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The Journal of Neuroscience, February 7, 2007, 27(6):1422-1433; doi:10.1523/JNEUROSCI.2382-06.2007

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Cellular/Molecular
Estrogen Regulates Bcl-w and Bim Expression: Role in Protection against ß-Amyloid Peptide-Induced Neuronal Death

Mingzhong Yao, Thuy-Vi V. Nguyen, and Christian J. Pike

Davis School of Gerontology, University of Southern California, Los Angeles, California 90089

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Estrogen is neuroprotective against a variety of insults, including ß-amyloid peptide (Aß); however, the underlying mechanism(s) is not fully understood. Here, we report that 17ß-estradiol (E2) selectively regulates neuronal expression of the Bcl-2 family (bcl-2, bcl-x, bcl-w, bax, bak, bad, bik, bnip3, bid, and bim). In primary cerebrocortical neuron cultures under basal conditions, we observe that E2 upregulates expression of antiapoptotic Bcl-w and downregulates expression of proapoptotic Bim in an estrogen receptor (ER)-dependent manner. In the presence of toxic levels of Aß, we observe that E2 attenuates indices of neuronal apoptosis: c-Jun N-terminal kinase (JNK)-dependent downregulation of Bcl-w and upregulation of Bim, mitochondrial release of cytochrome c and Smac, and cell death. These neuroprotective effects of E2 against Aß-induced apoptosis are mimicked by the JNK inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). In addition, E2 attenuates Aß-induced JNK phosphorylation in an ER-dependent manner, but does not affect basal levels of JNK phosphorylation. These results suggest that E2 may reduce Aß-induced neuronal apoptosis at least in part by two complementary pathways: (1) ER-dependent, JNK-independent upregulation of Bcl-w and downregulation of Bim under basal conditions, and (2) ER-dependent inhibition of Aß-induced JNK activation and subsequent JNK-dependent downregulation of Bcl-w and upregulation of Bim, resulting in mitochondrial release of cytochrome c and Smac and eventual cell death. These data provide new understanding into the mechanisms contributing to estrogen neuroprotection, a neural function with potential therapeutic relevance to Alzheimer's disease.

Key words: ß-amyloid; apoptosis; Bcl-w; Bim; c-Jun N-terminal kinase; estrogen

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Estrogen is an established modulator of neuron viability. Elevated estrogen levels are associated with decreased neuron death in specific sexually dimorphic nuclei during development (Forger, 2006) and in adulthood after toxic challenge (Wise et al., 2001). In cell culture models, 17ß-estradiol (E2) is neuroprotective against a variety of insults, including serum deprivation (Green et al., 1997), oxidative stress (Behl et al., 1995), excitotoxicity (Goodman et al., 1996; Singer et al., 1996), and ß-amyloid peptide (Aß) (Behl et al., 1995; Goodman et al., 1996; Green et al., 1996; Mook-Jung et al., 1997; Pike, 1999). The mechanisms underlying estrogen neuroprotection are not fully understood; however, several candidates have been identified. One putative mechanism of estrogen neuroprotection is regulation of the Bcl-2 family, pivotal regulators of apoptosis that include both proteins that promote cell survival (e.g., Bcl-2, Bcl-x_L, and Bcl-w) and others that antagonize it (e.g., Bax, Bak, Bad, Bik, BNIP3, Bid, and Bim) (Antonsson and Martinou, 2000). Upregulation of antiapoptotic proteins, such as Bcl-2 (Garcia-Segura et al., 1998; Singer et al., 1998; Dubal et al., 1999; Nilsen and Diaz Brinton, 2003) and Bcl-x_L (Patrone et al., 1999; Pike, 1999; Koski et al., 2004), or downregulation of proapoptotic Bad (Gollapudi and Oblinger, 1999) may contribute to the protective effects of estrogen. However, the relationship between estrogen and neuronal expression of Bcl-2 family members remains incompletely defined.

If members of the Bcl-2 family are important mediators of estrogen neuroprotection, then elucidating the underlying mechanism(s) will require identification of not only estrogen-regulated Bcl-2 family members but also the upstream and downstream signaling components in this pathway. One putative upstream component of regulation by estrogen of Bcl-2 family expression is c-Jun N-terminal kinase (JNK) signaling. JNK signaling is linked to transcriptional regulation of many genes (Ip and Davis, 1998), including members of the Bcl-2 family (Harris and Johnson, 2001; Bae and Song, 2003). Interestingly, JNK activation is observed in cultured neurons after Aß exposure, and its inhibition significantly attenuates Aß toxicity (Bozyczko-Coyne et al., 2001; Morishima et al., 2001; Troy et al., 2001; Yao et al., 2005). The mitochondrial localization of Bcl-2 proteins suggests that a downstream component of this pathway includes regulation of the proapoptotic molecules released from mitochondria, such as cytochrome c (Liu et al., 1996) and second mitochondrion-derived activator of caspase (Smac/DIABLO) (Du et al., 2000; Verhagen et al., 2000). Previous studies indicate that Aß causes mitochondrial release of both cytochrome c (Wang et al., 2001; Agostinho and Oliveira, 2003) and Smac (Yao et al., 2005) in cultured neurons. Whether the mechanism of estrogen neuroprotection against Aß involves regulation of JNK activation and its downstream effectors is unclear.

In this study, we examined the regulatory effects of E2 on neuronal expression of members of the Bcl-2 family under basal conditions and after insult with Aß. Furthermore, we investigated both the upstream (e.g., JNK signaling) and downstream (e.g., cytochrome c and Smac release) components of this antiapoptotic pathway.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell culture. Primary cultures of cerebrocortical neurons were prepared from embryonic (gestational day 18) Sprague Dawley rat pups, with minor modifications of a previously described protocol (Nguyen et al., 2005). In brief, dissected cerebral cortices were incubated 5 min in 0.125% trypsin at 37°C, followed by trypsin quenching with 1 vol of DMEM containing 20% fetal bovine serum. Cell suspensions were centrifuged (5 min at 200 x g), resuspended in serum-free DMEM, mechanically dissociated by repeated passage through a fire-polished Pasteur pipette, and then filtered through a sterile 40 µm nylon mesh (Falcon, Franklin Lakes, NJ). Cells were plated on to poly-L-lysine (0.05 mg/ml)-coated multiwell plates (Nunc, Naperville, IL) at either 5 x 10⁴ cells/cm² (cell viability analysis) or 1.5 x 10⁵ cells/cm² (Western blot and RT-PCR) in serum-free, phenol red-free DMEM buffered with 26 mM bicarbonate, 20 mM HEPES, and supplemented with 100 µg/ml transferrin, 5 µg/ml insulin, 100 µM putrescine, and 30 nM selenium. Cultures were maintained in a humidified incubator at 37°C with room air supplemented to 5% CO₂. These studies were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the University of Southern California.

