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. 2015 Mar 17;35(6):849–859. doi: 10.1007/s10571-015-0179-5

4-Phenylbutyric Acid (4-PBA) and Lithium Cooperatively Attenuate Cell Death during Oxygen–Glucose Deprivation (OGD) and Reoxygenation

Wai-Fai Tung 1, Wei-Jen Chen 3, Hui-Chih Hung 6, Guang-Yaw Liu 4, Jai-Nien Tung 2, Chien-Chih Huang 5, Chih-Li Lin 5,7,✉
PMCID: PMC11486266  PMID: 25776137

Abstract

Hypoxia is an important cause of brain injury in ischemic stroke. It is known that endoplasmic reticulum (ER) stress is an important determinant of cell survival or death during hypoxia. However, the signaling pathways and molecular mechanisms involved remain to be studied in more detail. To investigate whether inhibition of ER stress promotes neuroprotection pathways, we applied an in vitro oxygen–glucose deprivation (OGD) followed by reoxygenation model of human SK-N-MC neuronal cell cultures in this study. Our results showed that neuronal cell death was induced in this model during the OGD reoxygenation by the sustained ER stress, but not during OGD phase. However, treatment of the cultures with lithium with the OGD reoxygenation insult did not result in neuroprotection, whereas concomitant treatment of chemical chaperon 4-phenylbutyric acid (4-PBA) provides protective effects in ER stress-exposed cells. Moreover, 4-PBA rescued ER stress-suppressed Akt protein biosynthesis, which works cooperatively with lithium in the activation of Akt downstream signaling by inhibition of autophagy-induced cell death. Taken together, our finding provides a possible mechanism by which 4-PBA and lithium contribute to mediate neuroprotection cooperatively. This result may potentially be a useful therapeutic strategy for ischemic stroke.

Keywords: Akt, Endoplasmic reticulum stress, Lithium, Oxygen–glucose deprivation, 4-Phenylbutyric acid

Introduction

Cell hypoxia is a condition in which the cell does not receive enough oxygen supply. Although the brain represents only about 2 % of total body mass, it consumes almost 20 % of the body’s oxygen (Lin et al. 2013). For this reason, brain cells are extremely sensitive to hypoxia. Prolonged hypoxia induces an energy-depleted response that results in neuronal cell death. As a result, restoring oxygen supply as early as possible to prevent further damage is the basis of therapy in treating acute ischemic stroke. However, reoxygenation following hypoxia induces further injury by a sudden burst of oxidative stress. Current evidence indicates that hypoxia-reoxygenation triggers overproduction of reactive oxygen species accompanied by mitochondrial dysfunction and subsequent cell death (Manzanero et al. 2013). It is likely that Akt–glycogen synthase kinase 3β (GSK3β) pathway is involved in neuronal apoptosis and the pathogenesis of ischemic stroke (Pradeep et al. 2014). GSK3β is a serine/threonine kinase and regulates a variety of neuronal functions including neurotransmission, synaptic plasticity, neurogenesis, neuroinflammation, and apoptosis (Cole 2012; Perez-Alvarez et al. 2012). Particularly, the activity of GSK3β is mainly regulated by Akt, which leads to the inactivation of GSK3β by phosphorylation at Ser9. The relatively high activity of GSK3β promotes apoptotic signaling in response to various stresses such as hypoxia (Pradeep et al. 2014). In contrast, numerous studies have also demonstrated that inhibition of GSK3β protects neurons from hypoxic injury (Zhou et al. 2011), suggesting that GSK3β inhibitors may be relevant for the treatment of cellular hypoxia.

Increasing evidence shows that endoplasmic reticulum (ER) stress may play a crucial role in hypoxia-induced cell death (Yung et al. 2007). In most cases, the energy consumed by neurons is used to propagate action potentials and maintain the resting potential. It is therefore important that protein synthesis be kept at a high level so that neurons are capable of maintaining normal function. During hypoxia, the loss of energy disrupts the protein processing taking place in the ER, which results in the continuous accumulation of unfolded or misfolded proteins, and triggers an evolutionarily conserved cellular stress response termed as an unfolded protein response (UPR) (Hetz 2012). In general, UPR is known to transiently attenuate protein synthesis, allowing time to fix the damage. UPR also stimulates the production of ER chaperones such as GRP78, which acts to promote proper protein folding, maturation, secretion, and degradation (Mahadevan and Zanetti 2011). However, excessive and prolonged ER stress reduces the protein synthesis and folding capacity of the ER, and causes accumulation and aggregation of unfolded proteins and subsequent cell death (Schonthal 2012). Therefore, ER stress is thought as a target for therapy in hypoxia-induced neuronal death (Roussel et al. 2013).

