Estrogen Therapy Fails to Alter Amyloid Deposition in the PDAPP ...

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Endocrinology 146(6):2774 –2781 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2004-1433

Estrogen Therapy Fails to Alter Amyloid Deposition in the PDAPP Model of Alzheimer’s Disease Pattie S. Green, Kelly Bales, Steven Paul, and Guojun Bu Department of Pediatrics (P.S.G., G.B.), Washington University School of Medicine, St. Louis, Missouri 63110; Department of Medicine (P.S.G.), University of Washington, Seattle, Washington 98105; and Neuroscience Discovery Research (K.B., S.P.), Lilly Research Laboratories, Indianapolis, Indiana 46285 Epidemiological studies implicate estrogen deprivation as a risk factor for Alzheimer’s disease and postmenopausal estrogen replacement as protective factor. One potential mechanism involves estrogen attenuation of ␤-amyloid (A␤) peptide accumulation. We examined the effect of estrogen on amyloid accumulation in female PDAPP mice, which express human amyloid precursor protein (APP) with the V717F mutation. These animals deposit A␤ 1– 42 in the hippocampus and neocortex and develop Alzheimer-like neuropathology. Mice were subjected to ovariectomy, ovariectomy with estrogen replacement, or sham surgery at 3 months of age, and levels of cerebral A␤ 1– 40 and 1– 42 were determined after 5 months of treatment. Neither estrogen deprivation nor estrogen re-

placement altered A␤ accumulation in the hippocampus or neocortex. Similarly, immunoreactivity for full-length human APP and secreted APP␣ was unchanged. Estrogen status of the animals was confirmed using a variety of techniques, including uterine and pituitary weight, vaginal cytology, and plasma estradiol concentrations. There was no correlation between plasma estradiol levels and accumulation of either A␤ 1– 40 or A␤ 1– 42 in the brain. Our observations indicate that long-term estrogen therapy does not alter amyloid pathology in PDAPP mice, an animal model of Alzheimer’s disease, and question the role of estrogen in A␤ deposition in brain. (Endocrinology 146: 2774 –2781, 2005)

A

Extracellular accumulation of ␤-amyloid (A␤), the defining proteinaceous component of senile plaques, is strongly implicated in Alzheimer’s disease pathogenesis (14). A␤ is produced from the proteolytic processing of A␤ precursor protein (APP) by ␤- and ␥-secretase activities. Alternatively, ␣-secretase activity produces secreted APP␣ (sAPP␣) and precludes formation of A␤. In both nonneuronal (15) and neuronal cultures (16 –18), ␤-estradiol, the predominant circulating estrogen in premenopausal women, promotes processing of APP by the nonamyloidogenic ␣-secretory pathway and reduces cellular A␤ production (16 –17). In animal models, ovariectomy increased parenchymal A␤ levels in both guinea pigs (19) and transgenic mice overexpressing the Swedish mutation (K670N; M671L) of APP (APPsw) (20). In both studies, estrogen replacement reversed the ovariectomy-induced increase in A␤. The PDAPP mouse expresses human APP with a V717F mutation, one of several 717 mutations described in some families with early-onset Alzheimer’s disease (21). Although mutation of APP is a causative factor in relatively few human cases of Alzheimer’s disease, mice overexpressing APP mutations have proven useful models for studying mechanisms of A␤ deposition in vivo (22–25). The PDAPP mouse shows an age- and region-dependent deposition of A␤. Similar to human Alzheimer’s disease, A␤ 1– 42 is preferentially deposited in the hippocampus and neocortex of these mice (24 –25). In the current studies, we used ovary-intact, ovariectomized, and ovariectomized/estrogen-replaced PDAPP mice to study the effect of estrogen deprivation and replacement on A␤ accumulation in brain. Our observations indicate that estrogen status had no significant impact on the levels of amyloid protein in the brains of female PDAPP mice, an animal model of Alzheimer’s disease.

