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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: Neurobiol Learn Mem. 2008 Nov 8;91(3):315–322. doi: 10.1016/j.nlm.2008.09.017

THE EFFECTS OF ACUTE 17β-ESTRADIOL TREATMENT ON GENE EXPRESSION IN THE YOUNG FEMALE MOUSE HIPPOCAMPUS

Angela S Pechenino 1, Karyn M Frick 1,2
PMCID: PMC2674265  NIHMSID: NIHMS102743  PMID: 18938255

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 and 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 two-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.

Keywords: Estrogen, microarray, memory, Igfbp2, Hsp70, Actn4, Tubb2a, Snap25

1. Introduction

The loss of estrogens at menopause has been associated with increases in dementia and age-related memory decline observed in aging women (Yaffe et al., 2000; Wolf and Kirschbaum, 2002). 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 and Hampson, 2000), object memory (Duka et al., 2000), and verbal memory (Kampen and 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 (Rissanen et al., 1999; Heikkinen et al., 2002; Gresack and Frick, 2006a) and novel object recognition memory (Luine et al., 2003; Gresack and Frick, 2006b) in mice and rats. Spatial and object memory are also enhanced by pre-training E2 treatments administered systemically by injection or silastic capsules (Daniel et al., 1997; Luine et al., 1998; Bimonte and Denenberg, 1999; Sandstrom and Williams, 2001; Vaucher et al., 2002; Sandstrom and Williams, 2004). 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 et al., 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 (Woolley and McEwen, 1993; Frick et al., 2004) and synaptic protein expression (Stone et al., 1998) in the CA1 region of the hippocampus and enhance neurogenesis in the hippocampal dentate gyrus (Tanapat et al., 1999; Galea et al., 2006). Additionally, acute E2 administration can activate several intracellular kinase cascades, including phosphatidylinositol 3-kinase (PI3K; Cardona-Gomez et al., 2002; Yokomaku et al., 2003; Mannella and Brinton, 2006) and extracellular signal-regulated kinase (ERK; Fernandez et al., 2008; Kuroki et al., 2000; Fitzpatrick et al., 2002; 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 et al., 2004) and the hippocampus (Aenlle et al., 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 and Frick, 2006b) and activate the ERK cascade in the dorsal hippocampus (Fernandez et al., 2008; Lewis et al., 2008).

2. Methods

2.1. 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 and 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 Usage Committee of Yale University, and conformed to the guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Mice were randomly assigned to groups receiving i.p. injections of either 0.2 mg/kg 17β-estradiol (E2) conjugated to the solubility enhancer 2-hydroxypropyl-β-cyclodextrin (HBC) and dissolved in physiological saline (E2 group, n=3) or HBC dissolved in an equal volume of physiological saline containing the same amount of cyclodextrin present in the HBC-E2 solution (vehicle group, n=3). HBC is a solubility-enhancer for steroid hormones that does not alter the bioefficacy of the hormones (Pitha and Pitha, 1985), but allows them to successfully cross the blood-brain barrier and rapidly dissociate into the tissue while the HBC remains in the circulation (Taylor et al., 1989). This hormone preparation is metabolized within 24 hours (Pitha et al., 1986; Taylor et al., 1989) and our laboratory has shown that that a single i.p. injection of the 0.2 mg/kg dose given to young female mice immediately after training specifically enhances memory consolidation in both spatial Morris water maze and novel object recognition tasks (Gresack and Frick, 2006a). Previous studies have shown that a 1 μg dose of estradiol dissolved in oil produces levels similar to those seen in the estrus phase of the estrous cycle, and a 10 μg dose produces levels similar to those seen during the proestrus phase of the cycle (Akinci and Johnston, 1997). Given mean body weight for young ovariectomized females of 22 g, the approximate estradiol levels for the 0.2 mg/kg dose is 4.4 μg, and thus near the middle of the physiological range.

In the present study, injections of vehicle or 0.2 mg/kg E2 were given 1 hour before sacrifice, as our previous work found an increase in dorsal hippocampal p42 ERK phosphorylation at this time point (Fernandez et al., 2008; Lewis et al., 2008). One hour (for RNA analyses) or 3 and 4 hours (for protein analyses) after injection, mice were cervically dislocated, and dorsal hippocampi were bilaterally dissected and stored at -80°C until use.

