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. Author manuscript; available in PMC: 2017 Jun 22.
Published in final edited form as: J Alzheimers Dis. 2011;23(4):629–639. doi: 10.3233/JAD-2010-100993

17α-Estradiol Attenuates Neuron Loss in Ovariectomized Dtg AβPP/PS1 Mice

Kebreten F Manaye a,*, Joanne S Allard a,c, Sara Kalifa a, Amy C Drew a, Guang Xu a, Donald K Ingram b, Rafael de Cabo c, Peter R Mouton c,d
PMCID: PMC5481172  NIHMSID: NIHMS738747  PMID: 21157032

Abstract

Quantitative microanalysis of brains from patients with Alzheimer's disease (AD) find neuronal loss and neuroinflammation in structures that control cognitive function. Though historically difficult to recapitulate in experimental models, several groups have recently reported that by middle-age, transgenic mice that co-express high levels of two AD-associated mutations, amyloid-β protein precursor (AβPPswe) and presenilin 1 (PS1ΔE9), undergo significant AD-type neuron loss in sub-cortical nuclei with heavy catecholaminergic projections to the hippocampal formation. Here we report that by 13 months of age these dtg AβPPswe/PS1ΔE9 mice also show significant loss of pyramidal neuron in a critical region for learning and memory, the CA1 subregion of hippocampus, as a direct function of amyloid-β (Aβ) aggregation. We used these mice to test whether 17α-estradiol (17αE2), a less feminizing and non-carcinogenic enantiomer of 17β-estradiol, protects against this CA1 neuron loss. Female dtg AβPPswe/PS1ΔE9 mice were ovariectomized at 8–9 months of age and treated for 60 days with either 17αE2 or placebo via subcutaneous pellets. Computerized stereology revealed that 17αE2 ameliorated the loss of neurons in CA1 and reduced microglial activation in the hippocampus. These findings support the view that 17αE2, which may act through non-genomic mechanisms independent of traditional estrogen receptors, could prevent or delay the progression of AD in older men and women.

Keywords: Alzheimer's disease, amyloid-β, stereology, transgenic mice

INTRODUCTION

Alzheimer's disease (AD) is diagnosed at autopsy by the presence of gross and microscopic brain changes, including high densities of amyloid-β (Aβ)-containing plaques and neurofibrillary tangles in cortical tissue [1, 2], widespread neurogliosis [3, 4], marked cortical atrophy [5, 6], and severe neuron loss. Brain regions that show particularly severe neuron loss include pyramidal neurons in the CA1 subregion of hippocampus and noradrenergic neurons in the locus coeruleus (LC), two neuronal populations that mediate attention, learning, memory and other neural processes associated with cognitive function [1, 7–10]. The mechanisms that underlie this neuron loss are not understood, and there are no approved methods to slow this neurodegeneration.

Both estrogen and testosterone have been established as endogenous neuroprotective factors against AD-related neuropathology (for review, see [11]). The gonadal steroid 17β-estradiol (17βE2) mediates sexual differentiation of the developing brain [12, 13], improves spatial memory performance in middle-aged and aged female rodents [14–16], and provides neuroprotection against a range of age-related and experimental damage in adult brains [17–27]. Both receptor-dependent and receptor-independent mechanisms appear to mediate these effects [28–30]; however, in the past decade concerns about the safety of long-term 17βE2 treatment have raised serious questions about its future therapeutic potential [31–33].

A number of studies indicate that 17αE2, an enantiomer of 17βE2 with markedly lower feminizing and carcinogenic effects, could provide similar though potentially safer neuroprotective treatment against AD for aging men and women [22, 26, 27, 34–38]. Because of its low binding affinity to classical estrogen receptors, ERα and ERβ, and its inability to activate canonical genomic pathways at clinical doses, 17αE2 was long considered biologically inactive. Recent reports, however, indicate that 17αE2 stimulates rapid effects on a number of intracellular signaling cascades [22, 39] and exerts important neuroprotective effects in vitro [40–42]. The potential for protection against AD-type neuropathology, together with low feminization and carcinogenic side effects, suggests that 17αE2 offers a novel therapeutic option for prevention and management of AD, and possibly other neurodegenerative conditions. To date, few studies have investigated the potential of 17αE2 to protect against AD-type neuropathology using in vivo experimental models.

