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Hsp27 inhibits 6-hydroxydopamine-induced cytochrome c release and apoptosis in PC12 cells
BBRC Biochemical and Biophysical Research Communications 327 (2005) 801–810 www.elsevier.com/locate/ybbrc
Hsp27 inhibits 6-hydroxydopamine-induced cytochrome c release and apoptosis in PC12 cells Adrienne M. Gorman, Eva Szegezdi, Declan J. Quigney, Afshin Samali* Department of Biochemistry and the National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Received 9 December 2004 Available online 22 December 2005
Abstract Cellular stress may stimulate cell survival pathways or cell death depending on its severity. 6-Hydroxydopamine (6-OHDA) is a neurotoxin that targets dopaminergic neurons that is often used to induce neuronal cell death in models of ParkinsonÕs disease. Here we present evidence that 6-OHDA induces apoptosis in rat PC12 cells that involves release of cytochrome c and Smac/Diablo from mitochondria, caspase-3 activation, cleavage of PARP, and nuclear condensation. 6-OHDA also induced the heat shock response, leading to increased levels of Hsp25 and Hsp70. Increased Hsp25 expression was associated with cell survival. Prior heat shock or overexpression of Hsp27 (human homologue of Hsp25) delayed cytochrome c release, caspase activation, and reduced the level of apoptosis caused by 6-OHDA. We conclude that 6-OHDA induces a variety of responses in cultured PC12 cells ranging from cell survival to apoptosis, and that induction of stress proteins such as Hsp25 may protect cells from undergoing 6-OHDA-induced apoptosis. 2004 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Caspase; Heat shock response; Hsp27; ParkinsonÕs disease; Cytochrome c; Smac/Diablo; Rat
6-Hydroxydopamine (6-OHDA), a hydroxylated analogue of dopamine, is commonly used in model systems to mimic ParkinsonÕs disease, since it induces death of dopaminergic neurons both in vivo and in vitro [1,2]. The cellular events that occur as a result of 6-OHDA exposure are not entirely understood. Production of reactive oxygen species [3–5] and/or inhibition of complex I are both reported to mediate 6-OHDA-induced cell death [6]. There is general agreement, however, that the mode of cell death is by apoptosis in a variety of cell culture model systems, including primary mesencephalic dopaminergic neurons [7,8], MN9D [9], and PC12 cells [2,10]. Apoptosis is a highly regulated form of cell death that occurs under physiological and pathological conditions. The activation of caspase proteases is central to this pro*
Corresponding author. Fax: +353 91 750596. E-mail address: [email protected] (A. Samali).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.066
cess [11,12]. Caspase activation can be triggered by the release of cytochrome c from the mitochondria, which occurs in response to several apoptotic stimuli including neurotoxins [13,14]. In the cytosol, cytochrome c interacts with Apaf-1, which in the presence of dATP leads to clustering and autoactivation of caspase-9 [13,14]. Active caspase-9 causes cleavage and activation of downstream caspases, e.g., caspase-3. Caspases mediate the degradation of a number of proteins critical for cell homeostasis such as the cytoskeletal protein, fodrin, and the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP) [15]. While exposure of cells to severe stress can induce cell death, transient or milder stress conditions stimulate cells to activate protective strategies that involve the induction of prosurvival proteins [16,17]. For example, increased expression of heat shock protein 27 (Hsp27) and Hsp70 occurs after exposure of cells to non-lethal elevations in temperature or oxidants [18]. Hsp27
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belongs to a sub-family of stress proteins, the small Hsps, which are detectable in virtually all organisms. Increased expression of Hsp27 renders cells more resistant to lethal levels of a variety of toxic insults [19–21]. Hsp27 inhibits apoptosis [20], by interfering with caspase activation at several different levels [22–24]. It can interact directly with mitochondria, thereby preventing release of cytochrome c [23] and Smac/Diablo [25]. In the cytosol, it can also sequester both cytochrome c and pro-caspase-3, thus preventing the correct formation/function of the apoptosome [22,24]. The present study was undertaken to examine the effect of 6-OHDA on the induction of cell death and stress responses, and in particular, to examine the role of Hsp27 in 6-OHDA-induced cell death in PC12 cells.
Materials and methods Materials. All chemicals were purchased from Sigma unless indicated otherwise. Mouse monoclonal antibody against Hsp70 and rabbit polyclonal antibodies against Hsp25 (rat homologue of human Hsp27) were obtained from StressGen Biotechnologies. Mouse monoclonal antibodies against PARP were obtained from Biomol and cytochrome c was from BD Pharmingen. Rabbit polyclonal antibody against caspase-3 was obtained from Cell Signalling Technology and Smac/Diablo antibody was a kind gift from Prof. Seamus Martin, Dept. of Genetics, Trinity College, Dublin, Ireland. Goat secondary antibodies conjugated to horseradish peroxidase were from Pierce. Goat secondary anti-rabbit IgG conjugated to Alexa-546 was from Molecular Probes. Ac-Asp-Glu-Val-Asp-a-(4-methyl-coumaryl7-amide) (DEVD-MCA) was from the Peptide Institute, Osaka, Japan. Protein molecular weight markers were obtained from New England Biolabs. T4 polynucleotide kinase was from Promega. [c-32P]ATP was from ICN. Effectene transfection reagent was from Qiagen. PCI-neo Mammalian expression vector was obtained from Promega. Rat pheochromocytoma PC12 cells were obtained from the ECACC. Cell culture and treatments. PC12 cells were maintained in RPMI 1640 medium supplemented with 10% horse serum, 5% fetal calf serum, 50 U/ml penicillin, 50 lg/ml streptomycin, and 2 mM D -glutamine, at 37 C in a humidified 5% CO2 atmosphere. They were passaged once weekly. Experiments were performed using dishes coated with poly-L -lysine (10 lg/ml for 3 h) to assist cell adhesion. For determining Hsp induction cells were seeded at a density of 5 · 106 in a 25 cm2 culture flask and cultured for 24 h, prior to treatment (heat shock or addition of 6-OHDA). To subject the cells to heat shock, the culture flasks were sealed by wrapping parafilm around the lids and immersed in a water bath at 41.5 C for 1 h. Cells were then allowed to recover at 37 C in a humidified 5% CO2 atmosphere for various times. For treatment with 6-OHDA, stock solutions of 6-OHDA were made freshly in sodium metabisulphite (1 mg/ml) prior to each experiment. Cells were exposed to various concentrations of 6-OHDA as indicated in the figure legends. Analysis of cellular morphology. For morphological analysis, cells were scraped from flasks, and 100 ll of the cell suspension (approximately 50 · 104 cells) was cytocentrifuged onto glass slides. After airdrying the preparations were stained using RAPI-DIFF II stain pack (Triangle Biomedical Sciences). Cells were scored by counting at least 300 cells from each sample, from three different experiments. DNA fragmentation. Following experimental treatments, cells were removed from the culture flask by scraping. Cells were centrifuged at 775g for 5 min at 4 C. The pellet was washed once with phosphate-
