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Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice
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NEUROSCIENCE 1
RESEARCH ARTICLE F. Yu et al. / Neuroscience xxx (2018) xxx–xxx
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Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice
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Fan Yu, a1 Wei Han, b1 Gaofeng Zhan, c Shan Li, c Xiaohong Jiang, a Shoukui Xiang, a Bin Zhu, d Ling Yang, e Dongyu Hua, c Ailin Luo, c Fei Hua a* and Chun Yang c*
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a
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b
Department of Neurosurgery, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
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c
Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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d
Department of Critical Care Medicine, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
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e
Department of Cardiology, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
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Abstract—Increasing studies have revealed that metabolic disorders, especially diabetes, are high risk factors for the development of Alzheimer’s disease (AD) and other neurodegenerative diseases. It has been reported that patients with diabetes are prone to suffer from cognitive dysfunction (CD). Although abnormal glucose metabolism and deposition of amyloid b (Ab) are proven to have a closely relationship with diabetes-induced CD, its exact mechanism is still undetermined. In this study, a total of 14 mice were intraperitoneally injected with streptozotocin for 5 consecutive days to mimic diabetic models, and then hierarchical cluster analysis was adopted to classify the diabetic mice into CD and Non-CD phenotypes by the results of Morris water maze test (MWMT). Furthermore, we detected Hippo signaling including mammalian sterile 20-like protein kinases1 (MST1), large tumor suppressors 1 (LATS1), Yes-associated protein (YAP) and phosphorylation of YAP (p-YAP) in brain and peripheral tissues. As compared with control mice, the levels of MST1, LATS1 and p-YAP/YAP ratio were increased in medial prefrontal cortex (mPFC), striatum and hippocampus of CD mice, while these proteins were decreased in gut tissue of CD mice. Additionally, there were significant positive correlations between escape latency and p-YAP/YAP ratio in mPFC, anterior cingulate cortex (ACC) and hippocampus, as well as the level of LATS1 in liver, kidney and gut tissues. In conclusion, alterations in Hippo signaling may contribute to CD induced by diabetes. Therefore, therapeutic interventions improving Hippo signaling might be beneficial to the treatment of diabetes-induced CD and other neurodegenerative diseases. Ó 2019 IBRO. Published by Elsevier Ltd.
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Department of Endocrinology, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
All rights reserved.
key words: Diabetes, Cognitive dysfunction, Hippo signaling, Streptozotocin.
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INTRODUCTION
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It is estimated that there are approximately 435 million people living with diabetes worldwide and the number will reach over 642 million by 2040 (Ingelfinger et al., 2017; Ogurtsova et al., 2017). The rising prevalence of diabetes all over the world has gained public concerns largely ascribing to its relevant long-term complications.
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Although macrovascular and microvascular complications of diabetes are well recognized, there is a lack of attentions on diabetes-induced cognitive dysfunction (CD) (Munshi, 2017). Meanwhile, it has been reported that patients with diabetes during midlife have a higher incidence of 19% than healthy individuals to experience CD (Rawlings et al., 2014). Both type 1 and type 2 diabetes are predisposing factors for the onset of CD, ultimately leading to dementia in animal models and clinical studies (Wong et al., 2014; Li et al., 2017; Yuan et al., 2017). Alzheimer’s disease (AD) is clinically characterized by CD or even dementia, which is highly related to abnormal accumulation and deposition of amyloid plaques and neurofibrillary tangles in pathology (Ow et al., 2014; Lane et al., 2018). Several lines of evidence have suggested that abnormal Ab deposition and impaired glucose regulation might both underlie the mechanisms of comorbidity in AD and
*Corresponding authors. E-mail addresses: [email protected] (F. Hua), [email protected] (C. Yang). 1 These authors contributed equally to this study. Abbreviations: Ab, Amyloid b; ACC, anterior cingulate cortex; AD, Alzheimer’s disease; ANOVA, analysis of variance; CD, cognitive dysfunction; CONT, control; LATS1, large tumor suppressors 1; mPFC, medial prefrontal cortex; MST1, mammalian sterile 20-like protein kinases1; MWMT, Morris water maze test; NAc, nucleus accumbens; N.S., not significant; p-YAP, phosphorylation of YAP; STZ, streptozotocin; YAP, Yes-associated protein. https://doi.org/10.1016/j.neuroscience.2019.09.018 0306-4522/Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 1
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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Fig. 1. Comparisons of metabolic parameters and behaviors of MWMT among CONT, CD and Non-CD groups. (A) The schedule for the experiment. Mice were injected with STZ (55 mg/kg) intraperitoneally on Day 1 after seven days accommodation. From Day 6 to 61, metabolic parameters were measured after five consecutive days of STZ injection. Mice were scheduled for MWMT from Day 62 to 66, and that probe trial was performed on Day 67. On Day 68, tissues were collected for analysis. (B) Body weight (two-way repeated ANOVA). (C) Water intake (two-way repeated ANOVA). (D) Food intake (two-way repeated ANOVA). (E) Glucose (two-way repeated ANOVA). (F) Dendrogram of hierarchical clustering analysis. Mice after STZ exposure were divided into CD and Non-CD groups by MWMT results of hierarchical clustering analysis. (G) Representative trace graphs of CONT, CD and Non-CD mice in MWMT. (H) Escape latency (two-way repeated ANOVA). (I) Escape path length (two-way repeated ANOVA). (J) Platform crossing (Fisher’s exact test). (K) Time spent in each quadrant (two-way repeated ANOVA). Data are shown as mean ± S.E.M. (n = 6–8). *P < 0.05, **P < 0.01 or ***P < 0.001. ANOVA: analysis of variance; CONT: control; CD: cognitive dysfunction; MWMT: Morris water maze test; N.S.: not significant; STZ: streptozotocin.