2.2. RNA Isolation

RNA was isolated from hippocampal tissue using the Trizol reagent isolation protocol (Invitrogen, Carlsbad, CA). Briefly, 800 μL Trizol reagent was added to each tube, and the tissue was homogenized by 10 passes of a Dounce homogenizer (Kontes Glass Co, Vineland, NJ). The homogenate was incubated for 5 min at room temperature and subsequently extracted with 0.2 mL of chloroform to remove proteins. The aqueous phase was transferred to a fresh tube, and the RNA was precipitated using 0.5 mL of isopropanol and centrifuged to pellet the RNA. The RNA pellet was washed twice by resuspension in 1 mL of 75% ethanol. After the last wash, the RNA pellet was allowed to air dry and was then resuspended in DEPC-treated water. The RNA was further purified using the RNAeasy Mini Kit RNA Cleanup protocol (Qiagen, Valencia, CA) per manufacturer’s instructions. The RNA concentration and purity were measured by reading the absorbance at 260 nm and 280 nm on a SmartSpec 3000 Spectrophotometer (BioRad, Hercules, CA). The quality of the RNA was further assessed using the Agilent Bioanalyser (Agilent, Santa Clara, CA).

2.3. Microarray Analysis

The microarrays used were OMM25K arrays developed by the Yale University WM Keck Foundation Biotechnology Resource Laboratory. The arrays were fabricated from a 70mer oligo set consisting of 16,463 oligos from the Operon Mouse Version 2.0 set and 8,097 oligos from the Operon Mouse Version 3.0 set, both designed from the publicly available Ensembl Mouse 14.30 database (http://www.ensembl.org/index.html) and the Mouse Genome Sequencing Project (http://www.hgsc.bcm.tmc.edu/projects/mouse). Isolated RNA was labeled by either Cy5 (RNA from vehicle-treated mice, n=3) or Cy3 (RNA from E2-treated mice, n=3) dyes and hybridized onto the arrays using the Genisphere Array900 Expression Array Detection kit (Genisphere, Hatfield, PA) according to the manufacturer’s protocol. Hybridizations were performed on an Advalytix SlideBooster hybridization station (Olympus America Inc, Concord, MA), and slides were subsequently scanned on an Axon GenPix 4100 scanner and imaged using GenePix 5.0 software (Axon, Sunnyvale, CA). The data were then analyzed according to the Genespring program (Agilent), in collaboration with the Biostatistics Department of the Yale University WM Keck Foundation Biotechnology Resource Laboratory. The intensity values for each spot were averaged across the three arrays to give an average intensity of the spot for each group, and these values were normalized to background intensity. Only those spots whose intensity was twice the background value were considered in the analysis. The data were first subjected to multiple t-tests, and then to a false discovery rate (FDR) correction. Genes were considered to have an altered expression with E2 treatment if they showed a greater than 2-fold change in expression level, and had an FDR-adjusted p-value of less than 0.05.

2.4. Quantitative real-time PCR

RNA was isolated from dorsal hippocampi according to the protocol described above. Total RNA (1.5 μg) was reverse transcribed in the presence of random hexamers using the SuperScript First-Strand Synthesis kit (Invitrogen) to synthesize cDNA. One ng of cDNA from each sample (vehicle-treated, n = 3; E2-treated, n = 3) was used in analysis. Quantitative real-time PCR (QPCR) was performed using the QuantiTect SYBR Green PCR Kit, with the QuantiTect Primer Assay PCR primers (Qiagen). Quantitation of PCR products was performed using the relative standard curve method. The cDNA standards were prepared from the liver of a 4 month-old mouse (for all genes except Snap25) or mouse brain reference RNA (Applied Biosystems, Foster City, CA, for Snap25) in the concentrations of 100, 50, 25, 10, and 1 ng/μL. Standards were loaded onto the reaction plate in each experimental run with each primer set. Standards were run in duplicate and samples in triplicate, with the housekeeping gene GAPDH run on each plate for internal normalization. The PCR reactions were performed on an ABI 7900 Sequence Detection System (Applied Biosystems) for 40 cycles (15 s at 95°C, 30 s at the annealing temperature, 30 s at 70°C), followed by a dissociation step at 60°C to visualize the melt curve and determine the purity of the product. The threshold cycle (Ct) value was calculated as the cycle number in which the SYBR green fluorescent signal crossed detection threshold limit set by the instrument, which is the midpoint of the log phase of the amplification reaction. A standard curve for each primer set was generated by plotting the concentration versus the Ct value for the reference samples. The concentrations of unknown samples were determined by substituting the Ct values for each sample into the best fit line where y = mx + b, and solving for the concentration x. The concentrations of each sample were reported as concentration equivalents of reference sample cDNA. Using SPSS 14.0 (SPSS Inc., Chicago, IL), separate independent samples t-tests were run for each gene comparing vehicle and E2 groups.

