Corticosterone induces neurotoxicity in PC12 cells via disrupting autophagy flux mediated by AMPK/mTOR signaling

Abstract Aims Our previous study indicated that chronic stress caused autophagy impairment and subsequent neuron apoptosis in hippocampus. However, the mechanism underlying the stress‐induced damage to neurons is unclear. In present work, we investigated whether stress‐level glucocorticoids (GCs) GCs promoted PC12 cell damage via AMPK/mTOR signaling‐mediated autophagy. Methods Chronic stress‐induced PC12 cell injury model was built by treatment with high level corticosterone (CORT). Cell injury was evaluated by flow cytometry assay and transmission electron microscopy observation. Results Autophagy flux was measured based on the changes in LC3‐II and P62 protein expressions, and the color alteration of mCherry‐GFP‐LC3‐II transfection. Our results showed that CORT not only increased cell injury and apoptosis, but also dysregulated AMPK/mTOR signaling‐mediated autophagy flux, as indicated by the upregulated expression of LC3‐II and P62 proteins, and the lowered ration of autolysosomes to autophagosomes. Mechanistically, our results demonstrated that autophagy activation by AMPK activator metformin or mTOR inhibitor rapamycin obviously promotes cell survival and autophagy flux, improved mitochondrial ultrastructure, and reduced expression of Cyt‐C and caspase‐3 in CORT‐induced PC12 cells. Conclusion These results indicate that high CORT triggers PC12 cell damage through disrupting AMPK/mTOR‐mediated autophagy flux. Targeting this signaling may be a promising approach to protect against high CORT and chronic stress‐induced neuronal impairment.


| INTRODUC TI ON
Accumulated evidences have confirmed that elevated glucocorticoids (GCs), resulting from chronic stress and prolonged or excessive use of GCs, can induce neurotoxicity and cognitive dysfunction. [1][2][3][4] However, the underlying mechanisms for GCs-triggered these damaging effects have not been fully elucidated. To clarify the detrimental influence of high concentration of GCs on neuronal cells, increasing attention has been given to hippocampal neuron pathology. 5,6 It has been shown that stress-level of corticosterone (CORT), a major glucocorticoid, results in pathological damage to neurons in hippocampus. 7 Although our previous study indicated that chronic unpredictable mild stress (CUMS) significantly increased CORT level and neuron cell lost in the hippocampus CA1 region and contributed to cognition impairment of rats, the underlying mechanism by which stress-induced high GCs level exerts neurotoxicity on hippocampal neurons is still largely unknown. 8 Autophagy is an essential pathway for cell survival via degrading the dysfunctional cellular components and the damaged organelles. Autophagy flux, a dynamic process of autophagy, is featured by formatting autophagosomes (APs), fusing APs with lysosomes to form autolysosomes (ALs), and degrading the cargoes sequestered in ALs. 9,10 Thus, disrupted autophagy flux can result in aggregation of the damaged organelles, and thereby contributing to cell injury and death. Impaired autophagy flux is closely correlated with pathogenesis of neurodegenerative diseases. 11,12 In recent years, several studies have shown that abnormal autophagy is responsible for GCs-induced spinal cord and SH-SY5Y cell injury. 13,14 Our previous study found that CUMS promotes neuron apoptosis of hippocampal CA1 region via suppressing autophagy, but the relationship between stress-induced high GCs level and autophagy flux dysfunction in neuron cells has not been identified. 8 Therefore, further elucidating the mechanisms for these phenomena is beneficial to preventing neurotoxicity induced by high concentration of GCs.
AMP-activated protein kinase (AMPK), a upstream signaling molecule of rapamycin complex 1 (mTORC1), plays a critical role in regulating various cellular processes such as energy metabolism and autophagy. [15][16][17] The activation of AMPK depends on phosphorylation of its threonine 172. 15 Its activation facilitates autophagy through inhibiting mTORC1 activity. Several studies have indicated that excess glucocorticoids exposure significantly altered AMPK activity in a tissue-dependent manner. [18][19][20] Furthermore, inactivation of AMPK has been revealed to be associated with CORT-induced neurotoxicity. 21

| Cell culture
PC12 cells were cultured in 25 cm 2 flasks containing DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Then, cells were incubated in a humidified atmosphere containing 5% CO 2 at 37°C. For each experiment, PC12 cells in logarithmic phase were used.

