Estrogen and androgen protection of human neurons against intracellular amyloid beta1-42 toxicity through heat shock protein 70

Abstract

Intracellular amyloidbeta peptide (iAbeta1-42) accumulates in the Alzheimer's disease brain before plaque and tangle formation (Gouras et al., 2000) and is extremely toxic to human neurons (Zhang et al., 2002). Here, we investigated whether androgen and estrogen could prevent iAbeta1-4) toxicity, because both these hormones have a wide range of neuroprotective actions. At physiological concentrations, 17-beta-estradiol, testosterone, and methyl testosterone reduce iAbeta1-42-induced cell death by 50% in neurons treated after the injection and by 80-90% in neurons treated 1 hr before the injection. The neuroprotective action of the hormones is mediated by receptors, because the estrogen receptor (ER) antagonist tamoxifen and the androgen receptor (AR) antagonist flutamide completely block the estrogen- and androgen-mediated neuroprotection, respectively. Transcriptional activity is required for the neuroprotective action, because dominant negative forms of the receptors that block the transcriptional activity of the ER and AR prevent estrogen- and androgen-mediated neuroprotection. Proteomics followed by Western blot analyses identified increased levels of heat shock protein 70 (Hsp70) in testosterone- and estrogen-treated human neurons. Comicroinjection of Hsp70 with the iAbeta1-42 blocks the toxicity of iAbeta1-42. We conclude that estrogen and androgens protect human neurons against iAbeta1-42 toxicity by increasing the levels of Hsp70 in the neurons.

Figure 1.

Estrogen and androgens protect human neurons against intracellular Aβ_1-42 toxicity. A, B, TUNEL-positive cell death in human neurons in primary cultures injected with 10 nm Aβ_1-42 and incubated with 2, 4, and 10 nm 17-β-estradiol (17βE₂), 17-α-estradiol (17αE₂), BSA-17-β-estradiol (BSA-βE₂), testosterone (Test), epitestosterone (Epitest), or methyl testosterone (Methyl-test). Data represent the mean ± SEM of three independent experiments. ^*p < 0.002 compared with Aβ_1-42. C, TUNEL-positive cell death in human neurons preincubated with 10 nm estrogen or androgens for 1 hr before microinjections of iAβ_1-42 and further incubated with hormones for 24 hr after the injection. Data represent the mean ± SEM of three independent experiments. ^*p < 0.001 compared with iAβ_1-42 alone. D, Immunofluorescence micrographs of neurons injected with Aβ_1-42 and DTR and either left untreated (control) or treated with 2 and 4 nm 17βE₂ or testosterone (Test), respectively. The panels show the DTR-injected neurons stained with the DNA stain 4′,6′-diamidino-2-phenylindole (DAPI) or the cell death marker TUNEL. Ctl, Control.

Figure 2.

Estradiol and testosterone protect against iAβ_1-42 through their respective receptors. TUNEL-positive cell death in human neurons in primary cultures injected with Aβ_1-42 and incubated with TMX and Flut in the absence or presence of 10 nm 17-β-estradiol (17βE₂) or testosterone (Test) is shown. Data represent the mean ± SEM of three independent experiments. ^*p < 0.001 compared with the respective hormonal treatment. Ctl, Control.

Figure 3.

Estrogen requires transcriptional activation to protect against Aβ_1-42. A, Estrogen transcriptional activity assay using a luciferase reporter system in MCF-7 cells transfected with wild-type (WTER) or mutant (ΔDBDER) ER and treated without (control) or with 10 nm 17-β-estradiol (17βE₂) for 24 hr. Data represent the mean ± SEM of three independent experiments. ^*p < 0.001 compared with control values. B, TUNEL-positive cell death in primary neurons microinjected with iAβ_1-42 and WT-ER or ΔDBD-ER and treated without (control) or with 17βE₂. Data represent the mean ± SEM of three independent experiments. ^*p < 0.02 compared with vector-injected cells. Ctl, Control; RLU, relative light units.

Figure 4.

Testosterone requires transcriptional activation to protect against Aβ_1-42. A, Luciferase assay for androgen responsive element activity in AR24 cells cotransfected with the AR mutants 15579 and 12474 or empty pcDNA3 vector and treated with 10 nm testosterone for 24 hr. Data represent the mean ± SEM of three independent experiments. ^*p < 0.01 compared with control. B, TUNEL-positive cell death in neurons comicroinjected with Aβ_1-42 and wild-type AR (AR), 12474 AR, or 15279 AR and treated with 10 nm testosterone, methyl testosterone, or epitestosterone. ^*p < 0.001 compared with DTR-microinjected cells. Ctl, Control.

Figure 5.

Hsp70 is increased in estrogen- and testosterone-treated neurons and protects against iAβ_1-42. A, Western blot analysis of Hsp70 andβ-actin in 30 μg protein extracts from estrogen- or testosterone-treated neurons. B, TUNEL-positive neuronal cell death in neurons comicroinjected with iAβ_1-42 and recombinant Hsp70 or control BSA protein compared with testosterone treatment. Ctl, Control.