Neuregulin-1 Inhibits CoCl2-Induced Excitatory Amino Acid Carrier 1 Overexpression and Oxidative Stress in SH-SY5Y Cells

Excitatory amino acid carrier 1 (EAAC1) is an important subtype of excitatory amino acid transporters (EAATs) and is the route for neuronal cysteine uptake. CoCl 2 is not only a hypoxia-mimetic reagent but also an oxidative stress inducer. Here, we found that CoCl 2 induced signicant overexpression of EAAC1 in a dose- and time-dependent manner. We further demonstrated that pretreatment with NRG1 rescued the CoCl 2 -induced upregulation of EAAC1 and tau expression. Neuregulin-1 (NRG1) plays a protective role in the CoCl 2 -induced accumulation of reactive oxygen species (ROS) and reduction in antioxidative enzyme (SOD and Gpx) activity. Moreover, NRG1 attenuated CoCl 2 -induced apoptosis and cell death. NRG1 inhibited the CoCl 2 -induced release of cleaved caspase-3 and reduction in Bcl-X L . Our novel ndings suggest that NRG1 may play a protective role in oxidative stress and hypoxia through the regulation of EAAC1. analyzed by western blotting. Representative immunoblots showing SH-SY5Y cells treated with 100 µM CoCl2 and either PBS or NRG1 (5 nM or 10 M) for 36 hrs. d Quantitative analysis of the data in a. Treatment with 100 µM CoCl2 signicantly increased the expression of cleaved caspase-3. NRG1 attenuated the increase in cleaved caspase-3 expression, as shown by the densitometric values, which are shown as ratios relative to the values of the untreated control group (n=6, **P<0.01 versus the control group; #P<0.05, # #P<0.01 versus the CoCl2 alone group). e Quantitative analysis of the data in a. Treatment with 100 µM CoCl2 signicantly decreased the expression of Bcl-XL in SH-SY5Y cells. NRG1 inhibited the reduction in Bcl-XL expression, as shown by the densitometric values, which are shown as ratios relative to the values of the untreated control group (n=6, *P<0.01 versus the control group; #P<0.05 versus the CoCl2 alone group).


Introduction
Excitatory amino acid carrier 1 (EAAC1, also referred to as EAAT3) is one neuronal subtype of excitatory amino acid transporter (EAAT) that is ubiquitously expressed in the central nervous system (CNS). EAAC1 can also transport cysteine at a rate comparable to that of glutamate and is the primary route for the uptake of neuronal cysteine. Cysteine is a critically important substrate for the synthesis of glutathione (GSH), one of the most important intracellular antioxidants in the brain [1,2]. Mature neurons utilize cysteine but not cystine for GSH synthesis [3,4]. EAAC1-mediated uptake may be the major source of cysteine for GSH synthesis in mature neurons [5]. Oxidative stress is a general premonitory hallmark of numerous brain pathologies and largely contributes to the acute and chronic outcomes of CNS disorders, such as epilepsy, ischemic stroke, amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease [6]. Modulation of EAAC1 activity correlates with neuronal GSH levels [7]. Knockdown of EAAC1 reduces cysteine uptake and intracellular GSH levels [8].
The intracellular response to hypoxia is regulated by hypoxia inducible factor-1 (HIF-1). HIF-1 is a transcription factor, and a heterodimer consisting of an oxygen-dependent regulatory HIF-1α subunit and a constitutively expressed HIF-lβ subunit that acts as a master regulator of adaptation to a low oxygen environment in the cell [9]. Recent evidence suggests that the ROS produced in the mitochondria mediate HIF-1α stabilization during hypoxia [9]. Hypoxia leads to a rapid increase in spontaneous vesicular glutamate release [10] and impaired glutamate uptake [11][12][13]. EAAC1 was increased at the transcript level in C6 cells by hypoxia [14]. Oxygen-glucose deprivation (OGD) induced the protein expression of EAAC1 in pure and mixed neuronal cultures and promoted EAAT3 activity, which increased glutamate uptake into cultured neurons [15]. EAAC1 transcript levels were transiently upregulated during the reperfusion phase in ischemia-reperfusion models [15]. Ischemia-reperfusion leads to oxidative stress and an accompanying transient increase in EAAT3 immunoreactivity in the hippocampus [16].
Neuregulin-1 (NRG1) is a member of the NRG family of growth factors that play important roles in the developing and adult CNS [17]. Recently, accumulating evidence has collectively shown that NRG1 is a new regulator of injury and repair with multifaceted roles in neuroprotection, remyelination, and immunomodulation. NRG1 protects against a number of CNS pathological conditions, including ischemia, neurotrauma, and neurodegenerative diseases [18][19][20][21][22]. Our recent work showed that NRG1 regulated hypoxia-inducible factors such as HIF-1α and p53 [23]. NRG1/ErbB4 attenuates neuronal cell damage under OGD in primary hippocampal neurons [24]. These ndings suggest a correlation between NRG1 dysfunction and CNS pathology. Therefore, NRG1 may be a potential therapeutic target in the recovery of function after CNS injury.
Our study provides conclusive molecular evidence that CoCl 2 strongly induces EAAC1 expression in SH-SY5Y cells. These changes may alter homeostasis and enhance reactive oxidative stress in neurons.