| Cell viability assay
Cell viability of PC12 cells was performed by MTT method. The cells were seeded in 96-well plates at a density of 1 × 10 4 cells per well containing 10% FBS medium. After 24 hours incubation, the medium was replaced with FBS-free DMEM. Then, the cells were incubated with CORT at various concentrations 0.1, 1, and 10 μmol/L. After treatment with indicated agents for 24 hours, the cells were incubated with MTT solution (0.5 mg/mL, 30 μL per well) for additional 4 hours in the dark. Then, 100 μL DMSO was added to each well and the absorbance value was measured by spectrophotometer at 570 nm.

| Annexin V-FITC/PI double staining flow cytometry
Cell apoptosis was evaluated by flow cytometry with annexin V-FITC/PI double staining. 22 Briefly, PC12 cells were seeded in 6-well plates at density of 1 × 10 6 cells/well. After incubated with indicated agents for 24 hours, the cells were digested with trypsin-EDTA and

| Western blotting
Western blot analysis was carried out as previously described. 22 Briefly, after treating with indicated agents for 24 hours, PC12 cells were collected and washed three times with cold PBS. All the cells were lysed in ice-cold RIPA buffer (Cell Signaling Technology) with protease inhibitor/protein phosphatase inhibitors, and total protein was extracted by centrifugation. Protein contents were assessed by a bicinchoninic acid protein assay kit. Equal amounts of the dena-

| Transmission electron microscopy
The ultrastructural observation including mitochondrial morphometry and APs/ALs were performed by transmission electron microscopy (TEM) as previously described. 22,23 In brief, PC12 cells were treated with indicated agents for 24 hours. After removing culture medium and washing with PBS for three times, the cells were fixed with 2.5% glutaraldehyde for 1 hour at 4°C. After postfixation in 1% OsO4 for another 1h, the cells were dehydrated and embedded in Araldite. Ultra-thin sectioning (40-60 nm) was performed and the sections were double-stained with uranyl acetate and lead citrate.
The sections were then observed under a JEOL 1230 TEM.

| Statistical analysis
All the values are presented as the mean ± SEM and analysed by GraphPad Prism 7.0 (GraphPad Software, Inc). The statistical significance was determined by one-way analysis of variance (ANOVA) followed by a post hoc Dunnett's test to compare the means of individual groups. The difference was considered significant when P < .05.

| High concentration of CORT reduces the activity of PC12 cells
To confirm the cytotoxic effect of CORT on neuronal cells, PC12 cells were treated with CORT at a concentration of 0.1, 1, and 10 μmol/L for 24 hours, respectively. Then, cell survival rate was measured by MTT assay. As shown in Figure 1, there was no significant difference in cell viability between 0.1 μmol/L group and the control group, indicating that the physiological level of CORT had no significant damage to the cells. As expected, with the increase of CORT concentrations, the PC12 cell viability decreased dramatically. These results manifested that the stress-like levels of CORT significantly declined the activity of PC12 cells in a dose-dependent manner with the maximal influence at 10 μmol/L concentration.

| High concentration of CORT increases apoptosis rates of PC12 cells
To test the effects of CORT on PC12 cell apoptotic death, cell apoptosis rates were detected by flow cytometry. As shown in Figure 2A,B, there was no significant difference in the apoptosis rate between control group and 0.1 μmol/L CORT group. However, F I G U R E 1 Effects of CORT on cell viability of PC12 cells. PC12 cells were incubated with CORT at different concentrations (0.1, 1, and 10 μmol/L) for 24 h. Then, PC12 Cell viability was measured by MTT assay kit. All the data were presented as mean ± SEM of three dependent experiments. *P < .05, **P < .01 vs control group the apoptotic rates of PC12 cells were significantly elevated both in 1 μmol/L CORT group and 10 μmol/L CORT group as compared with those in the control group. The results indicate that physiological concentration CORT has no significant influence on apoptosis of PC12 cells, while high concentration of CORT profoundly promotes PC12 cell apoptosis in a dose-dependent manner.

