Salubrinal protects against rotenone-induced SH-SY5Y cell death via ATF4-parkin pathway

brain research 1549 (2014) 52–62

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Research Report

Salubrinal protects against rotenone-induced SH-SY5Y cell death via ATF4-parkin pathway Liang Wua, Na Luoa, Hong-Rui Zhaoa, Qing Gaoa, Jie Lub, Yang Panb, Jing-Ping Shib, You-Yong Tiana,n, Ying-Dong Zhanga,n a

Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu 210006, PR China Department of Neurology, Affiliated Nanjing Brain Hospital, Nanjing Medical University, Jiangsu 210029, PR China

b

ar t ic l e in f o

abs tra ct

Article history:

Parkinson0 s disease (PD) is a progressive neurodegenerative disorder, for which there are no

Accepted 4 January 2014

effective disease-modifying therapies. Growing evidence from studies in human PD brain,

Available online 10 January 2014

in addition to genetic and toxicological models, indicates that endoplasmic reticulum (ER) stress is a common feature of the disease and contributes to neurodegeneration. We examine

Keywords:

whether salubrinal, a ER stress inhibitor, can protect the rotenone-induced SH-SY5Y cell death

Salubrinal

and explore the mechanisms underlying this protection. Our results demonstrated that

Rotenone

rotenone induced a significant ER stress response and caused cell apoptosis, which was

ER stress

inhibited by salubrinal. Activating transcription factor 4 (ATF4), a member of the ATF/CREB

ATF4

family of basic leucine zipper transcription factors, has been implicated in the pathogenesis of

Parkin

neurodegeneration. We showed that salubrinal increased the up-regulation of ATF4 expression. An ATF4 siRNA significantly increased the rotenone cytotoxicity and decreased the salubrinal0 s protection. Further, we showed that ATF4 siRNA inhibited the expression of parkin, and parkin knockdown similarly aggravated the rotenone cytotoxicity and reduced the salubrinal0 s protection. Additionally, the protein level of parkin was declined after treatment with rotenone, whereas this reduction was rescued by salubrinal. These findings indicate ATF4-parkin pathway plays an important role in the salubrinal-mediated neuroprotection of rotenone-induced dopaminergic cell death. & 2014 Elsevier B.V. All rights reserved.

1.

in the substantia nigra (Martin et al., 2011). The mechanisms of neuronal loss remain unclear, and current therapies principally alleviate symptoms instead of targeting the underlying neuronal loss (Levy et al., 2009). Growing evidence

Introduction

Parkinson0 s disease (PD) is a progressive neurodegenerative disorder characterized by the death of dopaminergic neurons Abbreviations: ATF4,

activating transcription factor4; C/EBP,

protein homologous protein; ER, kinase; eIF2α,

endoplasmic reticulum; PERK,

CCAAT-enhancer-binding protein; CHOP,

CCAAT-enhancer-binding

ER membrane proteins double-stranded RNA-activated kinase-like

eukaryotic translation initiation factor 2 α; GRP78, glucose regulated protein 78; IRE1α, inositol requiring 1α enzyme;

MTT, 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide; PD, Parkinson0 s disease n Correspondence to: Department of Neurology, Affiliated Nanjing First Hospital, Nanjing Medical University, No. 68 Changle Road, Nanjing, Jiangsu 210006, PR China. Fax: þ86 25 52269924. E-mail addresses: [email protected] (Y.-Y. Tian), [email protected] (Y.-D. Zhang). 0006-8993/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2014.01.003

