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Chronic hyperglycemia induces tau hyperphosphorylation by downregulating OGT-involved O-GlcNAcylation in vivo and in vitro
Brain Research Bulletin 156 (2020) 76–85
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Chronic hyperglycemia induces tau hyperphosphorylation by downregulating OGT-involved O-GlcNAcylation in vivo and in vitro
T
Rong Huanga,b, Sai Tiana, Haoqiang Zhanga, Wenwen Zhua, Shaohua Wanga,* a b
Department of Endocrinology, Affiliated Zhongda Hospital of Southeast University, No. 87 DingJiaQiao Road, Nanjing, 210009, PR China Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, No. 68 Changle Road, Nanjing, 210006, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Type 2 diabetes mellitus Cognitive dysfunction O-GlcNAcylation Tau protein Phosphorylation
Objective: Diabetes mellitus (DM) can increase the risk of cognitive dysfunction, but its exact mechanisms remain unclear. The involvement of aberrant O-GlcNAcylation has been identified in hyperglycemia and DM, as well as the pathogenesis of Alzheimer’s disease via competition with tau phosphorylation. This study was designed to investigate the role of O-GlcNAcylation in diabetes-associated cognitive dysfunction (DACD). Methods: Fifteen-week old male KK-Ay mice were used as DACD models, and advanced glycation end product (AGE)-treated HT22 cells were used as a model of high glucose toxicity. Morris water maze tests, histological staining, real-time quantitative PCR, and Western blot were also applied. Results: Mice with DACD exhibited evident obesity, hyperinsulinemia, hyperglycemia, and impaired learning and memory function. O-GlcNAcylation levels decreased and tau phosphorylation levels at Ser396, Ser404, Thr212, and Thr231 increased in the hippocampus of mice with DACD, as well as in AGE-treated HT22 cells. Hypoglycemic therapy improved these anomalies and elevated O-GlcNAc transferase (OGT) levels in mice with DACD. OGT plasmid transfection in HT22 cells partially reversed AGE-induced decreases in O-GlcNAcylation levels and increased tau phosphorylation levels. Conclusions: Chronic hyperglycemia can induce tau hyperphosphorylation by downregulating OGT-involved OGlcNAcylation in vivo and in vitro, which mediates DACD.
1. Introduction The number of people worldwide diagnosed with diabetes in 2017 was estimated 451 million, and this number is expected to increase to 693 million by 2045 (Cho et al., 2018). The prevalences of diabetes and pre-diabetes in China are as high as 10.9% and 35.7%, respectively (Wang et al., 2017). Type 2 diabetes mellitus (T2DM) accounts for 90% of all reported diabetes cases and is characterized by insulin resistance (IR) and relative insulin deficiency, thereby resulting in chronic hyperglycemia (Gallwitz et al., 2013; Zheng et al., 2018). Accumulating evidence associates T2DM with increased risk of cognitive impairment and even dementia (Cheng et al., 2012; Li and Huang, 2016; Zhang et al., 2019). Several mechanisms have been demonstrated to contribute to diabetes-associated cognitive dysfunction (DACD), including abnormal glucose metabolism, impaired insulin signaling, blood brain barrier injury, β-amyloid (Aβ) accumulation, and tau hyperphosphorylation (Li et al., 2015; Gonzalez-Reyes et al., 2016; Bogush et al., 2017; Wu et al., 2017; Tumminia et al., 2018). However, the exact mechanisms of the disease remain unknown, and effective treatments
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are lacking. Chronic hyperglycemia, one of the main clinical manifestations of T2DM, is a major cause of diabetic chronic complications. Several metabolic pathways, including polyol pathway activation, advanced glycation end product (AGE) formation, oxidative stress injury and protein kinase C activation, may contribute to the development of DACD as the downstream consequence of hyperglycemia (Yagihashi, 2016). Abnormal activation of intracellular glucose metabolism pathways, such as the hexosamine biosynthesis pathway (HBP), may also mediate the toxic effects of hyperglycemia. Upon the entry of glucose into cells, approximately 2%–5% of this molecule enters the nutrient sensing HBP and is converted into uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), which is also the substrate for O-Linked β-N-acetylglucosamine (O-GlcNAc) modification (Wells et al., 2003). O-GlcNAcylation is a posttranslational modification that dynamically modifies serine (Ser) and threonine (Thr) through their hydroxyl moieties on nuclear and cytoplasmic proteins (Yang and Qian, 2017). O-GlcNAcylation is catalyzed by two key enzymes, namely, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which respectively add/
Corresponding author. E-mail address: [email protected] (S. Wang).
https://doi.org/10.1016/j.brainresbull.2020.01.006 Received 28 June 2019; Received in revised form 7 December 2019; Accepted 3 January 2020 Available online 10 January 2020 0361-9230/ © 2020 Elsevier Inc. All rights reserved.
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divided into three groups (n = 10), namely, the diabetes group (KK-Ay + Veh), the diabetes + 1 mg/(kg·d) glimepiride group (KK-Ay + Gmp1), and the diabetes + 2 mg/(kg·d) glimepiride group (KK-Ay + Gmp2); 10 age-matched C57BL/6 mice were used as controls (C57BL/ 6+Veh) (Takada et al., 1996; Tsukuda et al., 2007; Ishola et al., 2018). All mice in the intervention group were provided an equal volume of liquid gavage for 12 weeks. Before sacrifice, the body weight of the mice were measured by using an electronic scale, and fasting blood glucose (FBG) levels were measured by using a glucometer (ONETOUCH UltraVue, Johnson Medical Equipment Co., Ltd.). Fasting venous blood was obtained from the angular vein, and fasting insulin (FIN) levels were measured by a mouse insulin ELISA kit (Wuhan Liuhe Biotechnology Co., Ltd.).
remove UDP-GlcNAc to/from Ser and Thr residues of proteins (Joiner et al., 2019). As UDP-GlcNAc is derived from HBP, aberrant O-GlcNAcylation has been identified to be involved in hyperglycemia, IR, and T2DM (Park et al., 2010; Springhorn et al., 2012; Myslicki et al., 2014). O-GlcNAcylation levels of global and tau proteins in the postmortem brain tissue from frontal cortices of T2DM subjects are decreased (Liu et al., 2009). In the peripheral blood, OGA expression is significantly increased in erythrocytes of individuals with pre-diabetes and diabetes compared with normal control subjects (Park et al., 2010). Springhorn et al. also found that leukocyte, particularly granulocyte, O-GlcNAcylation may serve as a novel diagnostic tool for the early detection of T2DM (Springhorn et al., 2012). In vitro, SKOV-3 cells show diminished OGT mRNA levels 24, 48, and 72 h after culture in high glucose (HG) compared with cells in the control group (Rogalska et al., 2018). OGlcNAcylation in HK2 cells is elevated after 24 and 48 h of HG treatment (HG24 and HG48, respectively). In HG48 cells, full-length nucleocytoplasmic OGT (ncOGT) and mitochondrial OGT (mOGT) proteins are decreased compared with those in HG24 cells, and the mOGT protein is even lower in HG48 than in control cells. In addition, the long isoform OGA (OGA-L) protein is significantly higher in HG48 than in HG24 and even higher than that in the normal control group (Gellai et al., 2016). O-GlcNAcylation may compete with tau phosphorylation at Ser and Thr sites, thereby playing a role in the pathogenesis of Alzheimer’s disease (AD) (Zhu et al., 2014; Wani et al., 2017). Previous studies showed an imbalance of tau O-GlcNAcylation with phosphorylation in the hippocampus of a mouse model of AD (Gatta et al., 2016). Moreover, upon the inhibition of OGT, tau phosphorylation levels at Ser199 and Ser396 increase, thereby resulting in increased tau aggregation (Lim et al., 2015). By contrast, increasing O-GlcNAcylation by applying an OGA inhibitor can prevent Aβ plaque formation and tauopathy, thereby attenuating cognitive decline (Yuzwa et al., 2012; Kim et al., 2013; Graham et al., 2014; Yuzwa et al., 2014; Hastings et al., 2017). We speculate that chronic hyperglycemia may lead to an imbalance between O-GlcNAcylation and tau phosphorylation levels via regulation of the key enzymes OGT or OGA, including the effects of tau phosphorylation of certain sites, ultimately mediating DACD. In this study, we used 15-week-old KK-Ay mouse as a DACD model, because the KKAy mouse, a spontaneous animal model of T2DM, exhibits not only obesity, hyperglycemia, and hyperinsulinemia but also severe cognitive decline at 15 weeks of age (Iwatsuka et al., 1970; Tsukuda et al., 2007; Min et al., 2012). The present study aims to clarify the potential mechanisms of chronic hyperglycemia on DACD in spontaneous type 2 diabetic KK-Ay mice and AGE-treated mouse hippocampal neuron (HT22) cells.
