17-β-Estradiol (E2) is a steroid hormone involved in neuroprotection against excitotoxicity and other forms of brain injury. Through genomic and nongenomic mechanisms, E2 modulates neuronal excitability and signal transmission by regulating NMDA and non-NMDA receptors. However, the mechanisms and identity of the receptors involved remain unclear, even though studies have suggested that estrogen G-protein-coupled receptor 30 (GPR30) is linked to protection against ischemic injury. In the culture cortical neurons, treatment with E2 and the GPR30 agonist G1 for 45 min attenuated the excitotoxicity induced by NMDA exposure. The acute neuroprotection mediated by GPR30 is dependent on G-protein-coupled signals and ERK1/2 activation, but independent on transcription or translation. Knockdown of GPR30 using short hairpin RNAs (shRNAs) significantly reduced the E2-induced rapid neuroprotection. Patch-clamp recordings revealed that GPR30 activation depressed exogenous NMDA-elicited currents. Short-term GPR30 activation did not affect the expression of either NR2A- or NR2B-containing NMDARs; however, it depressed NR2B subunit phosphorylation at Ser-1303 by inhibiting the dephosphorylation of death-associated protein kinase 1 (DAPK1). DAPK1 knockdown using shRNAs significantly blocked NR2B subunit phosphorylation at Ser-1303 and abolished the GPR30-mediated depression of exogenous NMDA-elicited currents. Lateral ventricle injection of the GPR30 agonist G1 (0.2 μg) provided significant neuroprotection in the ovariectomized female mice subjected to middle cerebral artery occlusion. These findings provide direct evidence that fast neuroprotection by estradiol is partially mediated by GPR30 and the subsequent downregulation of NR2B-containing NMDARs. The modulation of DAPK1 activity by GPR30 may be an important mediator of estradiol-dependent neuroprotection.

CXCL12, a ligand for the chemokine receptor CXCR4, is well known in mediating neural progenitor cell (NPC) migration during neural development. However, the effects of CXCL12 on human NPC proliferation and its associated signaling pathways remain unclear. The transcription factor, FOXO3a, a downstream target of Akt‐1, is critical for cell cycle control and may also play an important role in regulating NPC proliferation. In this study, we found that CXCL12 promotes human NPC proliferation as determined by the proliferation marker Ki67 and BrdU incorporation. This CXCL12‐mediated NPC proliferation was associated with an increase in Akt‐1 and FOXO3a phosphorylation in a time‐ and dose‐dependent manner. The CXCR4 antagonist (T140) or inhibitors for G proteins (Pertussis toxin) and phosphoinositide 3‐kinase (PI3K) (LY294002) abolished CXCL12‐mediated NPC proliferation and phosphorylation of Akt‐1 and FOXO3a. The roles of Akt‐1 and FOXO3a in CXCL12‐mediated NPC proliferation were further investigated by using adenoviral over‐expression in NPCs. Over‐expression of dominant‐negative Akt‐1 or wild‐type FOXO3a in NPC abrogated CXCL12‐mediated proliferation. These data suggest that CXCL12‐mediated NPC proliferation is reliant upon the phosphorylation of Akt‐1 and FOXO3a and gives insight to an essential role of CXCL12 in neurogenesis. Understanding this mechanism may facilitate the development of novel therapeutic targets for NPC proliferation during neurogenesis.