Title

 

Authors

S.F. Spampinato

PhD Program in Neuropharmacology,
Department of Clinical and Molecular Biomedicine, section of Pharmacology and Biochemistry, University of Catania 

 

Abstract

Estrogen exerts its neuroprotective effect through interaction with estrogen receptors (ERalpha and ERbeta) that are localized in the intracellular compartment as well as at the plasma membrane. Rapid signaling activated by these membrane-localized ERs has been described and the ability of estrogen to transactivate other membrane receptors has also been reported. Specifically ERalpha and ERbeta are known to interact with metabotropic glutamate (mGlu) receptors and exploit their transduction mechanisms. Both ERaalpha and mGlu1 receptors are co-expressed in primary cultures of rat cortical neurons and co-immunoprecipitate upon estrogen stimulation. In mixed cortical cultures containing neurons and glial cells, activation of either ERalpha, by 10 nM 17beta-estradiol (17E2), or mGlu1 receptor, by 100 µM dihydroxyphenilglycine (DHPG), produced a neuroprotective effect against beta-amyloid (Abeta)1-42 (1 µM) or Abeta25-35 (25 µM) toxicity, an effect that was mimicked by stimulation of ERalpha by the specific agonist PPT (100 nM) or selective activation of membrane-localized receptors by the use of the cell impermeable analog BSA-conjugated 17E2 (100 nM). When cultures were co-stimulated with 17E2 and DHPG, the neuroprotection observed was comparable to that produced by each treatment alone. Maintenance of the neuroprotective effect under conditions of blockade of mGlu5 receptor, by the use of the selective antagonist MPEP, ruled out the possibility that the observed effect involved this receptor subtype. The neuroprotective effect of each agonist was prevented by the respective antagonist and, more interestingly, reduction of neuronal death induced by 17E2 treatment was counteracted by the mGlu1 receptor antagonist  JNJ16259685 (100 nM) whereas, the effect produced by DHPG treatment was attenuated by the ER antagonist ICI 182,780 (1 µM). The ability of the two antagonists to reduce 17E2- and DHPG-induced neuroprotection was observed also when the two drugs were added together. To assess the signaling pathway involved in this neuroprotective effect, attention was focused on the phosphatidylinositol kinase/Akt pathway. In Hek-293 cells transiently transfected with ERalpha and mGlu1 receptor, stimulation of both receptors by 10 nM 17E2 and 200 µM quisqualate produced increased Akt phosphorylation at serine 473 and concomitant stimulation of both receptors resulted in an effect that was greater than that produced by each treatment alone. A similar effect was observed in pure cultures of cortical neurons stimulated with 10 nM 17E2 and 100 µM DHPG. However, 17E2 effect was prevented by treatment with JNJ16259685 whereas, ICI 182,780 contrasted DHPG-induced AKT phosphorylation. Finally, treatment with the Akt inhibitor 10-DEBC hydrochloride (10 µM) attenuated the neuroprotective effect observed following treatment with 17E2, DHPG or both.  The present results suggest that the neuroprotective effect of 17E2 against Abeta toxicity involves an interaction between ERalpha  and mGlu1 receptor and activation of a common signaling pathway.