Abstract
The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstrate that corticosterone protects neurons from apoptosis by a mechanism involving the cyclin-dependent kinase inhibitor p21Waf1/Cip1. In primary cortical neurons, corticosterone leads to a dose- and Akt-kinase-dependent upregulation with enhanced phosphorylation and cytoplasmic appearance of p21Waf1/Cip1 at Thr 145. Exposure of neurons to the neurotoxin ethylcholine aziridinium (AF64A) results in activation of caspase-3 and a dramatic loss of p21Waf1/Cip1 preceding apoptosis in neurons. These effects of AF64A are reversed by pretreatment with corticosterone. Corticosterone-mediated upregulation of p21Waf1/Cip1 and neuroprotection are completely abolished by glucocorticoid and mineralocorticoid receptor antagonists as well as inhibitors of PI3- and Akt-kinase. Both germline and somatically induced p21Waf1/Cip1 deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21Waf1/Cip1 suffices to protect neurons from apoptosis. We identify p21Waf1/Cip1 as a novel antiapoptotic factor for postmitotic neurons and implicate p21Waf1/Cip1 as the molecular target of neuroprotection by high-dose glucocorticoids.
Introduction
Glucocorticoids act as a double-edged sword in the regulation of apoptosis. For example, glucocorticoids induce apoptosis in inflammatory and immune cells such as thymocytes, myeloma cells, and peripheral blood monocytes, whereas concomitantly they protect those cells and tissues in which the inflammation takes place (for review, see Amsterdam et al., 2002). The situation may be similar in the CNS [for review, see Ábrahám et al. (2001) and Lee et al. (2002)]: on the one hand, glucocorticoids enhance neuronal cell death (Behl et al., 1997a), and glucocorticoid receptor antagonists are protective, for example, during oxidative stress (Behl et al., 1997b; McCullers et al., 2002). On the other hand, high-dose glucocorticoid pretreatment interferes with apoptotic death in glioma cells and oligodendrocytes and reduces ischemic brain damage and retinal light damage (Tuor, 1997; Gorman et al., 2000; Melcangi et al., 2000; Wenzel et al., 2001; Limbourg et al., 2002).
Potential antiapoptotic mechanisms of glucocorticoids involve loss of mitochondrial membrane potential, upregulation of antiapoptotic proteins Bcl-2 and Bcl-xL, and activation of the phosphatidylinositol 3-Akt-kinase (PI3-Akt-kinase) pathway (Evans-Storms and Cidlowski, 2000; Gorman et al., 2000; Bailly-Maitre et al., 2001; Sasson et al., 2001, 2002). In line with the latter notion, the PI3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) completely abolishes any protective effect of glucocorticoids in models of stroke and myocardial infarction (Hafezi-Moghadam et al., 2002; Limbourg et al., 2002). Interestingly, glucocorticoids activate PI3-Akt-kinase in a ligand-dependent, nongenomic manner (Limbourg et al., 2002) similar to that of the estrogen receptor (Simoncini et al., 2000).
Interestingly, the cyclin-dependent kinase (cdk) inhibitor p21Waf1/Cip1 gene contains a glucocorticoid receptor response region in its promoter region, and dexamethasone significantly increases expression of p21Waf1/Cip1 in non-neuronal cells (Cha et al., 1998; Cram et al., 1998; Terada et al., 2001). Moreover, p21Waf1/Cip1 is phosphorylated by Akt-kinase at a consensus threonine residue (T145), leading to cytoplasmic localization and activation of p21Waf1/Cip1 (Zhou et al., 2001). Notably, p21Waf1/Cip1 functions not only as a cell cycle inhibitor but also as an inhibitor of caspase-3 and apoptotic cell death and hence is an attractive candidate for the antiapoptotic effects of glucocorticoids (Suzuki et al., 1998; Asada et al., 1999) [for review, see Gartel and Tyner (2002) and Coqueret (2003)]. Therefore, in the present study, we tested the hypothesis that corticosterone would protect neurons from apoptosis via both upregulation and PI3-Akt-kinase-dependent phosphorylation, activation, and cytoplasmic translocation of p21Waf1/Cip1.
Materials and Methods
Materials.