There is accumulating literature on neuroprotective effects of lithium (Li et al. 2002). Lithium is known to directly inhibit GSK3β, which is a highly conserved serine/threonine kinase that participates in the induction of apoptosis by destabilizing the anti-apoptotic Bcl2 family members (Chuang et al. 2011). These results highlight the therapeutic potential of lithium in preventing various neuronal injuries like hypoxia-induced damage (Shin et al. 2012). However, the molecular mechanism of the relationship between ER stress and lithium is still unclear. High levels of lithium were reported to increase the expression of ER stress-related proteins (Shao et al. 2006), which may deteriorate ER stress responses and reduce lithium’s neuroprotective effects (Nagy et al. 2013). Thus, designing new strategies that specifically target greater outcomes of lithium should be important. In the present study, we hypothesized that the suppression of the ER stress response may yield greater therapeutic effects of lithium, and provide additional protection against hypoxia-induced neuronal cell death. 4-phenylbutyric acid (4-PBA) is a low molecular weight fatty acid that has been approved for clinical use as an ammonia scavenger (Iannitti and Palmieri 2011). In addition, 4-PBA can also act as a chemical chaperone to alleviate ER stress by reducing the load of misfolded proteins retained in the ER (Kolb et al. 2015). Therefore, the use of 4-PBA may be a therapeutic approach for blocking the ER stress-induced neuronal death by ischemia. In the present study, we demonstrated that the combined treatment of lithium and 4-PBA exerts a neuroprotective role by restoring Akt Ser473 phosphorylation and intracellular total protein levels. These findings reveal a novel strategy for the attenuation of ER stress in mediating neuronal death during hypoxia injury.

Experimental Procedures

Materials

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LY294002, lithium bicarbonate, chloroquine, rapamycin, and 4-phenylbutyric acid (4-PBA) were purchased from Sigma-Aldrich (München, Germany). Antibodies against PERK, pPERK, eIF2α, peIF2α, pmTOR, mTOR, GSK3β, pGSK3β, Beclin 1, and poly(ADP-ribose) polymerase (PARP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and those against Caspase 12, Caspase 3, Akt, and pAkt were purchased from Millipore (Bedford, MA, USA). The β-actin antibody was from Novus Biologicals. (Littleton, CO, USA). Primary antibodies were used at a dilution of 1:1000 in 0.1 % Tween 20 and secondary antibodies were used at 1:5000 dilutions. All the chemicals were prepared by dissolving phosphate buffer saline solutions stored at −20 °C until needed for use in experiments.

Cell Culture and Oxygen–Glucose Deprivation (OGD) Treatment

Human SK-N-MC cells were obtained from the American Type Culture Collection (Bethesda, MD, USA), and maintained in Minimal Eagle’s medium (Gibco) supplemented with 10 % inactivated fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin equilibrated with 5 % CO2/95 % air at 37 °C. For the treatments of hypoxia, cells were transferred to a hypoxic chamber as described previously (Bando et al. 2000). The standard culture medium was replaced with an oxygen–glucose deprivation buffer (OGD buffer), and bubbled with an anaerobic gas mixture (95 % N2 and 5 % CO2) for at least 2 h before using. After that cells were treated in the presence or absence of indicated compounds and then placed into a hypoxic chamber for 20-h incubation at 37 °C, and following reoxygenation for an additional 24 h, the conditioned medium was rapidly exchanged with fresh standard culture medium in the presence or absence of compound as indicated. The oxygen level was about 18 % during reoxygenation at this stage.

Cell Viability Assay

Cells were seeded in 96-well plates at a density of 1 × 104 cells/well, and were incubated overnight. Next the cells were treated with indicated conditions. After treatments, the tetrazolium salt MTT was added to the medium according to the manufacturer’s instructions. Only viable cells metabolized MTT to a purple product formazan, which was quantified spectrophotometrically at 550 nm. The cell viability was determined as the percentage of control cells treated with the vehicle alone and in triplicate for each treatment condition, and calculated by A550 nm (sample)/A550 nm (control) × 100.

Measurement of Lactate Dehydrogenase (LDH) Activity

Cell death was quantified using the BioVision lactate dehydrogenase (LDH) cytotoxicity assay kit (BioVision, Inc., Mountain View, CA, USA), according to the manufacturer’s protocol and based on the measurement of LDH activity in the experimental media. Briefly, conditioned culture medium was clarified by centrifugation at 600×g for 10 min, and then the cell-free supernatant was transferred to a 96-well plate, mixed with LDH reaction reagent, incubated for 30 min, and the absorbance measured at 450 nm. LDH enzyme activity was measured using the test media and in triplicate for each treatment condition.

Western Blotting

Cells were harvested and homogenized in a protein extraction lysis buffer (50 mM Tris–HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5 % Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) and centrifuged at 12,000×g for 30 min at 4 °C. Equal protein amounts of the total cell lysates were resolved by 10 % SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Millipore), and then probed with primary antibody followed by secondary antibody conjugated with horseradish peroxidase. The immunocomplexes were visualized with enhanced chemiluminescence kits (Millipore). The relative expression of proteins was quantified densitometrically using the software QuantityOne (BioRad), and was calculated according to the reference bands of β-actin. All results were performed at least in three independent experiments.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis of XBP1 mRNA Splicing

The assay was adapted from Yung et al. (2007). In brief, total RNA was isolated from SK-N-MC cells using an RNeasy mini kit (Qiagen, UK) immediately after treatments. To amplify XBP-1 mRNA, PCR was carried out for 30 cycles using the following profile: 94 °C for 30 s; 58 °C for 30 s; 72 °C for 1 min. In the final cycle, PCR products were incubated at 72 °C for an additional 10 min with XBP-1 forward and reverse primers and Taq DNA polymerase (Invitrogen). Forward and reverse primer sequences were 5′-CTGGAACAGCAAGTGGTAGA-3′ and 5′-CTGGGTCCTTCTGGGTAGAC-3′, respectively.