buffered saline (PBS), and lysed in 300 ll of a buffer containing 100 mM Tris–HCl, pH 8.5, 5 mM EDTA, 0.2 M NaCl, 0.2% (w/v) SDS, and 0.0001% (w/v) proteinase K. The cell suspension was incubated for 3 h at 50 C, followed by addition of 30 ll of 3 M C2H3NaO2 and 660 ll of 96% ethanol. The cell suspension was kept overnight at 20 C. The lysate was then centrifuged at 21,000g for 10 min. The supernatant was discarded, the pellet was resuspended in 70% ethanol and centrifuged at 21,000g for 10 min. The pellet was air-dried at room temperature for 30 min. The pellet was resuspended in 50 ll buffer containing 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, and 100 lg/ml RNase. The sample was incubated for 1 h at 37 C. The sample was then centrifuged at 21,000g for 10 min, supernatant was removed, resuspended in 5· loading buffer (25% Ficoll and 0.25% bromophenol blue), and resolved on a 1.5% agarose gel for 2 h at 60 V. The bands were then visualised under UV light. Detection of caspase activity. The activity of group II caspases, DEVDases, was determined fluorometrically as developed by Nicholson et al. [26] with some modifications [27]. Briefly, lysate from 2.5 · 106 cells and substrate (DEVD-MCA) were combined in reaction buffer {100 M N-2-hydroxyethyl-piperazine-N 0 -2-ethanesulphonic acid (HEPES), pH 7.5, 10% sucrose, 0.1% 3[(3cholamidopropyl)-dimethylammonio]-1-propanesulphonate, 5 mM dithiothreitol, 104% Nonidet P40, and 50 lM DEVD-MCA} and added in triplicate to a microtitre plate. Substrate cleavage leading to the release of free MCA was monitored at 37 C using a Wallac Victor multilabel counter (excitation 355 nm, emission 460 nm). Fluorescent units were converted to micromoles of MCA released using a standard curve generated with free MCA and subsequently related to protein concentration. Preparation of whole cell extracts. Following experimental treatments the cells were removed from the culture flask by scraping. The cells were centrifuged at 775g for 5 min at 4 C. The pellet was washed once with PBS and lysed in 100 ll of a buffer containing 20 mM HEPES, pH 7.5, 350 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 0.5 mM dithiothreitol, 0.1% phenylmethylsulphonyl fluoride, and 1% aprotinin. The cell suspension was incubated on ice for 15 min and then centrifuged at 21,000g for 30 s. The supernatants were stored at 70 C until further analysis by Western blotting. Protein content was determined using a Bio-Rad protein assay kit with bovine serum albumin (BSA) as standard. Western blotting. Samples were resuspended in LaemmliÕs sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and boiled for 5 min. Proteins (20–25 lg per lane) were then resolved on 10–12% SDS–PAGE gels and electrophoretically transferred onto nitrocellulose for 1.5 h at 100 V. Membranes were blocked for 1 h in PBS containing 0.05% Tween 20 and 5% (w/v) nonfat dried milk. The membranes were then incubated for 1 h at room temperature with antibodies to Hsp25 (1:2000), Hsp27, cytochrome c, Hsp70 (1:1,000), Smac/Diablo (1:200) or actin (1:500). Alternatively, the membranes were incubated overnight at 4 C with antibodies to caspase-3 or PARP (1:1000). This was followed by 1 h incubation at room temperature with appropriate horseradish peroxidase-conjugated goat IgGs (1:10,000 or 1:2000 for detection of caspase-3). Protein bands were then visualised using Supersignal West pico Western blot detection kit (Pierce). Determination of cytochrome c and Smac/Diablo release. Cells were trypsinised and centrifuged at 150g for 5 min at 4 C. The pellet was washed once with PBS. The cells were then lysed using 100 ll cell lysis and mitochondria intact (CLAMI) buffer containing (250 mM sucrose and 70 mM KCl in PBS, 0.1 mM phenylmethylsulphonyl fluoride, 1 mM dithiothreitol, 5 lg/ml pepstatin, 10 lg/ml leupeptin, 2 lg/ml aprotinin, and 25 lg/ml calpain inhibitor 1). Digitonin (10 ll of 20 mg/ ml solution) was added to the samples on ice for 5 min and then the cell suspension was centrifuged at 3000g for 10 min at 4 C. The supernatant was removed and stored as the cytosolic fraction, at 20 C. The pellet was resuspended in 100 ll CLAMI buffer and stored as the mitochondrial fraction, at 20 C. Samples were analysed by Western blotting.
A.M. Gorman et al. / Biochemical and Biophysical Research Communications 327 (2005) 801–810 Electrophoretic mobility shift assay. Following appropriate treatments, cells were washed once in ice-cold PBS, centrifuged at 2000g, and the pellet was snap-frozen in liquid nitrogen. Whole cell extracts were prepared by resuspending the pellet with 50 ll of 20 mM HEPES, pH 7.9, containing 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulphonyl fluoride, and 25% glycerol. Samples were allowed to swell on ice for 5 min and subsequently centrifuged at 15,000g for 20 min at 4 C. A synthetic oligonucleotide corresponding to the proximal heat shock element (HSE) of the human Hsp70 promoter (5 0 -GCC TCG AAT GTT CGC GAA GTT TTC) was end labelled with [c-32P]ATP using T4 polynucleotide kinase. Whole cell extracts (10–15 lg) were incubated with the labelled oligonucleotide for 20 min in binding buffer containing 20 mM Tris, pH 7.5, 100 mM NaCl, 2 mM EDTA, and 10% glycerol. The protein–DNA complexes were resolved on a native 4% polyacrylamide gel using 0.25· TBE running buffer for 2 h at 150 V. The gel was dried under vacuum onto Whatmann 3MM filter paper and autoradiographed overnight with X-ray film. Hsp25 immunostaining. Cytocentrifuge preparations of PC12 cells were fixed in 3.7% formaldehyde for 5 min at room temperature and then permeabilised with 0.2% Triton X-100 for a further 5 min at room temperature. Free antibody binding sites were blocked for 1 h at room temperature with PBS containing 5% goat serum and 1% BSA followed by 1 h incubation with the primary antibody (rabbit anti-Hsp25, diluted 1:1000 with 1% BSA in PBS). Excess antibody was removed by 3 · 5 min washes in PBS. Then the slides were incubated with goat anti-rabbit IgG, Alexa-546 conjugate (diluted 1:200 in 1% BSA in PBS) for 1 h at room temperature. Unbound antibodies were removed by
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3 · 5 min washes in PBS before mounting the slides with 80% glycerol containing 3 lg/ml Hoechst 33342. Cells were visualised with a Zeiss, S100 fluorescence microscope. Cells were scored as dying if they stained intensely with Hoechst and showed evidence of nuclear condensation. Cells were scored as expressing high levels of Hsp25 if the staining with the antibody was comparable to that observed in cells that had been heat shocked (shown in Fig. 4). Transfection procedure. Transfection was performed using Effectene Transfection Reagent according to the manufacturerÕs protocol. Cells were transfected with either empty pCI-neo or human Hsp27-pCI-neo expression vector. Clones which exhibited stable expression of Hsp27 were selected with 800 lg/ml G418. Statistical analysis. Results are expressed as means ± standard error of the mean (SEM). Statistical analysis was made using ANOVA followed by post hoc tests indicated in the Figure legends.
Results Induction of apoptosis in PC12 cells following exposure to 6-OHDA To investigate the effect of 6-OHDA on PC12 cells, we exposed the cells for various periods of time and to a range of concentrations of 6-OHDA, and examined a variety of markers of apoptosis. Morphological
Fig. 1. Induction of apoptosis in PC12 cells treated with 6-OHDA. (A) The morphology of untreated cells (control) and cells treated for the indicated times (24 or 48 h) was examined by making cytocentrifuge preparations of the cells followed by staining with Rapi Diff. Filled arrows indicate apoptotic cells, which are shrunken with condensed nuclei. Open arrows indicate necrotic cells, which are swollen and lack intact plasma membrane. (B) The proportion of apoptotic and necrotic cells at each time point was calculated as a percentage of the total number of cells. Values represent means ± SEM of three separate determinations. Statistical analysis was performed with ANOVA followed by DunnettÕs multiple comparisons post hoc test. **p < 0.01 versus apoptosis at 0 h, *p < 0.05 versus apoptosis at 0 h. (C) PC12 cells were treated with 200 lM 6-OHDA for 0–48 h and the induction of a DNA ladder was shown by agarose gel electrophoresis of DNA extracted from the samples. This experiment was repeated twice with similar results.
