Neurochemical Research, Vol. 27, No. 9, September 2002 (© 2002), pp. 891–898

Neuronal Apoptosis Induced by Endoplasmic Reticulum Stress Lizhen Chen1 and Xiang Gao1,2,3 (Accepted June 12, 2002)

Apoptosis is a conserved active cellular mechanism occurring under a range of physiological and pathological conditions. In the nervous system, apoptosis plays crucial roles in normal development and neuronal degenerating diseases. Various deleterious conditions, including accumulation of the mutant proteins in the endoplasmic reticulum (ER) and inhibition of ER to Golgi transport of proteins, may result in apoptosis. In this study, we examined the downstream events of apoptosis in differentiated PC 12 cells under ER stress induced by brefeldin A, an inhibitor of ER to Golgi protein transport. Activation of NF-␬B and degradation of I-␬B were observed within 2 hours, followed by up-regulation of GRP78 protein level in treated cells. Caspase-12 only appeared around 24 hours after brefeldin A treatment, coincident with cell nuclei fragmentation. These results suggest that neuronal apoptosis may be induced by ER stress through a NF-␬B and caspase related pathway.

KEY WORDS: ER stress; apoptosis; NF-␬B; caspase; GRP78.

INTRODUCTION

and these components of apoptotic pathway, however, is still largely unclear. In this study, NF-␬B and caspase were chosen for examination of their roles in endoplasmic reticulum (ER) stress–induced apoptosis in neuronal cells. NF-␬B is an inducible transcription factor that is activated by a wide variety of agents. The major regulatory control of NF-␬B activation relies on nuclear translocation after dissociation from I-␬B␣. As a cytoplasmic protein, I-␬B␣ binds avidly to the p65 subunit of NF-␬B through the ankyrin repeats of I-␬B␣ and the nuclear localization signal and the lg-like domain of p65 (1,2). During activation of NF-␬B, numerous stimuli activate a complex of I-␬B kinase. I-␬B kinase then further catalyzes phosphorylation of several serine residues as a necessary step for I-␬B ubiquination and degradation (3,4). Degradation of I-␬B exposes the NF␬B nuclear localization signal, resulting in transportation of NF-␬B into the nucleus (5). Once activated, NF-␬B transcriptionally regulates many cellular genes related with apoptosis (3,6,7).

Apoptosis can be induced by many stimuli. Under deleterious conditions such as treatment with tumor necrosis factor or cytotoxic drugs, UV irradiation, heat shock, virus infection, and withdrawal of growth factors, cells may adopt this “active form” of death pathway. Apoptotic cells are characterized by distinct morphological changes of nucleus condensation/ fragmentation and formation of apoptotic bodies. Signals mediating these processes have been studied intensively, and several common components have been identified in the past few years. The functional relationship between a specific apoptosis-inducing signal 1

Model Animal Research Center, Institute of Molecular Medicine. State Key Lab of Biotechnology, Nanjing University, Nanjing, People’s Republic of China. 3 Address reprint requests to: Xiang Gao, Model Animal Research Center, Nanjing University, 22 Hankou Road, Nanjing 210093, People’s Republic of China. Tel: 86 25 3595577; Fax: 86 25 3595457; E-mail: [email protected] 2

891 0364-3190/02/0900–0891/0 © 2002 Plenum Publishing Corporation

892 Caspases are a family of cystine proteases critically mediating apoptotic process. Fourteen members of this family have been identified. Some of them mediate signal transduction downstream of death receptors located on the plasma membrane (8,9). Some mediate apoptotic signals after mitochondria damage (10). Recently, caspase-12 was found to localize to the ER and may be activated by ER stress (11). Endoplasmic reticulum is sensitive to homeostasis alterations induced by a variety of stimuli, such as glucose deprivation, perturbation of calcium homeostasis, and exposure to free radicals. Under these conditions, perturbation of protein folding or the accumulation of malfolded or mutated proteins can induce ER stress states (12,13). To mimic this process, we used brefeldin A, an inhibitor for the process of the protein transportation from the ER to the Golgi apparatus (11). Treatment with brefeldin A causes the accumulation of proteins in the ER, and ER stress in these cells ultimately leads to cell apoptosis. Several studies indicated ER stress involved in certain types of neuronal apoptosis, which might play an important role in brain degenerating diseases. For example, it is well know that disruption of normal folding structure can accumulate certain mutated proteins in ER and ultimately results in apoptosis in targeted cells (13). In this study, we chose differentiated PC 12 cells for examining the signaling pathway that involves apoptosis induced by ER stress. PC 12 cells were derived from pheochromocytoma and differentiated into a sympathetic neuron–like phenotype under induction of nerve growth factor (NGF) (14,15). EXPERIMENTAL PROCEDURE Cell Culture. Rat PC 12 cells were normally cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Life Technologies, Inc.) supplemented with 10% horse serum and 5% fetal calf serum at 37°C in a CO 2 incubator. For brefeldin A treatment, PC 12 cells (2 ⫻ 10 5 cells/ml) were seeded in DMEM containing 10% horse serum and 5% fetal calf serum and cultured for 6 h; then cells were starved for 12 h in DMEM containing 0.1% horse serum. The PC 12 cells then were induced to differentiate for 24 h with 50 ng/ml NGF (2.5S NGF, GIBCO Life Technologies). Finally, cells were treated with 10 ␮g/ml of brefeldin A (Sigma) for the time indicated. Staining of Apoptotic Neurons. PC 12 cells grown on cover glasses coated with polylysine were fixed with 4% paraformaldehyde in PBS for 15 min at 4°C and then permeabilized in 0.4% Triton X-100 for 10 min at room temperature. Cells were further stained with fluorescent bisbenzimide dye Hoechst 33258 (1 ␮g/ml, Sigma) for 4 min at room temperature. The cover glasses were washed, air-dried and mounted upside down on a microscope slide. Nuclear staining intensity and morphology were evaluated and

