Dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) regulate apoptosis during neurogenesis by triggering the Akt signaling pathway in opposing ways

Molecular Brain Research 98 (2002) 58–66 www.elsevier.com / locate / bres

Research report

Dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) regulate apoptosis during neurogenesis by triggering the Akt signaling pathway in opposing ways Lei Zhang a , *, Bing shen Li c , Wu Ma d , Jeffery L. Barker b , Yoong H. Chang b , Weiqin Zhao c , David R. Rubinow a a Behavioral Endocrinology Branch, National Institute of Mental Health, Building 36, Room 2 C02, Bethesda, MD 20892, USA Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda MD 20892, USA c Laboratory of Adaptive Systems, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA d Center for Bio /Molecular Science and Engineering, Naval Research Laboratory, Bethesda, MD 20892, USA b

Accepted 30 October 2001

Abstract Dehydroepiandrosterone (DHEA) can function to protect neural precursors and their progeny targeted with toxic insults; however, the molecular mechanisms underlying the neuroprotective effects of DHEA are not understood. We cultured neural precursors from the embryonic forebrain of rats and examined the effects of DHEA and its sulfated derivative (DHEAS) on the activation of the serine-threonine protein kinase Akt, which is widely implicated in cell survival signaling. We found that DHEA activated Akt in neural precursor culture, in association with a decrease in apoptosis. In contrast, DHEAS decreased activated Akt levels and increased apoptosis. The effects of DHEA on neural cell survival and activation of Akt were not blocked by the steroid hormone antagonists flutamide and tamoxifen, but both were blocked by a PI3-K inhibitor, LY294002. These findings suggest that during neurogenesis in the developing cortex, DHEA and DHEAS regulate the survival of neural precursors and progeny through the Akt signaling pathway.  2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Hormones and development Keywords: DHEA; DHEAS; Neural precursor, Akt; Kinase; Apoptosis

1. Introduction While DHEA and DHEAS represent the most abundant products of the adrenal cortex, they have, until recently, been regarded as relatively inert precursors of the sex steroids, estradiol and testosterone. However, following the work of Beaulieu et al., DHEA and DHEAS have been identified as both neurosteroids (i.e., synthesized de novo in the central nervous system [CNS] and independent, at least in part, of peripheral organ activity) and neuroactive steroids (i.e., steroid hormones possessing acute, nongenomic actions on membrane receptors) [5]. Several other *Corresponding author. Tel.: 11-301-402-1398; fax: 11-301-4021565. E-mail address: [email protected] (L. Zhang).

lines of evidence suggest the relevance of DHEA and DHEAS for neuronal function. In clinical studies, a number of neurobehavioral problems including mental disorders (e.g. depression), sleep disturbances and decreased feeling of well-being have been associated with lower levels of DHEA [24,28] and have been treated successfully with DHEA supplementation [6,24,28]. In several types of in vitro studies, DHEA and DHEAS exert neuroprotective effects. Firstly, DHEA and DHEAS enhance survival rates of dissociated embryonic mouse neurons in culture under well-established experimental conditions [7]. Secondly, DHEAS prevents neuronal death induced by inclusion of glutamate in the culture medium [18]. Thirdly, DHEA protects hippocampal cells from oxidative stress-induced damage [4]. Finally, DHEA antagonizes the neurotoxic effects of corticosterone and