brain research 1549 (2014) 52–62

from research in human PD brain, in addition to genetic and toxicological models, indicates that endoplasmic reticulum (ER) stress is a common feature of PD and contributes to dopaminergic neurodegeneration (Hoozemans et al., 2007; Colla et al., 2012; Chen et al., 2008). Therefore, therapeutic strategies that mitigate ER stress may be beneficial to PD patients. ER stress is known to activate a series of signals that comprise the unfolded protein response (UPR). The UPR is mediated through three signaling pathways under the control of the ER membrane proteins double-stranded RNA-activated kinase-like kinase (PERK), activating transcription factor (ATF)-6 and inositol requiring-1α (IRE1α) (Ron and Walter, 2007). These signals coordinate the cellular response to unfolded proteins, which includes (1) downregulation of protein translation via activation of PERK and subsequent phosphorylation of the eukaryotic translation initiation factor 2 α (eIF2α) at Ser51, (2) enhanced expression of ER chaperone proteins that promote protein refolding, and (3) activation of proteases involved in the degradation of misfolded proteins (Walter and Ron, 2011). However, prolonged or severe ER stress can lead to the activation of apoptotic cell death pathways. Rotenone is an environmental toxin used to induce experimental Parkinsonism in animals and cell cultures. Many studies reveal that rotenone induces ER stress (Chen et al., 2008; Ryu et al., 2002), and that eIF2α siRNA can decrease rotenone cytotoxicity in SK-N-MC cells (Chen et al.,

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2008), suggesting that ER stress mediates rotenone-induced cell death. The effects of ATF4, a member of the ATF/CREB family of basic leucine zipper transcription factors, on neuronal survival or death are complex. Previous studies found that rotenone induces PERK phosphorylation, which results in ATF4 and CHOP (CCAAT-enhancer-binding protein (C/EBP) homologous protein) protein induction in neuronal PC12 cells (Ryu et al., 2002). Indeed, in a previous study, we found that ATF4 may be involved in dopaminergic cell death in the rotenone rat model (Wu et al., 2013). However, many target genes are regulated by ATF4 (Singleton and Harris, 2012), including parkin, which is transcriptionally upregulated by ER stress and can protect cells from ER stress-induced cell death (Sun et al., 2013; Imai et al., 2000). Moreover, a dominant-negative ATF4 mutant prevents the ER stress-induced upregulation of parkin (Bouman et al., 2011). And a recent report showed that ATF4-parkin pathway protects against neuronal death induced by 6-hydroxydopamine (6-OHDA) and 1-methyl-4phenyl-pyridinium (MPPþ) (Sun et al., 2013). However, it is unknown whether ATF4-parkin pathway is beneficial or harmful in the rotenone model. Salubrinal, a phosphatase inhibitor, selectively inhibits dephosphorylation of the α subunit of eIF2 which is an upstream activator of ATF4. It has been shown that salubrinal can modulate the cellular stress response to protect against ER stress-induced apoptosis (Boyce et al., 2005). In the present

Fig. 1 – Effect of rotenone on ER stress and the death of SH-SY5Y cells. (A) SH-SY5Y cells were treated with rotenone (100 nM) or thapsigargin (Tg, 5 μM) for the specified times. The expression levels of GRP78, CHOP and caspase-3 were determined with immunoblotting. The relative amounts of GRP78 (B), CHOP (C) and cleaved caspase-3 (D) imaged on the films was measured by densitometry and normalized to the expression of β-actin. npo0.05, nnpo0.01, vs. control group, n ¼ 3.

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study, we use rotenone-induced apoptosis in SH-SY5Y cells as an in vitro model of dopaminergic neuronal degeneration. We investigate the effects and mechanisms of action of salubrinal on this model of neuronal cell death.

inducer, and observed robust elevation of GRP78 and CHOP expression. Similar to rotenone, thapsigargin induced death of SH-SY5Y cells.

2.2. Salubrinal attenuates rotenone-induced SH-SY5Y cells death

2.

