Ribosylation-Derived Advanced Glycation End Products Induce Tau Hyperphosphorylation Through Brain-Derived Neurotrophic Factor Reduction

Antibodies

The antibodies utilized in this study were directed against tau phosphorylated at the following epitopes: pThr181 (1:1,000 dilution, Signalway Antibody, USA); pSer214, pSer396 and AT8 (1:2,000, Invitrogen, USA); and Tau-1 (tau not phosphorylated at Ser199/202, 1:5,000, Millipore, USA). The total expression level of tau was detected using Tau-5 (1:2,000, Millipore, USA). The levels of kinase involved in tau phosphorylation were examined using antibodies against GSK-3β (1:2,000) and phospho-GSK-3β (Ser9, 1:2,000; Tyr216, 1:2,000) from Cell Signaling Technology (USA). Antibodies against BDNF (1:2,000, Abcam, USA), its receptor TrkB (1:1,000, Cell Signaling Technology, USA), AGEs (6D12, monoclonal antibody, Wako, Japan) and β-actin (1:5,000, Santa Cruz Biotechnology, USA) were also employed.

Preparation of ribosylated RSA/BSA

Rat serum albumin (RSA, Sigma, USA) and bovine serum albumin (BSA, AMRESCO, USA) were mixed with Rib or Glc prepared in 0.02 mM Tris-HCl (pH 8.0) to final concentrations of 10 mg/ml protein and 1 M sugar (named RRSA, GRSA, RBSA, and GBSA). Separate preparations of RSA and BSA without sugar were used as controls. The reaction mixtures were incubated at 37°C for 7 days. After incubation, the preparations were extensively dialyzed against Tris-HCl to remove free Rib/Glc before being used in cell cultures. RRSA and GRSA were not dialyzed before being administered to rats, and Rib and Glc alone were used as controls. All solutions were filtered with 0.22μm membranes (Millipore, USA). In the in vivo study, RRSA was used to reduce autoimmunity in rats. For the in vitro experiments involving N2a cells, because both RRSA and RBSA behaved the same in mouse-derived cells, inducing somewhat heterogeneous reactions, we chose BSA, which is much less expensive than RSA.

Ethics statement

The protocols for the handling of the rats and the experimental procedures were approved by the Animal Welfare and Research Ethics Committee of the Institute of Biophysics, Chinese Academy of Sciences (permit number: SYXK2013-77), and the methods were performed in accordance with the approved guidelines.

Animals and the administration of RRSA/GRSA

Male Sprague Dawley rats (8 weeks) were obtained from Vital River Laboratory Animal Technology Co. Ltd. (China). The rats were randomly divided into six groups and allowed to acclimatize to the cages for four days. Then, the rats received a daily tail intravenous injection of RRSA or GRSA for 30 days, and RSA, Rib, Glc, and saline were used as the controls. The administration dosage was 25 mg/kg·W. All rats were maintained under pathogen-free conditions.

Measurement of body weight and grip strength

Body weight and grip strength were recorded before and after tail intravenous injections as described previously [45, 46].

Open field test

Rats were subjected to an open field test in an apparatus that consisted of a 200×200 cm open arena with walls that were 40 cm high (Huaibeizhenghua Biological Instrument Equipment Co., LTD, China). The procedure was described previously [45].

Elevated plus maze

The elevated plus maze (EPM) was composed of four arms (50×10 cm, Huaibeizhenghua Biological Instrument Equipment Co., LTD, China) that were arranged in the shape of a ‘+’ sign, with two open arms across from each other that were perpendicular to two closed arms with a center platform (10×10 cm). The open arms had neither side nor end walls, whereas the closed arms had side and end walls (40 cm in height) but were open on the top. The procedure was described previously [45].

Morris water maze

The Morris water maze test was performed as described previously [45, 47]. Rats were placed on a visible platform before they were exposed to a hidden platform. All data were recorded with a computerized video system.

Sample collection from animals

After the behavioral tests, the rats were sacrificed, and the hippocampi and cortexes were quickly dissected for subsequent western blotting or fixed in 4% paraformaldehyde for immunohistochemistry.

Cell culture

Mouse neuroblastoma Neuro 2a (N2a) cells (obtained from Cell Resource Center, China) were cultured in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F12, Corning, USA) supplemented with 100 IU/ml penicillin and 100μg/ml streptomycin at 37°C in a humidified 5% CO₂ incubator. The medium contained 10% fetal bovine serum. Cells were grown to 70–80% confluence in 100 mm diameter dishes and provided with nutrients every three days. For all experiments, the culture medium was replaced with serum-free medium before the addition of RBSA/GBSA.