2.3. Cell culture, viability assay and treatments HT22 cells were purchased from iCell Bioscience Inc. (Shanghai, China) and cultured in HG Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C with 5% CO2 in a humidified atmosphere. The culture medium was replaced every 48 h, and the cells were passaged at a ratio of 1:3. Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay (Biosharp, China). HT22 cells were seeded into 96-well plates at a density of 5000 cells per well. After overnight incubation, the culture medium was replaced with fresh DMEM containing different concentrations of AGE (0, 100, 200, 400, 800, and 1600 μg/mL) for different time (24, 48, and 72 h). Subsequently, each culture well was added with CCK-8 reagent and incubated at 37 °C for 2 h. The absorbance of each well was detected at 450 nm using an automatic microplate reader (BioTek Instruments, Inc.). 2.4. Plasmid transfection pcDNA3.1(+)-OGT plasmid was purchased from Shanghai Generay Biotechnology Co., Ltd. HT22 cells were seeded into six-well plates at a density of 1 × 106 per well. pcDNA3.1(+)-OGT plasmid was transfected in the presence of 400 μg/mL AGE using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, USA), and the pcDNA3.1(+)-empty plasmid was used as control. After transfecting for 4 h, the medium was replaced with fresh DMEM containing 400 μg/mL AGE. 2.5. Morris water maze test Cognitive function was evaluated by the Morris water maze (MWM) test with 1 week time after the observational and interventional studies. All mice were allowed to swim freely for 1 min without the platform to adapt the environment on the first day of the test; those who floated or swam poorly were excluded. For the navigation test, each mouse was trained four times per day at 20 min intervals for following five consecutive days. The time each mouse took to find the platform and the total length of the path were recorded as escape latency and path length, respectively. If the mouse failed to find the platform within 60 s, it was guided to the platform by the technician for 10 s. The platform was removed, and mice were allowed to swim freely for 60 s in the probe trial. The percentage of time spent in the target quadrant and the frequency of crossing the platform area were recorded as a measure of spatial memory.
2. Materials and methods 2.1. Ethics statement This study was approved by the Animal Studies Committee of Southeast University. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institute of Health. 2.2. Animals and drug treatments Male KK-Ay and C57BL/6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China) and housed in a specific pathogen-free animal experiment center at Southeast University. The mice were kept in a room at 18−25 °C with 40%–60% humidity on a 12 h light/dark cycle and fed standard food and water ad libitum. Two sets of experiments were performed. In the observational experiment, 10 15-week-old KK-Ay mice were used as DACD models, and 10 age-matched C57BL/6 mice were used as controls. In the interventional experiment, 30 15-week old KK-Ay mice were randomly
2.6. Brain tissue preparation Following the behavioral test, all mice were anesthetized with 10% chloral hydrate (0.1 mL/10 g) and then perfused transcardially with 1 × phosphate buffered saline (PBS). The mice were sacrificed, and their brains were removed. Half of the samples were fixed with 4% 77
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Fig. 1. Mice with DACD exhibit worse spatial learning and memory performances. (A) Escape latency to search the platform during the navigation test. (B) Path length to search the platform during the navigation test. (C) Percentage of time spent in the target quadrant during the probe trial. (D) Frequency of crossing platform area during the probe trial. Data are represented as mean ± SEM; n = 10 per group. ** p < 0.01 versus C57BL/6 mice.
2.8. Western blot
Table 1 Body weight, FBG and FIN levels of C57BL/6 and KK-Ay mice. Group
Body weight (g)
FBG (mmol/L)
FIN (mIU/L)
C57BL/6 KK-Ay
26.86 ± 1.81 43.18 ± 3.97***
5.89 ± 0.78 13.49 ± 1.90***
78.81 ± 19.80 106.57 ± 29.81*
Hippocampal tissues and HT22 cells were homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology, China) supplemented with protease inhibitor phenylmethanesulfonyl fluoride and phosphatase inhibitor cocktail. Protein concentrations were determined by using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s protocol. Next, 20 μg of protein was loaded onto 8–10% SDS polyacrylamide gels and separated by electrophoresis at 80 V for 30 min, and then at 100 V for 1 h. The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h. The membranes were incubated overnight at 4 °C with the appropriate primary antibodies, including anti-RL2 (1:1000; Abcam, ab2739), anti-OGT (1:5000; Abcam, ab177941), anti-MGEA5 (1:5000; Abcam, ab124807), Tau-5 (1:800; Abcam, ab80579), anti-pTau (Ser396) (1:10,000; Abcam, ab109390), anti-pTau (Ser404) (1:1000; Abcam, ab92676), anti-pTau (Thr212) (1:1000; Abcam, ab4842), and anti-pTau (Tr231) (1:5000; Abcam, ab151559) with anti-GAPDH (1:3000; CMCTAG, AT0002) served as the control. Membranes were washed thrice with TBST and then incubated at room temperature for 1 h with the corresponding secondary antibodies, including goat antimouse IgG horseradish peroxidase (HRP) (1:50,000; MyBioScience, MKA001A) and goat anti-rabbit IgG HRP (1:25,000; MyBioScience, MKA003A). Membranes were washed another thrice with TBST, and signals were detected by chemiluminescence (Millipore, WBKLS0500) using a MiniChemi™610 imaging and analysis system (Beijing Sage Creation Science Co., Ltd.).
Data are represented as mean ± SD; n = 10 per group. * p < 0.05 and ***p < 0.001 versus C57BL/6 mice.
paraformaldehyde (PFA) for histological staining, and the rest were quickly dissected to isolate the hippocampus, which was stored in liquid nitrogen for Western blot.
2.7. Histological staining Brains fixed in 4% PFA were embedded in paraffin and 4−6 μm tissue sections were obtained for histological staining. First, sections were de-paraffinized with xylene, rehydrated through a series of decreasing percentages of ethanol, and then stained with hematoxylin and eosin (HE) or toluidine blue to observe cell morphologies or Nissl bodies. For immunofluorescence staining, antigen retrieval was performed by microwaving sections thrice in 1 mM ethylenediaminetetraacetic acid (pH 8.0) for 5 min. Next, the sections were washed thrice for 5 min with PBS and then incubated with 5% BSA at 37 °C for 30 min. The sections were treated with primary antibodies, including anti-RL2 (1:100; Abcam, ab2739), anti-neuronal nuclei (NeuN) (1:200; Servicebio, GB11138), anti-pTau (Ser396) (1:100; Santa Cruz Biotechnology, sc-101815), anti-pTau (Ser404) (1:100; Abcam, ab92676), anti-pTau (Thr212) (1:200; Invitrogen, 44−740 G) and antipTau (Thr231) (1:250; Millipore, AB9668), and incubated overnight at 4 °C. After three washes with PBS, the sections were incubated with the appropriate secondary antibodies, including FITC-conjugated goat antirabbit IgG (1:400; Servicebio, GB25303) and Cy3-conjugated goat antimouse IgG (1:300; Servicebio, GB21301), at 37 °C for 60 min. DAPI was used to stain cell nuclei at room temperature for 10 min. Immunofluorescence was observed with fluorescent microscope (OLYMPUS, Japan).
2.9. Real-time quantitative RT-PCR Total RNA was extracted from treated HT22 cells using RNA extraction solution (Wuhan Servicebio Biotechnology Co., Ltd.) according to the manufacturer’s protocol. cDNA was synthesized by using a HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotechnology Co., Ltd.). Real-time quantitative RT-PCR was performed with the ChamQ™ SYBR qPCR Master Mix (High ROX Premixed) kit (Vazyme Biotechnology Co., Ltd.). The PCR primers for OGT are as follows: 5′-GCCAAAGCACGCTGTTAG-3′ (forward) and 5′-GCAATGGA 78
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Fig. 2. Mice with DACD model exhibit hippocampal structure abnormalities. Representative photos showing histopathological changes in the hippocampal regions using HE staining (A) and Nissl staining (B) (scale bars: 500 μm for the first column, and 50 μm for the rest columns). (C) Quantification of Nissl bodies in different regions of the hippocampus. Data are represented as mean ± SEM; n = 3 per group. * p < 0.05 versus C57BL/6 mice.
MWM test) or one-way ANOVA (more than two groups) followed by the LSD post hoc multiple-comparison test. All statistical analyses were performed by using SPSS 22.0 (SPSS Inc., Chicago, IL, USA), and p < 0.05 was considered statistically significant.
CTGGCGACTA-3′ (reverse). The PCR primers for GAPDH are as follows: 5′-AAGAAGGTGGTGAAGCAGG-3′ (forward) and 5′-GAAGGTGGAAGA GTGGGAGT-3′ (reverse). Relative OGT gene expression level was calculated by using the comparative 2−△△CT method, and GAPDH was used as the control.