Results

2.1. Endoplasmic reticulum (ER) stress is involved in rotenone-induced death of SH-SY5Y cells Previously, it was shown that ER stress plays a critical role in rotenone-induced death in SK-N-MC neuroblastoma cells (Chen et al., 2008). Here, we sought to confirm and extend these observations by examining ER stress in rotenoneinduced SH-SY5Y cell death. The levels of cleaved caspase-3 increased significantly when SH-SY5Y cells were exposed to rotenone for 6 h or longer (Fig. 1A and D). And GRP78 (glucose regulated protein 78), a marker of ER stress, was upregulated significantly with rotenone0 s exposure periods of 6 h or longer (Fig. 1A and B). Moreover, rotenone had a modest effect on the expression of CHOP (Fig. 1A and C). As a positive control, we treated cells with thapsigargin, a well-known ER stress

To investigate whether salubrinal protects against rotenoneinduced dopaminergic cell death, SH-SY5Y cells were treated with rotenone in the presence or absence of salubrinal. We found that with 6- and 12-h treatment periods, rotenone caused dramatic activation of caspase-3, while salubrinal suppressed this rotenone-induced activation of caspase-3 (Fig. 2A and B). To further clarify the effect of salubrinal on rotenoneinduced apoptosis, SH-SY5Y cells were double-labeled for annexin V and PI, and analyzed on a FACSCalibur system. Rotenone treatment for a 12-h period induced apoptosis in 12.6771.74% of SH-SY5Y cells (Fig. 2C). In comparison, the apoptosis rate in SH-SY5Y cells treated with both rotenone and salubrinal was 7.7271.4% (Fig. 2C), consistent with the findings on caspase-3 activation. We also examined cell viability using the MTT assay. As shown in Fig. 2D, while

Fig. 2 – Effect of salubrinal on rotenone-induced neuronal death. (A) SH-SY5Y cells were treated with 100 nM rotenone (Rot), 40 μM salubrinal (Sal) or Rot plus Sal for the indicated time. Cleaved caspase-3 levels were determined by immunoblotting. (B) n The amounts of cleaved caspase-3 were measured by densitometry and normalized to β-actin levels. npo0.01, vs. matched ## control group, po0.01, n ¼3. (C) Apoptosis was investigated using flow cytometric analysis at 12 h after treatment with 100 nM rotenone (Rot), 40 μM salubrinal (Sal) or Rot plus Sal. nnpo0.01, vs. matched control group, #po0.05, ##po0.01, n ¼3. (D) Dose-dependent protection by salubrinal of SH-SY5Y cells treated for 12 h with 0, 5, 10, 20, 40, 80 or 160 μM salubrinal (Sal). n Quantitative analysis of cell viability using MTT assay is shown. po0.05, n ¼3.

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the viability of neurons decreased with 12 h of rotenone treatment, salubrinal significantly inhibited rotenone-induced neuronal cell death in a dose-dependent manner.

2.3. Salubrinal-mediated cytoprotection of SH-SY5Y cells is dependent on ATF4 Salubrinal is known to protect against ER stress-induced cytotoxicity by selectively inhibiting the dephosphorylation of eIF2α (Boyce et al., 2005). ATF4 is a downstream effector of phosphorylated eIF2α. We sought to determine whether salubrinal-mediated cytoprotection of SH-SY5Y cells is dependent on ATF4. Consistent with previous studies, salubrinal significantly elevated the levels of phosphorylated eIF2α, but did not affect the levels of total eIF2α (Fig. 3A, C and D). As shown in Fig. 4A and B, ATF4 levels modestly increased after treatment with rotenone for 6 or 12 h. When cells were incubated simultaneously with rotenone and salubrinal for 6 or 12 h, salubrinal enhanced the upregulation of ATF4 expression by 29.4% and 58.4%, respectively. These results were confirmed by immunofluorescence assay using an anti-ATF4 antibody; incubation with salubrinal and rotenone for 12 h markedly elevated ATF4 immunoreactivity in

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SH-SY5Y cells, although rotenone alone also increased immunoreactivity modestly (Fig. 4C). We further examined the effect of ATF4 on salubrinalmediated cytoprotection of SH-SY5Y cells. ATF4 siRNA was used to knock down the expression of ATF4 (Fig. 5C). ATF4 siRNA significantly decreased cell viability and enhanced rotenone-induced apoptosis (Fig. 5A and B), indicating that ATF4 protects against ER stress-induced cell death. The ATF4 siRNA also diminished salubrinal0 s protective effect against the rotenone-induced death of SH-SY5Y cells, suggesting that ATF4 is necessary for salubrinal to protect against ER stressinduced cell death. In addition, we found that ATF4 siRNA significantly decreased the expression of CHOP, which was not upregulated after treatment with rotenone or salubrinal (Fig. 5D and E).

