The effects of acute 17β-estradiol treatment on gene expression in the young female mouse hippocampus

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Abstract

Previous studies have demonstrated that treatment with 17β-estradiol (E2) improves both spatial and nonspatial memory in young female mice. Still unclear, however, are the molecular mechanisms underlying the beneficial effects of E2 on memory. We have previously demonstrated that a single post-training intraperitoneal (i.p.) injection of 0.2 mg/kg E2 can enhance hippocampal-dependent spatial and object memory consolidation (e.g., Gresack & Frick, 2006b). Therefore, in the present study, we performed a microarray analysis on the dorsal hippocampi of 4-month-old female mice injected i.p. with vehicle or 0.2 mg/kg E2. Genes were considered differentially expressed following E2 treatment if they showed a greater than 2-fold change in RNA expression levels compared to controls. Overall, out of a total of approximately 25,000 genes represented on the array, 204 genes showed altered mRNA expression levels upon E2 treatment, with 111 up-regulated and 93 down-regulated. Of these, 17 of the up-regulated and 6 of the down-regulated genes are known to be involved in learning and memory. mRNA expression changes in 5 of the genes were confirmed by real-time quantitative PCR analysis, and protein changes in these same genes were confirmed by Western blot analysis: Hsp70, a heat shock protein known to be estrogen responsive; Igfbp2, an IGF-I binding protein; Actn4, an actin binding protein involved in protein trafficking; Tubb2a, the major component of microtubules; and Snap25, a synaptosome-specific protein required for neurotransmitter release. The types of genes altered indicate that E2 may induce changes in the structural mechanics of cells within the dorsal hippocampus that could be conducive to promoting memory consolidation.

Introduction

The loss of estrogens at menopause has been associated with increases in dementia and age-related memory decline observed in aging women (Wolf and Kirschbaum, 2002, Yaffe et al., 2000). Despite the findings of the Women’s Health Initiative Memory Study (WHIMS), which demonstrated that giving estrogens alone or in combination with progesterone failed to prevent dementia in postmenopausal women (Shumaker et al., 2003, Shumaker et al., 2004), other evidence has shown that estrogen administration can have beneficial effects on memory. For example, giving estrogen to healthy postmenopausal women can improve spatial working memory (Duff & Hampson, 2000), object memory (Duka, Tasker, & McGowan, 2000), and verbal memory (Kampen & Sherwin, 1994). Research has also demonstrated the beneficial effects of the potent estrogen, 17β-estradiol (E2), in rodent models. For example, E2 administered intraperitoneally (i.p.) immediately post-training enhances both spatial reference memory in the Morris water maze (Gresack and Frick, 2006a, Heikkinen et al., 2002, Rissanen et al., 1999) and novel object recognition memory (Gresack and Frick, 2006b, Luine et al., 2003) in mice and rats. Spatial and object memory are also enhanced by pre-training E2 treatments administered systemically by injection or silastic capsules (Bimonte and Denenberg, 1999, Daniel et al., 1997, Luine et al., 1998, Sandstrom and Williams, 2001, Sandstrom and Williams, 2004, Vaucher et al., 2002). However, systemic hormone administration in women can lead to a host of physiological problems, including an increased incidence of coronary artery disease, stroke, and invasive breast cancer (Mastorakos, Sakkas, Xydakis, & Creatsas, 2006), calling into question whether the potential benefits of E2 on cognition outweigh the risks associated with hormone therapy.

An alternative approach to systemic hormone administration may be the specific targeting of proteins within the brain that are modulated by E2. For example, if the downstream effectors of E2 could be elucidated, then therapies that directly target these proteins could be developed that would enhance memory without the side effects of systemic hormone administration. E2 is known to increase dendritic spine density (Frick et al., 2004, Woolley and McEwen, 1993) and synaptic protein expression (Stone, Rozovsky, Morgan, Anderson, & Finch, 1998) in the CA1 region of the hippocampus and enhance neurogenesis in the hippocampal dentate gyrus (Galea et al., 2006, Tanapat et al., 1999). Additionally, acute E2 administration can activate several intracellular kinase cascades, including phosphatidylinositol 3-kinase (PI3K; Cardona-Gomez et al., 2002, Mannella and Brinton, 2006, Yokomaku et al., 2003) and extracellular signal-regulated kinase (ERK; Fernandez et al., 2008, Fitzpatrick et al., 2002, Kuroki et al., 2000, Wade and Dorsa, 2003), both of which can phosphorylate and activate CREB, an important protein involved in memory consolidation (Bozon et al., 2003). Previous microarray studies report that E2 alters expression of several genes in the hypothalamus (Malyala, Pattee, Nagalla, Kelly, & Ronnekleiv, 2004) and the hippocampus (Aenlle, Kumar, Cui, Jackson, & Foster, 2007), but these studies have been conducted using chronic E2 administration. No microarray study has yet examined the effects of a single, acute dose of E2 on gene transcription in the hippocampus. Such information is important to the development of hormone-based drug treatments in order to more closely link E2-induced changes in signal transduction to alterations in gene transcription. Therefore, the present study endeavored to identify genes whose mRNA and protein expression levels were altered by an acute dose of water-soluble E2 known in young ovariectomized mice to enhance spatial and object memory (Gresack & Frick, 2006b) and activate the ERK cascade in the dorsal hippocampus (Fernandez et al., 2008, Lewis et al., 2008).

Section snippets

Subjects

Four month old female C57BL/6 mice were obtained from Taconic (Germantown, NY). Mice were bilaterally ovariectomized 1 week after arrival as per previously published methods (Fernandez & Frick, 2004), and housed up to 5/cage in a room with a 12:12 light/dark cycle (lights on at 07:00) for at least a week before treatments. Animals were handled 5 min per day over the course of 5 days, and had ad libitum access to food and water. All procedures were approved by the Institutional Animal Care and

Results

In order to identify the effects of E2 on gene expression in the hippocampus of young adult mice, the dorsal hippocampi of 4-month-old mice were subjected to microarray analyses using DNA oligo microarrays containing oligos from 25,000 genes. Overall, 73 genes were up-regulated and 53 genes were down-regulated by E2 treatment (p < 0.05 by FDR multiple t-test correction). Of the genes up-regulated by E2, 17 are known to be specifically involved in learning and memory. These genes were divided into

Discussion

E2 administration led to increased RNA levels of 73 genes and decreased RNA levels of 53 genes. Of these, 17 of the up-regulated and 6 of the down-regulated genes have been previously associated with learning and memory as described above. Five of these genes were chosen for verification of RNA expression level changes by QPCR based on the availability of information in the literature relating to their involvement in learning and memory. The changes in mRNA and protein expression levels of all

Acknowledgments

This work was supported by NIH Grant RO1 AG022525 to K.M.F. and Yale University. The authors gratefully acknowledge Irina Tikhovna, Ainpin Lin, Sheila Westman, and the Yale University W.M. Keck Foundation Biotechnology Resource Laboratory for their assistance with the microarray studies and for use of their real-time PCR machine. The authors also thank Drs. Jonathan Ploski and Michael Lewis for their helpful comments on this manuscript.

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