Corticosterone, spironolactone, mifepristone, cycloheximide, DMSO, and enzyme standard for the kinetic lactate dehydrogenase (LDH) test were obtained from Sigma (Taufkirchen, Germany); LY294002 and SH6 were from Merck Biosciences (Bad Soden, Germany); Neurobasal medium and supplement B27 were from Invitrogen (Eggenstein, Germany); modified Eagle's medium, PBS, HEPES buffer, trypsin/EDTA, penicillin–streptomycin, l-glutamine, collagen-G, and poly-l-lysine were from Biochrom (Berlin, Germany); multiwell plates were from Falcon (Franklin Lakes, NJ); rabbit polyclonal antibodies raised against total p21Waf1/Cip1 (sc-397) or phospho-specific p21 [Thr 145] (sc-20220R), histone deacetylase inhibitor 1 (HDAC1) (sc-7872), and actin (sc-8432) were from Santa Cruz Biotechnology (Heidelberg, Germany); antibodies against total caspase-3 [catalog number (Cat. No.) 9662], cleaved caspase-3 (Cat. No. 9662), total Akt (Cat. No. 9272), and Pi-Akt [Ser 473] (Cat. No. 9271) were from Cell Signaling Technology (Frankfurt, Germany); mouse anti-microtubule-associated protein 2 (Map-2) antibody was obtained from Millipore (Hofheim, Germany), secondary anti-rabbit horseradish peroxidase-linked antibody was from GE Healthcare (Braunschweig, Germany); all other secondary antibodies [fluorescein isothiocyanate (FITC) or Rhodamine Red-X coupled] were obtained from Jackson ImmunoResearch (West Grove, PA); enhanced chemiluminescence kits were from GE Healthcare and Pierce Biotechnology (Rockford, IL); x-ray films were from Kodak (Stuttgart, Germany); Hoechst 33258 and high-range molecular-weight standard was from Sigma. Ethylcholine aziridinium (AF64A) was prepared from acetylethylcholine mustard (Sigma) according to Fisher et al. (1982). Fugene was obtained from Roche (Grenzach-Wyhlen, Germany).
Primary neuronal cell cultures.
Primary neuronal cultures of cerebral cortex were obtained from embryos [embryonic day 16 (E16) to E18] of Wistar rats (Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin, Berlin, Germany) or from mouse embryos from p21Waf1/Cip1 “knock-out” (p21Waf1/Cip1−/−) or their corresponding littermates (p21Waf1/Cip1+/+) obtained from Jax mice (strain B6; 129S2-Cdkn1atm1Tyj/J; stock number 003263; The Jackson Laboratory, Bar Harbor, ME) as described previously (Harms et al., 2000, 2004). Briefly, cerebral cortices were dissected, incubated for 15 min in trypsin/EDTA (0.05/0.02% w/v in PBS) at 36.5°C, rinsed twice with PBS and once with dissociation medium (modified Eagle's medium with 10% fetal calf serum, 10 mm HEPES, 44 mm glucose, 100 U of penicillin plus streptomycin per milliliter, 2 mm l-glutamine, 100 IE insulin/L), dissociated by Pasteur pipette in dissociation medium, pelleted by centrifugation (210 × g for 2 min at 21°C), redissociated in starter medium (Neurobasal medium with supplement B27, 100 U of penicillin plus streptomycin per milliliter, 0.5 mm l-glutamine, and 25 μm glutamate), and plated out in 24-well or six-well plates at a density of 150,000 or 100,000 (on coverslips for immunocytochemistry) cells/cm2. Wells were coated with poly-l-lysine for 1 h at room temperature (0.5% w/v in PBS), rinsed with PBS, and incubated with coating medium (dissociation medium with 0.03‰ w/v collagen G) for 1 h at 36.5°C with two rinsing steps with PBS. Cells were seeded in starter medium. Primary cortical neurons were cultivated with serum-free Neurobasal medium with B27 supplement for 10–14 d in vitro (DIV) before starting with the experiment. Supplement B27 contained ∼58 nm corticosterone.
Injury paradigm.
Serum-free primary neuronal cultures were used after 9–11 DIV. The condition of cells at various time points after induction of injury was determined morphologically by phase-contrast microscopy. The neurotoxin AF64A was added to reach the final concentration of 40 μm and remained in the medium throughout the observation period of 72 h instead of a 5 h exposure followed by replacement of conditioned medium as described previously (Harms et al., 2000, 2001). The effect of the toxin was comparable in both procedures. Controls were exposed an equivalent amount of vehicle.
Staurosporine was dissolved in DMSO (10 mm stock solution) and diluted with PBS to give the final concentration of 300 nm in culture. The vehicle-treated cultures received the same amount of DMSO in PBS.