Chen and Gao photographed with a fluorescence microscope (NIKON). Quantitative analysis of apoptotic cells was performed by SAS software. Flow Cytometry Analysis. Cells were washed with PBS and harvested by trypsinization. The Cycle Test™ Plus kit (Becton Dickinson) was used as recommended by the manufacturer. The DNA content per nucleus was analyzed by the FACS Calibur flow cytometer (Becton Dickinson). Detection of Caspase Activation. To analyze the activation of putative activation of caspases in situ, the CaspaTag™ The Fluorescein caspase (VAD) Activity Kit (Intergen) was used as recommended by the manufacturer. Isolation of Nuclear and Cytoplasmic Proteins. Nuclear and cytoplasmic proteins were isolated using a method described previously (16) with some modifications. Briefly, cells were washed twice with ice-cold PBS, scraped in ice-cold hypotonic buffer, and left on ice for 15 min. Hypotonic buffer was made of 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), with protease inhibitors, including aprotinin, pepstatin, leupeptin (10 ␮g/ml each), and 0.5 mM phenylmethylsulfonyl fluoride; and with phosphatase inhibitors, including 50 mM NaF, 30 mM ␤-glycerophosphate, 1 mM Na3VO4, and 20 mM ␳-nitrophenyl phosphate. Samples were added with 50 ␮l of 10% Nonidet P-40 and then centrifuged for 1 min at 4°C. Supernatants containing cytoplasmic proteins were collected and stored at ⫺80°C. The pellets, after a single wash with the hypotonic buffer without Nonidet P-40, were suspended in an ice-cold hypertonic salt buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, with protease and phosphatase inhibitors), incubated on ice for 30 min, and centrifuged for 15 min at 4°C. The supernatants were collected as nuclear extracts and stored at ⫺80°C. The concentration of total proteins in the samples was determined by the BCA assay reagent (Pierce). Western Blot Analysis. Cytoplasmic proteins (50 ␮g) from each sample were mixed with 4 ⫻ SDS sample buffer then heated at 95°C for 5 min, and separated by SDS-polyacrylamide gel electrophoresis. After electrophoresis on 12% polyacrylamide gels, the separated proteins were transferred from the gels onto PVDF membranes (Millipore). The membranes were blocked with 5% nonfat milk in TBS-0.05% Tween 20 for 2 h at room temperature, washed three times for 10 min each in TBS-0.05% Tween 20, and incubated with a primary I-␬B␣ antibody (Santa Cruz Biotechnology), goat anti-GRP78 (Santa Cruz), or rat anti-caspase-12 (a gift from Junying Yuan, Harvard Medical School) in TBS-0.05% Tween 20 containing 5% nonfat milk for 1 to 2 h at room temperature. After washed with TBS-0.05% Tween 20, the membranes were incubated with a specific peroxidase conjugated secondary antibody (Sigma) for 1 h at room temperature. After washing (six times for 5 min each), the membranes were analyzed by the Super Signal System (Pierce). Electrophoretic Mobility Shift Assay. NF-␬B binding activity was performed as described (16). Briefly, the binding reaction mixture contained 15 ␮g of nuclear proteins and 35 fmol([␥-32P] ATP labeled, about 50,000 cpm) of double-stranded NF-␬B consensus oligonucleotide (5⬘-AGT TGA GGG GAC TTT CCC AGG C-3⬘) in binding buffer. Binding buffer consists of 50 ␮g/ml of double-stranded poly (dI-dC), 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 1 mM MgCl 2, and 10% glycerol. The binding reaction mixture was incubated at room temperature for 20 min and analyzed by electrophoresis on 5% nondenaturing polyacrylamide gels. After electrophoresis, the gels were dried by Gel-Drier and exposed to Kodak X-ray films at ⫺80°C.