2.4.

Parkin mediates the cytoprotection of ATF4

To explore how ATF4 exerts its cytoprotective effect in the rotenone-induced death of SH-SY5Y cells, we examined the target genes of ATF4. Parkin is a component of the UPR in the PERK/ATF4 pathway, and protects cells from ER stressinduced cell death (Bouman et al., 2011). In our study, ATF4 siRNA significantly diminished the levels of parkin, but

Fig. 3 – Effect of salubrinal on the protein expression of eIF2α, p-eIF2α and CHOP in SH-SY5Y cells treated with rotenone. (A) SH-SY5Y cells were treated with 100 nM rotenone (Rot), 40 μM salubrinal (Sal) or Rot plus Sal for the indicated time. The expression levels of eIF2α, p-eIF2α and CHOP were determined with immunoblotting. The relative protein levels of eIF2α (B), p-eIF2α (C) and CHOP (D) imaged on the films were measured by densitometry and normalized to the expression of β-actin. n po0.05, nnpo0.01, vs. matched control group, ##po0.01, n ¼6.

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Fig. 4 – Effect of salubrinal on rotenone-mediated induction of ATF4 expression in SH-SY5Y cells. (A) SH-SY5Y cells were treated with 100 nM rotenone (Rot), 40 μM salubrinal (Sal) or Rot plus Sal for the indicated time. The expression of ATF4 was determined with immunoblotting. (B) The relative amount of ATF4 protein imaged on the films was measured by densitometry and normalized to the expression of β-actin. npo0.05, nnpo0.01, vs. control group, #po0.05, ##po0.01, n¼3. (C) Immunocytochemistry showing the effect of salubrinal on rotenone-induced ATF4 expression in SH-SY5Y cells. After treatment with rotenone (100 nM) for 12 h, SH-SY5Y cells were fixed and immunolabeled with anti-ATF4 antibodies, followed by FITC-conjugated secondary antibodies. The nucleus was counterstained with DAPI. Fluorescence images (40  magnification): green, ATF4; blue, DAPI. Scale bar, 50 μm.

scrambled siRNA did not change the expression of parkin (Fig. 6A). Similar to the findings in a previous study on 6-OHDA and MPPþ-induced death of neuronal PC12 cells (Sun et al., 2013), rotenone decreased the expression of parkin, but salubrinal inhibited this downregulation of parkin expression (Fig. 6A). To further investigate that the effect of parkin on salubrinal-mediated cytoprotection of SH-SY5Y cells, we knocked down the expression of parkin using parkin siRNA. As indicated in Fig. 6C and E, parkin siRNA significantly increased the apoptosis percent and inhibited the cell viability, and salubrinal did not reduce the apoptosis percent and increase the cell viability in parkin siRNA cells. This suggests that salubrinal may protect cells through the ATF4-parkin pathway.

2.5. Salubrinal reduces the expression of LIP and inhibits the CHOP levels in nucleus ATF4 induces expression of the transcription factor CHOP in many ER stress paradigms (Harding et al., 2000;

Averous et al., 2004). CHOP is considered a major mediator of apoptotic cell death (Silva et al., 2005). As shown in Figs. 3A and 5D, CHOP expression is upregulated significantly in rotenone-treated SH-SY5Y cells, but salubrinal did not decrease CHOP expression. Because the nuclear translocation of CHOP is a necessary step for the induction of apoptosis, we wondered the effect of salubrinal on CHOP levels in cell nucleus. We checked the CHOP expression in the nuclear extracts and detected that the increase in CHOP expression caused by rotenone was abolished by salubrinal (Fig. 7A). It has shown that the bZIP protein C/EBP isoform LIP is required for nuclear translocation of CHOP during ER stress, and the CHOP–LIP interaction plays a critical role in the induction of apoptosis (Chiribau et al., 2010). Therefore, we examined changes in LIP expression in rotenone-treated SH-SY5Y cells co-treated with salubrinal. As indicated in Fig. 7B, rotenone did not change the expression of LIP in SHSY5Y cells, but the levels of LIP decreased significantly upon treatment with salubrinal. This suggests that, although salubrinal increases CHOP levels in SH-SY5Y cells, CHOP may not exacerbate apoptosis.

