Compound 13 activates AMPK-Nrf2 signaling to protect neuronal cells from oxygen glucose deprivation-reoxygenation

Oxygen glucose deprivation-reoxygenation (OGD-R) causes the production of reactive oxygen species (ROS) and oxidative injury in neuronal cells. We tested the potential neuroprotective function of compound 13 (C13), a novel AMP-activated protein kinase (AMPK) activator, against OGD-R. We show that C13 pretreatment protected SH-SY5Y neuronal cells and primary hippocampal neurons from OGD-R. C13 activated AMPK signaling in SH-SY5Y cells and primary neurons. It significantly inhibited OGD-R-induced apoptosis activation in neuronal cells. Conversely, AMPKα1 shRNA or knockout reversed C13-mediated neuroprotection against OGD-R. C13 potently inhibited OGD-R-induced ROS production and oxidative stress in SH-SY5Y cells and primary neurons. Furthermore, C13 induced Keap1 downregulation and Nrf2 activation, causing Nrf2 stabilization, nuclear accumulation, and expression of Nrf2-dependent genes. Nrf2 silencing or knockout in SH-SY5Y cells abolished C13-mediated neuroprotection against OGD-R. In conclusion, C13 activates AMPK-Nrf2 signaling to protect neuronal cells from OGD-R.

AMP-activated protein kinase (AMPK) is a master energy sensor. Its activation is vital for energy and metabolism homeostasis [8]. AMPK is primarily composed of the catalytic α subunit along with the regulatory β and γ subunits [9,10]. Recent studies have shown that forced activation of AMPK, by genetic and/or pharmacological methods, will promote cell survival under different stress conditions [11]. The prosurvival function of AMPK is achieved through regulation of AMPK's downstream effectors.
The potential effect of C13 in the primary neurons was tested. As shown, OGD-R exposure similarly induced a reduction in CCK-8 OD ( Figure 1D) and LDH release ( Figure 1E) in primary murine hippocampal neurons. Pretreatment for 2h with the AMPK activator C13 (10 μM) potently inhibited the reduction in viability ( Figure  1D) and death ( Figure 1E) of primary neurons. C13 treatment by itself was ineffective ( Figure 1D and 1E). Therefore, C13 is neuroprotective against OGD-R.

C13 inhibited OGD-R-induced apoptosis activation in SH-SY5Y neuronal cells and primary neurons
The potential effect of C13 on the neuronal cell apoptosis was studied. First, we show that OGD-R induced caspase-3 activation in SH-SY5Y neuronal cells, and this was largely inhibited by pretreatment with C13 (5 or 10 μM, 2h; Figure 2A). The accumulation of histone-bound single strand DNA (ssDNA) is a characteristic marker of cell apoptosis. In OGD-R-treated SH-SY5Y neuronal cells, the amount of histone-bound ssDNA was significantly increased (Figure 2A), and again inhibited by C13 pretreatment ( Figure 2B). Furthermore, OGD-R increased the ratios of TUNEL-positive nuclei ( Figure 2C) and Annexin Vpositive cells (Figure2D), and these effects were also attenuated by C13 ( Figure 2C-2D). , together with/out OGD exposure for 4h, followed by 48h reoxygenation ("OGD-R"), cell viability was tested by CCK-8 assay (B and D); Cell death was examined by medium LDH release assay (C and E). "OGD-R" stands for OGD/re-oxygenation (Same for all figures). "Mock" stands for Mock control cells (no OGD-R, same for all Figures). "Vehicle" stands for the vehicle for C13 (0.1% DMSO, same for all Figures). For the OGD-R experiments, neuronal cells were always pretreated with C13 for 2h before OGD (Same for all figures). Bars stands for mean ± standard deviation (SD, n=5). * p<0.05 vs. "Mock" cells. # p<0.05 vs. cells with "OGD-R" treatment (no C13 pretreatment). Each experiment was repeated four times with similar results obtained. AGING In primary murine hippocampal neurons, OGD-R exposure induced the accumulation of histone-bound ssDNA ( Figure 2E) and increased the ratio of TUNELpositive nuclei ( Figure 2F), indicating the activation of apoptosis. Pretreatment with C13 (10 μM) potently inhibited the activation of OGD-R-induced apoptosis in the primary neurons ( Figure 2E and 2F). C13 treatment alone had no significant effect on apoptosis in SH-SY5Y cells (Figure 2A-2D) and primary neurons ( Figure 2E and 2F).

