Antibodies and dyes used for western blotting and/or immunostaining were obtained from the following resources: Anti-β-actin (ab6276), anti-PP2A (ab32141), anti-CaMKII (ab52476), anti-GSK3β (Y216) (ab85305), Goat anti-Mouse/ Donkey anti-Rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6789)/(ab6802) were purchased from Abcam (Cambridge, UK); anti-total tau [Tau5, a monoclonal antibody recognizing both phosphorylated and non-phosphorylated tau (MAB361)] was purchased from Merck Millipore (Massachusetts, U.S.A.); anti-phosphorylated tau at Thr181 (pT181) (SAB11107) was purchased from Signalway Antibody (Maryland, U.S.A.); anti-phosphorylated tau at Ser396 (pS396) (44752G) was purchased from Invitrogen (California, U.S.A.); anti-GSK-3β (Ser9) (#9322) was purchased from Cell Signaling Technology (Beverly, MA, U.S.A.); DAPI was purchased from Roche (Switzerland); anti-tubulin antibody (ab28035) was purchased from Abcam (U.S.A). Cy5-labeled Donkey anti-Rabbit/Donkey anti-Mouse secondary antibodies (711-175-152)/(715-175-150) were purchased from Jackson ImmunoResearch (West Baltimore Pike, PA, U.S.A.).

Chemicals: Dulbecco's Modified Eagle's Medium (DMEM)/F12 was purchased from Gibco (U.S.A.). Fetal bovine serum (FBS) was purchased from Hyclone (U.S.A.). The cell viability assay kit CCK8 was purchased from Dojindo Laboratories (Japan). The Annexin-V/PI cell apoptosis assay kit was purchased from KeyGen Biotech (China). Dimethyl sulfoxide (DMSO) was purchased from Amresco (U.S.A.). Trypsin was purchased from Life Technologies (U.S.A.). RIPA cell lysis buffer, BCA protein assay kit and penicillin-streptomycin solution were purchased from Beyotime (China). PVDF transfer membrane and chemiluminescent HRP substrate were purchased from Millipore (U.S.A.). Protease inhibitor cocktail (G6521) for cell lysates was purchased from Promega (U.S.A.). Methanol-free formaldehyde (16% w/v) was purchased from Thermo Scientific (U.S.A.). Resveratrol standard was purchased from Sigma (St. Louis, MO, U.S.A.) and was first dissolved in 50% DMSO and then diluted to various concentrations with culture medium immediately before use. Other reagents, unless otherwise noted, were of analytical grade and purchased from local distributors.

The mouse neuroblastoma N2a cell line was purchased from Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM)/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin in 100-mm culture dishes. Cells were incubated in a humidified incubator at 37°C with 5% CO₂.

N2a cells were divided into the following groups: blank control group (cells without any treatment) and vehicle control group (0.025% DMSO pre-treatment + FA treatment); FA group (FA applied at an appropriate concentration according to each experimental scale); Res group (Res applied at an appropriate concentration according to each experimental scale); and Res + FA group (Res pre-, simultaneous-, and post-treatment were performed according to each experimental scale). Cell viability, morphological changes and apoptosis rates were used to evaluate cytotoxicity. Expression levels of tau protein and candidate enzymes were measured by western blotting and immunofluorescence assays.

A FA concentration of 0.35 mM was selected to evaluate the timing of Res administration (pre-, simultaneous-, or post-treatments) because it was the LD₅₀ of FA in N2a cells found in preliminary studies (Figure 1A). In the western blotting and apoptosis studies, the concentration of FA was increased to 0.5 mM because this FA concentration had been shown to cause marked hyperphosphorylation of Tau protein in N2a cells (Lu et al., 2011; Tong et al., 2011). This increase served to both push out molecular indicators of FA toxicity to evaluate the neuroprotective effect of Res and to evaluate beyond the physiologically defined concentration levels of FA in the brain (0.2–0.4 mM; Chen et al., 2009) in accordance with the upper levels of FA concentrations (~0.5 mM) reported in the hippocampus of AD brains (Tong et al., 2013).

Cell viability was determined using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's instruction. Briefly, N2a cells were seeded at 1.5 × 10⁴ cells/well in a 96-well plate (Corning, U.S.A.) and cultured for 24 h to allow cell adhesion. Subsequently, the medium was replaced with FBS-free DMEM/F12 and cells were respectively treated with FA (0.1–0.9 mM), Res (2.5–50 μM), DMSO (0.1–0.5%,) for 24 h. To investigate whether Res could attenuate FA-induced cytotoxicity, cells were also pre- and post-treated with Res (0.1–10 μM) for 30 min before and after 24 h FA (0.35 mM; LD₅₀; Figure 1A) exposure as well as treated simultaneously with 0.35 mM FA for 24 h. The optical absorbance at 450 nm was measured on a spectrum plate reader (Synergy H1, BioTek, U.S.A.) and cell viability was expressed as the percentage of viable cells relative to untreated control cells.