Western blot analysis

Dorsal hippocampi were homogenized and Western blot analysis performed as previously described (Fernandez et al., 2008; Lewis et al., 2008). Briefly, six μg of protein from each homogenate (vehicle-treated, n = 3; E2-treated, n = 3) were run on a 10% SDS-PAGE (BioRad, Hercules, CA), transferred to a PVDF membrane (Millipore, Temecula, CA), blocked in a 5% non-fat dry milk dissolved in Tris-buffered saline containing 0.1% Tween-20 (TTBS), and probed with Hsp70 (1:2000, #sc-33575, Santa Cruz, Santa Cruz, CA), IGFBP2 (1:1000, #06-107, Upstate, Lake Placid, NY), Actn 4 (1:2000, #05-384, Millipore), Tubb (1:4000, #3146, Cell Signaling, Danvers, MA), or SNAP-25 (1:1000, #610366, BD Biosciences, San Jose, CA) diluted in 5% bovine serum albumin (BSA) in TTBS. Secondary antibodies were either anti-rabbit horse radish peroxidase (HRP)-conjugated (1:2000, #7074, Cell Signaling, used for Hsp70, IGFBP2, and Tubb2a) or anti-mouse-HRP (1:2000, #7076, Cell Signaling, used for Actn 4 and SNAP-25). Blots were stripped and then re-probed with GAPDH (1:2500, #ab9484, Abcam, Cambridge, MA) for normalization. Band intensity was measured by densitometry using Kodak 1D 3.6 software on the Kodak Image Station 440 CF. Data were analyzed as described above.

3. 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 functional categories by a PubMed search to elucidate their known activities in vivo. Two of the 17 genes encode for proteins that act as transcription factors, 3 encode proteins involved in metabolism, 6 encode structural proteins, 6 encode neuropeptides/growth factors/receptors, and 1 encodes a molecular chaperone (these 17 genes shown in bold and italics, Table 1). Functional categories for other up-regulated genes are also reported in Table 1. Of the genes down-regulated by E2, 6 are known to be specifically involved in learning and memory. Of the 6 genes known to be involved in learning and memory, 4 encode transcription factors, 1 encodes a transport protein, and 1 encodes an anti-apoptotic protein (these genes shown in bold and italics, Table 2). Functional categories for other down-regulated genes are also reported in Table 2.

Table 1. Genes whose expression was significantly up-regulated by E2 treatment.

The p-values listed in the table represent significance reported after the false discovery rate (FDR) analysis. Genes in bold and italics indicate those that are known to be involved in learning and memory based on a PubMed search. The genes marked with an asterisk indicate those that were chosen for further study by real-time PCR and Western blotting.