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analysis of cytocentrifuge preparations of PC12 cells that were exposed to 200 lM 6-OHDA for 24 h demonstrated nuclear condensation and fragmentation, typical hallmarks of apoptosis (Figs. 1A and B). Longer treatments (48 h) led to an increase in the proportion of necrotic cells in the culture (Figs. 1A and B). Apoptosis was accompanied by degradation of the chromatin into oligonucleosomal-sized fragments (Fig. 1C). There was a dose- and time-dependent increase in caspase activation as measured by an increase in DEVDase activity (Figs. 2A and B), processing of caspase-3 (Figs. 2C and D), and cleavage of the caspase substrate, PARP (Figs. 2C and D). Release of pro-apoptotic proteins from the mitochondria is one mechanism by which caspase activation is initiated [13,14]. In order to investigate the
mitochondria as a possible mediator of 6-OHDA-induced apoptosis, we examined changes in the localisation of mitochondrial pro-apoptotic factors. An early release of both cytochrome c and Smac/Diablo (a pro-apoptotic protein that binds to members of the inhibitors of apoptosis proteins and abrogates their anti-apoptotic effect [28,29]) into the cytosol was detected and this process occurred prior to caspase activation (Fig. 2E). Taken together, these results suggest that the mitochondrion is an early target in 6-OHDAinduced apoptosis. Induction of the heat shock response by 6-OHDA 6-OHDA is known to induce oxidative stress [3] and oxidative stress is a major inducer of stress proteins
Fig. 2. Caspase activation in PC12 cells treated with 6-OHDA. (A) PC12 cells were treated with 0–200 lM 6-OHDA for 24 h and caspase-3-like proteolytic activity, i.e., DEVD-MCA cleavage activity, was measured in whole cell extracts. Values are means ± SEM of three separate determinations. (B) PC12 cells were treated with 200 lM 6-OHDA for 0–48 h and DEVD-MCA cleavage activity was measured in whole cell extracts taken after 24 h of 6-OHDA treatment. Values are means ± SEM of 3 separate determinations. (C) PC12 cells were treated with 0–200 lM 6-OHDA for 24 h. Proteolytic processing of caspase-3 and PARP was analysed by Western blotting. The arrows point to pro-caspase-3 (32 kDa), the cleaved active fragment of caspase-3 (17 kDa), intact PARP (116 kDa), and the caspase-dependent cleavage product of PARP (85 kDa). The levels of actin in the samples were also analysed to demonstrate equal loading of the lanes. This experiment was repeated twice with similar results. (D) PC12 cells were treated with 200 lM 6-OHDA for 0–48 h. Proteolytic processing of caspase-3 and PARP was analysed by Western blotting. The levels of actin in the samples were also analysed to demonstrate equal loading of the lanes. This experiment was repeated 3 times with similar results. (E) The release of pro-apoptotic factors from the mitochondria was shown by examining the levels of cytochrome c and Smac/Diablo in cytosolic and mitochondrial fractions of PC12 cells that were exposed to 200 lM 6-OHDA for 0-24 h. This experiment was repeated 3 times with similar results.
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particularly heat shock proteins [30,31]. Since many of the cells in cultures treated with 6-OHDA survived the treatment, we hypothesised that induction of protective proteins may be facilitating the resistance of some cells to undergo cell death. Therefore, the ability of 6-OHDA to induce a heat shock response was examined. Western blot analysis showed that basal levels of both Hsp25 (the rat homologue of Hsp27) and Hsp70 were very low in unstressed PC12 cells. However, exposure to 6-OHDA induced a marked increase in Hsp25 at concentrations as low as 25 lM (Fig. 3A). The levels continued to rise until 200 lM 6-OHDA (Fig. 3A), with the maximal induction occurring at the highest concentration used (200 lM, Fig. 3A). There was also induction of Hsp70 expression, although to a lesser extent than Hsp25 (Fig. 3A). These low concentrations of 6-OHDA also induced activation of caspases in the cells as seen by an increase in DEVDase activity (Fig. 2A), caspase-3 and PARP cleavage (Fig. 2C). A time course of Hsp induction by 200 lM 6-OHDA showed that Hsp25 and Hsp70 were induced following 12 h of treatment and that Hsp expression remained elevated until at least
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24 h (Fig. 3B). This was preceded by the induction of heat shock factor 1 (HSF-1) DNA-binding activity which was detectable within 6 h of 6-OHDA treatment (Fig. 3C). These data show that a classical heat shock response occurs in PC12 cells exposed to 6-OHDA. Prior heat shock reduces 6-OHDA-induced cell death in PC12 cells Recent reports from our group showed that the exposure of PC12 cells to non-lethal elevations in temperature results in the synthesis and accumulation of Hsp25 and Hsp70 [27]. This has been shown to induce a state of thermotolerance and renders the cells resistant to subsequent treatment with 1-methyl-4-phenylpyridinium (MPP+) [27]. In order to determine whether the induction of Hsps and thermotolerance can protect against 6-OHDA-induced apoptosis, PC12 cells were heat shocked (or in the case of control cells kept at 37 C) for 1 h and allowed to recover at 37 C for 6 h. This treatment induced the synthesis of Hsp25 and Hsp70 (Fig. 4A). Cells were then treated with 200 lM 6-OHDA for a further 24 h. Prior heat shock treatment caused a significant decrease in the level of apoptosis in cells exposed to 6-OHDA compared with cells that did not undergo prior heat shock (p < 0.05, Fig. 4B). This was mirrored by a statistically significant reduction in 6-OHDA-induced DEVDase activity in cells that underwent prior exposure to heat shock (p < 0.05, Fig. 4C). Hsp25 expression in dying and surviving cells
Fig. 3. Expression of Hsp25 and Hsp70 following exposure to 6OHDA. (A) PC12 cells were treated with 0–200 lM 6-OHDA for 24 h. The expression of Hsp25 and Hsp70 was analysed by Western blotting. The levels of actin in the samples are also shown to demonstrate equal loading of the lanes. (B) PC12 cells were treated with 200 lM 6-OHDA for 0–24 h. A positive control (HS) to show induction of Hsps was included: cells were heat shocked at 41.5 C for 1 h and extracts were made immediately. The expression of Hsp25 and Hsp70 was analysed by Western blotting. The levels of actin in the samples are also shown to demonstrate equal loading of the lanes. (C) Cells were treated with 200 lM 6-OHDA for 0–24 h. Total protein extracts were incubated with a [c-32P]ATP-labelled oligonucleotide corresponding to the proximal HSE of human Hsp70 promoter and the protein–DNA complexes (HSF-1/HSE) were resolved on a native 4% polyacrylamide gel. These experiments were repeated twice with similar results.
Since there was a dual response, i.e., cell death and cell survival, observed in PC12 cell cultures exposed to 6OHDA (Figs. 1A and B), it was hypothesised that Hsp induction by 6-OHDA may correlate with cell survival and lack of induction of Hsp with cell death. Hsp25 was the main Hsp induced by 6-OHDA (Figs. 3A and B) and therefore, it was decided to examine levels of Hsp25 expression in the dying and surviving populations of cells exposed to 6-OHDA. The cells were either heat shocked (as a positive control for determining Hsp25 expression) or exposed to 150 lM 6-OHDA for 24 h and stained with Hoechst 33342 and anti-Hsp25. Fig. 5A shows a typical view of untreated, heat shocked, and 6-OHDA-treated cells. In untreated cultures the nuclei were not condensed and there were low levels of Hsp25 staining in all of the cells examined. In contrast, in heat shocked cultures there were high levels of Hsp25 immunostaining in a large proportion of the cells and the nuclei were not condensed. In cultures treated with 6-OHDA there was high level of Hsp25 staining in some but not all of the cells (Fig. 5A) and there was nuclear condensation (indicative of cell death) in a proportion of the cells. The cells were counted and it was found that in 6-OHDA-treated PC12 cells there
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Fig. 4. Effect of prior heat shock on 6-OHDA-induced apoptosis. (A) Cells were heat shocked at 41.5 C for 1 h and allowed to recover for 2, 4, 6, 8, and 24 h at 37 C. Total protein extracts (20 lg/lane) were subjected to 12% SDS–PAGE, followed by Western blot analysis, using monoclonal antibodies to Hsp25 and Hsp72. (B) Cells were either kept at 37 C (control) or heat shocked at 41.5 C for 1 h and allowed to recover for 6 h at 37 C (HS). They were then exposed to 200 lM 6-OHDA for 24 h. The morphology of the cells was assessed using cytocentrifuge preparations of the cells that were stained with Rapi Diff and the number of apoptotic cells was calculated as a percentage of the total number of cells. Values represent means ± SEM of three separate determinations. (C) Cells were treated as in (B) and DEVD-MCA cleavage activity was measured in whole cell extracts. Values are means ± SEM of three separate determinations. Statistical analysis was performed with repeated measures ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. *p < 0.05 versus 6-OHDA alone or heat shock alone.
was induction of high levels of Hsp25 in 34.0 ± 1.6% of the total cell population (Fig. 5B). In contrast, when the subpopulation of cells that were dying was examined it can be seen that there was induction of high levels of Hsp25 in 14.7 ± 2.5% of the dying cells (Fig. 5B) while the remaining 85.3% of the dying cells did not express Hsp25. The difference in the proportion of cells expressing high levels of Hsp25 in the dying population compared with the total population and with the subpopulation of cells that survived was statistically significant (p < 0.001).