3. Results 2.10. Statistical analysis 3.1. Mice with DACD exhibit abnormal hippocampal structure and imbalanced O-GlcNAcylation, and tau phosphorylation levels
All data are presented as mean ± standard deviation (SD) or standard error of measurement (SEM). Statistical differences were determined by using Student’s t-test (two-group comparison), two-way analysis of variance (ANOVA) (escape latency and path length in the
The spatial learning abilities of mice were evaluated after 5 consecutive days of the navigation test. Compared with age-matched 79
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Fig. 3. Mice with DACD exhibit imbalanced O-GlcNAcylation and tau phosphorylation levels. (A) Representative photos of immunofluorescence double staining with anti-RL2 and anti-NeuN antibodies (scale bars: 50 μm), and quantification of neurons and O-GlcNAc-positive neurons in the hippocampal CA3 region. (B) Representative photos of immunofluorescence double staining with anti-RL2 and anti-Tau antibodies (scale bars: 50 μm), and quantification of O-GlcNAcylation and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 sites in the hippocampal CA3 region. (C) Representative Western blot and quantitation of global OGlcNAcylation, OGT and OGA expression. (D) Representative Western blot and quantitation of total tau and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 sites. Data are represented as mean ± SEM; n = 3 per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus C57BL/6 mice.
number of Nissl bodies in the hippocampus, especially in the CA3 region (p < 0.05), is reduced in the DACD model (Fig. 2). Immunofluorescence double staining was conducted to clarify the localization of O-GlcNAcylation with anti-RL2 and anti-NeuN antibodies. The results showed that O-GlcNAcylation mainly occurs in hippocampal neuronal cells, and the number of neuronal cells modified by OGlcNAcylation (O-GlcNAc-positive neurons) decreases in the hippocampal CA3 region of KK-Ay mice (p < 0.05) (Fig. 3A). Moreover, OGlcNAcylation levels decreased (p < 0.01), while tau phosphorylation levels increased (p < 0.05, p < 0.01 or p < 0.001) in the hippocampus of DACD models; these results were determined by using immunofluorescence double staining, and confirmed by Western blot (Fig. 3B–D).
Table 2 Body weight, FBG and FIN levels of the interventional mice. Group
Body weight (g)
FBG (mmol/L)
FIN (mIU/L)
C57BL/6 + Veh KK-Ay + Veh KK-Ay + Gmp1 KK-Ay + Gmp2
33.88 ± 3.10 48.63 ± 3.26*** 46.88 ± 2.67 46.14 ± 3.22
5.93 ± 0.75 14.55 ± 4.04*** 10.28 ± 2.32## 7.98 ± 1.34###,&
82.76 ± 16.41 109.46 ± 31.16* 100.31 ± 20.41 97.37 ± 18.05
Data are represented as mean ± SD; n = 8–10 per group. * p < 0.05 and ***p < 0.001 versus the C57BL/6+Veh group. ## p < 0.01 and ###p < 0.001 versus the KK-Ay + Veh group. & p < 0.05 versus the KK-Ay + Gmp1 group.
C57BL/6 mice, KK-Ay mice showed increased escape latency and path length to find the platform from the 2nd to the 5th days of the test (p < 0.05) (Fig. 1A and B). Twenty-four hours after the last training test, probe trials were performed to evaluate the memory function of the trained mice. KK-Ay mice showed lower percentage of time spent in the target quadrant (50.68% ± 13.33% versus 28.86% ± 14.51%, p < 0.05) and frequency of crossing the platform area (4.10 ± 1.20 versus 2.40 ± 0.97, p < 0.05) compared with C57BL/6 mice (Fig. 1C and D), thus confirming that 15-week-old KK-Ay mice could be used as DACD models. The average body weight, FBG, and FIN levels of KK-Ay mice were significantly higher than those of C57BL/6 mice (p < 0.05 or p < 0.001) (Table 1). HE staining showed that several cells are loosely arranged or structurally destroyed. Nissl staining showed that the
3.2. Hypoglycemic therapy ameliorates spatial learning and memory dysfunction in KK-Ay mice After 12 weeks of treatment with glimepiride, FBG levels decreased in a dose-independent manner in the KK-Ay + Gmp group compared with the KK-Ay + Veh group (p < 0.05, p < 0.01 or p < 0.001) (Table 2). No significant differences in body weight and FIN level were observed between the KK-Ay + Gmp and KK-Ay + Veh groups (p > 0.05). The MWM test also showed that the KK-Ay + Gmp group exhibits better performance than the KK-Ay + Veh group from the 2nd to the 5th days (p < 0.05) (Fig. 4A and B). Moreover, mice treated with glimepiride showed more percentage of time spent in the target 80
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Fig. 4. Glimepiride ameliorates spatial learning and memory dysfunction in KK-Ay mice. (A) Escape latency to search the platform during the navigation test. (B) Path length to search the platform during the navigation test. (C) Percentage of time spent in the target quadrant during the probe trial. (D) Frequency of crossing platform area during the probe trial. Data are represented as mean ± SEM; n = 8–10 per group. *** p < 0.001 versus the C57BL/6 + Veh group; # p < 0.05 and ## p < 0.01 versus the KK-Ay + Veh group.
Fig. 5. Glimepiride ameliorates hippocampal structure abnormalities in KK-Ay mice. Representative photos showing histopathological changes in the CA3 region of the hippocampus using HE staining (A) and Nissl staining (B) (scale bars: 50 μm). (C) Quantification of Nissl bodies in the CA3 region of the hippocampus. Data are represented as mean ± SEM; n = 3 per group. *** p < 0.001 versus the C57BL/6 + Veh group; # p < 0.05 versus the KK-Ay + Veh group.
81
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Fig. 6. Glimepiride increases O-GlcNAcylation and decreases tau phosphorylation levels in the hippocampus of KK-Ay mice. (A) Representative photos of immunofluorescence double staining with anti-RL2 and anti-NeuN antibodies (scale bars: 50 μm), and quantification of neurons and O-GlcNAc-positive neurons in the hippocampal CA3 region. (B) Representative photos of immunofluorescence double staining with anti-RL2 and anti-Tau antibodies (scale bars: 50 μm), and quantification of O-GlcNAcylation and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 sites in the hippocampal CA3 region. (C) Representative Western blot and quantitation of global O-GlcNAcylation, OGT and OGA expression. (D) Representative Western blot and quantitation of total tau and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 sites. Data are represented as mean ± SEM; n = 3 per group. ** p < 0.01 and *** p < 0.001 versus the C57BL/6 + Veh group; # p < 0.05 and ## p < 0.01 versus the KK-Ay + Veh group; & p < 0.05 versus the KK-Ay + Gmp1 group.
glimepiride treatment significantly increased the number of Nissl bodies in the hippocampal CA3 region of mice with DACD (p < 0.05) (Fig. 5B and C). 3.4. Hypoglycemic therapy increases O-GlcNAcylation but decreases tau phosphorylation levels in KK-Ay mice Immunofluorescence double staining showed that the number of OGlcNAc-positive neurons is partially increased in the hippocampal CA3 region of DACD mice after glimepiride intervention (p < 0.05) (Fig. 6A). In addition, immunofluorescence double staining and Western blot demonstrated that O-GlcNAcylation level is increased (p, tau protein phosphorylation levels at Ser396 and Thr212 are decreased, and OGT levels are elevated after glimepiride treatment (Fig. 6B-D).
Fig. 7. Effects of AGE concentration and culture time on HT22 cells viability. The differences are compared between the control group and the treatment group with different concentrations of AGE (0, 100, 200, 400, 800, and 1600 μg/mL) after 12, 24 and 48 h. Cell viability was determinate by CCK8 assay. Data are represented as mean ± SEM; n = 3 per group. ∗ p < 0.05, ## p < 0.01, ### p < 0.001.
3.5. O-GlcNAcylation level is decreased whereas tau phosphorylation levels are increased in AGE-cultured HT22 cells According to the CCK8 results, an AGE concentration of 400 μg/mL after 48 h may be considered a chronic hyperglycemia model (AGE group); here, 400 μg/mL BSA was considered the control treatment (Con group) in this study (Fig. 7). Compared with the Con group, OGlcNAcylation levels decreased (p < 0.05) whereas tau phosphorylation levels at sites Ser396, Ser404, Thr212, and Thr231 increased (p < 0.05, p < 0.01 or p < 0.001) in the AGE group (Fig. 8A–D). OGT mRNA and protein levels were significantly decreased in AGE-treated HT22 cells (p < 0.05) (Fig. 8E–F).
quadrant (34.66% ± 7.85%, 41.18% ± 10.19% versus 25.39% ± 6.31%, p < 0.05) and frequency of crossing the platform area (2.78 ± 0.67, 3.00 ± 0.71 versus 2.00 ± 0.54, p < 0.05) compared with mice treated with the vehicle (Fig. 4C and D). These results indicate that hypoglycemic treatment could ameliorate spatial learning and memory dysfunction in KK-Ay mice.