Experimental treatment of cultures. Cortical neuron cultures were used for experimentation 3–6 d in vitro after plating. Cultures were treated with 0.001–1000 or 10 nM E2 (solubilized in 100% ethanol) (Sigma, St. Louis, MO), which in some experiments was followed 1 h later by exposure to 25 µM aggregated Aß_25–35 (Biochem, Torrance, CA) prepared as described previously (Pike et al., 1993). Estrogen receptor (ER) antagonist 7,17ß-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780) (1 µM; solubilized in 100% ethanol) (Tocris, Ellisville, MO) was added to cultures 1 h before E2. The JNK inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) (100 nM; solubilized in DMSO) (Calbiochem, La Jolla, CA) was added to cultures 1 h before Aß or E2. Final concentrations of drug vehicles were 0.1%; vehicle controls were added as appropriate.

Assessment of cell viability. Cell viability was assessed using calcein-AM and ethidium homodimer fluorescent staining (Invitrogen, Eugene, OR) as described previously (Pike, 1999). Briefly, live cells were counted in four fields per well, four to six wells per condition, in three or more independent culture preparations. The number of live cells counted per well in vehicle-treated controls ranged from 200 to 300. Cell viability is presented graphically as a percentage of live cells in the vehicle-treated, control condition.

RT-PCR. RT-PCR was performed using a standard protocol, as described previously (Yao et al., 2005). In brief, total cellular RNA was isolated using Trizol reagent (Invitrogen) and reverse transcribed into the cDNA using the Superscript first-strand synthesis system (Invitrogen). Next, 1 µl of reverse transcription product was mixed with 2.5 U of JumpStart TaqDNA polymerase (Sigma), 20 pmol each of sense and antisense primers in a buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, and 0.2 mM of each dNTP in a volume of 50 µl. The primers used in this experiment were as follows: 5'-CCGGGAGAACAGGGTATGAT-3', 5'-CAGGTATGCACCCAGAGTGA-3' for bcl-2; 5'-AGGCTGGCGATGAGTTTGAA-3', 5'-CGGCTCTCGGCTGCTGCATT-3' for bcl-x; 5'-AGCCTCAACCCCAGACACAC-3', 5'-AAGGCCCCTACAGTTACCAG-3' for bcl-w; 5'-TCAGCCCATCTTCTTCCAGATGGT-3', 5'-CCACCAGCTCTGAACAGATCATGA-3' for bax; 5'-ACTGCGATGAGGCCCTGTCT-3', 5'-GGCCCAACAGAACCACACCA-3' for bak; 5'-ATGGGAACCCCAAAGCAGCC-3', 5'-TCACTGGGAGGGAGTGGAGC-3' for bad; 5'-ATTTCATGAGGTGCCTGGAG-3', 5'-GGCTTCCAATCAAGCTTCTG-3' for bik; 5'-GAATCTGGACGAAGCAGCTC-3', 5'-AACATTTTCTGGCCGACTTG-3' for bnip3; 5'-ACTCTGAGGTCAGCAACGGT-3', 5'-CTAACCAAGTCCCTCACGTA-3' for bid; 5'-GCCCCTACCTCCCTACAGAC-3', 5'-CAGGTTCCTCCTGAGACTGC-3' for bim; and 5'-AGCCATGTACGTAGCCATCC-3', 5'-CTCTCAGCTGTGGTGGTGAA-3' for ß-actin (internal control). Primers were chemically synthesized (Integrated DNA Technologies, Coralville, IA). The PCR cycles consisted of initial incubation at 94°C for 1 min; denaturation at 94°C for 30 s; annealing at 52°C for 30 s; and extension at 72°C for 1 min, for 30 cycles, and final extension at 72°C for 3 min. RT-PCR products were electrophoresed on 1.7% agarose gels and visualized under UV light after ethidium bromide staining. Analysis of RT-PCR products for the bcl-x and bim primer sets was limited to the 337 bp bcl-x_L band and the 319 bp bim_EL band.

Design and transfection of small interfering RNAs. Small interfering RNA (siRNA) that targets bim was designed using the target finder and design tool (Ambion, Austin, TX). The target mRNA sequence of the siRNA is 5'-AAGAUCUUCUCUGCUGUCCCG-3', corresponding to nucleotides 240–260 of bim gene. As a negative control, a scrambled siRNA was designed consisting of the same nucleotide composition as the specific bim siRNA but lacking significant homology to the genome. As an additional negative control, a mismatched siRNA was used in which two bases in the specific bim siRNA were modified to make them noncomplementary to the target mRNA. The antisense and sense template DNA oligonucleotides for each siRNA, plus T7 promoter 5'-CCTGTCTC-3' to the 3' end, were chemically synthesized (Integrated DNA Technologies) and were as follows: siRNA targeting bim (sibim), 5'-AAGATCTTCTCTGCTGTCCCG-3', 5'-AACGGGACAGCAGAGAAGATC-3'; scrambled siRNA (ncbim), 5'-AATGCCTCCGCTTGTCATCTG-3', 5'-CAGATGACAAGCGGAGGCA-3'; mismatched siRNA (mmbim), 5'-AAGATCTTCTCGTCTGTCCCG-3', 5'-AACGGGACAGACGAGAAGATC-3'. The synthesized template DNA was in vitro transcribed into double-strand siRNA using the Silencer siRNA construction kit (Ambion). siRNA transfection with siPORT Amine (Ambion) was performed according to the manufacturer's instructions.

Western blot. Total cell lysates and mitochondrial and cytosolic extracts (prepared using a mitochondria/cytosol fractionation kit; Biovision, Mountain View, CA) were processed for Western blots using a standard protocol described previously (Pike, 1999). Briefly, lysate and extract samples were diluted into reducing sample buffer, electrophoresed for 1.5 h at 120 V in 15% polyacrylamide gels, and then transferred onto a polyvinylidene difluoride membrane (Millipore, Medford, MA) at constant voltage (100 V) for 1 h. After blocking of nonspecific binding (1 h incubation in 10 mm Tris, 100 mm NaCl, 0.1% Tween, 3% bovine serum albumin), membranes were incubated with primary antibody, which included goat anti-Bcl-w (Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-Bim (Santa Cruz Biotechnology), mouse anti-cytochrome c (Santa Cruz Biotechnology), goat-anti-Smac (Santa Cruz Biotechnology), or mouse anti-phospho-JNK (Thr183/Tyr185) (Cell Signaling Technology, Beverly, MA). After rinsing (5 min; six times; in 10 mm Tris, 100 mm NaCl, 0.1% Tween 20), membranes were incubated in the appropriate horseradish peroxidase-conjugated secondary antibody, followed by enhanced chemiluminescence detection (Amersham Biosciences, Arlington Heights, IL). To detect total JNK or verify equal loading of protein across conditions, membranes were stripped (5 min in 100 mm glycine, pH 2.5; and then 5 min in 62.5 mm Tris, 2% SDS, 0.7% 2-mercaptoethanol, pH 6.7, at 60°C) and reprobed with rabbit anti-JNK (Cell Signaling Technology) or mouse anti-ß-tubulin (Chemicon, Temecula, CA) antibody. Blots were quantified by band densitometry of scanned films using NIH Image 1.61 software. For JNK blots, the ratio of phospho:total JNK was determined and normalized to the vehicle control condition. For anti-Bim blots, the 24 kDa band corresponding to Bim_EL was quantified. Data are presented graphically as a percentage of control values.