CCK-8 assay

N2a cells were seeded on a 96-well plate at a concentration of 10⁴ cells per well and exposed to RBSA/GBSA (0, 0.1, 0.3, 0.5, 0.8, 1.0, 1.5, and 2 mg/ml) for 24 h, except for the control. The CCK-8 (Beyotime, China) reagent was added after 24 h. The plates were incubated at 37°C for 1 h, and the absorbance was read at 450 nm.

Lactate dehydrogenase cytotoxicity detection

Lactate dehydrogenase (LDH) cytotoxicity assays were performed according to the manufacturer’s protocol (Beyotime, China). This colorimetric assay quantifies the activity of LDH released from the cytosol of damaged cells into the supernatant and thus serves to quantify cell death.

Flow cytometric analysis

Cells undergoing apoptosis were detected by double staining with Annexin V-FITC/PI in the dark according to the manufacturer’s protocol (Beyotime, China). Cells were stained with Annexin V-FITC and PI and immediately analyzed by flow cytometry (BD FACS Calibur, USA).

Cell lysate protein extract preparation

After treatment, N2a cells were harvested in ice-cold WIP (Beijing Cellchip Biotechnology Co., Ltd., China) containing phosphatase and protease inhibitor (1 mM PMSF). The samples were then centrifuged at 20,000× g at 4°C for 15 min, and the supernatants were collected and analyzed for protein content using a bicinchoninate acid (BCA) assay kit (ThermoFisher Scientific, USA). The samples were stored at –80°C until further immunoblotting analysis.

Western blotting

Protein samples from brain tissues and cell lysates were mixed with 5× SDS-PAGE Sample Buffer (Genestar, China) and boiled for 10 min. Cell or brain lysate extracts containing ∼30μg of protein were separated on 12% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, USA). The nonspecific binding sites of the membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween-20 (PBST, pH 7.4) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with antibodies. The following day, the membranes were washed three times and then incubated for 1 h at room temperature with a horseradish peroxidase-linked secondary anti-mouse or anti-rabbit antibody (1:4000 dilution; ZSGB-BIO, China). The membranes were then washed three times with PBST. The immunoreactive bands were reacted with enhanced chemiluminescence detection reagents (Applygen, China), visualized after exposure of the membranes to Kodak X-ray film and quantified by Quantity One 1D analysis software 4.5.2 (Bio-Rad, USA).

Immunohistochemical analysis

Rat brains were processed for immunohistochemistry using standard protocols, as previously reported [45]. The primary antibodies used were rabbit anti-pS396 and rabbit anti-AT8 (both 1:500). The secondary antibody used was biotin-labeled goat anti-rabbit antibody. The proteins were detected using horseradish peroxidase-labeled antibodies. The staining was visualized with an AEC system (ZSGB-BIO, China), and light microscopy (Nikon Optical, Japan) was used to observe the staining.

Determination of reactive oxygen species in rat brain

Reactive oxygen species (ROS) concentrations in rat brains were measured with a rat ROS ELISA kit (Shanghaijining Co. Ltd., China). Lysates of hippocampus and cortex were used and experimental procedure was performed according to the manufacturer’s protocol.

Stereotactic BDNF injection

The hippocampi of Sprague Dawley rats (10 in each group) were stereotactically injected (–3.30; ±2.0; –4.0) with BDNF-expressing viruses (packaged by AAV9, 5μl, Vigene Biosciences, China). The titer of the purified BDNF-containing viral vector was >1×10¹³ vg/ml. Two weeks later, the rats were intravenously injected through the tail with RBSA or saline for 30 days.

Data analysis

All data were analyzed using SPSS software; one-way ANOVA or two-way ANOVA with a post hoc test were used. Differences with a probability level of 95% (p < 0.05) were considered significant.

Ribosylated products impair the cognitive ability of Sprague Dawley rats

To clarify whether ribosylated proteins affect cognitive functions in vivo, we incubated Rib with rat serum albumin (RSA) for 7 days to obtain ribosylated RSA (RRSA) [2]. Next, we treated Sprague Dawley rats (n = 10) with intravenous tail injections of RRSA (25 mg/kg·W, once daily) for 30 days. Glc and its modified RSA (GRSA) were employed for the purposes of comparison. No differences in weight or muscle strength were found among the groups (Supplementary Figure 1, p > 0.05). When the rats were tested in the EPM (Supplementary Figure 2) and the open field test (Supplementary Figure 3), we did not find that RRSA administration led to anxiety-related behavior in rats, as indicated by the absence of differences among the groups in the time spent in the open arm and open arm entries in the EPM or in the time spent in the center of the open field arena.