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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diabetes (Akter et al., 2011; Shinohara et al., 2017). However, the exact pathogenesis and interactions of the two diseases have not yet been fully elucidated (Huang et al., 2014). Therefore, studying the pathogenesis and exploring potential therapies of AD by investigating the mechanisms of CD caused by diabetes are greatly necessary. The Hippo signaling was first discovered in Drosophila using genetic manipulation and now has been successfully established and determined in mammals (Ji et al., 2017). The major physiologic functions of Hippo signaling are to limit tissue growth and control organ size, as well as to regulate metabolic homeostasis by modulating cellular proliferation, apoptosis and regeneration (Gumbiner et al., 2014; Li et al., 2017; Ardestani et al., 2018). In mammalian systems, Hippo signaling is composed of mammalian sterile 20-like protein kinases1 and 2 (MST1/2), large tumor suppressors1 and 2 (LATS1/2), Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) (Wang et al., 2015; Meng et al., 2016). Once the Hippo pathway is activated, upstream regulators directly initiate activation of MST1/2 and LATS1/2, and then active LATS1/2 phosphorylating YAP/TAZ at Serine 127, finally leading to YAP/TAZ cytoplasmic retention and promoting their ubiquitinproteasome degradation. By contrast, the inactivated state of Hippo pathway would help dephosphorylated YAP/TAZ to translocate into the nucleus, which is associated with neoplastic growth and occurrence of tumors (Plouffe et al., 2015; Yu et al., 2015; Ardestani et al., 2018). Hyper-activation of MST1 may have a significant impact on regulating proteolytic processing of the precursor of Ab (Jang et al., 2007; Tomiyama, 2010; Huang et al., 2012). Hence, excessive activation of Hippo signaling might contribute to the development of neurodegenerative diseases (Plouffe et al., 2015). Interestingly, recent findings have suggested that Hippo pathway has a bidirectional interaction with glucose metabolism (Wang et al., 2015; Peng et al., 2017). Collectively, we proposed that Hippo signaling pathway might play a vital role in the pathogenesis of diabetes-induced CD. For this end, we determined different expressions of Hippo signaling in selected brain and peripheral tissues
by Western blot analysis. Furthermore, correlations between escape latency of MWMT results and levels of Hippo signaling in selected brain and peripheral tissues were also performed to verify the causal linkage.
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EXPERIMENTAL PROCEDURES
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Animals
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All experimental protocols and animal handling procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publications No. 80-23, revised in 1996). This study was approved by the Experimental Animal Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Eight-week-old healthy male C57BL/6J mice weighing 20–25 g were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). A total of 26 mice used in this study were fed with food and water ad libitum, on a 12-h light/dark cycle schedule. The laboratory conditions were maintained with a consistent temperature at 22 °C ± 2 °C and a relative humidity of 60% ± 5%. All mice were allowed to acclimate for a week before experiments (Fig. 1A).
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MODELS OF TYPE 1 DIABETES MELLITUS
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A total of 20 mice were fasted for 12 h prior to treatment after 7 days accommodation to induce a model of type 1 diabetes (Kong et al., 2018). Subsequently, a fresh solution of 10 mg/ml STZ (Absin Bioscience Inc., Shanghai, China) that dissolved in 0.1 M sodium citrate buffer (pH4.5) was prepared, and then mice were injected intraperitoneally with STZ at a dose of 55 mg/kg for 5 consecutive days as previously described (Li et al., 2016). In addition, mice in control group were injected with the same volume of sodium citrate buffer. Metabolic parameters including body weight, water intake and food intake were recorded once a week, while fasting blood glucose was assessed every 2 weeks using a tail vein blood sample via OneTouchÒ Ultra blood glucose meter. Mice with fasting blood glucose levels more than 11.1 mmol/L were
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Table 1. Mean blood glucose. Week
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CONT (mmol/L) CD (mmol/L) Non-CD (mmol/L)
7.05 6.75 6.85
6.717 20.399 19.69
7.2 23.049 21.339
6.167 26.07 22.061
6.183 25.101 23.458
CD: cognitive dysfunction; CONT: control.
Table 2. Mean body weight. Week
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CONT (g) CD (g) Non-CD (g)
23.47 23.82 23.94
24.23 21.43 22.2
23.57 19.94 20.53
24.08 20.74 21.29
24.28 21.10 21.74
24.12 20.93 21.28
24.6 20.85 21.55
24.42 20.43 21.02
24.6 20.23 20.96
CD: cognitive dysfunction; CONT: control.
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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used in the following experiments (Zheng et al., 2018). Eight weeks later, mice were subjected to behavioral tests to evaluate cognitive function.
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Morris water maze test
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Spatial information acquisition and memory retention were assessed by the Morris water maze test (MWMT) after 8 weeks of the final STZ injection. A circular pool (diameter: 120 cm; height: 50 cm) was filled with warm (23 ± 1 °C) opaque water which was contained with nontoxic titanium white-colored dye. In the target quadrant, a movable clear platform with 15 cm in diameter was submerged 0.5–1 cm below water surface. The MWMT was performed 4 trials per day for consecutive 5 days to determine the ability of mice in spatial memory as previously described (Zhan et al., 2018, 2019). During each trial, all the mice were trained to find the hidden platform in 60 s on which they sat for 15 s before being removed from the pool. If a mouse did not find the platform within 60 s, it was gently guided to the platform and allowed to remain there for 15 s. For all training trials, time and distance taken to reach the platform were recorded. The less time it took a mouse to reach the platform, the better its learning ability (Han et al., 2013). On the day after finishing training, a probe test was conducted immediately to evaluate memory retention. The platform was removed from the pool and that mice were allowed to swim freely for 60 s. The number of platform crossing and time spent in each quadrant were recorded.
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Western blotting
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On day 68, all mice were anesthetized with 5% isoflurane and immediately sacrificed. The selected brain tissues and peripheral tissues of mice, including medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), nucleus accumbens (NAc), striatum, hippocampus, heart, liver, kidney, right anterior foot muscle and gut were dissected and collected. Samples were homogenized on ice in the presence of protease and phosphatase inhibitors that mixed in RIPA buffer (150 mM sodium chloride, Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50mMTris, pH 8.0) for 30 min, and then homogenates were centrifuged at 12,000 rpm at 4 °C for 15 min. Protein concentration in the supernatants was quantified via BCA protein assay kit (Boster, Wuhan, China). Proteins were analyzed by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes
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(Millipore, Bedford, MA, USA). Bands were blocked with 5% BSA in TBST (0.1% Tween 20 in Tris-buffered saline) at room temperature for 1 h. Appropriate primary antibodies were incubated at 4 °C overnight: rabbit antiMST1 (1:1000; Proteintech, Wuhan, China), rabbit antiLATS1 (1:1000; Absin Bioscience Inc., Shanghai, China), rabbit anti-p-YAP (1:1000; Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-YAP (1:1000; Proteintech, Wuhan, China). Afterwards, bands were washed with TBST and incubated with secondary antibodies at room temperature for 1.5 h: horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:5000; Affinity, Cincinnati, OH, USA). Finally, these bands were detected by enhanced chemiluminescence reagents (Abbkine, Wuhan, China) using the ChemiDocXRS chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA).
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Statistical analysis
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The data show as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Kolmogorov–Smirnov test was performed to test data normality, and that Levene test, Welch test or Brown-Forsythe test was used to test equality of variance. Data in this study were analyzed by one-way, two-way analysis of variance (ANOVA) or Fisher’s exact test, followed by Tukey’s multiple comparisons test or Sidak’s multiple comparisons test. In Hierarchical cluster analysis, the data were firstly standardized by z scores. Then, MWMT results (Escape latency on day 5 and platform crossing on the probe trial) were clustered via using Ward’s method and mice were classified as CD or Non-CD groups. A correlation analysis was conducted using Pearson’s product-moment coefficient. The Pvalues of less than 0.05 were considered statistically significant.