Fig. 5. Expression of Hsp25 in dying and surviving cells following 6OHDA treatment. (A) PC12 cells were either heat shocked or treated with 150 lM 6-OHDA for 24 h. Cytocentrifuge preparations of the cells were stained with anti-Hsp25 and Hoechst 33342. The cells were visualised by fluorescence microscopy and scored as dying (filled arrows; condensed nuclear staining indicative of apoptosis or diffuse nuclear staining indicative of necrosis) or surviving (open arrows). The dying cells were then scored as having high or low levels of Hsp25 expression. Representative images of untreated, heat shocked, and 6OHDA-treated PC12 cells stained with Hsp25 and Hoechst 33342. (B) The proportions of the total cell population, and the dying and surviving subpopulations of cells, that expressed high and low levels of Hsp25, were calculated and expressed as a percentage of the total number of cells in each of those groups (total, dying or surviving). The data are means ± SEM of three separate experiments. Statistical analysis was performed with repeated measures ANOVA followed by Student–Newman–Keuls multiple comparisons post hoc test. ***p < 0.001 versus dying, low Hsp25; versus surviving, high Hsp25; **p < 0.01 versus total, high Hsp25; and *p < 0.05 versus total, high Hsp25.
Overexpression of Hsp27 reduces 6-OHDA-induced cell death in PC12 cells The correlation between Hsp25 expression and cell survival in cultures treated with 6-OHDA suggested that Hsp25 provided a prosurvival advantage to the cells in which it was expressed. In order to determine whether Hsp25 would protect against 6-OHDA-induced apoptosis, PC12 cells were transfected with Hsp27, the human
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Fig. 6. Inhibition by Hsp27 of 6-OHDA-induced apoptosis. (A) The expression of Hsp27 in wild type PC12 cells (WT), cells transfected with an empty vector (Neo), and cells transfected with Hsp27 (Hsp27) was examined by Western blotting. (B) PC12 cells transfected with empty vector (Neo) or with Hsp27 (Hsp27) were exposed to 200 lM 6-OHDA for 24 h and the level of apoptosis was assessed by morphological criteria. Values are means ± SEM of 3 separate determinations. Statistical analysis was performed with repeated measures ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. *p < 0.01 versus 6-OHDA-treated neo-transfected cells. (C) Cells were treated as in (B) and caspase-3-like DEVDase activity was determined. Values are means ± SEM of three separate determinations. Statistical analysis was performed with repeated measures ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. *p < 0.01 versus 6-OHDA-treated neo-transfected cells. (D) PC12 cells transfected with empty vector (Neo) or with Hsp27 (Hsp27) were exposed to 200 lM 6-OHDA for 24 h and proteolytic processing of caspase-3 was analysed by Western blotting. This experiment was performed 3 times with similar results. (E) PC12 cells transfected with empty vector (Neo) or with Hsp27 (Hsp27) were exposed to 200 lM 6-OHDA for 0–12 h. The levels of cytochrome c and Smac/Diablo in the cytosolic fractions were analysed by Western blotting. This experiment was performed 3 times with similar results.
homologue of rat Hsp25. The expression of Hsp27 was detected using specific antibodies that did not cross-react with Hsp25 (Fig. 6A). In comparison to cells transfected with the empty vector, Hsp27-overexpressing cells were more resistant to treatment with 200 lM 6OHDA for 24 h (Fig. 6B). This coincided with a reduction in DEVDase activity (Fig. 6C) and a decrease in caspase-3 cleavage (Fig. 6D) in cells exposed to 6OHDA compared with cells that were transfected with a neo construct. The reduction in caspase activity and apoptosis was associated with a significant delay in the release of cytochrome c and Smac/Diablo into the cytosol (Fig. 6E).
Discussion The present study shows differential responses of individual cells in cultures exposed to 6-OHDA, ranging from the demise of cells by apoptosis to the induction of stress responses. In agreement with other reports, our
study shows that 6-OHDA-mediated toxicity in PC12 cells exhibits the morphological (nuclear condensation and cell shrinkage) and biochemical (caspase activation, PARP cleavage, and production of a DNA ladder) characteristics of apoptosis. This is in contrast to the induction of necrosis that we previously observed upon treatment of PC12 cells with another ParkinsonÕs disease mimetic, MPP+ [27]. Other groups have also observed a difference in the mode of cell death between these two neurotoxins in primary mesencephalic neurons [7], MN9D cells [9], and PC12 cells [2,10]. Concurrent with these apoptosis-associated changes, we report here for the first time the induction of a classical heat shock response by 6-OHDA. Treatment with 6-OHDA led to a robust induction in Hsp25 expression, with a relatively mild elevation in Hsp70. This was preceded by activation of HSF-1, the main transcription factor involved in classical Hsp induction. These data are in agreement with a recent report by Greene and co-workers [32], who examined Hsp27 and Hsp70 mRNA induction by 6-OHDA, and demonstrated a
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large induction in Hsp27 mRNA and a less marked increase in the levels of Hsp70 mRNA in PC12 cells exposed to 100 lM 6-OHDA. The relatively greater induction of Hsp25/27 may reflect a specific response to oxidative stress, since 6-OHDA is known to cause an increase in the level of reactive oxygen species [9]. Several studies have shown that small Hsps (including Hsp25/27) can protect against oxidative stress [19,33– 35], and antioxidants have also been shown to be more effective in blocking the induction of Hsp27 than Hsp70 in HL-60 cells [18]. Hsps are reported to promote cell survival by inhibiting apoptosis at several levels [20]. We hypothesise that the induction of Hsps by 6-OHDA reflects an attempt by the cell to protect itself. Indeed, following 6-OHDA treatment, the majority of dying cells (85.3%) did not express Hsp25. This is in agreement with previous findings that Hsps are not induced in cells that are committed to die, suggesting that the induction of the heat shock response and cell death are mutually exclusive events [12,18]. However, in the present study a proportion of the surviving cells did not express high levels of Hsp25, suggesting that it is not the only factor involved in protection against 6-OHDA. The induction of other stress proteins by 6-OHDA, such as Hsp70, may be involved in the survival of the cells that do not undergo apoptosis as a result of the treatment. In support of the hypothesis that induction of stress proteins by 6-OHDA could protect against induction of apoptosis by the compound, we showed for the first time that prior heat shock, which induces the expression of both Hsp25 and Hsp70, reduced the level of apoptosis due to 6-OHDA exposure. We and others have reported previously that prior heat shock can reduce the toxicity of another Parkinson mimetic, MPP+, in PC12 [27] and Chinese hamster ovary fibroblasts [36]. Since Hsp25 was the major Hsp induced by 6-OHDA we expected that it may play an important role in inhibiting apoptosis in the subpopulation that survives 6-OHDA treatment. This report demonstrates that overexpression of Hsp27 delays the release of pro-apoptotic factors, cytochrome c and Smac/Diablo, reduces caspase activity and apoptosis, thus providing protection to PC12 cells against 6OHDA toxicity. This lends support to the hypothesis that induction of Hsp25 by 6-OHDA could prevent apoptosis in those cells that express it. Previous publications from our group and others have shown that Hsp27 is a potent inhibitor of the mitochondrial pathway to caspase activation and apoptosis [22–24,37–39]. Hsp27 interferes with release of cytochrome c [23,39] and Smac/Diablo [25] from the mitochondria, formation of the apoptosome, and caspase activation [24]. The delay in cytochrome c release induced by Hsp27 has been reported to correlate with the level of its expression [39]. This suggests that there may be different levels of Hsp25 induced by 6OHDA and that the delay in cytochrome c release may
be more or less prolonged in different cells depending on the intracellular concentration of Hsp25. In conclusion, the relatively low level of death in the cultures reveals that there are different responses of the cells in the population with many of them surviving the stress. The induction of Hsps by the cells suggests that a proportion of the population mounts a protective response. There is mounting evidence for a role of stress responses in neurodegenerative disorders such as ParkinsonÕs disease and stroke [32,40–44]. However, with increasing age an organismÕs ability to induce heat shock proteins is reduced [45]. Therefore, if Hsp25 or another stress protein confers protection against the toxic effects of 6-OHDA, the prevalence of ParkinsonÕs disease in the aged population might be due, in part, to a decreased ability to induce protective proteins in response to a toxic insult. The identity of the endogenous toxin in sporadic forms of ParkinsonÕs disease is currently unknown, although 6-OHDA and other oxidized derivatives of dopamine are considered likely to play a role. Thus, understanding neuronal responses to 6-OHDA, both mechanisms of cell death and pro-survival strategies, may contribute towards our understanding of the disease.
