Distribution and Posttranslational Modification of Synaptic ERα in the Adult Female Rat Hippocampus

Abstract

Acute 17β-estradiol (E2) signaling in the brain is mediated by extranuclear estrogen receptors. Here we used biochemical methods to investigate the distribution, posttranslational modification, and E2 regulation of estrogen receptor-α (ERα) in synaptosomal fractions isolated by differential centrifugation from the adult female rat hippocampus. We find that ERα is concentrated presynaptically and is highly enriched with synaptic vesicles. Immunoisolation of vesicles using vesicle subtype-specific markers showed that ERα is associated with both glutamate and γ-aminobutyric acid-containing neurotransmitter vesicles as well as with some large dense core vesicles. Experiments using broad spectrum and residue-specific phosphatases indicated that a portion of ERα in synaptosomal fractions is phosphorylated at serine/threonine residues leading to a mobility shift in SDS-PAGE and creating a double band on Western blots. The phosphorylated form of ERα runs in the upper of the two bands and is particularly concentrated with synaptic vesicles. Finally, we used E2 with or without the acyl protein thioesterase 1 inhibitor, Palmostatin B, to show that 20 min of E2 treatment of hippocampal slices depletes ERα from the synaptosomal membrane by depalmitoylation. We found no evidence that E2 regulates phosphorylation of synaptosomal ERα on this time scale. These studies begin to fill the gap between detailed molecular characterization of extranuclear ERα in previous in vitro studies and acute E2 modulation of hippocampal synapses in the adult brain.

Sex steroid hormones matter for learning and memory: estrogenic regulation of hippocampal function in male and female rodents

“…Consistent with the localization of ERa to inhibitory interneurons in the hippocampus (Murphy et al 1998;Hart et al 2007;Tabatadze et al 2013), bath-applied E 2 suppresses synaptic inhibition in hippocampal slices from ovariectomized rats (Huang and Woolley 2012). Interestingly, this effect was not observed in slices from male rats (Huang and Woolley 2012), which may stem from sex differences in levels of extranuclear ERa found in the hippocampus (Mitterling et al 2010).…”

“…In embryonic hippocampal neuron cultures, ERa is also found in vesicle clusters within GABAergic interneurons in CA1, where it mediates an E 2 -induced decrease in hippocampal GABAergic neurotransmission that is thought to disinhibit pyramidal neurons (Murphy et al 1998;Hart et al 2007;Huang and Woolley 2012). Other work has localized ERa to glutamatergic and GABAergic synaptic vesicles in female rat hippocampal synaptosomes (Tabatadze et al 2013). Thus, similar to ERb in other brain regions, ERa in the hippocampus appears to regulate both inhibitory and excitatory tone.…”

Experience-dependent homeostasis of ‘noise’ at inhibitory synapses preserves information coding in adult visual cortex

“…Consistent with previous reports that ER-a is closely tied to the regulation of inhibitory synaptic function in rat hippocampus [39] and is located on presynaptic vesicles [41], acute application of ER-a selective agonist PPT (200 nM), but not ER-b agonist DPN (250 nM), for 10 min mimicked E2 by significantly reducing mIPSC frequency in P42 NR mice (figure 3d).…”

“…These effects were very rapidly induced with only a few minutes of acute application of E2 or ER-a agonist/antagonist, hence are likely mediated by non-genomic actions of oestrogen. Recently, studies showed that oestrogen receptors are localized on presynaptic vesicles and specifically, ER-a is associated with GABAergic vesicles [41,56]. Acute E2 application has been shown to depalmitoylate ER-a to dissociate it from presynaptic vesicles [41].…”

Insights into Rapid Modulation of Neuroplasticity by Brain Estrogens

“…In presynaptic structures, ERa-labeled unmyelinated axons and axon terminals containing synaptic vesicles form asymmetric (excitatory) and symmetric (inhibitory) synapses. A recent study supports a presynaptic localization of ERa in female hippocampus, where it localizes with both glutamate and GABA containing synaptic vesicles (Tabatadze et al, 2013). ERa has also been shown to be expressed in astrocytes that are often found near the spines of pyramidal cells (Kritzer, 2002;Milner et al, 2005) and microglia (Sierra et al, 2008).…”