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Oxidative Stress Induces p53-Mediated Apoptosis in Glia: p53 TranscriptionIndependent Way to Die
Paolo Bonini,1 Simona Cicconi,4 Alessio Cardinale,2 Cristiana Vitale,3 Anna Lucia Seraﬁno,4 Maria Teresa Ciotti,5 and Lionel N.J-L. Marlier4*
Department of Internal Medicine, University of Rome Tor Vergata, Rome, Italy Department of Neuroscience, University of Rome Tor Vergata, Rome, Italy 3 Department of Medical Science, San Raffaele Roma Hospital, Rome, Italy 4 Section of Molecular Medicine, Signal Transduction of Apoptotic Mechanisms Laboratory, National Research Council (CNR), Institute of Neurobiology and Molecular Medicine (INeMM), Rome, Italy 5 Section of Neuroscience, National Research Council (CNR), Institute of Neurobiology and Molecular Medicine (INeMM), Rome, Italy
Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the central nervous system (CNS). The tumor suppressor protein p53 plays a pivotal function in neuronal apoptosis triggered by oxidative stress. We investigated the role of p53 and related molecular mechanisms that support oxidative stress-induced apoptosis in glia. For this purpose, we exposed C6 glioma cells and primary cultures of rat cortical astrocytes to an H2O2-induced oxidative stress protocol followed by a recovery period. We evaluated the effects of piﬁthrin- (PF- ), which has been reported to protect neurons from ischemic insult by speciﬁcally inhibiting p53 DNA-binding activity. Strikingly, PF- was unable to prevent oxidative stress-induced astrocyte apoptosis. We demonstrate that p53 is able to mediate an apoptotic response by direct signaling at mitochondria, despite its transcriptional activity. The z-VAD-fmksensitive apoptotic response requires a caspasedependent MDM-2 degradation, leading to p53 mitochondrial targeting accompanied by cytochrome c release and nucleosomal fragmentation.
© 2003 Wiley-Liss, Inc.
Key words: astrocyte; C6 glioma; ischemia; MDM2; cytochrome; mitochondria; caspase
mediator of stress response because it plays an essential role in the death of many cell types, other than neurons. It is well known that p53 induces cell death by a multitude of molecular pathways involving activation of target genes (i.e., pro-apoptotic Bax) (Miyashita and Reed, 1995; Jordan et al., 1997; Brady et al., 1998; Xiang et al., 1998; Morrison et al., 2003). Nevertheless, evidence for transcription-independent pathways of p53-mediated apoptosis is accumulating (Gottlieb and Oren, 1998; Sansome et al., 2001), showing that p53 can contribute to apoptosis by direct signaling at the mitochondria (Marchenko et al., 2000) leading to cytochrome c (cyt-c) release and caspase activation (Schuler et al., 2000). A key player in p53 regulation is MDM-2 protein. MDM-2 can inhibit p53 binding within its transactivation domain interfering with recruitment of basal transcription machinery components. Moreover, MDM-2 can lead to complete elimination of p53 through the ubiquitinproteasome pathway (Momand et al., 1992, 2000; Barak et al., 1993; Wu et al., 1993; Zauberman et al., 1993; Chen et al., 1996; Haupt et al., 1996). In addition, p53 binds specifically to MDM-2 gene promoter establishing an important autoregulatory feedback loop (Barak et al., 1993; Wu et al., 1993; Haupt et al., 1997; Kubbutat et al., 1997). Recently, a small molecule was isolated for its ability to reversibly block p53 transcriptional activity. This compound,
Contract grant sponsor: San Raffaele Roma Hospital; Contract grant sponsor: CNR-INeMM; Contract grant sponsor: the Italian Ministry of University Research, and Technology (MURST). *Correspondence to: Lionel N.J-L. Marlier, National Research Council, Institute of Neurobiology and Molecular Medicine (INeMM), Viale Marx 15-43, 00137 Rome, Italy. E-mail: email@example.com Received 10 January 2003; Revised 15 August 2003; Accepted 17 August 2003
Apoptotic cell death plays an important role in brain development as well as in neuronal injury and disease. It is now generally accepted that massive neuronal death due to oxidative stress is a common characteristic of brain whether in stroke (Culmsee et al., 2001b; Cheng et al., 2003; Love, 2003) or in neurodegenerative diseases such as Alzheimer’s disease (de la Monte et al., 1997, 1998), Parkinson’s disease (Jenner, 2003; Tatton et al., 2003), and traumatic brain injury (Napieralski et al., 1999). The p53 tumor suppressor protein has been proposed as a key
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Bonini et al.
called piﬁthrin- (PF- ), protects neurons from apoptosis induced by ischemic injury and excitotoxic insults. Moreover, PF- protects mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors (Komarov et al., 1999). Protection by PF- was correlated with decreased p53 DNA-binding activity, decreased expression of the p53 target gene Bax and suppression of mitochondrial dysfunction and caspase activation (Culmsee et al., 2001a,b). The present study was undertaken to investigate the molecular mechanisms that support oxidative stressinduced apoptosis in glial cells. For this purpose, we designed an in vitro model of H2O2-induced oxidative stress using both rat primary cultures of astrocytes and C6 rat glioma cell line. We examined the role of p53 on the onset of apoptosis and investigated whether PF- was able to prevent glial cell death.