Acknowledgments The authors thank Dr. Una FitzGerald for critical reading of the manuscript. This work was financially supported by the Health Research Board of Ireland, the Higher Education Authority of Ireland, and the Millennium Fund of NUI, Galway.
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Hsp27 inhibits 6-hydroxydopamine-induced cytochrome c release and apoptosis in PC12 cells Adrienne M. Gorman, Eva Szegezdi, Declan J. Quigney, Afshin Samali* Department of Biochemistry and the National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Received 9 December 2004 Available online 22 December 2005
Abstract Cellular stress may stimulate cell survival pathways or cell death depending on its severity. 6-Hydroxydopamine (6-OHDA) is a neurotoxin that targets dopaminergic neurons that is often used to induce neuronal cell death in models of ParkinsonÕs disease. Here we present evidence that 6-OHDA induces apoptosis in rat PC12 cells that involves release of cytochrome c and Smac/Diablo from mitochondria, caspase-3 activation, cleavage of PARP, and nuclear condensation. 6-OHDA also induced the heat shock response, leading to increased levels of Hsp25 and Hsp70. Increased Hsp25 expression was associated with cell survival. Prior heat shock or overexpression of Hsp27 (human homologue of Hsp25) delayed cytochrome c release, caspase activation, and reduced the level of apoptosis caused by 6-OHDA. We conclude that 6-OHDA induces a variety of responses in cultured PC12 cells ranging from cell survival to apoptosis, and that induction of stress proteins such as Hsp25 may protect cells from undergoing 6-OHDA-induced apoptosis. 2004 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Caspase; Heat shock response; Hsp27; ParkinsonÕs disease; Cytochrome c; Smac/Diablo; Rat
6-Hydroxydopamine (6-OHDA), a hydroxylated analogue of dopamine, is commonly used in model systems to mimic ParkinsonÕs disease, since it induces death of dopaminergic neurons both in vivo and in vitro [1,2]. The cellular events that occur as a result of 6-OHDA exposure are not entirely understood. Production of reactive oxygen species [3–5] and/or inhibition of complex I are both reported to mediate 6-OHDA-induced cell death [6]. There is general agreement, however, that the mode of cell death is by apoptosis in a variety of cell culture model systems, including primary mesencephalic dopaminergic neurons [7,8], MN9D [9], and PC12 cells [2,10]. Apoptosis is a highly regulated form of cell death that occurs under physiological and pathological conditions. The activation of caspase proteases is central to this pro*
Corresponding author. Fax: +353 91 750596. E-mail address: [email protected] (A. Samali).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.066
cess [11,12]. Caspase activation can be triggered by the release of cytochrome c from the mitochondria, which occurs in response to several apoptotic stimuli including neurotoxins [13,14]. In the cytosol, cytochrome c interacts with Apaf-1, which in the presence of dATP leads to clustering and autoactivation of caspase-9 [13,14]. Active caspase-9 causes cleavage and activation of downstream caspases, e.g., caspase-3. Caspases mediate the degradation of a number of proteins critical for cell homeostasis such as the cytoskeletal protein, fodrin, and the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP) [15]. While exposure of cells to severe stress can induce cell death, transient or milder stress conditions stimulate cells to activate protective strategies that involve the induction of prosurvival proteins [16,17]. For example, increased expression of heat shock protein 27 (Hsp27) and Hsp70 occurs after exposure of cells to non-lethal elevations in temperature or oxidants [18]. Hsp27
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belongs to a sub-family of stress proteins, the small Hsps, which are detectable in virtually all organisms. Increased expression of Hsp27 renders cells more resistant to lethal levels of a variety of toxic insults [19–21]. Hsp27 inhibits apoptosis [20], by interfering with caspase activation at several different levels [22–24]. It can interact directly with mitochondria, thereby preventing release of cytochrome c [23] and Smac/Diablo [25]. In the cytosol, it can also sequester both cytochrome c and pro-caspase-3, thus preventing the correct formation/function of the apoptosome [22,24]. The present study was undertaken to examine the effect of 6-OHDA on the induction of cell death and stress responses, and in particular, to examine the role of Hsp27 in 6-OHDA-induced cell death in PC12 cells.
Materials and methods Materials. All chemicals were purchased from Sigma unless indicated otherwise. Mouse monoclonal antibody against Hsp70 and rabbit polyclonal antibodies against Hsp25 (rat homologue of human Hsp27) were obtained from StressGen Biotechnologies. Mouse monoclonal antibodies against PARP were obtained from Biomol and cytochrome c was from BD Pharmingen. Rabbit polyclonal antibody against caspase-3 was obtained from Cell Signalling Technology and Smac/Diablo antibody was a kind gift from Prof. Seamus Martin, Dept. of Genetics, Trinity College, Dublin, Ireland. Goat secondary antibodies conjugated to horseradish peroxidase were from Pierce. Goat secondary anti-rabbit IgG conjugated to Alexa-546 was from Molecular Probes. Ac-Asp-Glu-Val-Asp-a-(4-methyl-coumaryl7-amide) (DEVD-MCA) was from the Peptide Institute, Osaka, Japan. Protein molecular weight markers were obtained from New England Biolabs. T4 polynucleotide kinase was from Promega. [c-32P]ATP was from ICN. Effectene transfection reagent was from Qiagen. PCI-neo Mammalian expression vector was obtained from Promega. Rat pheochromocytoma PC12 cells were obtained from the ECACC. Cell culture and treatments. PC12 cells were maintained in RPMI 1640 medium supplemented with 10% horse serum, 5% fetal calf serum, 50 U/ml penicillin, 50 lg/ml streptomycin, and 2 mM D -glutamine, at 37 C in a humidified 5% CO2 atmosphere. They were passaged once weekly. Experiments were performed using dishes coated with poly-L -lysine (10 lg/ml for 3 h) to assist cell adhesion. For determining Hsp induction cells were seeded at a density of 5 · 106 in a 25 cm2 culture flask and cultured for 24 h, prior to treatment (heat shock or addition of 6-OHDA). To subject the cells to heat shock, the culture flasks were sealed by wrapping parafilm around the lids and immersed in a water bath at 41.5 C for 1 h. Cells were then allowed to recover at 37 C in a humidified 5% CO2 atmosphere for various times. For treatment with 6-OHDA, stock solutions of 6-OHDA were made freshly in sodium metabisulphite (1 mg/ml) prior to each experiment. Cells were exposed to various concentrations of 6-OHDA as indicated in the figure legends. Analysis of cellular morphology. For morphological analysis, cells were scraped from flasks, and 100 ll of the cell suspension (approximately 50 · 104 cells) was cytocentrifuged onto glass slides. After airdrying the preparations were stained using RAPI-DIFF II stain pack (Triangle Biomedical Sciences). Cells were scored by counting at least 300 cells from each sample, from three different experiments. DNA fragmentation. Following experimental treatments, cells were removed from the culture flask by scraping. Cells were centrifuged at 775g for 5 min at 4 C. The pellet was washed once with phosphate-