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Fig. 5 – Effect of ATF4 on rotenone-induced neuronal death in SH-SY5Y cells. (A) SH-SY5Y cells were transfected with ATF4 siRNA or a scrambled siRNA for 48 h, and then treated with 100 nM rotenone (Rot), 40 μM salubrinal (Sal) or Rot plus Sal for 12 h. Cell viability was determined by MTT assay and expressed as a percentage of the control. npo0.05, nnpo0.01, vs. matched group, &po0.05, vs. control group, #po0.05, ##po0.01, n ¼ 6. (B) SH-SY5Y cells were transfected with a scrambled siRNA or ATF4 siRNA for 48 h, and then treated with 100 nM rotenone (Rot), 40 μM salubrinal (Sal) or Rot plus Sal for 12 h. Apoptosis was investigated using flow cytometric analysis. npo0.05, nnpo0.01, vs. matched group, &&po0.01, vs. control group, #po0.05, ##po0.01, n ¼3. (C) SH-SY5Y cells were transfected with a scrambled siRNA or ATF4 siRNA for 48 h. The expression of ATF4 was determined with immunoblotting. (D) SH-SY5Y cells were transfected with ATF4 siRNA or a scrambled siRNA for 48 h. The expression of CHOP was determined with immunoblotting. (E) The relative amount of CHOP protein imaged on the films was measured by densitometry and normalized to the expression of β-actin.nnpo0.01, vs. matched group, ##po0.01, n ¼3.

3.

Discussion

In the present study, we provide data showing that treatment with salubrinal attenuates rotenone-induced neuronal death. We also demonstrate that ATF4 is necessary for the salubrinal-mediated cytoprotection. Moreover, we find that salubrinal protects against rotenone neurotoxicity through the ATF4-parkin pathway. Rotenone, a mitochondrial complex I inhibitor, possesses highly selective toxicity towards dopaminergic neurons in vitro (Hartley et al., 1994) and in vivo (Betarbet et al., 2000). And rotenone has been extensively used to experimentally model PD pathogenesis (Bove and Perier, 2012; Xiong et al., 2012). A recent study found that ER stress mediates rotenone-induced death of SK-N-MC neuroblastoma cells, and that ER stressmediated induction of the apoptotic process may precede oxidative stress within cells (Chen et al., 2008). In fact, accumulating evidence suggests that ER stress is a common pathological feature of PD, and is found in models of both the familial and sporadic forms of the disease (Colla et al., 2012; Holtz and

O0 Malley, 2003). ER stress markers have been reported in the substantia nigra pars compacta of human post-mortem sporadic PD cases (Hoozemans et al., 2007; Slodzinski et al., 2009). Numerous studies suggest that ER stress is an early component in PD pathogenesis, even preceding oxidative stress (Chen et al., 2008). Thus, potential therapeutic strategies that target ER stress may be beneficial for PD patients. Salubrinal is a selective inhibitor of eIF2α dephosphorylation that was recently found to protect against ER stress-mediated apoptosis (Boyce et al., 2005). Undifferentiated SH-SY5Y cells are identified as a suitable in vitro model for studying the molecular and cellular mechanisms underlying the pathophysiology of PD as well as for evaluating pharmacological interventions (Xie et al., 2012; Yong-Kee et al., 2012; Pan et al., 2009; Cheung et al., 2009). In our studies, salubrinal protected against rotenone-induced SH-SY5Y cell death. This finding is in line with that of a recent study showing that salubrinal attenuates β-amyloid-induced neuronal death (Huang et al., 2012). However, that study found that inhibition of the NF-κB pathway may be associated with salubrinal0 s protective effect.