3.3. Hypoglycemic therapy improves hippocampal structure abnormalities in KK-Ay mice
3.6. OGT is involved in the upregulation of O-GlcNAcylation and downregulation of tau phosphorylation in AGE-cultured HT22 cells
HE staining showed partial improvement of loosely arranged cells or the destroyed structure in the CA3 region of the hippocampus after treatment with glimepiride (Fig. 5A). In the Nissl staining assay,
We performed OGT plasmid transfection to demonstrate the involvement of OGT in the regulation of O-GlcNAcylation and tau 82
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Fig. 8. O-GlcNAcylation and tau phosphorylation levels in AGE-cultured HT22 cells. Representative Western blot (A) and quantitation (B) of global O-GlcNAcylation expression in HT22 cells treated with or without 400 μg/mL AGE. Representative Western blot (C) and quantitation (D) of total tau and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 expression in HT22 cells treated with or without 400 μg/mL AGE. (E) Real-time quantitative PCR of OGT expression in HT22 cells treated with or without 400 μg/mL AGE. (F) Representative Western blot (F) and quantitation (G) of OGT expression in HT22 cells treated with or without 400 μg/mL AGE. Data are represented as mean ± SEM; n = 3 per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the Con group.
et al., 2015; Kandimalla et al., 2018). Previous studies demonstrated that tau protein is hyperphosphorylated in the hippocampus of T2DM animals, particularly at Ser199, Ser202 and Ser396 (Ma et al., 2013; Wu et al., 2013; Xu et al., 2017; Zheng et al., 2017). Guo et al. found that significant increases in the levels of phosphorylated tau at Thr231, Thr205, Ser396, and Ser404 are evident in hyperglycemia-exposed Pdx1+/−/APP/PS1 mice compared with APP/PS1 mice (Guo et al., 2016). In the present study, we found that several AD-associated tau protein sites, including Ser396, Ser404, Thr212 and Thr231, are hyperphosphorylated in the hippocampus of mice with T2DM, thus suggesting that tau hyperphosphorylation at Ser396, Ser404, Thr212 and Thr231 is associated with DACD. Consistent with Gatta et al.’s study, we found an imbalance between O-GlcNAcylation and tau phosphorylation in the hippocampus of DACD mice (Gatta et al., 2016). Decreased O-GlcNAcylation levels and increased tau phosphorylation levels were also observed in HT22 cells cultured with AGE for 48 h. Previous studies showed that OGT inhibitor usage can increase tau phosphorylation levels at Ser199 and Ser396, thereby increasing tau aggregation (Lim et al., 2015); by contrast, treatment with an OGA inhibitor can increase tau O-GlcNAcylation and prevent the formation of pathological tau in the rTg4510 tau transgenic mouse model (Graham et al., 2014; Hastings et al., 2017). Yuzwa et al. found that an OGA inhibitor increases tau O-GlcNAc and hinders formation of tau aggregates other than altering tau phosphorylation (Yuzwa et al., 2012). Furthermore, pharmacological inhibition of OGA could prevent Aβ plague formation and rescue cognitive impairment in a mouse model of AD (Kim et al., 2013; Yuzwa et al., 2014). These findings support our position that decreased O-GlcNAcylation could contribute to cognitive impairment via increased tau phosphorylation and that increased O-GlcNAcylation may serve as a potential therapeutic target for the treatment of AD. OGT is a key enzyme that is responsible for addition of UDP-GlcNAc to the Ser or Thr residue of proteins; therefore it plays an important role
phosphorylation in AGE-cultured HT22 cells. After transfection, OGT mRNA and protein levels were significantly increased (p < 0.01 or p < 0.001) (Fig. 9A–C). We then detected O-GlcNAcylation and tau phosphorylation levels using Western blot, and results showed that OGlcNAcylation levels are upregulated (p < 0.05) whereas tau phosphorylation levels at Ser396, Ser404, Thr212, and Thr231 are downregulated (p < 0.05 or p < 0.01) (Fig. 9D–G). 4. Discussion DACD is one of the most common complications of diabetes. Despite previous findings, the mechanism underlying DACD remains unclear, and effective treatment strategies are scarce. Here, our study showed that 15-week-old spontaneous type 2 diabetic KK-Ay mice exhibit learning and memory dysfunction, hippocampal structure abnormalities, and imbalanced O-GlcNAcylation and tau phosphorylation levels and that hypoglycemic therapy with glimepiride could improve these abnormalities. In addition, OGT is involved in the upregulation of OGlcNAcylation and downregulation of tau phosphorylation in AGEcultured HT22 cells. Microtubule-associated protein tau, which is short for tau protein, plays important roles in the regulation of microtubule assembly and stabilization of cell structures in neurons, as well as regulation of axonal transport in vitro and cultured cells (Feinstein and Wilson, 2005; Utton et al., 2005; Dixit et al., 2008; Kanaan et al., 2011; Mandelkow and Mandelkow, 2012). When tau protein is hyperphosphorylated, it may accumulate abnormally and form neurofibrillary tangles, leading to cognitive dysfunction. Increased levels of total and phosphorylated tau at Thr181 and Thr231 could be observed in the hippocampal region of 12-month-old tau transgenic mice relative to age-matched wild-type mice (Kandimalla et al., 2018). Tau phosphorylation levels at Ser199/ Ser202, Ser262, Ser396, Thr205, and Thr231 are also significantly increased in streptozotocin-induced AD animals (Kosaraju et al., 2013; Du 83
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Fig. 9. OGT is involved in the regulation of O-GlcNAcylation and tau phosphorylation in AGE-cultured HT22 cells. (A) Real-time quantitative PCR of OGT expression in AGE-cultured HT22 cells transfected with pcDNA3.1 or pcDNA3.1-OGT plasmid. Representative Western blot (B) and quantitation (C) of OGT expression in AGEcultured HT22 cells transfected with pcDNA3.1 or pcDNA3.1-OGT plasmid. Representative Western blot (D) and quantitation (E) of global O-GlcNAcylation expression in AGE-cultured HT22 cells transfected with pcDNA3.1 or pcDNA3.1-OGT plasmid. Representative Western blot (F) and quantitation (G) of total tau and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 expression in AGE-cultured HT22 cells transfected with pcDNA3.1 or pcDNA3.1-OGT plasmid. Data are represented as mean ± SEM; n = 3 per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the pcDNA3.1 group.
Acknowledgement
in the regulation of O-GlcNAc modification. In our study, OGT protein decreased in DACD mice and increased after hypoglycemic treatment. A previous study showed decreased OGT mRNA expression in HG-treated SKOV-3 cells (Rogalska et al., 2018), and a time-dependent and isoform-specific alteration of OGT has been observed in HG-cultured HK2 cells (Gellai et al., 2016). Consistent with previous studies, we found decreased levels of OGT in AGE-cultured HT22 cells after 48 h. When AGE-treated HT22 cells were transfected with OGT plasmids, OGlcNAcylation levels increased and tau phosphorylation levels decreased, thus suggesting that OGT may mediate the imbalance between O-GlcNAcylation and tau phosphorylation under chronic hyperglycemia. In conclusion, our study demonstrates that hyperglycemia can induce tau hyperphosphorylation by downregulating OGT-involved OGlcNAcylation in vivo and in vitro, which mediates DACD. Our results provide additional insights into the mechanism of DACD. Further welldesigned clinical studies with a larger sample size are needed to confirm these findings.
This work was partially supported by the National Natural Science Foundation of China (No. 81570732, Shaohua Wang and No. 81870568, Shaohua Wang). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.brainresbull.2020.01. 006. References Bogush, M., Heldt, N.A., Persidsky, Y., 2017. Blood brain barrier injury in diabetes: unrecognized effects on brain and cognition. J. Neuroimmune Pharmacol. 12, 593–601. https://doi.org/10.1007/s11481-017-9752-7. Cheng, G., Huang, C., Deng, H., Wang, H., 2012. Diabetes as a risk factor for dementia and mild cognitive impairment: a meta-analysis of longitudinal studies. Intern. Med. J. 42, 484–491. https://doi.org/10.1111/j.1445-5994.2012.02758.x. Cho, N.H., Shaw, J.E., Karuranga, S., Huang, Y., da Rocha Fernandes, J.D., Ohlrogge, A.W., Malanda, B., 2018. IDF Diabetes Atlas: global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res. Clin. Pract. 138, 271–281. https:// doi.org/10.1016/j.diabres.2018.02.023. Dixit, R., Ross, J.L., Goldman, Y.E., Holzbaur, E.L., 2008. Differential regulation of dynein and kinesin motor proteins by tau. Science 319, 1086–1089. https://doi.org/10. 1126/science.1152993. Du, L.L., Chai, D.M., Zhao, L.N., Li, X.H., Zhang, F.C., Zhang, H.B., Liu, L.B., Wu, K., Liu, R., Wang, J.Z., Zhou, X.W., 2015. AMPK activation ameliorates Alzheimer’s diseaselike pathology and spatial memory impairment in a streptozotocin-induced Alzheimer’s disease model in rats. J. Alzheimers Dis. 43, 775–784. https://doi.org/ 10.3233/JAD-140564. Feinstein, S.C., Wilson, L., 2005. Inability of tau to properly regulate neuronal microtubule dynamics: a loss-of-function mechanism by which tau might mediate neuronal cell death. Biochim. Biophys. Acta 1739, 268–279. https://doi.org/10.1016/j.bbadis.
