Differential Effects of the G-Protein-Coupled Estrogen Receptor (GPER) on Rat Embryonic (E18) Hippocampal and Cortical Neurons

Abstract

Estrogen plays fundamental roles in nervous system development and function. Traditional studies examining the effect of estrogen in the brain have focused on the nuclear estrogen receptors (ERs), ERα and ERβ. Studies related to the extranuclear, membrane-bound G proteincoupled estrogen receptor (GPER/GPR30) have revealed a neuroprotective role for GPER in mature neurons. In this study, we investigated the differential effects of GPER activation in primary rat embryonic (E18) hippocampal and cortical neurons. Microscopy imaging, multielectrode array (MEA), and Ca 2+ imaging experiments revealed that GPER activation with selective agonist, G-1, and non-selective agonist, 17β-estradiol (E2), increased neural growth, neural firing activity, and intracellular Ca 2+ more profoundly in hippocampal neurons than in cortical neurons. The GPER-mediated Ca 2+ rise in hippocampal neurons involve internal Ca 2+ store release via activation of phospholipase C and extracellular entry via Ca 2+ channels. 3Immunocytochemistry results revealed no observable difference in GPER expression/localization in neurons, yet RT-qPCR and western blot showed a higher GPER expression in the cortex than hippocampus, implying that GPER expression level may not fully account for its robust physiological effects in hippocampal neurons. We used RNA sequencing data to identify distinctly enriched pathways and significantly expressed genes in response to G-1 or E2 in cultured rat E18 hippocampal and cortical neurons. In summary, the identification of differential effects of GPER activation on hippocampal and cortical neurons in the brain and the determination of key genes and molecular pathways are instrumental toward an understanding of estrogen's action in early neuronal development. Significance StatementStudies of estrogen function via a non-nuclear G protein-coupled estrogen receptor (GPER/GPR30) in the brain have primarily focused on mature neurons and neuroprotective actions with little investigation into the role of GPER in early neural development. In this work, we discover differential effects of GPER on early neurite outgrowth, neuronal activity, and intracellular calcium signaling in primarily cultured rat embryonic (E18) hippocampal and cortical neurons. This study further highlights distinct, transcriptomic genes and pathways that are regulated by GPER agonists in early developing hippocampal and cortical neurons. These results advance our fundamental understanding of estrogen functions via GPER signaling in different (hippocampal versus cortical) neurons during early neuronal development. This knowledge is also instrumental for therapeutics for GPER-related neurodevelopmental disorders.

Kif21B mediates the effect of estradiol on the morphological plasticity of mouse hippocampal neurons

“…However, in previous work, we revealed that classical estrogen receptors do not participate in the development of hippocampal neurons, but that estradiol modifies neuronal morphology by acting on GPER through a mechanism that involves activation of PI3K signaling and expression of Ngn3 ( Ruiz-Palmero et al, 2011 , 2013 ). A later study identified GPER as a neurotrophic promotor for neurite outgrowth of rat E18 hippocampal, but not cortical neurons, indicating that different molecular pathways may account for the neuritogenic effect of estradiol in different brain regions ( Pemberton et al, 2022 ). Similar results are observed with BDNF since it has been shown that during postnatal development BDNF regulates neuronal architecture and spine morphology of neurons within specific brain areas but not others ( Zagrebelsky et al, 2018 ).…”

Different Expression Pattern of G Protein-Coupled Estrogen Receptor GPER1 in Esophageal Squamous Cell Carcinoma and Adenocarcinoma

“…The streptavidin–peroxidase method was used for immunohistochemistry analysis of the tissue microarrays. The slides were stained with the GPER1 antibody (PA5-28647; Invitrogen, Carlsbad, CA, USA) [ 12 , 52 , 53 ] and visualized with DAB after incubation with the secondary antibody.…”

Emerging Evidence on Membrane Estrogen Receptors as Novel Therapeutic Targets for Central Nervous System Pathologies

“…Some mERs present distinct subcellular distributions: mERα is mainly expressed in endolysosomes, mERβ prevails in mitochondria, and the majority of GPER1 is in the plasma membrane (breast cancer cells), endoplasmic reticulum, or Golgi apparatus (hippocampus, cortex, striatum) [ 25 , 26 ]. In particular, mERs are present in cortico-limbic brain areas; e.g., ER-X is mainly localized in the neonatal hippocampus and neocortex, but it was recently also found in the adult brain after ischemic stroke [ 27 ].…”

“…The latest study by Pemberton et al (2022) shed light on the differential effects of GPER1 depending on brain structure. In rat embryonic cerebral tissue, the effects of GPER1 activation by a selective G1 agonist (e.g., neural growth, neural firing activity) were more profound in hippocampal neurons than in cortical neurons, independent of the higher GPER1 expression in the cortex compared to the hippocampus [ 26 ]. The lack of an obvious or inverse correlation between the GPER1 expression level and specific neuroprotective effects suggests that other mechanisms are involved, such as the stimulation of neurogenesis, the inhibition of neuroinflammation, or the modulation of nuclear ER-mediated effects [ 80 , 81 , 82 ].…”