| High levels of CORT dysregulates AMPK/ mTOR pathway and impairs autophagy flux in PC12 cells
Since AMPK/mTOR signaling plays a critical role in neuronal cell death and survival, 15,24 we hypothesized that this signaling may be implicated in neurotoxicity to PC12 cells induced by high concentrations of CORT. The expressions of phosphorylated AMPK (p-AMPK) and mTOR (p-mTOR) in PC12 cells treated with different doses of CORT were measured by Western blotting assay. As shown in Figure 3A Considering AMPK phosphorylation takes an important role in regulation of autophagy via negatively regulates mTOR activation, we then asked whether high levels of CORT influenced autophagy flux in PC12 cells. In present study, two classic autophagy-associated markers, LC3-II and p62 in PC12 cells, were determined by Western blotting analysis. As depicted in Figure 3A,D,E, CORT exposure caused an increase both in expressions of LC3-II and P62 with a dose-dependent manner. Given that P62 protein is degraded with LC3-II-formed APs and is inversely proportional to autophagy flux, the elevated LC3-II protein levels in PC12 cells exposed to CORT were resulted from autophagy flux blocking. Taken together, these results imply that stress-like levels CORT impair PC12 cell autophagy flux and cell viability, with a maximal impairment at 10 μmol/L. Therefore, 10 μmol/L CORT was selected for the subsequent experiments.

| High level of CORT disrupts autophagy flux via dysregulating AMPK/mTOR pathway in PC12 cells
To identify whether AMPK/mTOR pathway is responsible for the As expected, although yellow puncta were not augmented both F I G U R E 2 Effect of CORT on cell apoptosis of PC12 cells. PC12 cells were incubated with indicated concentrations of CORT (0.1, 1, and 10 μmol/L) in 6-well plates for 24 h. Then, cell apoptotic rates of PC12 cells were evaluated by flow cytometry with annexin V/PI double staining as described in Section 2. A, Representative images of cell apoptotic rates analysed by flow cytometry with annexin V/PI double staining. B, The percentage of PC12 cell apoptosis was quantified as described in Section 2. Data were presented as mean ± SEM of three dependent experiments. **P < .01, ***P < .001 vs control group in CORT + Met group and CORT + RAP group, red dots were notably increased as compared with those in the CORT group.
These results indicate that activation of AMPK or inhibition of mTOR can rescue the impaired autophagy flux induced by CORT in PC12 cells.

| CORT promotes the ultrastructure damage via impairing AMPK/mTOR-mediated autophagy in PC12 cells
To identity whether CORT exerted its neurotoxicity through AMPK/mTOR-mediated autophagy flux defect, PC12 cells were treated with CORT together with Met or RAP for 24 hours, and then, the morphological ultrastructural alterations were observed under the TEM. As shown in Figure 5A

| Restoring AMPK/mTOR pathway-mediated autophagy protects PC12 cells against cell apoptosis induced high CORT
To further clarify inhibition of autophagy by CORT contributing to neurotoxicity on PC12 cells, we investigated whether activation of autophagy by Met or RAP could reverse cell apoptosis in PC12 cells exposed to CORT. In present study, cell apoptotic rate of each group was analysed by flow cytometry. As shown in Figure 6A,B, cell apoptotic rates of CORT group were significantly increased as compared with those of the control group.
Interestingly, the cell apoptotic rates both in Met + CORT group and RAP + CROT group were significantly declined as compared with those in CORT group, indicating that activation of autophagy by co-treatment with Met or RAP profoundly protected PC12 cells against CORT-induced cell apoptosis. Collectively, these results clearly showed that activation of autophagy mediated by AMPK/mTOR signaling obviously abrogated CORT neurotoxicity on PC12 cells.

| Restoring AMPK/mTOR pathway-mediated autophagy reduces the release of Cyt-c and caspase-3 induced by CORT in PC12 cells
Considered that Cyt-c released from the damaged mitochondria exerts a critical role in cell apoptosis via activating caspase-3, 25 the expressions of Cyt-c and cleaved caspase-3 in PC12 cells were determined by Western blot assay. As shown in Figure 7A-C, the levels of Cyt-c and cleaved caspase-3 proteins in CORT group were elevated dramatically as compared with those of the control group.
As expected, the expressions of Cyt-c and cleaved caspase-3 were