MATERIALS AND METHODS Ethics Animals used for astrocyte preparations received humane care according to the standard criteria regarding animal use in EEC. C6 Cell Line C6 cells were maintained in log-phase growth by regular passage using Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 g/ml streptomycin (Invitrogen, Milan, Italy). Cells were subcultured twice a week and maintained at 37°C in an atmosphere of 95% humidiﬁed air and 5% CO2. Primary Cultures of Rat Astrocytes Rat primary cultures of astrocytes were prepared from cortex of embryonic Day 14 (E14) rat embryos as described previously in detail (Mercanti et al., 1987). Oxidative Stress/Recovery Protocol To mimic oxidative stress cells were exposed to 100 M H2O2/100 M FeSO4 (Fenton reagent) in Hank’s balanced saline solution (HBSS; Invitrogen) for 1 hr (the stress period) followed by a recovery period consisting of replacing cells in normal growth medium (Joseph et al., 1997; Richter-Landsberg and Vollgraf, 1998; Avshalumov and Rice, 2002). The basal point refers to the intact cells whereas the T0 time point refers to the end of the stress period/beginning of the recovery period. The cells were preincubated 3 hr with zVAD-fmk (20 M ﬁnal concentration; Biomol, Plymouth Meeting, PA) and PF- (100 nM ﬁnal concentration; Calbiochem, San Diego, CA) before the stress period and both zVAD-fmk and PFwere then replaced during the recovery period. PCR Analysis Total RNA was extracted using the TRIAzol reagent according to the manufacturer’s recommendation (Invitrogen) and extracted RNA was used for reverse transcriptionpolymerase chain reaction (RT-PCR) analysis. Brieﬂy, 1 g
RNA was reverse transcribed for 1 hr at 42°C using 200 U Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen) in the presence of 2.5 M random hexamers and 200 M nucleotides (Amersham Pharmacia Biotech, MI, Italy) in 20 L ﬁnal volume. Successively, 2 L of each cDNA were PCR-ampliﬁed using 2.5 U Platinum Taq (Invitrogen) in the presence of 15 pmol of speciﬁc primers as follows:
● Bcl-2 up: 5 -TAT AAG CTG TCA CAG AGG G; ● BclxL/S up: 5 -TCC CAG AAA GGA TAC AGC; ● Bad up: 5 -CCA GAT CCC AGA GTT TGA GC; ● Bak up: 5 -TCC TGC CTC TCA GGG CAA; down: ● Bax up: 5 -TTC ATC CAG GAT CGA GCA G; ● Bid up: 5 -ACT CTG AGG TCA GCA ATG GC; ● p53 up: 5 -GCC ATC TAC AAG AAG TCA CA;
down: 5 -GTC TTC CAG CGT GAT GAT G CCG; down: 5 -ACA ATG TGC TGC TGC TTC TC down: 5 -TAG GGA AGG ATG TCT TCA CCT C down: 5 -TTC TTC CAG ATG GTG AGC G 5 -AGT GGT CAG GGA GGC ACT T down: 5 -CTT TCC CCA AAT TTC GAT CC down: 5 -TCA CTT CCG ACT GAA GAG down: 5 -CTC CCC ACA CAC ATG ACC
● MDM-2 up: 5 -CCA ACA TGT CTG TGT CTA ● H4 up: 5 -GGT AAA GGG CTT GGG AAA G; ● GAPDH up: 5 -ACC ACA GTC CAT GCC ATC
AC; down: 5 -TCC ACC ACC CTG TTG CTG TA To normalize the cDNA amount to be used during the PCR ampliﬁcation, pilot experiments were carried out using histone 4 (H4) speciﬁc primers (not shown). For all templates, 30 cycles were used allowing us to consider the basal level as a real representation of relative expression levels of genes of interest. To verify whether after 24 hr reperfusion PF- remained able to block p53 transcriptional activity, RT-PCR analysis of mRNA expression levels for H4 (as a housekeeping control gene) and the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were carried out and the linear dynamic range analyzed by densitometry analysis using a computer-based imaging system (Fluor-S; Biorad, Hercules, CA). In all experiments, cDNAs were denatured initially for 5 min followed by 30 cycles consisting of: 45 sec denaturation; 30 sec annealing; and 50 sec extension (3 min ﬁnal extension) using a Perkin Elmer 9700 thermal cycler (Perkin Elmer, Foster City, CA). Cell Survival Analysis Astrocyte survival was assessed by counting the number of intact nuclei after lysing cells in detergent-containing solution as described initially by Soto and Sonnenschein (1985) and modiﬁed successively by Volonte et al. (1994). In all cases, 4 dishes were scored in 4 independent experiments. Immunoﬂuorescence Studies For immunoﬂuorescence studies, both C6 cells and primary astrocytes were seeded in poly-D-lysine-coated coverslips. Cells were washed three times in phosphate-buffered saline (PBS) and down: 5 -TTA ACC ACC GAA GCC GTA G
Astrocyte Apoptosis and p53
ﬁxed in freshly prepared 4% paraformaldehyde-PBS for 10 min at room temperature. Permeabilization was carried out in PBS/Triton X-100 (0.2%) for 5 min at room temperature. After three washes in PBS, cells were exposed to the appropriate dilution of primary antibodies in incubation buffer (0.2 mg/ml bovine serum albumin [BSA] in PBS) for 1 hr at room temperature. Cells were labeled for p53, cyt-c, and MDM-2. After three washes in PBS, cells were exposed to secondary antibody in the same buffer for 45 min at room temperature. Preparations were mounted with 70% glycerol in PBS. Samples were examined with a Leica ﬂuorescence microscope coupled to a CCD camera, equipped with a 100 oil immersion lens. The dye Hoechst 33258 (Sigma-Aldrich, MI, Italy) was used at 0.5 g/ml in PBS for 30 sec at room temperature before the cells were ﬁxed. Primary antibodies used were anti-p53 (1:200) and anti-cyt-c (1:200) from Pharmingen (Becton