LZHEIMER’S DISEASE IS an age-related neurodegenerative disorder afflicting an estimated four million Americans (1). Women are disproportionately at risk for developing this increasingly prevalent neurodegenerative disorder (2). Loss of ovarian steroids, particularly estrogens, at the menopause may increase the susceptibility of the aging brain to neurodegenerative disorders and be a risk factor for development of Alzheimer’s disease (3). In support of this hypothesis, several retrospective (4 – 6) and prospective (7–9) epidemiological studies correlate a decreased incidence of Alzheimer’s disease with postmenopausal estrogen replacement therapy, although other epidemiological studies find no beneficial effect of hormone use (10). The observed beneficial effect of estrogen therapy is dependent both on dose (4) and duration (4, 9) of estrogen replacement, and estrogen therapy may be more effective at preventing Alzheimer’s disease if given near the onset of menopause (9). In contrast to the epidemiological data, three recent randomized, double-blind clinical trials find no effect of estrogen therapy on the clinical course of the disease in patients with mild to moderate Alzheimer’s disease (11–13), suggesting a possible role for estrogen in the prevention, but not treatment, of the disease.

First Published Online February 24, 2005 Abbreviations: A␤, ␤-Amyloid; APP, amyloid precursor protein; APPsw, the Swedish mutation of APP; flAPP, full-length APP; Ovx, ovariectomized mice implanted with placebo; Ovx⫹E2, Ovx mice implanted with 17␤-estradiol; sAPP␣, secreted APP␣; SDS, sodium dodecyl sulfate; Sham, ovary-intact mice; WHIMS, Women’s Health Initiative Memory Study. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

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Green et al. • Estrogen Does Not Alter A␤ Deposition in Mice

Endocrinology, June 2005, 146(6):2774 –2781

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Materials and Methods Mouse treatment and analysis

(r2 ⫽ 0.83) and neocortex (r2 ⫽ 0.58), with 6.4 ⫾ 0.3% and 3.5 ⫾ 0.2% of the tissue wet weight accounted for by protein, respectively.

Transgenic mice overexpressing human APP with a V717F mutation under the control of a platelet-derived growth factor ␤ promoter (PDAPP mice) have been previously described (24). The mice were maintained on a hybrid background of C57BL/6, DBA, and SwissWebster strains. Mice were housed under a 12-h light, 12-h dark cycle, with food and water available ad lib; and, to exclude the presence of phytoestrogens in the diet, they were fed a casein-based, soy-free diet (Purina Mills, LLC, Richmond, IN) for the duration of the study. All procedures performed on animals were reviewed and approved by the Institutional Animal Care and Use Committee of Washington University before the initiation of the study. Mice were ovariectomized using a bilateral dorsal approach at 12 wk of age. At the time of surgery, mice were implanted with either 17␤estradiol (0.25 mg; Ovx⫹E2 group) or placebo (Ovx group) in a 90-d, timed-release pellet (Innovative Research of America, Sarasota, FL). Ovary-intact mice (Sham group) underwent a sham operation with placebo pellet implantation. The timed-release pellets were exchanged 12 wk after implantation, when mice were 24 wk of age. Two mice in the Ovx⫹E2 group were killed before the end of the study due to vaginal hyperplasia. Animals were killed at 32 wk of age using pentobarbital (200 mg/kg), followed by perfusion with cold normal saline (0.9%) and decapitation. Vaginal smears were obtained daily on female mice, for 3 wk starting at 9 wk of age, according to the method and criteria described by Nelson et al. (26). Mice with average cycle length greater than 6 d were excluded from the study. Vaginal smears were also obtained daily for the week before death, as a physiological confirmation of estrogen status.

