Neuregulin-1 inhibits CoCl2-induced upregulation of excitatory amino acid carrier 1 expression and oxidative stress in SH-SY5Y cells and the hippocampus of mice

Excitatory amino acid carrier 1 (EAAC1) is an important subtype of excitatory amino acid transporters (EAATs) and is the route for neuronal cysteine uptake. CoCl2 is not only a hypoxia-mimetic reagent but also an oxidative stress inducer. Here, we found that CoCl2 induced significant EAAC1 overexpression in SH-SY5Y cells and the hippocampus of mice. Transient transfection of EAAC1 reduced CoCl2-induced cytotoxicity in SH-SY5Y cells. Based on this result, upregulation of EAAC1 expression by CoCl2 is thought to represent a compensatory response against oxidative stress in an acute hypoxic state. We further demonstrated that pretreatment with Neuregulin-1 (NRG1) rescued CoCl2-induced upregulation of EAAC1 and tau expression. NRG1 plays a protective role in the CoCl2-induced accumulation of reactive oxygen species (ROS) and reduction in antioxidative enzyme (SOD and GPx) activity. Moreover, NRG1 attenuated CoCl2-induced apoptosis and cell death. NRG1 inhibited the CoCl2-induced release of cleaved caspase-3 and reduction in Bcl-XL levels. Our novel finding suggests that NRG1 may play a protective role in hypoxia through the inhibition of oxidative stress and thereby maintain normal EAAC1 expression levels.


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 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]23]. Our recent work showed that NRG1 regulated hypoxia-inducible factors such as HIF-1α and p53 [24]. NRG1/ErbB4 attenuates neuronal cell damage under OGD in primary hippocampal neurons [25]. These findings 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 and hippocampus of mice. These acute changes may response against of reactive oxidative stress. NRG1 reduced the CoCl 2 -induced oxidative and thereby rescue upregulation of EAAC1.

Animals and stereotaxic surgery
C57BL/6 (male, 10 weeks old, 24-27 g) mice were obtained from a laboratory animal supplier (Samtako Bio Korea) and were housed in cages under standard laboratory conditions with a 12-h light/12-h dark cycle. A total of twenty animals were randomly allocated to the following four groups: saline (n = 8), NRG1 (n = 8), CoCl 2 (n = 8), and CoCl 2 + NRG1 (n = 8). Experiments with animals were approved by the Institutional Animal Care and Use Committee of Eulji University (EUIACUC 19-08). All surgical procedures and perfusions were performed under anaesthesia via intraparietal injection of ketamine (100 mg/kg) with Rompun (10 mg/kg). The animals were subjected to a unilateral lesion by placing them in a stereotaxic apparatus. CoCl 2 (25 mM) was delivered in the ventral hippocampus of the right hemisphere (coordinates from bregma: anterior/posterior − 3.3 mm, medial/ lateral + 2.8 mm, dorsal/ventral − 4.0 mm). Each microinjection unit was attached to a 10-μl Hamilton microlitre syringe via a glass tube, and administration was controlled by the experimenter at a rate of 1 μl (volume injected) over a period of approximately 2 min 30 s.

Cell culture and transfection
SH-SY5Y human neuroblastoma cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified 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 humidified atmosphere containing 5% CO 2 . When the cells grew sufficiently in 100 mm culture dishes (SPL Life Sciences, Gyeonggi-do, Korea), they were subcultured in 6-well or 96-well plates. SH-SY5Y cells were transiently transfected with either 4 μg of plasmid pcDNA3.1 (Mock) or pcDNA3.1-EAAC1-myc and 10 μl of Lipofectamine 2000 (Invitrogen) in 250 μl of Opti-MEM without serum according to the manufacturer's instructions. Transient transfection efficiencies were confirmed by Western blot in SH-SY5Y cells.

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 min. The number of apoptotic cells was counted in five random fields 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.

ROS measurement
ROS generation in SH-SY5Y cells was analyzed using the dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Invitrogen, CA, USA). SH-SY5Y cells were washed three times with Dulbecco's phosphate-buffered saline (DPBS) and then incubated at 37 °C in DPBS containing 20 μM DCFH-DA for 30 min. Once inside the cells, DCFH-DA is hydrolyzed by esterase to form polar DCFH, which then interacts with ROS. Cells were subsequently washed three times with DPBS and visualized with a fluorescence microscope (EVOS M5000, Thermo Fisher Scientific, Eugene, OR, USA) at an excitation wavelength of 485 nm.

Glutathione peroxidase (GPx) activity assay
GPx activity was determined using a Biovision glutathione peroxidase activity assay kit (Cayman Chemical Company, MI, 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 min 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 min 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 min 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).

Immunofluorescence analysis
SH-SY5Y cells were fixed using 4% paraformaldehyde and 4% sucrose in DPBS (pH 7.4) for 20 min 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 min, 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 594 goat anti-chicken IgG (Jackson ImmunoResearch Laboratories, Inc., 1:200) for 2 h at RT. After counterstaining with DAPI (10 μM in DPBS), the cells were mounted in Vectorshield (Vector Laboratories). Fluorescent images were acquired with an LSM 5 LIVE confocal system (Carl Zeiss AG, Oberkochen, Germany).