Microscopic Observation and Nucleus Morphology

Changes in cell nucleus morphology, characteristic of apoptosis were examined in cells grown on coverslips, using a microscope. The cells were fixed in 4 % paraformaldehyde after 24 h of treatment with the indicated compounds. For phase-contrast inverted microscopy, images of cells were captured with no specific staining procedure. For nucleus morphology microscopy, cells were fixed in ice-cold methanol, and incubated for 15 min at room temperature with 1 ng/ml of 4′,6-diamidino-2-phenylindole (DAPI) stain, and observed under a fluorescence microscope (DP80/BX53, Olympus). Apoptosis was quantified by averaging cell counts in five random 400 × fields. Values were expressed as the percentage of apoptotic cells relative to total number of cells.

Apoptosis by Flow Cytometry

Cells were cultured as described above. The percentage of sub-G1 apoptotic cells was examined by the propidium iodide (PI) technique using flow cytometry. Briefly, SK-N-MC cells (1 × 106/ml) were fixed in 70 % methanol and resuspended in PBS incubated with 50 μg/ml of PI for 0.5 h, and analyzed by FACScan (Becton–Dickinson, San Jose, California, USA).

Protein Expression KnockDown by siRNA Technology

The Beclin1 gene silencer (human) dsRNA was obtained from Santa Cruz Biotechnology (sc-44286). Neuroblastoma cells were transfected with dsRNAs using siRNA Transfection Reagent (Santa Cruz), and incubated for 6 h. Thereafter, the cells were analyzed by immunoblot for protein expression.

Statistical Analysis

All data are presented as mean ± standard error of the means (SEM). Statistical analysis of data was performed using analysis of variance (ANOVA), followed by Dunnett’s post hoc test for multiple comparisons with SPSS statistical software (SPSS, Inc., Chicago, IL, USA). Differences were considered statistically significant at P < 0.05.

Results

Effects of OGD- and Reoxygenation-Induced Cell Death on SK-N-MC Neuronal Cells

To mimic neuronal insults during ischemia and reperfusion, we first cultured SK-N-MC neuronal cells exposed to OGD for 20 h, and followed by 24 h of reoxygenation. As shown in Fig. 1a, 20-h OGD treatment showed slightly decreased cell numbers and morphologically induced cell shrinkage. On the contrary, after an additional 24 h of reoxygenation, cells had markedly undergone cell death. In parallel, MTT assay showed that treatment of SK-N-MC cells with OGD for 6, 12, and 20 h decreased cell viability by 6, 14, and 21 %, respectively. Furthermore, exposure to additional reoxygenation for 12 and 24 h caused a significant 44 and 57 % reduction in cell viability, respectively (Fig. 1b). This indicates that cells undergo cell death slightly during 20 h of OGD; however, OGD-induced insults lead to further cell death by an additional 24 h of reoxygenation. To precisely determine which mode of cell death is induced by OGD, we measured the release of LDH in the culture supernatant, a stable cytoplasmic enzyme which is present in all cells but only released from non-apoptotic cells. As shown in Fig. 1c, only small amounts of LDH were released by 20 h of OGD; however, approximately a three-fold increase in LDH release into culture medium was detected after 24 h of additional reoxygenation. Thus, non-apoptotic cell death appears to be the dominant cell death pathway in the additional 24 h of reoxygenation. As mentioned above, lithium was previously shown to inhibit neuronal apoptosis in ischemic injury. To evaluate its protective effects, lithium bicarbonate (Li2CO3) was introduced into this study. As shown in Fig. 1d, 100 μM of lithium provided little protection during 20-h OGD, but no significant difference in protective effects after an additional 24-h reoxygenation. These data were also confirmed by DAPI staining, whereby treatment with lithium reduced nuclei fragmentation significantly in 20-h OGD, but no difference after an additional 24 h of reoxygenation (Fig. 1e). This indicates that lithium has no protective effects during the additional 24 h of reoxygenation.

Fig. 1.

Fig. 1

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Effects of OGD/reoxygenation on insults to SK-N-MC neuronal cells. a Treatment with oxygen–glucose deprivation (OGD) on SK-N-MC cells for 20 h shows slight morphological changes and decreased number of neuronal cells. However, the additional 24 h of reoxygenation markedly induces cell death. b Effects of cytotoxicity induced by exposure to OGD/reoxygenation determined by the MTT assay. c Quantify cell death by lactate dehydrogenase (LDH) activity assays. d Cell viability was evaluated by treatment with or without 100 μM of lithium bicarbonate (Li2CO3) under the OGD/reoxygenation process by MTT assay. e Apoptosis is determined by fragmented morphology in the nucleus for DAPI fluorescence. The number of apoptotic cells are quantified by averaging cell counts in five random 400 × fields. The other data were performed in three independent experiments, and values are presented as mean ± standard error of the means (SEM). Significant differences were determined using the multiple comparisons of Dunnett’s post hoc test for *P < 0.05 and **P < 0.01. Scale bar represents 50 μm. N.S. no significant difference