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Fig. 6 – Effect of Salubrinal on the expression of parkin in SH-SY5Y cells treated with rotenone. (A) SH-SY5Y cells were transfected with a scrambled siRNA or ATF4 siRNA for 48 h, and then treated with 100 nM rotenone (Rot), 40 μM salubrinal (Sal) or Rot plus Sal for 12 h. The expression of cleaved parkin was determined with immunoblotting. (B) The relative amount of parkin protein imaged on the films was measured by densitometry and normalized to the expression of β-actin. npo0.05, nn po0.01, vs. matched group; &po0.05, vs. control group, ##po0.01, vs. rotenone group, n ¼3. (C) SH-SY5Y cells were transfected with a scrambled siRNA or parkin siRNA for 48 h, and then treated with 100 nM rotenone (Rot), 40 μM salubrinal (Sal) or Rot plus Sal for 12 h. Apoptosis was investigated using flow cytometric analysis. npo0.05, nnpo0.01, vs. matched group, &&po0.01, vs. control group, #po0.05, ##po0.01, n ¼ 3. (D) SH-SY5Y cells were transfected with a scrambled siRNA or parkin siRNA for 48 h. The expression of parkin was determined with immunoblotting. (E) SH-SY5Y cells were transfected with a scrambled siRNA or parkin siRNA for 48 h. Cell viability was determined by MTT assay and expressed as a percentage of the control. npo0.05, nnpo0.01, vs. matched group, &po0.05, vs. control group, #po0.05, ##po0.01, n ¼6. Salubrinal blocks eIF2α dephosphorylation, thereby maintaining phosphorylation of the protein. Although global protein synthesis can be suppressed by phosphorylated eIF2α,

ATF4 expression was reported to be increased by eIF2α phosphorylation (Dever, 2002). In our present study, salubrinal enhanced the upregulation of ATF4 expression. Using

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Fig. 7 – Effect of Salubrinal on the nuclear protein levels of CHOP and the expression of LIP in SH-SY5Y cells treated with rotenone. SH-SY5Y cells were treated with 100 nM rotenone (Rot), 40 μM salubrinal (Sal) or Rot plus Sal for 12 h. (A) The protein levels of CHOP from nuclear extracts were determined with immunoblotting. (B) The relative level of CHOP imaged on the films was measured by densitometry and normalized to the expression of histone H3. nnpo0.01, vs. control group, ##po0.01, vs. rotenone group, n ¼ 3. (C) The protein levels of C/EBPβ were determined with immunoblotting. (D) The relative level of LIP imaged on the films was measured by densitometry and normalized to the expression of β-actin. npo0.05, vs. control group; # po0.05, vs. rotenone group, n ¼3. siRNA-mediated knockdown, we demonstrate that ATF4 is essential to salubrinal0 s protective effect. Our study is in agreement with a recent report showing that ATF4 protects against neuronal cell death induced by 6-OHDA and MPPþ (Sun et al., 2013). In addition, it has been reported that ATF4null neurons are more sensitive to DNA-damaging agents (Galehdar et al., 2010), and that activating mutations in ATF4 reduce glutamate toxicity (Lewerenz et al., 2012). Collectively, these studies and the present study demonstrate that ATF4 has a cytoprotective function. However, a couple of studies have suggested that ATF4 may exacerbate cell death (Galehdar et al., 2010; Lange et al., 2008; Han et al., 2013). ATF4-null mice exhibit significantly smaller infarcts and improved behavioral recovery compared with wild-type mice in a rodent model of ischemic stroke (Lange et al., 2008), and cortical neurons from ATF4-null mice are resistant to oxidative stress-induced cell death (Lange et al., 2008). Additionally, Galehdar et al. (2010) has found that the ATF4–CHOP– PUMA pathway is a key signaling pathway in ER stressinduced neuronal death, and that ATF4-deficient neurons are more resistant to ER stress. These contrasting findings indicate that the effects of ATF4 on neuronal survival are complex. ATF4 can promote either cell survival or death depending on the specific cell type or stressor. A factor likely