Several groups have reported middle-aged and older double transgenic (dtg) mice that overexpress two mutations related to familial AD, amyloid-β protein precursor (AβPPswe) and presenilin 1 (PS1ΔE9), undergo age-related neuron loss similar to that in AD. Using design-based stereological approaches, different groups have confirmed loss of tyrosine hydroxylase (TH) positive neurons in the LC, a region that provides heavy catecholarninergic projections to the hippocampal formation [43, 44], and alterations in synaptic numbers and size [45]. In the present study we report that at about 11 months of age (range 9–13 months), the brains of male and female dtg AβPPswe/PS1ΔE9 mice contain significantly fewer numbers of pyramidal neurons in the CA1 subregion of hippocampus. Furthermore, we report that treatment of female dtg AβPPswe/PS1ΔE9 mice with 60 days of 17αE2 via slow-release subcutaneous pellets prevents this loss of pyramidal CA1 neurons in hippocampus.

MATERIALS AND METHODS

Mice

Male and female dtg AβPPswe/PS1ΔE9 and non-transgenic littermate control (nou-Tg) mice were raised from birth in the vivarium at the Laboratory of Experimental Gerontology at the Gerontology Research Center (GRC, NIA/NIH, Baltimore, MD). These mice were raised from founders donated by Drs. David Borchelt and Michael Lee at the Johns Hopkins University School of Medicine that over-express AβPPswe and PS1ΔE9 mutations. Mice were group housed (n = 2–5) in plastic cages with corncob bedding with ad libitum access to food (NIH formula 07) and filtered water. Conditions within the vivarium were maintained at a 12:12 hour light:dark cycle and temperature of 22 ± 2°C. Animal husbandry and maintenance were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Study design

Effects of gender and transgene expression on CA1 neuron number

The initial phase of this study was designed to assess gender and transgene effects on total number of CA1 neurons in hippocampus. These experiments used a total of 24 mice, including 12 dtg AβPPswe/PS1ΔE9 mice (6 per gender, average age 13.5 ± 0.9 months) and 12 non-tg mice (6 per gender, average age 12.6 ± 0.9 months). All mice were sacrificed by anesthesia overdose and intracardial perfusion with aldehydes, with no significant age differences between groups, and brains were removed for histological processing and stereological quantification of the total number of pyramidal neurons in CA1, as detailed below. We have previously reported age-related increases and no gender differences in amyloid load or regional volume in hippocampus of both male and female dtg AβPPswe/PS1ΔE9 mice compared to age- and gender-matched non-tg controls [46–48]. Rather than repeat this analysis, in the present study amyloid load was analyzed only in one gender of dtg AβPPswe/PS1ΔE9 mice for the purpose of correlation analyses with loss of CA1 pyramidal neurons in these mice. The decision was made to analyze this correlation in female dtg AβPPswe/PS1ΔE9 mice because these mice were used to test the neuroprotective effects of 17αE2 on CA1 neuron loss, as detailed in the following section.

Neuroprotective effects of 17αE2 on CA1 neuron number

In the second phase of the study, female dtg AβPPswe/PS1ΔE9 mice were used to test whether 17αE2 protects against neuron loss in the CA1 sub-region of hippocampus. A total of 21 female mice aged 8–9 months were used in these studies, including 1) 16 female dtg AβPPswe/PS1ΔE9 mice; and 2) 5 age-matched female non-tg mice. Under anesthesia from a 100 mg/kg ketamine/10 mg/kg xylazine mixture, 16 female dtg AβPPswe/PS1ΔE9 mice underwent either bilateral ovariectomy (OVX) surgery (n = 10) or sham surgery (n = 6). The non-transgenic control mice did not receive any surgery. While under anesthesia, the 10 mice that received OVX were randomly assigned into two groups for implantation with dissolvable subcutaneous pellets (Innovative Research, Sarasota, F1) that deliver sustained release of either 1.5 mg 17αE2 (n = 5), or placebo (cholesterol, n = 5) over a 60-day period; the third group of dtg AβPPswe/PS1ΔE9 mice that received sham OVX surgery also underwent sham implantation surgery (see [21] for details of OVX, pellet implantation, and sham surgeries). All mice maintained normal consumption of food and water, with no weight differences or fluctuations during the 60-day treatment period. At 10–11 months of age, mice were sacrificed by anesthesia overdose and intracardial perfusion with 4% paraformaldehyde, and brains were removed for histological processing as detailed below. At the time of necropsy, the uterus from each mouse was removed and weighed to assess exposure to exogenous 17αE2. In addition, a group of 6 age-matched female dtg AβPPswe/PS1ΔE9 mice that underwent sham surgery for OVX+pellet implantation were included in the analysis of uterine weight. Because the uterine weights of the 17αE2-treated dtg AβPPswe/PS1ΔE9 mice were the same as those of the sham-treated dtg AβPPswe/PS1ΔE9 group, no further analysis, i.e., stereological studies, were carried on the brains from the sham surgery group.

