17β-Estradiol regulates insulin-degrading enzyme expression via an ERβ/PI3-K pathway in hippocampus: relevance to Alzheimer's prevention

Abstract

Insulin-degrading enzyme (IDE), an enzyme that primarily degrades insulin, has recently been demonstrated to play a significant role in the catabolism of amyloid β (Aβ) protein in the brain. Reduced IDE expression and/or activity have been associated with the etiology and development of Alzheimer's disease (AD). Using three model systems, the present investigation provides the first documentation indicating that estrogen robustly regulates the expression of IDE in normal, menopausal and early-stage AD brains. In vitro analyses in primary cultures of rat hippocampal neurons revealed that 17β-estradiol (17β-E2) increased IDE in both mRNA and protein levels in a time-dependent manner. Further pharmacological analyses indicated that 17β-E2-induced IDE expression was dependent upon estrogen receptor (ER) β and required activation of phosphatidylinositol 3-kinase (PI3-K). In vivo analyses in adult female rats revealed a brain region-specific responsive profile. Ovariectomy (OVX) induced a significant decline in IDE expression in the hippocampus, which was prevented by 17β-E2. Neither OVX nor 17β-E2 affected IDE expression in the cerebellum. In vivo analyses in triple transgenic AD (3xTg-AD) female mice revealed an inverse correlation between the age-related increase in Aβ load and the decrease in IDE expression in the hippocampal formation. Treatment with 17β-E2 attenuated Aβ accumulation/plaque formation and elevated hippocampal IDE expression in 12-month-old 3xTg-AD OVX mice. Collectively, these findings indicate that 17β-E2 regulates IDE expression in a brain region-specific manner and such a regulatory role in the hippocampus, mediated by an ERβ/PI3-K pathway, could serve as a direct mechanism underlying estrogen-mediated preventative effect against AD when initiated at the onset of menopause.

Figure 1

17β-E2 increased IDE expression via an ERβ/PI3-K pathway in rat hippocampal neurons. (A) Immunocytochemical staining revealed that IDE (green) was abundantly and primarily expressed in the cytoplasm and neural processes of rat hippocampal neurons. (B, C) Treatment with 17β-E2 (10 nM for 0–48 h) increased IDE expression in both mRNA and protein in a time-dependent manner in rat hippocampal neurons; * P < 0.05 compared to 0 h. (D) 17β-E2 (10 nM for 48 h)-induced increase in IDE protein expression was completely abolished in the presence of PI3-K inhibitors, wortmannin (50 nM) or LY-294002 (25 μM); ** P < 0.01 compared to vehicle alone-treated control cultures. (E, F) Treatment with the ERβ agonist, DPN (10 nM for 48 h), not the ERα agonist, PPT (10 nM for 48 h), increased IDE protein expression in rat hippocampal neurons, which was completely abolished in the presence of wortmannin (50 nM) or LY-294002 (25 μM); * P < 0.05 compared to control cultures. (G) Treatment with the ERα agonist/ERβ antagonist, (R,R)-THC (100 nM for 48 h), alone did not induce a significant effect; however, when co-administered, (R,R)-THC completely abolished DPN-induced increase in IDE protein expression in rat hippocampal neurons. 18s rRNA and β-tubulin were respectively used as RNA and protein loading controls. Data are presented as (B, C) percent of 0 h and (D–G) percent of vehicle alone-treated control groups and expressed as group mean ± S.E.M., n ≥ 3. Wort: wortmannin; LY: LY-294002.

