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. 2017 Oct 16;24(1):29–38. doi: 10.1111/cns.12771

Endoplasmic reticulum stress regulates oxygen‐glucose deprivation‐induced parthanatos in human SH‐SY5Y cells via improvement of intracellular ROS

Hai‐Feng Wang 1, Zong‐Qi Wang 1,2, Ye Ding 1,2, Mei‐Hua Piao 1,3, Chun‐Sheng Feng 3, Guang‐Fan Chi 4, Yi‐Nan Luo 1,2, Peng‐Fei Ge 1,2,✉
PMCID: PMC6490059  PMID: 29045036

Summary

Aims

Endoplasmic reticulum (ER) stress has been demonstrated to regulate neuronal death caused by ischemic insults via activation of apoptosis, but it still remains unclear whether ER stress participates in regulation of parthanatos, a new type of programmed cell death characterized by PARP‐1 overactivation and intracellular accumulation of PAR polymer.

Methods

we used oxygen‐glucose deprivation (OGD) and human SH‐SY5Y cells to simulate neuronal damage caused by ischemia.

Results

Oxygen‐glucose deprivation induced time‐dependent death in SH‐SY5Y cells, which was accompanied with upregulation of PARP‐1, accumulation of PAR polymer, decline of mitochondrial membrane potentials and nuclear translocation of AIF. Pharmacological inhibition of PARP‐1 with its specific inhibitor 3AB rescued OGD‐induced cell death, as well as prevented PAR polymer accumulation, mitochondrial depolarization, and AIF translocation into nucleus. Similar results could be found when PARP‐1 was genetically knocked down with SiRNA. These indicated that OGD triggered parthanatos in SH‐SY5Y cells. Then, we found inhibition of overproduction of ROS with antioxidant NAC attenuated obviously OGD‐induced parthanatos in SH‐SY5Y cells, suggesting ROS regulated OGD‐induced parthanatos. Additionally, OGD also induced upregulation of ER stress‐related proteins. Mitigation of ER stress with chemical chaperone 4‐PBA or trehalose suppressed significantly OGD‐induced overproduction of ROS, PARP‐1 upregulation, PAR polymer accumulation, and nuclear accumulation of AIF, and cell death in SH‐SY5Y cells.

Conclusion

Endoplasmic reticulum stress regulates OGD‐induced parthanatos in human SH‐SY5Y cells via improvement of intracellular ROS.

Keywords: endoplasmic reticulum stress, oxygen‐glucose deprivation, parthanatos, ROS, SH‐SY5Y cells

1. INTRODUCTION

Ischemic stroke is one of the primary factors causing adult death or disability in the world.1 Cerebral ischemia is found to trigger programmed neuronal death via induction of parthanatos, as well as apoptosis.2, 3 As a new modality of programmed cell death, parthanatos is characterized by a series of choreographed biochemical events including overactivation of poly (ADP‐ribose) synthetase 1 (PARP‐1), intracellular accumulation of PAR polymer, mitochondrial depolarization, and nuclear translocation of apoptosis‐inducing factor (AIF).4 The nuclear AIF would cause cell death via induction of chromatolysis or chromatin condensation.5 Parthanatos is different with apoptosis, considering that it is energy‐independent and does not lead to the formation of apoptotic body.6 However, parthanatos has some features similar to apoptosis, which include phosphatidylserine flipping onto the outer plasma membrane, mitochondrial membrane potential decline, and chromatin condensation.6 Besides cerebral ischemia, parthanatos has been reported to be involved in various pathological processes such as diabetes, inflammation, and brain trauma.7, 8, 9, 10 Despite DNA break is identified as the major initiator of parthanatos,4, 5, 6 the factors regulating parthanatos occurrence remain elusive. Thus, identification of the regulatory factors of parthanatos would benefit to develop new treatment strategy for these diseases.

Endoplasmic reticulum (ER) stress resulting from accumulation of unfolded and misfolded proteins within ER lumen has been reported to account for the neuronal death induced by cerebral ischemia, hypoxia, and trauma.11, 12, 13 Mild ER stress (unfolded protein response) exerts protection against cell damage, whereas sustained ER stress leads to cell death.14 Therefore, ER stress plays dual roles in regulation of cell destiny. Under condition of ER stress, GRP78, ATF6, phosphor‐PERK, and phosphor‐IRE1 are upregulated and thus regarded as the markers of ER stress.14, 15 Although accumulating evidence has shown that ER stress contributes to cell death via induction of apoptosis and lethal autophagy,16, 17 its role in the occurrence of parthanatos still remains unclear.