Tissue preparation

On the day of sacrifice, mice were anesthetized with ketamine and killed by intracardial perfusion with 0.9% saline followed by 4% paraformaldehyde. Brains were removed, postfixed in 4% paraformaldehyde, and cryoprotected in a 30% sucrose solution before being stored at −80°C until sectioning. For studies using computerized stereology analysis, each brain was serially sectioned in the coronal plane from the frontal pole through the brainstem at a microtome setting of 40 μm. Serial sections were sub-sampled in a systematic-random manner to generate 8–10 sections through each entire reference space (CA1, CA2/3). That is, the first section through each reference space was selected at random, and each successive section selected in a systematic manner. For example, for a total of 80 sections through CA1, taking every 10th section, with a random start in the interval 1 to 10, would generate a systematic-random sample of 8 sections.

Histology

To visualize pyramidal neurons in CA regions, sampled sections were stained in a 0.1% solution of cresyl violet, rinsed, dehydrated through an ascending graded series of alcohol, cleared in xylene and coverslipped. Furthermore, two adjacent sets of systematic-random sampled sections through the hippocampus were processed to reveal microglia cells and Aβ. The first set was immunostained with antibodies to ionized calcium binding adaptor molecule 1 (Iba1). The Iba1 antibody is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells, with no cross-reactivity to astrocytes or neurons (Chemicon International, Temecula, CA). The second set of systematic-random sections through the hippocampal formation was stained with Congo red to visualize amyloid deposits for determination of total amyloid volume (amyloid load), as detailed previously [43, 46]. The average section thickness after all tissue processing ranged from 14 to 17 μm.

Quantification of amyloid load and pyramidal cell numbers

With assistance from the Stereo Investigator system (MicroBrightField Inc., Williston, VT), design-based stereology was used to estimate total volume of amyloid plaques on Congo red sections (amyloid load) and mean total number of pyramidal cells in CA subregions. Estimates of amyloid load in molecular layers of the CA1 and CA2/3 regions of hippocampus were quantified using volume fraction and the Cavalieri-point counting method on Congo red-stained sections [43, 48]. Because previous studies have reported increased amyloid load in male dtg AβPPswe/PS1ΔE9 mice, studies of amyloid load in the present study were limited to the molecular layer in hippocampus of female dtg AβPPswe/PS1ΔE9 mice. The mean total numbers of pyramidal neurons in CA1 and CA2/3 regions of hippocampus were quantified using the optical fractionator method [49, 50], as previously reported [7, 21, 43, 44, 47, 51–54]. Briefly, the reference spaces on each sampled section were outlined under low power magnification (4X), and volume of Aβ and total number of cells quantified using a high resolution, oil immersion objective (60X, 1.4 numerical aperture). Deposits of Aβ appeared as pinkish red, while neurons were identified on the basis of a Nissl-stained neuronal phenotype, i.e., a dark violet blue nucleolus and a well-formed nuclear membrane, in CA subregions. Th avoid artifacts at the sectioning surface, e.g., lost caps, a guard volume was observed 2–3 μm above and below the disector where no cells were counted. Sampling of all parameters was continued to a mean coefficient of error (CE) of 0.05 to 0.10, according to Gundersen et al. [55]. All stereological parameters were quantified with assistance from a computerized stereology system by an operator blind to treatment and according to established principles, as detailed previously [21, 43, 47, 48, 51].