Statistical analyses. All experiments were repeated at least three times using independent culture preparations. Quantitative data were statistically analyzed by one-way ANOVA, followed by between-group comparisons using Fisher's least significant difference test. Statistical significance was concluded with a value of p < 0.01 for all analyses.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Estrogen reduces Aß-induced neuronal death
To confirm the established neuroprotective effect of E2 against Aß-induced neuronal death in our experimental system (Pike, 1999; Cordey et al., 2003; Cordey and Pike, 2006), primary cerebrocortical neuron cultures were pretreated with increasing concentrations (0.001–1000 nM) of E2 for 60 min, followed by exposure to 25 µM aggregated Aß_25–35 for 6–48 h. Cell viability assays revealed that nanomolar levels of E2 significantly reduced neuronal death induced by Aß_25–35 in a dose-dependent (Fig. 1A) and time-dependent (Fig. 1B) manner. The 10 nM dose of E2 yielded maximal protection and thus was the concentration used in subsequent experiments.

View larger version (20K): [in this window] [in a new window]   Figure 1. E2 reduces Aß-induced neuronal death in a dose- and time-dependent manner. A, Neuron cultures were pretreated with increasing concentrations (0.001–1000 nM) of E2 for 60 min, followed by exposure to 25 µM Aß25–35 for 48 h, and then assayed for cell viability. Data show mean cell viability (+SEM) from a representative experiment (n = 6). Significance is defined as follows: *p < 0.01 compared with Aß25–35 condition. B, Neuron cultures were pretreated with 0 nM (black bars) or 10 nM (gray bars) E2 for 60 min, followed by exposure to 25 µM Aß25–35 for the indicated times, and then assayed for cell viability. Data show mean cell viability (+SEM) from a representative experiment (n = 6). Significance is defined as follows: *p < 0.01 compared with Aß treatment at the matched time point.

 
Estrogen upregulates Bcl-w and downregulates Bim expression
To investigate the contributions of the Bcl-2 family to estrogen neuroprotection, we first assessed the effect of E2 on expression of bcl-2, bcl-x, bcl-w, bax, bak, bad, bik, bnip3, bid, and bim under basal culture conditions (i.e., in the absence of Aß challenge). Exposure of neuron cultures to 10 nM E2 for 6–72 h did not induce detectable changes in mRNA levels of several Bcl-2 family members, including bak, bad, bik, and bid. Modest alterations in mRNA levels were consistently observed for four genes 12–72 h after E2 exposure: bcl-x and to a lesser extent bcl-2 were mildly increased, and bax and bnip3 were slightly decreased. The most robust findings were that E2 increased expression of antiapoptotic bcl-w and decreased expression of proapoptotic bim at 12–72 h (Fig. 2A). Because bcl-w and bim were clearly the most strongly E2-regulated Bcl-2 family genes in our system, all subsequent studies focused on these two genes.

View larger version (39K): [in this window] [in a new window]   Figure 2. Estrogen upregulates Bcl-w and downregulates Bim expression. A, E2 induces a time-dependent increase of bcl-w and decrease of bim. Neuron cultures were treated with 10 nM E2 for 6–72 h, and mRNA expression levels of Bcl-2 family members (bcl-2, bcl-x, bcl-w, bax, bak, bad, bik, bnip3, bid, and bim) were detected by RT-PCR, followed by agarose gel electrophoresis. ß-actin served as an internal control. B, Representative Western blots show E2 regulation of Bcl-w (top panel) and Bim (middle panel) protein levels; ß-tubulin (bottom panel) was used as an internal control. C, D, Relative amounts of Bcl-w (C) and Bim (D) protein levels were determined by densitometric scanning of Western blots from three to five independent experiments. Data are represented as a mean (+SEM) percentage of control values. *p < 0.01 relative to vehicle-treated control group (Ctrl).

 
To confirm E2 regulation of bcl-w and bim gene expression at the protein level, Bcl-w and Bim were analyzed by Western blot. Results revealed a similar time-dependent upregulation of Bcl-w (Fig. 2B, top panel) and downregulation of Bim (Fig. 2B, middle panel). Densitometry measures indicated that 48 h after E2 treatment Bcl-w was increased to 175% of basal level (Fig. 2C), whereas Bim was decreased to 35% (Fig. 2D).

Estrogen attenuates Aß-induced Bcl-w downregulation and Bim upregulation
After determining the regulatory effects of E2 on expression of Bcl-w and Bim in neurons under basal conditions, we extended our investigation to evaluate E2 effects under Aß challenge. First, in agreement with our recent observations (Yao et al., 2005), we found that exposure of neuron cultures to 25 µM Aß_25–35 in the absence of E2 resulted in significant, time-dependent decrease of bcl-w mRNA (Fig. 3A) and modest increase of bim mRNA (Fig. 3B). Next, to explore whether E2 affects the Aß-induced changes in bcl-w and bim mRNAs, neuron cultures were pretreated with 10 nM E2 for 60 min, followed by exposure to 25 µM Aß_25–35 for 6–48 h. The results showed that E2 pretreatment attenuated both Aß-induced bcl-w downregulation (Fig. 3A) and bim upregulation (Fig. 3B). These mRNA observations were confirmed at the protein level 48 h after Aß exposure by Western blot using antibodies that detect Bcl-w (Fig. 3C, top panel) and Bim (Fig. 3C, middle panel). Densitometric analyses of Western blots showed that Aß significantly reduced Bcl-w expression to 45% of basal levels (Fig. 3D) and increased Bim expression to 125% of basal levels (Fig. 3E) (p < 0.01 in comparison with vehicle-treated control group). Importantly, E2 pretreatment significantly attenuated the effects of Aß on expression of Bcl-w (Fig. 3D) and Bim (Fig. 3E) (p < 0.01 relative to Aß_25–35-treated condition).

View larger version (28K): [in this window] [in a new window]   Figure 3. Estrogen attenuates Aß-induced Bcl-w downregulation and Bim upregulation. A, B, Neuron cultures were pretreated with 10 nM E2 for 60 min, followed by exposure to 25 µM Aß25–35 for indicated times, and analysis of bcl-w (A) and bim (B) mRNA expression by RT-PCR. ß-actin served as an internal control. C, Neuron cultures were pretreated with 10 nM E2 for 60 min followed by exposure to 25 µM Aß25–35 for 48 h. A representative Western blot of culture lysates shows protein levels of Bcl-w (top panel) and Bim (middle panel); ß-tubulin (bottom panel) was used as a control. D, E, Relative amounts of Bcl-w (D) and Bim (E) protein levels were determined by densitometric scanning of Western blots from three to four independent experiments. Data are represented as a mean (+SEM) percentage of control values. #p < 0.01 compared with vehicle-treated control group; *p < 0.01 relative to Aß25–35-treated condition.