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RESULTS
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Comparisons of metabolic parameters and behaviors among CONT, CD and Non-CD groups
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Mice were randomly selected to construct type 1 diabetes models. Two months after final exposure of streptozotocin at a dose of 55 mg/kg (Fig. 1A), a total of 14 diabetic models were successfully established versus agematched control mice by comparing body weight, water intake, food intake and blood glucose (Fig. 1B–E). Obviously, STZ-treated mice significantly decreased body weight than mice in CONT group (Time: F8,80 = 1.89, P = 0.0729; Group: F1,10 = 17.41,
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3 Fig. 2. Levels of Hippo signaling in selected brain tissues among CONT, CD and Non-CD groups. (A) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in mPFC. (B) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in ACC. (C) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in NAc. (D) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in striatum. (E) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in hippocampus. Data are shown as mean ± S.E.M. (n = 6). *P < 0.05, **P < 0.01 or ***P < 0.001. ANOVA: analysis of variance; ACC: anterior cingulate cortex; CONT: control; CD: cognitive dysfunction; LATS1: large tumor suppressors 1; mPFC: medial prefrontal cortex; MST1: mammalian sterile 20-like protein kinases1; NAc: nucleus accumbens; N.S.: not significant; p-YAP: phosphorylation of YAP; YAP: Yes-associated protein.
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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Interaction: F8,80 = 16.29, P < 0.001. Fig. 1C), food intake (Time: F8,80 = 2.307, P < 0.05; Group: F1,10 = 288.1, P < 0.001; Interaction: F8,80 = 2.526, P < 0.05. Fig. 1D) and blood glucose (Time: F4,40 = 27.65, P < 0.001; Group: F1,10 = 232.7, P < 0.001; Interaction: F4,40 = 31.23, P < 0.001. Fig. 1E) in STZ-treated mice. According to hierarchical clustering analysis of MWMT results, 14 mice confirmed as diabetic models were divided into CD (n = 6) and Non-CD (n = 8) groups (Fig. 1F). A notable difference of swimming traces in MWMT was represented among the three groups (Fig. 1G). Furthermore, there was a significant increase in escape latency (Time: F4,60 = 13.75, P < 0.001; Group: F2,15 = 8.466, P < 0.01; Interaction: F8,60 = 2.669, P < 0.05. Fig. 1H) and path length (Time: F4,60 = 11.17, P < 0.001; Group: F2,15 = 2.209, P > 0.05; Interaction: F8,60 = 3.085, P < 0.01. Fig. 1I) in CD group than those of CONT or Non-CD group on day 5. In platform crossing, CD mice showed a significant decrease than CONT or Non-CD mice (F2,15 = 9.286, P < 0.01. Fig. 1J). Additionally, mice in CD group spent significant less time in the target quadrant as compared to CONT or Non-CD mice (Time: F3,45 = 3.201, P < 0.05; Group: F2,15 = 5.672, P < 0.05; Interaction: F6,45 = 5.253, P < 0.001. Fig. 1K). Interestingly, we found that the mean blood glucose in CD mice was higher than that of Non-CD mice in 2–8 weeks (Table 1). Besides, the mean body weight in CD mice was slightly decreased than that of Non-CD mice (Table 2). It is therefore likely that STZ-treated mice with a higher level of blood glucose were more likely to suffer from CD.
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Differential levels of Hippo signaling in selected brain tissues among CONT, CD and Non-CD groups
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We performed Western blot analysis to determine the levels of Hippo signaling including MST1, LATS1, pYAP and YAP in selected brain tissues of CONT, CD and Non-CD mice (Fig. 2A–E). Compared with CONT group, mice in CD group showed a significant increase
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Fig. 3. Correlations between escape latency and levels of Hippo signaling in brain tissues (n = 14). (A) MST1, LATS1 and p-YAP/YAP ratio in mPFC. (B) MST1, LATS1 and p-YAP/YAP ratio in ACC. (C) MST1, LATS1 and p-YAP/YAP ratio in NAc. (D) MST1, LATS1 and p-YAP/YAP ratio in striatum. (E) MST1, LATS1 and p-YAP/YAP ratio in hippocampus. ACC: anterior cingulate cortex; LATS1: large tumor suppressors 1; mPFC: medial prefrontal cortex; MST1: mammalian sterile 20-like protein kinases1; NAc: nucleus accumbens; p-YAP: phosphorylation of YAP; YAP: Yes-associated protein.
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P < 0.01; Interaction: F8,80 = 1.89, P < 0.001. Fig. 1B). Two weeks after final STZ exposure, there was a palpable increase in water intake (Time: F8,80 = 16.29, P < 0.001; Group: F1,10 = 127.2, P < 0.001;
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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Fig. 4. Levels of Hippo signaling in peripheral tissues among CONT, CD and Non-CD groups. (A) MST1, LATS1 and YAP (one-way ANOVA) in heart. (B) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in liver. (C) MST1, LATS1, p-YAP/YAP ratio, YAP (one-way ANOVA) in kidney. (D) MST1, LATS1, p-YAP/YAP ratio, YAP (one-way ANOVA) in muscle. (E) MST1, LATS1, p-YAP/YAP ratio, YAP (one-way ANOVA) in gut. Data are shown as mean ± S.E.M. (n = 6). *P < 0.05, **P < 0.01 or ***P < 0.001. ANOVA: analysis of variance; CONT: control; CD: cognitive dysfunction; LATS1: large tumor suppressors 1; MST1: mammalian sterile 20-like protein kinases1; N.S.: not significant; p-YAP: phosphorylation of YAP; YAP: Yes-associated protein.
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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those of CD group (Fig. 2A). In ACC, the levels of MST1 (F2,15 = 6.032, P < 0.05) and p-YAP/YAP ratio (F2,15 = 9.253, P < 0.01), but not LATS1 (F2,15 = 0.4474, P > 0.05), in CD mice were both higher than mice in CONT or Non-CD group (Fig. 2B). However, the levels of all proteins were not significantly different in NAc among CONT, CD and Non-CD groups (MST1: F2,15 = 0.0669, P > 0.05; LATS1: F2,15 = 0.5167, P > 0.05; p-YAP/ YAP ratio: F2,15 = 0.7625, P > 0.05. Fig. 2C). The levels of MST1 (F2,15 = 7.246, P < 0.01), LATS1 (F2,15 = 4.639, P < 0.05) and pYAP/YAP ratio (F2,15 = 3.272, P = 0.0554) were significantly higher in the striatum of CD mice compared to CONT mice. Although the level of MST1 was significantly increased in CD group than Non-CD group, no distinct difference was detected in the levels of LATS1 and p-YAP/YAP ratio (Fig. 2D). In addition, there was a significant increase in the levels of MST1 (F2,15 = 5.348, P < 0.05), LATS1 (F2,15 = 5.61, P < 0.05) and p-YAP/YAP ratio (F2,15 = 8.809, P < 0.01) in the hippocampus of CD mice as compared with CONT mice (Fig. 2E).