Common Name Fold change p-value PubMed Number
Transcription Junb 2.35 0.0465 NM_008416
Pik3cb 2.13 0.0368 AK003230
Tcf4 2.24 0.0368 NM_013685
Eef1d 2.07 0.0491 NM_023240
Metabolism Odc 2.42 0.0487 NM_013614
Got2 2.35 0.0368 NM_010325
Atp1b3 2.28 0.0463 NM_007502
Sult4a1 2.16 0.0368 NM_013873
ATP F1 3.38 0.0203 BC013607
Dlat 2.03 0.0203 BC003202
Nec1 2.01 0.0368 AK018159
Atp6b2 2.07 0.0435 BC012497
Pmca1b 2.63 0.0398 AK013291
E2RG 10.42 0.0368 AK019895
Agk 2.05 0.0368 BC019145
Hatpaseb1 2.06 0.0464 BC017127
Structure Myo6 2.13 0.0435 NM_008662
Tubb2a* 2.67 0.042 M28739
Actn4* 2.63 0.0368 NM_021895
Tmsb4x 2.29 0.0368 NM_021278
Dctn4 2.4 0.0491 BC006677
Snap25* 2.84 0.0487 BC018249
Myh2 9.73 0.0368 BC008538
Esm1 12.28 0.0435 BC020038
Arfl10C 2.66 0.0465 BC013719
Tpk2 2.45 0.0464 BC019149
Structure Mtrp6 2.3 0.0368 BC020019
Cct6a 2.26 0.0203 NM_009838
Ccol 2.28 0.0368 NM_177177
NP/GF/Rec Rfrp 2.03 0.0465 NM_021892
Hgfl 3.69 0.0471 NM_008243
Erbb4 2.32 0.0402 AF059177
Neurod2 2.54 0.0368 NM_010895
Pafah1b1 2.4 0.0368 NM_013625
Kcna1 2.04 0.0471 NM_010595
Olfr, put 2.01 0.0368 X89678
Olfr67 2.78 0.0491 NM_013619
Gpr124 2.05 0.0368 NM_054044
lgsf1 2.35 0.0464 AY227771
Scube1 2.34 0.046 NM_022723
Eva 5 09 0.0368 BC015076
Cd209e 2.39 0.0368 AF373412
Neph 2.36 0.0471 NM_025684
Chaperone HSP70-1* 2.11 0.0368 M12573
Differentiation Morf 2.04 0.0464 NM_017479
Cd81 2.21 0.049 BC011433
Gcm2 2.5 0.0368 NM_008104
Ctf1 2.84 0.0368 NM_007795
Alcam 2.07 0.0435 U95030
Mmd 2.37 0.0368 NM_026178
Nuf2r 8.42 0.0368 NM_023284
Ash1l 2.11 0.0389 AF247132
Oxidative Stress Pxf 2.56 0.0368 AK012785
Inflammation Tcra 2.49 0.0368 X72904
Aldh1a7 2 0.0465 NM_011921
Egln1 2.07 0.0386 BC006903
CS1 2.23 0.0368 AK014517
Protein deg Rbx1 2.05 0.0435 AK005127
Rnf5 2.1 0.0398 NM_019403
Bean 21.34 0.0368 AF240460
Pep4 6.54 0.0368 NM_008820
Serp23 2.18 0.0311 BC018517
Psmd1 2.1 0.0368 AK010596
Psmb2 2.06 0.0498 NM_011970
Transport SV2 2.13 0.0399 AK013742
Rab1b 2.54 0.0368 BC016408
MLC1 2.11 0.0466 AF449425
Snta1 2.17 0.0465 NM_009228
Slc14a1 2.08 0.0389 NM_028122
Rcc1 2.36 0.0465 BC019807
Itgb4bp 2.22 0.0368 BC015274
Ergic1 2.33 0.0464 NM_026170
Ssra 2.07 0.0386 NM_025965
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Abbreviations: Transcription, transcription factor; NP/GF/Rec, neuropeptides, growth factors, or receptors; Protein deg, involved in protein degradation.

Table 2. Genes whose expression was significantly down-regulated by E2 treatment.

The p-values listed in the table represent significance reported after the false discovery rate (FDR) analysis. Genes in bold and italics indicate those that are known to be involved in learning and memory based on a PubMed search. The genes marked with an asterisk indicate those that were chosen for further study by real-time PCR and Western blotting.