Activation of ER Stress-Mediated Cell Death Signaling During Additional Reoxygenation

Since lithium showed limited protective effects of OGD/reoxygenation-induced cell death, we therefore focused our attention on what mechanisms diminished lithium’s protective efficacy. It has been suggested that sustained ER stress is implicated in the pathology of ischemia–reperfusion injury (Yung et al. 2007). To elucidate the role of ER stress on OGD/reoxygenation-induced cell death, three typical markers including double-stranded RNA-activated protein kinase-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), and splicing of X-box binding protein 1 (XBP-1) mRNA were investigated. As shown in Fig. 2a, cells exposed to OGD exhibited an increased level of phospho-PERK and phospho-eIF2α in a time-dependent manner, and these increases were further up-regulated by a further 24 h of reoxygenation. Evidence of ER stress was also confirmed by an increase in the spliced variant of XBP-1 mRNA upon OGD treatment (Fig. 2b). Numerous studies have revealed that cell death induced by ER stress depends on activation of caspase 12 (Hetz 2007). As shown in Fig. 2c, OGD exposures markedly elevated caspase 12, and its downstream effectors such as caspase 3 and PARP. Apoptotic cells were simultaneously quantified by flow cytometry, and there was a significant increase in the percentage of cells in sub-G1 phase after OGD/reoxygenation (Fig. 2d). These results suggest that the activation of ER stress-mediated cell death signaling is an important determinant of OGD-induced neuronal death after an additional 24 h of reoxygenation.

Fig. 2.

Fig. 2

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SK-N-MC cells respond to OGD/reoxygenation through the activation of ER stress. a Western blots showing the activation of ER stress by monitoring the phosphorylation levels of PERK and eIF-2α. b RT-PCR analysis of the splicing of XBP-1 mRNA for the indicated time during OGD and reoxygenation. c Caspase 3, 12, and PARP activation were determined by immunoblotting. d Apoptotic cells were measured by flow cytometry analysis. Results are shown from a single representative of three independent experiments, and values are presented as mean ± SEM

4-Phenylbutyric Acid (4-PBA) Attenuates ER Stress Cooperatively with Lithium in Reducing OGD/Reoxygenation-Induced Cell Death

Previous results have demonstrated that sustained ER stress during reoxygenation enhances neuronal cell death. Since ER stress has been implicated by the accumulation of misfolded protein aggregates, it is likely that the alleviation of ER stress responses by small molecular chaperon compounds such as 4-phenylbutyric acid (4-PBA) may be beneficial for OGD/reoxygenation-induced cell injury or death. As shown in Fig. 3a, the elevation of ER stress markers such as phosphorylated PERK and eIF-2α was significantly inhibited by 4-PBA at concentrations ranging from 10 to 200 μM. The maximum reduction in ER stress markers was found at 100 μM 4-PBA; in subsequent experiments, we therefore investigated the action of 4-PBA at a concentration of 100 μM. To further confirm these findings, we evaluated the levels of phospho-PERK and phospho-eIF2α at different stages of OGD/reoxygenation. As shown in Fig. 3b, the levels of phosphorylated PERK and eIF2α were suppressed by 4-PBA in both OGD and at the additional reoxygenation stage. Similarly, 4-PBA-inhibited ER stress was further confirmed by LDH cytotoxicity assay, which demonstrated a significant reduction in LDH release, especially in the stage of reoxygenation (Fig. 3c). This suggests that 4-PBA is able to protect neuronal cells against OGD/reoxygenation-elicited ER stress and subsequent cell death. Lithium is known for neuroprotective activity involving both Akt activation and GSK3β inhibition (Freland and Beaulieu 2012); however, our results showed that treating cells with lithium alone did not effectively suppress OGD/reoxygenation-induced cell death. As a result, we next examined whether 4-PBA may display cooperative effects that can enhance lithium’s neuroprotection by alleviating ER stress. As shown in Fig. 3d, treatment with 100 μM of 4-PBA protected cell viability to 47.0 ± 11.4 % compared to OGD/reoxygenation only groups (33.3 ± 7.6 %). However, 67.0 ± 8.5 % cells remained alive under a combination of 4-PBA (100 μM) and lithium (100 μM) after OGD/reoxygenation. Furthermore, co-treatment of 4-PBA and lithium not only prevented ER stress-induced activation of PERK and eIF2α but also reduced the amounts of cleaved caspase 3, caspase 12, and PARP by OGD/reoxygenation (Fig. 3e). This indicates that 4-PBA acts as a chaperone in alleviating sustained ER stress, and enhances lithium’s neuroprotection during OGD/reoxygenation.

Fig. 3.