Dickinson, MI, Italy) and anti-MDM-2 (1:200) from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibody was a Texas Redconjugated anti-mouse IgG (1:300; Calbiochem). Confocal Fluorescent Imaging For confocal microscopy analyses, primary astrocyte cultures were incubated for 30 min with MitoTracker Red 580 (200 nM ﬁnal concentration; Molecular Probes Europe BV, Poortgebouw, The Netherlands) at the end of the recovery period then ﬁxed with freshly prepared 2.5% glutaraldehyde in PBS (Sigma-Aldrich) for 30 min at 4°C. Successively glutaraldehyde-induced autoﬂuorescence was quenched by incubating cultures in sodium borohydride (2 mg/ml for 3 10 min; Sigma-Aldrich). Cells were then permeabilized and processed for p53 immunoﬂuorescence using a ﬂuorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (1:500; Sigma-Aldrich) as above. Fluorescently labeled preparations were observed using a Leica TCS 4D confocal laser scanning microscope supplemented with an argon/krypton laser and equipped with 40 1.00 – 0.5 and 100 1.3– 0.6 oil immersion lenses. Confocal sections were taken at intervals of 0.5 m. Excitation/emission wavelengths employed were 488 nm/510 nm, for FITC labeling, and 568 nm/590 nm, for MitoTracker dye. The acquisitions were recorded, employing pseudo-color representation. Nucleosomal Formation Assay Both C6 cells and primary astrocytes were seeded in 24-well plates. After 48 hr and 8–10 days, respectively, the culture medium was removed and cells were exposed to the oxidative stress protocol detailed above. Control cells were incubated with HBSS only during the stress period. After recovery, cells were lysed and nucleosomal formation was assayed by quantitative in vitro measurement of cytoplasmic histone-associated DNA fragments (Cell Death Detection ELISAPLUS; Roche, MI, Italy), according to the manufacturer’s recommendations. Results are expressed as the enrichment factor obtained according to the formula:
ODexperimental ODbackground / ODcontrol ODbackground
basal apoptosis level, i.e., the assay of nucleosomes present in the culture immediately before stress (also termed basal). Data are presented as means SEM. Each experiment was repeated at least 3 times using different batches of cells. Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc comparison test. Results were considered statistically signiﬁcant when P value was below 0.05.
RESULTS Analysis of Cell Death, Nuclear Morphology, and Nucleosomal Formation Assay in Glial Cells After Reactive Oxygen Species Exposure Cell survival was analyzed in primary cultures of rat astrocytes (Fig. 1A). This analysis demonstrated that HBSS exposure during the stress period did not induce cell death even after 24 hr recovery. On the contrary, cultures exposed to reactive oxygen species (ROS) exhibited cell death: after 6 hr in untouched control cultures, there was 53.98 3.98% viable cells; after 12 hr, 45.43 7.41%; and after 24 hr, 28.91% 7.67. This demonstrated that after 24 hr recovery, more than 70% of the cells were dead. Similar data were obtained in C6 glioma cells (not shown). To determine whether cell death was due to apoptosis, a time course analysis of nuclear morphology using Hoechst 33258 labeling (Fig. 1B) and a nucleosomal formation assay (Fig. 1C) was carried out. Both C6 glioma cells and primary astrocytes exposed to HBSS during the stress period did not enter apoptosis, even after 24 hr recovery, as demonstrated by the absence of chromatin condensation and the lack of nucleosomal enrichment (Fig. 1B,C). On the contrary, after 12 hr recovery, apoptotic nuclei could be seen clearly in both cell cultures exposed to ROS, with a marked increase after 24 hr reperfusion (Fig. 1B). The same results were conﬁrmed by increased nucleosomal formation (Fig. 1C): the enrichment factor increased signiﬁcantly to 3.20 0.22 in C6 cells (P 0.05 vs. HBSS) and 2.07 0.36 in primary cultures of astrocytes (P 0.05 vs. HBSS) after 12 hr reperfusion. This increase was much more pronounced after 24 hr of reperfusion, reaching 8.90 0.45 and 6.69 1.30, respectively (P 0.01 vs. HBSS in both cases). Time Course Semiquantitative RT-PCR Analysis of Various Bcl-2 Family Members and p53Regulated Genes To test for possible involvement of p53 transcriptional activity in the onset of oxidative stress-induced apoptosis and, eventually, a relationship with Bcl-2 family member mRNA expression levels, we carried out a time course semiquantitative RT-PCR analysis on several potential targets (Fig. 2). This analysis demonstrated that in C6 glioma cells, Bcl-2, Bcl-xL, Bax, and Bak mRNA expression remained almost unchanged throughout the experiment. On the contrary, Bcl-xS and Bid transcripts were upregulated already during the stress period and their expression levels remained significantly higher compared to baseline during the recovery period. In addition, Bad mRNA tended to increase during recovery but this increase was more evident for MDM-2 and
where the experimental point refers to the assay of nucleosomes present at a given time point during stress or recovery periods, background consists of OD from ELISA plate dishes where cellular extracts were omitted, and the control point refers to the