Common Name Fold change p-value PubMed ID
Transcription Plc 16.67 0.0466 AK011892
lnpp5d 3.57 0.0468 NM_010566
Pla2g1b 3.85 0.0465 NM_011107
lgfbp2* 2.38 0.0389 NM_008342
AF-9 20 0.0368 AK008707
Brd7 25 0.0465 NM_012047
Ikbkb 10 0.0363 AF088910
Rbm8 3.23 0.0368 NM_025875
Smarca3 6.67 0.0368 AF165911
Lztfl1 2.13 0.0466 NM_033322
Transport Konj8 2.13 0.0389 NM_008428
Apoptosis Birc4 7.14 0.0368 NM_009688
Ca2+-assoc Calb2 2.5 0.0368 BC017646
Cacng3 2.56 0.0491 NM_019430
Calmbp1 5 0.0402 NM_009791
Cacybp 11.11 0.0368 U97327
Trpv6 2.17 0.0368 NM_022413
Pln 3.85 0.0466 AK002622
Metabolism Anpep 2.38 0.0368 NM_008486
Lrp6 2.17 0.0368 NM_008514
Atic 2.56 0.0465 AK010611
Fabp4 11.11 0.0465 BC002148
Cyp40 50 0.0368 AB006034
Nit1 3.03 0.0465 AK004988
Ethi6 20 0.0435 AK010551
Pit1 50 0.0389 AF196476
Structure Lox 11.11 0.0368 NM_010728
Siat8d 2.17 0.0368 NM_009183
Dpt 100 0.0368 NM_019759
Mtap4 50 0.0435 AK019611
Pxn10 2.17 0.0203 -
Cypt6 2.22 0.0368 NM_025738
Adam18 2.63 0.049 NM_010084
Adam rep 2.17 0.0491 AB112362
Differentiation Rho 7.14 0.0368 BC013125
Wnt4 3.85 0.0368 NM_009523
Gli5 3.33 0.0368 BC021517
Pcdh15 2.86 0.0435 NM_023115
Pax5 8.33 0.0464 NM_008782
Pax9 2.78 0.0468 NM_011041
Ccna1 4.35 0.0203 NM_007628
Pol theta 2.7 0.0368 AK020790
Cdc2l2 2.22 0.0368 NM_007661
GF/Rec Pglp 50 0.0368 AJ293619
Cpr2 6.67 0.0401 BC016483
Gadd45a 20 0.0464 NM_007836
Protein deg Arih2 2.86 0.0495 NM_011790
Psg4-1 2.08 0.0464 AK017550
Immune sys Cd2 2.38 0.0384 NM_013486
Itgae 4.17 0.0435 NM_008399
Tslp 2.22 0.0471 NM_021367
Fcnb 3.7 0.0368 AK010913
BCA3 2.13 0.0465 NM_020616
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Abbreviations: Transcription, transcription factor; Ca2+-assoc, involved in calcium signaling or processing; GF/Rec, growth factors or receptors; Protein deg, involved in protein degradation; Immune sys, genes related to the immune system.

Of the genes whose expression was altered by E2, 5 were selected for further analysis by quantitative real-time PCR (QPCR), followed by Western blot analysis based on reports in the literature strongly linking them with learning and memory. The first chosen was heat shock protein 70 (Hsp70-1), as it is not only associated with increased memory retention in the Morris water maze (Pizarro et al., 2003), but is also known to be up-regulated by E2 (Olazabal et al., 1992). Hsp70-1 mRNA levels increased 3-fold by QPCR (t(4) = 3.731, p = 0.02; Figure 2A), matching the 2.1-fold increase seen on the microarray (Table 1). Additionally, when protein levels of Hsp70-1 were measured by Western blot analysis, there was a 1.4-fold increase at 4 hours after treatment with E2 (t(5) = 4.418, p = 0.007; Figure 2A).

Figure 2.

Figure 2

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IGFBP2 gene and protein expression levels following E2 treatment. A. Igfbp2 mRNA expression levels significantly decreased 1 hour after E2 treatment. B. IGFBP2 protein levels were also significantly lower than vehicle 4 hours after E2 treatment. *p < 0.05 relative to vehicle.

The next gene chosen was the transcription factor-associated gene insulin-like growth factor binding protein 2 (Igfbp2). IGFBP2 is a serum protein secreted by a variety of cells, and is a part of the insulin-like growth factor (IGF-I) signaling cascade. IGF-I is known to be involved in memory, as long-term IGF-I treatment in aged rats improves learning and memory (Sonntag et al., 2005), and is thought to exert its memory-enhancing affects by promoting adult neurogenesis (Aberg et al., 2000; Perez-Martin et al., 2003) or neuronal survival (Subramaniam et al., 2005). IGFBP2 binds IGF-I in the serum and prevents it from binding its receptor and activating the cellular signaling cascade (Chesik et al., 2007); additionally, the serum levels of IGFBP2 are down- regulated in humans after conjugated equine estrogen treatment (Heald et al., 2005). The mRNA levels of Igfbp2 were down-regulated 2.4-fold on the microarrays (Table 2), a finding which was verified by QPCR, with a significant decrease of 1.5-fold relative to vehicle (t(4) = 3.90, p = 0.18; Figure 2B). Additionally, protein levels of IGFBP2 showed a 2-fold decrease 4 hours after E2 treatment (t(2.145) = 5.402, p = 0.028; Figure 2B), further confirming the mRNA data and indicating that IGFBP2 levels were decreased by E2 treatment.