Determination of APP levels

Brain tissue preparation The neocortex and hippocampus were dissected from the left hemisphere of the brain, quickly frozen, and maintained at ⫺80 C. For determination of total A␤ levels, tissue was homogenized in 10 vol of 5 m guanidine in 50 mm Tris (pH 8.0), with protease inhibitors and rocked at room temperature for 2 h. Homogenates were centrifuged at 14,000 ⫻ g for 15 min, and the supernatant was then diluted in 1% BSA in PBS for analysis by ELISA. Differential extraction of brain tissue was initiated by homogenization in 10 vol of 50 mm Tris, 150 mm NaCl (pH 7.5) with protease inhibitors. Homogenate was centrifuged at 100,000 ⫻ g for 90 min. This supernatant (soluble fraction) was used to detect soluble, nonmembrane-associated proteins. The pellet was sonicated in 10 vol of 50 mm Tris, 150 mm NaCl, 2% Triton X-100, 2% sodium dodecyl sulfate (SDS) (pH 7.5) with protease inhibitors and centrifuged at 100,000 ⫻ g for 90 min. The second supernatant (membrane fraction) was used for the detection of membrane associated proteins. The second pellet was sonicated in 10 vol of 5 m guanidine, 50 mm Tris (pH 8.0) with protease inhibitors and was cleared by centrifugation at 14,000 ⫻ g for 15 min. This final supernatant (insoluble fraction) was used for the detection of insoluble A␤. The right hemisphere of the brain was fixed for 24 h in 4% paraformaldehyde and cytopreserved in 30% sucrose for 3 d. Tissue was then frozen in dry ice, cut into 50-␮ slices, and stored in a 15% sucrose, 30% ethylene glycol, 30 mm phosphate buffer (pH 7.4) at ⫺20 C.

A␤ quantitation A␤ was quantitated using a sandwich ELISA as previously described (25). Briefly, monoclonal antibodies 21F12 and 2G3 were used as capture antibodies for A␤1– 42 and A␤1– 40, respectively. The reporter antibody for both ELISAs was biotinylated 3D6. Avidinhorseradish peroxidase (Vector Laboratories, Inc., Burlingame, CA), followed by slow TMB (Pierce Biotechnology Inc., Rockford, IL), was used for color development. Color development was monitored at 630 nm every 15 sec for 10 min. All samples were analyzed at two different dilutions in the linear range of the standard curve, in duplicates; and analyses were repeated on two independent ELISA experiments. Values presented are the averages of these results and are normalized to tissue wet weight. Protein concentration in the homogenates strongly correlated with tissue wet weight in both the hippocampus

sAPP␣ was measured in the soluble fraction of the brain homogenate by immunoblot analysis using monoclonal antibody 6E10 (Signet Laboratories, Inc., Dedham, MA), which reacts with full-length APP (flAPP) and sAPP␣ but not sAPP␤. flAPP was measured in the membrane fraction of the brain homogenate by immunoblot analysis using a rabbit polyclonal C-terminal flAPP antibody (Zymed Laboratories, Inc., San Francisco, CA); flAPP was not detected in the soluble fraction or the insoluble fraction of the brain homogenate. Bands immunoreactive with the 6E10 antibody were detected in both the membrane fraction and soluble fraction, consistent with 6E10 recognizing both sAPP␣ and flAPP. However, another transmembrane protein, lipoprotein-related receptor protein, was absent from the soluble fraction (data not shown). All sAPP␣ blots were stripped and reprobed using the C-terminal antibody for flAPP, to ensure integrity of the tissue preparations. For both analyses, 5 ␮g protein was loaded for each lane. Immunoblots were developed using ECL plus reagent (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ). Linearity of band density was verified by multiple dilutions of one sample on each blot. All samples were analyzed on two different blots.

Tissue staining of A␤ deposits Every sixth section was blocked with normal goat sera for 1 h, then stained using a pan-A␤ antibody (Biosource International, Camarillo, CA) at 4 C overnight. The antirabbit ABC kit (Vector Laboratories, Inc.,) was used according to the manufacturer’s directions for antibody visualization. Cell bodies were counterstained using cresyl violet.