Oxygen and glucose deprivation (OGD) due to lack of cerebral blood supply is the common features of global and focal cerebral ischemia, and is regarded as a major pathological basis leading to neuronal death.15 SH‐SY5Y cells are human neuroblastoma cells sharing similar properties with neurons in morphology, neurochemistry, and electrophysiology.18 Therefore, OGD of SH‐SY5Y cells is often used to study neuronal injury or death caused by ischemic insults. Although animal studies suggest ischemia induces neuronal death via overactivation of PARP‐1,6 the role of OGD in PARP‐1 activation remains unclear. Additionally, whether ER stress participates in regulation of parthanatos are needed to be addressed despite ER stress has been demonstrated to account for OGD‐induced death in SH‐SY5Y cells.15, 19 Thus, in this study, we use OGD and SH‐SY5Y cells to mimic cerebral ischemia and investigate the above‐mentioned two issues.

2. MATERIALS AND METHODS

2.1. Reagents

4‐Phenylbutyric acid (4‐PBA), N‐acetyl‐L‐cysteine (NAC), trehalose, and 3AB were purchased from Sigma‐Aldrich Company (St. Louis, MO). Anti‐PAR polymer (AM80) was from Calbiochem company (Danvers, MA). PARP‐1(14‐6667) was from eBioscience company (San Diego, CA). Anti‐IRE1(ab37037), anti‐GRP78(ab12685), anti‐ATF6(ab11909), antiphospho‐IRE1(S724)(ab124945), anti‐Histone‐H3(ab1791), and anti‐AIF(ab32516) were from Abcam company (Cambridge, MA). Antiphospho‐eIF2α(Ser51)(#9721), anti‐ATF4(#11815), anti‐eIF2α(#5324), anti‐PERK(#3192), horseradish‐peroxidase‐conjugated anti‐mouse IgG (#7076), and horseradish‐peroxidase‐conjugated anti‐rabbit IgG (#7074) were from Cell Signaling Technology (Danvers, MA). Antiphospho‐PERK(Thr981)(sc‐32577) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti‐β‐actin (AA128), lipid peroxidation MDA assay kit and glutathione assay kit were from Beyotime Biotech (Nanjing, China). The SiRNA was purchased from GenePharma Company (Suzhou, China).

2.2. Cell culture and oxygen‐glucose deprivation

Human SH‐SY5Y cells were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine (Gibco, Grand Island, NY, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml), and maintained at 37°C and 5% CO2 in a humid environment. The medium was replaced twice each week.

Oxygen‐glucose deprivation was carried out when the SH‐SY5Y cells were seeded for 24 hours. As described previously,20 the cells were placed in a modular incubator chamber (Billups‐Rothenberg, Del Mar, USA) after the culture medium was replaced with oxygen‐glucose‐free DMEM, which was produced by 5 minutes of bubbling glucose‐free DMEM with a mixture of 95% N2 and 5% CO2. Then, the chamber was sealed and flushed with a mixture of 95% N2 and 5% CO2 for 5 minutes at a flow rate of 25 L/min. Finally, the chamber was placed at 37°C incubator for 24 hours to produce lethal OGD after the two ports were sealed.

2.3. LDH release cell death assay

SH‐SY5Y cells were seeded onto 96‐well microplate and cultured 24 hours, and then treated with OGD for indicated periods. The cell death due to cell membrane damage was examined by measuring the levels of lactate dehydrogenase (LDH) in the culture medium via using a detection kit (Beyotime Biotech, Nanjing, China).21The levels of LDH in the cultured medium were determined using a colorimetric reaction reading of absorbance at 490 nm according to manufacturer's protocol.

2.4. Measurement of intracellular ROS

SH‐SY5Y cells were seeded onto 96‐well microplate and cultured 24 hours, and then treated with OGD for indicated periods. The levels of intracellular ROS were measured using ROS probe DCFH‐DA (Beyotime Biotech, Nanjing, China) as described previously.21 All the experimental cells were washed twice in PBS and stained 30 minutes with 20 μmol/L DCFH‐DA in a dark room. Following the cells were dissolved with 1% Triton X‐100, the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength 530 nm using a fluorescence spectrometer (HTS 7000, Perkin Elmer, Boston, MA). The ROS levels were expressed as the folds of control.

Other groups of SH‐SY5Y cells seeded onto a culture dish with 3 cm diameter were treated with OGD and stained with DCFH‐DA as described above. Then, the cells were observed under fluorescence microscope (Olympus IX71, Tokyo, Japan).