The last 3 genes analyzed by QPCR were all structural proteins: α-actinin 4 (Actn 4), tubulin β2 (Tubb), and synaptosomal-associated protein 25 (Snap-25). Because estradiol is known to increase dendritic spine density (Woolley and McEwen, 1993) and enhance neurogenesis (Tanapat et al., 1999; Galea et al., 2006), these genes were predicted to be up-regulated by E2 treatment. Actn 4, an actin binding protein that regulates hippocampus spine morphology and density (Nakagawa et al., 2004), showed a 2.6-fold increase in mRNA levels upon microarray analysis (Table 1), a smaller but significant 0.5-fold increase relative to vehicle with QPCR (t(4) = 3.562, p = 0.024; Figure 2C), and 1.3-fold increase in protein levels 3 hours after treatment (t(5) = 3.603, p = 0.015; Figure 2C). Additionally, Tubb, an isoform of the microtubule-forming protein tubulin that is used as a marker for neuronal differentiation and whose expression increases with isoflavone treatment (Bu and Lephart, 2005), showed a 2.7-fold increase in mRNA levels on the microarrays (Table 1), a significant 2-fold increase relative to vehicle upon QPCR analysis (t(4) = 3.069, p = 0.037; Figure 2C), and a 1.7–fold increase in protein levels 4 hours after E2 treatment (t(5) = 2.568, p = 0.049; Figure 2C). Lastly, SNAP-25, a protein that localizes to synapses and may be required for long-term memory formation (Hou et al., 2006), showed a 2.8-fold increase in expression on the microarrays (Table 1), a significant 1.5-fold increase relative to vehicle upon QPCR analysis (t(4) = 4.623, p = 0.01; Figure 2C), and a 1.9–fold increase in protein levels 4 hours after E2 treatment (t(5) = 2.887, p = 0.034; Figure 2C).

4. 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 5 of the genes were confirmed by QPCR and Western blot analysis: Hsp70-1, Actn 4, Tubb, and Snap-25 were up-regulated with E2 treatment, and Igfbp2 was down-regulated with E2 treatment. The increase in Hsp70 mRNA and protein levels was consistent with previous work, as estradiol has been shown to enhance Hsp70 expression (Olazabal et al., 1992). The decrease in IGFBP2 mRNA and protein levels may suggest a role for the IGF-I signaling pathway in mediating the mnemonic response to E2, whereas the increases in mRNA and protein levels of Actn 4, Tubb, and Snap-25 suggest that E2-induced alterations in the expression of these structural genes may play a role in the beneficial effects of E2 on memory consolidation in the dorsal hippocampus. Interestingly, of the 5 genes examined, only one, Hsp70, contains an estrogen response element in its promoter (Hamilton et al., 2004), which suggests that most of the gene expression changes observed were likely mediated primarily by mechanisms other than the classical estrogen receptor binding. Although the present analyses cannot directly link E2-induced alterations in gene and protein expression to those in learning and memory, the fact that a behaviorally effective dose of E2 was used in this study lends strength to the correlative evidence linking E2-induced changes in the hippocampus to memory. More study will be needed to determine the extent to which the molecular alterations observed in the present study are necessary for E2 to modulate hippocampal memory function.

Hsp70-1 belongs to the extensive heat shock protein family, and is responsible for assisting in protein folding (Burston and Clarke, 1995). Hsp70 is also an integral part of the cytosolic estrogen receptor (ER) protein complex that keeps ER in an inactive state until it binds its estrogens and translocates to the nucleus (Whitesell and Lindquist, 2005). Hsp70 expression has been well-documented to increase with E2 treatment in a wide variety of cell types, such as breast cancer cells (Takahashi et al., 1994), heart (Hamilton et al., 2004), and brain, where it may show differential expression based on sex (Olazabal et al., 1992). Hsp70 isoforms have also been implicated in learning. Hsp70-1 mRNA and protein levels are increased in the hippocampus of rats trained in the spatial Morris water maze (Pizarro et al., 2003), and the inducible form of Hsp70, Hsp72, is increased in the cerebellum during acquisition of a two-way avoidance task in rats (Ambrosini et al., 1999). Further, when Hsp72 is knocked out in mice, this deletion prevents spatial memory acquisition in the radial arm maze (Ambrosini et al., 2005), suggesting an active and potentially critical role for Hsp70 isoforms in learning and memory beyond their traditional function of simply complexing with ER in the cytoplasm and maintaining it in the inactive state.