ER Stress Induced Neuronal Apoptosis RESULTS Apoptosis of PC 12 Cell Was Induced by Brefeldin A. NGF induces neuronal-like morphological changes in PC 12 cells (14,15). Fibroblast-like parental PC 12 cells became bipolar shape with long axon-like structures after treatment with 50 ng/ml NGF for 24 h (data not shown). Treatment of brefeldin A (10 ␮g/ml), an inhibitor of the protein transportation from the ER to the Golgi apparatus, induced apoptosis both in parental cells and differentiated cells (Fig. 1). Evaluated with fluorescent DNA binding dye Hoechst 33258 for chromatin condensation and nuclear mor-

893 phology, parental and differentiated PC 12 cells showed significant increase number of apoptotic cells with condensed or fragmented nucleus (Fig. 1). Interestingly, differentiated PC 12 cells were much more sensitive than undifferentiated parental cells (Fig. 1E). The induction of apoptosis and the susceptibility difference between parental and differentiated cells were confirmed by FACS results (Fig. 2). However, FACS data demonstrated much higher DNA fragment rates in both parental cells and differentiated cells after befeldin A treatment. One possibility is that many apoptotic cells detached from cover glasses during Hoechst 33258 staining procedure, whereas

Fig. 1. Brefeldin A induces apoptosis in PC 12 cells, detected by histochemical staining of Hoechst 33258. Arrows indicate typical apoptotic nuclei. (A) Rat PC 12 cells were initially treated with NGF for 24 h, then with NGF and brefeldin A for 24 h (N&B24h). (B) PC 12 cells were initially treated with NGF for 24 h, then with NGF and brefeldin A for 24 h (NGF48h). (C) PC 12 cells were treated with brefeldin A for 24 h (C&B24h). (D) Control PC 12 cells (control). (E) Quantitative analysis of apoptosis induced by brefeldin A (P ⬍ 0.01, n ⫽ 42).

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Fig. 2. FACS analysis of the DNA content of PC 12 cells after being treated with NGF or/and brefeldin A. (A) N&B24h, apoptosis ⫽ 39.85%. (B) NGF48h, apoptosis ⫽ 0.51%. (C) Control ⫹ B, apoptosis ⫽ 4.53%. (D) Control, apoptosis ⫽ 0.49%.

FACS gave more accurate results. Alternatively, the initial phase of DNA fragmentation during apoptosis may be too subtle to be detected by Hoechst 33258 staining. Brefeldin A Results in ER Stress and NF-kB Activation in Differentiated PC 12 Cells. Brefeldin A blocks the transportation of proteins from the ER to the Golgi apparatus and therefore causes the accumulation of proteins in the ER. The 78 kDa glucoseregulated protein GRP78, also known as an immunoglobulin heavy chain–binding protein (BiP), acts as a molecular chaperone located in the ER and may be induced by accumulation of naïve, aberrantly

folded or mutated proteins in ER (17). In this study, ER stress in brefeldin A treated cells was confirmed by the up-regulation of GRP78 protein in differentiated PC 12 cells (Fig. 3A). A time-course study indicated that the GRP78 level increased more than 5 folds after 12 h of treatment. The elevated level of GRP78 in PC 12 cells holds up to 24 h exposure to brefeldin A. This result indicates a continuous stress state in these cells. Parallel to GRP78, I-␬B levels in cytoplasm of differentiated PC 12 cells were significantly lower after only a 2-h treatment with brefeldin A (Fig. 3B). Other researchers have reported that I-␬B␣, the inhibitor of NF-␬B, may be phosphorylated, ubiquinated, and rap-

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Fig. 3. Activation of the NF␬B pathway during ER stress. (A) Induction of GRP78 protein in PC 12 cell with ER stress. (B) Reduction of I-␬B␣ in PC 12 cell with ER stress. Rat lung was used as positive control. (C) Electrophoretic mobility shift assay of NF-␬B binding activity, indicated by the shifted band. NGF24h: PC 12 cells were treated with NGF for 24 h. NGF48h: PC 12 cells were treated with NGF for 48 h. N&B0.5h, N&B2h, N&B12h, N&B24h, N&B36h: PC 12 cells were initially treated with 50 ng/ml NGF for 24 h, then cultured for 0.5, 2, 12, 24, or 36 h in presence of brefeldin A. All experiments were repeated at least three times at each time point.