C13 activates AMPK signaling in SH-SY5Y neuronal cells and primary neurons
C13 is an AMPK activator, we therefore analyzed AMPK signaling in C13-treated neuronal cells. As shown in Figure 3A, in SH-SY5Y cells, C13 dosedependently increased the phosphorylation of AMPKα1 (at the Thr-172 residue), and the total level of AMPKα1 was unchanged. By using the AMPK activity assay, we show that C13 (5-25 μM) treatment potently increased the activity of AMPK in SH-SY5Y cells ( Figure 3B). Thus, C13 activated AMPK signaling in SH-SY5Y cells. In the primary murine hippocampal neurons, the phosphorylation of AMPKα1 ( Figure 3C) and the activity of AMPK ( Figure 3D) were significantly increased following treatment with C13 (10 μM, 1h).

Figure 2. Compound 13 inhibited OGD-R-induced apoptosis activation in SH-SY5Y neuronal cells and primary neurons. SH-
SY5Y human neuronal cells (A-D) or the primary murine hippocampal neurons (E and F) were pretreated (for 2h) with applied concentration (5/10 μM) of compound 13 ("C13"), followed by OGD-R stimulation for applied time periods, cell apoptosis was tested by the assays mentioned in the text. Bars stands for mean ± standard deviation (SD, n=5). * p<0.05 vs. "Mock" cells. # p<0.05 vs. cells with "OGD-R" treatment (no C13 pretreatment). Each experiment was repeated four times with similar results obtained. AGING

C13 activated AMPK downstream Nrf2 signaling in neuronal cells
Nrf2 signaling is the key endogenous antioxidant signaling cascade [25,26]. Once activated, Nrf2 will separate from Keap1, causing Nrf2 protein stabilization, accumulation, and activation [25,26]. Activated Nrf2 translocates to and accumulates in cell nuclei where it binds to ARE (antioxidant response element) to initiate transcription and the expression of antioxidant genes, including HO1, NQO1, and GCLC [25,26]. Existing studies have established that there is a crosstalk between AMPK and Nrf2 signaling [14,15,27]. Therefore, we tested whether C13 could also activate Nrf2 signaling in neuronal cells.
The results above show that C13 activated Nrf2 signaling in SH-SY5Y cells and primary murine hippocampal neurons ( Figure 5A-5E). Importantly, in Figure 5F and 5G, C13-induced mRNA expression of HO1, NQO1, and GCLC was almost completely blocked in "sh-AMPKα1" and "ko-AMPKα1" cells (see Figure 3). These results imply that activation of AMPK mediates the activation of C13-induced Nrf2 signaling in SH-SY5Y cells.