Author statement Shaohua Wang and Rong Huang contributed to the study conception and design. Rong Huang and Sai Tian performed the experiments. Rong Huang and Haoqiang Zhang analyzed the data. Rong Huang wrote the manuscript. Shaohua Wang and Wenwen Zhu revised the manuscript. All authors approved the final version to be published. Declaration of Competing Interest None. 84
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Chronic hyperglycemia induces tau hyperphosphorylation by downregulating OGT-involved O-GlcNAcylation in vivo and in vitro
T
Rong Huanga,b, Sai Tiana, Haoqiang Zhanga, Wenwen Zhua, Shaohua Wanga,* a b
Department of Endocrinology, Affiliated Zhongda Hospital of Southeast University, No. 87 DingJiaQiao Road, Nanjing, 210009, PR China Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, No. 68 Changle Road, Nanjing, 210006, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Type 2 diabetes mellitus Cognitive dysfunction O-GlcNAcylation Tau protein Phosphorylation
Objective: Diabetes mellitus (DM) can increase the risk of cognitive dysfunction, but its exact mechanisms remain unclear. The involvement of aberrant O-GlcNAcylation has been identified in hyperglycemia and DM, as well as the pathogenesis of Alzheimer’s disease via competition with tau phosphorylation. This study was designed to investigate the role of O-GlcNAcylation in diabetes-associated cognitive dysfunction (DACD). Methods: Fifteen-week old male KK-Ay mice were used as DACD models, and advanced glycation end product (AGE)-treated HT22 cells were used as a model of high glucose toxicity. Morris water maze tests, histological staining, real-time quantitative PCR, and Western blot were also applied. Results: Mice with DACD exhibited evident obesity, hyperinsulinemia, hyperglycemia, and impaired learning and memory function. O-GlcNAcylation levels decreased and tau phosphorylation levels at Ser396, Ser404, Thr212, and Thr231 increased in the hippocampus of mice with DACD, as well as in AGE-treated HT22 cells. Hypoglycemic therapy improved these anomalies and elevated O-GlcNAc transferase (OGT) levels in mice with DACD. OGT plasmid transfection in HT22 cells partially reversed AGE-induced decreases in O-GlcNAcylation levels and increased tau phosphorylation levels. Conclusions: Chronic hyperglycemia can induce tau hyperphosphorylation by downregulating OGT-involved OGlcNAcylation in vivo and in vitro, which mediates DACD.
1. Introduction The number of people worldwide diagnosed with diabetes in 2017 was estimated 451 million, and this number is expected to increase to 693 million by 2045 (Cho et al., 2018). The prevalences of diabetes and pre-diabetes in China are as high as 10.9% and 35.7%, respectively (Wang et al., 2017). Type 2 diabetes mellitus (T2DM) accounts for 90% of all reported diabetes cases and is characterized by insulin resistance (IR) and relative insulin deficiency, thereby resulting in chronic hyperglycemia (Gallwitz et al., 2013; Zheng et al., 2018). Accumulating evidence associates T2DM with increased risk of cognitive impairment and even dementia (Cheng et al., 2012; Li and Huang, 2016; Zhang et al., 2019). Several mechanisms have been demonstrated to contribute to diabetes-associated cognitive dysfunction (DACD), including abnormal glucose metabolism, impaired insulin signaling, blood brain barrier injury, β-amyloid (Aβ) accumulation, and tau hyperphosphorylation (Li et al., 2015; Gonzalez-Reyes et al., 2016; Bogush et al., 2017; Wu et al., 2017; Tumminia et al., 2018). However, the exact mechanisms of the disease remain unknown, and effective treatments
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are lacking. Chronic hyperglycemia, one of the main clinical manifestations of T2DM, is a major cause of diabetic chronic complications. Several metabolic pathways, including polyol pathway activation, advanced glycation end product (AGE) formation, oxidative stress injury and protein kinase C activation, may contribute to the development of DACD as the downstream consequence of hyperglycemia (Yagihashi, 2016). Abnormal activation of intracellular glucose metabolism pathways, such as the hexosamine biosynthesis pathway (HBP), may also mediate the toxic effects of hyperglycemia. Upon the entry of glucose into cells, approximately 2%–5% of this molecule enters the nutrient sensing HBP and is converted into uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), which is also the substrate for O-Linked β-N-acetylglucosamine (O-GlcNAc) modification (Wells et al., 2003). O-GlcNAcylation is a posttranslational modification that dynamically modifies serine (Ser) and threonine (Thr) through their hydroxyl moieties on nuclear and cytoplasmic proteins (Yang and Qian, 2017). O-GlcNAcylation is catalyzed by two key enzymes, namely, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which respectively add/
Corresponding author. E-mail address: [email protected] (S. Wang).
https://doi.org/10.1016/j.brainresbull.2020.01.006 Received 28 June 2019; Received in revised form 7 December 2019; Accepted 3 January 2020 Available online 10 January 2020 0361-9230/ © 2020 Elsevier Inc. All rights reserved.
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divided into three groups (n = 10), namely, the diabetes group (KK-Ay + Veh), the diabetes + 1 mg/(kg·d) glimepiride group (KK-Ay + Gmp1), and the diabetes + 2 mg/(kg·d) glimepiride group (KK-Ay + Gmp2); 10 age-matched C57BL/6 mice were used as controls (C57BL/ 6+Veh) (Takada et al., 1996; Tsukuda et al., 2007; Ishola et al., 2018). All mice in the intervention group were provided an equal volume of liquid gavage for 12 weeks. Before sacrifice, the body weight of the mice were measured by using an electronic scale, and fasting blood glucose (FBG) levels were measured by using a glucometer (ONETOUCH UltraVue, Johnson Medical Equipment Co., Ltd.). Fasting venous blood was obtained from the angular vein, and fasting insulin (FIN) levels were measured by a mouse insulin ELISA kit (Wuhan Liuhe Biotechnology Co., Ltd.).
remove UDP-GlcNAc to/from Ser and Thr residues of proteins (Joiner et al., 2019). As UDP-GlcNAc is derived from HBP, aberrant O-GlcNAcylation has been identified to be involved in hyperglycemia, IR, and T2DM (Park et al., 2010; Springhorn et al., 2012; Myslicki et al., 2014). O-GlcNAcylation levels of global and tau proteins in the postmortem brain tissue from frontal cortices of T2DM subjects are decreased (Liu et al., 2009). In the peripheral blood, OGA expression is significantly increased in erythrocytes of individuals with pre-diabetes and diabetes compared with normal control subjects (Park et al., 2010). Springhorn et al. also found that leukocyte, particularly granulocyte, O-GlcNAcylation may serve as a novel diagnostic tool for the early detection of T2DM (Springhorn et al., 2012). In vitro, SKOV-3 cells show diminished OGT mRNA levels 24, 48, and 72 h after culture in high glucose (HG) compared with cells in the control group (Rogalska et al., 2018). OGlcNAcylation in HK2 cells is elevated after 24 and 48 h of HG treatment (HG24 and HG48, respectively). In HG48 cells, full-length nucleocytoplasmic OGT (ncOGT) and mitochondrial OGT (mOGT) proteins are decreased compared with those in HG24 cells, and the mOGT protein is even lower in HG48 than in control cells. In addition, the long isoform OGA (OGA-L) protein is significantly higher in HG48 than in HG24 and even higher than that in the normal control group (Gellai et al., 2016). O-GlcNAcylation may compete with tau phosphorylation at Ser and Thr sites, thereby playing a role in the pathogenesis of Alzheimer’s disease (AD) (Zhu et al., 2014; Wani et al., 2017). Previous studies showed an imbalance of tau O-GlcNAcylation with phosphorylation in the hippocampus of a mouse model of AD (Gatta et al., 2016). Moreover, upon the inhibition of OGT, tau phosphorylation levels at Ser199 and Ser396 increase, thereby resulting in increased tau aggregation (Lim et al., 2015). By contrast, increasing O-GlcNAcylation by applying an OGA inhibitor can prevent Aβ plaque formation and tauopathy, thereby attenuating cognitive decline (Yuzwa et al., 2012; Kim et al., 2013; Graham et al., 2014; Yuzwa et al., 2014; Hastings et al., 2017). We speculate that chronic hyperglycemia may lead to an imbalance between O-GlcNAcylation and tau phosphorylation levels via regulation of the key enzymes OGT or OGA, including the effects of tau phosphorylation of certain sites, ultimately mediating DACD. In this study, we used 15-week-old KK-Ay mouse as a DACD model, because the KKAy mouse, a spontaneous animal model of T2DM, exhibits not only obesity, hyperglycemia, and hyperinsulinemia but also severe cognitive decline at 15 weeks of age (Iwatsuka et al., 1970; Tsukuda et al., 2007; Min et al., 2012). The present study aims to clarify the potential mechanisms of chronic hyperglycemia on DACD in spontaneous type 2 diabetic KK-Ay mice and AGE-treated mouse hippocampal neuron (HT22) cells.