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translocation of stress-activated protein kinase 3 in primary cultures of hippocampal neurons [15]. Potential mechanisms for the neuroprotective effects are suggested by the observation that DHEA stimulates specific signal transduction pathways involved in cell survival [18]. For example, DHEA-induced neuroprotection is consistent with its ability to elevate an NF-kappaB-dependent transcription factor activity associated with protection of hippocampal neurons from glutamate cytotoxicity [18]. The serine-threonine protein kinase Akt (also called protein kinase B) is implicated in survival signaling in a wide variety of cells including fibroblasts, epithelial cells, and neurons [16]. For example, insulin-like growth factor I regulates cell growth through an Akt-dependent survival signaling pathway [1,16]; growth arrest-specific gene 6 (Gas6 ) / adhesion related kinase (Ark)-induced extracellular signal-regulated kinase (ERK) and Akt signaling enhance gonadotropin-releasing hormone-induced neuronal survival [2]; and cytokine IL-3 induced-cell survival also is mediated by Akt activation [26]. These findings prompted the following questions: firstly, could the neuroprotective effects of DHEA be mediated through the Akt signaling pathway; secondly, could Akt play a role in the survival of neural precursors and their progeny as it does in mature well-differentiated neurons [14,16]. Finally, while DHEA and DHEAS are interconversion products (DHEAS serving as the major source (60–80%) of DHEA and DHEA serving as a minor source (5–7%) of DHEAS), their actions appear to differ in several systems. For example, DHEAS is a much more potent allosteric inhibitor of the GABAA receptor [21] than DHEA. Additionally, Compagnone and Mellon [9] demonstrated in primary cultures of mouse embryonic neocortical neurons that DHEA (but not DHEAS) increased the length of neurites containing the axonal marker Tau-1, while DHEAS (but not DHEA) increased the length of dendritic labeled (microtubuleassociated protein-2) neurites. Therefore, we asked, in addition, whether the observed effects of DHEA on survival are altered by sulfation at the C3 position (DHEAS) as occurs with some of its neuromodulatory effects. Here, we show that DHEA stimulates, while DHEAS inhibits, survival of neural precursors and progeny and that these opposite effects are associated with those on the Akt signaling pathway.

2. Materials and methods

2.1. Cell culture Timed pregnant Sprague–Dawley rats (Taconic Farms, Germantown, NY) were anesthetized with sodium pentobarbital (40 mg / kg body weight, ip). The pups were removed from the dams and placed into Hank’s buffered saline solution (HBSS). Embryonic day 1 (E1) was defined as the day of conception, established by the presence of a

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vaginal plug. The crown-rump length was also measured to confirm the embryonic age. The telencephalic neuroepithelium was dissected from rats of embryonic days 13–13.5. Dissected areas corresponded to the formative dorsal telencephalon according to the atlas of the prenatal rat brain by Altman and Bayer [3]. Tissue was dissociated by brief mechanical trituration in HBSS. The cells were collected by centrifugation and resuspended in a serumfree medium containing MEM / N3, glutamine and 10 ng / ml of recombinant human basic FGF (fibroblast growth factor) (Intergen, Purchase, NY). Cells (1310 6 ) were plated in 35 mm plastic dishes precoated with poly-Llysine (10 mM) and 1 mg / ml of human recombinant fibronectin (GIBCO, Rockville, MD). Basic FGF was added daily, and the medium was changed every 2 days until day 6. Then, the cell cultures [19] were maintained in serum- and bFGF-free medium and incubated for 72 h with three concentrations (1, 10 and 100 nM) of DHEA or DHEAS (Sigma, St. Louis, MO), which were dissolved in DMSO (0.2%). As described elsewhere [19] and below, most of the cells were TuJ1 (neuronal marker) negative at day 6 and were TuJ1 positive at day 9. Control cultures were treated with equal concentrations of DMSO only (see cell death assay). To evaluate the possible role of DHEA metabolites in the observed effects of DHEA, tamoxifen (100 nM, an estrogen receptor blocker) (Sigma, St. Louis, MO) or flutamide (100 nM, an androgen receptor blocker) (Sigma, St. Louis, MO) was added 1 h prior to treatment with 100 nM DHEA. LY294002 (10 mM, (Sigma, St. Louis, MO) a phosphotidylinositol 3-kinase [PI3-K] inhibitor) (Upstate Biotechnology, Lake Placid, NY) was also added 1 h prior to treatment to determine whether the effects of DHEA on apoptosis required activation of the PI3-K /Akt signaling pathway.