DISCUSSION
AMPK participates in a number of key cellular behaviors, from energy metabolism, cell mitosis, cell survival, and cell growth to apoptosis and autophagy [13,28]. Studies have demonstrated that AMPK activation could promote cell survival under stress conditions [29,30]. AMPK is reported to be overactivated in the brains of patients with neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis [31,32]. The exact function of AMPK in neurological diseases remains to be clarified, but existing studies have reported the involvement of AMPK in various signaling pathways that are important for the survival of neuronal cells [31,32].  F) were pretreated with C13 (10 μM) for 2h, followed by OGD-R stimulation for applied time, ROS contents were tested by the appropriate assays (A, B and E); Mitochondrial depolarization and lipid peroxidation levels were tested by JC-1 dye assay (C and F) and TBAR activity assay (D), respectively. Bars stands for mean ± standard deviation (SD, n=5). * p<0.05 vs. "Mock" cells. # p<0.05 vs. cells with "OGD-R" treatment (no C13 pretreatment). Each experiment was repeated three times with similar results obtained.
Here, we show that the activation of AMPK by C13 protected SH-SY5Y cells and primary hippocampal neurons from OGD-R. Conversely, AMPKα1 shRNA or KO reversed C13-mediated neuroprotection. Thus, C13induced AMPK activation offers significant protection to neuronal cells against OGD-R.
Studies have reported on the potent antioxidant activity mediated by activated AMPK [30]. In addition to its role in ATP homeostasis, forced activation of AMPK could attenuate oxidative injury by promoting NADPH synthesis and/or inhibiting NADPH consumption [30]. Studies have also established that there is a crosstalk between AMPK and Nrf2 signaling cascades [14,15,33]. Joo et al., show that AMPK directly phosphorylates Nrf2 at Serine 550 to promote Nrf2 nuclear translocation and activation [14]. The results of the present study show that C13 activated AMPK downstream Nrf2 signaling to inhibit OGD-R-induced ROS production and oxidative stress, thus protecting neuronal cells from OGD-R. Importantly, we proposed a novel mechanism for AMPK-induced Nrf2 signaling activation, that is, downregulation of Keap1.  E) and proteins (in both total lysates and nuclear fraction lysates, B-D) were shown. The stable SH-SY5Y cells with AMPKα1 shRNA ("sh-AMPKα1") and CRISPR/Cas-9 AMPKα1-KO construct ("ko-AMPKα1"), as well as the parental control cells ("Pare"), were treated with C13 (10 μM) for indicated time, expression of listed mRNAs were shown (F and G). The stable SH-SY5Y cells with Nrf2 shRNA ("sh-Nrf2" cells) and CRISPR/Cas-9 Nrf2-KO construct ("ko-Nrf2" cells), as well as the parental control cells ("Pare"), were treated with C13 (10 μM) for indicated time, listed mRNAs were shown (H and I); Cells were also pretreated with C13 (10 μM) for 2h, followed by OGD-R stimulation for 36/48h, cell viability (CCK-8 assay, J) and apoptosis (Histone DNA ELISA assay, K) were tested. Expression of listed proteins were quantified and normalized to the loading controls (B-D). Bars stands for mean ± standard deviation (SD, n=5). * p<0.05 vs. "Vehicle" control cells (A and E). # p<0.05 vs. "Pare" cells (F-K). Each experiment was repeated three times with similar results obtained.
Under resting conditions, Nrf2 binds to Keap1, causing Cul3-dependent Nrf2 ubiquitination and proteasomal degradation. We showed that C13 downregulated Keap1 and caused Nrf2 accumulation and activation of Nrf2 cascade; the latter was evidenced by Nrf2 protein stabilization, nuclear accumulation, and expression of Nrf2-depenedent genes (HO1, NQO1, and GCLC) in neuronal cells. Significantly, Nrf2 shRNA or KO in SH-SY5Y cells almost abolished C13-mediated neuroprotection against OGD-R. These results suggest that Nrf2 activation, downstream of AMPK, mediates C13induced neuroprotection from OGD-R-induced oxidative injury. Indeed, we show that C13 potently inhibited OGD-R-induced ROS production, mitochondrial depolarization, and lipid peroxidation in SH-SY5Y cells and primary neurons. The under-lying mechanisms of C13-induced Nrf2 signaling activation needs further investigation.
The present study has shown that C13 activated AMPK-Nrf2 signaling to protect the neuronal cells from OGD-R, a cellular model of ischemic stroke. It would be interesting to further test the potential of this compound against ischemic stroke.

SH-SY5Y cells
The human neuroblastoma cell line SH-SY5Y was purchased from the Cell Bank of Fudan University (Shanghai, China). SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and penicillin/streptomycin (1:100, Sigma-Aldrich). For neuronal cell differentiation, SH-SY5Y cells were cultured for seven days in serum-free DMEM medium with brain-derived neurotrophic factor and glutamine. Only differentiated SH-SY5Y cells were subjected to the OGD-R procedure.

Murine hippocampal neurons
Cultures of primary murine hippocampal neurons have been reported previously [35]. Briefly, primary neurons of CA1 hippocampus of E12-E14 embryos of C57 mice were separated and the CA1 hippocampal neurons were plated onto serum-free neuron basal medium with 2% B27 supplement and 2 mM glutamine. At day-8 in vitro, neurons were subjected to the OGD-R procedure.

OGD-R
The detailed procedures of OGD-R have been previously described [3]. Briefly, neuronal cells were placed in an airtight chamber and equilibrated for 10-12 min with a continuous flux of gas (95% N2and 5% CO2). The chamber was sealed and placed in an incubator for 4h under OGD. Cells were then reoxygenated. "Mock" cells were placed in DMEM containing glucose under normal oxygenation.

Cell survival assay
The Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) was employed to test cell viability, with its optical density (OD) values recorded at a wavelength of 450 nm.

Cell death assay
Cell death was examined by measuring lactate dehydrogenase (LDH) medium release via a two-step easy enzymatic reaction LDH assay kit (Takara, Tokyo, Japan). The LDH content in medium was normalized to the total LDH content.

Apoptosis assays
Routine apoptosis assays, including caspase-3 activity assay, Histone DNA ELISA assay, TUNEL staining assay, and the Annexin V FACS assay have been described in detail in previous studies [36][37][38].

