Antibodies against LC3, P62, Beclin-1, β-actin, AMPK, and p-AMPK (Thr172), were obtained from Cell Signaling Technology. Antibodies against Homer1a were obtained from Synaptic Systems. Rapamycin, STF-62247, compound C, 3-MA, and chloroquine were purchased from Sigma-Aldrich.

HT-22 cells (Institute of Biochemistry and Cell Biology, SIBS, CAS.) were grown in DMEM (Gibco, Frederick, MD, USA) plus 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The cells were cultured in a humidified atmosphere at 37°C under 5% CO2. Before the experiments, HT-22 cells were seeded in 6-well culture dishes (10⁶ per well) and incubated until they reached 70–80% confluency. Rapamycin (5 μM), STF-62247 (10 μM), 3-MA (2 mM), chloroquine (10 μM), and compound C (20 μM) were added to the cultures for 24 h before H₂O₂ treatment.

The preparation of lentivirus for the overexpression experiments was performed as previously described (Luo et al., 2014). The lentivirus overexpression system was developed by removing the EGFP open reading frame from the pGC-FU-EGFP-3FLAG construct (GeneChem Co., Shanghai, China) with an AgeI/NheI digestion and replacing this cassette with Homer1a cDNA.HT-22 cells were transfected with lentivirus vectors at a multiplicity of infection (MOI) of 30 for 48 h.

After each treatment, the cells were lysed in RIPA buffer with protease inhibitor cocktail (Roche Applied Bioscience, Indianapolis, IN, USA) and subjected to western blotting as described previously (Rao et al., 2016). Briefly, protein concentrations were assessed with a BCA Kit (Pierce, Rockford, IL, USA), and equivalent amounts of protein (30 μg) were separated by 8–15% SDS-PAGE gels, followed by transfer onto PVDF membranes. The membranes were then soaked in 5% skim milk in Tris-phosphate buffer containing 0.05% Tween 20 (TBST) for 1 h and further incubated overnight at 4°C with the appropriate primary antibody (Homer1a 1:1000; LC3II 1:1000; Beclin-1 1:1000; P62 1:8000; β-actin 1:5000; AMPK 1:1000; p-AMPK 1:1000). After three washes for 8 min in TBST, the blots were incubated with HRP-conjugated secondary antibodies for 1–2 h. The target protein signal was detected by SuperSignal West Pico Chemiluminescent Substrate (Thermos Scientific). The optical densities of the bands were quantified by ImageJ (Scion Corporation, Torrance, CA, USA).

The intracellular ROS was measured by 2,7-dichlorodihydrofluoresceindiacetate (H₂DCFDA) (Molecular Probe), as previously reported (Luo et al., 2012). HT-22 cells were incubated with H₂DCFDA (10 mM) for 1 h at 37°C in dark and then resuspended in phosphate-buffered saline (PBS). The fluorescence intensity was read with a fluorescence plate reader (excitation wavelength of 480 nm and an emission wavelength of 530 nm).

Malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE), two indexes of lipid peroxidation, were detected using a Lipid Peroxidation 4-HNE Assay Kit (Beyotime, Shanghai, China) and a Lipid Peroxidation MDA Assay Kit (Beyotime, Shanghai, China), according to the manufacturer's instructions. The absorbance was read at 450 nm using a ELISA reader.

MMP was monitored using the fluorescent dye rhodamine 123 (Rh 123). Rh 123 was added to cultures to achieve a final concentration of 10 mM for 30 min at 37°C after the HT-22 cells had been treated and washed with PBS. Fluorescence was acquired using a fluorescence plate reader (excitation wavelength of 480 nm, emission wavelength of 530 nm).

The intracellular ATP levels were measured using a firefly luciferase based ATP assay kit (Beyotime, China), strictly following the manufacturer's protocol. The ATP levels of each group were calculated as a percentage of the control.

HT-22 cells were fixed in 4% paraformaldehyde for 20 min, washed in PBS, and then permeabilized with 0.2% Triton X-100 for 10 min. Next, the cells were incubated with rabbit anti-LC3 antibody (1:200) at 4°C overnight, followed by incubation with Alexa 488 donkey-anti-rabbit IgG (1:400) for 2 h at room temperature. The nuclei were counter-stained with DAPI for 10 min (Sigma). All the images were acquired using a confocal microscope (FV10i, Olympus, Tokyo, Japan) with the same exposure time, light sensitivity and laser power.

The cell viability assay was performed using the Cell Counting Kit-8 (CCK-8) (#CK04; Dojindo, Japan), following the manufacturer's instructions. Normal cells or lentivirus-infected cells were seeded in 96-well plates with 5,000 cells per well. After treatment, 10 μl/well of CCK-8 solution was added into each well and incubated for 4 h. The absorbance was measured at 450 nm using a microplate reader. (Bio-Rad, Hercules, CA, USA).

The release of LDH was evaluated using the Cytotoxicity Detection Kitplus (Roche Applied Bioscience, Indianapolis, IN, USA), following the manufacturer's protocol. After subtracting the background values in the medium, the percentage cytotoxicity was calculated with the following equation: LDH release (% of Max) = 100 × (experimental value–low control)/(high control–low control). Experimental value, LDH values in the experimental groups; Low control, LDH values in the untreated normal cells; High control, the maximum releasable LDH values in the untreated normal cells.