To observe cellular morphology changes, cells (1.5 × 10⁵ cells/well) were seeded in a 12-well plate and after a 24 h attachment period the cells were treated with Res (0.1–10 μM) for 30 min followed by a 4 h FA (0.5 mM) exposure. The cells were fixed with 4% paraformaldehyde (PFA) for 10 min and observed with a Fluorescent Cell Imager (ZOE, Bio-Rad, U.S.A.).

Cell apoptosis was detected with an Annexin-V/PI Staining Kit according to the manufacturer's instructions. Briefly, cells (1.5 × 10⁵) were seeded in a 12-well plate, after treatment with Res (0.1–10 μM, 30 min) and FA (0.5 mM, 4 h), cells were digested with 0.25% EDTA-free Typsin. The cells were then collected and washed with cold phosphate-buffered saline (PBS). After washing, the cells were re-suspended in 500 μl of binding buffer containing 5 μl Propidium Iodide (PI) and 5 μl Annexin V-FITC, and incubated for 15 min at room temperature in the dark. Cells were then quantitatively analyzed by Flow cytometer (Influx, BD, U.S.A.). Ten thousand cells were counted one time for each sample. The apoptosis rate was analyzed using the Flowjo 7.6.1 (FlowJo, LLC, U.S.A.) software. The following controls were used to set up compensation and quadrants: (1) unstained cells, (2) cells stained with FITC Annexin V (no PI), and (3) cells stained with PI (no FITC Annexin V). The unstained cells (cells without any treatment) were used to measure the forward scatter (FSC) and side scatter (SSC) signals, which were used to find viable cells and remove signals for debris and dead cells. Cellular debris was considered FSC-low and dead cells were considered to have low FSC and high SSC. A gate was set to exclude debris and dead cells from the analysis by drawing polygons that included the events with high FSC and low SSC (viable cells). Once the gate was set, viable cells with either FITC or PI single staining were measured to generate the FITC positive (vertical axis) or PI positive (horizontal axis) lines, respectively. The boundary of each line was referenced to the unstained viable cells. These two parameters were used to produce a bivariate histogram, which divided the plot into the four quadrants displaying the four possible combinations: double positive (Q2, Annexin V⁺/PI⁺, late apoptotic cells), single positive for each dye (Q1, Annexin V⁻/PI⁺, necrotic or mechanically damaged cells; Q3, Annexin V⁺/PI⁻, early apoptotic cells), and negative for both (Q4, Annexin V⁻/PI⁻, non-apoptotic cells). The four quadrant gates were then applied to the other samples to quantify the percentage of apoptotic cells. The cell apoptotic rate was calculated as Q2+Q3.

For western blotting assays, cells were seeded at 1.5 × 10⁵ cells/well in a 12-well plate and were pre- and post-treated with 10 μM Res for 30 min before and after 4 h FA (0.5 mM) treatment as well as simultaneously treated with 10 μM Res and 0.5 mM FA. Moreover, pre-treatments of 0.1–10 μM Res for 30 min before 4 h FA (0.5 mM) exposure were also performed. Western blotting procedures were performed as described in the literature (Lu et al., 2013) with small modifications. Briefly, whole-cell lysates were prepared by incubation of cells in RIPA buffer supplemented with protease inhibitor cocktail according to the manufacturer's instructions. The protein content was determined using a BCA protein assay kit. Then samples (20 μg of protein) were mixed with loading buffer, heated at 98°C for 10 min, separated on 12% SDS-PAGE gels, and transferred to PVDF membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies (β -actin, 1:5000; Tau5, 1:2500; pT181, 1:4000; GSK-3β , 1:1000; GSK-3β (Ser-9, phosphor-Serine 9), 1:1000; GSK-3β (Y216, phosphor-tyrosine 216), 1:250; CaMKII, 1:5000; PP2A, 1:1000) at room temperature for 1.5 h or at 4°C overnight. The membranes were then washed three times with TBST for 10 min each, followed by incubation with the corresponding anti-rabbit/anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) at room temperature for 1 h. The membranes were again washed three times with TBST (10 min each time) and visualized with chemiluminescent HRP substrate according to the manufacturer's instructions. Luminescence was captured in the ImageQuant LAS 4000 digital imaging system (GE Healthcare Bio-Sciences, Pisacataway, U.S.A.). The individual band intensities were quantified with the Image J software package (National Institute of Health, Besthada, U.S.A.) and normalized to the intensity of β-actin bands. This allowed individual bands to be compared to the total protein on each membrane. The quantification of GSK3β-Y216 band intensity was measured as the total of all bands, as per a previous study of GSK3β post-translational measurements (Beurel et al., 2015). The intensity of the lowest band alone was also quantified.