Glutamate exposure was performed with 100 μm glutamate for 30 min with subsequent rinsing and reapplication of conditioned medium.
Treatment with corticosterone, spironolactone, mifepristone, LY294002, and cycloheximide.
Corticosterone was dissolved in DMSO (250 mm stock solution) and added to the cell cultures in a dose range of 1–50 μm (final concentration in the medium) at different time points before and after the initiation of the injury. Spironolactone was dissolved in DMSO (50 mm stock solution), and mifepristone was dissolved in DMSO (10 mm stock solution; higher concentrations were not soluble in DMSO) and diluted in medium. The final concentration in the medium was 10 μm. Consequently, corticosterone was used in equimolar concentrations. LY294002 was dissolved in DMSO (65 mm stock solution) with 5 μm as final concentration. SH6 was dissolved in DMSO (1 mm stock solution) with 5 μm as final concentration. Cycloheximide, dissolved in medium, was added to give a final concentration of 500 ng/ml medium. Spironolactone, mifepristone, LY294002, or cycloheximide was added at the same time point as corticosterone.
Cell death assays.
Neuronal injury was quantitatively assessed by the measurement of LDH in the medium (Koh and Choi, 1987) at 48 or 72 h after AF64A application or by measurement of caspase-3 activity (Harms et al., 2004). Cell viability was assessed after staining of naive cell cultures with propidium iodide to distinguish living and dead cells (0.001 mg/ml for 5 min with subsequent rinsing) as described previously (Endres et al., 2004). Pictures from both phase-contrast images and fluorescent pictures were taken using a digital camera. Pictures were merged, and viable and dead neuronal cells were counted and presented as a percentage of viable neurons of all cells. To assess neurons with morphological features of apoptosis with chromatin condensation, nuclear membrane blebbing, retraction of dendrites, and apoptotic bodies, cultures were subjected to indirect fluorescence immunocytochemistry against the cytoplasmic neuronal marker Map-2 (1:500; developed with tetramethylrhodamine isothiocyanate-conjugated secondary antibody) and a DNA counterstain with bis-benzimide (Hoechst 33258) as described previously (Harms et al., 2004). Neurons (200) were counted for each condition, and the number of healthy neurons was calculated as a percentage of control.
Quantitative real-time reverse transcription-PCR of p21Waf1/Cip1 mRNA.
Total cellular RNA from primary cortical neurons was isolated, and RNA preparation and cDNA synthesis were performed as described previously. Expression of each sample was normalized for RNA preparation and reverse transcriptase reaction on the basis of β-actin mRNA content (Ruscher et al., 2002, Prass et al., 2003). For detection of the amplification products in β-actin and p21Waf1/Cip1 reverse transcription-PCR, we used the LightCycler and FastStart DNA Master kit (Roche Molecular Biochemicals, Penzberg, Germany), as recommended by the manufacturers. Thermal cycling started with 10 min at 95°C and proceeded with 55 cycles of 95°C for 15 s, 68°C for 10 s, and 72°C for 15 s (amplification product data acquisition at 90°C for p21Waf1/Cip1 and 86°C for β-actin). For amplification and detection, we used the LightCycler Relative Quantification software (Roche Molecular Biochemicals). The following sequence-specific primers (Tibmolbiol, Berlin, Germany) were used: β-actin forward (fwd), 5′-ACCCACACTGTGCCCATCTA-3′; β-actin reverse (rev), 5′-GCCACAGGATTCCATACCCA-3′; p21Waf1/Cip1 fwd, 5′-AGAGTGCAAGACAGCGACAAGG-3′; and p21Waf1/Cip1 rev, 5′-GGTGATGTCCGACC-TGTTCCG-3′.
Immunoblots.
Immunoblots were performed as described previously (Katchanov et al., 2001). NP-40 buffer was used for whole cellular extracts of cortical neuron (50 mm Tris-HCl, pH 7.5, 250 mm NaCl, 0.5% NP-40, 5 mm EDTA, pH 8.0, 1 mm phenylmethylsulfonylfluoride, 20 mm NaF, 1 mm Na3VO4, and protease inhibitor mixture; Roche). Samples containing 30 μg of protein or the equivalent of 25,000 cells in lysis buffer were subjected to SDS-PAGE (4–20% gradient Precise Protein Gels; Pierce Biotechnology) and immunoblotting procedure. Primary antibodies were used in a concentration of 0.2 μg/ml overnight at 4°C on a platform with gentle agitation, and horseradish peroxidase-linked secondary antibodies were used at 1:5000 for 1 h at room temperature. Detection was performed using the enhanced chemiluminescence assay (GE Healthcare).