 
Reduction of Bim expression attenuates Aß toxicity
If regulation by estrogen of Bcl-w and Bim expression is important to estrogen neuroprotection against Aß, then levels of both Bcl-w and Bim should significantly contribute to Aß toxicity. We recently demonstrated that Aß toxicity is reduced by increased Bcl-w expression and potentiated by decreased Bcl-w expression (Yao et al., 2005). Previous research indicates that the proapoptotic Bim can contribute to Aß toxicity (Yin et al., 2002). To confirm this finding in our culture paradigm, we evaluated the prediction that decreasing Bim expression using specific siRNA should reduce Aß toxicity. Cultures were transfected for 1 or 3 d with siRNA directed against bim or with mismatched or scrambled bim siRNA. RT-PCR analyses show that bim mRNA was significantly reduced by the specific siRNA but by neither of the control siRNAs (Fig. 4A). Western blot analysis revealed similar effects of the siRNA on Bim protein expression (Fig. 4B). Quantitation of Western blots indicated that bim siRNA decreased Bim levels by 76% 1 d after transfection and by up to 81% 3 d after transfection in comparison with scrambled siRNA control (Fig. 4C). These results demonstrate that the designed bim siRNA had strong inhibitory effects on Bim expression at the mRNA and protein levels.

View larger version (25K): [in this window] [in a new window]   Figure 4. Suppression of endogenous Bim expression decreases Aß-induced neuronal death. Primary neuron cultures were transfected for 1 or 3 d with bim-specific siRNA (sibim), scrambled siRNA (ncbim), mismatched siRNA (mmbim), or siPORT amine vehicle (siPORT), or were untreated (Ctrl). A, Representative agarose gels of RT-PCR products show endogenous bim mRNA expression decreased only by specific bim siRNA (top) with no change in levels of ß-actin, an internal control (bottom). B, Protein levels of Bim were similarly affected by the siRNA treatments, as determined by Western blot (top), with no change in ß-tubulin levels (bottom). C, Relative amounts of Bim were determined by densitometric scanning of Western blots from three independent experiments. Data are represented as a mean (+SEM) percentage of vehicle-treated control (Ctrl) values. *p < 0.01 relative to 1 d ncbim control group; #p < 0.01 relative to 3 d ncbim control group. D, Neuron cultures were exposed for 48 h to 25 µM Aß25–35 24 h after transfection with siRNA. Data show mean (+SEM) cell viability from a representative experiment (n = 4). *p < 0.01 relative to respective ncbim condition.

 
To examine the effect of Bim suppression on Aß-induced neuron death, cultures were treated with 25 µM Aß_25–35 1 d after siRNA transfection, and cell viability was assessed 2 d later. In comparison with both the mismatched and scrambled siRNA conditions, cultures transfected with the bim siRNA showed significantly decreased Aß-induced cell death (p < 0.01, compared with scrambled siRNA) (Fig. 4D). This finding suggests that Bim plays a significant role in regulating apoptosis pathways involved in Aß toxicity.

Estrogen regulation of Bcl-w and Bim expression is ER dependent
To determine whether the regulatory effects of E2 on Bcl-w and Bim expression are dependent on ERs, neuron cultures were pretreated with ICI 182,780, an ER antagonist (Wakeling et al., 1991). ICI 182,780 (1 µM; effective concentration determined in Fig. 9A) exposure had no effect on basal expression of either bcl-w (Fig. 5A) or bim (Fig. 5C), but blocked E2 regulation of both bcl-w (Fig. 5A) and bim expression (Fig. 5C). Furthermore, ICI 182,780 also blocked the inhibitory effects of E2 on Aß-induced bcl-w downregulation (Fig. 5B) and bim upregulation (Fig. 5D). These mRNA observations were confirmed at the protein level 48 h after Aß exposure by Western blot using antibodies directed against Bcl-w (Fig. 5E, top panel) and Bim (Fig. 5E, middle panel). Quantitative analysis of blots confirmed that ICI 182,780 not only blocked E2 regulation of basal Bcl-w (Fig. 5F) and Bim expression (Fig. 5G), but also E2 inhibition of Aß-induced Bcl-w downregulation (Fig. 5F) and Bim upregulation (Fig. 5G).

View larger version (40K): [in this window] [in a new window]   Figure 5. Estrogen regulation of Bcl-w and Bim expression is ER dependent. A, C, Representative agarose gels of RT-PCR products using bcl-w (A) or bim (C) primers show that the ER antagonist ICI 182,780 blocks E2 regulation of bcl-w and bim under basal conditions. Neuron cultures were pretreated with 1 µM ICI 182,780 for 60 min, followed by 10 nM E2 for 24 and 48 h, respectively. ß-actin served as an internal control. B, D, Representative agarose gels of RT-PCR products using bcl-w (B) or bim (D) primers show that the ER antagonist ICI 182,780 blocks E2 inhibition of Aß-induced changes in bcl-w and bim expression. Neuron cultures were pretreated with 1 µM ICI 182,780 for 60 min, followed by 10 nM E2 for 60 min, and then exposed to 25 µM Aß25–35 for 24 and 48 h. ß-actin served as an internal control. E, ER dependence of E2 bcl-w and bim regulation was extended to protein expression using Western blots. A representative Western blot shows Bcl-w (top panel), Bim (middle panel), and ß-tubulin (bottom panel) expression in lysates from neuron cultures that were pretreated with 1 µM ICI 182,780, followed 60 min later with 10 nM E2 treatment, and then 60 min later exposed to 25 µM Aß25–35 for 48 h. F, G, Relative protein levels of Bcl-w (F) and Bim (G) were determined by densitometric scanning of Western blots from four independent experiments. Data show mean (+SEM) percentages of control values. *p < 0.01 relative to E2-treated condition; #p < 0.01 compared with E2 plus Aß25–35 treatment.

 
Effect of JNK inhibition on estrogen regulation of Bcl-w and Bim expression
JNK signaling is an established signaling pathway in the regulation of Bcl-2 family expression (Harris and Johnson, 2001; Bae and Song, 2003; Yao et al., 2005). To begin investigating the role of JNK signaling in E2 regulation of Bcl-2 family members, we assessed the effect of the specific JNK inhibitor SP600125 (Bennett et al., 2001) on E2 induced bcl-w upregulation and bim downregulation under nonchallenged conditions. Neuron cultures were pretreated for 60 min with 100 nM SP600125 followed by treatment with 10 nM E2 for 24 and 48 h. RT-PCR analyses showed that basal mRNA levels of bcl-w (Fig. 6A) and bim (Fig. 6C) were not affected by pharmacological inhibition of JNK. Similarly, E2-induced bcl-w upregulation (Fig. 6A) and bim downregulation (Fig. 6C) also were not altered by JNK inhibition, suggesting that the regulation of E2 on basal expression of bcl-w and bim is not dependent on JNK signaling.