2.5. Mitochondrial membrane potential (JC‐1) assay

The SH‐SY5Y cells treated with OGD alone or treated 1 hour with NAC or 3AB prior to OGD were collected and stained with JC‐1 according to manufacture's instruction (Beyotime Biotech, Nanjing, China). JC‐1 is a dye that aggregates within normal mitochondria, and an increase in JC‐1 monomers indicates a loss of mitochondrial membrane potential. The percent of cells with a loss in mitochondrial membrane potential was determined by gating the population of cells with a decrease in JC‐1 aggregates but increase in JC‐1 monomers. After being stained with JC‐1, the ells were analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, CA). The excitation wavelength of JC‐1 is 488 nm, and the approximate emission wavelength of the monometric, and J‐aggregate forms are 529 and 590 nm, respectively.

Another group of cells seeded onto a culture dish with 3 cm diameter were treated with OGD and stained with JC‐1 as described above, and then were observed under fluorescence microscope (Olympus IX71, Tokyo, Japan).

2.6. Transfection of small interfering RNA (siRNA)

The SH‐SY5Y cells (4 × 105) were seeded onto a culture dish with a diameter of 10 cm. According to the manufacturer's instructions, transfection of siRNA was performed using Lipofectamine 2000 (Invitrogen, USA). The siRNAs targeting PARP‐1 were siRNA‐1: 5′‐GAGACCCAAUAGGCUUAAUTT‐3′, siRNA‐2: 5′‐GAGCACUUCAUGAAAUUAUTT‐3′, and siRNA‐3: 5′‐GAGGAAGGUAUCAACAAAUTT‐3′. Scrambled siRNA was 5′‐UUCUCCGAACGUGUCACGUTT‐3′. After overnight of siRNA transfection, the cells were treated with OGD for 24 hours for subsequent experiments.

2.7. Protein isolation

As described previously,19 the SH‐SY5Y cells were homogenized after being collected by centrifugation and suspended in ice‐cold lysis buffer. The homogenates were centrifuged at 10 000 g for 10 minutes at 4°C to isolate the supernatants containing cytoplasm fraction and the pellets including nucleus fraction. The protein concentrations of both supernatants and pellets were determined using Bio‐Rad protein assay kit.

2.8. Gel electrophoresis and Western blotting

Equal protein amounts were electrophoresed on 10% SDS gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 3% bovine serum albumin in TBS for 30 minutes at room temperature, and incubated overnight at 4°C with the following primary antibodies including: anti‐PAR polymer (1:500), anti‐AIF(1:1000), antiphosphor‐IRE1(1:1000), antiphosphor‐EIF2α(1:1000), anti‐GRP78(1:1000), anti‐PARP‐1(1:1000), anti‐Histone‐3 (1:2000), and anti‐β‐actin (1:1000). Then, the membranes were then incubated with horseradish‐peroxidase‐conjugated anti‐mouse (1:2000), or horseradish‐peroxidase‐conjugated anti‐rabbit IgG (1:2000) for 1 hour at room temperature. The immunoreactive proteins were visualized on a Kodak X‐omat LS film (Eastman Kodak Company, New Haven, CT, USA) with an enhanced chemiluminescence. Densitometry was performed with Kodak ID image analyses software (Eastman Kodak Company).

2.9. Immunocytochemical staining

The SH‐SY5Y cells (4 × 105) were seeded onto a culture dish with a diameter of 3 cm and cultured 24 hours. After being treated with OGD for 24 hours, the cells were fixed in 4.0% paraformaldehyde, washed with PBS, and incubated with 1% Triton X‐100 for 10 minutes. After the nonspecific antibody binding sites were blocked by 10% goat serum in PBS containing 0.3% Triton X‐100 and 0.5% BSA (bovine serum albumin), the cells were incubated with a antibody against AIF (1:100) followed by incubation in Alexa‐Fluor 594‐conjugated goat anti‐rabbit IgG (1:200) for 30 minutes at room temperature, and then incubated with Heochst33342 for 30 minutes. Finally, the cells were visualized under laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan).