Fig. 3

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4-Phenylbutyric acid (4-PBA) attenuates ER stress during additional reoxygenation. a Western blotting reveals that phosphorylation of Thr980-PERK and Ser51-eIF2α is elicited when cells are exposed to OGD/reoxygenation, and this up-regulation is effectively blocked by 4-PBA in a dose-dependent manner. b The levels of phosphorylated PERK and eIF2α are suppressed by 4-PBA in both OGD and additional reoxygenation treatments. c 4-PBA displays a significant inhibition in LDH release in the stage of reoxygenation. d Cell viability assay demonstrates that lithium (100 μM) combined with 4-PBA (100 μM) significantly reduces OGD/reoxygenation-induced cell death. e Western blots showing ER stress and apoptosis induction by monitoring pPERK, p-eIF2α, cleaved caspase 3, cleaved caspase 12, and cleaved PARP levels in SK-N-MC cells treated with 4-PBA (100 μM) alone, lithium (100 μM) alone, or in combination with 4-PBA and lithium during OGD/reoxygenation. All data were performed in three independent experiments, and values are presented as mean ± SEM. *P < 0.05 and **P < 0.01 compared with the H/R-only group for multiple comparisons of Dunnett’s post hoc test

Lithium Combined with 4-PBA Significantly Reduces OGD/Reoxygenation-Induced Cell Death by Suppressing Autophagy

Previous studies have reported that Akt signaling is the key regulator of neuron survival during ischemia–reperfusion (Mullonkal and Toledo-Pereyra 2007). As shown in Fig. 4a, 20 h of OGD suppressed most pSer473-Akt, and an additional 24 h of reoxygenation further suppressed both total protein levels and phosphorylation of Akt. This suggests that reduced Akt signaling may contribute to OGD/reoxygenation-induced cell death. To verify the obtained results, Western immunoblotting was performed to probe Akt downstream of GSK3β. In Fig. 4b, OGD/reoxygenation decreased Akt activities and suppressed GSK3β Ser9 phosphorylation, resulting in up-regulation of GSK3β enzyme activity, which has been shown to trigger cell death signal such as caspases. However, co-treating with lithium and 4-PBA returned both total amount and pSer473 levels of Akt to normal, which results in the inhibition of GSK3β. This indicates that lithium and 4-PBA may exert cooperative neuroprotective roles against OGD/reoxygenation-induced damage by up-regulation of pSer473 and total protein levels of Akt simultaneously. Emerging data indicate ER stress may trigger autophagy-dependent cell death by degradation of survival proteins (Liu and Levine 2014). To further confirm the observations, immunoblotting was performed to probe the autophagy-related proteins. As shown in Fig. 4b, lithium combined with 4-PBA significantly up-regulated phospho-mTOR (Ser2448), which subsequently inhibits downstream autophagy hallmark LC3-II formation. Furthermore, this Akt-mTOR suppressed autophagy was reversed by LY294002. Because up-regulation of Beclin 1 is a principle mechanism contributing to autophagy-like cell death (Rami et al. 2008), to further confirm this result, knockdown of Beclin1 was conducted using target-specific siRNAs and cells exposed with or without OGD/reoxygenation. As shown in Fig. 4c, the Beclin1 knockdown resulted in inhibition of cell death marker PARP in response to OGD/reoxygenation treatment. Similarly, co-treatment of 4-PBA and lithium significantly enhanced the neuroprotective effect, whereas the suppression of Akt activity by LY294002 or the induction of autophagy by rapamycin abolished this protective benefit. On the contrary, cells treated with the autophagy inhibitor chloroquine are partially protected against the cytotoxicity effect of rapamycin (Fig. 4d). This indicates that OGD/reoxygenation-induced ER stress may induce neuronal death via down-regulation of Akt signaling, and lithium combined with 4-PBA returned the intracellular levels of Akt to normal, resulting in suppression of autophagy cell death.

Fig. 4.

Fig. 4

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The combination of lithium and 4-PBA significantly reduces OGD/reoxygenation-induced autophagic cell death. a Immunoblotting was probed by specific antibodies against pAkt and total Akt during OGD and reoxygenation. b Western blot analysis shows pAkt/Akt, pGSK3β/GSK3β, p-mTOR/mTOR, and LC3 in SK-N-MC cells treated with indicated conditions. c Beclin1 siRNA (20 nmol/l pool) knockdown was confirmed via Western blot, and autophagy blockade was validated using LC3-II immunoblotting. Enhanced cell death in response to OGD/reoxygenation was determined via PARP cleavage. d Both the LY294002 (20 μM) and rapamycin (200 nM) treatments for 24 h blocked 4-PBA and lithium to prevent cell death significantly. However, treatment with chloroquine (50 μM) partially returned this inhibition to that in the LY294002-treated groups. LY294002, a specific inhibitor of PI3-kinase. Mean ± SEM of three independent experiments are shown. *P < 0.05 and **P < 0.01 when compared with 4-PBA+ lithium-treated cells using multiple comparisons of Dunnett’s post hoc test