Immunofluorescence assays were performed to further confirm the presence of phosphorylated tau (pT181) and related enzymes according to a previously established protocol (Wei et al., 2016). In brief, cells were seeded at 1.5 × 10⁴ cells/well on sterile coverslips placed in 24-well culture plates. Cells were pre-treated with 10 μM Res for 30 min before a 0.35 mM FA treatment for 4 h. After treatment, cells were washed three times with PBS (5 min each time) and fixed with 4% PFA for 20 min at 4°C, then permeabilized with 0.4% Triton X-100 for 30 min at room temperature. The coverslips were then blocked with 5% bovine serum albumin (BSA) for 1 h, followed by an incubation in 1% blocking buffer with primary antibodies (tublin, 1:1000, pT181, 1:100; GSK-β, 1:1000; GSK-β (Y216), 5 μg/ml; GSK-3β (Ser9), 1:100; CaMKII, 1:250; PP2A, 1:250; Tau2, 1:500) at 4°C overnight. Cells were washed three times with PBS (5 min each time) to remove residual primary antibodies. Cy5-labeled Donkey anti-Rabbit/Donkey anti-Mouse secondary antibodies (1:200) were added and allowed to bind at 37°C for 1 h in the dark. The coverslips were then washed as mentioned earlier and were incubated with DAPI (1:1000) for 10 min in the dark. Following three 5-min washings with PBS, the slides were mounted with 90% glycerol (Solarbio, China), then sealed with nail polish. Slides were observed under a laser scanning confocal microscope (A1MP+, Nikon, Japan).

Cell viability and western blot data analysis were conducted using the SPSS software package (SPSS Inc., Chicago, IL, U.S.A.). The normality of the data was determined using the Kolmogorov-Smirnov test. All the data were normally distributed (Kolmogorov-Smirnov tests: all p > 0.05) and were analyzed by one-way ANOVA. The alpha level was set at p = 0.05 and all p-values were generated using two-sided tests. Tukey's tests were used for post-hoc comparisons. Results were calculated over at least three independent experiments and were presented as the mean ± SEM (standard error of the mean).

Slow progressive neuronal death is a major characteristic of AD (Shimohama, 2000). In this study, the protective effect of Res against FA-induced neuronal toxicity was studied in N2a cells. FA induced cell death in a dose-dependent manner with an LD₅₀ of ~0.35 mM (Figure 1A). Data from the CCK-8 assays showed that Res alone (2.5–50 μM) did not cause any loss of N2a cell viability after 24 h treatments (Figure 1B). Similar results were observed when N2a cells were treated with different concentrations of DMSO (0.1–0.5%), the vehicle used for Res (Figure 1C). To determine whether Res attenuates FA-induced cytotoxicity, cell viability assays were performed with pre- and post-treatments of Res (0.1–10 μM) for 30 min, as well as with simultaneous-treatments of Res, with FA for 24 h. Pre-treatment with Res dose-dependently attenuated FA-induced cytotoxicity (Figure 1D), with a significant effect observed at a concentration of 2.5 μM (p = 6.728 × 10⁻⁵). The viability of N2a cells was not influenced by post-treatments (Figure 1E, p = 0.497) or simultaneous-treatments (Figure 1F, p = 0.371) of Res.

In addition, an Annexin V-FITC/PI double staining assay was used to briefly evaluate the respective effect of FA and Res on N2a cell apoptosis. The experiments found that 0.5 mM FA increased the apoptotic rate to 32.7% in comparison to the control group (0.9%) (Figure 2). However, pre-treatment with Res (0.1, 1, 2.5, 5, and 10 μM) given prior to FA (0.5 mM) exposure decreased the percentage of apoptotic cells to 14.5, 14.4, 11.6, 10.2, and 6.9%, respectively (Figure 2). These results were consistent with the findings from the cell viability assays, and, together, showed that pre-treatment of Res protected N2a cells against FA-induced cytotoxicity.

Cellular morphology was also observed using a microscope at different magnifications. Compared with controls, FA-treated cells were smaller and atrophied with irregular cell borders and reduced cellular processes, while cells pre-treated with Res showed a higher cell density and a more natural cell morphology. Moreover, cell neurites were partially retained in comparison with cells treated with FA alone (Figure 3).