buffered saline (PBS), and lysed in 300 ll of a buffer containing 100 mM Tris–HCl, pH 8.5, 5 mM EDTA, 0.2 M NaCl, 0.2% (w/v) SDS, and 0.0001% (w/v) proteinase K. The cell suspension was incubated for 3 h at 50 C, followed by addition of 30 ll of 3 M C2H3NaO2 and 660 ll of 96% ethanol. The cell suspension was kept overnight at 20 C. The lysate was then centrifuged at 21,000g for 10 min. The supernatant was discarded, the pellet was resuspended in 70% ethanol and centrifuged at 21,000g for 10 min. The pellet was air-dried at room temperature for 30 min. The pellet was resuspended in 50 ll buffer containing 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, and 100 lg/ml RNase. The sample was incubated for 1 h at 37 C. The sample was then centrifuged at 21,000g for 10 min, supernatant was removed, resuspended in 5· loading buffer (25% Ficoll and 0.25% bromophenol blue), and resolved on a 1.5% agarose gel for 2 h at 60 V. The bands were then visualised under UV light. Detection of caspase activity. The activity of group II caspases, DEVDases, was determined fluorometrically as developed by Nicholson et al. [26] with some modifications [27]. Briefly, lysate from 2.5 · 106 cells and substrate (DEVD-MCA) were combined in reaction buffer {100 M N-2-hydroxyethyl-piperazine-N 0 -2-ethanesulphonic acid (HEPES), pH 7.5, 10% sucrose, 0.1% 3[(3cholamidopropyl)-dimethylammonio]-1-propanesulphonate, 5 mM dithiothreitol, 104% Nonidet P40, and 50 lM DEVD-MCA} and added in triplicate to a microtitre plate. Substrate cleavage leading to the release of free MCA was monitored at 37 C using a Wallac Victor multilabel counter (excitation 355 nm, emission 460 nm). Fluorescent units were converted to micromoles of MCA released using a standard curve generated with free MCA and subsequently related to protein concentration. Preparation of whole cell extracts. Following experimental treatments the cells were removed from the culture flask by scraping. The cells were centrifuged at 775g for 5 min at 4 C. The pellet was washed once with PBS and lysed in 100 ll of a buffer containing 20 mM HEPES, pH 7.5, 350 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 0.5 mM dithiothreitol, 0.1% phenylmethylsulphonyl fluoride, and 1% aprotinin. The cell suspension was incubated on ice for 15 min and then centrifuged at 21,000g for 30 s. The supernatants were stored at 70 C until further analysis by Western blotting. Protein content was determined using a Bio-Rad protein assay kit with bovine serum albumin (BSA) as standard. Western blotting. Samples were resuspended in LaemmliÕs sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and boiled for 5 min. Proteins (20–25 lg per lane) were then resolved on 10–12% SDS–PAGE gels and electrophoretically transferred onto nitrocellulose for 1.5 h at 100 V. Membranes were blocked for 1 h in PBS containing 0.05% Tween 20 and 5% (w/v) nonfat dried milk. The membranes were then incubated for 1 h at room temperature with antibodies to Hsp25 (1:2000), Hsp27, cytochrome c, Hsp70 (1:1,000), Smac/Diablo (1:200) or actin (1:500). Alternatively, the membranes were incubated overnight at 4 C with antibodies to caspase-3 or PARP (1:1000). This was followed by 1 h incubation at room temperature with appropriate horseradish peroxidase-conjugated goat IgGs (1:10,000 or 1:2000 for detection of caspase-3). Protein bands were then visualised using Supersignal West pico Western blot detection kit (Pierce). Determination of cytochrome c and Smac/Diablo release. Cells were trypsinised and centrifuged at 150g for 5 min at 4 C. The pellet was washed once with PBS. The cells were then lysed using 100 ll cell lysis and mitochondria intact (CLAMI) buffer containing (250 mM sucrose and 70 mM KCl in PBS, 0.1 mM phenylmethylsulphonyl fluoride, 1 mM dithiothreitol, 5 lg/ml pepstatin, 10 lg/ml leupeptin, 2 lg/ml aprotinin, and 25 lg/ml calpain inhibitor 1). Digitonin (10 ll of 20 mg/ ml solution) was added to the samples on ice for 5 min and then the cell suspension was centrifuged at 3000g for 10 min at 4 C. The supernatant was removed and stored as the cytosolic fraction, at 20 C. The pellet was resuspended in 100 ll CLAMI buffer and stored as the mitochondrial fraction, at 20 C. Samples were analysed by Western blotting.
A.M. Gorman et al. / Biochemical and Biophysical Research Communications 327 (2005) 801–810 Electrophoretic mobility shift assay. Following appropriate treatments, cells were washed once in ice-cold PBS, centrifuged at 2000g, and the pellet was snap-frozen in liquid nitrogen. Whole cell extracts were prepared by resuspending the pellet with 50 ll of 20 mM HEPES, pH 7.9, containing 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulphonyl fluoride, and 25% glycerol. Samples were allowed to swell on ice for 5 min and subsequently centrifuged at 15,000g for 20 min at 4 C. A synthetic oligonucleotide corresponding to the proximal heat shock element (HSE) of the human Hsp70 promoter (5 0 -GCC TCG AAT GTT CGC GAA GTT TTC) was end labelled with [c-32P]ATP using T4 polynucleotide kinase. Whole cell extracts (10–15 lg) were incubated with the labelled oligonucleotide for 20 min in binding buffer containing 20 mM Tris, pH 7.5, 100 mM NaCl, 2 mM EDTA, and 10% glycerol. The protein–DNA complexes were resolved on a native 4% polyacrylamide gel using 0.25· TBE running buffer for 2 h at 150 V. The gel was dried under vacuum onto Whatmann 3MM filter paper and autoradiographed overnight with X-ray film. Hsp25 immunostaining. Cytocentrifuge preparations of PC12 cells were fixed in 3.7% formaldehyde for 5 min at room temperature and then permeabilised with 0.2% Triton X-100 for a further 5 min at room temperature. Free antibody binding sites were blocked for 1 h at room temperature with PBS containing 5% goat serum and 1% BSA followed by 1 h incubation with the primary antibody (rabbit anti-Hsp25, diluted 1:1000 with 1% BSA in PBS). Excess antibody was removed by 3 · 5 min washes in PBS. Then the slides were incubated with goat anti-rabbit IgG, Alexa-546 conjugate (diluted 1:200 in 1% BSA in PBS) for 1 h at room temperature. Unbound antibodies were removed by
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3 · 5 min washes in PBS before mounting the slides with 80% glycerol containing 3 lg/ml Hoechst 33342. Cells were visualised with a Zeiss, S100 fluorescence microscope. Cells were scored as dying if they stained intensely with Hoechst and showed evidence of nuclear condensation. Cells were scored as expressing high levels of Hsp25 if the staining with the antibody was comparable to that observed in cells that had been heat shocked (shown in Fig. 4). Transfection procedure. Transfection was performed using Effectene Transfection Reagent according to the manufacturerÕs protocol. Cells were transfected with either empty pCI-neo or human Hsp27-pCI-neo expression vector. Clones which exhibited stable expression of Hsp27 were selected with 800 lg/ml G418. Statistical analysis. Results are expressed as means ± standard error of the mean (SEM). Statistical analysis was made using ANOVA followed by post hoc tests indicated in the Figure legends.
Results Induction of apoptosis in PC12 cells following exposure to 6-OHDA To investigate the effect of 6-OHDA on PC12 cells, we exposed the cells for various periods of time and to a range of concentrations of 6-OHDA, and examined a variety of markers of apoptosis. Morphological
Fig. 1. Induction of apoptosis in PC12 cells treated with 6-OHDA. (A) The morphology of untreated cells (control) and cells treated for the indicated times (24 or 48 h) was examined by making cytocentrifuge preparations of the cells followed by staining with Rapi Diff. Filled arrows indicate apoptotic cells, which are shrunken with condensed nuclei. Open arrows indicate necrotic cells, which are swollen and lack intact plasma membrane. (B) The proportion of apoptotic and necrotic cells at each time point was calculated as a percentage of the total number of cells. Values represent means ± SEM of three separate determinations. Statistical analysis was performed with ANOVA followed by DunnettÕs multiple comparisons post hoc test. **p < 0.01 versus apoptosis at 0 h, *p < 0.05 versus apoptosis at 0 h. (C) PC12 cells were treated with 200 lM 6-OHDA for 0–48 h and the induction of a DNA ladder was shown by agarose gel electrophoresis of DNA extracted from the samples. This experiment was repeated twice with similar results.