Statistical analysis

One-way ANOVA analyses and the Bonferroni/Dunn post hoc test were carried out to assess the effects of gender on total number of neurons in the CA1 region in male and female dtg AβPP/PS1 mice. Analysis of covariance (ANCOVA) was carried out with assistance from SAS statistical software (JMP, Cary, NC) to assess the influence of age at sacrifice (Age) on total number of CA1 neurons (Total n) in female mice for two independent variables: Transgene (wt vs. dtg AβPPswe/PS1ΔE9 mice + placebo); and Treatment (dtg AβPPswe/PS1ΔE9 mice + placebo vs. dtg AβPPswe/PS1ΔE9 mice +17αE2).

RESULTS

We investigated gender and transgene effects on neuron loss in hippocampal CA regions of 9 to 13 month-old male and female dtg AβPPswe/PS1ΔE9 mice and age- and gender-matched non-tg mice. Based on the finding of significantly fewer CA1 pyramidal neurons in female dtg AβPPswe/PS1ΔE9 mice compared to female non-tg controls, in a follow-up study we tested the hypothesis that this neuron loss in the CA regions of hippocampus could be mitigated by long-term (60 days) subcutaneous 17αE2 treatment of female dtg AβPPswe/PS1ΔE9 mice with bilateral OVX.

Gender and transgene effects on neuron number in hippocampal CA1 region

Loss of CA1 neurons and amyloid load

No gender differences were observed in total number of CA1 neurons for non-tg mice [male (n = 6) mean 80,781 (SEM 7758); female (n = 6) mean 84,173 (SEM 7651); p = 0.72]; or dtg AβPPswe/PS1ΔE9 mice [male mean 45,525 (SEM 4326), female mean 54,231 (SEM 4780) p = 0.20]. Comparison of data from non-tg and dtg AβPPswe/PS1ΔE9 mice revealed a significant 37% reduction in mean total number of CA1 neurons (F = 13.01; p < 0.01), as shown in Fig. 1.

Fig 1.

Fig 1

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The total number of CA1 neurons in hippocampus of 9–13-month-old dtg AβPPswe/PS1ΔE9 mice (n = 12) was significantly lower than that in age- and gender-matched non-transgenic littermate controls (n = 12); * p < 0.01.

ANCOVA confirmed that the effect of Transgene and Age on Total n was due to a significant effect of Transgene (F = 9.22; p < 0.03) with no effect of Age (0.05; p < 0.83). Furthermore, there was a significant correlation between increasing amyloid load in hippocampal molecular layers of female dtg AβPPswe/PS1ΔE9 and the loss of CA1 pyramidal neurons (n = 6; R2 = 0.914, p < 0.01).

Stereological analysis showed no differences in the total numbers of pyramidal cells in the CA2/3 hippocampal subregion of dtg AβPPswe/PS1ΔE9 mice [mean 62,000 (SEM 3650) cells] compared to non-tg mice [76,500 (SEM 4650) cells; p = 0.14]. There was no correlation between CA2/3 pyramidal neuron number and Aβ load in molecular layers of the hippocampus (p = 0.88; data not shown).

Neuroprotective effect of 17αE2

Female dtg AβPPswe/PS1ΔE9 mice maintained normal weight and food/water consumption during the 60 days of treatment with 17αE2 and placebo. Uteri were removed and weighed blind to treatment history. As shown in Fig. 2, the two groups that did not receive OVX, i.e., non-tag or sham dtg AβPPswe/PS1ΔE9 mice, showed no significant differences in uterine weight; in contrast, uterine weights for placebo-treated dtg AβPPswe/PS1ΔE9 mice were significantly lower than that for the three other groups.

Fig. 2.

Fig. 2

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Weight values of uteri from OVX female dtg AβPPswe/PS1ΔE9 mice treated with 17αE2 or placebo, non-OVX dtg AβPPswe/PS1ΔE9 mice (sham), and non-tg mice. The weight of dtg uteri was significantly different among the treatment groups; n = 5–6/group; *p < 0.005.

Neuroprotective effects of 17αE2

There was a significant effect of 17αE2 on neuron number in the CA1 subregion of hippocampus of female dtg AβPPswe/PS1ΔE9 mice (F = 7.66;p < 0.02), as shown in Figs 3 and 4.