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Genomic Effects of PF- and Role in Apoptotic Response We monitored after 24 hr of recovery the genomic effects of PF- on p53 transcriptional activity. We carried out a step-cycle RT-PCR analysis of mRNA expression levels in C6 cells for H4 (as a housekeeping control gene) and GAPDH was used as a positive control because it is known to be regulated by p53 (Chen et al., 1999). This experiment demonstrated that whereas the H4 housekeeping gene expression level remained unaffected by PF- treatment, the GAPDH expression level was decreased in a dose-dependent manner (20 –500 nM; Fig. 3A). Similar results were obtained on primary astrocytes (not shown). We next carried out a nucleosomal formation assay in parallel on C6 glioma cells and primary cultures of astrocytes exposed or not exposed to PFtreatment (100 nM) after 24 hr recovery (Fig. 3B). In both experimental models, 1 hr oxidative stress followed by 24 hr recovery markedly induced nucleosomal formation. This increase reached 8.56 0.45 in C6 cells and 7.33 0.75 in primary cultures of glia. In the presence of PF- , the enrichment factors in C6 cells and primary cultures of astrocytes were 7.65 0.65 and 6.54 0.60, respectively. No signiﬁcant differences were noted cells treated with PF- and untreated cells were compared. Immunoﬂuorescence Analysis of p53, Cyt-c, and MDM-2 Intracellular Distribution Time-course immunoﬂuorescence analysis of p53, cyt-c, and MDM-2 protein expression combined with Hoechst labeling of the nuclei showed that p53 was detectable almost exclusively in the nucleus until the end of the stress period in both experimental models (Fig. 4, top). The expression level of p53 increased progressively over the 24-hr recovery period, showing mainly a cytoplasmic localization of the protein in both experimental systems. The increase in p53 expression level was more pronounced in C6 cells when compared to that in primary astrocytes. Cyt-c distribution analysis in both experimental models (Fig. 4, center) evidenced a typical mitochondrial localization in intact cells after 1 hr stress; ﬁlamentous mitochondria were discerned clearly. After 3– 6 hr recovery, mitochondrial structure was altered profoundly, showing the typical apoptotic organization known as megamitochondria (MG). After 12 and 24 hr of recovery, changes in mitochondrial structure and apoptotic changes of cells were evidenced clearly and probably reﬂected cyt-c release. Hoechst labeling also evidenced chromatin condensation in both cell systems after 12–24 hr of recovery. Time course immunoﬂuorescence analysis of MDM-2 (Fig. 4, bottom), showed in both models a very low expression level in control cells and at the end of the stress period. After 3 hr of recovery, MDM-2 seemed strongly upregulated, showing a marked nuclear localization. In both experimental models, MDM-2 immunoreactivity started to decrease after 6 hr of recovery and after 12 hr was almost undetectable.
Fig. 1. A: Cell viability assay in primary astrocytes exposed to HBSS or ROS for 1 hr and followed by a 6-, 12-, or 24-hr recovery period. After ROS exposure, dead cells represent almost 50% of the population after 6 hr and reach more than 70% after 24 hr. B: C6 cells and primary astrocytes exposed to oxidative stress (0.1 mM FeSO4/H2O2 for 1 hr) undergo apoptosis only during the recovery period. Both cell types were exposed to ROS (or HBSS alone as control) for 1 hr and then allowed to recover for 12 and 24 hr. Hoechst staining shows that real apoptotic signs, such as nuclear shrinking and chromatin condensation, can be observed only after ROS exposure and 24 hr of recovery. Rare apoptotic nuclei were also observed in primary astrocytes after 12 hr of recovery. Arrows point to apoptotic nuclei. C: Nucleosomal formation assay demonstrating severe DNA fragmentation in both experimental models, particularly after 24 hr of recovery. *P 0.05; **P 0.01.
p53 mRNA; the latter was upregulated transiently but signiﬁcantly during recovery. In primary cultures of astrocytes, similar results occurred for all transcripts studied except Bcl-2, Bcl-xL, and MDM-2. Indeed, during 24 hr of recovery, Bcl-2 expression was reduced slightly compared to a progressive increase in Bcl-xL expression. MDM-2 expression levels remained unchanged.
Astrocyte Apoptosis and p53
Fig. 2. Semiquantitative PCR analysis of p53 and Bcl-2 family member mRNA expression levels. This analysis shows that concomitantly with p53, several proapoptotic genes such as Bad, Bid and Bcl-xS are upregulated during oxidative stress and recovery periods. A concomitant slight decrease was observed in expression levels of anti-apoptotic genes such as Bcl-2 and Bcl-xL.
Fig. 3. A: Step-cycle RT-PCR analysis of GAPDH and H4 histone. PF- treatment leads to a marked shift of the ampliﬁcation curve, compared to that of untreated cells, starting at a concentration of 20 nM. On the contrary, the H4 housekeeping gene proﬁle is not affected by PF- treatment. B: Nucleosomal formation assay. Both cell types were exposed to 1 hr ROS application followed by 24 hr of recovery in the presence (ROS PF- ) or absence (ROS) of PF- . Control cells were exposed to HBSS alone. It seems that PF- does not prevent DNA fragmentation.