In this study, the Morris water maze was employed to assess spatial learning and memory. Tensile test has shown no difference in animal muscle strength among rats of these groups, which excluded the interference of motor abilities on the behavioral tests. During the training session, all rats improved their performance as indicated by a shortened escape latency over successive days (Fig. 1a). There were no significant differences in the performance of the rats among the groups after a 5-day training period; however, on day 3, the escape latency of rats injected with RRSA was higher than that of control rats (p = 0.0228), and the escape latencies of rats injected with RSA (p = 0.0103) and rats injected with Rib (p = 0.0092) were shorter than those of control rats on day 2 and day 4. No significant differences were found between the Glc- and GRSA-injected groups and the control in the 5-day training period. Together, these data showed that glycated products (regardless of whether they were ribosylated or glucosylated) did not impair spatial learning in Sprague Dawley rats under our experimental conditions.

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Fig.1

Impaired spatial memory in rats intravenously injected with ribosylated proteins demonstrated by the Morris water maze test. Rats were injected with RRSA (25 mg/kg·W, once daily) for 30 days. GRSA, RSA, Rib, Glc, and saline were used as controls. The length of time needed to find the hidden platform was recorded as the escape latency on each of the five training days (a). The percentages of the search time and the distance traveled spent in the quadrant from which the platform was removed during the probe trial are shown (b), as is the number of platform crossings in the probe trial (c). Rib, D-ribose; Glc, D-glucose; RSA, rat serum albumin; RRSA, ribosylated RSA; GRSA, glucosylated RSA. All values are expressed as the mean±SD. ^*p < 0.05, ^**p < 0.01 versu control.

After withdrawing the platform (probe trial), the time and distance the RRSA-injected rats spent swimming in the target quadrant were significantly shorter than those of the control group (p = 0.0247 and p = 0.0302, respectively) (Fig. 1b). No significant differences were observed between any other treatment group and the control group. The number of platform crossings was also lower in the RRSA-treated group (p = 0.0463) than in the other groups (Fig. 1c). This finding indicates that it is memory, but not learning ability, that is impaired in the rats treated with ribosylated protein products.

Ribosylated products induce tau hyperphosphorylation in the rat brain

It has been shown that the presence of hyperphosphorylated tau is closely related to cognitive impairment. Therefore, we determined the level of tau phosphorylation in the brain by western blotting and immunohistochemistry.

The total tau level (detected with Tau-5) in the rat brain (cortex and hippocampus) did not change after RRSA treatment (Fig. 2a, c). The levels of nonphosphorylated tau in the hippocampi assayed with the monoclonal antibody Tau-1 (recognizing Ser199/202) were also not significantly different among the groups, with the exception of a lower level observed in the RRSA-treated group than in the non-RRSA-treated group (p = 0.0377) (Fig. 2a, b). The levels of phosphorylated Ser396 and AT8 (phosphorylated at epitope Ser199/202) of tau were increased in the hippocampi of RRSA-injected rats compared with the control group (p = 0.0181 and p = 0.0095, respectively), although rats in the RSA, Rib, and Glc groups also showed significantly higher AT8 levels in their hippocampi compared with the control. Among all groups, there were no significant differences in the levels of nonphosphorylated or phosphorylated tau in the cortex, with the exception of an increased level of AT8 in the RRSA group (Fig. 2c, d).

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Fig.2

Ribosylated products induced tau hyperphosphorylation in the rat brain. The conditions for the treatment were the same as in Fig. 1. The phosphorylation levels of tau in the hippocampus and cortex were detected by western blotting using the anti-pSer396, anti-AT8, anti-Tau-1, and anti-Tau-5 antibodies a, c). β-Actin was used as a loading control. The quantification is shown in b and d. The value of the saline control was designated as 1.0. The levels of phosphorylated tau are expressed as the ratio of phospho-site to total tau. All values are expressed as the mean±SEM. ^*p < 0.05, ^**p < 0.01 versus control unless indicated otherwise in the Figure.