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analysis of cytocentrifuge preparations of PC12 cells that were exposed to 200 lM 6-OHDA for 24 h demonstrated nuclear condensation and fragmentation, typical hallmarks of apoptosis (Figs. 1A and B). Longer treatments (48 h) led to an increase in the proportion of necrotic cells in the culture (Figs. 1A and B). Apoptosis was accompanied by degradation of the chromatin into oligonucleosomal-sized fragments (Fig. 1C). There was a dose- and time-dependent increase in caspase activation as measured by an increase in DEVDase activity (Figs. 2A and B), processing of caspase-3 (Figs. 2C and D), and cleavage of the caspase substrate, PARP (Figs. 2C and D). Release of pro-apoptotic proteins from the mitochondria is one mechanism by which caspase activation is initiated [13,14]. In order to investigate the
mitochondria as a possible mediator of 6-OHDA-induced apoptosis, we examined changes in the localisation of mitochondrial pro-apoptotic factors. An early release of both cytochrome c and Smac/Diablo (a pro-apoptotic protein that binds to members of the inhibitors of apoptosis proteins and abrogates their anti-apoptotic effect [28,29]) into the cytosol was detected and this process occurred prior to caspase activation (Fig. 2E). Taken together, these results suggest that the mitochondrion is an early target in 6-OHDAinduced apoptosis. Induction of the heat shock response by 6-OHDA 6-OHDA is known to induce oxidative stress [3] and oxidative stress is a major inducer of stress proteins
Fig. 2. Caspase activation in PC12 cells treated with 6-OHDA. (A) PC12 cells were treated with 0–200 lM 6-OHDA for 24 h and caspase-3-like proteolytic activity, i.e., DEVD-MCA cleavage activity, was measured in whole cell extracts. Values are means ± SEM of three separate determinations. (B) PC12 cells were treated with 200 lM 6-OHDA for 0–48 h and DEVD-MCA cleavage activity was measured in whole cell extracts taken after 24 h of 6-OHDA treatment. Values are means ± SEM of 3 separate determinations. (C) PC12 cells were treated with 0–200 lM 6-OHDA for 24 h. Proteolytic processing of caspase-3 and PARP was analysed by Western blotting. The arrows point to pro-caspase-3 (32 kDa), the cleaved active fragment of caspase-3 (17 kDa), intact PARP (116 kDa), and the caspase-dependent cleavage product of PARP (85 kDa). The levels of actin in the samples were also analysed to demonstrate equal loading of the lanes. This experiment was repeated twice with similar results. (D) PC12 cells were treated with 200 lM 6-OHDA for 0–48 h. Proteolytic processing of caspase-3 and PARP was analysed by Western blotting. The levels of actin in the samples were also analysed to demonstrate equal loading of the lanes. This experiment was repeated 3 times with similar results. (E) The release of pro-apoptotic factors from the mitochondria was shown by examining the levels of cytochrome c and Smac/Diablo in cytosolic and mitochondrial fractions of PC12 cells that were exposed to 200 lM 6-OHDA for 0-24 h. This experiment was repeated 3 times with similar results.
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particularly heat shock proteins [30,31]. Since many of the cells in cultures treated with 6-OHDA survived the treatment, we hypothesised that induction of protective proteins may be facilitating the resistance of some cells to undergo cell death. Therefore, the ability of 6-OHDA to induce a heat shock response was examined. Western blot analysis showed that basal levels of both Hsp25 (the rat homologue of Hsp27) and Hsp70 were very low in unstressed PC12 cells. However, exposure to 6-OHDA induced a marked increase in Hsp25 at concentrations as low as 25 lM (Fig. 3A). The levels continued to rise until 200 lM 6-OHDA (Fig. 3A), with the maximal induction occurring at the highest concentration used (200 lM, Fig. 3A). There was also induction of Hsp70 expression, although to a lesser extent than Hsp25 (Fig. 3A). These low concentrations of 6-OHDA also induced activation of caspases in the cells as seen by an increase in DEVDase activity (Fig. 2A), caspase-3 and PARP cleavage (Fig. 2C). A time course of Hsp induction by 200 lM 6-OHDA showed that Hsp25 and Hsp70 were induced following 12 h of treatment and that Hsp expression remained elevated until at least
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24 h (Fig. 3B). This was preceded by the induction of heat shock factor 1 (HSF-1) DNA-binding activity which was detectable within 6 h of 6-OHDA treatment (Fig. 3C). These data show that a classical heat shock response occurs in PC12 cells exposed to 6-OHDA. Prior heat shock reduces 6-OHDA-induced cell death in PC12 cells Recent reports from our group showed that the exposure of PC12 cells to non-lethal elevations in temperature results in the synthesis and accumulation of Hsp25 and Hsp70 [27]. This has been shown to induce a state of thermotolerance and renders the cells resistant to subsequent treatment with 1-methyl-4-phenylpyridinium (MPP+) [27]. In order to determine whether the induction of Hsps and thermotolerance can protect against 6-OHDA-induced apoptosis, PC12 cells were heat shocked (or in the case of control cells kept at 37 C) for 1 h and allowed to recover at 37 C for 6 h. This treatment induced the synthesis of Hsp25 and Hsp70 (Fig. 4A). Cells were then treated with 200 lM 6-OHDA for a further 24 h. Prior heat shock treatment caused a significant decrease in the level of apoptosis in cells exposed to 6-OHDA compared with cells that did not undergo prior heat shock (p < 0.05, Fig. 4B). This was mirrored by a statistically significant reduction in 6-OHDA-induced DEVDase activity in cells that underwent prior exposure to heat shock (p < 0.05, Fig. 4C). Hsp25 expression in dying and surviving cells
Fig. 3. Expression of Hsp25 and Hsp70 following exposure to 6OHDA. (A) PC12 cells were treated with 0–200 lM 6-OHDA for 24 h. The expression of Hsp25 and Hsp70 was analysed by Western blotting. The levels of actin in the samples are also shown to demonstrate equal loading of the lanes. (B) PC12 cells were treated with 200 lM 6-OHDA for 0–24 h. A positive control (HS) to show induction of Hsps was included: cells were heat shocked at 41.5 C for 1 h and extracts were made immediately. The expression of Hsp25 and Hsp70 was analysed by Western blotting. The levels of actin in the samples are also shown to demonstrate equal loading of the lanes. (C) Cells were treated with 200 lM 6-OHDA for 0–24 h. Total protein extracts were incubated with a [c-32P]ATP-labelled oligonucleotide corresponding to the proximal HSE of human Hsp70 promoter and the protein–DNA complexes (HSF-1/HSE) were resolved on a native 4% polyacrylamide gel. These experiments were repeated twice with similar results.
Since there was a dual response, i.e., cell death and cell survival, observed in PC12 cell cultures exposed to 6OHDA (Figs. 1A and B), it was hypothesised that Hsp induction by 6-OHDA may correlate with cell survival and lack of induction of Hsp with cell death. Hsp25 was the main Hsp induced by 6-OHDA (Figs. 3A and B) and therefore, it was decided to examine levels of Hsp25 expression in the dying and surviving populations of cells exposed to 6-OHDA. The cells were either heat shocked (as a positive control for determining Hsp25 expression) or exposed to 150 lM 6-OHDA for 24 h and stained with Hoechst 33342 and anti-Hsp25. Fig. 5A shows a typical view of untreated, heat shocked, and 6-OHDA-treated cells. In untreated cultures the nuclei were not condensed and there were low levels of Hsp25 staining in all of the cells examined. In contrast, in heat shocked cultures there were high levels of Hsp25 immunostaining in a large proportion of the cells and the nuclei were not condensed. In cultures treated with 6-OHDA there was high level of Hsp25 staining in some but not all of the cells (Fig. 5A) and there was nuclear condensation (indicative of cell death) in a proportion of the cells. The cells were counted and it was found that in 6-OHDA-treated PC12 cells there
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Fig. 4. Effect of prior heat shock on 6-OHDA-induced apoptosis. (A) Cells were heat shocked at 41.5 C for 1 h and allowed to recover for 2, 4, 6, 8, and 24 h at 37 C. Total protein extracts (20 lg/lane) were subjected to 12% SDS–PAGE, followed by Western blot analysis, using monoclonal antibodies to Hsp25 and Hsp72. (B) Cells were either kept at 37 C (control) or heat shocked at 41.5 C for 1 h and allowed to recover for 6 h at 37 C (HS). They were then exposed to 200 lM 6-OHDA for 24 h. The morphology of the cells was assessed using cytocentrifuge preparations of the cells that were stained with Rapi Diff and the number of apoptotic cells was calculated as a percentage of the total number of cells. Values represent means ± SEM of three separate determinations. (C) Cells were treated as in (B) and DEVD-MCA cleavage activity was measured in whole cell extracts. Values are means ± SEM of three separate determinations. Statistical analysis was performed with repeated measures ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. *p < 0.05 versus 6-OHDA alone or heat shock alone.
was induction of high levels of Hsp25 in 34.0 ± 1.6% of the total cell population (Fig. 5B). In contrast, when the subpopulation of cells that were dying was examined it can be seen that there was induction of high levels of Hsp25 in 14.7 ± 2.5% of the dying cells (Fig. 5B) while the remaining 85.3% of the dying cells did not express Hsp25. The difference in the proportion of cells expressing high levels of Hsp25 in the dying population compared with the total population and with the subpopulation of cells that survived was statistically significant (p < 0.001).