Confocal Microscopy Analysis of p53 Intracellular Distribution To document further the intracellular distribution of p53, we carried out confocal microscopy analysis of p53
labeling after MitoTracker labeling of mitochondria (Fig. 5). For this purpose, primary astrocytes were exposed to 1 hr oxidative stress followed by 6 or 24 hr of recovery. Confocal microscopy analysis evidenced a clear mitochon-
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Fig. 4. Top: Immunoﬂuorescence analysis of p53 during oxidative stressinduced injury. In both C6 and primary astrocytes, p53 is expressed at very low levels under basal conditions but increases during stress periods, showing a nuclear localization. After 24 hr of recovery, p53 localization changes markedly, evidencing a translocation to the cytoplasm. Center: Immunoﬂuorescence analysis of cyt-c during oxidative stress-induced injury. Mitochondrial structure remodeling and cyt-c release in primary astrocytes and C6 cells exposed to oxidative stress-induced injury. Under basal conditions, mitochondria display a healthy morphology characterized by ﬁlamentous structures. Within 3– 6 hr of reperfusion, megamitochondria formation is evident. Subsequently (12–24 hr of recovery), mitochondrial disruption and chromatin condensation can be observed. Bottom: Immunoﬂuorescence analysis of MDM-2 during oxidative stress-induced injury. In both C6 and primary astrocytes, MDM-2 shows a low expression level at the end of the stress period and a marked increase is detectable after 3 hr of recovery, showing a strong nuclear localization. Subsequently, MDM-2-like immunoreactivity decreases (see the 6-hr recovery point), becoming almost undetectable after 12 hr of recovery until the end of the experiment.
drial localization of p53 after ROS exposure compared to that in HBSS treated cells. Effects of zVAD-fmk and PF- on p53 and MDM-2 Distribution After 6 Hours of Recovery The p53 regulator MDM-2 contains a caspase-3-like cleavage site (Chen et al., 1997). We therefore wanted to monitor whether zVAD-fmk treatment could modulate MDM-2 expression in our experimental paradigm. For this purpose, C6 cells and primary astrocytes were exposed to 1 hr of oxidative stress followed by 6 hr of recovery in presence or absence of the broad-spectrum caspase inhibitors zVAD-fmk or PF- . In both experimental models,
zVAD-fmk, but not PF- inhibited MDM-2 degradation (Fig. 6, bottom). Conversely, a marked reduction of p53 protein level was observed in zVAD-fmk but not in PFtreated cells (Fig. 6, top). Effects of zVAD-fmk on Nucleosomal Formation Because caspase inhibition was able to modulate both p53 and MDM-2 expression levels, we investigated whether this could inhibit apoptosis. We monitored the effects of zVAD-fmk on nucleosomal formation in both experimental models after 24 hr recovery (Fig. 7). This assay demonstrated that in both C6 cells and primary astrocytes, zVAD-fmk protected cells from
Astrocyte Apoptosis and p53
Fig. 5. Confocal microscopy analysis of p53 redistribution during oxidative stress-induced injury in primary cultures of astrocytes after mitochondrial staining (MitoTracker). HBSS control is presented on the ﬁrst column after 24 hr of recovery. Second and third column show primary astrocytes exposed to 1 hr ROS, after 6 and 24 hr recovery, respectively. Immunolabeling of p53 is barely detectable in HBSS-treated cells compared to that in ROS-exposed astrocytes, where p53 can be seen in the nucleus after 6 hr of recovery. Please note on overlay lane the almost complete overlay for MitoTracker and p53 labeling in ROS-treated cells, documenting the p53 mitochondrial localization.
oxidative stress-induced apoptosis: the enrichment factor decreased from 7.54 0.70 to 2.93 0.35 (P 0.01 vs. HBSS) in C6 cells and from 5.26 0.65 to 1.84 0.75 (P 0.01 vs. HBSS) in primary astrocytes after 24 hr of recovery. Effects of zVAD-fmk and PF- on Cyt-c Distribution After 24 Hours of Recovery To document further the effects of caspase blockade on apoptosis with respect to PF- , cyt-c immunolocalization was monitored in both C6 cells and primary astrocytes exposed to 1 hr of oxidative stress followed by 24 hr recovery, treated or not treated with zVAD-fmk and compared to those treated with PF- (Fig. 8). This analysis evidenced that mitochondrial disruption, cyt-c release, and chromatin condensation were completely abrogated by zVAD-fmk treatment, whereas PF- treatment did not impede these events.
