IGF-I signaling prevents dehydroepiandrosterone (DHEA)-induced apoptosis in hypothalamic neurons

Molecular and Cellular Endocrinology 214 (2004) 127–135

IGF-I signaling prevents dehydroepiandrosterone (DHEA)-induced apoptosis in hypothalamic neurons Shuo-Yen J. Lin a,1 , Hong Cui a,1 , Bernardo Yusta b,c , Denise D. Belsham a,b,d,∗ a

Department of Physiology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ont., Canada M5S 1A8 Division of Cellular and Molecular Biology, Toronto General Research Institute, University Health Network, Toronto, Ont., Canada M5S 1A8 Toronto General Hospital Research Institute, The Banting and Best Research Centre, University Health Network, Toronto, Ont., Canada M5S 1A8 d Departments of Obstetrics & Gyneacology and Medicine, University of Toronto, Toronto, Ont., Canada M5S 1A8 b

c

Received 3 September 2003; accepted 27 October 2003

Abstract Dehydroepiandrosterone (DHEA) is synthesized in the brain, but whether DHEA is involved in modulating neuronal cell survival is not yet fully understood. Herein we show that when deprived of trophic support, GT1-7 hypothalamic neurons undergo apoptosis following exposure to DHEA, as demonstrated both by morphological and biochemical criteria. This proapoptotic effect appeared to be specific to DHEA itself, and not through conversion of DHEA to other steroids such as androgen or estrogen. Importantly, we determined that IGF-I protects GT1-7 neurons from DHEA-induced cell death. DHEA-induced apoptosis was associated with increased activation of caspase 3 and decreased PARP, which were both attenuated with addition of IGF-I. Addition of DHEA prevented phosphorylation of both Akt and glycogen synthase kinase-3 beta (GSK-3␤), downstream effector molecules of the phosphatidylinositol 3-kinase (PI3K) pathway. Further IGF-I was able to sustain Akt activity and thus preventing GSK-3␤ activation in the presence of DHEA. On the other hand, the MAP kinases, ERK, p38, and JNK, were not affected by DHEA. These findings suggest that in GT1-7 hypothalamic neurons, DHEA acts detrimentally to induce cell death and IGF-I is able to rescue the neurons by preserving the activity of Akt, and therefore maintaining the proapoptotic kinase GSK-3␤, in a phosphorylated catalytically inactive state. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: GT1-7 neurons; Dehydroepiandrosterone; Apoptosis; Insulin-like growth factor I; Akt; Glycogen synthase kinase 3

1. Introduction The mechanisms underlying neuronal cell death have been the focus of active research for the past decade. Various pathological conditions of the brain, most notably neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease, have all been linked to neuronal cell death (reviewed in Yuan and Yankner, 2000). Programmed cell death has also been associated to aging in the brain. During reproductive aging, the function of gonadotropin-releasing Abbreviations: GnRH, gonadotropin-releasing hormone; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; E2, 17␤-estradiol; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; DAPI, 4,6diamidin-2-phenylindole; IGF-I, insulin-like growth factor I; GSK-3␤, glycogen synthase kinase-3beta; PARP, poly(ADP-ribose) polymerase ∗ Corresponding author. Tel.: +1-416-946-7646; fax: +1-416-978-4940. E-mail address: [email protected] (D.D. Belsham). 1 Equal contributing authors.

hormone (GnRH) neurons becomes compromised. The exact mechanism involved in this process is not yet understood, but changes in afferent drive to the GnRH neuron, hormone levels, and neurotransmitter responses have all been suggested (Rubin, 2000; Gore, 2001; Le et al., 2001). Interestingly, a decrease in GnRH neuron number during aging has been documented (Miller et al., 1990; Funabashi and Kimura, 1995). However, it is currently not known whether increased GnRH neuronal cell death contributes to reproductive aging, and only a few studies have approached the loss of GnRH neuronal function from this perspective. Previous studies have involved several intracellular signaling cascades in the regulation of neuronal apoptosis. Activation of the extracellular signal-regulated kinase (ERK) pathway has been shown to promote growth and survival in PC12 cells (Foncea et al., 1997). On the contrary, activation of other MAP kinases, namely the JNK and p38 MAP kinases, have been associated with induction of neuronal cell death (Xia et al., 1995; Kummer et al., 1997). Other studies

