Dorsal Hippocampal Actin Polymerization Is Necessary for Activation of G-Protein-Coupled Estrogen Receptor (GPER) to Increase CA1 Dendritic Spine Density and Enhance Memory Consolidation.

Activation of the membrane estrogen receptor G-protein-coupled estrogen receptor (GPER) in ovariectomized mice via the GPER agonist G-1 mimics the beneficial effects of 17β-estradiol (E2) on hippocampal CA1 spine density and memory consolidation, yet the cell-signaling mechanisms mediating these effects remain unclear. The present study examined the role of actin polymerization and c-Jun N-terminal kinase (JNK) phosphorylation in mediating effects of dorsal hippocampally infused G-1 on CA1 dendritic spine density and consolidation of object recognition and spatial memories in ovariectomized mice. We first showed that object learning increased apical CA1 spine density in the dorsal hippocampus (DH) within 40 min. We then found that DH infusion of G-1 increased both CA1 spine density and phosphorylation of the actin polymerization regulator cofilin, suggesting that activation of GPER may increase spine morphogenesis through actin polymerization. As with memory consolidation in our previous work (Kim et al., 2016), effects of G-1 on CA1 spine density and cofilin phosphorylation depended on JNK phosphorylation in the DH. Also consistent with our previous findings, E2-induced cofilin phosphorylation was not dependent on GPER activation. Finally, we found that infusion of the actin polymerization inhibitor, latrunculin A, into the DH prevented G-1 from increasing apical CA1 spine density and enhancing both object recognition and spatial memory consolidation. Collectively, these data demonstrate that GPER-mediated hippocampal spinogenesis and memory consolidation depend on JNK and cofilin signaling, supporting a critical role for actin polymerization in the GPER-induced regulation of hippocampal function in female mice.SIGNIFICANCE STATEMENT Emerging evidence suggests that G-protein-coupled estrogen receptor (GPER) activation mimics effects of 17β-estradiol on hippocampal memory consolidation. Unlike canonical estrogen receptors, GPER activation is associated with reduced cancer cell proliferation; thus, understanding the molecular mechanisms through which GPER regulates hippocampal function may provide new avenues for the development of drugs that provide the cognitive benefits of estrogens without harmful side effects. Here, we demonstrate that GPER increases CA1 dendritic spine density and hippocampal memory consolidation in a manner dependent on actin polymerization and c-Jun N-terminal kinase phosphorylation. These findings provide novel insights into the role of GPER in mediating hippocampal morphology and memory consolidation, and may suggest first steps toward new therapeutics that more safely and effectively reduce memory decline in menopausal women.

Kim J ，Schalk JC ，Koss WA ，Gremminger RL ，Taxier LR ，Gross KS ，Frick KM ... -  《-》

17β-Estradiol and Agonism of G-protein-Coupled Estrogen Receptor Enhance Hippocampal Memory via Different Cell-Signaling Mechanisms.

The ability of 17β-estradiol (E2) to enhance hippocampal object recognition and spatial memory depends on rapid activation of extracellular signal-regulated kinase (ERK) in the dorsal hippocampus (DH). Although this activation can be mediated by the intracellular estrogen receptors ERα and ERβ, little is known about the role that the membrane estrogen receptor GPER plays in regulating ERK or E2-mediated memory formation. In this study, post-training DH infusion of the GPER agonist G-1 enhanced object recognition and spatial memory in ovariectomized female mice, whereas the GPER antagonist G-15 impaired memory, suggesting that GPER activation, like E2, promotes hippocampal memory formation. However, unlike E2, G-1 did not increase ERK phosphorylation, but instead significantly increased phosphorylation of c-Jun N-terminal kinase (JNK) in the DH. Moreover, DH infusion of the JNK inhibitor SP600125 prevented G-1 from enhancing object recognition and spatial memory, but the ERK inhibitor U0126 did not. These data suggest that GPER enhances memory via different cell-signaling mechanisms than E2. This conclusion was supported by data showing that the ability of E2 to facilitate memory and activate ERK signaling was not blocked by G-15 or SP600125, which demonstrates that the memory-enhancing effects of E2 are not dependent on JNK or GPER activation in the DH. Together, these data indicate that GPER regulates memory independently from ERα and ERβ by activating JNK signaling, rather than ERK signaling. Thus, the findings suggest that GPER in the DH may not function as an estrogen receptor to regulate object recognition and spatial memory. Although 17β-estradiol has long been known to regulate memory function, the molecular mechanisms underlying estrogenic memory modulation remain largely unknown. Here, we examined whether the putative membrane estrogen receptor GPER acts like the classical estrogen receptors, ERα and ERβ, to facilitate hippocampal memory in female mice. Although GPER activation did enhance object recognition and spatial memory, it did so by activating different cell-signaling mechanisms from ERα, ERβ, or 17β-estradiol. These data indicate that 17β-estradiol and GPER independently regulate hippocampal memory, and suggest that hippocampal GPER may not function as an estrogen receptor in the dorsal hippocampus. These findings are significant because they provide novel insights about the molecular mechanisms through which 17β-estradiol modulates hippocampal memory.