2.2. Western blot assay Cell lysates were analyzed by Western blot for Akt levels. Cells were washed with ice-cold PBS, harvested by scraping from dishes, lysed in ice-cold high salt RAB buffer (containing 0.1 M HEPES, 0.5 mM MgSO 4 , 1 mM EDTA, 2 mM dithiothreitol, and 0.75 M NaCl, pH 6.8, supplemented with 0.1% Triton X-100 and a mixture of protease inhibitors) by passing through a 21-gauge needle several times, and incubated for 30 min on ice. Lysates were then clarified at 14 000 g for 10 min. Protein concentration in the supernatant was determined by the Bio-Rad Protein Concentration Reagent (Hercules, CA). Equal amounts of total protein (20 mg protein per lane) were resolved on S.D.S. / 10% polyacrylamide gels and blotted onto PVDF membranes for immunoblotting analysis with phospho-Akt antibodies. Due to the importance of Ser-473 for Akt activation and regulation [1], we used an antibody (1:1000, Sigma, St. Louis, MO) that specifically recognizes Akt only when that residue becomes phosphorylated. We also used an antibody that recognizes total

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(unactivated) Akt (New England Biolabs, Beverly, MA). After identifying the optimal dose of DHEA for stimulating the activation of (phosphorylated) Akt, this dose (100 nM) was employed in a time course study (10 min to 1 h) to determine whether the effect occurred with a latency compatible with activation of a signal transduction pathway. Western blots were examined by using the Amersham ECL kit following the manufacturer’s instructions (Amersham, Chicago, IL). The mean optical density (average gray value in the pixels) was measured with NIH Imaging 1.60 software. The mean densities of DHEA- or DHEAStreated cultures were divided by the mean density of control cultures, yielding a relative density. The relative density is presented as the mean6S.D. from three experiments. Statistical significance was determined by Student’s t-test, with an alpha of 0.05.

2.3. Akt activity assay The physiological significance of alterations in phosphoAkt levels induced by DHEA was examined by determining the ability of phospho-Akt immunoprecipitates to phosphorylate histone H2B (H2B), a substrate for activated Akt. For kinase activity assays, neural cells were grown in 35-mm dishes for 6 days with bFGF (10 ng / ml) then deprived of bFGF and treated for 72 h with 100 nM DHEA or DHEAS. To determine whether DHEA-induced increases in Akt occur through activation of the Akt pathway, LY294002 (10 mM) was also added into some dishes 1 h before DHEA or DHEAS. After treatment, cells were scraped into 0.1 ml of lysis buffer (10 mM Tris–HCl, pH 7.5, 1% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl, protease and phosphatase inhibitors). Extracts were then sonicated and centrifuged for 5 min at 14 0003 g. Immunoprecipitations were performed by adding the phosphorylation-dependent Akt antibody to cell lysates and incubating overnight at 4 8C with constant rotation. Protein G-Sepharose beads, 30 ml of 50% slurry (Amersham Pharmacia Biotech, Piscataway, NJ), were then added and incubated under the same conditions for an additional 4 h. Immunocomplexes were centrifuged for 2 min at 14 0003 g and washed twice in lysis buffer, twice in kinase buffer (25 mM Tris–HCl, pH 7.5, 5 mM b-glycerol phosphate, 0.1 mM sodium orthovanadate, 2 mM dithiothreitol, and 10 mM magnesium chloride), and matched for protein content before being used in each specific kinase reaction. Immunoprecipitates were used in an immunocomplex kinase assay in the presence of H2B and [g 32 P]ATP. Phosphorylated H2B was separated on an 8–16% S.D.S. polyacrylamide gel. The graph represents the mean6S.D. of three independent experiments.

and absence of DHEA (10–100 nM). Apoptosis was determined as follows: Neural cells were grown in 35 mm dishes for 6 days in the presence of bFGF and then for 3 days in the absence of bFGF with addition of either DHEA (100 nM) or DHEA (100 nM) plus LY294002 (10 mM). (Pretreatment with LY294002 was performed to determine the role of Akt in DHEA-induced neuroprotection.) Fragmented DNA was then measured in cultures from the three conditions: DHEA, DHEA1LY294002, or control (no supplement). Fragmented DNA was visualized by the terminal deoxynucleotidytransferase-mediated dUTP nick end labeling (TUNEL) procedure using an in situ cell death detection kit from Boehringer Manheim (Scotts Valley, CA) following the manufacturer’s instructions. The labeled nuclei and total number of cells were counted in three independent fields for each dish by a blinded observer. Three dishes per condition were selected and experiments were performed three times for each treatment. The data are presented as mean6S.D. Statistical significance was determined by Student’s t-test, with an alpha of 0.05. Results of the TUNEL procedure were confirmed with the fluorescence dye propidium iodide (PI) (Sigma Aldrich, Deisenhofen, Germany) used according to the manufacturer’s instructions. The extent of cell death was estimated by assessing the extent of neuronal uptake of PI. Briefly, the culture medium was replaced with a phenol red-free, HEPES-buffered Hanks-based medium, pH 7.4, containing PI (4.5 mg / ml). After a one-step wash, PI uptake into dead cells of each well was automatically quantified (excitation5485 nm; emission wavelength at 640 nm) using a fluorescence plate reader. Three dishes per-condition were selected and experiments were conducted three times for each treatment. Values are expressed as a percentage of the control and are the mean6S.D. of three independent experiments.