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have also shown the importance of phosphatidylinositol 3-kinase (PI3K)/Akt pathway in mediating protection from apoptosis. A role for PI3K in the regulation of cell death was first indicated by experiments showing that activation of this kinase by nerve growth factor (NGF) was required to prevent apoptosis of PC12 cells (Yao and Cooper, 1995). This observation has been later extended to other immortalized cell lines, particularly those dependent on insulin-like growth factor-I (IGF-I) for survival (Kulik et al., 1997; Kauffmann-Zeh et al., 1997; Kennedy et al., 1997; Parrizas et al., 1997). More recently, studies have also shown the importance of Akt (also known as PKB), a key downstream effector of PI3K, in mediating cell survival (Crowder and Freeman, 1998; Dudek et al., 1997; Franke et al., 1997). Akt activation prevents apoptosis by means of its ability to phosphorylate and inactivate several pro-apoptotic molecules, including Bad (Datta et al., 1997), forkhead transcription factors (Brunet et al., 1999), caspase 9 (Cardone et al., 1998) and glycogen synthase kinase-3 (GSK-3) (Cross et al., 1995). Akt is also able to phosphorylate and activate a number of pro-survival factors, such as cyclic AMP response element binding protein (CREB) and IkappaB kinase (IKK). Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS), the most abundant adrenal steroids in the blood, are present in all regions of the human brain at concentrations above those in the plasma (Majewska, 1995), and are synthesized de novo and metabolized by brain neurons and astrocytes (Zwain and Yen, 1999a,b). The high rates of production and metabolism of DHEA by hypothalamic astrocytes have led us to investigate the effects of this hormone in the regulation of hypothalamic neuronal function, particularly GnRH neurons. Using the well-characterized GT1-7 cells as a model for GnRH neurons, we recently reported that DHEA represses GnRH gene expression via regulation of the GnRH gene 5 flanking region (Cui et al., 2003). In the current study, we used the same model system to address the question of whether DHEA has any effects on GnRH neuron survival. Previous studies on the effect of DHEA in neuronal viability have been controversial; using different experimental paradigms, DHEA has been shown to be neuroprotective (Aragno et al., 2000; Bastianetto et al., 1999; Compagnone and Mellon, 1998; Li et al., 2001; Karishma and Herbert, 2002; Zhang et al., 2002) and neurotoxic (Gil-ad et al., 2001; Yang et al., 2000). We report here that DHEA exhibits neurotoxic effects in the absence of trophic support and induces hypothalamic neuronal apoptosis. Co-treatment with IGF-I, and the consequent activation of the prosurvival kinase Akt, however, was able to attenuate DHEA-induced cell death.

2. Materials and methods 2.1. Cell culture and reagents GT1-7 cells were grown in monolayer in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corpo-