Figure 2

17β-E2 reversed OVX-induced decline in IDE protein expression in the hippocampus of adult female rats. (A) Immunohistochemical triple staining revealed that IDE (red) was co-localized with the neuronal marker, MAP2 (green), and not with the glial marker, GFAP (blue), indicating that IDE was expressed in neurons and not in glial cells of adult female rats. (B, C) OVX induced a significant reduction in IDE protein expression in the hippocampus, and not in the cerebellum, of adult female rats; ** P < 0.01 compared to sham-OVX rats. (D) Treatment with 17β-E2 (30, 70, and 300 μg/kg BW, once daily for 2 d) increased, in a dose-dependent manner, IDE protein expression in the hippocampus of OVX rats; ** P < 0.01 compared to vehicle alone-treated OVX rats. (E, F) Treatment with 17β-E2 (70 μg/kg BW, once daily for 2 d) increased IDE protein expression in the hippocampus, and not in the cerebellum, of OVX rats; ** P < 0.01 compared to vehicle alone-treated OVX rats. β-tubulin was used as the loading control. Data are presented as (B, C) percent of sham-OVX (D–F) percent of vehicle alone-treated control groups and expressed as group mean ± S.E.M., n ≥ 4.

Figure 3

The age-related increase in Aβ_1–42 accumulation and plaque formation was accompanied with the decrease in IDE protein expression in the hippocampus of 3xTg-AD female mice. (A) A total of 20 hippocampal sections collected at 210 μM intervals from one hemisphere of 3, 6, 9, 12 and 18-month-old 3xTg-AD female mice were stained with the Campbell-Switzer silver stain, which labels diffuse plaques in black and a more mature form of plaques in amber. (Left) Montage images revealed the temporal-spatial pattern of plaque formation that began and was concentrated in the subicular region in 12-month-old AD mice, which was significantly increased in 18-month-old AD mice. (Right) Upper panel: 10x views of plaque formation in the subicular region; Lower panel: 40x close-up views of a mature form of plaques stained in amber observed in 18-month-old AD mice. (B) ELISA data revealed an age-related increase in Aβ_1–42 accumulation in the hippocampus of 3xTg-AD female mice; ** P < 0.01. (C) Western blot data revealed an age-related overall reduction in IDE protein expression in the hippocampus of 3xTg-AD female mice. Data are presented as the percent of IDE expression in 3-month-old AD mice; ^# P < 0.05 and ^## P < 0.01 compared to 18-month-old mice; * P < 0.05 compared to 12-month-old mice; ** P < 0.01 compared to 3, 6, and 9-month-old mice; (D) Immunohistochemical staining revealed changes in immunoreactivity for Aβ (green) and IDE (red) that are consistent with observations from ELISA and Western blot analyses. β-tubulin was used as the loading control. Data are expressed as group mean ± S.E.M., n ≥ 4.

Figure 4

17β-E2 attenuated Aβ_1–42 accumulation and plaque formation, which was accompanied with an increase in IDE protein expression, in the hippocampus of 12-month-old 3xTg-AD female OVX mice. (A) A total of 20 hippocampal sections collected at 210 μM intervals were stained with the Campbell-Switzer silver stain. The staining revealed a significant reduction in plaque formation in OVX mice treated with 17β-E2 compared to vehicle alone-treated OVX mice. (B) A significantly lower percentage of sections was affected by plaques in OVX mice treated with 17β-E2 compared to vehicle alone-treated OVX mice; * P < 0.05. (C) A significantly smaller area in the subicular region (defined by box 1) was occupied by plaques in OVX mice treated with 17β-E2 compared to vehicle alone-treated OVX mice; * P < 0.05. (D) ELISA data revealed a significant reduction in Aβ_1–42 accumulation in the hippocampus of OVX mice treated with 17β-E2 compared to vehicle alone-treated OVX mice; * P < 0.05. (E) Western blot data revealed a significant increase in IDE protein expression in the hippocampus of OVX mice treated with 17β-E2 compared to vehicle alone-treated OVX mice; ** P < 0.01. (F) Consistent with ELISA and Western blot data, immunohistochemical staining revealed a lower intensity of Aβ immunoreactivity (green) and a higher intensity of IDE immunoreactivity (red) in hippocampal sections of OVX mice treated with 17β-E2 than showed in sections of vehicle alone-treated OVX mice. β-tubulin was used as the loading control. Data are expressed as group mean ± S.E.M., n ≥ 4.