Reagents and antibodies
Recombinant β-type NRG1 was purchased from ProSpec (East Brunswick, NJ, USA). Antibodies were Cell culture SH-SY5Y human neuroblastoma cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modi ed Eagle's medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and a penicillin-streptomycin-amphotericin B mixture (Invitrogen) at 37 °C in a humidi ed atmosphere containing 5% CO 2 . When the cells grew su ciently in 100 mm culture dishes (SPL Life Sciences, Gyeonggi-do, Korea), they were subcultured in 6-well or 96-well plates.

Assessment of cell death
Cell death after CoCl 2 treatment was assessed by determining the release of lactate dehydrogenase (LDH) into the culture medium, thereby indicating a loss of membrane integrity. LDH activity was measured using a commercial kit (Cytotox 96 nonradioactive cytotoxicity assay kit, Promega, Madison, WI, USA) according to the manufacturer's protocol. The absorbance was measured at 490 nm using a VICTOR X3 multilabel plate reader (PerkinElmer, Shelton, USA).

TUNEL staining
In situ DNA fragmentation was assessed using a terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) staining kit (Roche Diagnostics) according to the manufacturer's instructions. Images were captured after counterstaining with 10 µM 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) for 30 minutes. The number of apoptotic cells was counted in ve random elds using a Zeiss LSM 5 LIVE confocal microscope (Carl Zeiss AG, Oberkochen, Germany). The apoptotic cells are expressed as the percentage of TUNEL-positive cells in the total number of DAPI-stained cells. Glutathione peroxidase (GPx) activity assay GPx activity was determined using a Biovision glutathione peroxidase activity assay kit (CA, USA) according to the manufacturer's protocol. SH-SY5Y cells were homogenized on ice in cold assay buffer and then centrifuged at 10,000 × g for 15 minutes at 4 °C. Then, 50 µl of cell supernatant was added to a 96-well plate with 50 µl of assay buffer. The reaction mixture was added to each sample and incubated for 15 minutes to deplete all GSSG in the samples. Ten microliters of cumene hydroperoxide substrate was subsequently added to initiate the enzymatic reaction. The absorbance was immediately measured at a wavelength of 340 nm using a VICTOR X3 multilabel plate reader (PerkinElmer, Shelton, USA). GPx activity was calculated using an NADPH standard curve.
Superoxide dismutase (SOD) activity assay SOD activity was measured using a commercially available kit (Cayman Chemical Company, MI, USA) according to the manufacturer's protocol. SH-SY5Y cells were homogenized in cold 20 mM HEPES buffer (pH 7.2) and centrifuged at 1,500 × g for 5 minutes at 4 °C. Each sample (10 µl) was added to a 96-well plate with 200 µl of the diluted radical detector. Then, 20 µl of diluted xanthine oxidase was added to initiate the enzymatic reaction. The absorbance was immediately measured at a wavelength of 450 nm using a VICTOR X3 multilabel plate reader (PerkinElmer, Shelton, USA).
Immuno uorescence analysis SH-SY5Y cells were xed using 4% paraformaldehyde and 4% sucrose in DPBS (pH 7.4) for 20 minutes at room temperature (RT). Next, the cells were permeabilized and blocked using DPBS containing 1% BSA and 0.1% Triton X-100 at RT for 30 minutes, and then primary antibodies (mouse anti-EAAC1 (1:100) and rabbit anti-tau (1:100)) were added and incubated overnight at 4 °C. The cells were then washed three times in PBS and incubated with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 595 goat antichicken IgG (Jackson ImmunoResearch Laboratories, Inc., 1:200) for 2 hrs at RT. After counterstaining with DAPI (10 µM in PBS), the cells were mounted in Vectorshield (Vector Laboratories). Fluorescent images were acquired with an LSM 5 LIVE confocal system (Carl Zeiss AG, Oberkochen, Germany).