DISCUSSION Glial Cells Enter Apoptosis During Recovery After ROS Exposure To mimic oxidative stress injury, cultured rat cortical astrocytes and C6 glioma cells were exposed to ROS in HBSS for 1 hr followed by a recovery period in which cells were placed in complete growth medium. Time course analysis of cell viability, nuclear morphology, (Hoechst 33258 labeling) and a nucleosomal formation assay demonstrated that both C6 glioma cells and primary astrocytes die when exposed to ROS but not when exposed to HBSS during the stress period. Cell death involved up to 70% of the cells within the 24 hr of recovery and was identiﬁed clearly as apoptotic cell death based on nuclear morphology after Hoechst staining and enrichment of nucleosome formation. After 12 hr of recovery, apoptotic nuclei could be seen clearly in both cell cultures exposed to ROS, with a marked increase after 24 hr of recovery. This induction of apoptosis due to
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Fig. 6. Immunoﬂuorescence analysis of p53 and MDM-2: effects of zVAD-fmk and PF- treatments. C6 and primary astrocytes were exposed to ROS followed by 6 hr of recovery (second column) compared to cells exposed to oxidative stress but treated with zVAD-fmk (third column) or PF- (fourth column). Basal (ﬁrst column) refers to intact cells. MDM-2 degradation is inhibited by zVAD-fmk, leading to concomitant p53 degradation. No p53 immunoreactivity can be detected in cytosol of cells treated with zVAD-fmk. PF- did not interfere with MDM-2 distribution or with p53 mitochondria-like accumulation.
ROS exposure was conﬁrmed by the increase in nucleosomal formation. Expression Levels of Bcl-2 Family Member mRNA Are Modulated During Recovery: Possible Involvement of p53 In recent years, a critical role for mitochondria in apoptosis induction has been demonstrated (Matsuyama and Reed, 2000). In particular, these organelles release proteins into the cytosol such as cyt-c and apoptosisinducing factor (AIF), triggering caspase-dependent and independent apoptotic mechanisms (Green, 1998; Reed et al., 1998; Susin et al., 1998, 1999). Several pro-apoptotic proteins of Bcl-2 family, such as Bid, Bak, and Bax play a pivotal role in this process (Eskes et al., 1998, 2000). In addition, expression of many members of the Bcl-2 proteins family are regulated by p53 (Kannan et al., 2000).
Semiquantitative RT-PCR analysis (Fig. 2) showed that in both experimental models p53 mRNA was upregulated transiently but signiﬁcantly during recovery with a parallel upregulation of pro-apoptotic Bax and Bad genes. Moreover, two other pro-apoptotic members of the Bcl-2 family, Bcl-xS and Bid, were upregulated already during the oxidative period. The anti-apoptotic genes Bcl-2 and Bcl-xL remained almost unchanged in C6 glioma cells whereas in primary astrocytes, during 24 hr of recovery, Bcl-2 expression was reduced slightly compared to a progressive increase in Bcl-xL expression. Finally, MDM-2 mRNA was upregulated in C6 cells, but not in primary astrocytes, during recovery. The expression proﬁle of pro-apoptotic genes observed in our system was in agreement with a previous large-scale expression analysis of nonneuronal models of p53induced apoptosis (Polyak et al., 1997).
Astrocyte Apoptosis and p53
Interestingly, although most pro-apoptotic genes were upregulated during oxidative stress simulation (T0 time point, Fig. 2), cells exhibited real apoptotic signs (i.e., nuclear condensation) only after several hours of recovery (Fig. 1A). These data conﬁrm observations published previously regarding p53 induction in the human glioblastoma cell line A172 after exposure to oxidative stress; however, these authors reported an increase in Bak expression, which did not occur in our system (Kitamura et al., 1999). All these ﬁndings suggest a possible role for p53 transcriptional activity on apoptosis onset, leading the bal-
ance of pro-apoptotic versus anti-apoptotic genes towards pro-apoptotic genes. PF- Inhibits p53 Transcriptional Activity but Does Not Protect Glial Cells Against Oxidative Stress-Induced Apoptosis Recent preclinical studies have demonstrated the efﬁcacy of a p53 inhibitor in models of stroke and neurodegenerative disorders, and have suggested that drugs that inhibit p53 may reduce the extent of brain damage in related human neurodegenerative conditions (Culmsee et al., 2001a,b; Lakkaraju et al., 2001; Chung et al., 2002). Based on these data, we tested the potential protective properties of PF- . We ﬁrst carried out a pilot experiment using the nucleosomal formation assay, to evaluate a potential proapoptotic effect of PF- treatment. We showed that, as reported previously in neurons (Culmsee et al., 2001b), PF- was without signiﬁcant effect on glial cell survival at concentrations up to 200 nM, although higher concentrations slightly induced apoptosis, as revealed by nucleosomal formation assay (data not shown). To verify whether after 24 hr of recovery PFremained able to block p53 transcriptional activity, we used step-cycle RT-PCR analysis to monitor in C6 cells the mRNA expression levels for H4, as a housekeeping control gene, and GAPDH, because its expression is modulated by p53 as mentioned previously (Chen et al., 1999). The dose-dependent decrease in GAPDH expression compared to the lack of H4 housekeeping gene expression modulation conﬁrmed PF- efﬁcacy at THE genomic level (Fig. 3A). We therefore decided to use 100 nM PF- to test its putative protective effect against oxidative stress-induced apoptosis, in both rat primary cultures of cortical astrocytes and C6 glioma cells.
Fig. 7. Nucleosomal formation assay in zVAD-fmk treated cultures. Both cell types were exposed to 1 hr oxidative stress followed by 24 hr of recovery in the presence (ROS zVAD-fmk) or absence (ROS) of zVAD-fmk. Control cells were exposed to HBSS alone. Nucleosomal formation was prevented signiﬁcantly by zVAD-fmk.