Discussion

Ischemic stroke is a major cause of death and disability in adults. As a result, there is a pressing need for the development of effective drugs or strategies for this devastating disease. In this study, we showed the combination of lithium and 4-PBA protecting cells from OGD/reoxygenation-induced cell death. Although the role of ER stress in neuronal OGD/reoxygenation remains undetermined, it is known that pronounced hypoxia causes a significant lack of energy and oxygen, which initiates a defect in protein-folding processes (Tagliavacca et al. 2012). To avoid the excessive accumulation of unfolded proteins, the ER-resident PERK was activated upon hypoxia, which leads to the phosphorylation of eIF2α and halts global protein synthesis (Donnelly et al. 2013). Although ER stress is an adaptive response to increase cell survival during severe hypoxia, the additional reoxygenation may prolong ER stress, and exacerbates cell death by reducing de novo synthesis of survival-mediating proteins (Duvigneau et al. 2010). This indicates that severe ER stress may exacerbate neuronal cell death during reoxygenation. By contrast, our results showed that 4-PBA rescues ER stress-suppressed Akt protein biosynthesis, which works cooperatively with lithium in the activation of Akt downstream signaling to promote cell survival. Thus, we believe that suppression of ER stress-mediated signaling by chemical chaperons such as 4-PBA may be a neuroprotective strategy to protect OGD/reoxygenation-induced insults and treating ischemic stroke. These results support the idea that Akt is a major switch involved in regulating hypoxia-induced cell death, and inhibition of ER stress is beneficial to OGD-induced cellular damage during reoxygenation.

Although 4-PBA has been reported to protect against neuronal ischemia through the inhibition of ER stress-mediated apoptosis (Qi et al. 2004; Mizukami et al. 2010), the molecular mechanism by which ER stress induces apoptosis has remained unclear. However, there is increasing evidence that Akt plays a crucial role in ER stress-elicited cell dysfunction (Wang et al. 2012). The mechanism of the loss of Akt proteins in late hypoxia is still not fully elucidated, although it appears to be a result of protein degradation by a non-proteasome-dependent pathway (Box et al. 2010). Autophagy is an evolutionarily conserved process involved in the proteolysis of intracellular material within lysosomes. As autophagy is a major quality control mechanism to maintain homeostasis of the cell, it has been suggested that autophagy could promote cell survival under stresses such as hypoxia (Dohi et al. 2012). However, excessive levels of autophagy have also been observed in association with various forms of cell death. For example, it has been found that inhibition of autophagy by pharmacological inhibitors such as 3-methyladenine significantly protects against ischemia-induced neuronal death, suggesting that autophagy may function as a pro-death factor (Puyal et al. 2009). This observation is consistent with our findings that significant up-regulation of Beclin1 and LC3-II conversion during reoxygenation, indicating apoptotic processes are suppressed by reoxygenation. Particularly, Beclin 1 is a principle mechanism contributing to autophagosome formation, indicating an essential role of autophagy in OGD/reoxygenation-induced cell death (Liu and Levine 2015). In contrast, 4-PBA and lithium block GSK3β apoptotic signaling and inhibit autophagy by up-regulation of the Akt-mTOR pathway, resulting in regulation of the over-expressed UPR toward cell survival. This result is consistent with those of previous studies in which ER stress inhibitor salubrinal displays neuroprotective effects by inhibiting the activation of autophagy in brain ischemia (Gao et al. 2013). However, autophagy is a complex cellular mechanism to regulate cell survival or death, indicating that this process remains to be elucidated.

It is unclear whether the process of lithium-activating Akt activity is related to its therapeutic efficacy for the treatment of bipolar disorder. However, Akt activation is related to lithium’s neuroprotective effects in post-stroke impairment (Gold et al. 2010). There are two known ways to explain how lithium regulates the Akt/GSK3β pathway. First, lithium may cause subunit dissociation of phosphatase 2A (PP2A), a negative regulator of Akt, and results in a sustained activation of Akt (Harrison et al. 2013). Second, lithium has also been shown to directly inhibit GSK3β due to competitive binding for magnesium that disrupts the catalytic activity of GSK3β (Brown and Tracy 2013). Therefore, a cooperative effect of lithium combined with 4-PBA between Akt, ER stress, and GSK3β is thought to be protective against OGD/reoxygenation. Recent evidence indicates that transient hypoxia induces activation of PI3K-Akt survival signaling (Zhao 2009). Once Akt signaling is activated, it phosphorylates a variety of downstream regulators of cell survival. However, reoxygenation-sustained ER stress impairs survival signaling by reducing Akt protein de novo synthesis. Similarly, previous studies have claimed that Akt is a major switch regulating cell death during hypoxia. If Akt activity is lost by hypoxia treatment, cells may be destined to undergo hypoxia-induced cell death. However, cells in which Akt activity was not lost were destined to survive (Box et al. 2010). This result is consistent with our findings, and reinforces the importance of the Akt signaling pathway for ischemic insults, and implies Akt signaling as a possible target for the therapy of ischemic brain stroke. In conclusion, our findings first provide a possible mechanism about how lithium and 4-PBA mediate their neuroprotective effects cooperatively, and these results might be used to develop a novel strategy for ischemia-induced neuronal damage.

Acknowledgments

This work was supported by Grants from the Chung Shan Medical University-Tungs’ Taichung Metroharbor Hospital (CSMU-TTM-098-003), and from the Ministry of Science and Technology (101-2320-B-040-015-MY3). The fluorescence microscope and imaging analyzer were performed at the Instrument Center of Chung Shan Medical University, which is supported by the Ministry of Science and Technology, Ministry of Education, and Chung Shan Medical University.