Cellular fractionation.
Cellular fractionation was performed 24 h after exposure to AF64A using ice-cold cell lysis buffer (10 mm HEPES, pH 7.5, 2 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 10 mm KCl, 10 mm NaF, and 0.1 mm Na3VO4 with one tablet of protease inhibitor mixture per 25 ml of buffer and freshly added 1 mm DTT) to harvest cells using a cell scraper and 100 μl of buffer per six wells. For each condition, six wells were pooled. Cells were incubated for 15 min on ice, and 10 μl of 10% NP-40 was added followed by centrifugation for 1 min at 13,000 rpm at 4°C. The supernatant was taken as the cytoplasmic fraction. The pellet was washed twice using cell lysis buffer, and 15 μl of nuclear extraction buffer per well was added (25 mm HEPES, pH 7.5, 500 mm NaCl, 10 mm NaF, 10% glycerol, 0.2% NP-40, 5 mm MgCl2, and freshly added protease inhibitors and 1 mm DTT) followed by sonification and centrifugation at 13,000 rpm for 5 min. The supernatant was taken as the nuclear fraction. Protein content was determined, and equal loading and fractionation were evaluated using antibodies raised against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Millipore, MAB374 as a cytoplasmic marker, and HDAC1 (sc-7872; Santa Cruz Biotechnology) as a nuclear marker.
Immunocytochemistry.
For immunocytochemical analysis of cell cultures, cells were seeded onto glass coverslips at a density of 100,000 cells/cm2, fixed with 3.7% formaldehyde in PBS for 8.5 min, permeabilized with 0.1% Triton X-100 in PBS (8.5 min), and exposed to blocking solution (PBS containing 5% goat serum and 0.2% Tween 20) for 1 h at room temperature. Cultures were then incubated with primary antibodies and developed with Rhodamine Red-X- or FITC-labeled secondary antibody. Hoechst dye was used for genomic DNA counterstain. Control slides were treated the same way except that primary antibodies were omitted, resulting in no visible staining.
Confocal pictures and three-dimensional rendering of confocal images.
Confocal pictures and three-dimensional rendering of confocal images were performed with Velocity imaging software (Improvision, Coventry, UK). All confocal microscopy was performed using a spectral confocal microscope (TCS SP2; Leica, Nussloch, Germany). Appropriate gain and black level settings were determined on control slides stained with secondary antibodies alone. Colocalization images were processed with Photoshop version 6.0 (Adobe Systems, San Jose, CA).
Antisense transfections.
The following synthetic HPLC-purified oligodeoxynucleotides (BioTez, Berlin-Buch, Germany) were used to decrease expression of rat p21Waf1/Cip1 (GenBank U24174): 5′-GGCAGGCGCCGATCCACAGCG-3′ (sense), 5′-CGCTGTGGAT CGGCGCCTGCC-3′ (antisense). These sequences were not significantly homologous to genes other than rat p21 by BLAST (basic local alignment search tool) search (National Center for Biotechnology Information). Cortical neurons were seeded on glass coverslips at a density of 150,000 cells/cm2 and transfected with p21 sense or antisense oligonucleotides or fugene (Roche) alone on DIV 9 24 h before pretreatment with 50 μm corticosterone. Optimal conditions for transfection were achieved according to the manufacturer's instructions. Briefly, 1 μl of distilled water containing 1 μg of oligodeoxynucleotides and 2 μl of fugene were incubated separately in 20 μl of OptiMEM for 20 min, incubated for an additional 30 min before addition of 300 μl of Neurobasal medium, and added to a 24-well plate with cortical neurons. The medium was replaced after 6 h with Neurobasal medium from sister cultures. Cells were fixed with paraformaldehyde 48 h after AF64A, and immunocytochemistry was performed with antibodies against p21Waf1/Cip1 (green) to demonstrate endogenous levels of p21Waf1/Cip1 or Map-2 (red) to reveal neuronal origin. “Knock-down” efficiency was determined by endogenous levels of p21Waf1/Cip1 and cell counts. Sister cultures were treated with fugene alone. Nuclear morphology was revealed by Hoechst staining. A total of 200 Map-2-positive neurons from five high-power fields taken from three different slides per group were evaluated as viable/healthy or dead/severely damaged and presented as a percentage of all Map-2-positive neurons. Experiments were performed in triplicate.