ration, Burlington, Ont.) supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT), 4.5 mg/ml glucose and penicillin/streptomycin and maintained at 37 ◦ C in an atmosphere of 5% CO2 as previously described (Mellon et al., 1990). DHEA, 17␤-estradiol (E2), dihydrotestosterone (DHT), recombinant insulin-like growth factor (IGF-I), 4,6-diamidin-2-phenylindole (DAPI), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), protease inhibitor cocktail, and phosphatase inhibitor cocktail were all purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ont.). Steroid stock solutions were prepared in absolute ethanol. The final ethanol concentration in the treatments was 20 nM. 2.2. Cell viability assay Cells were exposed to either DHEA or vehicle in the presence or absence of IGF-I following an 8 h period in phenol-red free DMEM with either 5% charcoal-stripped FBS or no serum. At the indicated time points, the number of viable cells in each condition was assessed by monitoring the bioreduction of 3-[4,5-Dimethylthiazol-2-y l]-2,5-diphenyltetrazolium bromide (MTT) into blue formazan. Cultures were incubated with MTT (0.3 mg/ml) in phenol-red free DMEM for 1 h at 37 ◦ C. The insoluble formazan was then solubilized with dimethyl sulfoxide and quantified spectrophotometrically at 540 nm. 2.3. Visualization of nuclear morphology and chromatin condensation GT1-7 neurons were fixed with 2% paraformaldehyde, followed by staining with 1 ␮g/ml DAPI in PBS for 1 min. The cells were then washed once with PBS to remove excess DAPI, mounted with 50% glycerol, and viewed under an Olympus BX60 System fluorescence microscope. Images were recorded using a CoolSNAP CCD video camera (Photometrics GmbH, Munich) and CoolSNAP software. 2.4. Analysis of genomic DNA fragmentation by agarose gel electrophoresis For the detection of genomic DNA fragmentation associated with apoptosis, GT1-7 neurons were lysed in 10 mM Tris–HCl (pH 7.5), 1 mM EDTA and 0.2% Triton X-100. The lysate was centrifuged at 12,000 × g for 10 min, and the resulting supernatant was treated with proteinase K (100 ␮g/ml; Invitrogen Corporation, Burlington, Ont.) at 37 ◦ C for 16 h and then with RNase A (5 ␮g/ml; Qiagen Inc., Mississauga, Ont.) for 1 h. Following phenol/chloroform (1:1 v/v) extraction, DNA was precipitated with 2.5 volumes of absolute ethanol in the presence of 5 M NaCl. After centrifugation, DNA pellets were washed with 70% ethanol, air dried, and subsequently dissolved in 10 mM Tris–HCl (pH 7.5), 1 mM EDTA. DNA samples were size

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fractionated by electrophoresis in 1.6% agarose gel, and visualized by UV light after ethidium bromide staining. 2.5. SDS–Polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis Cells were washed with ice-cold PBS and lysed in high-salt buffer (0.4 M NaCl, 20 mM HEPES (pH 8.0), 1 mM MgCl2 , 0.5 mM EDTA, 0.5 mM DTT, 1 mM PMSF, 0.1% NP-40, 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail) for 40 min on ice. Lysates were cleared at 12,000 × g for 20 min at 4 ◦ C and then treated for 4 min at 96 ◦ C with SDS–PAGE sample buffer containing ␤-mercaptoethanol. Protein concentration was determined by the BCA protein assay kit (Pierce Biotechnology, Rockford, IL). 40 ␮g of lysate protein were resolved on SDS–PAGE gels and blotted onto Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech, Baie d’Urfe, PQ). The resulting blot was blocked with 5% skim milk in PBS containing 0.2% Tween 20, and incubated with primary antibody overnight at 4 C. Proteins were detected with a secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence (ECL) kit from Amersham Pharmacia Biotech. Anti-Akt (1:1000), anti-phosho-Akt (Ser473 ) (1:1000), anti-phospho-GSK-3␤ (Ser9 ) (1:1000) and anti-active caspase-3 p17 subunit (1:1000) antibodies were purchased from Cell Signaling Technology (Beverly, MA). The anti-poly(ADP-ribose) polymerase (PARP; 1:2000) antibody was from BD Pharmingen Canada (Mississauga, Ont.). Polyclonal antibodies raised against ERK1/2 (1:500), phospho-ERK1/2 (Tyr204 ) (1:500), p38 MAPK (1:200), phospho-p38 (Tyr182 ) MAPK (1:200), JNK (1:500), and phospho-JNK(Thr183 /Tyr185 ) (1:500) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Loading and transfer conditions were monitored by reprobing the blots with an anti-G␤ (1:1000) antibody from Santa Cruz Biotechnology. Densitometry was performed on blots exposed to Kodak X-OMAT film using a UMAX Astra 1220 Scanner and the NIH Image software. 2.6. Statistical analysis Comparisons of results between treatments over different times, were done using either one- or two-way analysis of variance (ANOVA), as appropriate. The statistical significance of the results between individual pairs was determined using the Tukey’s multiple comparison test. Data were analyzed using the statistical program package SPSS 10.0 for Windows (University of Toronto site license).