In consideration of the functions of the tau protein to promote the assemble of microtubules and maintenance of the microtubule system in neurons (Drechsel et al., 1992; Götz et al., 2006), as well as findings that abnormal phosphorylation of tau protein leads to microtubule disassembly, cytoskeleton destabilization and eventually cell death (Iqbal et al., 2005), it was hypothesized that the protective effect of Res against FA-induced neurotoxicity and morphological changes was likely related to the modulation of tau protein phosphorylation.

Previous studies indicated that FA triggered abnormal tau protein hyperphosphorylation in vitro and in vivo (Nie et al., 2007; Lu et al., 2013). In this study, tau protein hyperphosphorylation at Thr181 (p = 6.965 × 10⁻⁹) and Ser396 (p = 2.750 × 10⁻⁷) were significantly increased after treatment with 0.5 mM FA (Figure 4A). Pre-treatment with 10 μM Res significantly reduced FA-induced tau phosphorylation at Thr181 (p = 7.456 × 10⁻⁹), but not at the Ser396 site, which did not change significantly (p = 1.000). As in the cell viability and apoptosis studies above, post- (p = 0.930 for Thr181 and p = 0.991 for Ser396) or simultaneous-treatment (p = 0.994 for Thr181 and p = 1.000 for Ser396) of 10 μM Res did not influence tau phosphorylation levels at either the Thr181 or Ser396 loci (Figure 4A). It was further elucidated that pre-treatment with 0.1–10 μM Res reduced FA-induced tau hyperphosphorylation at Thr181 in a dose-dependent manner (p = 0.007 at 1 μM of Res; Figure 4B). The expression level of total tau protein did not show a significant change with (p = 0.979 at 10 μM of Res) or without (p = 0.321) Res treatment (Figure 4B), which indicated an overall net effect of Res that suppressed FA induced tau protein hyperphosphorylation. All told, these results suggested that Res significantly inhibited tau protein hyperphosphorylation at Thr181, but not at Ser396, in a dose-dependent manner.

Kinases and phosphatases, such as glycogen synthase kinase (GSK-3β), calmodullin-dependent protein kinase II (CaMKII) and phosphoseryl/ phosphothreonyl protein phosphatase-2A (PP2A), are important enzymes involved in the regulation of hyperphosphorylation of tau protein in the development of AD (Gong et al., 1993, 1995; Pei et al., 1999; Vogelsberg-Ragaglia et al., 2001; Wang et al., 2005). In the present study, the effects of Res on the regulation of GSK-3β, CaMKII, and PP2A activities were investigated. It was found that the level of phosphorylation on GSK-3β at Y216 (activation site) was significantly increased after treatment with 0.5 mM FA for 4 h (p = 1.974 × 10⁻⁵; Figure 5), while phosphorylation at Ser9 (inhibition site) did not exhibit a significant change (p = 1.000). This suggested an increased activity of total GSK-3β (p = 0.005). Conversely, pre-treatment with Res significantly decreased the level of phosphorylation on GSK-3β at Y216 (p = 1.722 × 10⁻⁴ at 2.5 μM of Res). This indicated that the phosphorylation of GSK-3β at the activation site was inhibited by Res treatment (Figure 5). However, phosphorylation of GSK-3β at the Ser9 site was not found to change significantly with Res pre-treatment prior to FA exposure (Figure 5, p = 0.997 at 10 μM of Res).

An analogous result was found with CaMKII. The protein level of CaMKII was significantly increased under FA stress (p = 0.002), while pre-treatment with Res significantly reduced the level of CaMKII in a dose-dependent manner (Figure 5, p = 0.005 at 1 μM of Res).

With respect to the phosphatase PP2A, a significantly decrease in the protein level of PP2A was found after FA exposure (p = 0.008), while pre-treatment with Res significantly increased the protein levels (Figure 5, p = 0.020 at 1 μM of Res). These results suggest that Res protects N2a cells from FA-induced tau protein hyperphosphorylation by decreasing the activities of GSK-3β and CaMKII as well as increasing the activity of PP2A. Interestingly, no significant changes on the regulation of GSK-3β, CaMKII and PP2A activities and/or expression were found after 30 min of Res treatment alone (data not shown).

Alterations to tau protein hyperphosphorylation, kinase, and phosphatase levels and protein localizations were further investigated by immunofluorescence staining. An increased staining signal of tau (pT181), GSK-3β, and CaMKII as well as a decreased staining pattern of PP2A were detected after FA treatment (Figure 6). These staining patterns were also ameliorated with pre-treatments of Res and were consistent with the findings of the Western blot analysis.

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. The reviewer RJR and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.