Insulin-like growth factor binding protein 2 (Igfbp2) is the predominant IGF-I binding protein secreted by neuronal and glial cells in the brain (Bezchlibnyk et al., 2006). Igfbp2 binds IGF-I in the serum and prevents it from activating the IGF-I receptor (IGF-IR) and initiating intracellular signaling cascades such as PI3K and ERK (Aberg et al., 2006; Chesik et al., 2007). Thus, an E2-induced down-regulation of Igfbp2 may lead to greater IGF-I availability, and subsequently to increased activation of the PI3K and ERK cascades. Links between the IGF-I signaling cascade and E2 are well-established. For example, treatment with E2 can activate IGF-IR signaling, and IGF-I has been shown to regulate the transcriptional activation of the classical estrogen receptors (ER) alpha and beta (Garcia-Segura et al., 2006). Administration of the ER antagonist ICI 182,780 blocks IGF-I-induced neurogenesis in female rats (Perez-Martin et al., 2003), suggesting that interactions with classical ERs are important in mediating the effects of IGF-I on hippocampal plasticity. Previous microarray analyses have shown a 1.7-fold increase in mRNA levels of another IGF-I binding protein, Igfbp6, in the hippocampus of male intact middle-aged rats relative to young rats (Blalock et al., 2003), and a decrease in the expression of Igfbp2 mRNA in the dentate gyrus of rats that have undergone passive avoidance training relative to untrained controls (O’Sullivan et al., 2007). An age-related increase in Igfbp6 mRNA could suggest that less IGF-I is available for binding to its receptor, which would block downstream signaling cascades. In contrast, the decrease in Igfbp2 mRNA observed after passive avoidance and in the present study could suggest more IGF-I available for binding, and may indicate an activity- or E2-induced increase in the activity of the downstream signaling cascades associated with IGF-I, as we have previously observed with ERK after 0.2 mg/kg E2 treatment (Fernandez et al., 2008).

Cytoskeletal structural genes are involved in several aspects of cellular function in the brain, including transport of proteins to various cellular locations (Antar et al., 2005), endocytosis and exocytosis of neurotransmitters (Chin et al., 2000), cell division during neurogenesis (German and Eisch, 2004), neuronal plasticity (Bianchi et al., 2005), and dendritic spine morphogenesis (Sekino et al., 2007). Several microarray studies have found alterations in the expression patterns of structural genes with learning. For example, synaptojanin II, a protein required for synaptic vesicle recycling, is up-regulated in aged rats who were classified as superior learners in the spatial Morris water maze task as compared to age-matched control learners (Burger et al., 2007). In addition, the mRNA levels of procollagen-type I, collagen type III and coronin, an actin binding protein, were down-regulated with age in the rat hippocampus, although vimentin and α-tubulin were both up-regulated with age (Blalock et al., 2003). Fewer structural genes appear to be influenced by E2 alone in array studies. The vesicle-associated membrane protein 2 and synaptogyrin were up-regulated in the basal hypothalamus of female guinea pigs following E2 treatment (Malyala et al., 2004). In non-array based studies, however, α-actinin 4 (Actn 4), a structural protein that binds to the actin cytoskeleton and is involved in protein trafficking within the cell, was reportedly up-regulated by the synthetic estrogen clomiphene citrate in rat uteri (Hosie et al., 2008) and was up-regulated by E2 treatment in the present study as well. Actn 4 mRNA levels are also increased in the amygdala during fear conditioning (Ressler et al., 2002), suggesting a role for this structural protein both in response to E2 and in learning and memory.