Recombinant adenoviral constructs and transfections.
Ad-p21Waf1/Cip1 and Ad-β-galactosidase (β-Gal) were used as described previously (Hauck et al., 2002). Virus propagation and purification were performed as described previously (von Harsdorf et al., 1999). At 24 h after adenoviral infection (100 plaque-forming units/cell) cortical neurons were exposed to AF64A for 48 h. The expression and nuclear localization of ectopically expressed proteins were confirmed by fluorescence microscopy of fixed cells using immunostaining with specific antibodies (data not shown).
Results
Corticosterone protects neurons from apoptosis via activation of the PI3-Akt-kinase pathway
Differentiated rat primary cortical neurons were exposed to AF64A, leading to full cell disintegration by 48–72 h. As reported previously, neuronal cells react to AF64A with a delayed, caspase-dependent apoptotic cell death. The caspase dependence has been proven both by the broad-spectrum caspase inhibitor Z-VAD-FMK and the caspase-3-specific inhibitor Z-DQMD-FMK (Harms et al., 2000, 2004). The apoptotic cell death has been confirmed previously by DNA laddering, α-fodrin cleavage, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, Hoechst 33258 staining, and morphological appearance in phase-contrast microscopy (Harms et al., 2000, 2001, 2004; Lautenschlager et al., 2000). Release of LDH into the culture medium served as an indirect marker of cell death. Pretreatment with high-dose corticosterone (1–50 μm) protected cortical neurons in a time- and dose-dependent manner from AF64A and also completely reduced activation and cleavage of caspase-3 (maximum protection achieved with 20 h pretreatment) (see supplemental Table 1, available at www.jneurosci.org as supplemental material; Fig. 1A,C,F). Cotreatment with antagonists of either glucocorticoid or mineralocorticoid receptor subtypes at concentrations equimolar to that of corticosterone completely bypassed the neuroprotective effect of corticosterone (Fig. 1B). Pretreatment of receptor antagonist without corticosterone did not alter AF64A toxicity significantly (data not shown).
The neuroprotective effect of corticosterone (50 μm) was tested in two additional models of neurodegeneration, including exposure to staurosporine or glutamate. A significant decrease in LDH release was achieved 24 and 48 h after staurosporine. After glutamate exposure, the acute excitotoxic damage at 24 and 48 h was not antagonized by corticosterone, whereas the late increase between 48 and 72 h was prevented (supplemental Table 2, available at www.jneurosci.org as supplemental material).
To test whether the protective effect of corticosterone was mediated via the PI3-Akt-kinase-pathway, we coadministered either the PI3-kinase inhibitor LY294002 or the Akt-kinase inhibitor SH6. Both inhibitors completely reversed the neuroprotective effect of corticosterone (Fig. 1D). Moreover, corticosterone treatment induced transient (i.e., 30–180 min, maximum after 90 min) phosphorylation of Akt-kinase at serine 473, which was completely blocked by SH6 (Fig. 1E). This increase in phosphorylation was not associated with an increase in total Akt expression. Together, these results demonstrate that corticosterone protects from AF64A-induced apoptosis via activation of the PI3-Akt-kinase pathway.