2.5. Hoechst staining of apoptotic nuclei To further confirm the effects of DHEA and DHEAS on cell death, the A-T base-pair-specific dye, Hoechst 33258 (bis-benzimide; Sigma), was used to stain cell nuclei under identical conditions as above. Following overnight fixation in 4% paraformaldehyde at 4 8C, cells grown on German glass were washed three times with PBS and labeled with 1 mg / ml of the DNA dye Hoechst 33258 in PBS for 5–10 min at room temperature, using enough solution to cover the cells completely. The cells were rinsed twice with PBS and then mounted with Crystal-mount medium (Biomeda, Foster City, CA). Cells were observed and photographed on a phase contrast and fluorescence microscope with a UV2A filter.

2.4. Cell death assay

2.6. Double-immunostaining for BrdU incorporation and neuron-specific b -tubulin ( TuJ1)

To determine whether DHEA influenced cell death, the percentage of apoptotic cells was measured in the presence

In order to identify actively proliferating cells and differentiating neurons, cells cultured for 6 and 9 days

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were characterized by double-immunostaining for BrdU incorporation and neuron-specific b-tubulin, identified by the monoclonal antibody TuJ1 [17,20]. A total of 10 mmol BrdU was included for the last 6 h prior to fixation with 70% ethanol. Cells incorporating BrdU were identified using the FITC-conjugated mouse class IgG 1 anti-BrdU antibody (Bectin Dickinson, Mountain View, CA). Differentiating neurons were revealed using a monoclonal mouse class IgG 2a anti-TuJ1 antibody [17,20]. Secondary antibody was rhodamine-conjugated donkey anti-mouse IgG 2a (Southern Biotechnology Associates, Birmingham, AL). Finally, studies of Akt and apoptosis were repeated under identical conditions (see above) with substitution of identical doses of DHEAS (Sigma, St. Louis, MO) for DHEA.

3. Results Consistent with others [19], our cultures consisted of three populations of cells: TuJ1 1 / BrdU 2 , TuJ1 2 / BrdU 1 and TuJ1 1 / BrdU 1 , which were 5, 75 and 10%, respectively, at day 6 and 54, 32 and 12%, at day 9. Western blots of phosphorylated Akt antibody – and control (phosphorylation-independent) antibody – labeled proteins are shown in the upper and lower panels of Fig. 1A. DHEA doses of 50 nM (lane three) and 100 nM (lane

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four) resulted in a significant increase (160 and 180%, respectively) in activation of Akt compared with controls (lane one) and cells exposed to 10 nM DHEA (lane two). (200 nM DHEA significantly increased [172%] activation of Akt compared with controls, but to a lesser degree than 100 nM DHEA). The total amount of control Akt detected with phosphorylation-independent Akt antibody was constant across samples at all concentrations of DHEA and at all post-treatment times, indicating that the increase in phosphorylation of Akt could not be attributed to either an increase in total Akt or to the amount of protein loaded into each column. The time course of expression of phosphorylated Akt following DHEA (100 nM) treatment is shown in Fig. 1B. Akt activation was already detectably increased at 10 min and reached a plateau at 30 min. Immunocytochemistry showed that Akt was phosphorylated by DHEA (100 nM) in both BrdU 1 and TuJ1 1 cells (data not shown). Because the Akt pathway is thought to promote cell survival [2,16,26], it is possible that the DHEA-induced increase in phosphorylated Akt could result in enhanced survival of precursor and differentiated neurons. Therefore, we examined apoptosis in the cultures by using TUNEL procedures. DHEA at both 50 and 100 nM significantly decreased the percentage of apoptotic cells (Fig. 2A–C) measured with TUNEL. The extent of cell death was also estimated by assessing the extent of neuronal uptake of propidium iodide. Treatment with 50–100 nM DHEA