We further performed immunohistochemical staining of brain sections. An increase in the immunoreactivity of tau phosphorylated at Ser396 and Ser199/202 was observed in the hippocampi of rats treated with RRSA (Fig. 3). The levels of immunoreactivity were less pronounced in the other groups. No differences in immunoreactivity in the cortex were observed among all the groups (data not shown). Combining the data, this indicated that ribosylated products are able to induce tau hyperphosphorylation in the brains of rats, especially in the hippocampus.

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Fig.3

Immunohistochemical staining of phosphorylated tau in the hippocampus. The conditions for the treatment were the same as in Fig. 1. In the hippocampus, the levels of tau phosphorylated at Ser396 and Ser199/202 were detected by immunohistochemistry using the anti-pSer396 or anti-AT8 antibodies (a). The quantification of the relative intensity is shown (b). All values are expressed as the mean±SEM. ^**p < 0.01 versus control.

High cytotoxicity and tau hyperphosphorylation induced by ribosylated proteins in cells

To investigate the effects of ribosylated products on tau phosphorylation in cells, we used the mouse neuroblastoma neuro-2a cell line (N2a) for in vitro experiments. To this end, N2a cells were incubated with RBSA for 24 h. First, cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay (Fig. 4a). Only RBSA was found to induce decreased cell viability. Activity assays measuring the release of LDH also showed that RBSA was highly cytotoxic to N2a cells (Fig. 4b). Furthermore, a flow cytometric analysis using Annexin V-FITC/propidium iodide (PI) staining also showed a high level of cytotoxicity (Fig. 4c and Supplementary Figure 5). RBSA treatments resulted in a clear increase in both the upper right and lower right (indicating early and late apoptosis, respectively) populations after 24 h. These data demonstrate that ribosylated protein products exert a highly cytotoxicity effect on N2a cells.

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Fig.4

Cell viability was measured using the CCK-8 assay, the LDH cytotoxicity assay and the detection of apoptosis in N2a cells cultured with RBSA. RBSA (0, 0.1, 0.3, 0.5, 0.8, 1.0, 1.5, and 2 mg/ml) was added to cells on a 96-well plate. The CCK-8 assay was performed 24 h later (a). The LDH assay was also used to measure the cytotoxicity of RBSA (0.5 and 1.0 mg/ml) after incubation for 24 h. GBSA and BSA were used as controls. Cells incubated without proteins were used as a negative control (b). Apoptosis was measured by flow cytometry using Annexin V-FITC/PI staining, and the statistical results from Supplementary Figure 4 are shown here (c). All values are expressed as the mean±SEM. ^**p < 0.01, ^***p < 0.001 versus control unless indicated otherwise in the Figure. LR, lower right, UR, upper right.

To determine whether ribosylated products also trigger tau hyperphosphorylation in N2a cells, we determined the phosphorylation of tau at Thr181, Ser214, Ser396, and Ser199/202 (AT8) epitopes in N2a cells. Incubation with RBSA (1 mg/ml) resulted in a significant increase in tau phosphorylation at Thr181, Ser214, Ser396, and Ser199/202, along with a decrease in tau nonphosphorylation (Tau-1) (Fig. 5a, b). Incubation with RBSA at a lower concentration (0.5 mg/ml) also induced tau hyperphosphorylation at Thr181, Ser396, and Ser199/202, but not at Ser214. GBSA, in contrast, did not exert the same effect, indicating that ribosylated but not glucosylated products induced tau hyperphosphorylation in N2a cells under our experimental conditions.

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Fig.5

RBSA activated GSK-3β, promoted tau hyperphosphorylation, and reduced BDNF and TrkB levels in N2a cells. Cells were incubated with different concentrations of RBSA (0.5 and 1.0 mg/ml) for 24 h on 6-well plates. GBSA and BSA were employed as controls. The levels of phosphorylated tau, tau kinase, BDNF, and TrkB in cells were determined by western blotting (a). β-Actin was used as a loading control. The quantification is shown in b, c, and d. The value of the saline control value was designated as 1.0. All values are expressed as the mean±SEM. ^*p < 0.05, ^***p < 0.001 versus control.