Corticosterone confers enhanced phosphorylation and cytoplasmic appearance of p21Waf1/Cip1 via Akt-kinase
The cell cycle inhibitor p21Waf1/Cip1 has an Akt-kinase-specific phosphorylation site at threonine 145 (Zhou et al., 2001). Using an antibody directed against the p21-Thr 145 phosphorylation, we tested whether corticosterone (via Akt-kinase) would confer phosphorylation and increase the cytoplasmic appearance of phosphorylated p21Waf1/Cip1 (Pi-p21). Confocal images, including multiple z-plane images and three-dimensional reconstruction demonstrated that Pi-p21 [Thr 145] was present at low levels in the nucleus of naive Map-2-stained neurons but was hardly visible in the cytoplasm (Fig. 2A,B). After treatment with corticosterone, Pi-p21 [Thr 145] was upregulated and expressed in both nucleus and cytoplasm, which was completely reversed by cotreatment with SH6 (Fig. 2C). Treatment with AF64A resulted in a profound downregulation of Pi-p21 [Thr 145] in untreated but not in corticosterone-treated neurons (Fig. 2A). Immunoblotting showed an increase of Pi-p21 [Thr 145] 1 h after corticosterone treatment, whereas total p21Waf1/Cip1 remained unaffected (Fig. 2C). After AF64A, both total p21Waf1/Cip1 and Pi-p21 [Thr 145] levels markedly declined, which was prevented in the presence of corticosterone. In contrast, cotreatment with the Akt-kinase inhibitor SH6 resulted in a time-dependent marked loss of both total and Thr 145-phosphorylated p21Waf1/Cip1 (Fig. 2C). For further identification of the cellular localization of Pi-p21, we performed fractionation of nucleus and cytosol followed by Western blot analysis of Pi-p21. Under control conditions, Pi-p21 was exclusively detectable in the nucleus fraction, whereas after treatment with corticosterone, Pi-p21 was distributed both in the nucleus and cytosol. After AF64A treatment, Pi-p21 almost completely disappeared in the nucleus and was not detectable in the cytosol. After pretreatment with corticosterone, the AF64A-induced loss of Pi-p21 was partly prevented and Pi-p21 appeared in the cytosol. Moreover, in the presence of SH6, Pi-p21 was almost not detectable in the nuclear fraction under basal conditions, and the corticosterone-induced increase in the cytosol was completely prevented (Fig. 2D). Together, these results indicate that corticosterone induces phosphorylation at T145, and cytoplasmic shuttling of p21Waf1/Cip1 is mediated by Akt-kinase. Interestingly, phosphorylation of Akt-kinase was a transient event with a maximum at 60–90 min after addition of corticosterone (Fig. 1E), whereas increased cytoplasmic and nuclear expression of Pi-p21 [Thr 145] was still evident 44 h after corticosterone (Fig. 2A,B).
Corticosterone induces p21Waf1/Cip1-gene transcription
The p21Waf1/Cip1 promoter region contains a glucocorticoid receptor response region. Therefore, we tested whether, in addition to increased phosphorylation, corticosterone treatment would increase p21Waf1/Cip1 gene and protein expression. Real-time PCR, immunoblots, and immunocytochemistry demonstrated that corticosterone dose- and time-dependently induced p21Waf1/Cip1 mRNA synthesis and protein expression (Fig. 3A–D). In the physiological range (0.1 μm), corticosterone did not affect the level of p21Waf1/Cip1 mRNA. However, in the dose range of 1–50 μm corticosterone, the mRNA levels dose-dependently increased 24 h after treatment, correlating well with the neuroprotective potency of corticosterone (Fig. 1A). The increase in p21Waf1/Cip1 mRNA was completely abolished by cotreatment with antagonists of either glucocorticoid or mineralocorticoid receptor subtypes (Fig. 3B). Moreover, inhibition of Akt-kinase by SH6 not only reversed the corticosterone-induced increase in p21Waf1/Cip1 mRNA, but even decreased its level under basal conditions (Fig. 3C). Similar to the findings with Pi-p21 [Thr 145] immunocytochemistry, p21Waf1/Cip1 was mainly present in the nucleus of naive neurons but was found in both nucleus and cytoplasm of corticosterone-treated neurons. AF64A conferred a profound loss of p21Waf1/Cip1, which was prevented by corticosterone pretreatment (Fig. 4A,B). The protein synthesis inhibitor cycloheximide completely blocked the neuroprotective effects of corticosterone (Fig. 4C), which suggests that upregulation of p21Waf1/Cip1 expression (possibly along with other proteins) is necessary for corticosterone-mediated neuroprotection.
An alternative explanation for the neuroprotective effects of corticosterone would be inhibition of cdk activity, because nuclear p21Waf1/Cip1 inhibits cdk2. Indeed, AF64A significantly activated cdk2 (see also Katchanov et al., 2001); cotreatment with corticosterone, however, did not inhibit cdk2 activation induced by AF64A as shown in a preliminary study (data not shown).
p21Waf1/Cip1 is essential for the neuroprotective effect of corticosterone
To test whether p21Waf1/Cip1 is essential for the neuroprotective effect of corticosterone, we transfected neurons with antisense deoxynucleotide to reduce the amount of p21Waf1/Cip1 (Fig. 5A,B). Successful knock-down of p21Waf1/Cip1 protein expression was confirmed by immunostaining of fixed cells (Fig. 5B). Transfection with p21Waf1/Cip1 antisense oligonucleotides, but not with sense oligonucleotides, abrogated the neuroprotective action of corticosterone as assessed by cell counts of viable Map-2-positive neurons (Fig. 5A).