Statistical Analysis
The data are presented as the means ± SEM of three or more independent experiments. For the data of more than two groups, statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. A value of P < 0.05 was considered statistically signi cant.

Discussion
In the present study, we assessed the effects and mechanisms of NRG1 on CoCl 2 -induced oxidative stress in SH-SY5Y cells. First, we demonstrated that CoCl 2 dramatically increased EAAC1 protein expression. EAAT1 and EAAT2 are mainly expressed in glial cells [26][27][28], whereas EAAT3 is exclusively expressed in neurons [29][30][31][32]. The EAAC1 protein is abundantly expressed in the hippocampus, cerebellum, and midbrain areas [30]. In general, EAAC1 activity is considered to be the main mechanism responsible for glutamatergic transmission [2], and EAAC1 also transports cysteine into neurons [33,34].
Modulation of EAAC1 activity correlates with neuronal GSH levels [7] and the rate-limiting substrate for neuronal synthesis of GSH [35]. EAAC1 may be the major contributor to GSH synthesis [5] in neurons. Interestingly, Rossi et al. reported that glutamate release is largely mediated by reversed activity of the neuronal glutamate transporter in severe brain ischemia. The glutamate transporter plays a key role in generating anoxic depolarizations in hippocampal neurons [36]. These results suggest that the abnormal activity abolished information processing in the CNS within minutes of ischemia [37]. EAAC1-de cient mice showed that the delayed anoxic overexpression of EAAC1 could contribute to the reversed activity in neurons.
Hypoxic conditions have been extensively studied for their potential to regulate glutamate transporters, as this putative regulation could have important consequences for brain pathologies. A previous study reported that chronic hypoxia upregulates EAAC1 expression in PC12 cells [38]. CoCl 2 was reported to be a widely used hypoxia mimetic in a large variety of cells and is known to both inhibit prolyl hydroxylases, leading to HIF-1α stabilization, and induce ROS formation under normoxic conditions [39,40]. Furthermore, we examined the effect of NRG1 on CoCl 2 -induced EAAC1 and hypoxia-related protein.
Several lines of evidence collectively suggest that NRG1 plays a neuroprotective role in the brain against neurotoxic substances related to apoptosis and oxidative damage in neurons [41][42][43][44]. In this study, we showed that NRG1 could prevent CoCl 2 -induced upregulation of EAAC1 levels in SH-SY5Y cells. We also con rmed that NRG1 could attenuate the CoCl 2 -induced accumulation of HIF-1α and p53 [23].
Immuno uorescence analysis also showed that NRG1 signi cantly inhibited CoCl 2 -induced overexpression of EAAC1 in SH-SY5Y cells. Tau protein is a soluble microtubule-associated protein that is abundant in neurons and plays a role in neurite outgrowth and axonal transport [45,46]. Additionally, the level of Tau increased in cells after CoCl 2 treatment, suggesting that hypoxia or oxidative stress can lead to alterations in cell structure. Previously, there was a report showing that hypoxia promoted the phosphorylation and total expression of tau protein [38,47]. Moreover, we found that NRG1 prevented the CoCl 2 -induced upregulation of EAAC1 and Tau.
Next, we examined whether NRG1 protects against CoCl 2 -induced ROS generation. Numerous studies have suggested that hypoxia induces increased production of ROS in the brain [48][49][50]. When we treated the cells with CoCl 2 , ROS levels were increased. According to our results, NRG1 attenuated the CoCl 2induced generation of ROS in SH-SY5Y cells. There is a balance between the generation of ROS and their clearance by antioxidant networks, mainly by GPx, SOD, and catalase under physiological conditions [51,52]. In the present study, CoCl 2 reduced the activity of Gpx and SOD in SH-SY5Y cells. We found that NRG1 had a protective effect on the CoCl 2 -induced reduction in Gpx and SOD enzymatic activity.
ROS is a powerful initiator of apoptosis, which also contributes to hypoxia-mediated neuronal cell death [53]. We also found that NRG1 signi cantly reduced CoCl 2 -induced apoptosis and cell death in SH-SY5Y cells.
In the intrinsic pathway, ROS induce mitochondria-dependent apoptosis. This process can be modulated by the release of cytochrome c and the downstream activation of caspases. We next focused on whether NRG1 could protect SH-SY5Y cells against the activation of caspase-3 after CoCl 2 treatment. Our results veri ed that NRG1 signi cantly reduced the expression of cleaved caspase-3, which may have prevented hypoxia-induced apoptosis and cell death in SH-SY5Y cells. Immunoblot analysis also con rmed the effect of NRG1 on the CoCl 2 -induced activation of caspase-3. Bcl-2 family members act as critical regulators of the intrinsic apoptotic pathway. The antiapoptotic Bcl-2 family protein Bcl-X L predominantly localizes to the outer mitochondrial membrane, whereas other members indirectly interact with mitochondria [54]. We further con rmed that NRG1 inhibited the CoCl 2 -induced reduction in Bcl-xL expression.
Taken together, our data suggest that NRG1 protects against CoCl 2 -induced overexpression of EAAC1.
The abnormal overexpression of EAAC1 by CoCl 2 may accelerate ROS generation and hypoxic injury.
Pretreatment with NRG1 could activate these cellular defense mechanisms to mimic hypoxic preconditioning. NRG1 exerts its biological effects by activating a family of ErbB tyrosine kinase receptors. NRG1 can trigger signaling pathways such as Raf-MEK-ERK and PI3K-Akt-S6K. Further study is needed to clarify the underlying pathway associated with NRG1 in these effects.