contributing to the differential effects of ATF4 on neuronal survival is the set of transcriptional targets regulated by ATF4. Specially, ATF4 regulates the expression of parkin, which is transcriptionally upregulated by ER stress and can protect cells from ER stress-induced cell death (Imai et al., 2000). In fact, a dominant-negative ATF4 mutant prevents ER stress-induced upregulation of parkin (Imai et al., 2000). However, ATF4 overexpression had little effect on parkin mRNA. These findings indicated that ATF4 is necessary but not sufficient for transactivation of parkin (Sun et al., 2013). Consistent with the previous study, ATF4 knockdown led to a decrease in parkin protein and increase rotenone induced apoptosis in our study. In our studies, rotenone led to a substantial fall in parkin protein levels. Sun et al. (2013) observed this effect in both differentiated PC12 cells and primary cortical neurons and suggested parkin levels decreased via enhanced proteasomal degradation. The parkin has a protective role in PD cellular and animal models (Sun et al., 2013; Lewerenz et al., 2012; Yasuda et al., 2011), and recent study demonstrated that an increase in parkin expression could slow aging both at the level of biochemical and molecular marker of aging (Rana et al., 2013). Of note, enhancing ATF4-parkin pathway represents a potential neuroprotective strategy in PD (Sun et al.,

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2013). The same opinion has been confirmed in our present study. Apoptosis percent was enhanced in parkin-siRNA SHSY5Y cells. Similar to ATF4, the ER stress inhibitor salubrinal increased the protein levels of parkin, which may be caused by inhibiting proteasomal degradation. Interestingly, ATF4 also regulates the expression of CHOP, which was found to mediate apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of Parkinsonism (Shin et al., 2011). However, it has also been reported that CHOP possesses neuroprotective effects (Halterman et al., 2010). In present study, we observed that salubrinal increases protein expression of CHOP, but salurinal abolished the increase in CHOP expression of the cell nucleus. LIP, which is one of the three functionally relevant isoforms of C/EBP, interacts with CHOP during ER stress and transports it into the nucleus (Chiribau et al., 2010). CHOP is then able to modulate the levels of its target genes by repressing the expression of Bcl-2 and other antiapoptotic genes (Huang et al., 2009; Su and Kilberg, 2008). We found that LIP levels, which did not change in the rotenone-treated neurons, were decreased significantly by salubrinal. Taken together, our findings suggest that CHOP does not translocate to the nucleus during early ER stress, and therefore cannot induce apoptosis, in our model. In summary, we demonstrate that salubrinal protects against rotenone-induced dopaminergic neuron death, and that the role of the cytoprotection may depend on the activation of ATF4. Although the effect of ATF4 activation on neuronal survival is complex, our study provides an insight into the potential involvement of ATF4-parkin pathway in the salubrinal-mediated neuroprotection of rotenone-induced dopaminergic cell death.

4.

Experimental procedures

4.1.

Chemicals and antibodies

Salubrinal was obtained from Tocris BioScience (R&D, Ellisville, MO, USA). Rotenone was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used in this study were anticleaved caspase-3, anti-eIF2α, anti-phospho-eIF2α (Ser51), antiATF4, anti-GRP78, anti-CHOP, anti-parkin from Cell Signaling Technology (Danvers, MA, USA), anti-C/EBPβ from Abcam (Cambridge, MA, USA), anti-histone H3 and anti-β-actin from Santa Cruz (Dallas, TX, USA), and. FITC-conjugated secondary antibody was purchased from Zhongshan Inc. (Beijing, China).

4.2.

Cell cultures

Human neuroblastoma SH-SY5Y cells (ATCC) were cultured in RPMI 1640 medium supplemented with 10% heatinactivated fetal calf serum containing 2 mM glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin under a humidified atmosphere of 95% air–5% CO2 at 37 1C.