View larger version (36K): [in this window] [in a new window]   Figure 6. Estrogen regulation of Bcl-w and Bim expression is JNK independent under basal conditions but involves JNK signaling under Aß challenge. A, C, Representative agarose gels of RT-PCR products using bcl-w (A) or bim (C) primers show that, under basal conditions, the JNK inhibitor SP600125 neither blocks E2 regulation of bcl-w and bim nor independently affects expression of bcl-w and bim. Neuron cultures were pretreated with 100 nM SP600125 for 60 min, followed by 10 nM E2 for 24 and 48 h. ß-actin served as an internal control. B, D, Representative agarose gels of RT-PCR products using bcl-w (B) or bim (D) primers show that the JNK inhibitor SP600125 both independently and additively with E2 blocks Aß-induced changes in bcl-w and bim expression. Neuron cultures were pretreated with 100 nM SP600125 for 60 min, followed by 10 nM E2 for 60 min, and then exposed to 25 µM Aß25–35 for 24 and 48 h. ß-actin served as an internal control. Effects of JNK inhibition on E2 regulation of bcl-w and bim were extended to protein expression using Western blots. E, F, Representative blots show Bcl-w (E) and Bim (F) and expression in lysates from neuron cultures that were pretreated with 100 nM SP600125, followed 60 min later with 10 nM E2 treatment, and then 60 min later exposed to 25 µM Aß25–35 for 48 h. G, H, Relative protein levels of Bcl-w (G) and Bim (H) were determined by densitometric scanning of Western blots from three independent experiments. *p < 0.01 relative to Aß25–35 condition; #p < 0.01 compared with E2 plus Aß25–35 treatment; p < 0.01 relative to Aß25–35 plus SP600125 condition.

 
Our recent data indicate that JNK signaling contributes to the mechanism by which Aß regulates expression of Bcl-2 family members (Yao et al., 2005). We confirm here that inhibition of JNK signaling by pretreatment with 100 nM SP600125 mostly prevents Aß-induced downregulation of bcl-w (Fig. 6B) and upregulation of bim (Fig. 6D). Although the regulation of E2 on basal expression of bcl-w and bim is not dependent on JNK signaling (Fig. 6A,C), we investigated whether JNK signaling may contribute to the inhibitory effect of E2 on Aß-induced regulation of bcl-w and bim. Neuron cultures were pretreated with 100 nM SP600125 for 60 min, followed by treatment with 10 nM E2 for 60 min and exposure to 25 µM Aß_25–35 for 48 h. RT-PCR analyses showed that the inhibitory effects of E2 on Aß-induced downregulation of bcl-w (Fig. 6B) and upregulation of bim (Fig. 6D) appeared to be enhanced by cotreatment with SP600125. These mRNA observations were evaluated at the protein level with Bcl-w (Fig. 6E) and Bim (Fig. 6F) Western blots. Densitometric analyses of blots confirmed that JNK inhibition attenuated Aß-induced downregulation of Bcl-w (Fig. 6G) (*p < 0.01 relative to Aß treatment) and upregulation of Bim (Fig. 6H) (*p < 0.01 relative to Aß treatment). However, the effect of estrogen and SP600125 cotreatment was not significantly greater than the effect of SP600125 alone for either Aß-induced downregulation of Bcl-w (Fig. 6G) or upregulation of Bim (Fig. 6H). These data suggest the possibility that the inhibitory effect of E2 on Aß-induced changes of Bcl-w and Bim may involve not only a JNK-independent regulatory action on Bcl-w and Bim expression, but also inhibition of Aß-induced JNK signaling.

Estrogen reduces Aß-induced JNK activation
To explore whether E2 regulates JNK signaling, we first determined the effect of E2 on activation of JNK under basal conditions. Neuron cultures were treated with 10 nM E2 for 5 min to 48 h and whole-cell extracts were analyzed by Western blot using antibodies that recognize total JNK and phosphorylated JNK, an indicator of activation. Results showed that two isoforms of JNK (46 and 54 kDa) were detected. E2 treatment affected neither phosphorylated (Fig. 7A, top panel) nor total (Fig. 7A, bottom panel) JNK protein levels, indicating that E2 does not significantly affect JNK signaling under nonchallenge conditions (Fig. 7B).

View larger version (38K): [in this window] [in a new window]   Figure 7. Estrogen reduces Aß-induced JNK activation. A, Neuron cultures were treated with 10 nM E2 for the indicated times and then assessed by Western blot with phospho-JNK (p-JNK) (top panel) and pan-JNK (bottom panel) antibodies. B, Blots were quantified by band densitometry for both the 54 kDa (black bars) and 46 kDa (gray bars) JNK forms. C, D, E2 reduces Aß-induced JNK activation, as shown by both representative Western blots (C) and densitometric quantification of blots (D), in neuron cultures that were pretreated with 10 nM E2 for 60 min, followed by exposure to 25 µM Aß25–35 for the indicated times. E, F, Representative Western blots probed with phospho-JNK (top panel) and pan-JNK (bottom panel) antibodies (E) and densitometric quantification of blots (F) show that pretreatment of neuron cultures with 1 µM ICI 182,780 for 60 min attenuates the inhibitory effect of 10 nM E2 treatment on JNK phosphorylation induced by exposure to 25 µM Aß25–35 for 6 h. G, H, The JNK inhibitor SP600125 reduces Aß-induced JNK activation, as shown by representative Western blots (G) and densitometric quantification of blots (H) from lysates of neuron cultures that were pretreated with 100 nM SP600125 for 60 min, followed by exposure to 25 µM Aß25–35 for the indicated times. All experiments were repeated in three or more independent culture preparations. Data show mean phospho-JNK:total JNK ratios, normalized to the vehicle control condition. *p < 0.01 relative to matched Aß25–35 condition at the same time point; #p < 0.01 relative to vehicle control (Ctrl) condition.

 
Next, we determined the effect of E2 on Aß-induced JNK activation. Neuron cultures were pretreated with 10 nM E2 for 60 min, followed by exposure to 25 µM Aß_25–35. Western blot results show that Aß_25–35 triggers JNK phosphorylation that was detectable as early as 3 h after Aß treatment and persisted at least through 48 h (Fig. 7C). Both the 46 and 54 kDa JNK bands showed an Aß-induced increase in phosphorylation, with the 54 kDa band showing the more robust increase (Fig. 7D). E2 pretreatment incompletely blocked the Aß-induced increase in JNK activation at most time points (Fig. 7C, top panel; D) but did not alter total JNK protein levels (Fig. 7C, bottom panel). E2 similarly inhibited Aß-induced phosphorylation of the 46 and 54 kDa JNK bands. To assess the role of ER in this effect, neuron cultures were pretreated with ER antagonist ICI 182,780 (1 µM) for 60 min, followed by treatment with 10 nM E2 for 60 min, and then exposed to 25 µM Aß_25–35 for 6 h. Western blots indicated that ICI 182,780 blocked the inhibitory effect of E2 on Aß-induced JNK phosphorylation (Fig. 7E,F), demonstrating that the regulatory effect of E2 on Aß-induced JNK activation occurs in an ER-dependent manner. For comparison, we confirmed our previous finding (Yao et al., 2005) that the JNK inhibitor SP600125 both reduces basal levels of JNK phosphorylation and blocks Aß-induced JNK phosphorylation of the 46 and 54 kDa JNK bands (Fig. 7G,H).

