Perimembrane Localization of the Estrogen Receptor α Protein in Neuronal Processes of Cultured Hippocampal Neurons

Abstract

There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) α or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERα were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERα, rabbit polyclonal IgG antibody which does not recognize ERβ was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERα, but abundant labeling for ERα throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 µM antisense oligonucleotide directed against the translation start site of ERα reduced ERα immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERα. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERα in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERα as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.

Estradiol Targets Synaptic Proteins to Induce Glutamatergic Synapse Formation in Cultured Hippocampal Neurons: Critical Role of Estrogen Receptor-

“…The intensity of staining qualitatively varied among the neurons in our dispersion cultures. Our results corroborate previous findings that hippocampal neurons express extranuclear ER-␣ (Clarke et al, 2000;Milner et al, 2001;Adams et al, 2002;Kalita et al, 2005;Romeo et al, 2005) and extranuclear ER-␤ (Mitra et al, 2003;Kalita et al, 2005;Milner et al, 2005). We focused specifically on cultured hippocampal neurons expressing the NR1 protein and analyzed the pattern and distribution of the punctate extranuclear immunostaining of ER proteins present along dendrites.…”

“…ER-␣ and ER-␤ have been found at extranuclear sites in the hippocampus and cortex (Dubal et al, 2006). Their presence in dendritic spines and axons of hippocampal and cortical neurons (Clarke et al, 2000;Adams et al, 2002;Kalita et al, 2005;Romeo et al, 2005;Hu et al, 2007) suggests a role for the ER as a transducer of rapid, nongenomic estrogen-mediated events during synapse formation.…”

Modulation of αCaMKII signaling by rapid ERα action

“…Our ERα localization data in NLT neurons corresponds with other groups in that "classical" ERs are positioned not only in the nucleus, but in the cytoplasm and outgrowths as well (Clarke, 2000;Hart, 2007) where they are available to interact with cytoplasmic signaling cascades. Immunofluorescence and cell fractionation showed that E2-induced αCaMKII autophosphorylation in NLT neurons occurs specifically in the cell body and processes, suggesting that the extranuclear pool of ER is responsible for mediating this event.…”

Estrogen Facilitates both Phosphatidylinositol 3-Kinase/Akt and ERK1/2 Mitogen-Activated Protein Kinase Membrane Signaling Required for Long-Term Neuropeptide Y Transcriptional Regulation in Clonal, Immortalized Neurons

“…These studies have revealed that classical nuclear ERs are located at the plasma membrane of a variety of cell types, including endothelial cells, spermatozoa, and neurons in the brain (Pappas et al, 1995;Clarke et al, 2000), hence proving that membrane estrogen signaling through nuclear receptors is a more common phenomenon than originally thought. However, an issue that should be addressed is which of the nuclear ERs is responsible for the rapid events of membrane signaling and how it is connected to E 2 -mediated signal transduction systems.…”