Sex-specific Regulation of Spine Density and Synaptic Proteins by G-protein-coupled Estrogen Receptor (GPER)1 in Developing Hippocampus

Highlights

• Early developmental sex difference with respect to GPER1 signaling in hippocampus.

• GPER1 regulates spine density in hippocampal stratum lacunosum-moleculare sex-specifically.

• GPER1 regulates synaptic proteins in developing hippocampus sex-specifically.

Abstract

G-protein-coupled-estrogen-receptor 1 (GPER1) is a membrane-bound receptor that mediates estrogen signaling via intracellular signaling cascades. We recently showed that GPER1 promotes the distal dendritic enrichment of hyperpolarization activated and cyclic nucleotide-gated (HCN)1 channels in CA1 stratum lacunosum-moleculare (SLM), suggesting a role of GPER1-mediated signaling in neuronal plasticity. Here we studied whether this role involves processes of structural plasticity, such as the regulation of spine and synapse density in SLM. In organotypic entorhino-hippocampal cultures from mice expressing eGFP, we analyzed spine densities in SLM after treatment with GPER1 agonist G1 (20 nM). G1 significantly increased the density of “non-stubby” spines (maturing spines with a spine head and a neck), but did so only in cultures from female mice. In support of this finding, the expression of synaptic proteins was sex-specifically altered in the cultures: G1 increased the protein (but not mRNA) expression of PSD95 and reduced the p-/n-cofilin ratio only in cultures from females. Application of E2 (2 nM) reproduced the sex-specific effect on spine density in SLM, but only partially on the expression of synaptic proteins. Spine synapse density was, however, not altered after G1-treatment, suggesting that the increased spine density did not translate into an increased spine synapse density in the culture model. Taken together, our results support a role of GPER1 in mediating structural plasticity in CA1 SLM, but suggest that in developing hippocampus, this role is sex-specific.

Introduction

It has long been recognized that 17β-estradiol (E2) has important neuromodulatory functions in the hippocampus, including the regulation of spine and spine synapse density (Woolley et al., 1990, Kretz et al., 2004, MacLusky et al., 2005, Murakami et al., 2006, Mukai et al., 2007, Vierk et al., 2012, Phan et al., 2015, Kim et al., 2019, Brandt et al., 2020a), synaptic protein expression (Prange-Kiel et al., 2006, Liu et al., 2008, Waters et al., 2009, Vierk et al., 2012, Lu et al., 2019, Zhang et al., 2019) and long-term potentiation (Warren et al., 1995, Foy et al., 1999, Smith and McMahon, 2005, Srivastava et al., 2008, Kramar et al., 2009, Grassi et al., 2011, Vierk et al., 2012). E2 mediates its functions via two ways of intracellular signaling: classical estrogen signaling involves cytosol-based receptors, which translocate to the nucleus upon ligand activation and bind to specific estrogen response elements (EREs) in target genes to induce gene transcription, whereas non-classical signaling involves receptors which activate intracellular signaling cascades capable of mediating structural changes in neurons on a more rapid time scale (for a recent review, see: Taxier et al., 2020). Remarkably, several E2-mediated neuromodulatory effects have been shown to be sex-dependent, as they were detected in females but were less pronounced or absent in males, suggesting that sex differences with respect to E2 signaling exist (Vierk et al., 2012, Tabatadze et al., 2015, Bender et al., 2017, Wang et al., 2018).

Among the putative estrogen receptors (ERs) that may convey non-classical estrogen signaling, G-protein-coupled estrogen receptor 1 (GPER1; previously termed GPR30) has received considerable interest in recent years. GPER1 is a seven-transmembrane receptor that signals via G_ɑ/s-subunits causing activation of a variety of signaling pathways (for review, see Srivastava and Evans, 2013, Alexander et al., 2017, Hadjimarkou and Vasudevan, 2018). It was first identified in breast tissue (Carmeci et al., 1997), but was later shown to be expressed also in several regions of the brain, including the hippocampus (Funakoshi et al., 2006, Brailoiu et al., 2007, Matsuda et al., 2008, Hazell et al., 2009, Akama et al., 2013, Waters et al., 2015, Meseke et al., 2018, Llorente et al., 2020). In the hippocampus, robust GPER1 expression is observed in the apical dendritic layers of CA1–CA3 (Akama et al., 2013, Waters et al., 2015), and particularly in the stratum lacunosum-moleculare of CA1 (Meseke et al., 2018), suggesting that GPER1 could be involved in the regulation of neural transmission at Schaffer collateral and temporoammonic path (TA) synapses. In support, application of the selective GPER1-agonist G1 (Bologa et al., 2006) rapidly enhanced Schaffer collateral synaptic transmission in CA1 stratum radiatum (SR) in acute slices from adult mice (Kumar et al., 2015) and increased spine density in SR after systemic (Gabor et al., 2015) or dorsal hippocampal infusion (Kim et al., 2019). Moreover, G1 promoted the enrichment of hyperpolarization-activated HCN1 channels in the distal dendrites of CA1 pyramidal cells, which receive TA input, in organotypic entorhino-hippocampal slice cultures deriving from developing rats (Meseke et al., 2018).

As our previous findings indicate that in SLM GPER1 signaling is contributing to processes of molecular plasticity, involving the subcellular trafficking of ion channels (Meseke et al., 2018), we hypothesized that GPER1 also mediates processes of structural plasticity, such as the new formation of spines and spine synapses. To address this hypothesis, we employed mice which express eGFP under the control of the Thy1-promoter (Feng et al., 2000), enabling identification of individual dendrites and thus to analyze their spine density. From these mice organotypic entorhino-hippocampal slice cultures were prepared, to which G1 or E2 were applied, in order to study effects on spine density in vitro. Accounting for potential sex differences, cultures from both female and male mice were analyzed. We report a stimulating effect of both G1 and E2 on spine density in SLM, which, however, was sex-dependent and only observed in the tissue from females. Similarly, expression levels of synaptic proteins were altered only in females, suggesting that GPER1-signaling in developing hippocampus is sex-specific.