References

  1. Bando Y, Ogawa S, Yamauchi A, Kuwabara K, Ozawa K, Hori O, Yanagi H, Tamatani M, Tohyama M (2000) 150-kDa oxygen-regulated protein (ORP150) functions as a novel molecular chaperone in MDCK cells. Am J Physiol Cell Physiol 278(6):C1172–1182 [DOI] [PubMed] [Google Scholar]
  2. Box AH, Kim SM, Demetrick DJ (2010) AKT loss in human epithelial cells treated with severe hypoxia. Biochim Biophys Acta 1803(8):951–959. doi:10.1016/j.bbamcr.2010.03.011 [DOI] [PubMed] [Google Scholar]
  3. Brown KM, Tracy DK (2013) Lithium: the pharmacodynamic actions of the amazing ion. Ther Adv Psychopharmacol 3(3):163–176. doi:10.1177/2045125312471963 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chuang DM, Wang Z, Chiu CT (2011) GSK-3 as a target for lithium-induced neuroprotection against excitotoxicity in neuronal cultures and animal models of ischemic stroke. Front Mol Neurosci 4:15. doi:10.3389/fnmol.2011.00015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cole AR (2012) GSK3 as a sensor determining cell fate in the brain. Front Mol Neurosci 5:4. doi:10.3389/fnmol.2012.00004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dohi E, Tanaka S, Seki T, Miyagi T, Hide I, Takahashi T, Matsumoto M, Sakai N (2012) Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival. Neurochem Int 60(4):431–442. doi:10.1016/j.neuint.2012.01.020 [DOI] [PubMed] [Google Scholar]
  7. Donnelly N, Gorman AM, Gupta S, Samali A (2013) The eIF2alpha kinases: their structures and functions. Cell Mol Life Sci 70(19):3493–3511. doi:10.1007/s00018-012-1252-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duvigneau JC, Kozlov AV, Zifko C, Postl A, Hartl RT, Miller I, Gille L, Staniek K, Moldzio R, Gregor W, Haindl S, Behling T, Redl H, Bahrami S (2010) Reperfusion does not induce oxidative stress but sustained endoplasmic reticulum stress in livers of rats subjected to traumatic-hemorrhagic shock. Shock 33(3):289–298. doi:10.1097/SHK.0b013e3181aef322 [DOI] [PubMed] [Google Scholar]
  9. Freland L, Beaulieu JM (2012) Inhibition of GSK3 by lithium, from single molecules to signaling networks. Front Mol Neurosci 5:14. doi:10.3389/fnmol.2012.00014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gao B, Zhang XY, Han R, Zhang TT, Chen C, Qin ZH, Sheng R (2013) The endoplasmic reticulum stress inhibitor salubrinal inhibits the activation of autophagy and neuroprotection induced by brain ischemic preconditioning. Acta Pharmacol Sin 34(5):657–666. doi:10.1038/aps.2013.34 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gold AB, Herrmann N, Lanctot KL (2010) Lithium and its neuroprotective and neurotrophic effects: potential treatment for post-ischemic stroke sequelae. Curr Drug Targets 12:243–255 [DOI] [PubMed] [Google Scholar]
  12. Harrison LM, Muller SH, Spano D (2013) Effects of the Ras homolog Rhes on Akt/protein kinase B and glycogen synthase kinase 3 phosphorylation in striatum. Neuroscience 236:21–30. doi:10.1016/j.neuroscience.2012.12.062 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hetz CA (2007) ER stress signaling and the BCL-2 family of proteins: from adaptation to irreversible cellular damage. Antioxid Redox Signal 9(12):2345–2355. doi:10.1089/ars.2007.1793 [DOI] [PubMed] [Google Scholar]
  14. Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13(2):89–102. doi:10.1038/nrm3270 [DOI] [PubMed] [Google Scholar]
  15. Iannitti T, Palmieri B (2011) Clinical and experimental applications of sodium phenylbutyrate. Drugs R&D 11(3):227–249. doi:10.2165/11591280-000000000-00000 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kolb PS, Ayaub EA, Zhou W, Yum V, Dickhout JG, Ask K (2015) The therapeutic effects of 4-phenylbutyric acid in maintaining proteostasis. Int J Biochem Cell Biol 61:45–52. doi:10.1016/j.biocel.2015.01.015 [DOI] [PubMed] [Google Scholar]
  17. Li X, Bijur GN, Jope RS (2002) Glycogen synthase kinase-3beta, mood stabilizers, and neuroprotection. Bipolar Disord 4(2):137–144 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lin L, Bivard A, Parsons MW (2013) Perfusion patterns of ischemic stroke on computed tomography perfusion. J Stroke 15(3):164–173. doi:10.5853/jos.2013.15.3.164 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu Y, Levine B (2014) Autosis and autophagic cell death: the dark side of autophagy. Cell Death Differ. doi:10.1038/cdd.2014.143 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liu Y, Levine B (2015) Autosis and autophagic cell death: the dark side of autophagy. Cell Death Differ 22(3):367–376. doi:10.1038/cdd.2014.143 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mahadevan NR, Zanetti M (2011) Tumor stress inside out: cell-extrinsic effects of the unfolded protein response in tumor cells modulate the immunological landscape of the tumor microenvironment. J Immunol 187(9):4403–4409. doi:10.4049/jimmunol.1101531 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Manzanero S, Santro T, Arumugam TV (2013) Neuronal oxidative stress in acute ischemic stroke: sources and contribution to cell injury. Neurochem Int 62(5):712–718. doi:10.1016/j.neuint.2012.11.009 [DOI] [PubMed] [Google Scholar]