Conclusion
Our study suggests that CoCl 2 signi cantly increases EAAC1 expression in SH-SY5Y cells.

Declarations
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Consent for publication
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Availability of data and materials
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Con ict of Interest
The authors declare no competing nancial interests.  The effects of NRG1 on the CoCl2-induced protein levels of EAAC1. a Representative immunoblots of EAAC1, HIF-1a, and p53 in SH-SY5Y cells in the presence or absence of 5 nM or 10 nM NRG1 following treatment with 100 µM CoCl2 for 36 hrs are shown. b Quantitative analysis of the data in a. Treatment with 100 µM CoCl2 signi cantly increased the expression of EAAC1. CoCl2-induced EAAC1 overexpression was attenuated by 5 nM or 10 nM NRG1 treatment. The densitometry values are shown as ratios relative to the values of the control group (n=8, *P < 0.05, ***P < 0.001 versus the control group;   NRG1 reduced the increase in ROS accumulation and decreased oxidative stress-related enzyme activity induced by CoCl2 in SH-SY5Y cells. a After 24 hrs, intracellular ROS levels were measured by uorescence microscopy using DCFH-DA dye that was administered to 100 µM CoCl2-treated SH-SY5Y cells that had been pretreated with NRG1 (5 or 10 nM) for 15 minutes. b Bar graph summarizing the data in a (n=6, ***P<0.001 versus the control group; #P<0.05, ##P<0.01 versus the CoCl2 alone group). c SH-SY5Y cells were treated with 100 µM CoCl2 alone or with PBS or NRG1 (5 nM or 10 nM) for 36 hrs. GPx activity was