Estrogen attenuates JNK-dependent mitochondrial cytochrome c and Smac release induced by Aß
Bcl-2 family members exert their effects in part through regulating mitochondrial release of cytochrome c and Smac into the cytosol (Kuwana and Newmeyer, 2003), an event that is a general feature of the mitochondrial pathway of apoptosis (Liu et al., 1996; Du et al., 2000; Verhagen et al., 2000). In this paradigm, we previously reported that Bcl-w overexpression effectively inhibited Aß-induced Smac release from mitochondria, whereas knock-down of Bcl-w expression increased Smac release (Yao et al., 2005). On the contrary, Bim can increase mitochondrial cytochrome c (Putcha et al., 2001; Whitfield et al., 2001) and Smac (Yin et al., 2002) release. Because we observed that E2 increases Bcl-w and decreases Bim expression, E2 may regulate Aß-induced mitochondrial cytochrome c and Smac release. To investigate this possibility and the potential involvement of JNK signaling, we analyzed by Western blot mitochondrial and cytosolic extracts of neuron cultures treated with 25 µM Aß_25–35 in the presence and absence of either 100 nM SP600125 or 10 nM E2. We observed that, 48 h after Aß_25–35 treatment, cytochrome c (Fig. 8A) and Smac (Fig. 8B) levels decreased in the mitochondrial fraction and increased in the cytosolic fraction. Pretreatment with JNK inhibitor SP600125 mostly prevented Aß-induced cytochrome c and Smac release, suggesting a JNK-dependent mechanism. Similarly, E2 also attenuated Aß-induced mitochondrial release of both cytochrome c (Fig. 8C) and Smac (Fig. 8D). Densitometric analyses of blots showed that E2 significantly reduced Aß-induced depletion of cytochrome c (Fig. 8E) and Smac (Fig. 8F) from mitochondria and accumulation of cytochrome c (Fig. 8G) and Smac (Fig. 8H) in cytosol (*p < 0.01 relative to 24 h Aß treatment; ^#p < 0.01 relative to 48 h Aß treatment).

View larger version (32K): [in this window] [in a new window]   Figure 8. Estrogen reduces JNK-dependent mitochondrial cytochrome c and Smac release induced by Aß. A, B, Neuron cultures were pretreated with 100 nM JNK inhibitor SP600125 for 60 min, followed by exposure to 25 µM Aß25–35 for 48 h, and then analyzed for cytochrome c (Cyt C) (A) and Smac (B) content in mitochondrial (Mt) and cytosolic (Cyt) extracts by Western blot. Neuron cultures were pretreated with 10 nM E2 for 60 min, followed by exposure to 25 µM Aß25–35 for 24 and 48 h. C, D, Levels of cytochrome c (C) and Smac (D) in mitochondrial and cytosolic extracts were analyzed by Western blot. E–H, Relative protein levels of cytochrome c, in the mitochondrial fraction (E) and cytosolic fraction (G), and Smac, in the mitochondrial fraction (F) and cytosolic fraction (H), were determined by densitometric scanning of Western blots from three independent experiments. Data are presented as mean (+SEM) percentages of vehicle-treated control (Ctrl) values. *p < 0.01 relative to Aß25–35 condition at 24 h; #p < 0.01 relative to Aß25–35 condition at 48 h.

 
Estrogen neuroprotection against JNK-dependent Aß toxicity is mediated by estrogen receptor
The above studies suggest that E2 antagonizes Aß-induced apoptosis signaling by an ER-dependent mechanism that operates at least in part by inhibiting JNK activation. Next, we investigated the effects of ER antagonism on the ability of E2 to attenuate Aß-induced neuron death. To verify that the neuroprotective effect of E2 on Aß toxicity is mediated by ER, neuron cultures were pretreated with increasing concentrations (0.01–10 µM) of the ER antagonist ICI 182,780, followed by 48 h exposure to 25 µM Aß_25–35 in the presence or absence of 10 nM E2. ICI 182,780 alone had no effect on cell viability under basal conditions and after Aß challenge (Fig. 9A). However, at concentrations of 1 µM and above, ICI 182,780 almost completely blocked E2-mediated neuroprotection against Aß toxicity (p < 0.01 in comparison with E2 plus Aß treatment) (Fig. 9A).

View larger version (21K): [in this window] [in a new window]   Figure 9. Aß toxicity is JNK dependent and attenuated by ER-dependent estrogen actions. A, E2-induced neuroprotection against Aß is ER dependent. Primary neuron cultures were pretreated with ICI 182,780 (ICI) at concentrations ranging from 0.01 to 10 µM for 60 min, followed by treatment with 0 nM (black bars) or 10 nM (gray bars) E2 for 60 min, and then exposed to 25 µM Aß25–35 for 48 h. Controls groups with vehicle treatment (Veh), 10 nM E2 alone, and 1 µM ICI alone are shown in white bars. Data show mean cell viability (+SEM) from a representative experiment (n = 4). *p < 0.01 compared with E2 plus Aß25–35 and 0 µM ICI treatments. B, Inhibition of JNK signaling reduces Aß toxicity and increases estrogen neuroprotection. Neuron cultures were pretreated with 100 nM JNK inhibitor SP600125 (SP) for 60 min, followed by 10 nM E2 for 60 min, and then exposure to 25 µM Aß25–35 for 48 h. Data show mean cell viability (+SEM) from a representative experiment (n = 4). Significance is defined as follows: #p < 0.01 compared with Aß treatment; *p < 0.01 compared with E2 plus Aß25–35 treatment.

 
We observed that E2 inhibits JNK signaling and that JNK signaling contributes to Aß-induced changes in Bcl-w and Bim expression and mitochondrial release of cytochrome c and Smac. Here, we extended these observations to neuronal survival. Neuron cultures were exposed for 48 h to 25 µM Aß_25–35 with or without pretreatment with 100 nM SP600125 and or 10 nM E2. Cell viability assays showed that independent treatment with either SP600125 or E2 significantly attenuated neuronal death induced by Aß_25–35 (Fig. 9B). Cotreatment with SP600125 and E2 resulted in a modest increase in neuroprotection that was significantly greater than E2 treatment alone but not significantly greater than SP600125 treatment alone (Fig. 9B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although abundant evidence has established estrogen as a neuroprotective factor that is relevant across the life span, elucidation of its protective mechanisms has proven challenging. One compelling mechanism involves regulation of the Bcl-2 family. Findings from this study not only further define regulation by estrogen of Bcl-2 family members, they also identify two separate pathways of regulation: (1) increased expression of antiapoptotic Bcl-w and decreased expression of proapoptotic Bim under basal conditions, and (2) attenuation of JNK-dependent changes in expression of Bcl-2 family that occurs under apoptotic challenge (Fig. 10).

View larger version (21K): [in this window] [in a new window]   Figure 10. Estrogen neuroprotection may involve two complementary pathways. Under basal conditions (solid lines), E2 increases expression of antiapoptotic Bcl-w and decreases expression of proapoptotic Bim, events that counteract the mitochondrial pathway of apoptosis. This may represent an estrogen maintenance pathway of neuron survival. Under toxic challenges such as Aß exposure (dashed lines), E2 can inhibit activation of JNK, functionally mimicking the pharmacological JNK inhibitor SP600125. JNK activation induces the mitochondrial pathway of apoptosis involving decreased expression of Bcl-w and increased expression of Bim, followed by mitochondrial release of cytochrome c and Smac, and eventually neuron death. Because E2 does not alter basal JNK activity, such a protective mechanism may represent an estrogen response pathway that is activated after injury.