2.3. Cell culture, viability assay and treatments HT22 cells were purchased from iCell Bioscience Inc. (Shanghai, China) and cultured in HG Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C with 5% CO2 in a humidified atmosphere. The culture medium was replaced every 48 h, and the cells were passaged at a ratio of 1:3. Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay (Biosharp, China). HT22 cells were seeded into 96-well plates at a density of 5000 cells per well. After overnight incubation, the culture medium was replaced with fresh DMEM containing different concentrations of AGE (0, 100, 200, 400, 800, and 1600 μg/mL) for different time (24, 48, and 72 h). Subsequently, each culture well was added with CCK-8 reagent and incubated at 37 °C for 2 h. The absorbance of each well was detected at 450 nm using an automatic microplate reader (BioTek Instruments, Inc.). 2.4. Plasmid transfection pcDNA3.1(+)-OGT plasmid was purchased from Shanghai Generay Biotechnology Co., Ltd. HT22 cells were seeded into six-well plates at a density of 1 × 106 per well. pcDNA3.1(+)-OGT plasmid was transfected in the presence of 400 μg/mL AGE using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, USA), and the pcDNA3.1(+)-empty plasmid was used as control. After transfecting for 4 h, the medium was replaced with fresh DMEM containing 400 μg/mL AGE. 2.5. Morris water maze test Cognitive function was evaluated by the Morris water maze (MWM) test with 1 week time after the observational and interventional studies. All mice were allowed to swim freely for 1 min without the platform to adapt the environment on the first day of the test; those who floated or swam poorly were excluded. For the navigation test, each mouse was trained four times per day at 20 min intervals for following five consecutive days. The time each mouse took to find the platform and the total length of the path were recorded as escape latency and path length, respectively. If the mouse failed to find the platform within 60 s, it was guided to the platform by the technician for 10 s. The platform was removed, and mice were allowed to swim freely for 60 s in the probe trial. The percentage of time spent in the target quadrant and the frequency of crossing the platform area were recorded as a measure of spatial memory.
2. Materials and methods 2.1. Ethics statement This study was approved by the Animal Studies Committee of Southeast University. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institute of Health. 2.2. Animals and drug treatments Male KK-Ay and C57BL/6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China) and housed in a specific pathogen-free animal experiment center at Southeast University. The mice were kept in a room at 18−25 °C with 40%–60% humidity on a 12 h light/dark cycle and fed standard food and water ad libitum. Two sets of experiments were performed. In the observational experiment, 10 15-week-old KK-Ay mice were used as DACD models, and 10 age-matched C57BL/6 mice were used as controls. In the interventional experiment, 30 15-week old KK-Ay mice were randomly
2.6. Brain tissue preparation Following the behavioral test, all mice were anesthetized with 10% chloral hydrate (0.1 mL/10 g) and then perfused transcardially with 1 × phosphate buffered saline (PBS). The mice were sacrificed, and their brains were removed. Half of the samples were fixed with 4% 77
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Fig. 1. Mice with DACD exhibit worse spatial learning and memory performances. (A) Escape latency to search the platform during the navigation test. (B) Path length to search the platform during the navigation test. (C) Percentage of time spent in the target quadrant during the probe trial. (D) Frequency of crossing platform area during the probe trial. Data are represented as mean ± SEM; n = 10 per group. ** p < 0.01 versus C57BL/6 mice.
2.8. Western blot
Table 1 Body weight, FBG and FIN levels of C57BL/6 and KK-Ay mice. Group
Body weight (g)
FBG (mmol/L)
FIN (mIU/L)
C57BL/6 KK-Ay
26.86 ± 1.81 43.18 ± 3.97***
5.89 ± 0.78 13.49 ± 1.90***
78.81 ± 19.80 106.57 ± 29.81*
Hippocampal tissues and HT22 cells were homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology, China) supplemented with protease inhibitor phenylmethanesulfonyl fluoride and phosphatase inhibitor cocktail. Protein concentrations were determined by using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s protocol. Next, 20 μg of protein was loaded onto 8–10% SDS polyacrylamide gels and separated by electrophoresis at 80 V for 30 min, and then at 100 V for 1 h. The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h. The membranes were incubated overnight at 4 °C with the appropriate primary antibodies, including anti-RL2 (1:1000; Abcam, ab2739), anti-OGT (1:5000; Abcam, ab177941), anti-MGEA5 (1:5000; Abcam, ab124807), Tau-5 (1:800; Abcam, ab80579), anti-pTau (Ser396) (1:10,000; Abcam, ab109390), anti-pTau (Ser404) (1:1000; Abcam, ab92676), anti-pTau (Thr212) (1:1000; Abcam, ab4842), and anti-pTau (Tr231) (1:5000; Abcam, ab151559) with anti-GAPDH (1:3000; CMCTAG, AT0002) served as the control. Membranes were washed thrice with TBST and then incubated at room temperature for 1 h with the corresponding secondary antibodies, including goat antimouse IgG horseradish peroxidase (HRP) (1:50,000; MyBioScience, MKA001A) and goat anti-rabbit IgG HRP (1:25,000; MyBioScience, MKA003A). Membranes were washed another thrice with TBST, and signals were detected by chemiluminescence (Millipore, WBKLS0500) using a MiniChemi™610 imaging and analysis system (Beijing Sage Creation Science Co., Ltd.).
Data are represented as mean ± SD; n = 10 per group. * p < 0.05 and ***p < 0.001 versus C57BL/6 mice.
paraformaldehyde (PFA) for histological staining, and the rest were quickly dissected to isolate the hippocampus, which was stored in liquid nitrogen for Western blot.
2.7. Histological staining Brains fixed in 4% PFA were embedded in paraffin and 4−6 μm tissue sections were obtained for histological staining. First, sections were de-paraffinized with xylene, rehydrated through a series of decreasing percentages of ethanol, and then stained with hematoxylin and eosin (HE) or toluidine blue to observe cell morphologies or Nissl bodies. For immunofluorescence staining, antigen retrieval was performed by microwaving sections thrice in 1 mM ethylenediaminetetraacetic acid (pH 8.0) for 5 min. Next, the sections were washed thrice for 5 min with PBS and then incubated with 5% BSA at 37 °C for 30 min. The sections were treated with primary antibodies, including anti-RL2 (1:100; Abcam, ab2739), anti-neuronal nuclei (NeuN) (1:200; Servicebio, GB11138), anti-pTau (Ser396) (1:100; Santa Cruz Biotechnology, sc-101815), anti-pTau (Ser404) (1:100; Abcam, ab92676), anti-pTau (Thr212) (1:200; Invitrogen, 44−740 G) and antipTau (Thr231) (1:250; Millipore, AB9668), and incubated overnight at 4 °C. After three washes with PBS, the sections were incubated with the appropriate secondary antibodies, including FITC-conjugated goat antirabbit IgG (1:400; Servicebio, GB25303) and Cy3-conjugated goat antimouse IgG (1:300; Servicebio, GB21301), at 37 °C for 60 min. DAPI was used to stain cell nuclei at room temperature for 10 min. Immunofluorescence was observed with fluorescent microscope (OLYMPUS, Japan).
2.9. Real-time quantitative RT-PCR Total RNA was extracted from treated HT22 cells using RNA extraction solution (Wuhan Servicebio Biotechnology Co., Ltd.) according to the manufacturer’s protocol. cDNA was synthesized by using a HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotechnology Co., Ltd.). Real-time quantitative RT-PCR was performed with the ChamQ™ SYBR qPCR Master Mix (High ROX Premixed) kit (Vazyme Biotechnology Co., Ltd.). The PCR primers for OGT are as follows: 5′-GCCAAAGCACGCTGTTAG-3′ (forward) and 5′-GCAATGGA 78
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Fig. 2. Mice with DACD model exhibit hippocampal structure abnormalities. Representative photos showing histopathological changes in the hippocampal regions using HE staining (A) and Nissl staining (B) (scale bars: 500 μm for the first column, and 50 μm for the rest columns). (C) Quantification of Nissl bodies in different regions of the hippocampus. Data are represented as mean ± SEM; n = 3 per group. * p < 0.05 versus C57BL/6 mice.
MWM test) or one-way ANOVA (more than two groups) followed by the LSD post hoc multiple-comparison test. All statistical analyses were performed by using SPSS 22.0 (SPSS Inc., Chicago, IL, USA), and p < 0.05 was considered statistically significant.
CTGGCGACTA-3′ (reverse). The PCR primers for GAPDH are as follows: 5′-AAGAAGGTGGTGAAGCAGG-3′ (forward) and 5′-GAAGGTGGAAGA GTGGGAGT-3′ (reverse). Relative OGT gene expression level was calculated by using the comparative 2−△△CT method, and GAPDH was used as the control.