3. Results Depletion of trophic factors through serum deprivation is a standard procedure to enhance cell susceptibility to proapoptotic stimuli. In order to determine the effect of DHEA, if

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any, on cell survival, serum-starved, quiescent, GT1-7 neurons were exposed to increasing concentrations of DHEA (10, 50 and 100 ␮M) over a 48 h timecourse. Cell viability was then assessed by a standard tetrazolium salt bioreduction assay. GT1-7 neurons are remarkably resilient to serum deprivation and cell number remains constant for at least 48 h under these conditions (Fig. 1A). In contrast, the addition of DHEA to serum-deprived GT1-7 neurons significantly increased cell loss at 48 h (Fig. 1A). Since we detect a significant amount of cell death at 48 h with serum deprivation and 100 ␮M DHEA, this experimental paradigm was chosen for the remainder of the analysis. As was previously reported, DHEA did not affect cell viability when GT1-7 neurons were maintained in the presence of serum (Cui et al., 2003; Fig. 1A). Analysis of nuclear morphology in GT1-7 neurons treated for 48 h with DHEA revealed a significant number of condensed and fragmented cell nuclei, features typically associated with cells undergoing apoptosis (Fig. 1B). Consistent with the changes in nuclear morphology, analysis of genomic DNA integrity revealed the DNA fragmentation (laddering) pattern characteristic of apoptosis in the neurons treated with DHEA (Fig. 1C). These results indicate that DHEA induces apoptosis when GT1-7 neurons are deprived of trophic support. The physiological actions of DHEA have largely been attributed to the metabolism of DHEA to other steroidal compounds, such as androgen and estrogen (Labrie et al., 2001). We have previously established that conversion of DHEA to estrogen is unlikely, as the GT1-7 neurons do not express aromatase (Cui et al., 2003), and do not exhibit aromatase activity (Poletti et al., 1994). We have also reported that in GT1-7 neurons DHEA is not converted into either androgens, testosterone nor DHT (Cui et al., 2003). However, in order to exclude that conversion of DHEA to androgen or estrogen may be involved in the induction of cell death by DHEA in the serum-starved conditions, we treated the cells with E2 or DHT for 48 h. As no changes in cell viability were detected using the MTT assay in the GT1-7 neurons exposed to either estradiol or DHT (Fig. 2), our results suggest that DHEA itself and not its primary metabolites account for its proapoptotic activity in GT1-7 neurons. Activation of growth factor receptor signaling coupled to kinase cascades represents an established paradigm for controlling cell growth and apoptosis in a number of model systems, including GT1-7 neurons (Sortino and Canonico, 1996). The GT1-7 cells have been shown to contain functional receptors for a number of growth factors, including IGF-I (Olson et al., 1995; Ochoa et al., 1997; Longo et al., 1998; Anderson et al., 1999). In order to determine if IGF-I was able to modulate DHEA-induced GT1-7 apoptosis, cells were exposed for 48 h to DHEA in the absence or presence of 100 ng/ml IGF-I. In agreement with a previous report (Olson et al., 1995), IGF-I alone increased cell number in vehicle-treated serum starved GT1-7 neurons but, more

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Fig. 2. The DHEA metabolites 17␤-estradiol (E2) and dihydrotestosterone (DHT) do not promote cell death in GT1-7 neurons. GT1-7 neurons were incubated in serum-free medium with DHEA (100 ␮M), E2 (10 nM, 10 ␮M), or DHT (10 nM, 10 ␮M) for 48 h. Cell viability was assessed as described in Fig. 1 and expressed as a percentage of the corresponding vehicle-treated neurons at 48 h. Data are mean ± S.E. from three independent experiments, each performed in triplicate, ∗ P < 0.05.

