrosine 1472 of NR2B—both of which are documented markers for synaptically-localized NMDAR complex. These findings agree well with a growing literature on the dichotomous role of synaptic and extra-synaptic NMDAR on neuronal survival vs. excitotoxicity, respectively.
Furthermore, two non-receptor tyrosine kinases known to modulate NMDAR synaptic activity and localization, Src and Pyk2, were shown to be phospho-activated in the cerebellar cultures at a timepoint that correlated with the increases in PSD-95 and NR2B-pY1472, implicating them in the targeting of the NMDAR to the synapse. These findings are intriguing, since PSD-95 has been shown to target Pyk2 to the synapse and provide Src with molecular scaffolding. Src can then interact with multiple MAPK pathways.
It has been recently reported that synaptic NMDAR activity enhances resistance of neurons from oxidative stress by upregulating the peroxiredoxin-thioredoxin antioxidant pathway (Papadia et al., 2008). In exploring whether or not peroxiredoxins were increased in association with augmented NMDAR activity in the MEP model, initial evidence indicated that cytosolic peroxiredoxin-2 is increased by day 6, but not earlier—correlating with the appearance of statistically significant MEP neuroprotection. This result suggests a previously unappreciated “effector” in preconditioning neuroprotection. Since Aβ is know to induce oxidative stress, this finding may partially explain why MEP reduces Aβ neurotoxicity.
Summarizing, the results show that MEP acts through a sensor, NMDAR, to promote neuroprotection. They provide insights into the molecular mechanism by which a receptor once thought responsible for the neurotoxicity of AD pathophysiology actually is modulated in a prosurvival way to induce neuroprotection. Hopefully, future research based on these preconditioning mechanisms might lead to new avenues of therapy that might be useful in AD, age-related cognitive decline, and potentially even vascular dementia (stroke). (Abstract shortened by UMI.)