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Correlations between escape latency and levels of Hippo signaling in selected brain tissues
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Here we speculated that the incidence of CD in STZ-induced diabetic mice might be related to the alterations in Hippo signaling, including MST1, LATS1 and p-YAP. Given the important role of escape latency in MWMT, correlations between the escape latency and the expression of these proteins were analyzed (Fig. 3A–E). Consequently, there were significant positive correlations between the escape latency and the expression of LATS1 Fig. 5. Correlations between escape latency and levels of Hippo signaling in peripheral tissues (n = 14). (A) MST1, LATS1 and p-YAP/YAP ratio in heart. (B) MST1, LATS1 and p-YAP/YAP ratio (r = 0.5791, P < 0.05) and p-YAP/ in liver. (C) MST1, LATS1 and p-YAP/YAP ratio in kidney. (D) MST1, LATS1 and p-YAP/YAP ratio YAP ratio (r = 0.6078, P < 0.05) in in muscle. (E) MST1, LATS1 and p-YAP/YAP ratio in gut. LATS1: large tumor suppressors 1; mPFC (Fig. 3A), as well as MST1 MST1: mammalian sterile 20-like protein kinases1; p-YAP: phosphorylation of YAP; YAP: Yes(r = 0.6683, P < 0.05) and p-YAP/ associated protein. YAP ratio (r = 0.611, P < 0.05) in ACC (Fig. 3B). By contrast, there in the levels of MST1 (F2,15 = 6.237, P < 0.05) and was no correlation between the LATS1 (F2,15 = 5.706, P < 0.05), as well as in p-YAP/ escape latency and all proteins in NAc (MST1: YAP ratio (F2,15 = 9.449, P < 0.01). By contrast, the r = 0.08041, P > 0.05; LATS1: r = 0.3086, P > 0.05; levels of MST1, LATS1 and p-YAP/YAP ratio in the p-YAP/YAP ratio: r = 0.09736, P > 0.05. Fig. 3C). mPFC of Non-CD group were significantly lower than Moreover, a positive correlation between the escape
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Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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latency and the level of MST1 (r = 0.6314, P < 0.05. Fig. 3D) was exhibited in striatum, as well as the ratio of p-YAP/YAP in the hippocampus (r = 0.6351, P < 0.05. Fig. 3E).
Differential levels of Hippo signaling in peripheral tissue among CONT, CD and Non-CD groups In peripheral tissues (heart, liver, kidney, skeletal muscle and gut), Western blot analysis was also adopted to evaluate the levels of MST1, LATS1 and p-YAP/YAP ratio (Fig. 4A–E). Obviously, a significant decrease in the expression of LATS1 (F2,15 = 1.003, P < 0.05) was noted in liver of CD group, but not in CONT and NonCD groups (Fig. 4B). The levels of LATS1 (F2,15 = 14.17, P < 0.001) and p-YAP/YAP ratio (F2,15 = 12.21, P < 0.001) were lower in kidney of CD mice than those in CONT, but no statistical difference in the ratio of p-YAP/YAP between CD and Non-CD groups. Interestingly, the expression of p-YAP/YAP ratio in Non-CD group was significantly lower than CONT group, associated with a downward trend towards the LATS1 expression in Non-CD group than CONT group (Fig. 4C). In skeletal muscle, there was a significant decrease in the levels of LATS1 (F2,15 = 3.469, P = 0.0578) and p-YAP/YAP ratio (F2,15 = 9.385, P < 0.01) in CD mice as compared with CONT mice, while the ratio of p-YAP/YAP was lower in Non-CD mice than that of CONT mice (Fig. 4D). As to the Hippo signaling in gut, mice in CD group showed a significant decrease in the levels of MST1 (F2,15 = 4.796, P < 0.05) and LATS1 (F2,15 = 6.251, P < 0.05) than CONT group, as well as in the ratio of p-YAP/YAP (F2,15 = 3.876, P < 0.05. Fig. 4E). Nevertheless, there were no significant changes in the levels of Hippo signaling in heart tissue (MST1: F2,15 = 0.1847, P > 0.05; LATS1: F2,15 = 0.003881, P > 0.05; p-YAP/ YAP ratio: F2,15 = 0.5127, P > 0.05. Fig. 4A).
Correlations between escape latency and levels of Hippo signaling in peripheral tissues We performed correlation analysis between escape latency of MWMT and levels of Hippo signaling in peripheral tissues. Similarly, correlations between the escape latency and the expressions of MST1, LATS1 and p-YAP/YAP ratio were analyzed (Fig. 5A–E). The results showed a significant positive correlation between the escape latency and the level of LATS1 in liver (r = 0.7414, P < 0.01. Fig. 5B), kidney (r = 0.6248, P < 0.05. Fig. 5C) and gut tissues (r = 0.7122, P < 0.01. Fig. 5E). However, there were no significant correlations between the escape latency and the levels of Hippo signaling in heart (MST1: r = 0.1291, P > 0.05; LATS1: r = 0.114, P > 0.05; p-YAP/YAP ratio: r = 0.0529, P > 0.05. Fig. 5A) and muscle tissues (MST1: r = 0.09339, P > 0.05; LATS1: r = 0.174, P > 0.05; p-YAP/YAP ratio: r = 0.4758, P > 0.05. Fig. 5D).