2.10. Detection of lipid peroxidation

The malondialdehyde (MDA) content of SH‐SY5Y cells in each group was detected to assess the levels of membrane lipid peroxidation according to the manufacture's instruction. Briefly, the cells were lysed in the buffer containing 20 mmol/L Tris‐HCl (pH7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L NaVO4, 50 mmol/L NaF, 1% Triton X‐100, 1 mmol/L PMSF, 0.7 μg/ml aprotinin, and 0.5 μg/ml leupeptin. Cell lysates were homogenized, sonicated, and centrifuged at 15 000 g for 15 minutes at 4°C. Then, 100 μl supernatant was mixed with 200 μl MDA working solution and boiled at 100°C for 15 minutes. Samples were cooled down to room temperature in a water bath and then centrifuged at 1000 g for 10 minutes. The absorbance at 532 nm was measured in a 96‐well plate (200 μl/well) with a universal microplate spectrophotometer. The results of MDA assay were expressed as micromoles of MDA per milligram of protein, and then finally expressed as a ratio to the absorbance value at 532 nm of the control cells.

2.11. Measurement of intracellular glutathione (GSH)

Intracellular total GSH was measured using the DTNB‐GSSH reductase recycling assay kit (Beyotime Biotechnology, Nanjing, China) as described by the manufacture. Briefly, the cells in each group were collected by centrifugation at 800 g for 5 minutes and washed twice with PBS. The cell pellets were resuspended in protein‐removing buffer S and lysed by repeated cycles of freezing and thawing under liquid nitrogen. The cell lysates were centrifuged at 10 000 g for 10 minutes at 4°C to get the supernatant used for intracellular total GSH assay. GSH content was expressed as a ratio to the absorbance value at 412 nm of the control cells.

2.12. Statistical analysis

All data represent at least 4 independent experiments and are expressed as mean±SD. Statistical comparisons were made using one‐way ANOVA. P‐values of less than 0.05 were considered to represent statistical significance.

3. RESULTS

3.1. OGD inhibited the viabilities and induced death of SH‐SY5Y cells

To evaluate the effect of OGD on SH‐SY5Y cells, MTT assay was used to examine cellular viability. As shown in Figure 1A, the cellular viabilities decreased significantly at 3 hours after being exposed to OGD, and further decreased with the extension of OGD time. To clarify whether OGD induces death in SH‐SY5Y cells, we measured the LDH levels in culture medium on the basis that dying cells would release LDH. When compared with that in the control group, the LDH levels in the medium of OGD group increased significantly at 24 hours (Figure 1B). Therefore, these results suggested that OGD induced death in SH‐SY5Y cells, as well as inhibits their viabilities.

Figure 1.

Figure 1

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OGD inhibited the viabilities and induced death of SH‐SY5Y cells. A, MTT assay showed that the viabilities of SH‐SY5Y cells decrease at 3 h of GGD treatment, and further reduced with the extension of OGD time. B, LDH release assay showed that OGD induced death in SH‐SY5Y cells at 24 h

3.2. OGD induced activation of PARP‐1 and nuclear translocation of AIF

To demonstrate whether PARP‐1 is involved in the cell death caused by OGD, we assayed its activity by detecting the levels of PAR polymer, which is a product of activated PARP‐1. As Western blotting showed PAR polymer increased significantly in both cytoplasm and nucleus with the elongation of OGD time (Figure 2A). Meanwhile, either cytoplasmic or nuclear PARP‐1 was also upregulated markedly at each OGD time point (Figure 2B). These indicated that OGD triggered activation and expressional upregulation of PARP‐1 in a time‐dependent manner in SH‐SY5Y cells.

Figure 2.

Figure 2

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OGD induced activation of PARP‐1 and nuclear translocation of AIF. A, Western blotting showed that OGD induced accumulation of PAR polymer in either nucleus and cytoplasm in a time‐dependent manner. B, Western blotting proved as well that OGD treatment resulted in expressional upregulation of PARP and nuclear translocation of AIF. C, Fluorescence microscopy combined with JC‐1 staining revealed the red fluorescence weakened but the green one enhanced at OGD 12 h, and became more apparent at OGD 24 h. D, Flow cytometry analysis with JC‐1 staining proved that mitochondrial membrane potential decreased at OGD 12 h and further declined at OGD 24 h. E, The images under confocal microscope showed that AIF accumulated apparently within nucleus at OGD 24 h

Because abnormally increased PAR polymer results in cell death via causing mitochondrial dysfunction,4 we examined mitochondrial membrane potentials using JC‐1 staining. JC‐1 presents red fluorescence in normal cells but emits green fluorescence in dying cells. The images under fluorescence microscope revealed the red fluorescence weakened but the green one enhanced at OGD 12 hours, and became more apparent at OGD 24 hours (Figure 2C). Consistently, flow cytometry analysis with JC‐1 staining demonstrated that mitochondrial membrane potential decreased at OGD 12 hours and further declined at OGD 24 hours (Figure 2D). Moreover, as shown by Western blotting, the nuclear levels of AIF increased significantly at each OGD time points (Figure 2B), which was confirmed by confocal microscopic images showing that AIF accumulated apparently within nucleus at OGD 24 hours (Figure 2E). Thus, these results indicated OGD‐induced mitochondria damage and nuclear translocation of AIF.