Fig. 8. Immunoﬂuorescence analysis of cyt-c: effects of zVAD-fmk and PFtreatment. C6 cells and primary astrocytes were exposed to ROS followed by 24 hr of recovery (second column) compared to cells exposed to oxidative stress but treated with zVAD-fmk (third column) or PF- (fourth column). Basal (ﬁrst column) refers to intact cells. In both primary astrocytes and C6 cells treated with zVAD-fmk (third column), mitochondrial structure is preserved, cyt-c immunoreactivity is not altered, and chromatin condensation cannot be observed. In cells treated with PF(fourth column), no peculiar difference can be observed when compared to cells exposed to oxidative stress (second column).
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We analyzed nucleosomal formation in both experimental cell models after 24 hr of recovery in the presence or absence of PF- (Fig. 3B). Strikingly, we observed that PF- failed to protect both primary astrocytes and C6 cells against oxidative stress-induced apoptosis, despite its ability to inhibit p53 genomic effects. This was an unexpected result because in neurons, this compound was able to prevent apoptosis induced by ischemic insult, as reported previously (Komarov et al., 1999). In addition, the lack of protection against apoptosis after PF- treatment further supports the hypothesis that discrepancies in various gene expression noticed between C6 cells and primary astrocytes may not have a primary importance for cell survival. Intracellular Redistribution of p53 Toward Mitochondria After Oxidative Stress Injury Nuclear bypass experiments demonstrated that mitochondrial localization of p53 is sufﬁcient to induce p53dependent apoptosis, indicating dual action of this protein during apoptosis (Ding and Fisher, 1998; Ding et al., 1998; Marchenko et al., 2000). Time-course immunoﬂuorescence analysis of p53 protein combined with Hoechst labeling of the nuclei (Fig. 4), showed that p53 was detectable in both experimental models almost exclusively in the nucleus until the end of the stress period. The p53 expression level increased progressively over the 24-hr recovery period, showing an almost cytoplasmic localization of the protein in both experimental systems. The p53 expression level seemed higher in C6 when compared to that in primary astrocytes, in agreement with RT-PCR analysis. It has been reported that when present in cytoplasm, most p53 protein is localized within the mitochondrial membrane compartment, although a small fraction of the protein can be found in other organelles. This mitochondrial localization of p53 was conﬁrmed by confocal microscopy analysis in primary astrocytes (Fig. 5). At the mitochondrial level, p53 exerts its proapoptotic function by favoring cyt-c release and subsequent caspase activation (Marchenko et al., 2000; Sansome et al., 2001; Schuler et al., 2000; Schuler and Green, 2001). To investigate further such a mechanism, we carried out a time course immunoﬂuorescence analysis of cyt-c distribution. Both C6 cells and primary astrocytes were exposed to ROS for 1 hr and analyzed within 24 hr of recovery. As shown in Figure 4 in control cells (basal point), ﬁlamentous mitochondria were discerned clearly whereas between 3– 6 hr of recovery and concomitantly with p53 redistribution toward mitochondria (see Fig. 4 at 6 hr recovery), the mitochondrial structure was altered profoundly, showing a typical apoptotic organization known as megamitochondria (MG). At this point, cyt-c release was not yet detectable, leading to a situation that usually, but not necessarily, precedes apoptosis (Karbowski et al., 1999; Teranishi et al., 2000). After 24 hr recovery, changes in mitochondrial structure and apoptotic changes in cells were marked and showed the swelling of free radical-induced megamitochondria, which in turn were responsible for cyt-c release and nuclear shrinking. In
agreement with previous ﬁndings, these data suggested that p53 was able to induce apoptosis through its translocation to mitochondria and the subsequent release of cyt-c (Schuler et al., 2000). Mitochondrial Localization of p53 Requires MDM-2 Degradation via Caspase Activity MDM-2 protein is a key player in the regulation of p53. Thus, MDM-2 binds to p53 within its transactivation domain, interfering with recruitment of basal transcription machinery components, and within the nucleus MDM-2 can bind p53 targeting newly formed complexes toward the cytoplasm due to a nuclear export sequence (NES) present in the MDM-2 protein sequence. This leads to the degradation of p53 by the ubiquitin-proteasome pathway (Haupt et al., 1997; Kubbutat et al., 1997). To investigate the involvement of MDM-2 in the apoptotic response in our experimental model, we carried out a time course immunoﬂuorescence analysis. As shown in Figure 4, MDM-2 was expressed in both C6 and primary cultures of rat astrocytes at very low levels until the end of ROS exposure, whereas p53 was upregulated already. MDM-2 reached its maximum expression level after 3 hr of recovery with a marked nuclear localization and seemed barely detectable after 6 hr of recovery. Simultaneously, p53 immunoreactivity increased strongly, showing a marked cytosolic signal. After 12 hr of recovery MDM-2 immunoreactivity decreased strongly becoming almost undetectable until the end of the recovery period. These data suggested a direct relationship between the increase in cytoplasmic p53 expression, and its mitochondrial redistribution, and the corresponding MDM-2 expression levels. MDM-2 contains a caspase-3-like cleavage site that is evolutionarily conserved (Chen et al., 1997), and in vitro experiments carried out in H1299 cells, an epithelial cell line expressing a temperature-sensitive human p53, demonstrated that caspase-3 is able to cleave MDM-2. The cleaved MDM-2 loses the ability to promote p53 degradation and potentially functions in a dominantnegative fashion to stabilize p53 (Pochampally et al., 1999). To verify a possible relationship between MDM-2 degradation and p53 expression levels, we exposed both C6 cells and primary astrocytes to oxidative stress injury in the presence or absence of the broad-spectrum caspase inhibitor zVAD-fmk. We also analyzed the effect of PFtreatment. We demonstrated that p53 and MDM-2 expression levels displayed a complementary but opposite proﬁle after 6 hr of recovery (Fig. 4). As shown in Figure 6, in both C6 cells and primary astrocytes, zVAD-fmk, but not PF- inhibited MDM-2 degradation, leading to a marked reduction of p53 protein level and prevention of its translocation toward mitochondria. These ﬁndings suggest a cause-and-effect relationship between caspase activity, MDM-2 degradation, and p53 protein stabilization.