Fig. 1. DHEA increases Akt phosphorylation in neural precursor cell culture. (A) Cell cultures were incubated with increasing concentrations of DHEA from 0 to 100 nM, assayed by Western blot using Akt phosphoserine 473-specific antibody (upper panel), and probed with control (phosphorylationindependent) Akt antibody (lower panel). Treatment of neural cells with 50–100 nM DHEA for 72 h produces significant increases in the relative density of phosphorylated Akt compared to control values (bottom panel). (B) Time course of DHEA effects on Akt activation. At 30 min and 1 h, the phosphorylation of Akt is significantly increased by treatment with DHEA (100 nM). Quantified results in the bottom panels of A and B are the means6S.D. from three independent experiments. *,0.05 and ***P,0.001.

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Fig. 2. The effect of DHEA on apoptosis in cultured neural precursors examined by TUNEL (A–C) and PI (D) procedures. Cultured neural cells were treated with vehicle (A) and 100 nM DHEA (B) for 72 h. The arrows indicate the apoptotic cells. A and B show a decrease in the number of apoptotic cells in the DHEA-treated group (B) compared with vehicle-treated groups (A). The percentages of apoptotic cells in each treated group shown in C are the means6S.D. from three independent experiments examined by TUNEL procedure. Similar results were obtained by PI assay (D). Treatment with DHEA 50–100 nM decreased neural cell death, compared with controls. The percentages of controls are the means6S.D. from three independent experiments. *,0.05 and ***P,0.001.

decreased neural cell death by about 30–50%, compared with controls (Fig. 2D). These findings were confirmed by Hoechst staining (Fig. 3). To identify the role of Akt activation in DHEA-enhanced neural cell survival, Akt activation was blocked with a PI3-K inhibitor. Pretreatment with the PI3-K inhibitor LY294002 significantly decreased the DHEAinduced activation of Akt (Fig. 4A) and blocked the inhibitory effect of DHEA on apoptosis (Fig. 4B). LY294002 decreased DHEA-induced phosphorylated Akt by .50% and more than doubled the number of apoptotic cells. This latter effect occurred whether or not the cells were exposed to DHEA. Thus, the PI3-K inhibitor in-

Fig. 3. Effects of DHEA on apoptosis detected by Hoechst 33258 staining. (A) Control culture. (B) and (C) Cells were treated with 100 nM DHEA (B) and 100 nM DHEAS (C). The apoptotic cells possess irregularly shaped nuclei with condensed chromatin, which is brightly stained with Hoechst dye (arrows). Neural cell death was decreased by DHEA and increased by DHEAS compared with controls.

creased the spontaneous accumulation of neural cells undergoing apoptosis. This implies that the PI3-K-pathway is constitutively active in these cells. Pretreatment with tamoxifen or flutamide was without effect on DHEAinduced Akt increase. To examine the effect of DHEAS, studies of Akt and apoptosis were repeated under identical conditions (see above) with substitution of identical doses of DHEAS for DHEA. In contrast to DHEA, both 50 and 100 nM DHEAS significantly decreased phospho Akt after only 10 min, with maximal decreases seen by 60 min (Fig. 5A,B). (Increasing the dose of DHEAS to 200 nM did not result in further decreases in Akt.) The decreased Akt activity was associated with significant increases in the percentage of apoptotic cells measured by TUNEL (Fig. 6A) and PI (Fig. 6B), again in contrast to effects seen with DHEA. Finally, we also examined Akt activation with kinase assays, in which cells were treated with vehicle, DHEA (100 nM), DHEA (100 nM) and LY294002 (10 mM), or DHEAS (100 nM). Consistent with data from Western