Apoptosis in HT-22 cells was detected with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) In Situ Cell Death Detection Kit (#11684795910; Roche, Mannheim, Germany). The cells were fixed with freshly prepared 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.2% Triton X-100 for 5 min. The cells were then incubated with 50 μl TUNEL reagent mixture for 60 min at 37°C following the manufacturer's protocol. The images were captured with a fluorescence microscope, the TUNEL-positive cells were counted and the ratio of TUNEL-positive cells/total cells was calculated. DAPI (10 μg/ml) was used to stain nuclei.

The HT-22 cells were fixed in 2% paraformaldehyde-2% glutaraldehyde buffered with 0.1 mol/L phosphate buffer at room temperature. The cells were post-fixed with 1% osmium tetroxide, dehydrated in acetone and immersed in resin. After hardening, the samples were cut into 50 nm thick slices, stained with lead citrate and observed on an electron microscope.

All the experiments were performed a minimum of three times. The statistical analyses were conducted using the GraphPad Prism software, version 6.0 (GraphPad, San Diego, CA, USA). Significance between experiments was assessed by univariate analysis of variance (ANOVA; more than two groups), followed by Bonferroni's multiple comparisons or unpaired t-test (two groups).

HT-22 cells were exposed to an increasing concentration of H₂O₂ (200, 400, 600, 800, 1000 μM) for 24 h. The results showed that H₂O₂ decreased cell viability and induced LDH release in a dose-dependent manner (Figures 1A,B). An exposure to 600 μM H₂O₂ for 24 h was used in the following experiments given that exposure to the cell insult induced nearly 50% cell death. To investigate the effect of H₂O₂ on Homer1a expression, HT-22 cells were incubated in the presence of H₂O₂ (600 μM) for different periods of time (control, 8, 16, and 24 h). The results indicated that H₂O₂ significantly increased the levels of Homer1a within 24 h (Figure 1C).

To identify the effect of Homer1a on H₂O₂-induced oxidative stress, HT-22 cells were transfected with lentivirus carrying Homer1a (LV-Homer1a) or a negative control lentivirus (LV-NC). Immunoblot analysis showed that lentiviral transduction of LV-Homer1a increased the expression of Homer1a protein (Figure 1D). After treatment with H₂O₂ for 24 h, the viability of HT-22 cells transfected with LV-Homer1a was higher than the cells transfected with LV-NC (Figure 1E). Furthermore, the overexpression of Homer1a clearly decreased LDH release after H₂O₂ treatment (Figure 1F).

To test whether the Homer1a regulates autophagy following oxidative stress, we transfected HT-22 cells with LV-Homer1a and cultured the cells for 2 days before adding H₂O₂. Our results showed that the overexpression of Homer1a increased protein levels of LC3II and Beclin-1 and decreased the expression of p62 (Figures 2A–D). The immunofluorescent results indicated that the overexpression of Homer1a significantly increased the number of LC3-positive puncta after H₂O₂ treatment compared to the LV-NC group (Figures 2E,F). In addition, ultrastructural studies clearly revealed more autophagosomes in the LV-Homer1a group after H₂O₂ treatment compared to the LV-NC group (Figures 2G,H).

To determine the role of autophagy in H₂O₂-induced oxidative damage, we evaluated cell injury after HT-22 cells were treated with H₂O₂ and pharmacological agents that modulated autophagy (Figure 3A). The results indicated that rapamycin, a classical inducer of autophagy, reduced the number of TUNEL-positive cells and decreased LDH release after oxidative stress (Figures 3B–D). To identify whether Homer1a conferred protection through modulation of autophagy, HT-22 cells were transfected with LV-Homer1a and/or treated with the autophagy inhibitor 3-MA. The results showed that the increased expression of LC3II induced by Homer1a overexpression was decreased by 3-MA (Figure 3E). Moreover, treatment with 3-MA or chloroquine (CQ), another autophagy inhibitor, partially reversed the protective effects of Homer1a against H₂O₂-induced injury (Figures 3F–H).

To assess the relationship between autophagy, H₂O₂-induced oxidative stress and mitochondrial damage, HT-22 cells were pretreated with rapamycin or STF-62247, another autophagy activator before H₂O₂ treatment. The results showed that rapamycin and STF-62247 both significantly reduced H₂O₂-induced ROS production, lipid peroxidation (MDA and 4-HNE), loss of MMP and ATP production (Figures 4A–E). To further investigate the role of Homer1a-induced autophagy in regulating oxidative stress and mitochondrial function, HT-22 cells were transfected with LV-Homer1a or LV-NC and treated with 3-MA and H₂O₂. We observed that H₂O₂-induced ROS production and lipid peroxidation decreased in HT-22 cells transfected LV-Homer1a (Figures 4A–C). Moreover, the overexpression of Homer1a prevented the H₂O₂-induced loss of MMP and reduction of ATP production (Figures 4D,E). However, the Homer1a-mediated protection against H₂O₂-induced oxidative stress and mitochondrial damage were partially abolished by 3-MA (Figures 4A–E).

To further clarify the molecular mechanisms through which Homer1a mediates autophagy and protection, we tested whether Homer1a protects HT-22 cells through the AMPK pathway. The results indicated that the overexpression of Homer1a markedly increased the phosphorylation of AMPK after H₂O₂ treatment (Figure 5A). In addition, the increased levels of p-AMPK induced by Homer1a was partially prevented by the AMPK inhibitor compound C (Figure 5A). The western blot results showed that the increased expression of Beclin-1 and LC3II induced by Homer1a overexpression after oxidative stress was reversed by compound C treatment (Figures 5B–D). Moreover, the Homer1a-induced decreased LDH release, ROS production and increase in MMP levels upon H₂O₂ challenge were partially abolished by compound C, suggesting that AMPK was involved in Homer1a-induced protection (Figures 5E–G).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.