DISCUSSION
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Currently, a sharp increase in the prevalence of diabetesinduced CD has gained widespread attentions owing to its ambiguous pathogenesis and a lack of effective therapeutic strategies (Dik et al., 2007; Tanaka et al., 2019). In the present study, we observed the crucial role of Hippo signaling in the pathogenesis and progress of CD induced by diabetes. Differential expressions of Hippo signaling in selected brain and peripheral tissues, including increased levels of MST1 or p-YAP/YAP ratio in mPFC, ACC, striatum and hippocampus, while decreased levels of LATS1 in liver, kidney, skeletal muscle and gut tissues in diabetes-induced CD, implying a clue in the effective treatment of AD, dementia or other symptoms with CD. Accumulating studies have revealed that both T1DM and T2DM are high risk factors in the development of AD, accompanied by attenuated performance on multiple aspects of cognitive function (Karan et al., 2012). It is well recognized that individuals with T1DM are prone to have mild to moderate CD compared with non-diabetic controls (Li et al., 2017). In addition, abnormalities in glucose metabolism related to impaired insulin signaling in AD might suggest that diabetes-induced CD and neurodegenerative diseases may share a common underlying pathologic mechanism (Banks et al., 2012). We here successfully constructed type 1 diabetic rodent models with an intraperitoneal injection of STZ at a dose of 55 mg/kg for 5 consecutive days as previously described (Li et al., 2016). During the eight weeks of observation, STZ-treated mice significantly increased fluid intake, food intake and blood glucose levels, while decreased body weight, which is consistent with the classic features of type 1 diabetes in the clinic. Several lines of evidence support that the core kinase in Hippo signaling, MST1, is highly associated with the neuronal cell death, and that it is defined as an apoptosis-promoting kinase (Li et al., 2018a,b). On the other hand, the downstream mediator of Hippo pathway, YAP, is widely expressed in human brain tumors and promotes glioblastoma growth. Therefore, YAP is proven to play a critical role in the normal human brain development (Orr et al., 2011). To further confirm the change of Hippo signaling in the central nervous system, we detected the expressions of MST1, LATS1 and p-YAP/AYP ratio in selected brain tissues among the groups. We here demonstrated that the levels of both MST1 and p-YAP/ YAP ratio were increased significantly in mPFC, ACC, striatum and hippocampus of diabetes-induced CD mice, but not Non-CD or control mice. We also found positive correlations between escape latency of MWMT results and levels of MST1 or p-YAP/YAP ratio in mPFC, ACC, striatum and hippocampus tissues. These interesting results provided a novel perspective to study the pathogenesis and further develop therapeutic strategies of CD caused by diabetes. Peripheral tissues, such as skeletal muscle and liver, are the emerging key roles in regulating the intake and utilization of blood glucose (Yamanaka et al., 2007). Severe lesions in peripheral tissues are tightly associated with the development of insulin resistance, and then trigger the
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onset of diabetes (Sugimoto et al., 2016). Although few studies about Hippo signaling pathway in peripheral tissues of CD rodents has been reported, this pathway is established to regulate peripheral insulin pathway and maintain glucose homeostasis by mediating the distinct expression of MST1, LATS1 or YAP (Iglesias et al., 2017). An in-vivo study shows that exceedingly active YAP could reduce plasma glucose levels via suppressing gluconeogenic gene expression and increase the size of liver (Hu et al., 2017). Given the fact that diabetic cardiomyopathy and diabetic nephropathy are two major pathological changes (Li et al., 2012), along with the strong association between gut-brain axis and cognitive impairment in our previous studies (Zhan et al., 2018, 2019), and the key role of energy synthesis and metabolism in skeletal muscle and liver tissues, we therefore enrolled peripheral tissues, including kidney, heart, liver, muscle, and gut to study Hippo signaling pathway in this study. Interestingly, we determined decreased expression of LATS1 in liver, kidney, skeletal muscle and gut tissues of mice in CD group, as well as p-Yap/YAP ratio. Positive correlations between escape latency and the level of LATS1 in liver, muscle and gut tissues were also exhibited. Intriguingly, we observed that lower level of p-YAP/ YAP ratio in kidney and muscle tissues of Non-CD mice than the controls. This finding is consistent with the role of Hippo signaling in glucose metabolism by mediating the down regulation of YAP (Hu et al., 2017). Collectively, different levels of Hippo signaling pathway in selected brain and peripheral tissues might have a causal linkage in the occurrence and progression of CD induced by diabetes. There are several limitations in this study. First, largesize number of mice is needed to diminish the discrepancy among the groups. Second, we did not adopt inhibitors of Hippo signaling, or interference plasmid with lentivirus vectors into mice to knock down the expression of Hippo signaling. Considering the pivotal role of Hippo signaling in diabetes-induced CD, further studies are required. In conclusion, our results suggest that alterations of Hippo signaling in selected brain and peripheral tissues may contribute to the incidence of CD caused by diabetes. Hence, therapeutic interventions improving Hippo signaling might be beneficial to the treatment of AD and other disorders characterized by cognitive function, which provides a new insight into the investigations of neurodegenerative diseases in the future.
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ACKNOWLEDGMENTS
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We thank the Beijing Genomics Institute for providing assistance with the data analysis of 16S rRNA sequencing.
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DECLARATION OF COMPETING INTEREST
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All the authors declare no conflicts of interest.
FUNDING
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This study was supported by grants from the National Natural Science Foundation of China (to A.L., 81974160, 81771159 and 81571047; to C.Y., 81974171 and 81703482), and was partially supported by the Program of Bureau of Science and Technology Foundation of Changzhou (to B.Z., CJ20159022; to L. Y., CJ20160030) and Major Science and Technology Projects of Changzhou Municipal Committee of Health and Family Planning (to B.Z., ZD201505; to L.Y., ZD201407) and Changzhou High-Level Medical Talents Training Project (to F.H., 2016ZCL J020).
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(Received 12 May 2019, Accepted 12 September 2019) (Available online xxxx)
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RESEARCH ARTICLE F. Yu et al. / Neuroscience xxx (2018) xxx–xxx
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Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice
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Fan Yu, a1 Wei Han, b1 Gaofeng Zhan, c Shan Li, c Xiaohong Jiang, a Shoukui Xiang, a Bin Zhu, d Ling Yang, e Dongyu Hua, c Ailin Luo, c Fei Hua a* and Chun Yang c*
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a
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b
Department of Neurosurgery, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
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c
Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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d
Department of Critical Care Medicine, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
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e
Department of Cardiology, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
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Abstract—Increasing studies have revealed that metabolic disorders, especially diabetes, are high risk factors for the development of Alzheimer’s disease (AD) and other neurodegenerative diseases. It has been reported that patients with diabetes are prone to suffer from cognitive dysfunction (CD). Although abnormal glucose metabolism and deposition of amyloid b (Ab) are proven to have a closely relationship with diabetes-induced CD, its exact mechanism is still undetermined. In this study, a total of 14 mice were intraperitoneally injected with streptozotocin for 5 consecutive days to mimic diabetic models, and then hierarchical cluster analysis was adopted to classify the diabetic mice into CD and Non-CD phenotypes by the results of Morris water maze test (MWMT). Furthermore, we detected Hippo signaling including mammalian sterile 20-like protein kinases1 (MST1), large tumor suppressors 1 (LATS1), Yes-associated protein (YAP) and phosphorylation of YAP (p-YAP) in brain and peripheral tissues. As compared with control mice, the levels of MST1, LATS1 and p-YAP/YAP ratio were increased in medial prefrontal cortex (mPFC), striatum and hippocampus of CD mice, while these proteins were decreased in gut tissue of CD mice. Additionally, there were significant positive correlations between escape latency and p-YAP/YAP ratio in mPFC, anterior cingulate cortex (ACC) and hippocampus, as well as the level of LATS1 in liver, kidney and gut tissues. In conclusion, alterations in Hippo signaling may contribute to CD induced by diabetes. Therefore, therapeutic interventions improving Hippo signaling might be beneficial to the treatment of diabetes-induced CD and other neurodegenerative diseases. Ó 2019 IBRO. Published by Elsevier Ltd.