3.3. PARP‐1 contributed to OGD‐induced SH‐SY5Y cell death

To elucidate the role of PARP‐1 in OGD‐induced death in SH‐SY5Y cells, we used PARP‐1 inhibitor 3AB to incubate the cells for 1 hour and then exposed the cells to OGD for 24 hours. LDH release assay showed that pretreatment with 3AB at 500 μmol/L significantly prevented OGD‐induced cell death (Figure 3A). Then, we found that OGD‐induced increase in PAR polymer and upregulated expression of PARP‐1 were both obviously inhibited in the presence of 3AB (Figure 3B,C). Moreover, 3AB partially reversed OGD‐induced decline of mitochondrial membrane potential and nuclear translocation of AIF (Figure 3C,D). Thus, these results indicated that PARP‐1 participated in regulation of OGD‐induced mitochondrial dysfunction, AIF translocation into nucleus and cell death.

Figure 3.

Figure 3

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PARP‐1 contributed to OGD‐induced SH‐SY5Y cell death. A, LDH release assay showed that pretreatment with 3AB at 500 μmol/L for 1 h significantly prevented OGD‐induced cell death. B, Western blotting proved that 3AB obviously prevented OGD‐induced production of PAR polymer in either nucleus or cytoplasm. C, Western blotting demonstrated that 3AB inhibited OGD‐induced expressional upregulation of PARP‐1 and nuclear accumulation of AIF. D, Flow cytometry analysis with JC‐1 staining showed that pretreatment with both 3AB or antioxidant NAC could effectively prevented OGD‐induced mitochondrial depolarization. E, Western blotting proved that knockdown of PARP‐1 with siRNA attenuated OGD‐induced upregulation of PARP‐1 and nuclear accumulation of AIF. F, Western blotting demonstrated OGD‐induced overproduction of PAR polymer was attenuated when PARP‐1 was knocked down with siRNA. G, LDH release assay showed that knockdown of PARP‐1 rescued OGD‐induced death in SH‐SY5Y cells

To further verify the role of PARP‐1 in OGD‐induced SH‐SY5Y cell death, we knocked down its levels using small interfering RNA (SiRNA). Western blotting proved that OGD‐induced upregulation of PARP‐1, PAR polymer production, and AIF accumulation in nucleus was all mitigated in the cells transfected with PARP‐1 SiRNA, when compared with the cells transfected with scrambled SiRNA (Figure 3E,F). Notably, as revealed by LDH release assay, the cells became resistant to OGD‐induced death when PARP‐1 was knocked down by SiRNA (Figure 3G). Therefore, these data suggested PARP‐1 contributed to OGD‐induced death in SH‐SY5Y cells.

3.4. ROS regulated OGD‐induced PARP‐1 overactivation

To clarify why OGD could cause PARP‐1 overactivation, we examined OGD‐induced changes in intracellular ROS levels. Fluorescence microscopy showed the green fluorescence detected by ROS probe DCFH‐DA was much brighter in the cells exposed to OGD than that in the control cells (Figure 4A). Statistical analysis of the fluorescence intensity proved that ROS was excessively produced in the cells stressed with OGD for 12 hours, and arrived at a higher level at 24 hours (Figure 4B). Consistently, MDA which is a marker of lipid peroxidation increased correspondingly with the elevation of ROS (Figure 4C). In contrast, intracellular antioxidant GSH decreased significantly at each time point (Figure 4D). These results indicated that OGD induced oxidative stress in SH‐SY5Y cells. However, either the increases of ROS and MDA or the decrease in GSH resulting from OGD exposure were significantly suppressed when the cells were pretreated with antioxidant NAC at 3 mmol/L for 1 hour (Figure 4B‐D). Moreover, LDH release assay showed as well that the cell death caused by OGD was inhibited accordingly in the presence of NAC (Figure 4E). Thus, these data indicated that ROS regulated OGD‐induced death in SH‐SY5Y cells.

Figure 4.

