Previous estradiol treatment during midlife maintains transcriptional regulation of memory-related proteins by ERα in the hippocampus in a rat model of menopause

Abstract

Previous midlife estradiol treatment, like continuous treatment, improves memory and results in lasting increases in hippocampal levels of estrogen receptor (ER) α and ER-dependent transcription in ovariectomized rodents. We hypothesized that previous and continuous midlife estradiol act to specifically increase levels of nuclear ERα, resulting in transcriptional regulation of proteins that mediate estrogen effects on memory. Ovariectomized middle-aged rats received estradiol or vehicle capsule implants. After 40 days, rats initially receiving vehicle received another vehicle capsule (ovariectomized controls). Rats initially receiving estradiol received either another estradiol (continuous estradiol) or a vehicle (previous estradiol) capsule. One month later, hippocampi were dissected and processed. Continuous and previous estradiol increased levels of nuclear, but not membrane or cytosolic ERα and had no effect on Esr1. Continuous and previous estradiol impacted gene expression and/or protein levels of mediators of estrogenic action on memory including ChAT, BDNF, and PSD-95. Findings demonstrate a long-lasting role for hippocampal ERα as a transcriptional regulator of memory following termination of previous estradiol treatment in a rat model of menopause.

Keywords: BDNF; ChAT; Estrogen; Estrogen receptor; Hippocampus; Menopause.

Figure 1.. Transcriptional Regulation of Esr1 and subcellular localization of ERα in the hippocampus of ovariectomized rats following continuous or previous exposure to estradiol in midlife.

Middle-aged female rats were ovariectomized and treated to one of three hormone conditions via Silastic capsule: Vehicle (Veh), which received a vehicle capsule; Continuous Estradiol (Cont E), which received an estradiol capsule for the duration of the experiment; or Previous Estradiol (Prev E), which received an estradiol capsule for 40 days followed by a vehicle capsule for the duration of the experiment. One month later, hippocampi were processed for either RNA extraction and RT-PCR using primers for Esr1 and housekeeping gene Gapdh or for subcellular fractionation and western blotting for ERα, measured by density x area of ERα/loading control proteins. A) There was no significant effect of hormone treatment on Esr1 expression in the hippocampus relative to Gapdh expression. B-C) There was no significant effect of hormone treatment on cytosolic (B) or membrane (C) ERα. D) There was a main effect of hormone treatment on levels of nuclear ERα (p<.05). Post hoc testing revealed increased levels in the Cont E and Prev E groups relative to the Veh group. Data are presented as means ± SEM normalized to percent Vehicle group. *p<.05 vs. Veh

Figure 2.. Verification of subcellular compartment fractionation.

Hippocampal tissue was processed for subcellular fractionation in order to separate the cytosolic, membrane, and nuclear compartments of cells via consecutive centrifugation steps using a commercially available kit. Compartment separation was verified using western blotting for cytosolic marker enolase, membrane marker ATP1A1, and nuclear marker CREB on samples from all compartments.

Figure 3.. Hippocampal transcriptional regulation and protein expression of genes that contain ERE sequences following continuous or previous midlife estradiol exposure.

Middle-aged female rats were ovariectomized and treated to one of three hormone conditions via Silastic capsule: Vehicle (Veh), which received a vehicle capsule; Continuous Estradiol (Cont E), which received an estradiol capsule for the duration of the experiment; or Previous Estradiol (Prev E), which received an estradiol capsule for 40 days followed by a vehicle capsule for the duration of the experiment. One month later, hippocampi were processed for RNA extraction and RT-PCR using primers for Bdnf, Chat, and Gapdh, or for western blotting for BDNF, ChAT, and β-actin. RT-PCR data were normalized to housekeeping gene Gapdh and western blot data were normalized to loading control protein β-actin. A-B) There was a main effect of hormone treatment (p<.05) on Bdnf RNA expression (A) and BDNF protein levels (B) in the hippocampus. Post hoc testing revealed increased Bdnf expression in the Cont E and Prev E groups and increased BDNF protein levels in the Prev E group as compared to the Veh group. C-D) There was a main effect of hormone treatment (p<.05) on ChAT RNA expression (C) and ChAT protein levels (D) in the hippocampus. Post hoc testing revealed increased Chat expression and increased ChAT protein levels in the Cont E and Prev E groups as compared to the Veh group. *p<.05 vs. Veh

Figure 4.. Hippocampal transcriptional regulation and protein expression of gene that does not contain an ERE sequence following continuous or previous midlife estradiol exposure.

Middle-aged female rats were ovariectomized and treated to one of three hormone conditions via Silastic capsule: Vehicle (Veh), which received a vehicle capsule; Continuous Estradiol (Cont E), which received an estradiol capsule for the duration of the experiment; or Previous Estradiol (Prev E), which received an estradiol capsule for 40 days followed by a vehicle capsule for the duration of the experiment. One month later, hippocampi were processed for RNA extraction and RT-PCR using primers for Dlg4 and Gapdh, or for western blotting for PSD-95 and β-actin. RT-PCR data were normalized to housekeeping gene Gapdh and western blot data were normalized to loading control protein β-actin. A) There was a main effect of hormone treatment (p<.05) on Dlg4 RNA expression in the hippocampus. Post hoc testing revealed increased Dlg4 expression in the Cont E and Prev E groups as compared to the Veh group. B) There was no effect of hormone treatment on PSD-95 protein levels. *p<.05 vs. Veh