Fig. 5. Expression of Hsp25 in dying and surviving cells following 6OHDA treatment. (A) PC12 cells were either heat shocked or treated with 150 lM 6-OHDA for 24 h. Cytocentrifuge preparations of the cells were stained with anti-Hsp25 and Hoechst 33342. The cells were visualised by fluorescence microscopy and scored as dying (filled arrows; condensed nuclear staining indicative of apoptosis or diffuse nuclear staining indicative of necrosis) or surviving (open arrows). The dying cells were then scored as having high or low levels of Hsp25 expression. Representative images of untreated, heat shocked, and 6OHDA-treated PC12 cells stained with Hsp25 and Hoechst 33342. (B) The proportions of the total cell population, and the dying and surviving subpopulations of cells, that expressed high and low levels of Hsp25, were calculated and expressed as a percentage of the total number of cells in each of those groups (total, dying or surviving). The data are means ± SEM of three separate experiments. Statistical analysis was performed with repeated measures ANOVA followed by Student–Newman–Keuls multiple comparisons post hoc test. ***p < 0.001 versus dying, low Hsp25; versus surviving, high Hsp25; **p < 0.01 versus total, high Hsp25; and *p < 0.05 versus total, high Hsp25.
Overexpression of Hsp27 reduces 6-OHDA-induced cell death in PC12 cells The correlation between Hsp25 expression and cell survival in cultures treated with 6-OHDA suggested that Hsp25 provided a prosurvival advantage to the cells in which it was expressed. In order to determine whether Hsp25 would protect against 6-OHDA-induced apoptosis, PC12 cells were transfected with Hsp27, the human
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Fig. 6. Inhibition by Hsp27 of 6-OHDA-induced apoptosis. (A) The expression of Hsp27 in wild type PC12 cells (WT), cells transfected with an empty vector (Neo), and cells transfected with Hsp27 (Hsp27) was examined by Western blotting. (B) PC12 cells transfected with empty vector (Neo) or with Hsp27 (Hsp27) were exposed to 200 lM 6-OHDA for 24 h and the level of apoptosis was assessed by morphological criteria. Values are means ± SEM of 3 separate determinations. Statistical analysis was performed with repeated measures ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. *p < 0.01 versus 6-OHDA-treated neo-transfected cells. (C) Cells were treated as in (B) and caspase-3-like DEVDase activity was determined. Values are means ± SEM of three separate determinations. Statistical analysis was performed with repeated measures ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. *p < 0.01 versus 6-OHDA-treated neo-transfected cells. (D) PC12 cells transfected with empty vector (Neo) or with Hsp27 (Hsp27) were exposed to 200 lM 6-OHDA for 24 h and proteolytic processing of caspase-3 was analysed by Western blotting. This experiment was performed 3 times with similar results. (E) PC12 cells transfected with empty vector (Neo) or with Hsp27 (Hsp27) were exposed to 200 lM 6-OHDA for 0–12 h. The levels of cytochrome c and Smac/Diablo in the cytosolic fractions were analysed by Western blotting. This experiment was performed 3 times with similar results.
homologue of rat Hsp25. The expression of Hsp27 was detected using specific antibodies that did not cross-react with Hsp25 (Fig. 6A). In comparison to cells transfected with the empty vector, Hsp27-overexpressing cells were more resistant to treatment with 200 lM 6OHDA for 24 h (Fig. 6B). This coincided with a reduction in DEVDase activity (Fig. 6C) and a decrease in caspase-3 cleavage (Fig. 6D) in cells exposed to 6OHDA compared with cells that were transfected with a neo construct. The reduction in caspase activity and apoptosis was associated with a significant delay in the release of cytochrome c and Smac/Diablo into the cytosol (Fig. 6E).
Discussion The present study shows differential responses of individual cells in cultures exposed to 6-OHDA, ranging from the demise of cells by apoptosis to the induction of stress responses. In agreement with other reports, our
study shows that 6-OHDA-mediated toxicity in PC12 cells exhibits the morphological (nuclear condensation and cell shrinkage) and biochemical (caspase activation, PARP cleavage, and production of a DNA ladder) characteristics of apoptosis. This is in contrast to the induction of necrosis that we previously observed upon treatment of PC12 cells with another ParkinsonÕs disease mimetic, MPP+ [27]. Other groups have also observed a difference in the mode of cell death between these two neurotoxins in primary mesencephalic neurons [7], MN9D cells [9], and PC12 cells [2,10]. Concurrent with these apoptosis-associated changes, we report here for the first time the induction of a classical heat shock response by 6-OHDA. Treatment with 6-OHDA led to a robust induction in Hsp25 expression, with a relatively mild elevation in Hsp70. This was preceded by activation of HSF-1, the main transcription factor involved in classical Hsp induction. These data are in agreement with a recent report by Greene and co-workers [32], who examined Hsp27 and Hsp70 mRNA induction by 6-OHDA, and demonstrated a
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large induction in Hsp27 mRNA and a less marked increase in the levels of Hsp70 mRNA in PC12 cells exposed to 100 lM 6-OHDA. The relatively greater induction of Hsp25/27 may reflect a specific response to oxidative stress, since 6-OHDA is known to cause an increase in the level of reactive oxygen species [9]. Several studies have shown that small Hsps (including Hsp25/27) can protect against oxidative stress [19,33– 35], and antioxidants have also been shown to be more effective in blocking the induction of Hsp27 than Hsp70 in HL-60 cells [18]. Hsps are reported to promote cell survival by inhibiting apoptosis at several levels [20]. We hypothesise that the induction of Hsps by 6-OHDA reflects an attempt by the cell to protect itself. Indeed, following 6-OHDA treatment, the majority of dying cells (85.3%) did not express Hsp25. This is in agreement with previous findings that Hsps are not induced in cells that are committed to die, suggesting that the induction of the heat shock response and cell death are mutually exclusive events [12,18]. However, in the present study a proportion of the surviving cells did not express high levels of Hsp25, suggesting that it is not the only factor involved in protection against 6-OHDA. The induction of other stress proteins by 6-OHDA, such as Hsp70, may be involved in the survival of the cells that do not undergo apoptosis as a result of the treatment. In support of the hypothesis that induction of stress proteins by 6-OHDA could protect against induction of apoptosis by the compound, we showed for the first time that prior heat shock, which induces the expression of both Hsp25 and Hsp70, reduced the level of apoptosis due to 6-OHDA exposure. We and others have reported previously that prior heat shock can reduce the toxicity of another Parkinson mimetic, MPP+, in PC12 [27] and Chinese hamster ovary fibroblasts [36]. Since Hsp25 was the major Hsp induced by 6-OHDA we expected that it may play an important role in inhibiting apoptosis in the subpopulation that survives 6-OHDA treatment. This report demonstrates that overexpression of Hsp27 delays the release of pro-apoptotic factors, cytochrome c and Smac/Diablo, reduces caspase activity and apoptosis, thus providing protection to PC12 cells against 6OHDA toxicity. This lends support to the hypothesis that induction of Hsp25 by 6-OHDA could prevent apoptosis in those cells that express it. Previous publications from our group and others have shown that Hsp27 is a potent inhibitor of the mitochondrial pathway to caspase activation and apoptosis [22–24,37–39]. Hsp27 interferes with release of cytochrome c [23,39] and Smac/Diablo [25] from the mitochondria, formation of the apoptosome, and caspase activation [24]. The delay in cytochrome c release induced by Hsp27 has been reported to correlate with the level of its expression [39]. This suggests that there may be different levels of Hsp25 induced by 6OHDA and that the delay in cytochrome c release may
be more or less prolonged in different cells depending on the intracellular concentration of Hsp25. In conclusion, the relatively low level of death in the cultures reveals that there are different responses of the cells in the population with many of them surviving the stress. The induction of Hsps by the cells suggests that a proportion of the population mounts a protective response. There is mounting evidence for a role of stress responses in neurodegenerative disorders such as ParkinsonÕs disease and stroke [32,40–44]. However, with increasing age an organismÕs ability to induce heat shock proteins is reduced [45]. Therefore, if Hsp25 or another stress protein confers protection against the toxic effects of 6-OHDA, the prevalence of ParkinsonÕs disease in the aged population might be due, in part, to a decreased ability to induce protective proteins in response to a toxic insult. The identity of the endogenous toxin in sporadic forms of ParkinsonÕs disease is currently unknown, although 6-OHDA and other oxidized derivatives of dopamine are considered likely to play a role. Thus, understanding neuronal responses to 6-OHDA, both mechanisms of cell death and pro-survival strategies, may contribute towards our understanding of the disease.
Acknowledgments The authors thank Dr. Una FitzGerald for critical reading of the manuscript. This work was financially supported by the Health Research Board of Ireland, the Higher Education Authority of Ireland, and the Millennium Fund of NUI, Galway.
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