A. Cardounel, W. Regelson, M. Kalimi
Nov 1, 1999
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Influential Citations
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Experimental Biology and Medicine
Abstract
Dehydroeplandrosterone (DHEA), an adrenal cortex hormone secreted in large quantities In humans, protects cells of the clonal mouse hippocampal cell line HT-22against the excitatory amino acid glutamate (5 mM), and amyloid (3-proteln (2 ~M) toxicity In a dose-dependent manner with optimum protection obtained at 5 ~M concentration of DHEA. The protective effects of DHEA appear to be specific In that other related steroids and metabolites of DHEA, such as 5-androstene-3(3,17(3-dlol, etlocholan-3a-ol-17-one, etiocholan-3(3-01-17-one, testosterone, and 5a-androstane3,17-dlone, offered no protection even at 50 ~M concentrations. In addition, using Immunocytochemical techniques, we observed that 20 hr of treatment with 5 mM glutamate remarkably increased glucocorticoid receptor (GR) nuclear localization In neuronal cells. Interestingly, 5 ~M DHEAtreatment for 24 hr, followed by 5 mM glutamate treatment for 20 hr almost completely reversed the copious nuclear localization of GR observed by glutamate treatment alone. Results obtained suggest that DHEA protects hippocampal neurons, at least in part, by its antlglucocorticoid action via decreasing hippocampal cells nuclear GR levels. [P.S.E.B.M. 1999, Vol 222] Received December 4. 1998. [P.S.E.B.M. 1999. Vol 222) Accepted June 7. 1999. 0037·9727/99/2222-0145$14.00/0 Copyright © 1999 by the Society for Experimental Biology and Medicine This work was supported in part by a grant from the Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust. 1 To whom requests for reprints should be addressed at Department of Physiology. MCVNCU. Richmond. VA 23298"'{)55I. E-mail: MKalimi@hsc.vcu.edu 2 Part of this study was presented at the Experimental Biology '98 meeting held in April 1998 in San Francisco. California DehYdroePiandrosterone (DHEA) is an adrenal steroid secreted in large quantities in humans (30 mg! daily) and also synthesized in the human brain (1). For many years, the role of DHEA has focused on its place as an intermediate in sex steroid synthesis. More recently, DHEA has been shown to possess anticancer, antidiabetogenic, antiobesity and antiaging properties (2, 3). In addition, the antiglucocorticoid effects of DHEA have been observed by many investigators (3, 4). However, the precise physiological role and the mechanism of action of this hormone remains largely unknown. It is proposed that excitatory amino acids such as glutamate, as well as amyloid l3-protein, are neurotoxic to cultured cells through their effects on antioxidant systems and a reduction in intracellular glutathione levels, leading to intracellular accumulation of peroxides and ultimately death (5). Interestingly, several groups have recently shown that estrogen protects neurons against glutamate and amyloid l3-protein toxicity in vitro (6-8). These findings have generated considerable excitement in the scientific community and have raised suggestions that estrogen may not only be beneficial for the treatment of Alzheimer's disease but may also provide benefit to normal age-related memory loss (9). Since estrogen has been shown to have neuroprotective effects, in the present investigation we asked the question whether a neurosteroid, DHEA-like estrogen could provide protection against glutamate and amyloid l3-protein-induced neurotoxicity. Since glucocorticoids are known to enhance oxidative stress-induced neuronal cell death (10), we have tested the hypothesis that DHEA's protective effects are mediated at least in part, through the modulation of glucocorticoid receptor. Materials and Methods Cell Culture and Chemicals. The cell line HT-22 is a subclone of the HT4 hippocampal cell line. HT-22 cells MECHANISMOF NEUROPROTECTIVE ACTION OF DEHYDROEPIANDROSTERONE 145 50..1------------------' 100 -r-----------------, (ANOVA) and by Student's t test (two-tailed). A P value less than 0.05 was considered significant. B ---.,. l..-_--~A 90 60 ~ 80 1'5 o s at 70 Results First, we determined the optimum dose of glutamate, or amyloid /3-protein required for 40%-50% of HT-20 cell death as assessed by MIT assay. Data presented in Figure 1 showed that 5 mM glutamate, or 2 ILMamyloid ~-protein was needed to obtain approximately 40% neuronal death of HT-22 cells. Therefore, these concentrations of neurotoxins were used for subsequent experiments. Figure 2 represents the dose-response curve of DHEA Figure 1. Glutamate or amyloid 13-protein-induced neurotoxicity using HT-22 cells. Cells were treated with (A) 5 mM glutamate or (8) 2 IJM amyloid 13-protein for 20 hr. Cell viability was determined using MTT assays as described in Methods. Percentage viability is expressed as 100% of control. Results presented are average of at least three experiments each done in triplicate and expressed as the mean ± SEM. 'Significantly different from control levels (P < 0.05). 