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Fig. 4. Opposite effects of DHEA and LY294002 on Akt activation (A) and apoptosis (B) in cultured neural precursors and their progeny, and lack of effects of flutamide or tamoxifen (C). (A) Akt protein levels were determined by Western blot as described in the text. The Western blot shown was derived from a typical experiment (top panel). Lane 1, vehicle; Lane 2, 100 nM DHEA; Lane 3, LY294002 (10 mM)1DHEA (100 nM); Lane 4, LY294002 (15 mM)1DHEA (100 nM); Lane 5, LY294002 (15 mM) alone. Cultured neural cells were treated for 72 h. Quantified results (relative density) are shown in the lower panel. (B) The effect of an inhibitor of Akt activation (LY294002) on apoptosis. The addition of LY294002 (10 mM) reverses the decrease in apoptosis seen with DHEA, and LY294002 alone significantly increases the percentage of apoptotic cells seen relative to baseline. (C) Lack of effect of steroid receptor blockade [flutamide (Flu), tamoxifen (Tam)] on DHEA-induced changes in Akt. Pretreatment with tamoxifen (100 nM) or flutamide (100 nM) does not affect DHEA-induced increase of Akt. Neither flutamide nor tamoxifen alone affects the levels of Akt. Data shown are the means6S.D. from three independent experiments. ***P,0.001.

blots, DHEA and DHEAS increased and decreased, respectively, the kinase activity of Akt as measured by phosphorylation of histone H2B (Fig. 7). The stimulatory effects of DHEA on Akt-dependent phosphorylation of histone H2B were blocked by LY294002.

4. Discussion The molecular mechanisms underlying the neuroprotective role of DHEA have not been elucidated. Here, we have investigated the effects of DHEA during neurogenesis using a model neuroepithelium composed primarily of proliferating neural precursors and post-mitotic neurons (described elsewhere) [19]. We demonstrated the ability of DHEA to increase the activation of Akt (between 160 and

180% of control values) in these neural cells. This effect occurred relatively rapidly (within 30 min), suggesting involvement of a ‘non-genomic’ mechanism, and was accompanied by a decrease in the percentage of apoptotic cells. Taken together, the results suggest that DHEA may exert a neuroprotective effect during neurogenesis via an Akt-induced inhibition of cell apoptosis. The observation that both the DHEA-induced activation of Akt and reduction in apoptosis were reversed by pretreatment with an inhibitor of Akt activation (PI3-K) demonstrates the relevance of this signal transduction pathway in the effects of DHEA on cell survival. The discovery that the specific PI3-K inhibitor promoted apoptosis in the absence of DHEA indicates that the Akt pathway is constitutively active. Further, the lack of effect of pretreatment with androgen or estrogen receptor antagonists indicates that

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Fig. 5. DHEAS decreases Akt phosphorylation in neural precursors and their progeny. (A) Neural cells were incubated with increasing concentrations of DHEAS from 0 to 100 nM for 72 h, assayed by Western blot using Akt phosphoserine 473-specific antibody (upper panel), and probed with control (phosphorylation-independent) Akt antibody (lower panel). DHEAS significantly decreases the activation of Akt. Treatment of neural cells with 50–100 nM DHEAS produces significant decreases in the relative density of Akt compared to control (bottom panel). (B) Time course of DHEAS effects on Akt activation. At 10 min, 30 min and 1 h, the activation of Akt is significantly decreased by treatment with DHEAS (100 nM). Quantified results in the bottom panels of A and B are the means6S.D. from three independent experiments. *,0.05, **,0.01 and ***P,0.001.

DHEA-mediated Akt signaling does not involve these steroid receptors. A number of studies support the conclusion that DHEA-triggered Akt signaling underlies its antiapoptotic activity: (1) treatment of neurons with nerve growth factor (NGF) activates endogenous Akt protein kinase and promotes cell survival [10]; (2) expression of constitutively active Akt in neurons efficiently prevents death after NGF withdrawal [10]; (3) decreased Akt kinase activity is associated with C2-ceramide-induced apoptosis in HMN1 motor neurons [29]; (4) growth factor activation of the PI3-K /Akt signaling pathway culminates in the phosphorylation of the Bcl-2 family member Bad (Bcl 2 / Bcl-XL-associated death promoter), thereby suppressing apoptosis and promoting cell survival [11]; (5) activated Akt increases survival of differentiating neuronal (hippocampal H19-7) cells [13]. It should be noted that while the effects of LY294002 and DHEA on apoptosis were balanced, the effects of LY294002 on Akt overwhelmed those of DHEA. This suggests that the effects of DHEA on neural cell survival may be mediated through several pathways, similar to the multiple pathways observed to mediate the neuroprotective effects of estradiol [22]. Further supportive of a pivotal role for Akt activation in the anti-apoptotic effects of DHEA are the polar opposite effects of DHEAS; i.e., increased apoptosis in association with decreased phosphorylated Akt. While the opposite effects of DHEA and DHEAS may appear counter-intuitive, particularly since DHEAS functions as a stable reservoir for potential conversion to DHEA in the periphery, recent electrophysiological data demonstrate that substitution of a negatively charged moiety at the C3