 
Consistent with an emerging literature, we found that, in the absence of toxic challenge, estrogen regulates Bcl-2 family members in a manner that favors antagonism of apoptosis. Our most robust findings are the novel observations that E2 significantly increases basal expression of antiapoptotic Bcl-w and decreases basal expression of proapoptotic Bim. Bcl-w is widely expressed in mammalian tissues, including CNS (Print et al., 1998; Hamnér et al., 1999; O'Reilly et al., 2001), and functions as a negative regulator of neuronal apoptosis (Gibson et al., 1996; Hamnér et al., 2001; Middleton et al., 2001). Neural expression of Bcl-w is highest in the mature brain (Hamnér et al., 1999), suggesting that Bcl-w function may be particularly important in adulthood. In a previous study, we reported that Aß-induced neuronal death was reduced by Bcl-w overexpression and potentiated by Bcl-w suppression (Yao et al., 2005), findings that suggest a critical role for Bcl-w in Aß-induced apoptosis.

In contrast to Bcl-w, Bim is a proapoptotic protein that exists in several isoforms: Bim_EL, Bim_L, Bim_S, and BimAD (O'Connor et al., 1998; U et al., 2001; Marani et al., 2002). In the CNS, Bim expression is localized primarily in neurons (O'Reilly et al., 2000) and is upregulated in a variety of neuron death paradigms (Putcha et al., 2001; Whitfield et al., 2001; Becker et al., 2004; Biswas et al., 2005). Inhibition of Bim by antisense and genetic knock-out approaches can significantly reduce neuronal apoptosis (Whitfield et al., 2001; Becker et al., 2004). In cultured cerebral endothelial cells, Aß toxicity is associated with increased Bim expression and is suppressed by Bim knock-down (Yin et al., 2002). Consistent with our observations, these results implicate Bim in the mechanism of Aß-induced apoptosis. Bim produces its proapoptotic effects by interacting with and neutralizing antiapoptotic Bcl-2 family proteins such as Bcl-w (O'Connor et al., 1998; Puthalakath et al., 1999; Wilson-Annan et al., 2003; Shinoda et al., 2004). Thus, E2-induced upregulation of Bcl-w and downregulation of Bim should result in complementary inhibition of neuronal apoptosis induced by toxins such as Aß.

In addition to E2 regulation of Bcl-w and Bim, we observed relatively modest elevations in mRNA levels of antiapoptotic Bcl-2 family members bcl-2 and bcl-x, and small but consistent reductions in mRNA levels of proapoptotic bax and bnip3. Consistent with our findings are previous results in neuronal cultures showing that estrogen increases basal expression of Bcl-2 (Singer et al., 1998; Honda et al., 2001; Nilsen and Diaz Brinton, 2003; Zhao et al., 2004) and Bcl-x_L (Patrone et al., 1999; Pike, 1999; Koski et al., 2004) and decreases expression of proapoptotic bnip2 (Belcredito et al., 2001). Similar results have been observed in rodent brain under nonchallenge conditions. For example, accumulating evidence links developmental sex differences in E2 exposure to differences in Bcl-2 family expression and neuron survival in sexually dimorphic brain regions (for review, see Forger, 2006). In adult female rats, E2 positively regulates expression of Bcl-2 in hypothalamus (Garcia-Segura et al., 1998) and bcl-x in hippocampus (Stoltzner et al., 2001).

One interpretation of the current data is that estrogen functions as an endogenous, homeostatic regulator of apoptosis signaling. Under normal conditions in estrogen-responsive brain regions, estrogen may help maintain long-term neuronal viability by regulating the expression of several Bcl-2 family members. Because apoptosis is affected by the net interactions of proapoptotic and antiapoptotic Bcl-2 members, we speculate that neuroprotective actions of estrogen reflect additive regulatory effects on Bcl-w, Bim, and other Bcl-2 proteins. This "E2 maintenance pathway" (Fig. 10) appears to be ER dependent, but it is unclear whether the mechanism involves predominantly classic genomic pathways (i.e., interaction of activated ER with estrogen response elements on target genes) (Pike, 1999; Perillo et al., 2000) and/or indirect genomic pathways activated by cell signaling pathways [e.g., CREB (cAMP response element-binding protein) signaling] (Honda et al., 2001; Wu et al., 2005). Although activation of JNK signaling is linked to regulation of Bcl-2 family members (Harris and Johnson, 2001; Bae and Song, 2003), our data do not implicate JNK signaling in the E2 maintenance pathway, because under basal conditions E2 did not affect JNK phosphorylation and JNK inhibition did not affect E2 regulation of Bcl-w and Bim.

In addition to regulating expression of Bcl-2 family members under basal conditions, our data show that E2 antagonizes proapoptotic changes in Bcl-2 family expression induced by toxic challenge. Consistent with our previous report (Yao et al., 2005), we found that Aß-induced neuronal apoptosis involves JNK-dependent alterations in Bcl-2 family expression: downregulation of Bcl-w and upregulation of Bim. Other members of the Bcl-2 family may also play a significant role in Aß toxicity. We observed that E2 significantly attenuated Aß-induced changes in Bcl-w and Bim expression. Notably, E2 also significantly reduced Aß-induced JNK phosphorylation, suggesting that E2 attenuation of Aß-induced changes in Bcl-w and Bim expression involves inhibition of JNK activation.

Activation of JNK signaling has been closely linked to a variety of apoptotic stimuli, whereas inhibition of JNK signaling provides protection against neuronal apoptosis in multiple paradigms, including Aß neurotoxicity (Bozyczko-Coyne et al., 2001; Morishima et al., 2001; Troy et al., 2001). It appears that JNK signaling promotes apoptosis at least in part via both transcriptional and posttranslational regulation of Bcl-2 family members (Sanchez and Yuan, 2001). Consequently, JNK inhibition can block alterations in Bcl-2 family expression induced during apoptosis (Harris and Johnson, 2001; Linseman et al., 2002; Schuster et al., 2002; Lei and Davis, 2003; Okuno et al., 2004; Yao et al., 2005; Papadakis et al., 2006). Our results show that inhibition of JNK phosphorylation by both the pharmacological inhibitor SP600125 and E2 attenuated Aß-induced changes in Bcl-w and Bim expression, mitochondrial cytochrome c and Smac release, and neuron death. Consistent with our data are findings in non-neural cell types that E2 can reduce JNK activation, which results in attenuation of JNK-dependent gene expression (Srivastava et al., 1999, 2001) and/or increased cell survival (Razandi et al., 2000; Eckhoff et al., 2003). Interestingly, despite significantly attenuating indices of apoptosis, E2 provided only partial protection against Aß-induced cell death. Incomplete neuroprotection in primary neuron cultures is commonly observed with not only estrogen (Singer et al., 1996; Pike, 1999; Harms et al., 2001; Honda et al., 2001) but also androgens (Ahlbom et al., 2001; Hammond et al., 2001; Pike, 2001) and progesterone (Nilsen and Brinton, 2002), suggesting that sex steroid hormones act as partial modulators of neuronal apoptosis pathways.