Measurement of plasma estradiol levels Blood samples were obtained by cardiac puncture before perfusion at time of death. Plasma was stored at ⫺20 C until assayed using the ultrasensitive E2 RIA kit from Diagnostic Systems Laboratories, Inc. (Los Angeles, CA) according to the manufacturer’s instructions, with the exception that 75 ␮l plasma was analyzed rather than 100 ␮l. This reduced the sensitivity of the assay to 10 pg/ml but allowed duplicate determinations with the plasma available.

Determination of uterine wet weight and pituitary weight. At the time of death, uteri and pituitaries were excised, extraneous tissue gently removed, and wet weight immediately determined.

Statistical analyses Data are presented in the text as mean ⫾ sem. Statistical difference between groups was determined by one-way ANOVA, with planned comparisons between groups (Sham vs. Ovx and Ovx vs. Ovx⫹E2) determined by Dunnett’s multiple comparison test when P ⬍ 0.05. Relationships between multiple variables were determined by linear regression analysis. A ␹2 analysis was used to determine the statistical significance of the tissue A␤ staining. For all analyses, P ⬍ 0.05 was considered significant. Statistical analyses were performed using Prism (GraphPad Software, Inc., San Diego, CA).

Results Physiological variables in Sham, Ovx, and Ovx⫹E2 PDAPP mice

PDAPP mice underwent ovariectomy, ovariectomy with estradiol replacement, or sham surgery to determine the effects of endogenous and exogenous estrogen on A␤ levels in this model of amyloidogenesis. Before randomization into the treatment groups for the study, the average estrus cycle length was determined for all female mice over a 3-wk period. Three mice were excluded from the study for an average cycle length

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greater than 6 d. There was no difference in presurgical estrus cycle lengths between the treatment groups (Table 1; P ⫽ 0.18). As previous described (26 –27), housing density significantly correlated with estrus cycle length (P ⫽ 0.002; r2 ⫽ 0.59), with a housing density of five mice per cage increasing average estrus cycle length by 19% over singly housed mice. Mice were housed in pairs with a member of the same treatment group, after surgery, for the duration of the experiment. Ovariectomy reduced circulating plasma ␤-estradiol from 31.9 ⫾ 6.4 pg/ml in the Sham group (Table 1) to less than 10 pg/ml (Ovx group; below the detectability range of 10 pg/ml in seven of nine mice). Replacement of ovariectomized mice with ␤-estradiol in a timed-release pellet resulted in plasma ␤-estradiol concentrations of 67.8 ⫾ 7.2 pg/ml when determined at time of kill. Uterine atrophy and acellular or thin leukocytic vaginal smears confirmed estrogen deprivation in the Ovx mice. Physiological verification of estrogen replacement in the Ovx⫹E2 mice included uterine hypertrophy (Table 1; P ⬍ 0.001), increased pituitary weight (Table 1; P ⬍ 0.001), and persistently cornified vaginal smears. Additionally, ovariectomy resulted in a trend toward increased body weight, which was countered by estrogen therapy (P ⬍ 0.05). Hippocampal A␤ accumulation in Sham, Ovx, and Ovx⫹E2 PDAPP mice

PDAPP mice demonstrate age-dependent A␤ accumulation in both the hippocampus and neocortex, with the highest levels in the hippocampus (24 –25). In agreement with previous reports (25), we found the A␤ level in the hippocampus to be approximately 4-fold that of the neocortex, with A␤ 1– 42 representing 85.6 ⫾ 1.4% and 83.5 ⫾ 0.9% of the total A␤ in each region, respectively. The hippocampal levels of A␤ 1– 42 were unchanged among ovary-intact mice (37.9 ⫾ 6.5 pmol/g), ovariectomized mice (35.0 ⫾ 16.2 pmol/g), and estrogen-replaced ovariectomized mice (30.4 ⫾ 12.7 pmol/g; P ⫽ 0.91; Fig. 1A). Similarly, there was no statistical difference among A␤ 1– 40 levels in the three treatment groups (Fig. 1B; P ⫽ 0.73). The ratio of hippocampal A␤ 1– 40 to A␤ 1– 42 was not significantly altered with estrogen manipulation (Fig. 1C; P ⫽ 0.18). The levels of A␤ 1– 40 strongly correlated with the levels of A␤ 1– 42 in the hippocampus (r2 ⫽ 0.57; P ⬍ 0.0001). TABLE 1. Physiological variables in ovariectomized and estrogenreplaced PDAPP mice Sham

