Preconditioning refers to the phenomenon by which a brief, sublethal stress confers protection against subsequent, lethal insult. Previous studies in our laboratory have demonstrated that moderate ethanol preconditioning (MEP; 20-30 mM, 6 days) confers neuroprotection against potent neurotoxins such as HIV-1 gp120 and Alzheimer's Aβ. MEP mechanism may involve sensors, transducers and effectors. Initial studies indicated that the heat shock proteins (HSPs), especially HSP70 and HSP27, are necessary effectors in MEP-induced neuroprotection against HIV-1 gp120.

This study identified two prosurvival enzymes, protein kinase C (PKC) and focal adhesion kinase (FAK), as potential transducers in this mechanism. MEP increased PKC activity in mixed cerebellar cell cultures after 2 and 6 days. At the 6 day timepoint, expression levels of PKCϵ, PKCα and PKCδ—but not PKCβ—were increased, as was the membrane translocation (activation) of the three increased isoforms. MEP also increased the total levels and the levels of phosphorylated (activated) FAK after 6 days. FAK induction and activation correlated with HSP27/70 induction and HSP27 phosphorylation (activation), which is known to cause prosurvival actin cytoskeletal changes in other preconditioning paradigms. The induction of PKC and FAK, and the activation of FAK and HSP27 were also replicated in ethanol-preconditioned rat organotypic hippocampal entorhinal cortical slice cultures.

The sequence of the signaling events involving PKC, FAK and HSPs was then examined. PKC inhibition with GF109203X abrogated ethanol-dependent induction/activation of FAK and the two HSPs. Adenoviral infection of the mixed cerebellar cell cultures with dominant negative PKCϵ and PKCα indicated that PKCϵ was the isoform involved in the induction and activation of FAK and HSPs. FAK inhibition with dominant negative FAK (FRNK) abrogated the induction and activation of HSPs, while abolishing MEP-induced neuroprotection against Aβ. Since FAK's role in preconditioning has been little studied and since ethanol's effect on FAK activation has not been explored to date, the aforementioned reports suggest a novel mechanism of preconditioning-induced neuroprotection. Elucidation of this newly defined MEP-dependent upregulation/activation cascade progressing from PKCϵ to FAK (transducers) to HSP27 and HSP70 (effectors) could enable the development of novel therapeutic targets to treat neuropathological conditions and insults.