  23. Mizukami T, Orihashi K, Herlambang B, Takahashi S, Hamaishi M, Okada K, Sueda T (2010) Sodium 4-phenylbutyrate protects against spinal cord ischemia by inhibition of endoplasmic reticulum stress. J Vasc Surg 52(6):1580–1586. doi:10.1016/j.jvs.2010.06.172 [DOI] [PubMed] [Google Scholar]
  24. Mullonkal CJ, Toledo-Pereyra LH (2007) Akt in ischemia and reperfusion. J Investig Surg 20(3):195–203. doi:10.1080/08941930701366471 [DOI] [PubMed] [Google Scholar]
  25. Nagy T, Frank D, Katai E, Yahiro RK, Poor VS, Montsko G, Zrinyi Z, Kovacs GL, Miseta A (2013) Lithium induces ER stress and N-glycan modification in galactose-grown Jurkat cells. PLoS One 8(7):e70410. doi:10.1371/journal.pone.0070410 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Perez-Alvarez MJ, Maza Mdel C, Anton M, Ordonez L, Wandosell F (2012) Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia. J Neuroinflamm 9:157. doi:10.1186/1742-2094-9-157 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pradeep H, Densi A, Rajanikant GK (2014) Glycogen synthase kinase-3beta in ischemic neuronal death. Curr Neurovasc Res 12:243–255 [DOI] [PubMed] [Google Scholar]
  28. Puyal J, Vaslin A, Mottier V, Clarke PG (2009) Postischemic treatment of neonatal cerebral ischemia should target autophagy. Ann Neurol 66(3):378–389. doi:10.1002/ana.21714 [DOI] [PubMed] [Google Scholar]
  29. Qi X, Hosoi T, Okuma Y, Kaneko M, Nomura Y (2004) Sodium 4-phenylbutyrate protects against cerebral ischemic injury. Mol Pharmacol 66(4):899–908. doi:10.1124/mol.104.001339 [DOI] [PubMed] [Google Scholar]
  30. Rami A, Langhagen A, Steiger S (2008) Focal cerebral ischemia induces upregulation of Beclin 1 and autophagy-like cell death. Neurobiol Dis 29(1):132–141. doi:10.1016/j.nbd.2007.08.005 [DOI] [PubMed] [Google Scholar]
  31. Roussel BD, Kruppa AJ, Miranda E, Crowther DC, Lomas DA, Marciniak SJ (2013) Endoplasmic reticulum dysfunction in neurological disease. Lancet Neurol 12(1):105–118. doi:10.1016/s1474-4422(12)70238-7 [DOI] [PubMed] [Google Scholar]
  32. Schonthal AH (2012) Endoplasmic reticulum stress: its role in disease and novel prospects for therapy. Scientifica 2012:857516. doi:10.6064/2012/857516 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shao L, Sun X, Xu L, Young LT, Wang JF (2006) Mood stabilizing drug lithium increases expression of endoplasmic reticulum stress proteins in primary cultured rat cerebral cortical cells. Life Sci 78(12):1317–1323. doi:10.1016/j.lfs.2005.07.007 [DOI] [PubMed] [Google Scholar]
  34. Shin WJ, Gwak M, Baek CH, Kim KS, Park PH (2012) Neuroprotective effects of lithium treatment following hypoxic-ischemic brain injury in neonatal rats. Childs Nerv Syst 28(2):191–198. doi:10.1007/s00381-011-1627-2 [DOI] [PubMed] [Google Scholar]
  35. Tagliavacca L, Caretti A, Bianciardi P, Samaja M (2012) In vivo up-regulation of the unfolded protein response after hypoxia. Biochim Biophys Acta 1820(7):900–906. doi:10.1016/j.bbagen.2012.02.016 [DOI] [PubMed] [Google Scholar]
  36. Wang Z, Zhang H, Xu X, Shi H, Yu X, Wang X, Yan Y, Fu X, Hu H, Li X, Xiao J (2012) bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways. Toxicol Lett 212(2):137–146. doi:10.1016/j.toxlet.2012.05.006 [DOI] [PubMed] [Google Scholar]
  37. Yung HW, Korolchuk S, Tolkovsky AM, Charnock-Jones DS, Burton GJ (2007) Endoplasmic reticulum stress exacerbates ischemia-reperfusion-induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells. FASEB J 21(3):872–884. doi:10.1096/fj.06-6054com [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhao H (2009) Ischemic postconditioning as a novel avenue to protect against brain injury after stroke. J Cereb Blood Flow Metab 29(5):873–885. doi:10.1038/jcbfm.2009.13 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhou X, Zhou J, Li X, Guo C, Fang T, Chen Z (2011) GSK-3beta inhibitors suppressed neuroinflammation in rat cortex by activating autophagy in ischemic brain injury. Biochem Biophys Res Commun 411(2):271–275. doi:10.1016/j.bbrc.2011.06.117 [DOI] [PubMed] [Google Scholar]

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