In addition, we prepared primary cortical neurons from mice deficient in p21Waf1/Cip1 gene expression (p21Waf1/Cip1 knock-out mice) along with wild-type littermates, which showed no obvious differences in cellular viability at baseline or after AF64A (as determined by LDH, phase-contrast morphology, and propidium iodide staining). Corticosterone pretreatment provided significant protection in wild-type neurons; however, it did not protect p21Waf1/Cip1 knock-out neurons at all (Fig. 5C–E). Similarly, the protection achieved by corticosterone against staurosporine- or glutamate-induced neuronal damage was not detectable in p21Waf1/Cip1 knock-out neurons (supplemental Table 3, available at www.jneurosci.org as supplemental material). Together, these experiments demonstrate that p21Waf1/Cip1 is essential for the neuroprotective effects of corticosterone.
Ectopic expression of p21Waf1/Cip1 confers neuroprotection
Next, we tested whether ectopic expression of p21Waf1/Cip1 by adenovirus delivery would protect neurons from AF64A. Transduction efficiency was close to 100%, as determined by β-galactosidase staining and colocalization with Map-2 (data not shown). Adenovirally delivered p21Waf1/Cip1 protected neurons from AF64A-induced cell death to a degree similar to corticosterone. Cotreatment of corticosterone plus adenoviral delivery of p21Waf1/Cip1 conferred a small but significant additional protective effect compared with p21Waf1/Cip1 or corticosterone alone (Fig. 6A,B). Interestingly, in these neurons, retraction of dendrites and nuclear membrane blebbing in response to AF64A were also inhibited (Fig. 6A).
Discussion
Our study has the following major findings: (1) high-dose corticosterone protects primary cortical neurons from apoptotic cell death induced by the neurotoxin AF64A in vitro, and this appears to involve both glucocorticoid and mineralocorticoid receptor signaling. Neuroprotection by corticosterone could also be demonstrated in staurosporine-induced apoptosis and in the delayed cell death after glutamate exposure. (2) Neuroprotection by corticosterone is dependent on the activation of both PI3-kinase and Akt-kinase. (3) Via PI3-Akt-kinase activation, corticosterone mediates phosphorylation at Thr 145 and cytoplasmic appearance of the cdk inhibitor p21Waf1/Cip1. In addition, corticosterone dose-dependently increases total p21Waf1/Cip1 gene and protein expression. (4) Inhibition of Akt-kinase-dependent phosphorylation and subsequent cytoplasmic appearance of Pi-p21 completely blocks the neuroprotective effect of corticosterone. (5) p21Waf1/Cip1 is essential for neuroprotection by glucocorticoids, because corticosterone did not protect neurons at all from damage induced by AF64A, staurosporine, or glutamate, when p21Waf1/Cip1 gene expression was abolished either by oligonucleotide treatment or gene deletion. (6) Last, ectopic expression of p21Waf1/Cip1 provides neuroprotection independent of (but similar to) corticosterone treatment. Together, this study identifies p21Waf1/Cip1 as a novel antiapoptotic therapeutic target in postmitotic neurons and demonstrates that it serves as the molecular mechanism of neuroprotection by high-dose glucocorticoids.
Activation of PI3-Akt-kinase is required for the neuroprotective effects of corticosterone
PI3-Akt-kinase activation is necessary for the antiapoptotic effects of corticosterone. Neuroprotection was completely abolished by LY294002, an inhibitor of PI3 kinase, and by SH6, an Akt-kinase inhibitor. Akt-kinase became active as early as 30 min after corticosterone treatment. Indeed, rapid, nongenomic effects of high-dose glucocorticoid treatments have been described in non-neuronal cells. For example, nontranscriptional activation of endothelial nitric oxide synthase in endothelial cells by high-dose glucocorticoids confers acute cardioprotective effects, and this is mediated via the glucocorticoid receptor and PI3-Akt-kinase (Hafezi-Moghadam et al., 2002). Surprisingly, then, in our study maximal neuroprotection was achieved only after 20 h corticosterone pretreatment. This and the fact that neuroprotection was abolished by the protein synthesis inhibitor cycloheximide indicate that (in addition to PI3-Akt-kinase activation) synthesis of new proteins is necessary for corticosterone-induced neuroprotection. The potential of our evidence for the involvement of PI3-Akt-kinase in neuroprotection is limited, however, because most of the results refer to the use of SH6 only.