Thus, E2 may regulate Bcl-2 family members not only by an E2 maintenance pathway but also by an "E2 response pathway" (Fig. 10). In the latter case, E2 can respond to at least some toxic challenges by an ER-dependent pathway to inhibit activation of cell signaling pathways that induce proapoptotic changes in Bcl-2 family expression (e.g., JNK signaling). Importantly, the literature indicates several mechanisms that contribute to estrogen neuroprotection (Green and Simpkins, 2000). We speculate that the pathways identified in this study likely function in conjunction with other mechanisms of estrogen neuroprotection.

Recent observations in animal models are consistent with an E2 response pathway in which E2 responds to neural injury by regulating expression of Bcl-2 family members in a way that reduces apoptosis. For example, in ischemic brain injury models, E2 is neuroprotective and attenuates injury-induced downregulation of Bcl-2 (Dubal et al., 1999; Alkayed et al., 2001; Zhang et al., 2004) and, in some reports, upregulation of Bax (Won et al., 2006). Similarly, E2 neuroprotection is associated with increased Bcl-2 expression after traumatic brain injury (Soustiel et al., 2005) and bcl-2 and bcl-x levels after spinal cord injury (Yune et al., 2004). Such an E2 response pathway may be particularly important for resistance to neuronal death associated with stroke, Alzheimer's disease, and other age-related disorders.

Together, our results are consistent with the hypothesis that E2 attenuates Aß-induced neuronal death, at least in part, by ER-dependent upregulation of Bcl-w and downregulation of Bim, as well as by inhibition of Aß-induced JNK activation, subsequent JNK-dependent downregulation of Bcl-w and upregulation of Bim, and mitochondrial release of cytochrome c and Smac. Furthermore, our findings suggest that these neuroprotective actions of E2 reflect concurrent effects of at least two different, ER-dependent signaling pathways: (1) an E2 maintenance pathway of JNK-independent regulation of Bcl-2 family members under basal, nonchallenge conditions, and (2) an E2 response pathway of inhibition of JNK-dependent regulation of Bcl-2 family members associated with neural injury. These data provide new understanding into the mechanisms contributing to estrogen neuroprotection, a function with potential therapeutic relevance to Alzheimer's disease and other age-related neurodegenerative disorders.

	Footnotes

 
Received June 5, 2006; revised Dec. 6, 2006; accepted Dec. 8, 2006.

This work was supported by National Institutes of Health Grants AG26752 and AG23739.

Correspondence should be addressed to Dr. Christian J. Pike, Davis School of Gerontology, University of Southern California, 3715 McClintock Avenue, Los Angeles, CA 90089-0191. Email: cjpike{at}usc.edu

Copyright © 2007 Society for Neuroscience 0270-6474/07/271422-12$15.00/0

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

Agostinho P, Oliveira CR (2003) Involvement of calcineurin in the neurotoxic effects induced by amyloid-beta and prion peptides. Eur J Neurosci 17:1189–1196.[CrossRef][ISI][Medline]

Ahlbom E, Prins GS, Ceccatelli S (2001) Testosterone protects cerebellar granule cells from oxidative stress-induced cell death through a receptor mediated mechanism. Brain Res 892:255–262.[CrossRef][ISI][Medline]

Alkayed NJ, Goto S, Sugo N, Joh HD, Klaus J, Crain BJ, Bernard O, Traystman RJ, Hurn PD (2001) Estrogen and Bcl-2: gene induction and effect of transgene in experimental stroke. J Neurosci 21:7543–7550.[Abstract/Free Full Text]

Antonsson B, Martinou JC (2000) The Bcl-2 protein family. Exp Cell Res 256:50–57.[CrossRef][ISI][Medline]

Bae MA, Song BJ (2003) Critical role of c-Jun N-terminal protein kinase activation in troglitazone-induced apoptosis of human HepG2 hepatoma cells. Mol Pharmacol 63:401–408.[Abstract/Free Full Text]

Becker EB, Howell J, Kodama Y, Barker PA, Bonni A (2004) Characterization of the c-Jun N-terminal kinase-BimEL signaling pathway in neuronal apoptosis. J Neurosci 24:8762–8770.[Abstract/Free Full Text]

Behl C, Widmann M, Trapp T, Holsboer F (1995) 17-ß Estradiol protects neurons from oxidative stress-induced cell death in vitro. Biochem Biophys Res Commun 216:473–482.[CrossRef][ISI][Medline]

Belcredito S, Vegeto E, Brusadelli A, Ghisletti S, Mussi P, Ciana P, Maggi A (2001) Estrogen neuroprotection: the involvement of the Bcl-2 binding protein BNIP2. Brain Res Brain Res Rev 37:335–342.[CrossRef][Medline]

Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y, Bhagwat SS, Manning AM, Anderson DW (2001) SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 98:13681–13686.[Abstract/Free Full Text]

Biswas SC, Liu DX, Greene LA (2005) Bim is a direct target of a neuronal E2F-dependent apoptotic pathway. J Neurosci 25:8349–8358.[Abstract/Free Full Text]

Bozyczko-Coyne D, O'Kane TM, Wu ZL, Dobrzanski P, Murthy S, Vaught JL, Scott RW (2001) CEP-1347/KT-7515, an inhibitor of SAPK/JNK pathway activation, promotes survival and blocks multiple events associated with Abeta-induced cortical neuron apoptosis. J Neurochem 77:849–863.[CrossRef][ISI][Medline]

Cordey M, Pike CJ (2006) Conventional protein kinase C isoforms mediate neuroprotection induced by phorbol ester and estrogen. J Neurochem 96:204–217.[CrossRef][ISI][Medline]

Cordey M, Gundimeda U, Gopalakrishna R, Pike CJ (2003) Estrogen activates protein kinase C in neurons: role in neuroprotection. J Neurochem 84:1340–1348.[CrossRef][ISI][Medline]

Du C, Fang M, Li Y, Li L, Wang X (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102:33–42.[CrossRef][ISI][Medline]

Dubal DB, Shughrue PJ, Wilson ME, Merchenthaler I, Wise PM (1999) Estradiol modulates bcl-2 in cerebral ischemia: A potential role for estrogen receptors. J Neurosci 19:6385–6393.[Abstract/Free Full Text]

Eckhoff DE, Smyth CA, Eckstein C, Bilbao G, Young CJ, Thompson JA, Contreras JL (2003) Suppression of the c-Jun N-terminal kinase pathway by 17beta-estradiol can preserve human islet functional mass from proinflammatory cytokine-induced destruction. Surgery 134:169–179.[CrossRef][ISI][Medline]

Forger NG (2006) Cell death and sexual differentiation of the nervous system. Neuroscience 138:929–938.[CrossRef][ISI][Medline]

Garcia-Segura LM, Cardona-Gomez P, Naftolin F, Chowen JA (1998) Estradiol upregulates Bcl-2 expression in adult brain neurons. NeuroReport 9:593–597.[ISI][Medline]

Gibson L, Holmgreen SP, Huang DC, Bernard