Ovx

Ovx ⫹ E2

No. of mice 10 8 7 Estrus cycle length before 5.0 ⫾ 0.8 4.4 ⫾ 0.7 5.2 ⫾ 0.8 surgery (d) Plasma estradiol (pg/ml) 31.9 ⫾ 6.4a ⬍10.1 67.8 ⫾ 9.2ab Body weight (g) 31.0 ⫾ 1.0 32.3 ⫾ 1.0 28.8 ⫾ 0.6c Uterine weight (mg) 178 ⫾ 17c 89 ⫾ 7 323 ⫾ 36ab Pituitary weight (mg) 1.5 ⫾ 0.15 1.1 ⫾ 0.19 5.4 ⫾ 0.61ab Values are presented as mean ⫾ SEM. Estrus cycle length was determined before surgery and randomization into treatment groups. All other values were determined at time of death. a P ⬍ 0.001 vs. Ovx. b P ⬍ 0.01 vs. Sham. c P ⬍ 0.05 vs. Ovx.

Neocortical A␤ accumulation in Sham, Ovx, and Ovx⫹E2 PDAPP mice

Neocortical A␤ 1– 42 levels did not vary significantly with estrogen status (Fig. 2A; P ⫽ 0.43). Levels of A␤ 1– 40 were similarly unchanged among Sham (1.4 ⫾ 0.2 pmol/g), Ovx (1.68 ⫾ 0.4 pmol/g), and Ovx⫹E2 (1.23 ⫾ 0.2 pmol/g) groups (Fig. 2B; P ⫽ 0.57). Likewise, the ratio of A␤ 1– 40 to A␤ 1– 42 was similar among the treatment groups (Fig. 2C; P ⫽ 0.56). Similar to the hippocampus, the levels of A␤ 1– 40 strongly correlated with those of A␤ 1– 42 (r2 ⫽ 0.68; P ⬍ 0.001) in the neocortex. SDS-insoluble A␤ 1– 42 was examined in the cortical tissue for a subset of animals. A strong positive correlation existed between SDS-insoluble A␤ 1– 42 and total A␤ 1– 42 (r2 ⫽ 0.73; P ⬍ 0.001). There was no statistical difference in the amount of SDS-insoluble A␤ 1– 42 among treatment groups (Fig. 3A; P ⫽ 0.90). Similarly, there was no effect of treatment group on the ratio of SDS-insoluble A␤ 1– 42 to total A␤ 1– 42 (Fig. 3B; P ⫽ 0.75). Relationship among plasma estradiol concentrations, A␤ levels, and physiological variables

There was no linear relationship between plasma estradiol concentrations and levels of either hippocampal A␤ 1– 42 (Fig. 4A; r2 ⫽ 0.011; P ⫽ 0.63) or A␤ 1– 40 (Fig. 4A; r2 ⫽ 0.003; P ⫽ 0.79). Similarly, there was no statistical correlation between plasma estradiol concentrations and either A␤ 1– 42 (Fig. 4B; r2 ⫽ 0.026; P ⫽ 0.44) or A␤ 1– 40 (Fig. 4B; r2 ⫽ 0.008; P ⫽ 0.66) in the neocortex. In contrast, uterine wet weight showed a strong positive correlation with plasma estradiol concentrations (Fig. 4C; r2 ⫽ 0.58; P ⬍ 0.0001). Pituitary weight also correlated positively with estradiol levels (Fig. 4D; r2 ⫽ 0.51; P ⫽ 0.0002). Tissue staining of A␤ in Sham, Ovx, and Ovx⫹E2 PDAPP mice