Ribosylated products induce tau hyperphosphorylation through BDNF-mediated activation of GSK-3β

To clarify the mechanism by which ribosylated products induce tau hyperphosphorylation, we examined changes in the activation of the specific tau kinase GSK-3β, which is involved in the regulation of major phospho-epitopes of tau phosphorylation [48]. In the rat hippocampus (but not the cortex) and N2a cells, treatment with ribosylated products (either RRSA or RBSA) resulted in a significant increase in the active form (pY216) of GSK-3β but not the inhibitory form (pS9) compared with the levels in the other groups (Figs. 5a, c and 6a, b, d, e), indicating that GSK-3β might play a role in tau hyperphosphorylation. Since RSA and BSA have highly conserved protein sequences, it is reasonable to expect that ribosylated RSA and BSA would have similar effects on tau phosphorylation and GSK-3β activation.

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Fig.6

RRSA activates GSK-3β and reduces BDNF and TrkB in the rat brain. Conditions for the treatment were the same as in Fig. 2, except that the levels of tau kinase, BDNF, and TrkB in the hippocampus and cortex were detected by western blotting (a, d). β-Actin was used as a loading control. The quantification is shown in b, c, e, and f. The value of the saline control value was designated as 1.0. The levels of phosphorylated GSK-3β are expressed as the ratio of phospho-site to total GSK-3β. All values are expressed as the mean±SEM. ^*p < 0.05 versus control unless specific indication in the Figure.

We next inhibited GSK-3β using a specific inhibitor, CHIR99021 [49] (Fig. 7). This markedly rescued tau phosphorylation (Thr181, Ser214, Ser396, AT8, and Tau-1) in a concentration-dependent manner, whereas there were no significant changes in the total level of tau (Tau-5). These data confirm that GSK-3β is involved in tau hyperphosphorylation in the presence of ribosylated products.

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Fig.7

GSK-3β catalyzed the hyperphosphorylation of tau in the presence of RBSA. The GSK-3β-specific inhibitor CHIR99021 (1μM and 2μM) was added to the cells in the presence of 1.0 mg/ml RBSA and incubated for 24 h on 6-well plates. The levels of phosphorylated tau were determined by western blotting (a). β-Actin was used as a loading control. The quantification is shown in b. The value of the saline control value was designated as 1.0. All values are expressed as the mean±SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus control.

It is known that the BDNF-TrkB pathway regulates GSK-3β activity, as described previously [32]. Therefore, we assessed the levels of BDNF and TrkB in both the rat brain and N2a cells (Figs. 5, 6). In the hippocampus of the rat brain, there were significant decreases in BDNF and TrkB levels in the RRSA-treated group compared with the other groups, whereas no differences were detected in the cortex among all groups (Fig. 6a, c, d, f). An analysis of the N2a cell experiments yielded the same result (Fig. 5a, d). The BDNF and TrkB levels in N2a cells were significantly decreased under RBSA treatment in a concentration-dependent manner. This suggests that the BDNF-TrkB pathway is inactivated by ribosylated products and could therefore be involved in tau hyperphosphorylation.

To determine whether the BDNF-TrkB pathway is involved in the hyperphosphorylation of tau induced by ribosylation-derived AGEs, we added extracellular BDNF to the cultured cells to determine whether there was a rescue effect on tau phosphorylation. The addition of BDNF 2 h before RBSA treatment significantly suppressed tau phosphorylation at Thr181, Ser214, Ser396, and Ser199/202 compared with RBSA treatment alone (Fig. 8). The effect was concentration-dependent. Additionally, BDNF preincubation decreased the active form of GSK-3β, indicating that BDNF acts upstream of GSK-3β. Furthermore, the rescue effect of BDNF treatment was also observed in an in vivo study. When RBSA-treated rats were given BDNF by stereotactic injection of the AAV9 virus containing the BDNF sequence, their spatial memory ability increased significantly compared with that of RBSA-treated rats in the Morris water maze test. They spent more time swimming in the target quadrant than control or RBSA-treated rats (Supplementary Figure 5). These data together demonstrated that ribosylated products induced tau hyperphosphorylation through BDNF-mediated GSK-3β activation. BDNF treatment can rescue the memory impairment caused by ribosylated products.

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Fig.8

The effect of preincubation with BDNF on the level of tau phosphorylation in N2a cells. Cells were cultured with BDNF (0.5 ng/ml and 2.5 ng/ml) for 2 h before RBSA (1.0 mg/ml) was added and then incubated for 24 h on 6-well plates. The levels of GSK-3β, phosphorylated tau, BDNF, and TrkB were determined by western blotting (a). β-Actin was used as a loading control. The quantification is shown in b, c, and d. The value of the saline control was designated as 1.0. All values are expressed as the mean±SEM. ^*p < 0.05, ^**p < 0.01 versus control.