Kim J ，Szinte JS ，Boulware MI ，Frick KM ... -  《-》

G protein-coupled estrogen receptor (GPER) in the dorsal hippocampus regulates memory consolidation in gonadectomized male mice, likely via different signaling mechanisms than in female mice.

Studies in ovariectomized (OVX) female rodents suggest that G protein-coupled estrogen receptor (GPER) is a key regulator of memory, yet little is known about its importance to memory in males or the cellular mechanisms underlying its mnemonic effects in either sex. In OVX mice, bilateral infusion of the GPER agonist G-1 into the dorsal hippocampus (DH) enhances object recognition and spatial memory consolidation in a manner dependent on rapid activation of c-Jun N-terminal kinase (JNK) signaling, cofilin phosphorylation, and actin polymerization in the DH. However, the effects of GPER on memory consolidation and DH cell signaling in males are unknown. Thus, the present study first assessed effects of DH infusion of G-1 or the GPER antagonist G-15 on object recognition and spatial memory consolidation in gonadectomized (GDX) male mice. As in OVX mice, immediate post-training bilateral DH infusion of G-1 enhanced, whereas G-15 impaired, memory consolidation in the object recognition and object placement tasks. However, G-1 did not increase levels of phosphorylated JNK (p46, p54) or cofilin in the DH 5, 15, or 30 min after infusion, nor did it affect phosphorylation of ERK (p42, p44), PI3K, or Akt. Levels of phospho-cAMP-responsive element binding protein (CREB) were elevated in the DH 30 min following G-1 infusion, indicating that GPER in males activates a yet unknown signaling mechanism that triggers CREB-mediated gene transcription. Our findings show for the first time that GPER in the DH regulates memory consolidation in males and suggests sex differences in underlying signaling mechanisms.

Machado GDB ，Schnitzler AL ，Fleischer AW ，Beamish SB ，Frick KM ... -  《-》

Estradiol-Mediated Spine Changes in the Dorsal Hippocampus and Medial Prefrontal Cortex of Ovariectomized Female Mice Depend on ERK and mTOR Activation in the Dorsal Hippocampus.

Dendritic spine plasticity underlies the formation and maintenance of memories. Both natural fluctuations and systemic administration of 17β-estradiol (E2) alter spine density in the dorsal hippocampus (DH) of rodents. DH E2 infusion enhances hippocampal-dependent memory by rapidly activating extracellular signal-regulated kinase (ERK)-dependent signaling of mammalian target of rapamycin (mTOR), a key protein synthesis pathway involved in spine remodeling. Here, we investigated whether infusion of E2 directly into the DH drives spine changes in the DH and other brain regions, and identified cell-signaling pathways that mediate these effects. E2 significantly increased basal and apical spine density on CA1 pyramidal neurons 30 min and 2 h after infusion. DH E2 infusion also significantly increased basal spine density on pyramidal neurons in the medial prefrontal cortex (mPFC) 2 h later, suggesting that E2-mediated activity in the DH drives mPFC spinogenesis. The increase in CA1 and mPFC spine density observed 2 h after intracerebroventricular infusion of E2 was blocked by DH infusion of an ERK or mTOR inhibitor. DH E2 infusion did not affect spine density in the dentate gyrus or ventromedial hypothalamus, suggesting specific effects of E2 on the DH and mPFC. Collectively, these data demonstrate that DH E2 treatment elicits ERK- and mTOR-dependent spinogenesis on CA1 and mPFC pyramidal neurons, effects that may support the memory-enhancing effects of E2. Although systemically injected 17β-estradiol (E2) increases CA1 dendritic spine density, the molecular mechanisms regulating E2-induced spinogenesis in vivo are largely unknown. We found that E2 infused directly into the dorsal hippocampus (DH) increased CA1 spine density 30 min and 2 h later. Surprisingly, DH E2 infusion also increased spine density in the medial prefrontal cortex (mPFC), suggesting that estrogenic regulation of the DH influences mPFC spinogenesis. Moreover, inhibition of ERK and mTOR activation in the DH prevented E2 from increasing DH and mPFC spines, demonstrating that DH ERK and mTOR activation is necessary for E2-induced spinogenesis in the DH and mPFC. These findings provide novel insights into the molecular mechanisms through which E2 mediates dendritic spine density in CA1 and mPFC.

Tuscher JJ ，Luine V ，Frankfurt M ，Frick KM ... -  《-》

Chemogenetic Suppression of Medial Prefrontal-Dorsal Hippocampal Interactions Prevents Estrogenic Enhancement of Memory Consolidation in Female Mice.

Tuscher JJ ，Taxier LR ，Schalk JC ，Haertel JM ，Frick KM ... -  《eNeuro》