Figure 4

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ROS regulated OGD‐induced PARP‐1 overactivation. A, Fluorescence microscopy with ROS probe DCFH‐DA staining showed that green fluorescence was much brighter in the cells exposed to OGD than that in the control cells (×10). B, Statistical analysis of the fluorescence intensity proved that ROS was excessively produced in the cells stressed with OGD for 12 h, and reached a higher level at 24 h. However, pretreatment with NAC for 1 h significantly prevented OGD‐induced overproduction of ROS. C, Measurement of MDA levels showed that lipid peroxidation increased with the extension of OGD time and could be prevented by NAC pretreatment. D, GSH assay proved that OGD exposure resulted in decrease of intracellular levels of GSH in a time‐dependent manner, which were partially reversed by prior administration of NAC. E, LDH release assay showed that NAC obviously rescued OGD‐induced SH‐SY5Y cell death. F, Western blotting proved that NAC pretreatment prevented OGD‐induced generation of PAR polymer. G, Western blotting demonstrated that NAC attenuated OGD‐induced upregulation of PARP‐1 levels and nuclear accumulation of AIF

Then, we examined the effect of NAC on OGD‐induced changes in PARP‐1, and found that the increased levels of PAR polymer and PARP‐1 due to OGD exposure were both mitigated by NAC (Figure 4F,G). Consistently, mitochondrial membrane decline and AIF translocation which were the downstream biochemical events of PARP‐1 activation were partially reversed (Figures 3D and 4G). Therefore, these indicated that ROS was involved in regulation of PARP‐1 activation and expressional upregulation caused by OGD.

3.5. ER stress contributed to OGD‐induced overproduction of ROS

Given that ROS is an upstream regulator of PARP‐1‐dependent death and could be produced during the process of ER stress, we tested whether OGD could activate ER stress. As revealed by Western blotting, the cytoplasmic levels of GRP78, ATF6, IRE‐1, phospho‐IRE‐1, PERK, phosphor‐PERK, eIF2α, phosphor‐eIF2α, and ATF4, which are markers of ER stress, were time‐dependently upregulated in the cells stressed with OGD (Figure 5A). Moreover, the nuclear levels of ATF4 and ATF6 were also increased with the extension of OGD time (Figure 5B). These indicated that OGD triggered ER stress in SH‐SY5Y cells.

Figure 5.

Figure 5

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ER stress contributed to OGD‐induced overproduction of ROS. A, Western blotting showed OGD upregulated the expression of ER stress maker proteins in a time‐dependent manner. B, Western blotting proved that OGD induced nuclear translocation of both ATF4 and ATF6. C, Western blotting revealed pretreatment with chemical chaperones 4‐PBA or trehalose could markedly inhibited OGD‐induced upregulation of ER stress‐related proteins. D, Western blotting demonstrated that the nuclear translocation of both ATF4 and ATF6 due to OGD exposure was effectively prevented in the presence of 4‐PBA or trehalose. E, Statistical analysis of the fluorescence intensity detected using ROS probe DCFH‐DA proved that pretreatment with chemical chaperons 4‐PBA or trehalose prevented OGD‐induced abnormal increase of ROS. F, LDH release assay showed that pretreatment with 4‐PBA or trehalose rescued OGD‐induced death in SH‐SY5Y cells. G, Western blotting proved that 4‐PBA or trehalose inhibited OGD‐induced generation of PAR polymer, upregulation of PARP‐1 and nuclear translocation of AIF

In contrast, pretreatment with chemical chaperone 4‐PBA at 3.0 mmol/L for 1 hour or trehalose at 5.0 mmol/L for 48 hours, significantly prevented OGD‐induced expressional upregulation of GRP78, phospho‐IRE‐1, and phosphor‐PERK (Figure 5C). Consistently, both the cytoplasmic and the nuclear levels of ATF6 and ATF4 were also mitigated in the presence of 4‐PBA or trehalose (Figure 5D). These indicated that chemical chaperones 4‐PBA and trehalose could effectively inhibit OGD‐induced ER stress in SH‐SY5Y cells. Notably, we found that inhibition of ER stress with either 4‐PBA or trehalose obviously mitigated OGD‐induced overproduction of ROS (Figure 5E). This indicated that the overproduced ROS due to OGD exposure was partially from ER stress. Moreover, LDH release assay demonstrated that both 4‐PBA and trehalose protected the cells against OGD‐induced death (Figure 5F). Further, Western blotting analysis revealed that 4‐PBA and trehalose significantly suppressed OGD‐induced excessive production of PAR polymer and expressional upregulation of PARP‐1 (Figure 5G). Accordingly, the nuclear accumulation of AIF caused by OGD was alleviated (Figure 5G). Thus, these data indicated that ER stress participated in the regulation of OGD‐induced PARP‐1‐dependent cell death.