3. Results 2.10. Statistical analysis 3.1. Mice with DACD exhibit abnormal hippocampal structure and imbalanced O-GlcNAcylation, and tau phosphorylation levels
All data are presented as mean ± standard deviation (SD) or standard error of measurement (SEM). Statistical differences were determined by using Student’s t-test (two-group comparison), two-way analysis of variance (ANOVA) (escape latency and path length in the
The spatial learning abilities of mice were evaluated after 5 consecutive days of the navigation test. Compared with age-matched 79
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Fig. 3. Mice with DACD exhibit imbalanced O-GlcNAcylation and tau phosphorylation levels. (A) Representative photos of immunofluorescence double staining with anti-RL2 and anti-NeuN antibodies (scale bars: 50 μm), and quantification of neurons and O-GlcNAc-positive neurons in the hippocampal CA3 region. (B) Representative photos of immunofluorescence double staining with anti-RL2 and anti-Tau antibodies (scale bars: 50 μm), and quantification of O-GlcNAcylation and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 sites in the hippocampal CA3 region. (C) Representative Western blot and quantitation of global OGlcNAcylation, OGT and OGA expression. (D) Representative Western blot and quantitation of total tau and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 sites. Data are represented as mean ± SEM; n = 3 per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus C57BL/6 mice.
number of Nissl bodies in the hippocampus, especially in the CA3 region (p < 0.05), is reduced in the DACD model (Fig. 2). Immunofluorescence double staining was conducted to clarify the localization of O-GlcNAcylation with anti-RL2 and anti-NeuN antibodies. The results showed that O-GlcNAcylation mainly occurs in hippocampal neuronal cells, and the number of neuronal cells modified by OGlcNAcylation (O-GlcNAc-positive neurons) decreases in the hippocampal CA3 region of KK-Ay mice (p < 0.05) (Fig. 3A). Moreover, OGlcNAcylation levels decreased (p < 0.01), while tau phosphorylation levels increased (p < 0.05, p < 0.01 or p < 0.001) in the hippocampus of DACD models; these results were determined by using immunofluorescence double staining, and confirmed by Western blot (Fig. 3B–D).
Table 2 Body weight, FBG and FIN levels of the interventional mice. Group
Body weight (g)
FBG (mmol/L)
FIN (mIU/L)
C57BL/6 + Veh KK-Ay + Veh KK-Ay + Gmp1 KK-Ay + Gmp2
33.88 ± 3.10 48.63 ± 3.26*** 46.88 ± 2.67 46.14 ± 3.22
5.93 ± 0.75 14.55 ± 4.04*** 10.28 ± 2.32## 7.98 ± 1.34###,&
82.76 ± 16.41 109.46 ± 31.16* 100.31 ± 20.41 97.37 ± 18.05
Data are represented as mean ± SD; n = 8–10 per group. * p < 0.05 and ***p < 0.001 versus the C57BL/6+Veh group. ## p < 0.01 and ###p < 0.001 versus the KK-Ay + Veh group. & p < 0.05 versus the KK-Ay + Gmp1 group.
C57BL/6 mice, KK-Ay mice showed increased escape latency and path length to find the platform from the 2nd to the 5th days of the test (p < 0.05) (Fig. 1A and B). Twenty-four hours after the last training test, probe trials were performed to evaluate the memory function of the trained mice. KK-Ay mice showed lower percentage of time spent in the target quadrant (50.68% ± 13.33% versus 28.86% ± 14.51%, p < 0.05) and frequency of crossing the platform area (4.10 ± 1.20 versus 2.40 ± 0.97, p < 0.05) compared with C57BL/6 mice (Fig. 1C and D), thus confirming that 15-week-old KK-Ay mice could be used as DACD models. The average body weight, FBG, and FIN levels of KK-Ay mice were significantly higher than those of C57BL/6 mice (p < 0.05 or p < 0.001) (Table 1). HE staining showed that several cells are loosely arranged or structurally destroyed. Nissl staining showed that the
3.2. Hypoglycemic therapy ameliorates spatial learning and memory dysfunction in KK-Ay mice After 12 weeks of treatment with glimepiride, FBG levels decreased in a dose-independent manner in the KK-Ay + Gmp group compared with the KK-Ay + Veh group (p < 0.05, p < 0.01 or p < 0.001) (Table 2). No significant differences in body weight and FIN level were observed between the KK-Ay + Gmp and KK-Ay + Veh groups (p > 0.05). The MWM test also showed that the KK-Ay + Gmp group exhibits better performance than the KK-Ay + Veh group from the 2nd to the 5th days (p < 0.05) (Fig. 4A and B). Moreover, mice treated with glimepiride showed more percentage of time spent in the target 80
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Fig. 4. Glimepiride ameliorates spatial learning and memory dysfunction in KK-Ay mice. (A) Escape latency to search the platform during the navigation test. (B) Path length to search the platform during the navigation test. (C) Percentage of time spent in the target quadrant during the probe trial. (D) Frequency of crossing platform area during the probe trial. Data are represented as mean ± SEM; n = 8–10 per group. *** p < 0.001 versus the C57BL/6 + Veh group; # p < 0.05 and ## p < 0.01 versus the KK-Ay + Veh group.
Fig. 5. Glimepiride ameliorates hippocampal structure abnormalities in KK-Ay mice. Representative photos showing histopathological changes in the CA3 region of the hippocampus using HE staining (A) and Nissl staining (B) (scale bars: 50 μm). (C) Quantification of Nissl bodies in the CA3 region of the hippocampus. Data are represented as mean ± SEM; n = 3 per group. *** p < 0.001 versus the C57BL/6 + Veh group; # p < 0.05 versus the KK-Ay + Veh group.
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Fig. 6. Glimepiride increases O-GlcNAcylation and decreases tau phosphorylation levels in the hippocampus of KK-Ay mice. (A) Representative photos of immunofluorescence double staining with anti-RL2 and anti-NeuN antibodies (scale bars: 50 μm), and quantification of neurons and O-GlcNAc-positive neurons in the hippocampal CA3 region. (B) Representative photos of immunofluorescence double staining with anti-RL2 and anti-Tau antibodies (scale bars: 50 μm), and quantification of O-GlcNAcylation and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 sites in the hippocampal CA3 region. (C) Representative Western blot and quantitation of global O-GlcNAcylation, OGT and OGA expression. (D) Representative Western blot and quantitation of total tau and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 sites. Data are represented as mean ± SEM; n = 3 per group. ** p < 0.01 and *** p < 0.001 versus the C57BL/6 + Veh group; # p < 0.05 and ## p < 0.01 versus the KK-Ay + Veh group; & p < 0.05 versus the KK-Ay + Gmp1 group.
glimepiride treatment significantly increased the number of Nissl bodies in the hippocampal CA3 region of mice with DACD (p < 0.05) (Fig. 5B and C). 3.4. Hypoglycemic therapy increases O-GlcNAcylation but decreases tau phosphorylation levels in KK-Ay mice Immunofluorescence double staining showed that the number of OGlcNAc-positive neurons is partially increased in the hippocampal CA3 region of DACD mice after glimepiride intervention (p < 0.05) (Fig. 6A). In addition, immunofluorescence double staining and Western blot demonstrated that O-GlcNAcylation level is increased (p, tau protein phosphorylation levels at Ser396 and Thr212 are decreased, and OGT levels are elevated after glimepiride treatment (Fig. 6B-D).
Fig. 7. Effects of AGE concentration and culture time on HT22 cells viability. The differences are compared between the control group and the treatment group with different concentrations of AGE (0, 100, 200, 400, 800, and 1600 μg/mL) after 12, 24 and 48 h. Cell viability was determinate by CCK8 assay. Data are represented as mean ± SEM; n = 3 per group. ∗ p < 0.05, ## p < 0.01, ### p < 0.001.
3.5. O-GlcNAcylation level is decreased whereas tau phosphorylation levels are increased in AGE-cultured HT22 cells According to the CCK8 results, an AGE concentration of 400 μg/mL after 48 h may be considered a chronic hyperglycemia model (AGE group); here, 400 μg/mL BSA was considered the control treatment (Con group) in this study (Fig. 7). Compared with the Con group, OGlcNAcylation levels decreased (p < 0.05) whereas tau phosphorylation levels at sites Ser396, Ser404, Thr212, and Thr231 increased (p < 0.05, p < 0.01 or p < 0.001) in the AGE group (Fig. 8A–D). OGT mRNA and protein levels were significantly decreased in AGE-treated HT22 cells (p < 0.05) (Fig. 8E–F).
quadrant (34.66% ± 7.85%, 41.18% ± 10.19% versus 25.39% ± 6.31%, p < 0.05) and frequency of crossing the platform area (2.78 ± 0.67, 3.00 ± 0.71 versus 2.00 ± 0.54, p < 0.05) compared with mice treated with the vehicle (Fig. 4C and D). These results indicate that hypoglycemic treatment could ameliorate spatial learning and memory dysfunction in KK-Ay mice.