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Department of Endocrinology, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
All rights reserved.
key words: Diabetes, Cognitive dysfunction, Hippo signaling, Streptozotocin.
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INTRODUCTION
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It is estimated that there are approximately 435 million people living with diabetes worldwide and the number will reach over 642 million by 2040 (Ingelfinger et al., 2017; Ogurtsova et al., 2017). The rising prevalence of diabetes all over the world has gained public concerns largely ascribing to its relevant long-term complications.
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Although macrovascular and microvascular complications of diabetes are well recognized, there is a lack of attentions on diabetes-induced cognitive dysfunction (CD) (Munshi, 2017). Meanwhile, it has been reported that patients with diabetes during midlife have a higher incidence of 19% than healthy individuals to experience CD (Rawlings et al., 2014). Both type 1 and type 2 diabetes are predisposing factors for the onset of CD, ultimately leading to dementia in animal models and clinical studies (Wong et al., 2014; Li et al., 2017; Yuan et al., 2017). Alzheimer’s disease (AD) is clinically characterized by CD or even dementia, which is highly related to abnormal accumulation and deposition of amyloid plaques and neurofibrillary tangles in pathology (Ow et al., 2014; Lane et al., 2018). Several lines of evidence have suggested that abnormal Ab deposition and impaired glucose regulation might both underlie the mechanisms of comorbidity in AD and
*Corresponding authors. E-mail addresses: [email protected] (F. Hua), [email protected] (C. Yang). 1 These authors contributed equally to this study. Abbreviations: Ab, Amyloid b; ACC, anterior cingulate cortex; AD, Alzheimer’s disease; ANOVA, analysis of variance; CD, cognitive dysfunction; CONT, control; LATS1, large tumor suppressors 1; mPFC, medial prefrontal cortex; MST1, mammalian sterile 20-like protein kinases1; MWMT, Morris water maze test; NAc, nucleus accumbens; N.S., not significant; p-YAP, phosphorylation of YAP; STZ, streptozotocin; YAP, Yes-associated protein. https://doi.org/10.1016/j.neuroscience.2019.09.018 0306-4522/Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 1
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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Fig. 1. Comparisons of metabolic parameters and behaviors of MWMT among CONT, CD and Non-CD groups. (A) The schedule for the experiment. Mice were injected with STZ (55 mg/kg) intraperitoneally on Day 1 after seven days accommodation. From Day 6 to 61, metabolic parameters were measured after five consecutive days of STZ injection. Mice were scheduled for MWMT from Day 62 to 66, and that probe trial was performed on Day 67. On Day 68, tissues were collected for analysis. (B) Body weight (two-way repeated ANOVA). (C) Water intake (two-way repeated ANOVA). (D) Food intake (two-way repeated ANOVA). (E) Glucose (two-way repeated ANOVA). (F) Dendrogram of hierarchical clustering analysis. Mice after STZ exposure were divided into CD and Non-CD groups by MWMT results of hierarchical clustering analysis. (G) Representative trace graphs of CONT, CD and Non-CD mice in MWMT. (H) Escape latency (two-way repeated ANOVA). (I) Escape path length (two-way repeated ANOVA). (J) Platform crossing (Fisher’s exact test). (K) Time spent in each quadrant (two-way repeated ANOVA). Data are shown as mean ± S.E.M. (n = 6–8). *P < 0.05, **P < 0.01 or ***P < 0.001. ANOVA: analysis of variance; CONT: control; CD: cognitive dysfunction; MWMT: Morris water maze test; N.S.: not significant; STZ: streptozotocin.
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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diabetes (Akter et al., 2011; Shinohara et al., 2017). However, the exact pathogenesis and interactions of the two diseases have not yet been fully elucidated (Huang et al., 2014). Therefore, studying the pathogenesis and exploring potential therapies of AD by investigating the mechanisms of CD caused by diabetes are greatly necessary. The Hippo signaling was first discovered in Drosophila using genetic manipulation and now has been successfully established and determined in mammals (Ji et al., 2017). The major physiologic functions of Hippo signaling are to limit tissue growth and control organ size, as well as to regulate metabolic homeostasis by modulating cellular proliferation, apoptosis and regeneration (Gumbiner et al., 2014; Li et al., 2017; Ardestani et al., 2018). In mammalian systems, Hippo signaling is composed of mammalian sterile 20-like protein kinases1 and 2 (MST1/2), large tumor suppressors1 and 2 (LATS1/2), Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) (Wang et al., 2015; Meng et al., 2016). Once the Hippo pathway is activated, upstream regulators directly initiate activation of MST1/2 and LATS1/2, and then active LATS1/2 phosphorylating YAP/TAZ at Serine 127, finally leading to YAP/TAZ cytoplasmic retention and promoting their ubiquitinproteasome degradation. By contrast, the inactivated state of Hippo pathway would help dephosphorylated YAP/TAZ to translocate into the nucleus, which is associated with neoplastic growth and occurrence of tumors (Plouffe et al., 2015; Yu et al., 2015; Ardestani et al., 2018). Hyper-activation of MST1 may have a significant impact on regulating proteolytic processing of the precursor of Ab (Jang et al., 2007; Tomiyama, 2010; Huang et al., 2012). Hence, excessive activation of Hippo signaling might contribute to the development of neurodegenerative diseases (Plouffe et al., 2015). Interestingly, recent findings have suggested that Hippo pathway has a bidirectional interaction with glucose metabolism (Wang et al., 2015; Peng et al., 2017). Collectively, we proposed that Hippo signaling pathway might play a vital role in the pathogenesis of diabetes-induced CD. For this end, we determined different expressions of Hippo signaling in selected brain and peripheral tissues
by Western blot analysis. Furthermore, correlations between escape latency of MWMT results and levels of Hippo signaling in selected brain and peripheral tissues were also performed to verify the causal linkage.
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EXPERIMENTAL PROCEDURES
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Animals
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All experimental protocols and animal handling procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publications No. 80-23, revised in 1996). This study was approved by the Experimental Animal Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Eight-week-old healthy male C57BL/6J mice weighing 20–25 g were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). A total of 26 mice used in this study were fed with food and water ad libitum, on a 12-h light/dark cycle schedule. The laboratory conditions were maintained with a consistent temperature at 22 °C ± 2 °C and a relative humidity of 60% ± 5%. All mice were allowed to acclimate for a week before experiments (Fig. 1A).