Tubulin-β (Tubb) is the major structural component of the microtubule network inside of cells. Column retention studies using hippocampal cell lysate have shown that tubulin-β binds directly to E2 (Ramirez et al., 2001). Additionally, yeast two-hybrid screens using MCF-7 cells indicate that microtubules may mediate the response of E2 by binding to and helping to shuttle the ERs to various locations within the cell (Manavathi et al., 2006). On the other hand, studies in breast cancer cells show that E2 actually disrupts microtubule formation (Aizu-Yokota et al., 1994), casting some doubt on whether there is a positive interaction between Tubb and E2. Tubulin-β is often used as a marker for neurogenesis (Rai et al., 2007), indicating that the increase in Tubb mRNA levels seen in this study may be due to cells preparing for future cell division and neurogenesis. Given that E2 has been shown to enhance neurogenesis in the hippocampal dentate gyrus (Tanapat et al., 1999; Galea et al., 2006), the increase in Tubb may not reflect a direct interaction between E2 and tubulin-β, but rather be an indirect result of E2-induced neurogenesis.

The final gene examined that was up-regulated by the microarray and QPCR analysis, as well as the Western blot analysis, was the synaptosomal-associated protein of 25 kD (Snap-25). SNAP-25 is a part of the synaptic vesicle docking and fusion process and is essential for neurotransmitter release (Wang and Tang, 2006). Interestingly, previous in situ hybridization data indicated that E2 treatment of ovariectomized rats decreased Snap-25 mRNA levels in the pituitary (Jacobsson et al., 1998), and another microarray study indicated that Snap-25 mRNA levels decrease after passive avoidance learning in the dentate gyrus of male rats (O’Sullivan et al., 2007). Although both of these findings conflict with the present data, other studies have shown that SNAP-25 is required for memory formation. For example, granule cells of the dentate gyrus show an increase in Snap-25 mRNA expression 2 hours after LTP stimulation (Roberts et al., 1998), and a Snap-25 antisense oligonucleotide added to either rat cortical neurons or PC12 cells impairs axon growth (Osen-Sand et al., 1993). Male intact rats infused with a SNAP-25 antisense oligonucleotide directly into either CA1 (Hou et al., 2004) or CA3 (Hou et al., 2006) exhibited impaired consolidation of contextual fear memory and spatial memory, suggesting that SNAP-25 is involved in memory consolidation in the hippocampus. As such, the increase in Snap-25 mRNA levels observed in our study indicate that treatment with a dose of E2 that enhances hippocampal memory consolidation (Gresack and Frick, 2006b) has a beneficial effect on the expression of this important gene, perhaps by influencing an increase in neurotransmitter release.

In conclusion, this study demonstrates that a single i.p. injection of a dose of E2 that enhances spatial and object memory consolidation (Gresack and Frick, 2006b) can influence the mRNA and protein expression levels of genes involved in learning and memory within 60 minutes of injection. We have found that E2 can enhance the expression of genes that act as chaperone proteins, such as Hsp70; transcription factor-related genes such as Igfbp2; genes involved in intracellular trafficking pathways, such as α-actinin 4 and tubulin-β; and synaptosome-associated proteins, such as Snap-25. These expression changes demonstrate that E2 may exert its effects on a wide variety of intracellular mechanisms and render cells within the hippocampus more amenable to the physical changes necessary to consolidate memories. More work is needed to understand if these gene expression changes specifically affect memory consolidation within the hippocampus. Nevertheless, the present data provide information that will be critical to understanding how E2 and similar hormones modulate memory.

Figure 1.

Figure 1

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Heat-shock protein 70-1 (Hsp70-1) gene and protein expression levels following E2 treatment. A. Hsp70-1 mRNA expression levels significantly increased 1 hour after E2 treatment. B. Hsp70-1 protein levels were also significantly higher than vehicle 4 hours after E2 treatment. *p < 0.05 relative to vehicle.

Figure 3.

Figure 3

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Structural protein gene and protein expression levels following E2 treatment. A. Actn4, Tubb, and Snap-25 mRNA expression levels were all significantly increased 1 hour after E2 treatment. B. Actn4 protein levels were increased 3 hours after E2 treatment, whereas, SNAP-25 and Tubb protein levels were significantly higher than vehicle 4 hours after treatment. *p < 0.05 relative to vehicle.

Acknowledgments

This work was supported by NIH grant RO1 AG022525 to KMF 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 would also like to thank Drs. Jonathan Ploski and Michael Lewis for their helpful comments on this manuscript.

Footnotes

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