position of certain neurosteroids reverses the ‘polarity’ of neuromodulation from positive to negative (e.g., at the GABA receptor) or negative to positive (e.g., at the NMDA receptor) [21]. In this regard, DHEAS (but not DHEA) binds to the picrotoxin site of the GABAA receptor [27] and is a much more powerful inhibitor of GABAA receptor chloride ion channel activity than DHEA [21]. Of note, our data differ from those of two other studies showing that both DHEA and DHEAS promote neuronal survival (but not proliferation) in embryonic mouse brain [7] and protect hippocampal neurons against excitatory amino acid-induced neurotoxicity in embryonic (day 18) rat brain. These differences in the effects of DHEAS may reflect differences in cell type (differentiated neurons vs. proliferating cells), species (mouse vs. rat), brain regions (hippocampus vs. forebrain), or procedure (amino acid-induced neurotoxicity vs. starvation). Our data add to existing knowledge regarding cell survival in the brain during development. Firstly, these are the first data demonstrating a role of the Akt signaling pathway in the survival of neural precursor and differentiated neurons, suggesting a larger role both for DHEA and Akt in the developmental organization of the brain than previously demonstrated. Secondly, while clearly speculative, DHEA may function like a growth factor in promoting the survival of cells from embryonic forebrain, since, like estradiol and progesterone [25], it displays cross talk with growth factor signaling pathways. While DHEA has previously been shown to affect neurons directly through modulation of GABA or excitatory aminoacid receptors or indirectly through its metabolism to androgens or estrogen,

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Fig. 7. Kinase activity parallels effects of DHEA and DHEAS on Akt phosphorylation. DHEA alone for 72 h significantly increases kinase activity. In contrast, 100 nM DHEAS significantly decreases kinase activity, and DHEA (100 nM)-induced kinase activity is significantly inhibited by addition of the specific PI3-K inhibitor, LY2940002 (10 mM). **P,0.01, ***P,0.001; (data are from three independent experiments).

Fig. 6. The pro-apoptotic effects of DHEAS on cultured neural precursors and their progeny. In cultured cells, DHEAS (50 and 100 nM for 72 h) induces a significant increase in the percentage of apoptotic cells examined by TUNEL procedures. Similar results were obtained by PI assay (B). *P,0.05, **P,0.01; (data are from three independent experiments).

our data provide an additional means, i.e., regulation of cell survival, by which DHEA may influence neuronal function. Finally, it remains to be determined whether signaling pathways other than the Akt cascade are involved in the cell survival-promoting effects of DHEA, as well as whether the effects involve transcription-dependent (e.g., increased transcription of Bcl-2) or transcription-independent (e.g., phosphorylation and inactivation of Bad) mechanisms, as have been shown by Bonni et al. [8] to be involved in MAPK regulation of cell survival. This study describes for the first time the ability of DHEA to activate, and DHEAS to inhibit, a signaling pathway (Akt) associated with survival of neural precursors and progeny in culture. These data are consistent with previous results showing that Akt is a critical mediator of growth factor-induced neuronal survival [12]. One possible mechanism for DHEA-induced neuroprotection is that, similar to several growth factors, DHEA activation of the Akt signaling pathway culminates in the phosphorylation of the Bcl-2 family member Bad, thereby

suppressing apoptosis and promoting cell survival [8]. Alternatively, Akt may promote survival through crosstalk with other signaling pathways, as has been shown in muscle cells in which Akt promotes survival by inhibiting activation of the Raf-MEK-ERK pathway [23]. Our data suggest that in the developing CNS, the balance of DHEA and DHEAS may help to determine neural precursor cell development through the Akt signaling pathway.

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