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MODELS OF TYPE 1 DIABETES MELLITUS
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A total of 20 mice were fasted for 12 h prior to treatment after 7 days accommodation to induce a model of type 1 diabetes (Kong et al., 2018). Subsequently, a fresh solution of 10 mg/ml STZ (Absin Bioscience Inc., Shanghai, China) that dissolved in 0.1 M sodium citrate buffer (pH4.5) was prepared, and then mice were injected intraperitoneally with STZ at a dose of 55 mg/kg for 5 consecutive days as previously described (Li et al., 2016). In addition, mice in control group were injected with the same volume of sodium citrate buffer. Metabolic parameters including body weight, water intake and food intake were recorded once a week, while fasting blood glucose was assessed every 2 weeks using a tail vein blood sample via OneTouchÒ Ultra blood glucose meter. Mice with fasting blood glucose levels more than 11.1 mmol/L were
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Table 1. Mean blood glucose. Week
0
2
4
6
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CONT (mmol/L) CD (mmol/L) Non-CD (mmol/L)
7.05 6.75 6.85
6.717 20.399 19.69
7.2 23.049 21.339
6.167 26.07 22.061
6.183 25.101 23.458
CD: cognitive dysfunction; CONT: control.
Table 2. Mean body weight. Week
0
1
2
3
4
5
6
7
8
CONT (g) CD (g) Non-CD (g)
23.47 23.82 23.94
24.23 21.43 22.2
23.57 19.94 20.53
24.08 20.74 21.29
24.28 21.10 21.74
24.12 20.93 21.28
24.6 20.85 21.55
24.42 20.43 21.02
24.6 20.23 20.96
CD: cognitive dysfunction; CONT: control.
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used in the following experiments (Zheng et al., 2018). Eight weeks later, mice were subjected to behavioral tests to evaluate cognitive function.
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Morris water maze test
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Spatial information acquisition and memory retention were assessed by the Morris water maze test (MWMT) after 8 weeks of the final STZ injection. A circular pool (diameter: 120 cm; height: 50 cm) was filled with warm (23 ± 1 °C) opaque water which was contained with nontoxic titanium white-colored dye. In the target quadrant, a movable clear platform with 15 cm in diameter was submerged 0.5–1 cm below water surface. The MWMT was performed 4 trials per day for consecutive 5 days to determine the ability of mice in spatial memory as previously described (Zhan et al., 2018, 2019). During each trial, all the mice were trained to find the hidden platform in 60 s on which they sat for 15 s before being removed from the pool. If a mouse did not find the platform within 60 s, it was gently guided to the platform and allowed to remain there for 15 s. For all training trials, time and distance taken to reach the platform were recorded. The less time it took a mouse to reach the platform, the better its learning ability (Han et al., 2013). On the day after finishing training, a probe test was conducted immediately to evaluate memory retention. The platform was removed from the pool and that mice were allowed to swim freely for 60 s. The number of platform crossing and time spent in each quadrant were recorded.
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Western blotting
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On day 68, all mice were anesthetized with 5% isoflurane and immediately sacrificed. The selected brain tissues and peripheral tissues of mice, including medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), nucleus accumbens (NAc), striatum, hippocampus, heart, liver, kidney, right anterior foot muscle and gut were dissected and collected. Samples were homogenized on ice in the presence of protease and phosphatase inhibitors that mixed in RIPA buffer (150 mM sodium chloride, Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50mMTris, pH 8.0) for 30 min, and then homogenates were centrifuged at 12,000 rpm at 4 °C for 15 min. Protein concentration in the supernatants was quantified via BCA protein assay kit (Boster, Wuhan, China). Proteins were analyzed by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes
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(Millipore, Bedford, MA, USA). Bands were blocked with 5% BSA in TBST (0.1% Tween 20 in Tris-buffered saline) at room temperature for 1 h. Appropriate primary antibodies were incubated at 4 °C overnight: rabbit antiMST1 (1:1000; Proteintech, Wuhan, China), rabbit antiLATS1 (1:1000; Absin Bioscience Inc., Shanghai, China), rabbit anti-p-YAP (1:1000; Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-YAP (1:1000; Proteintech, Wuhan, China). Afterwards, bands were washed with TBST and incubated with secondary antibodies at room temperature for 1.5 h: horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:5000; Affinity, Cincinnati, OH, USA). Finally, these bands were detected by enhanced chemiluminescence reagents (Abbkine, Wuhan, China) using the ChemiDocXRS chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA).
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Statistical analysis
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The data show as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Kolmogorov–Smirnov test was performed to test data normality, and that Levene test, Welch test or Brown-Forsythe test was used to test equality of variance. Data in this study were analyzed by one-way, two-way analysis of variance (ANOVA) or Fisher’s exact test, followed by Tukey’s multiple comparisons test or Sidak’s multiple comparisons test. In Hierarchical cluster analysis, the data were firstly standardized by z scores. Then, MWMT results (Escape latency on day 5 and platform crossing on the probe trial) were clustered via using Ward’s method and mice were classified as CD or Non-CD groups. A correlation analysis was conducted using Pearson’s product-moment coefficient. The Pvalues of less than 0.05 were considered statistically significant.
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RESULTS
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Comparisons of metabolic parameters and behaviors among CONT, CD and Non-CD groups
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Mice were randomly selected to construct type 1 diabetes models. Two months after final exposure of streptozotocin at a dose of 55 mg/kg (Fig. 1A), a total of 14 diabetic models were successfully established versus agematched control mice by comparing body weight, water intake, food intake and blood glucose (Fig. 1B–E). Obviously, STZ-treated mice significantly decreased body weight than mice in CONT group (Time: F8,80 = 1.89, P = 0.0729; Group: F1,10 = 17.41,
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3 Fig. 2. Levels of Hippo signaling in selected brain tissues among CONT, CD and Non-CD groups. (A) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in mPFC. (B) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in ACC. (C) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in NAc. (D) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in striatum. (E) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in hippocampus. Data are shown as mean ± S.E.M. (n = 6). *P < 0.05, **P < 0.01 or ***P < 0.001. ANOVA: analysis of variance; ACC: anterior cingulate cortex; CONT: control; CD: cognitive dysfunction; LATS1: large tumor suppressors 1; mPFC: medial prefrontal cortex; MST1: mammalian sterile 20-like protein kinases1; NAc: nucleus accumbens; N.S.: not significant; p-YAP: phosphorylation of YAP; YAP: Yes-associated protein.