idly degraded during ER stress in other types of cells (18–20). After release from I-␬B binding, NF-␬B enters the nucleus and regulates the target gene expression. Many downstream genes are crucial for further activation of the apoptosis pathway. By the electrophoretic mobility shift assay, we confirmed activation of NF-␬B in cells exposed to brefeldin A cells as early as 30 min after treatment with brefeldin A (Fig. 3C). Caspase-12 Was Activated during ER Stress in Differentiated PC 12 Cells. To determine whether the caspase pathway may be activated in apoptosis of differentiated PC 12 cells treated with brefeldin A, the CaspaTag™ Kit was used to check the activation of putative caspase in situ. The cells with active caspases were detected under a fluorescent microscope with EX of 330–380 nm and BA of 420 nm. We found that caspases were activated in most of the differentiated cells treated with brefeldin A for 24 h, while little caspase activity was detected in cells without brefeldin A treatment (Fig. 4A–D). Furthermore, treatment of

parental PC 12 cells or differentiated PC 12 cells with brefeldin A induced effective cleavage of procaspase-12 (Fig. 4E).

DISCUSSION Apoptosis is a conserved active cellular mechanism functioning in many important biological processes. In the nervous system, various deleterious conditions could trigger the apoptosis response in neurons and glial cells. Some of these conditions activate the apoptotic pathway though ER stress. For instance, misfolded proteins encoded by a mutated PLP gene fail to be transported from the ER to the Golgi apparatus and subsequently cause cell death induced by ER stress in Pleizaeus-Merzbacher disease (21). Some recent studies also indicated that ER stress might also play a crucial role in the development of Alzheimer’s disease (11). The molecular mechanism that underlies this

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Fig. 4. CaspaTag™ Kit staining of PC 12 cells detecting the activation of caspases. (A) Nuclei staining of Hoechst 33258 in differentiated PC 12 cells treated with brefeldin A for 24 h (EX: 380–420 nm, BA: 450 nm). (B) The same field as in A, showing activation of caspases (EX: 330–380 nm, BA: 420 nm). (C) Nuclei staining of Hoechst 33258 in differentiated PC 12 cells without brefeldin A treatment. (D) The same field of vision as in C, showing activation of caspases. (E) Caspase-12 is cleaved in cells that are undergoing ER stress-induced apoptosis. 1. Untreated PC 12 cells. 2. PC 12 cells treated with brefeldin A for 24 h. 3. Untreated differentiated PC 12 cells. 4. Differentiated PC 12 cells treated with brefeldin A for 24 h.

ER Stress Induced Neuronal Apoptosis phenomenon may provide some insight on the later onset of most neuronal degenerative diseases. However, the signaling pathway from the ER to cell degeneration is still largely unclear. Utilizing the in vitro culture system, we confirmed that ER stress induced by inhibition of ERGolgi protein transportation could trigger massive apoptosis in both parental and differentiated PC 12 cells. Interestingly, we found that differentiated PC 12 cells (cells with neuronal properties) are much more sensitive to ER stress, as indicated by nuclei staining and FACS analysis. This may reflect the heavier workload for protein transportation of differentiated cells, which have higher membrane/plasma ratios. In oligodendrocytes, Baerwald and coworkers (22) also found that differentiated oligodendrocytes were more susceptible to ER stress induced apoptosis than progenitor cells. NF-␬B has been shown to play a crucial role in ER stress. In PC 12 cells, we observed rapid activation of NF-␬B and degradation of I-␬B within 2 h of exposure to brefeldin A. The definitive ER stress marker, up-regulation of GRP78, was only detected 10 h later. Is NF-␬B activation the upstream event of GRP78 up-regulation? What is the functional significance of GRP78 up-regulation? These are questions that need to be address in the future. Caspases are activated in many types of cells during apoptosis. Positive cells containing activated caspases were detected in ER stressed PC 12 cells using the CaspaTag™ Kit. Further analysis suggested that at least caspase-12, a member of the caspase family, was activated in these PC 12 cells. Our results are consistent with the early report that caspase-12 is localized in the ER and may involve ER stress. Moreover, caspase-12 activation was only observed around 24 h after brefeldin A treatment, suggesting that caspase activation is a downstream event in apoptotic signaling pathway. In conclusion, brefeldin. A treatment inhibits the ER-Golgi protein transportation and induces apoptosis of both parental and differentiated PC 12 cells. The ER stress-induced apoptosis in these cells may be regulated by the pathway related to activation of NF-␬B and caspase-12.

ACKNOWLEDGMENTS This study was supported by funds from the Chinese Ministry of Science and Technology and from Nanjing University (X.G.). We thank Dr. Junying Yuan for providing caspase-12 antibody.

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