Fig. 3.

Fig. 3

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Low-magnification micrograph images of Congo red staining of Aβ plaques in the hippocampus region of dtg AβPPswe/PS1ΔE9 mice treated with either placebo (A) or 17αE2 (B). High-magnification images of pyramidal neurons stained with violet in the CA1 region of the hippocampus in placebo-treated (C) and 17αE2-treated (D) dtg AβPPswe/PS1ΔE9 mice.

Fig. 4.

Fig. 4

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Total number of pyramidal cells in the CA1 hippocampal layer in brains from non-tg mice and ovariectomied (OVX) female dtg AβPPswe/PS1ΔE9 mice sacrificed at about 11 months of age (9 to 13 months) following treatment with either 17αE2 or placebo. The number of CA1 pyramidal cells in dtg AβPPswe/PS1ΔE9 mice treated with placebo was significantly decreased compared to non-tg mice. The number of CA1 neurons in 17αE2-treated mice was significantly elevated (39%) compared to placebo treated animals, and not statistically diiferent from non-tg mice. n = 5/group; * p < 0.05.

The significant effect of Treatment (placebo or 17αE2) and Age on Total N in CA1 was due to a significant effect of Treatment (F = 13.8; p < 0.0075) and not Age (F = 0.126; p < 0.73), as shown by ANCOVA. The finding of no significant differences in the mean total number of CA1 neurons in the hippocampus of non-tg female mice compared to that in the 17αE2-treated female mice indicates that 17αE2 treatment prevented the loss of CA1 neurons observed in the placebo-treated female AβPPswe/PS1ΔE9 mice.

As shown in Fig. 3A and B, amyloid load in dtg AβPPswe/PS1ΔE9 mice treated with 17αE2 was reduced by over 25% compared to that in placebo-treated dtg AβPPswe/PS1ΔE9 mice [6965 (SEM 720) mm3 vs. 9770 (SEM 1525) mm3]; however, this effect did not reach statistical significance (p = 0.17; data not shown).

Figure 5 shows low and high magnification views of Iba1-positive microglia and presents results for the mean total number of Iba1-immunopositive microglia in hippocampus. The images at high magnification in the inset shows examples of a less activated phenotype, with more numerous immunoreactive processes on microglia following treatment with 17αE2 for 60 days. The mean total number of Iba1-positive microglia was significantly higher in the placebo-treated dtg AβPPswe/PS1ΔE9 mice compared to both non-tg and 17αE2-treated mice (p < 0.0001). There was no significant difference in number of Iba1-positive microglia between the non-tg and the 17αE2-treated mice. Thus, 60 days of 17αE2 treatment appeared to have attenuated microglial activation in the molecular layers of hippocampus of female dtg AβPPswe/PS1ΔE9 mice.

Fig. 5.

Fig. 5

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Images of Iba1-positive microglia in the hippocampus of non-tg mice (A), OVX female dtg AβPPswe/PS1ΔE9 mice treated with placebo (B) or 17αE2 (C). Upper panel shows lower magnification (4X) views of immunostained microglia in (60X). D) Mean total numbers of Iba1-positive microglia in hippocampal molecular layers of non-tg mice and OVX: female dtg AβPPswe/PS1ΔE9 mice sacrificed about 11 months of age following 60 days of treatment with 17αE2 or placebo. The mean total number of Iba1-positive microglia in placebo-treated mice was significantly elevated compared to both non-tg and 17αE2-treated mice. There was no statistical difference between non-tg and 17αE2-treated mice. n = 5/group; * p < 0.05.

DISCUSSION

Previous studies in single tg AβPP mice [52, 53, 60] and double transgenic APP670-671/PS1M146L mice that express relatively low levels of mutant Aβ, and especially low levels of the more fibrillar Aβ1–42 peptide, report relatively minor or no AD-type neuron loss in brain regions associated with cognitive function [61, 68]. Transgenic mice that express higher levels of the more fibrillar Aβ species following co-expression of two AβPP mutations (APP751 and APP670-671) and a PS1 knock-in reportedly show loss of pyramidal cells in CA1/2 but not in CA3 or dentate gyrus [69]. Other studies in mice that express five AD-associated mutations report qualitative evidence of reduced numbers of large pyramidal neurons in layer V of neocortex and subiculum at 9 months of age [70]. Mice expressing multiple AβPP mutations (APPK670N, M671L, APPV717I, under the mouse Thy1 promoter) and mutant PS1 (PS-1M146L under the pHMG promoter) show qualitative evidence only for neuronal loss in the hippocampal pyramidal layers [71].