3.3. Hypoglycemic therapy improves hippocampal structure abnormalities in KK-Ay mice
3.6. OGT is involved in the upregulation of O-GlcNAcylation and downregulation of tau phosphorylation in AGE-cultured HT22 cells
HE staining showed partial improvement of loosely arranged cells or the destroyed structure in the CA3 region of the hippocampus after treatment with glimepiride (Fig. 5A). In the Nissl staining assay,
We performed OGT plasmid transfection to demonstrate the involvement of OGT in the regulation of O-GlcNAcylation and tau 82
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Fig. 8. O-GlcNAcylation and tau phosphorylation levels in AGE-cultured HT22 cells. Representative Western blot (A) and quantitation (B) of global O-GlcNAcylation expression in HT22 cells treated with or without 400 μg/mL AGE. Representative Western blot (C) and quantitation (D) of total tau and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 expression in HT22 cells treated with or without 400 μg/mL AGE. (E) Real-time quantitative PCR of OGT expression in HT22 cells treated with or without 400 μg/mL AGE. (F) Representative Western blot (F) and quantitation (G) of OGT expression in HT22 cells treated with or without 400 μg/mL AGE. Data are represented as mean ± SEM; n = 3 per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the Con group.
et al., 2015; Kandimalla et al., 2018). Previous studies demonstrated that tau protein is hyperphosphorylated in the hippocampus of T2DM animals, particularly at Ser199, Ser202 and Ser396 (Ma et al., 2013; Wu et al., 2013; Xu et al., 2017; Zheng et al., 2017). Guo et al. found that significant increases in the levels of phosphorylated tau at Thr231, Thr205, Ser396, and Ser404 are evident in hyperglycemia-exposed Pdx1+/−/APP/PS1 mice compared with APP/PS1 mice (Guo et al., 2016). In the present study, we found that several AD-associated tau protein sites, including Ser396, Ser404, Thr212 and Thr231, are hyperphosphorylated in the hippocampus of mice with T2DM, thus suggesting that tau hyperphosphorylation at Ser396, Ser404, Thr212 and Thr231 is associated with DACD. Consistent with Gatta et al.’s study, we found an imbalance between O-GlcNAcylation and tau phosphorylation in the hippocampus of DACD mice (Gatta et al., 2016). Decreased O-GlcNAcylation levels and increased tau phosphorylation levels were also observed in HT22 cells cultured with AGE for 48 h. Previous studies showed that OGT inhibitor usage can increase tau phosphorylation levels at Ser199 and Ser396, thereby increasing tau aggregation (Lim et al., 2015); by contrast, treatment with an OGA inhibitor can increase tau O-GlcNAcylation and prevent the formation of pathological tau in the rTg4510 tau transgenic mouse model (Graham et al., 2014; Hastings et al., 2017). Yuzwa et al. found that an OGA inhibitor increases tau O-GlcNAc and hinders formation of tau aggregates other than altering tau phosphorylation (Yuzwa et al., 2012). Furthermore, pharmacological inhibition of OGA could prevent Aβ plague formation and rescue cognitive impairment in a mouse model of AD (Kim et al., 2013; Yuzwa et al., 2014). These findings support our position that decreased O-GlcNAcylation could contribute to cognitive impairment via increased tau phosphorylation and that increased O-GlcNAcylation may serve as a potential therapeutic target for the treatment of AD. OGT is a key enzyme that is responsible for addition of UDP-GlcNAc to the Ser or Thr residue of proteins; therefore it plays an important role
phosphorylation in AGE-cultured HT22 cells. After transfection, OGT mRNA and protein levels were significantly increased (p < 0.01 or p < 0.001) (Fig. 9A–C). We then detected O-GlcNAcylation and tau phosphorylation levels using Western blot, and results showed that OGlcNAcylation levels are upregulated (p < 0.05) whereas tau phosphorylation levels at Ser396, Ser404, Thr212, and Thr231 are downregulated (p < 0.05 or p < 0.01) (Fig. 9D–G). 4. Discussion DACD is one of the most common complications of diabetes. Despite previous findings, the mechanism underlying DACD remains unclear, and effective treatment strategies are scarce. Here, our study showed that 15-week-old spontaneous type 2 diabetic KK-Ay mice exhibit learning and memory dysfunction, hippocampal structure abnormalities, and imbalanced O-GlcNAcylation and tau phosphorylation levels and that hypoglycemic therapy with glimepiride could improve these abnormalities. In addition, OGT is involved in the upregulation of OGlcNAcylation and downregulation of tau phosphorylation in AGEcultured HT22 cells. Microtubule-associated protein tau, which is short for tau protein, plays important roles in the regulation of microtubule assembly and stabilization of cell structures in neurons, as well as regulation of axonal transport in vitro and cultured cells (Feinstein and Wilson, 2005; Utton et al., 2005; Dixit et al., 2008; Kanaan et al., 2011; Mandelkow and Mandelkow, 2012). When tau protein is hyperphosphorylated, it may accumulate abnormally and form neurofibrillary tangles, leading to cognitive dysfunction. Increased levels of total and phosphorylated tau at Thr181 and Thr231 could be observed in the hippocampal region of 12-month-old tau transgenic mice relative to age-matched wild-type mice (Kandimalla et al., 2018). Tau phosphorylation levels at Ser199/ Ser202, Ser262, Ser396, Thr205, and Thr231 are also significantly increased in streptozotocin-induced AD animals (Kosaraju et al., 2013; Du 83
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Fig. 9. OGT is involved in the regulation of O-GlcNAcylation and tau phosphorylation in AGE-cultured HT22 cells. (A) Real-time quantitative PCR of OGT expression in AGE-cultured HT22 cells transfected with pcDNA3.1 or pcDNA3.1-OGT plasmid. Representative Western blot (B) and quantitation (C) of OGT expression in AGEcultured HT22 cells transfected with pcDNA3.1 or pcDNA3.1-OGT plasmid. Representative Western blot (D) and quantitation (E) of global O-GlcNAcylation expression in AGE-cultured HT22 cells transfected with pcDNA3.1 or pcDNA3.1-OGT plasmid. Representative Western blot (F) and quantitation (G) of total tau and phosphorylated tau at Ser396, Ser404, Thr212 and Thr231 expression in AGE-cultured HT22 cells transfected with pcDNA3.1 or pcDNA3.1-OGT plasmid. Data are represented as mean ± SEM; n = 3 per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the pcDNA3.1 group.
Acknowledgement
in the regulation of O-GlcNAc modification. In our study, OGT protein decreased in DACD mice and increased after hypoglycemic treatment. A previous study showed decreased OGT mRNA expression in HG-treated SKOV-3 cells (Rogalska et al., 2018), and a time-dependent and isoform-specific alteration of OGT has been observed in HG-cultured HK2 cells (Gellai et al., 2016). Consistent with previous studies, we found decreased levels of OGT in AGE-cultured HT22 cells after 48 h. When AGE-treated HT22 cells were transfected with OGT plasmids, OGlcNAcylation levels increased and tau phosphorylation levels decreased, thus suggesting that OGT may mediate the imbalance between O-GlcNAcylation and tau phosphorylation under chronic hyperglycemia. In conclusion, our study demonstrates that hyperglycemia can induce tau hyperphosphorylation by downregulating OGT-involved OGlcNAcylation in vivo and in vitro, which mediates DACD. Our results provide additional insights into the mechanism of DACD. Further welldesigned clinical studies with a larger sample size are needed to confirm these findings.
This work was partially supported by the National Natural Science Foundation of China (No. 81570732, Shaohua Wang and No. 81870568, Shaohua Wang). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.brainresbull.2020.01. 006. References Bogush, M., Heldt, N.A., Persidsky, Y., 2017. Blood brain barrier injury in diabetes: unrecognized effects on brain and cognition. J. Neuroimmune Pharmacol. 12, 593–601. https://doi.org/10.1007/s11481-017-9752-7. Cheng, G., Huang, C., Deng, H., Wang, H., 2012. Diabetes as a risk factor for dementia and mild cognitive impairment: a meta-analysis of longitudinal studies. Intern. Med. J. 42, 484–491. https://doi.org/10.1111/j.1445-5994.2012.02758.x. Cho, N.H., Shaw, J.E., Karuranga, S., Huang, Y., da Rocha Fernandes, J.D., Ohlrogge, A.W., Malanda, B., 2018. IDF Diabetes Atlas: global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res. Clin. Pract. 138, 271–281. https:// doi.org/10.1016/j.diabres.2018.02.023. Dixit, R., Ross, J.L., Goldman, Y.E., Holzbaur, E.L., 2008. Differential regulation of dynein and kinesin motor proteins by tau. Science 319, 1086–1089. https://doi.org/10. 1126/science.1152993. Du, L.L., Chai, D.M., Zhao, L.N., Li, X.H., Zhang, F.C., Zhang, H.B., Liu, L.B., Wu, K., Liu, R., Wang, J.Z., Zhou, X.W., 2015. AMPK activation ameliorates Alzheimer’s diseaselike pathology and spatial memory impairment in a streptozotocin-induced Alzheimer’s disease model in rats. J. Alzheimers Dis. 43, 775–784. https://doi.org/ 10.3233/JAD-140564. Feinstein, S.C., Wilson, L., 2005. Inability of tau to properly regulate neuronal microtubule dynamics: a loss-of-function mechanism by which tau might mediate neuronal cell death. Biochim. Biophys. Acta 1739, 268–279. https://doi.org/10.1016/j.bbadis.
Author statement Shaohua Wang and Rong Huang contributed to the study conception and design. Rong Huang and Sai Tian performed the experiments. Rong Huang and Haoqiang Zhang analyzed the data. Rong Huang wrote the manuscript. Shaohua Wang and Wenwen Zhu revised the manuscript. All authors approved the final version to be published. Declaration of Competing Interest None. 84
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