Fig. 1. DHEA induces apoptosis in serum-deprived GT1-7 neurons. (A) Following an 8 h serum deprivation period (t = 0), GT1-7 neurons were grown in medium with (S) or without (SS) serum, in the absence or presence of DHEA (10, 50 or 100 ␮M) over a 48 h timecourse. Cell viability was assessed at each timepoint using a tetrazolium salt bioreduction assay. Data are expressed as a percentage of control at t = 0 and are the mean ± S.E. from three independent experiments, each performed in triplicate, ∗ P < 0.05 (SS/vehicle vs. SS/DHEA). (B) Serum-deprived GT1-7 neurons were incubated with 100 ␮M DHEA or vehicle alone for 48 h. Nuclear morphology was examined by fluorescence microscopy following DAPI staining. Images shown correspond to a 400× magnification. Healthy nuclei show pale blue fluorescence whereas apoptotic nuclei exhibit an intense, bright blue fluorescence due to chromatin condensation (online version of the figure is in colour). (C) Agarose gel electrophoresis of GT1-7 cell genomic DNA extracted following 48 h incubation with 100 ␮M DHEA or vehicle alone in serum-free medium. In both (B) and (C), “control” correspond to GT1-7 neurons cultured for 8 h in serum-free medium prior to any treatment.

remarkably, it fully prevented DHEA-induced decrease in MTT bioreduction (Fig. 3A). To define whether the effect in cell viability we detect with IGF-I in DHEA exposed cells was due to increased

Fig. 3. IGF-I protects GT1-7 neurons from DHEA-induced cell death. (A) GT1-7 neurons were incubated in serum-free medium with either 100 ␮M of DHEA or vehicle alone in the presence or absence of 100 ng/ml IGF-I for 48 h. Cell viability was assessed as described in Fig. 1 and expressed as a percentage of corresponding vehicle-treated neurons at 48 h. Data are mean ± S.E. from three independent experiments, each performed in triplicate, ∗ P < 0.05. (B) Following the same treatment scheme as in panel A, cell nuclei were visualized by fluorescence microscopy after DAPI staining (online version of the figure is in colour). Magnification: 400×.

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proliferation or an actual IGF-I-mediated rescue from DHEA-induced cell death, we assessed nuclear morphology with the treatment paradigms. As was shown before in cells exposed to DHEA, a significant number of nuclei exhibited features consistent with apoptosis, including chromatin condensation, as well as nuclear fragmentation (Fig. 3B). A 48 h serum starvation induced a slight increase in pycnotic nuclei compared to the 0 h control, as expected. Remarkably, nuclear morphology and chromatin condensation in the cells exposed to both DHEA and IGF-I were indistinguishable from those of serum-deprived cells exposed to vehicle alone (Fig. 3B). These studies suggest that inhibition of apoptosis accounts for the ability of IGF-I to prevent DHEA-induced loss of cell viability in GT1-7 neurons. Further support for our proposal that IGF-I is able to prevent DHEA-induced apoptosis was obtained when the activation of caspase-3, a key executioner caspase, was monitored following 24 and 48 h of DHEA treatment in the presence or absence of IGF-I. Whereas DHEA substantially increased the appearance of the active caspase-3 p17 subunit, as detected by Western blot analysis (Fig. 4A), cotreatment of the cells with both DHEA and IGF-I attenuated the increase in caspase 3 p17 subunit. This result was evident at 24 h but substantially more pronounced at the 48 h treatment. PARP is one of the best characterized downstream targets of activated executioner caspases. In agreement with the increase in active caspase-3 p17 subunit, DHEA treatment resulted in the virtual disappearance

Fig. 4. IGF-I attenuates DHEA-induced caspase 3 activation and PARP cleavage in GT1-7 neurons. GT1-7 neurons were treated for 24 and 48 h either in 5% serum supplemented medium or in serum-free medium with 100 ␮M DHEA or vehicle alone in the presence or absence of 100 ng/ml IGF-I, as indicated. Cell extracts were analyzed by Western blot using antibodies raised against (A) the active p17 subunit of caspase 3 and (B) intact PARP. Equal loading was verified by reprobing the blots with an anti-G␤ antibody. Results are representative of three independent experiments.