Here we report ~37% loss of CA1 neurons in dtg AβPPswe/PS1ΔE9 mice sacrificed in the age range of 9–13 months. Correlational analysis in female dtg AβPPswe/PS1ΔE9 mice revealed a strong correlation between loss of pyramidal cells in CA1 and increasing amyloid load. In these mice, the prion promoter (PrP) is used to co-express a Swedish mutation of AβPPswe and PS1ΔE9 that leads to the progressive accumulation of mutant Aβ peptides, with a high percentage of the more fibrillar Aβ1–42 peptide beginning at age 4–5 months [62, 63]. By middle age, these dtg AβPPswe/PS1ΔE9 mice manifest a range of AD-like characteristics, including significant loss of catecholaminergic neurons in the LC, dorsal raphe, and ventral tegmental area [43, 44]; stability in the number of TH-positive neurons in substantia nigra [43]; alterations in numbers of synapses in the stratum radiata of the CA1 [44]; reduced branching of cerebral capillaries [64]; and abnormalities in anxiety behavior, spatial and episodic memory [44, 65–67]. These findings have strong relevance to AD because they provide a link between neuron loss in brain regions that project to limbic and associated cortical regions, which are especially vulnerable to amyloid deposition in humans. Thus, the identification of dtg AβPPswe/PS1ΔE9 mice with loss of pyramidal cells in the CA1 region, along with neuronal loss in LC [43, 44] and altered synaptic contacts in CA1 molecular layer [45], highlights a potentially powerful tool to test novel sttategies for the therapeutic management of older men and women with AD.

Based on the finding of significant loss of CA1 neurons in middle-aged dtg AβPPswe/PS1ΔE9 mice, we used these mice to assess the potential of 17αE2 to mitigate this neuronal loss, plaque burden and activation of microglia. These results build on the findings from our earlier studies using 17βE2 in female C57/B16 (B6) mice [21, 54]. In a previous study of female non-tg C57/B16 mice, we reported that age-related loss of circulating estrogen (estrapause) is associated with a significant elevation of microglia in hippocampal molecular layers [54]. To test the hypothesis that this microglia activation may be under the control of circulating 17βE2, we report that 60 days of exogenous 17βE2 treatment via subcutaneous pellets reduces the total number of microglia to a level that is comparable to that of young female mice. Our earlier studies used radioimmunoassay to confirm that the delivery system, i.e., slow-release subcutaneous pellets, does indeed maintain plasma 17βE2 at physiological levels for at least 60 days [21], as claimed by the manufacturer (Innovative Research, Sarasota, FL). A third finding from this earlier study [21] is that subcutaneous delivery of 1.5 mg of 17βE2 over 60 days is associated with minimal morbidity or mortality. In a previous study using this method of delivery in aged female C57BL/6 mice [21], analysis of plasma confirmed that physiological levels of 17βE2 equivalent to that in young female C57BL/6 mice were maintained throughout the treatment period. In the present study, mean uterine weight of 17αE2 treated mice was similar to that of non-OVX (sham-treated) mice, while the uteri weight of OVX mice treated with placebo was siguificantly lower than that for sham mice. This indirect, yet convincing evidence supports the view that slow-release pellets maintained physiological levels of circulating 17αE2 during the treatment period.

The few studies that have investigated the neuro-protective potential of 17βE2 to attenuate AD-type neuropathology in transgenic mouse models have reported conflicting results. Among the likely factors that contribute to these discrepancies is the type of AD-associated mutations expressed, the timing of expression, and the maguitude and type of different Aβ peptides deposited in cortical brain regions. No significant effects of 17βE2 were reported in PDAPP [56], APP670-671/PS1M146L [57], or 3xTg [58] mice, while other studies report significant reductions in amyloid burden in both single and double transgenic mice [36, 59] following 17βE2 treatment. In addition to differences in treatment regimens and choice of transgenic mice, the difficulty in assessing the protective effects of 17βE2 has been hampered by dependence on amyloid load as the primary or sole endpoint [56, 57, 60, 61].