50nM O.5mM 5 mM 10mM Figure 2. Dose response curve. HT-22 cells were treated with increasing concentrations of DHEA for 24 hr followed by 5 mM glutamate (A) or 2 IJM amyloid 13-protein (8) for 20 hr. Cell viability was determined using MTT assays as described in Methods. Percentage viability is expressed as 100% of control. Results presented are average of at least three experiments each done in triplicate and expressed as the mean ± SEM. were a gift from Dr. David Schubert (The Salk Institute, San Diego California). The cell were cultured in DMEM supplemented with 10% FCS at 37°C, 10% CO2, Amyloid ~25-35' monosodium glutamate, and all steroids were purchased from Sigma Chemical Co. (St. Louis, MO). All media, serum, and supplements were purchased from Gibco BRL (Grand Island, NY). All other chemicals used were of analytical grade. Cell Survival Assays. Neuronal cell death was estimated using (i) microscopic examination of cells using phase contrast microscopy to evaluate morphological changes, and (ii) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT) assay. The MIT assays were performed in 96-well microtiter plates containing 100 ILl of media per well. Following experimental treatments, 10 ILl of a 5-mg/ml MIT stock in PBS was added to each well, and the incubation was continued for 4 hr. Finally, 100 ILl of a solubilization solution containing 50% dimethylformamide, 20% SDS (pH 4.8) was added. The following day, absorption values at 570 nm were determined with an automatic microtiter reader. Phase contrast microscopy and cell counting were performed to assess morphological changes. All assays were performed in triplicate at least three times each. Hormone and Toxin Treatment. Briefly, HT-22 cells (3000 cells/well) were plated in 96-well plates, and left untreated overnight. The medium was removed, and DMEM with 2% fetal calf serum was added. Hormones were added, and incubation proceeded for a period of 24 hr, followed by addition of toxin. After an additional 20 hr, cell viability was assessed by one of the previously described methods. Stock solutions of steroids (10-) were prepared in ethanol with a final ethanol concentration of 0.1%. Glutamate, and amyloid ~-protein solutions were prepared in PBS. Immunocytochemistry. Cells were plated on cover slips and treated with various experimental protocols. Slides were washed three times in PBS and fixed for 10 min at room temperature with 2% paraformaldehyde. The cells were washed three times in PBS and the permeabilized in PBS, containing 0.1% saponin and 0.25% gelatin, for 30 min and washed three times in PBS. The cells were then incubated for 30 min at room temperature in 0.2% normal goat serum followed by overnight incubation at 4°C with GR antibody diluted (1:200) in PBS. Cells were then treated for 30 min with biotinylated goat anti-rabbit IgG at a dilution of 1:200. Avidin-biotin peroxidase (1:200) was added, incubated for 30 min, and then treated for 10 min with diamino benzidine-hydrogen peroxide solution. Various controls, such as using nonspecific purified mouse IgM and IgG antibodies andpreabsorbing GR antibody with partially purified GR receptor and preimmune rabbit serum (l :50 dilution), were used to assure glucocorticoid receptor specificity. Statistical Analysis. Data were expressed as mean ± SEM. Data were analyzed by one-way analysis of variance 146 MECHANISM OF NEUROPROTECTIVE ACTION OF DEHYDROEPIANDROSTERONE Figure 3. Steroid Specificity. HT-22 cells were treated with 5 ~M of indicated steroids for 24 hr followed by 5 mM glutamate for 20 hr. Cell viability was determined by MIT assays and expressed as 100% of control. (A) control, (8) DHEA, (C) 5-androstene 3[3,17[3diol, (D) eticchotan-ao-ol-tz-one, (E) etocnotan-aa-ot-tz-one. (F) testosterone, (G) 5cx-androstane-3,17-dione, and (H) 17[3 estradiol. Results are average of three experiments done each time in triplicates and presented as the mean ± SEM. 'Significantly different from control levels (P < 0.05). protection against neuronal death induced by glutamate, or amyloid r3-protein. DHEA protects HT-22 cells against the various neurotoxins tested in a dose-dependent manner, and optimum protection was observed at 5 fLM concentration of DHEA (Fig. 2). Since DHEA is effective against both neurotoxins using HT-22 cells, we have used glutamate toxicity using HT-22 cells in all the subsequent experiments for simplicity. Data presented in Figure 3 demonstrates the specificity of this observed effect. Several DHEA analogs were probed for their structure/function relationship in protecting against glutamate-induced neuronal death. Interestingly, 5 fLM of testosterone significantly induced cell death as compared to control, untreated cells. All other androgen-related steroids or DHEA metabolites, such as 5-androstene-3r3,17r3-diol; etiocholan-3a-ol-17-one; eriocholan-Sjs-ol-l?-one; and Soandrostane-3,17-dione, at 5 f-LM concentrations were without any neuroprotective effect. Following the glutamate challenge, phase contrast microscopy revealed significant changes in the general cellular morphology of hippocampal neurons evidenced