Estrogen receptor alpha and G-protein coupled receptor 30 mediate the neuroprotective effects of 17β-estradiol in novel murine hippocampal cell models

The hippocampus is a multifaceted, complex brain structure considered to be the learning center. The use of primary hippocampal cell cultures has uncovered important cellular mechanisms involved in overall physiological function. Yet, the use of primary culture is inherently difficult, and the lack of immortalized cell lines from the murine hippocampus for mechanistic studies at the molecular level is evident. We have immortalized cell lines from embryonic (E18) and adult-derived hippocampal primary cell culture using retroviral infection of SV40 T-antigen. Four clonal embryonic lines, mHippoE-2, mHippoE-5, mHippoE-14, mHippoE-18, and one mixed adult line, mHippoA-mix, exhibited neuronal morphologies with neurite extensions and expression of neuronal markers, with unique gene expression profiles. We used these cell models to study the neuroprotective effects of 17β-estradiol (E2) on glutamate-induced neurotoxicity. The cell lines express a relevant array of genes and receptors suggested to play a role in neuroprotection, including estrogen receptors ERα, ERβ, and GPR30. We find that pretreatment with E2 (10 or 100 nM) for 24 h significantly reduced cell death induced by glutamate mHippoE-14 and mHippoE-18 cells, but not the mHippoA-mix. Using 24 h pretreatment with the specific estrogen receptor (ER) agonists, 4,4′,4′′-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) and diarylpropionitrile, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), we linked the E2-mediated neuroprotection to ERα, but only in the mHippoE-18 cells. Since E2 activated both PI3K/Akt and STAT3 signaling pathways, we also tested whether the membrane-bound E2 receptor GPR30 was involved in its neuroprotective action. Pretreatment with the GPR30 agonist G-1 (10 and 100 nM) for 1 h, but not 24 h, significantly attenuated cell death in both mHippoE-14 and mHippoE-18 cells. The use of specific ER antagonist ICI 182780 and GPR30 antagonist G-15 linked these effects to both ER and GPR30 receptors. This is the first evidence that GPR30 may play a role in the protective effects of estrogen in hippocampal neurons.