4. DISCUSSION

In this study, we found that PARP‐1 played a crucial role in regulation of OGD‐induced death in SH‐SY5Y cells. PARP‐1 and its product PAR polymer were both upregulated in a time‐dependent manner under the condition of OGD, which was accompanied with mitochondrial depolarization and nuclear translocation of AIF. Pharmacological inhibition of PARP‐1 with its specific inhibitor 3AB not only rescued OGD‐induced cell death, but also prevented PAR polymer elevation, mitochondrial membrane potential decline, and AIF translocation into nucleus. Similar results could be found when PARP‐1 was genetically knocked down with SiRNA. Then, we found OGD also induced excessive generation of intracellular ROS and upregulation of ER stress‐related proteins GRP78, phosphor‐EIF2α, and phosphor‐IRE1. Inhibition of ROS with antioxidant NAC and mitigation of ER stress with chemical chaperone 4‐PBA or trehalose obviously suppressed OGD‐induced PARP‐1 upregulation, PAR polymer production, and nuclear translocation of AIF. Notably, the abnormal levels of intracellular ROS caused by OGD were significantly alleviated when ER stress was inhibited by 4‐PBA or trehalose. Therefore, on the basis of these findings, we think that ER stress participates in regulation of OGD‐induced PARP‐1 dependent death in SH‐SY5Y cells via improvement of intracellular ROS (Figure 6).

Figure 6.

Figure 6

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Schematic illustration of the role of ER stress in regulation of OGD‐induced parthanatos in SH‐SY5Y cells

PARP‐1 is an enzyme mainly responsible for chromosomal DNA repair after being mildly activated by DNA strand nicks and breaks. However, overactivated PARP‐1 could transform nicotinamide adenine dinucleotide (NAD+) into long PAR polymers, which produces cytotoxic effects after translocation from nucleus into cytoplasm.5 Two main criteria are often used to determine the occurrence of parthanatos. One is excessively generated PAR polymers accompany cell death; the other is the cell death should be completely or partially prevented when PARP‐1 is inhibited or deleted.5 In this study, we found that OGD induced cell death in a time‐dependent manner, which was accompanied with time‐dependent expressional upregulation of PARP‐1 and increase in PAR polymer. However, when PARP‐1 was pharmacologically inhibited by its specific inhibitor 3AB or genetically knocked down with SiRNA, the death of SH‐SY5Y cells due to OGD exposure was prevented significantly. Because these findings were consistent with the criteria for defining parthanatos, we think that parthanatos is also involved in OGD‐induced cell death.

PAR polymer is thought to be an executioner of parthanatos on the basis that exogenous delivery of purified PAR polymer could kill cells and the toxic potential of PAR polymer increases with the polymer complexity.22 Mitochondrion is a downstream target of cytoplasmic PAR polymer. PAR polymer induces mitochondrial membrane potential collapse and AIF release from the outer mitochondrial membrane pool.6 Moreover, recent studies have shown that nuclear AIF leads to cell death via causing chromatin condensation and large‐scale DNA fragmentation after translocation into nucleus.6, 23 We found in this study that suppression of the production of PAR polymer via inhibition of PARP1 with 3AB or SiRNA not only decrease OGD‐induced nuclear accumulation of AIF, but also rescued cell death. Moreover, pretreatment with 3AB partially reversed OGD‐induced mitochondrial depolarization. These suggested that PAR polymer was involved in OGD‐induced mitochondrial membrane potential decline and subsequent AIF translocation to nucleus. Additionally, PAR polymer could bind with intracellular proteins to damage cellular physiological function. Andrabi et al found that the glycolysis dysfunction during the course of parthanatos was due to the binding to PAR polymer with hexokinase.24 In this study, the smear bands detected by anti‐PAR antibody on Western blotting membrane suggested that PAR polymer might bind to cellular proteins when SH‐SY5Y cells were exposed to OGD.