Dihydroethidium (DHE) staining
To assess superoxide production, the brain was immediately frozen in embedding medium [22]. Briefly, postfixed cryosections (15 µm) were incubated in DPBS containing 10 μM DHE (Invitrogen CA, USA) for 30 min at 37 °C in the dark room. The sections were then washed thrice with DPBS and mounted in Vectorshield (Vector Laboratories). Fluorescent images were acquired with an LSM 5 LIVE confocal system (Carl Zeiss AG, Oberkochen, Germany). Images were obtained using an excitation wavelength of 561 nm and an emission wavelength of 640 nm.

Statistical analysis
The data are presented as the means ± SEM of three or more independent experiments. Student's paired t-test was used for comparisons of the means between two groups of cells in a single experiment. 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 significant.

Discussion
In the present study, we assessed the effects and mechanisms of NRG1 on CoCl 2 -induced oxidative stress in SH-SY5Y cells and the hippocampus of mice. First, we demonstrated that CoCl 2 dramatically increased EAAC1 protein expression in SH-SY5Y cells. We also confirmed the increased EAAC1 expression by CoCl 2 microinjection in the VH in mice. EAAT1 and EAAT2 are mainly expressed in glial cells [27][28][29], whereas EAAT3 is exclusively expressed in neurons [30][31][32][33]. The EAAC1 protein is abundantly expressed in the hippocampus, cerebellum, and midbrain areas [31]. In general, EAAC1 activity is considered to be the main mechanism responsible for glutamatergic transmission [2], and EAAC1 also transports cysteine into neurons [34,35]. Modulation of EAAC1 activity correlates with neuronal GSH levels [7] and the rate-limiting substrate for neuronal synthesis of GSH [36]. 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 depolarization in hippocampal neurons [37]. These results suggest that the abnormal activity abolished information processing in the CNS within minutes of ischemia EAAC1-deficient mice showed that the delayed anoxic depolarization [38], overexpression of EAAC1 could contribute to the reversed activity in neurons. SLC1A1 encodes EAAC1, a SLC1A1 polymorphism highly replicated in obsessive-compulsive disorder studies that is associated with increased transcript levels in human brain tissue [39,40]. Mice with EAAC1 overexpression displayed increased anxiety-like and repetitive behaviours and synaptic alterations [41]. Even if our data demonstrate that the transient transfection of EAAC1myc reduced CoCl 2 -induced cell death and oxidative stress in SH-SY5Y cells, the abnormal overexpression of EAAC1 by chronic hypoxic stress might alter synaptic function and neuronal circuits in animal models.
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 [42]. 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 [43,44]. In addition, direct CoCl 2 brain microinjection provides a valuable animal model to develop focal ischemia in selected brain regions to study their functional consequences and potential pharmacological therapies.
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 [45][46][47][48]. In this study, we showed that NRG1 could prevent CoCl 2 -induced upregulation of EAAC1 levels in SH-SY5Y cells and the hippocampus of brain. We also confirmed that NRG1 could attenuate the CoCl 2 -induced accumulation of HIF-1α and p53 [24]. Immunofluorescence analysis also showed that NRG1 significantly 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 [49,50]. Additionally, the level of Tau and phospho-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 [42,51]. Additional evidence suggests that hypoxic and ischaemic brain damage in humans and animals may contribute to tau protein dysfunction, which is proposed as a risk factor for developing Alzheimer's disease (AD) [52]. The model generated using the hypoxia-mimicking agent CoCl 2 excluded environmental and vascular factors; thus, it could be useful to investigate the correlation between cellular hypoxia and AD. Moreover, we found that NRG1 prevented the CoCl 2 -induced upregulation of EAAC1, Tau and phospho-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 [53][54][55]. When we treated the cells with CoCl 2 , ROS levels were increased. According to our results, NRG1 attenuated the CoCl 2 -induced 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 [56,57]. 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. Furthermore, we confirmed that NRG1 reduced superoxide generation induced by microinjection of CoCl 2 into the VH of brain. ROS is a powerful initiator of apoptosis, which also contributes to hypoxia-mediated neuronal cell death [58]. We also found that NRG1 significantly reduced CoCl 2 -induced apoptosis and cell death in SH-SY5Y cells.
In the intrinsic pathway, ROS induce mitochondriadependent 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 verified that NRG1 significantly reduced the expression of cleaved caspase-3, which may have prevented hypoxia-induced apoptosis and cell death in SH-SY5Y cells. Immunoblot analysis also confirmed 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 [59]. We further confirmed 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.
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 significantly increases EAAC1 expression in SH-SY5Y cells and the hippocampus of mice. NRG1 attenuates the CoCl 2 -induced overexpression of EAAC1 and reduces CoCl 2 -induced oxidative stress and apoptotic signaling. NRG1 potentially plays a protective role in hypoxia through the inhibition of oxidative stress and maintains normal EAAC1 expression levels.
These results may show a new path toward understanding the pathogenesis and treatment of hypoxia and oxidative stress-related neurological diseases.