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of the intact PARP protein band at 48 h (Fig. 4B). Furthermore, PARP cleavage was clearly decreased to the levels of serum deprivation alone when IGF-I was present during the DHEA treatment. Similarly, the presence of 5% serum abrogated any signs of DHEA-induced apoptosis (Fig. 4A and B). These results confirm that indeed DHEA triggers apoptosis in GT1-7 neurons through a mechanism that can be inhibited by activation of IGF-I signaling. Akt, also referred to as PKB, is a pivotal component of the PI3K pathway that is known to be critically involved in regulating cell survival (Crowder and Freeman, 1998; Vincent and Feldman, 2002; Eves et al., 1998). As PI3K-dependent phosphorylation at Thr303 and Ser473 is necessary and sufficient for the activation of Akt, we used phosphorylation site-specific antibodies to monitor possible changes in catalytically active Akt in GT1-7 neurons in response to DHEA. No change in Akt phosphorylation was noticeable after 24 h of DHEA treatment, however, a reduction in the level of phospho-Akt relative to vehicle-alone

Fig. 5. IGF-I prevents DHEA-induced decreases in total Akt and subsequently GSK-3␤ and stimulates Akt (Ser473 ) phosphorylation in serum-deprived GT1-7 neurons. GT1-7 neurons were treated for 24 and 48 h with either 100 ␮M DHEA or vehicle alone in the presence or absence of 100 ng/ml IGF-I in serum-free medium, or with 100 ␮M DHEA in medium containing 5% serum. (A) Cell extracts were analyzed by Western blot analysis using an Akt phospho-Ser473 -specific antibody, a phosphorylation state-independent Akt antibody, or a phospho-GSK-3␤ (Ser9 ) antibody, as indicated. Equal loading was verified by reprobing the blot with an anti-G␤ antibody. Illustrated Western blots are representative of four independent experiments. (B) The phospho-Akt to total Akt ratio at the 48 h timepoint was calculated following densitometry and normalized to the ratio for vehicle-treated SS control. Results are mean ± S.E.M. (n = 3), ∗ P < 0.01, DHEA/IGF-I (SS) vs. DHEA alone (SS).

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treated neurons was apparent following 48 h of DHEA (Fig. 5A). Similarly, total Akt levels were decreased 48 h following addition of DHEA, consistent with the fact that Akt is, as happens with many other signaling molecules, degraded by executioner caspases during apoptosis. When the ratio of phospho-Akt/Akt was calculated no difference was detected in serum-starved cells exposed to either vehicle or DHEA, suggesting that the decrease in phospho-Akt in DHEA-treated neurons is secondary to the loss of total Akt protein and not to DHEA-mediated regulation of Akt activity (Fig. 5B). Both IGF-I and 5% serum prevented DHEA-induced decrease in total Akt and furthermore stimulated Akt Ser473 phosphorylation, an observation in agreement with their ability to overcome the proapoptotic activity of DHEA. To determine the mechanisms underlying the reduced cell survival in DHEA-treated neurons, we examined the phosphorylation state of GSK3, a downstream target of the PI3K/Akt antiapoptotic signaling pathway whose activity suppresses proliferation and induces cell death. In mammalian cells, activation of Akt has been demonstrated to phosphorylate GSK3␤ at Ser9 resulting in inhibition of GSK3 kinase activity and promotion of survival. Changes in the levels of catalitically inactive GSK3␤ phosphorylated at Ser9 mirrored those of Akt Ser473 phosphorylation (Fig. 5A): strong activation of GSK3␤ in cells exposed to

Fig. 6. DHEA treatment does not affect ERK1/2, p38, or JNK phosphorylation in GT1-7 neurons. GT1-7 neurons were treated over a 12 h timecourse in serum-free medium with 100 ␮M DHEA or vehicle alone in the presence or absence of 100 ng/ml IGF-I, as indicated. Cell extracts were analyzed by Western blot using antibodies raised against (A) phospho-ERK1/2 (Tyr204 ) and ERK1/2, (B) phospho-p38 (Tyr182 and p38, and (C) phospho-JNK (Thr183 /Tyr185 ) and JNK. Illustrated Western blots are representative of three independent experiments.