Every sixth section from a subset of mice was stained for deposits immunoreactive with a pan-A␤ antibody. Of the 18 mouse brains examined, 10 demonstrated A␤ immunoreactive deposits. In agreement with previous reports, the deposits that were present at this age were predominately in the cingulate cortex and the hippocampus (Fig. 5). There was no difference among treatment groups in the number of brains showing A␤ deposits (four of seven in Sham group, three of five in Ovx group, three of six in Ovx⫹E2 group; P ⫽ 0.98). flAPP and sAPP␣ levels in Sham, Ovx, and Ovx⫹E2 PDAPP mice

Brain tissue was differentially extracted, as described in Materials and Methods, to give three fractions of protein: soluble, membrane, and SDS-insoluble. The membrane fraction was used for determination of flAPP levels and the soluble fraction used for immunodetection of sAPP␣. The levels of flAPP were unchanged among treatment groups (Fig. 6, A and B; P ⫽ 0.40), suggesting equal expression of the transgene among groups. Similarly, the ratio of sAPP␣ to flAPP was unchanged among estrogen-deprived, intact, and estrogen-supplemented groups (Fig. 6, A and C; P ⫽ 0.24).


FIG. 1. Hippocampal A␤ levels in ovariectomized and estrogenreplaced PDAPP mice. Twelve-week-old PDAPP mice were Sham, OVX, or Ovx⫹E2. At 32 wk, mice were killed and A␤ levels determined in guanidine extracts of the hippocampus by ELISA. A and B, A␤ 1– 42 and A␤ 1– 40 levels, respectively. C, Ratio of A␤ 1– 40 to A␤ 1– 42. Bar, Mean value for the treatment group. All values plotted represent the average of four sets of duplicate determinations.

Discussion

Estrogen deprivation has been proposed to be a major risk factor for development of Alzheimer’s disease in humans. One potential mechanism involves accentuated amyloid accumulation in the brain (28). Using the PDAPP mouse model, we determined the effect of estrogen on A␤ levels in the brain using female mice that were intact, ovariectomized, or ovariectomized and subsequently treated with estrogen. Estrogen status of all treatment groups was verified by plasma ␤estradiol concentrations, uterine weight, pituitary weight, and vaginal cytology. Neither estrogen deprivation nor estrogen supplementation of ovariectomized mice altered the amount of amyloid in either the neocortex or hippocampus. There was no association between plasma levels of estradiol and levels of A␤ 1– 40 or A␤ 1– 42 in brain tissue. Additionally, the ratio of A␤ 1– 40 to A␤ 1– 42 remained unchanged among treatment groups in both the neocortex and hippocampus. Moreover, amyloid deposition, as determined both by the amount of SDS-insoluble A␤ and by the number of animals with immunohistochemically detectable amyloid deposits, was also unaffected by estrogen. Collectively, our observations provide strong evidence that neither estrogen


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FIG. 2. Neocortical A␤ levels in ovariectomized and estrogenreplaced PDAPP mice. Twelve-week-old PDAPP mice were Sham, OVX, or Ovx⫹E2. At 32 wk, mice were killed and A␤ levels determined in guanidine extracts of the neocortex by ELISA. A and B, A␤ 1– 42 and A␤ 1– 40 levels, respectively. C, Ratio of A␤ 1– 40 to A␤ 1– 42. Bar, Mean value for the treatment group. All values plotted represent the average of four sets of duplicate determinations.