| D ISCUSS I ON
In the present study, we investigated the influences of stress-level GCs on PC12 cell injuries and the underlying mechanism. We found that high level CORT exposure decreased cell viability and increased It has been known that AMPK/mTOR signaling mediates cell survival and plays a critical role in regulating autophagy. 15,17,28 AMPK, a metabolic sensor, which favors neuronal survival through guarding mitochondrial homeostasis and energy balance. Several studies have implied that high CORT promotes neuronal cell death via inhibiting the activation of AMPK. 21,29 In present study, our results revealed a key role of this signaling in CORT-induced PC12 cell damage. 10 µmol/L CORT significantly lowered AMPK phosphorylation and enhanced mTOR phosphorylation, and increased cell apoptosis of PC12 cells. Met is a major therapeutic agent for treating patients with type 2 diabetes. The ability of Met to treat this common metabolic disorder requires activation of AMPK signals, but its exact molecular mechanism is still a mystery. 30 In this work, Met treatment not only reversed the influences of CORT on AMPK activation, but also attenuated PC12 cell death and mitochondrial structure damage resulting from CORT. Same neuroprotective effects were observed in PC12 cells treatment with mTORC1 inhibitor RAP. These results demonstrate that high CORT-induced neurotoxicity to PC12 cell is via dysregulating AMPK/mTOR signaling pathway.
Autophagy is essential for cell survival via removing the damaged/senescence organelles and degenerating abnormal folded proteins. 31,32 It has been confirmed that activation of AMPK inhibits mTOR and induces autophagy in neurons. 33 Therefore, we further evaluated the alterations of autophagic flux in PC12 cells for determining whether AMPK/mTOR-mediated autophagy impairment accounted for CORT-induced neurotoxicity. Changes in expression of LC3-II and p62 protein are often used to measure autophagic flux.
The results of Western blotting analysis indicated that CORT markedly increased LC3-II and p62 expression. Since the intracellular protein levels of p62 are negatively correlated with autophagy flux, 34,35 the results implied that autophagy flux was impaired in PC12 cells incubated with CORT. Moreover, dual fluorescence mRFP-GFP-LC3-II F I G U R E 7 Effects of treatment with Met or RAP on proapoptotic protein Cyt-c and cleaved caspase-3 expression in PC12 cells exposed to high CORT. PC12 cells were administrated with CORT (10 μmol/L) in the presence of Met (2 mmol/L) or RAP (200 nmol/L) for 24 h. All the cells were collected, and then, proteins were extracted from cell lysates for Western blot analysis. Each lane was loaded with 20 μg of proteins for all experiments. A, Representative blots of GAPDH (36 kDa), Cyt-c (14 kDa), and cleaved caspase-3 (17 kDa) in PC12 cells. B, C, Quantitative analyses of the relative optical density values of Cyt-c and cleaved caspase-3 to GAPDH were performed by Sigma Scan Pro5 software. All the data were expressed as mean ± SEM. *P < .05 vs control group, # P < .05 vs CORT group plasmids transfection and TEM assay results further confirmed that CORT severely disrupted autophagy flux in PC12 cells. Of note, both Met and RAP treatment abolished the autophagic flux impairment induced by CORT on PC12 cells. Our findings are consistent with GY Yang's study that autophagy activation by glycyrrhizic acid alleviates CORT-induced neurotoxicity in SH-SY5Y cells. 14 These results indicate that CORT impaired autophagy flux of PC12 cells through dysregulating AMPK/mTOR pathway.
Mitochondria not only regulates cell metabolism and energy balance, but also control cell death. 36,37 Damaged mitochondrial results in proapoptotic protein Bax translocating from the cytoplasm to its outer membrane, causing mitochondrial membrane potential decline. Subsequently, Cyt-C is released from mitochondria into the cytosol. 38 The released Cyt-C activates caspase-3 in an Apaf-1 and caspase 9-dependent manner. 39 In present work, our TEM results indicated that CORT-treated PC12 cells exhibited obvious mitochondrial ultrastructure damage, including mitochondria swelling and the mitochondrial cristae fragmented. Consistent with TEM results, Western blot analysis dada revealed that CORT exposure significantly enhanced Cyt-C release and caspase-3 activation, implying that CROT-induced PC12 cell apoptosis via mitochondrial apoptotic pathway. Interesting, the above proapoptotic effects of CORT on PC12 cells were canceled by Met and RAP treatment, respectively.
In conclusion, CORT-induced neurotoxicity on PC12 cells is a possible mechanism for hippocampal neuron injuries under chronic stress. This neurotoxic effects of CORT are via disrupting AMPK/ mTOR-mediated autophagy flux, which results in aggregation of the damaged mitochondrial and activation of mitochondria-mediated apoptotic pathway. Regulation of AMPK/mTOR signaling to restore neuronal cell autophagy flux may represent a potential novel therapeutic approach for CORT and chronic stress-induced neurodegenerative diseases.

ACK N OWLED G M ENTS
This work was supported by grants from the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.