p21Waf1/Cip1 as a novel antiapoptotic target of glucocorticoids
Corticosterone induced phosphorylation of p21Waf1/Cip1 at Thr 145 and subsequent cytoplasmic appearance of Pi-p21, which could be completely blocked by Akt-kinase inhibition. Although phosphorylation of Akt-kinase was transient, increased levels of Pi-p21 [Thr 145] and its cytoplasmic translocation persisted over time. Indeed, in non-neuronal cells, p21Waf1/Cip1 after phosphorylation and cytoplasmic shuttling exerts antiapoptotic effects (Porter, 1999; Zhou et al., 2001; Li et al., 2005). Moreover, corticosterone induced a dose-, glucocorticoid and mineralocorticoid receptor-, and Akt-kinase-dependent upregulation of p21Waf1/Cip1 gene and protein expression in postmitotic neurons. An upregulation of p21Waf1/Cip1 has previously been demonstrated in proliferating cells only (Corroyer et al., 1997; Ramalingam et al., 1997; Cha et al., 1998; Cram et al., 1998; Terada et al., 2001). Akt-kinase controls p21Waf1/Cip1 at both the transcriptional and posttranslational levels (Rössig et al., 2002; Maddika et al., 2007). Our finding that the corticosterone-induced increase in p21Waf1/Cip1 mRNA was reversed both by receptor antagonism and Akt-kinase inhibition indicates a cooperative interaction of both systems at the p21Waf1/Cip1 gene.
Corticosterone also prevented the dramatic loss of p21Waf1/Cip1 after AF64A preceding neuronal apoptosis. Activation of cdks and cell cycle reentry has been identified as a pathway for neuronal apoptosis [for review, see Becker and Bonni (2004) and Greene et al. (2004)]. Nuclear p21Waf1/Cip1 acts as a cdk2 inhibitor; hence, upregulation of p21Waf1/Cip1 by corticosterone would provide an attractive mechanism of action for the neuroprotective effects of glucocorticoids. However, our results indicate that corticosterone treatment did not result in a decline of cdk2 activation induced by AF64A as shown in a preliminary study.
p21Waf1/Cip1 is essential/required for corticosterone-mediated neuroprotection: the neuroprotective effect of corticosterone was abolished when neurons were transfected with p21Waf1/Cip1 antisense oligonucleotides and also in neurons obtained from p21Waf1/Cip1−/− mice. Moreover, ectopic expression of p21Waf1/Cip1 provided neuroprotection that was comparable with that of corticosterone. Together, these results indicate that upregulation, phosphorylation of p21Waf1/Cip1, and cytoplasmic appearance of Pi-p21 are essential for neuroprotection mediated by corticosterone.
Corticosterone inhibits cleavage of caspase-3
The inhibition of caspase-3 cleavage appears to play a prominent role in neuroprotection mediated by corticosterone. Cytoplasmic p21Waf1/Cip1 may directly inactivate caspase-3 by forming a complex with procaspase-3 in mitochondria that is dependent on phosphorylation (Suzuki et al., 1998, 1999a,b, 2000). Indeed, corticosterone significantly reduced caspase-3 cleavage and enzymatic activity after AF64A, which was antagonized by Akt-kinase inhibition (Fig. 1).
In conclusion, we identify p21Waf1/Cip1 as the molecular mediator of neuroprotection by glucocorticoids in primary cortical neurons. High-dose corticosterone confers phosphorylation and possibly cytoplasmic translocation of Pi-p21 via PI3-Akt-kinase signaling as well as upregulation of p21Waf1/Cip1 gene and protein expression. p21Waf1/Cip1 emerges as a novel molecular target for the treatment of neurodegenerative diseases.
Footnotes
-
This work was supported by VolkswagenStiftung (Lichtenberg Program; M.E.), Deutsche Forschungsgemeinschaft (M.E., U.D.), Hermann and Lilly Schilling Stiftung (U.D.), and Charité Universitätsmedizin Berlin. We are grateful to Anny Kretschmer, Claudia Muselmann, Daniela Grothe, and Astrid Arnswald for excellent technical assistance.
- Correspondence should be addressed to either of the following: Dr. Matthias Endres, Klinik und Poliklinik für Neurologie, Charitéplatz 1, D-10117 Berlin, Germany, matthias.endres{at}charite.de; or Dr. Heide Hörtnagl, Institut für Pharmakologie und Toxikologie, Charité, Dorotheenstrasse 94, D-10117 Berlin, Germany, heide.hoertnagl{at}charite.de