deficiency nor estrogen replacement therapy has a major impact on the deposition of A␤ protein in female PDAPP mice. Our findings differ markedly from previous reports in which estrogen treatment attenuated amyloid accumulation in two different mouse models overexpressing the APPsw of APP: Tg2576 mice (20) and TgC3–3 mice (29). The failure to observe a difference in the levels of amyloid protein accumulation in estrogen-deficient and -replete PDAPP animals is not likely to reflect a lack of statistical power of our studies. Previous studies have shown that estrogen therapy reduces cerebral amyloid levels by approximately 50% in mice (20, 29), and our sample size had a power greater than 0.90 to detect such a change in amyloid levels in the brain. Supporting an effect of gonadal steroids on amyloid deposition, Callahan et al. (30) report a sex difference in amyloid plaque area in the Tg2576 mice, with aged females showing greater deposition than males of the same age. In contrast, we found no differences in hippocampal amyloid levels between 8-month-old female PDAPP mice and age-matched male littermates (Green, P. S., and G. Bu, unpublished observations; n ⫽ 6; P ⫽ 0.39 for A␤40, and P ⫽ 0.76 for A␤42). It is possible

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FIG. 3. SDS-insoluble A␤ in ovariectomized and estrogen-replaced PDAPP mice. Twelve-week-old PDAPP mice were Sham, OVX, or Ovx⫹E2. At 32 wk, mice were killed, and total A␤ 1– 42 and SDS-insoluble A␤ 1– 42 levels were determined in neocortical extracts by ELISA. Bar, Mean for the treatment group and all values plotted represent the average of two to four sets of duplicate determinations. A, Measured levels of SDS-insoluble A␤1– 42. B, Ratio of SDS-insoluble to total A␤ 1– 42.

that an effect of sex on amyloid accumulation would be apparent in older PDAPP mice. A number of factors might account for the variable effect of estrogen on the accumulation of brain amyloid in the different mouse studies. One key feature is likely to be the differences in the APP mutations expressed in the different mouse models of Alzheimer’s disease that have been used (20, 29). Our studies focused on the V717F mutation in female PDAPP mice, which alters the ␥-secretase cleavage and results in increased levels of the more amyloidogenic A␤ 1– 42 in both cell culture (31) and mouse models (32). As a result, the PDAPP mouse model primarily deposits A␤ 1– 42 (24) in

an age- and region-dependent manner consistent with the human disease (25). In contrast, the APPsw expressed in the Tg2576 mice (22) and the TgC3–3 (23) mice decreases ␣-secretase processing of APP. This results in increased production of total A␤ without affecting the ratio of A␤1– 42 to A␤ 1– 40 (32–33). Both the Tg2576 and TgC3–3 lines deposit primarily A␤ 1– 40 (22, 23). The Tg2576 mice coexpressing a presenilin 1 mutation (20) have increased A␤ 1– 42 production, resulting in approximately equal levels of A␤ 1– 42 and A␤ 1– 40. It is interesting to note that the estrogen effect on amyloid levels was less pronounced in the Tg2576/PS1 mice (20), which have a decreased A␤ 1– 40 to A␤ 1– 42 ratio compared

FIG. 4. Correlation between plasma estradiol concentrations and (A) A␤ levels in the hippocampus, (B) A␤ levels in the neocortex, (C) uterine wet weight, and (D) pituitary weight. Twelve-week-old PDAPP mice were Sham, OVX, or Ovx⫹E2. At 32 wk, mice were killed, and plasma ␤-estradiol concentrations were determined by RIA. Additionally, A␤ levels in the hippocampus and neocortex, uterine wet weight, and pituitary weight were determined. Correlations between these variables and plasma ␤-estradiol concentrations were determined by linear regression analysis. Plasma ␤-estradiol values less than 10 pg/ml were treated as equal to 10 pg/ml in the regression analysis.