Astrocyte Apoptosis and p53
Fig. 9. Proposed mechanism of oxidative stress-induced apoptosis in glia cells. Dashed arrows represent the physiological pathway in which p53 contributes to MDM-2 transcription. When exported into the cytoplasm, MDM-2 protein binds p53, driving its degradation through ubiquitin-proteasome pathway. This contributes to maintain low p53 expression level. In the presence of ROS (large arrows), partial mitochondrial membrane depolarization leads to cyt-c release and caspase activation, which in turn contributes to MDM-2 degradation. p53 can translocate toward mitochondria, leading to a massive cyt-c release and establishing an apoptotic loop. Treatment with zVAD-fmk abolishes onset of the apoptotic loop. PF- is unable to protect cells from apoptosis, indicating that p53 transcriptional activity is not the main pathway involved in our model of oxidative stress-induced apoptosis.
Pro-apoptotic Mitochondrial Function of p53 Is Abrogated Completely by zVAD-fmk but not by PFWe demonstrated that, in our models, p53 redistribution within the cytosol is related to cyt-c release from mitochondria and cell death execution (Fig. 4). In the presence of zVAD-fmk, p53 mitochondrial targeting was abrogated completely by preventing MDM-2 caspasedependent degradation. The question then raised was whether this process was sufﬁcient to prevent apoptotic
cell death. For this purpose, we monitored nucleosomal formation in both experimental models after to 24 hr of recovery in the presence or absence of zVAD-fmk (Fig. 7). This assay demonstrated that in both C6 cells and primary astrocytes, zVAD-fmk protected cells from ROS exposure-induced apoptosis; indeed, the enrichment factor was decreased strongly in both cell cultures after 24 hr of recovery. Finally, we investigated whether zVAD-fmk protection was related to the inhibition of p53-mediated cyt-c
Bonini et al.
release. Both C6 cells and primary astrocytes were exposed to ROS, treated or not with zVAD-fmk and compared to sisters cultures exposed to ROS not treated with PF- . This analysis evidenced that after 24 hr of recovery, no cyt-c released could be observed and mitochondria displayed a ﬁlamentous and healthy morphology in zVADfmk treated cells only. In conclusion, we have found that in a glial model of oxidative stress-induced apoptosis, p53 plays a pivotal role by directly signaling mitochondria despite its transcriptional activity. PF- fails to protect glial cells from ROS exposure despite its ability to inhibit p53 genomic effects, contrary to what has been observed in neuronal cells (Komarov et al., 1999). Our data suggest that in the apoptotic cascade, three fundamental steps are involved. The ﬁrst step, engagement, involves low cyt-c release from mitochondria within ﬁrst 3 hr of reperfusion (probably due to a partial mitochondrial membrane depolarization, as evidenced by JC-1 mitochondrial potential sensor analysis; data not shown). This is followed by partial caspase activation, which in turn acts on early targets such as MDM-2, leading to a redistribution of p53 toward mitochondria. In the second step, commitment, concomitantly with p53 redistribution into the cytosol, mitochondria modify their structure by forming megamitochondria (MG) within 6 hr of recovery. MG formation may be an adaptive and reversible response to unfavorable environments at the subcellular level that leads to a decrease in oxygen consumption and ROS production (Karbowski et al., 1999). The third and last step, death-warrant, occurs after 24 hr of reperfusion when a complete disruption of free radical-induced MG leads to cyt-c release, caspase activation, and cell death. It is now accepted generally that massive neuronal death due to oxidative stress is a regular feature in neurodegenerative diseases of the brain. Much less attention, however, has been given to glial cells death. Several studies suggest that glial cells in neurodegenerative diseases (i.e., Alzheimer’s disease) are affected more than neurons by apoptotic cell death (Lassmann et al., 1995; Smale et al., 1995). In our glial system, we highlight for the ﬁrst time that glial cells respond to oxidative stress undergoing apoptosis via p53 transcription independent pathways, which is summarized in Figure 9. Based on these results, drugs such as PF- that selectively protect neurons from apoptotic stimuli would not be sufﬁcient in the treatment of neurodegenerative diseases. Therefore, therapeutic approaches based on caspase inactivation (Cheng et al., 1998; Robertson et al., 2000, 2001) are likely a more efﬁcient strategy against neurodegenerative diseases because in cell death execution, both glial cells and neurons exhibit a ﬁnal common pathway in which caspases are the main actors. ACKNOWLEDGMENTS We thank Dr. S. Biocca (University of Rome Tor Vergata) for the gift of C6 cells and for critical discussions. This work was supported in part by research fellowships
(to P.B., S.C., and A.C. from Ministry of Education, University and Research, Miur-Italy). REFERENCES
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