Western blotting assay
The RIPA lysis buffer (Biyuntian Co., Wuxi, China) was added to neuronal cells. After quantification, 30 µg of lysate proteins per treatment was separated by 10-12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride blots (Merck Millipore, Darmstadt, Germany). After blocking in 10% non-fat milk in phosphatebuffered saline with Tween 20 (PBST), the blots were incubated with the appropriate primary antibodies. After three washes with PBST, the secondary antibodies were added and detected using an enhanced chemiluminescence system (Amersham, Little Chalfont, UK). The intensity (total gray area) of each band was quantified via using ImageJ software (National Institutes of Health, Bethesda, MD), and the value was normalized to the corresponding loading control.

ROS assay
As previously described [21], ROS contents were tested by the carboxy-H2DCFDA dye assay. Following the applied treatment, neuronal cells were stained with carboxy-H2-DCFDA (5 μM) for 30 min in the dark. DCF fluorescence was tested by a fluorescence spectrofluorometer (Thermo Fisher Scientific) at 485 nm excitation and 525 nm emission.

Superoxide assay
A superoxide assay kit (Beyotime, Wuhan, China) was utilized to examine the cellular superoxide contents based on the attached protocol. Briefly, neuronal cells with the applied treatments were incubated with the superoxide detection reagent (100 µL/well) for 30 min in the dark at room temperature. Superoxide absorbance was tested by a spectrophotometer at a wavelength of 450 nm.

Lipid peroxidation assay
Following the applied treatment, thiobarbituric acid reactive substances (TBAR) activities were examined to quantify the cellular lipid peroxidation levels using a reported protocol [21,39].

Mitochondrial depolarization assay
As previously described [40], in mitochondrial depolarization, the JC-1 red fluorescence aggregates to form green monomers [41]. Briefly, after the applied treatment, neuronal cells were stained with JC-1 (5 μg/mL) for 20 min in the dark at room temperature. After three washes, JC-1 green intensity was examined immediately at 550 nm via a fluorescence spectrofluorometer.

Quantitative real-time PCR assay
Cellular RNA was extracted via TRIzol reagents (Promega, Madison, WI). For quantitative real-time PCR, a SYBR Green PCR kit (Applied Biosystems, Foster City, CA) was utilized for reverse transcription under the ABI Prism7600 Fast Real-Time PCR system. The primers of human GCLC, Nrf2, HO1, NQO1, and GAPDH have been described previously [42]. Primers of murine GCLC, Nrf2, HO1, NQO1, and GAPDH have also been described [43].
AMPKα1or Nrf2 short hairpin RNA Lentiviral particles, encoding AMPKα1 short hairpin RNA (shRNA; sc-44281-V), Nrf2 shRNA (sc-44332-V) or control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology. SH-SY5Y cells were plated onto six-well plates at a density of 2 × 10 5 cells/mL in the polybrene-containing complete medium. Lentivirus particles were added to SH-SY5Y cells for 12h. Puromycin (3.0 μg/mL) was then added to select stable cells for a total of six passages (10-12 days). AMPKα1or Nrf2 silencing in stable cells was tested by western blotting.

CRISPR/Cas9-induced knockout of AMPKα1 or Nrf2
Small guide RNA against human AMPKα1 or human Nrf2 was selected from Optimized Crispr Design software from Dr. Zhang's laboratory (http:// crispr.mit.edu/), and individually inserted into the lentiCRISPR-green fluorescent protein (GFP) plasmid with a puromycin selection gene (Addgene, Watertown, MA). SH-SY5Y cells were plated onto six-well plates at a density of 2 × 10 5 cells/mL in polybrene-containing complete medium. The lentiCRISPR AMPKα1knockout (KO) plasmid or the lentiCRISPR Nrf2-KO plasmid was transfected to SH-SY5Y cells by Lipofectamine 2000 reagent. The transfected cells were then subjected to FACS-mediated GFP sorting and selected by puromycin (3.0 μg/mL) for six passages. The resulting stable cells were assayed by western blotting of AMPKα1 or Nrf2 expression. Control cells were transfected with the empty vector.

Statistical analysis
Data are expressed as the mean ± standard deviation. Statistically significant differences were tested by oneway analysis of variance using Tukey's post hoc multiple comparisons tests (SPSS version 21.0, SPSS Inc., Chicago, IL). Two group comparisons were performed using Student's t test (Excel 2007). P< 0.05 was considered statistically significant.

AUTHOR CONTRIBUTIONS
All listed authors designed the study, performed the experiments and the statistical analysis, and wrote the manuscript. All authors have read the manuscript and approved the final version.

CONFLICTS OF INTEREST
Authors have no conflicts of interest.