The generation of PAR polymer is the result of overactivation of PARP‐1. Chromosomal DNA strand nick and break are a primary factor accounting for the overactivation of PARP‐1. Chiu et al proved that PARP‐1‐dependent death could be induced in mouse embryonic fibroblasts (MEFs) by alkylating agent MNNG which could damage DNA directly.25 However, recent studies have shown that ROS also participate in regulation of PARP‐1 overactivation. Ma et al reported that inhibition of ROS with NAC effectively attenuated deoxypodophyllotoxin‐induced intracellular accumulation of PAR polymer in glioma cells.26 Particularly, as a member of ROS, H2O2 played an important role in regulation of PARP activity. Zheng et al reported that exogenous H2O2 induced expressional upregulation of PARP‐1 and obvious accumulation of PAR polymer in glioma cells.27 Moreover, Akhiani et al found that exogenous H2O2 could trigger PARP‐1‐dependent accumulation of PAR polymer in myeloid cells.28 Although it is still elusive why ROS could activate PARP‐1, previous studies suggest ROS might overactivate PARP‐1 via two pathways. On the one hand, ROS could lead to DNA strand breaks via causing oxidative damage of nucleic acids.29 On the other hand, ROS could induce ERK phosphorylation, and the phosphorylated ERK has direct regulatory effect on DNA damage‐induced PARP‐1 activation.28, 30 In this study, we found that OGD induced time‐dependent overproduction of intracellular ROS, and suppression of ROS prevented markedly expressional upregulation of PARP‐1 and PAR polymer. Thus, we think that OGD induced PARP‐1 in SH‐SY5Y cells via improvement of intracellular ROS levels.

ER provides an exclusive oxidizing‐folding environment to the proteins to facilitate disulfide bond formation, and this process is believed to contribute to 25% of ROS generated by cell.31 When proteins are overloaded, ROS are excessively generated in the ER as a part of an oxidative folding process of the overloaded proteins during electron transfer between protein disulfide isomerase and endoplasmic reticulum oxidoreductin‐1.32 It has been found that the process of ER stress is accompanied with excessive generation of intracellular ROS. Kim et al reported that treatment with ER stress inducer thapsigargin or tunicamycin significantly increased intracellular ROS levels in liver HepG2 cells.33 In contrast, mitigation of ER stress with interior chaperone or supplement of exterior chaperone could result in decrease in intracellular ROS levels. Liu et al found that expression of interior ER chaperones GRP78 and calreticulin prevented oxidative stress in renal epithelial cells.34 Luo et al also demonstrated that inhibition of ER stress with chemical chaperone 4‐PBA obviously attenuated renal oxidative stress induced by streptozotocin in diabetic rats.35 Moreover, Li et al reported that mitigation of OGD‐induced ER stress with chaperone trehalose significantly alleviated intracellular ROS levels in SH‐SY5Y cells.36 ER stress could be induced by OGD in SH‐SY5Y cells, but its role in OGD‐induced overproduction of ROS remains unclear. The factors accounting for the contribution of ER stress to OGD‐induced accumulation of intracellular ROS were investigated previously by other researchers, and the findings showed that ER stress leads to abnormal increase in intracellular ROS via causing mitochondrial dysfunction and NADPH oxidase 4 activation.37 Notably, OGD was reported to induce ROS overproduction via causing mitochondrial dysfunction and activation of NADPH oxidase.38, 39 Additionally, the ER stress‐related PERK and IRE‐1 pathways play a role in regulation of intracellular ROS levels. The PERK pathway has negative regulation of intracellular ROS levels, because the activated PERK could free Nrf2 by phosphorylation of Keap1 and facilitate Nrf2 translocation into nucleus to express antioxidant proteins.40 However, activated IRE‐1 could promote intracellular ROS levels given that the IRE‐1‐dependent activation of ASK and P38 MARK contributes to ROS generation via further activation of CHOP.41 In this study, we proved that chemical chaperones trehalose and 4‐PBA not only mitigated OGD‐induced ER stress, but also suppressed the abnormal increase in intracellular ROS caused by OGD. Therefore, we think that activation of ER stress is a pathway contributing to the generation of excessive ROS caused by OGD in the SH‐SY5Y cells.

In conclusion, we demonstrated that ER stress regulates OGD‐induced parthanatos in SH‐SY5Y cells via improvement of intracellular ROS.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

This work was supported by National Nature and Science Foundation of China (81372697, 81271215, 81772669, and 81771141), Changbaishan Scholar Project of Jilin Province (2013026), Scientific Research Foundation of Jilin province (20150414013GH and 20160101127JC).

Wang H‐F, Wang Z‐Q, Ding Y, et al. Endoplasmic reticulum stress regulates oxygen‐glucose deprivation‐induced parthanatos in human SH‐SY5Y cells via improvement of intracellular ROS. CNS Neurosci Ther. 2018;24:29–38. 10.1111/cns.12771

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