DHEA alone and conversely, increased levels of inactive GSK3␤, when either IGF-I or 5% serum were present with the DHEA. These results suggest that IGF-I might enhance cell survival in the presence of DHEA via Akt activation and subsequently phosphorylation and inactivation of GSK3␤. Because mitogen-activated protein (MAP) kinase signaling pathways have also been linked to regulation of cell death or survival in many cell types, we examined whether the activity of MAP kinases was modified by treatment with DHEA. We used phosphorylation site-specific antibodies, as the activity of these kinases in vivo closely correlates with the phosphorylation state of specific amino acid residues. Studies have previously shown the importance of the extracellular signal-regulated kinase (ERK1/2) pathway in protection from cell death (Xia et al., 1995). Exposure of the GT1-7 neurons to DHEA over a 12 h timecourse did not induce any changes in phospho-Tyr204 ERK1/2 levels (Fig. 6A). Similarly, levels of phospho-Tyr182 p38 and phospho-Thr183 /Tyr185 JNK, both considered proapoptotic MAP kinases, were not changed by DHEA treatment in the GT1-7 neurons (Fig. 6B and C). These results exclude any role of MAP kinases in DHEA induction of cell death as well as their involvement in IGF-I-mediated protection.

4. Discussion Although DHEA and DHEAS are abundant steroids produced by the human adrenal (reviewed in Kroboth et al., 1999), these steroids are also present in relatively high concentrations in the brain of many mammalian species (Baulieu and Robel, 1996), and have recently been found to be synthesized de novo in neurons and astrocytes (Zwain and Yen, 1999a,b). However, despite links to several physiological processes, receptor candidates for these steroids are still speculative, and their function has not yet been clearly delineated (Kroboth et al., 1999; Baulieu and Robel, 1996). Previous studies have suggested both neuroprotective (Aragno et al., 2000; Bastianetto et al., 1999; Compagnone and Mellon, 1998; Li et al., 2001; Karishma and Herbert, 2002; Zhang et al., 2002) as well as neurotoxic (Gil-ad et al., 2001) effects of DHEA. The disparity in the effect of DHEA may reflect differences in cell models (differentiated versus proliferating cells); brain regions (hippocampus versus forebrain); or experimental paradigm (oxidative stress-induced versus serum starvation-induced cell death). To complicate matters even further, the observed effects of DHEA may be indirect as previous studies of DHEA on neuronal viability were performed in mixed primary cultures. This concern was validated by recent reports suggesting that DHEA can be metabolized to other steroidal products by brain neurons and glial cells (Zwain and Yen, 1999a,b). To address the question of whether DHEA has any direct effects on hypothalamic neuronal viability, we used a model of the GnRH neuron, the GT1-7 cell line, developed through targeted tumorigenesis of the murine GnRH neurons via SV-40 T-antigen (Mellon

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et al., 1990). By having a homogenous cell population, we were able to determine any direct effects of DHEA at the level of the GnRH neuron without the confounding glia or afferent neurons present in a mixed culture system. The GT1-7 cell line has previously been used to study cell death caused by oxidative stress (Sortino and Canonico, 1996; Kane et al., 1993). Further, the GnRH-secreting immortalized cell lines, GT1-7 and Gn11, have also been used to delineate the mechanisms involved in programmed cell death during neuronal migration early in development (Allen et al., 1999). In this study, serum starvation was also used as the proapoptotic stimulus and GT1-7 cells were found to be comparably resistant to cell death following removal of serum. Interestingly, this paper suggests that both ERK and PI3K, via Akt, are involved in the protection of migrating GnRH neurons from apoptosis (Allen et al., 1999). In the current study, we found that when deprived of trophic support, DHEA is able to specifically activate pro-apoptotic pathways in the GT1-7 neurons. This is substantiated by the observation of both a significant number of condensed and fragmented cell nuclei typically associated with cells undergoing apoptosis, and the appearance of internucleosomal DNA fragmentation (laddering pattern) following DHEA exposure. When the GT1-7 neurons are treated with DHEA, a critical executioner caspase, caspase-3, was activated. In agreement with the increase in active caspase-3 p17 subunit, DHEA treatment resulted in the virtual disappearance of the intact PARP protein band, one of the best characterized downstream targets of activated executioner caspases. Activation of IGF-I signaling is sufficient to prevent all the morphological (chromatin condensation and nuclear fragmentation) and biochemical changes (caspase 3 activation, internucleosomal DNA fragmentation, and PARP degradation) associated to DHEA-induced apoptosis and in consequence also prevents DHEA-induced loss of cell viability in serum-deprived GT1-7 neurons. While the concentration of DHEA used in this study is in the pharmacological range, such concentrations have been used by other studies (Bastianetto et al., 1999; Yang et al., 2000), and may not be unusually high considering the rates of DHEA production by hypothalamic neurons and astrocytes, postulated to be as high as the micromolar range (Zwain and Yen, 1999a). Furthermore, when 5% serum was present, cell growth was unaffected by 100 ␮M DHEA, suggesting that DHEA is not neurotoxic at this concentration. Even more convincing is the fact that when used at a concentration of 100 ␮M in hippocampal cells undergoing apoptosis due to oxidative stress, DHEA was found to have neuroprotective properties, indicating that DHEA itself in high concentrations is not inherently toxic (Bastianetto et al., 1999). We examined the effect of DHEA on the PI3K/Akt pathway, as well as on the main MAP kinase cascades (ERK, p38, and JNK). DHEA may be able to induce cell death by inhibiting the activation of pro-survival pathways (PI3K/Akt or ERK) or by activating the pro-apoptotic pathways (p38