FIG. 5. A␤ staining in the brains of ovariectomized (A and C) and estrogen-replaced (B and D) PDAPP mice. Twelve-week-old PDAPP mice were OVX or Ovx⫹E2. At 32 wk, mice were killed and the right hemisphere of the brain fixed, cytopreserved, and stained for A␤ deposits as described in Materials and Methods. A␤ deposits were primarily observed in the cingulate cortex (A and B) and the hippocampus (C and D). Arrowheads, A␤ deposits.

with the Tg2576 mice. Thus, the relative levels of A␤ 1– 40 and A␤ 1– 42 may be one important factor modulating the deposition of amyloid protein in the brain of estrogen-replete and -deficient mice. Another important issue is the level and duration of estrogen replacement employed in the different studies. In our studies, plasma levels of ␤-estradiol during estrogen


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replacement ranged from proestrus levels in ovary-intact mice (⬃45 pg/ml) to slightly supraphysiological (up to 100 pg/ml). These levels were well within the range of plasma estradiol values that have been reported to be effective at reducing A␤ levels in APPsw mice (20). Our study focused on long-term (5 months) estrogen-deprivation and/or replacement. These mice were sexually mature at the time of ovariectomy. The studies of APPSW mice used short-term treatment protocols ranging from 6 wk (20, 29) to 3 months (20). These different treatment lengths could contribute to the disparate findings as short-term (2 wk) estrogen treatment enhances high-affinity choline uptake, although longer (4 wk to 6 months) had no effect (34). Similar results have been observed in clinical trials of estrogen in Alzheimer’s disease, where estrogen treatment may improve cognitive function for short terms (less than 3 months) (11, 35–36) but is not efficacious when used for longer periods (11–13). The long-term estrogen exposure may alter estrogen receptor levels in the brain. However, we observed no difference in neocortical levels of either estrogen receptor ␣ or estrogen receptor ␤ levels by immunoblot among the treatment groups (Green, P. S., and G. Bu, unpublished observations). Epidemiological studies indicate that estrogen replacement therapy lowers the risk of developing Alzheimer’s disease (4 –9). Estradiol has numerous effects on the brain that may contribute to a decreased disease risk, including neuroprotective effects (37– 40), stimulation of neurotrophin expression and signaling (41), induction of neurite outgrowth (42– 44), and modulation of cholinergic function (34). Studies of estrogen-deficient and -replete APPsw mice suggest that one protective mechanism involves decreased accumulation of amyloid protein (20, 29). However, not all epidemiology studies find a beneficial effect of estrogen therapy (10). Moreover, the Women’s Health Initiative Memory Study (WHIMS) (a prospective, randomized trial) failed to find a beneficial effect of chronic therapy, with conjugated equine estrogens either alone (45) or in combination with progestin therapy (46), on the risk of dementia. Our studies indicate that alterations in estrogen status fail to effect amyloid deposition or accumulation in the hippocampus and cerebrum of PDAPP animals. The applicability of these findings to human, either with or without the 717 mutation, remains to be determined. Future studies are needed to explore the role

FIG. 6. sAPP␣ and flAPP in ovariectomized and estrogen-replaced PDAPP mice. Twelve-week-old PDAPP mice were Sham, OVX, or Ovx⫹E2. At 32 wk, mice were killed and sAPP␣ and flAPP determined as described in Materials and Methods. A, Representative immunoblots. B, Relative band density for flAPP immunoreactivity. C, Ratio of relative band densities of sAPP␣ to flAPP. Bar, Mean for the treatment group and all values plotted represent the average of two sets of duplicate determinations.

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of estrogen in regulating amyloid protein deposition in other animal models of Alzheimer’s disease, and to determine whether estrogen replacement therapy alters amyloid deposition and the risk of dementia in humans suffering from Alzheimer’s disease.

19. 20. 21.

Acknowledgments The authors thank Eli Lilly for the PDAPP mice and antibodies used in the ␤ ELISA measurements. Received November 2, 2004. Accepted February 14, 2005. Address all correspondence and requests for reprints to: Pattie S. Green, Division of Gerontology and Geriatric Medicine, Box 356426, University of Washington, Seattle, Washington 98195. Email: psgreen@ u.washington.edu. This work was supported by the Alzheimer’s Association NIRG-013137 (to P.S.G.) and National Institutes of Health P50 AG05681 (to G.B.).

22. 23.

24.

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