We report that 60 days of high circulating levels of 17αE2, a less feminizing and non-carcinogenic analog of 17βE2, significantly reduced neuronal loss in CA1 in dtg AβPPswe/PS1ΔE9 mice. The present findings support the view that 17αE2 may be at least as neuroprotective as 17βE2, in agreement with the relatively few in vivo studies carried out on 17αE2 [34]. Among those studies is a semi-quantitative study of hippocampal spine densities in rats that indicates 17αE2 stimulates synapse formation on CA1 spines to a greater extent than 17βE2, paralleling previous data on the effects of these two steroids on spatial memory [72]. Importantly, studies that use a single-mutation transgenic mouse model of AD (AβPPswe) demonstrated that 17αE2 is equally or more effective than 17βE2 in altering AβPP processing towards non-amyloidogenic, α-secretase products, leading to a reduction in cerebral Aβ levels as measured by ELISA [36]. In support of these neuroprotective effects in vivo, 17αE2 significantly attenuates the GABAA receptor-induced reduction in hippocampal volume and impaired spatial memory in a neonatal model of early brain injury [24]. As reported by previous studies with 17βE2 [56, 57], we did not find a robust treatment effect on amyloid load. Future studies will be carried out to determine if 17αE2 has a weaker effect on amyloid burden that may have been masked by the limited statistical power or age-range of the mice in this study.

Like 17βE2, 17αE2 appears to protect against a diverse array of lethal and etiologically relevant stressors, including amyloid toxicity, serum withdrawal, oxidative stress, excitotoxicity, and mitochondrial inhibition, among others [73] (for review, see [74]). Unlike 17βE2, however, 17αE2 does not stimulate proliferation of ER-positive tumors.

Hamsters treated with 17βE2 or 17αE2 for seven months showed widespread growth and proliferation of renal tumors after 17βE2 treatment, while hamsters treated with αE2 showed no tumors [75]. Although 17βE2 and 17αE2 share a close structural similarity and some of the same effects on tissue, the fact that these analogs act through different mechanisms and at different cellular locations stresses the need to focus less on the differences between 17βE2 and 17αE2, and rather on the potential of either epimer to provide a safe and effective treatment for middle-aged men and women against the onset and progression of AD.

In addition to protection against neuron loss, 17αE2 treatment appears to have attenuated microglial activation, as evidenced by both quantitative and qualitative endpoints. In female C57BL/6 mice, normal brain aging is associated with activation of neuroglia, possibly due to the loss of estrogen activity during estrapause [25, 51, 54]. Treatment of adult B6 mice with 17βE2 significantly diminishes this age-related activation of neuroglia [21, 51], and reduces neuronal loss through apoptosis [44]. Moreover, the formation of amyloid plaques in AD and dtg AβPPswe/PS1ΔE9 mice are closely associated with activated microglia [3, 76–78]. In response to this deposition of Aβ peptides in brain parenchyma, the activation of microglia leads to the release of pro-inflammatory cytokines [3, 79, 80]. Through a mechanism that is independent of actions on classical ERα and ERβ, 17αE2 inhibits microglia-mediated inflammatory mechanisms in-vivo, which in turn may block the degenerative processes leading to neuronal loss in middle-aged dtg AβPPswe/PS1ΔE9 mice, and perhaps older humans. Understanding these mechanisms and the steps necessary to block them in the brains of middle-aged humans is a critical area for further study. The possibility that 17αE2 may be as neuroprotective as 17βE2, but with over 200-fold less hormonal activity, encourages the development of other E2 analogs with low risk and more potent protection against the onset and progression of AD.

ACKNOWLEDGMENTS

The authors wish to acknowledge support from NIH/NINDS; Grant number: U54 NS42867, R01 AG026478 (RST), the Public Health Service (NIH extramural grants MH076541-02A1, NS039407-06A1), Drs. David R. Borchelt and Michael K. Lee for donation of founders for this study, and the NIA Intramural Program in Baltimore, Maryland. We would also like to acknowledge Dr. John Kwagyan for his assistance with the statistical analysis.

Footnotes

Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=655).

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