4.3.

MTT assay for cell viability

Cell viability was assessed using MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide). Briefly, SH-SY5Y cells were collected and seeded in 96-well plates at a density of 1  105 cells/well. After the indicated treatment, 20 μL of MTT tetrazolium salt (Sigma-Aldrich) was added to each well for 3 h at 37 1C. Afterwards, the growth medium was replaced with dimethyl sulfoxide, and the absorbance of each well was measured with a plate reader using a test wavelength of 490 nm and a reference wavelength of 630 nm.

4.4.

Apoptosis assay

Quantitative evaluation of apoptosis was performed using flow cytometry after double labeling with an FITC Annexin V Apoptosis Detection kit (BD Biosciences, NJ, USA) to discriminate between early apoptotic (annexin V-positive) and necrotic (annexin V-propidium iodide (PI) double-positive) cells. SH-SY5Y cells were cultured in 6-well plates, and were then treated with 100 nM rotenone, 40 μM salubrinal, or rotenone plus salubrinal for different periods of time. The cells were harvested after treatment with 0.25% trypsin, washed with phosphate-buffered saline (PBS) and incubated in 1  Binding Buffer containing FITC annexin V and PI at 37 1C in the dark for 15 min. The apoptosis rate¼[annexin V(þ)PI ( ) cellsþannexin V(þ)PI (þ) cells]/total cells  100%. The specific fluorescence of 10,000 cells was analyzed with a FACSCalibur system (BD Biosciences) within 1 h after the antigen–antibody reaction. Data were analyzed using FSC express version 3.0 software (De Novo Software, Los Angeles, CA, USA).

4.5.

Immunofluorescence staining

After the indicated experimental treatment, SH-SY5Y cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were then incubated in blocking solution followed by overnight incubation with monoclonal rabbit anti-ATF4 (1:200). After washing with PBS, the cells were incubated with FITC-conjugated goat-anti-rabbit IgG (1:100). The nuclei were counterstained with DAPI using Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Immunostaining was visualized at 40  magnification under a fluorescence microscope.

4.6.

Small interfering RNA transfection

ATF4 or parkin small interfering RNA (siRNA) and its scrambled siRNA (Santa Cruz Biotechnology, Santa Cruz, TX, USA) were transfected into SH-SY5Y cells with Lipofectamine 2000 reagent (Invitrogen, Life Technologies, USA) according to the manufacturer0 s protocol. At 48 h after transfection, the cells were subjected to immunoblotting analysis for ATF4 expression.

4.7.

Western blot analysis

Western blot analysis was performed as described previously (Wu et al., 2013). Briefly, total protein extracts (20 μg) or nuclear protein extracts (10 μg) were separated by

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electrophoresis on 10–15% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels, transferred to polyvinylidene fluoride (PVDF) membranes, and blocked in 5% bovine serum albumin in 1  Tris-buffered saline containing 0.1% Tween 20 (1  TBST). The membranes were sequentially incubated with primary antibodies and horseradish peroxidase (HRP)conjugated secondary antibodies. After washing with 1  TBST, protein bands were detected with chemiluminescence HRP substrate (SuperSignal West Pico; Thermo Scientific Inc., Pittsburgh, USA) and exposed to X-ray film (Fujifilm Inc., Tokyo, Japan). Band intensities were analyzed using Image J 1.44 (NIH, Bethesda, MD, USA).

4.8.

Statistical analysis

All data are presented as mean7standard error of the mean (SEM). The alterations were analyzed using analysis of variance (ANOVA) with SPSS software (version 13.0, SPSS, Chicago, IL, USA). P-valueso0.05 were considered statistically significant.

Acknowledgments This study was supported by grants from National Natural Science of China (No. 81271418), Natural Science Foundation of Jiangsu Province (No. BK2012524), Six talent Summit of Jiangsu Province (No. 2012-WS-086), and the Research and Innovation Project for College Graduates of Jiangsu Province (No. CXZZ12_0576).

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