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and JNK). While no significant changes in phosphorylation status were observed for the three MAP kinases examined, Akt phosphorylation levels were found to be significantly reduced by DHEA treatment. Although this seems to result from the apoptosis-induced disappearance of total Akt protein, the almost complete loss of catalytically active Akt in DHEA-treated cells resulted in a strong activation of the Akt target GSK3. Although little is known about how GSK-3␤ is able to achieve its effects on programmed cell death, as no further components downstream have been definitively confirmed, it has been determined that activation of GSK-3␤ is sufficient for cellular apoptosis (Pap and Cooper, 1998). GSK-3␤ is a multifunctional enzyme and some speculation has been put forth as to how it achieves its apoptotic effects at the molecular level (Grimes and Jope, 2001). Through its phosphorylation ability, GSK-3␤ downregulates the activities of several transcription factors, including several that are known to promote cell survival, such as NF␬B, AP-1, ␤-catenin, myc, and CREB (Grimes and Jope, 2001). Further studies on the effect of DHEA on these cellular components is underway in order to delineate the downstream components of DHEA-enhanced cell death in GT1-7 neurons. We do not yet have a clear picture of the proximal events involved in the DHEA-mediated induction of apoptosis in GT1-7 neurons, but it seems clear from our study that loss of active Akt is instrumental in the triggering process, as preventing such a loss with IGF-I is enough to inhibit the cell death program initiated by DHEA. IGF-I signaling has been shown to be involved in the regulation of GnRH gene expression and secretion (Longo et al., 1998; Anderson et al., 1999; Zhen et al., 1997). Importantly, IGF-I has also been previously demonstrated to be involved in the protection of GT1-7 cells from oxidative stress-induced cell death (Sortino and Canonico, 1996; Heck et al., 1999), and was found to be associated with activation of NF␬B specifically through the PI3K pathway (Heck et al., 1999). As mentioned above, GSK-3␤ is also known to decrease the activity of NF␬B, therefore since IGF-I is able to override the DHEA-induced activation of GSK-3␤, this transcription factor may be a good candidate as one of the downstream effector molecules of IGF-I signaling in the GT1-7 neurons. We speculate that under conditions of stress, such as nutrient deprivation, the GT1-7 GnRH neurons may be more susceptible to cell death and that the IGF-I signaling pathway allows these neurons to tolerate potential apoptotic insults, such as the high levels of DHEA synthesized in the brain. It is established that changing gonadal hormone levels during aging in the rodent contribute to GnRH neuronal senescence (Rubin, 2000; Gore, 2001), therefore these levels may be influenced by the overall synthesis of DHEA in the brain as well. IGF-I has also been implicated as a key regulator of reproductive function, as IGF-I knockout mice are infertile (Baker et al., 1996). Interestingly, GnRH neurons themselves are immunopositive for IGF-I and it has been found that hypothalamic IGF-I levels decrease during reproductive aging (Miller and Gore, 2001). Since we propose that increased

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apoptosis may contribute to loss of GnRH neuronal function with aging, these findings may advance to our current understanding of how reproductive aging is manifested.

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