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Interaction: F8,80 = 16.29, P < 0.001. Fig. 1C), food intake (Time: F8,80 = 2.307, P < 0.05; Group: F1,10 = 288.1, P < 0.001; Interaction: F8,80 = 2.526, P < 0.05. Fig. 1D) and blood glucose (Time: F4,40 = 27.65, P < 0.001; Group: F1,10 = 232.7, P < 0.001; Interaction: F4,40 = 31.23, P < 0.001. Fig. 1E) in STZ-treated mice. According to hierarchical clustering analysis of MWMT results, 14 mice confirmed as diabetic models were divided into CD (n = 6) and Non-CD (n = 8) groups (Fig. 1F). A notable difference of swimming traces in MWMT was represented among the three groups (Fig. 1G). Furthermore, there was a significant increase in escape latency (Time: F4,60 = 13.75, P < 0.001; Group: F2,15 = 8.466, P < 0.01; Interaction: F8,60 = 2.669, P < 0.05. Fig. 1H) and path length (Time: F4,60 = 11.17, P < 0.001; Group: F2,15 = 2.209, P > 0.05; Interaction: F8,60 = 3.085, P < 0.01. Fig. 1I) in CD group than those of CONT or Non-CD group on day 5. In platform crossing, CD mice showed a significant decrease than CONT or Non-CD mice (F2,15 = 9.286, P < 0.01. Fig. 1J). Additionally, mice in CD group spent significant less time in the target quadrant as compared to CONT or Non-CD mice (Time: F3,45 = 3.201, P < 0.05; Group: F2,15 = 5.672, P < 0.05; Interaction: F6,45 = 5.253, P < 0.001. Fig. 1K). Interestingly, we found that the mean blood glucose in CD mice was higher than that of Non-CD mice in 2–8 weeks (Table 1). Besides, the mean body weight in CD mice was slightly decreased than that of Non-CD mice (Table 2). It is therefore likely that STZ-treated mice with a higher level of blood glucose were more likely to suffer from CD.
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Differential levels of Hippo signaling in selected brain tissues among CONT, CD and Non-CD groups
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We performed Western blot analysis to determine the levels of Hippo signaling including MST1, LATS1, pYAP and YAP in selected brain tissues of CONT, CD and Non-CD mice (Fig. 2A–E). Compared with CONT group, mice in CD group showed a significant increase
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Fig. 3. Correlations between escape latency and levels of Hippo signaling in brain tissues (n = 14). (A) MST1, LATS1 and p-YAP/YAP ratio in mPFC. (B) MST1, LATS1 and p-YAP/YAP ratio in ACC. (C) MST1, LATS1 and p-YAP/YAP ratio in NAc. (D) MST1, LATS1 and p-YAP/YAP ratio in striatum. (E) MST1, LATS1 and p-YAP/YAP ratio in hippocampus. ACC: anterior cingulate cortex; LATS1: large tumor suppressors 1; mPFC: medial prefrontal cortex; MST1: mammalian sterile 20-like protein kinases1; NAc: nucleus accumbens; p-YAP: phosphorylation of YAP; YAP: Yes-associated protein.
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P < 0.01; Interaction: F8,80 = 1.89, P < 0.001. Fig. 1B). Two weeks after final STZ exposure, there was a palpable increase in water intake (Time: F8,80 = 16.29, P < 0.001; Group: F1,10 = 127.2, P < 0.001;
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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Fig. 4. Levels of Hippo signaling in peripheral tissues among CONT, CD and Non-CD groups. (A) MST1, LATS1 and YAP (one-way ANOVA) in heart. (B) MST1, LATS1, p-YAP/YAP ratio and YAP (one-way ANOVA) in liver. (C) MST1, LATS1, p-YAP/YAP ratio, YAP (one-way ANOVA) in kidney. (D) MST1, LATS1, p-YAP/YAP ratio, YAP (one-way ANOVA) in muscle. (E) MST1, LATS1, p-YAP/YAP ratio, YAP (one-way ANOVA) in gut. Data are shown as mean ± S.E.M. (n = 6). *P < 0.05, **P < 0.01 or ***P < 0.001. ANOVA: analysis of variance; CONT: control; CD: cognitive dysfunction; LATS1: large tumor suppressors 1; MST1: mammalian sterile 20-like protein kinases1; N.S.: not significant; p-YAP: phosphorylation of YAP; YAP: Yes-associated protein.
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those of CD group (Fig. 2A). In ACC, the levels of MST1 (F2,15 = 6.032, P < 0.05) and p-YAP/YAP ratio (F2,15 = 9.253, P < 0.01), but not LATS1 (F2,15 = 0.4474, P > 0.05), in CD mice were both higher than mice in CONT or Non-CD group (Fig. 2B). However, the levels of all proteins were not significantly different in NAc among CONT, CD and Non-CD groups (MST1: F2,15 = 0.0669, P > 0.05; LATS1: F2,15 = 0.5167, P > 0.05; p-YAP/ YAP ratio: F2,15 = 0.7625, P > 0.05. Fig. 2C). The levels of MST1 (F2,15 = 7.246, P < 0.01), LATS1 (F2,15 = 4.639, P < 0.05) and pYAP/YAP ratio (F2,15 = 3.272, P = 0.0554) were significantly higher in the striatum of CD mice compared to CONT mice. Although the level of MST1 was significantly increased in CD group than Non-CD group, no distinct difference was detected in the levels of LATS1 and p-YAP/YAP ratio (Fig. 2D). In addition, there was a significant increase in the levels of MST1 (F2,15 = 5.348, P < 0.05), LATS1 (F2,15 = 5.61, P < 0.05) and p-YAP/YAP ratio (F2,15 = 8.809, P < 0.01) in the hippocampus of CD mice as compared with CONT mice (Fig. 2E).
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Correlations between escape latency and levels of Hippo signaling in selected brain tissues
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Here we speculated that the incidence of CD in STZ-induced diabetic mice might be related to the alterations in Hippo signaling, including MST1, LATS1 and p-YAP. Given the important role of escape latency in MWMT, correlations between the escape latency and the expression of these proteins were analyzed (Fig. 3A–E). Consequently, there were significant positive correlations between the escape latency and the expression of LATS1 Fig. 5. Correlations between escape latency and levels of Hippo signaling in peripheral tissues (n = 14). (A) MST1, LATS1 and p-YAP/YAP ratio in heart. (B) MST1, LATS1 and p-YAP/YAP ratio (r = 0.5791, P < 0.05) and p-YAP/ in liver. (C) MST1, LATS1 and p-YAP/YAP ratio in kidney. (D) MST1, LATS1 and p-YAP/YAP ratio YAP ratio (r = 0.6078, P < 0.05) in in muscle. (E) MST1, LATS1 and p-YAP/YAP ratio in gut. LATS1: large tumor suppressors 1; mPFC (Fig. 3A), as well as MST1 MST1: mammalian sterile 20-like protein kinases1; p-YAP: phosphorylation of YAP; YAP: Yes(r = 0.6683, P < 0.05) and p-YAP/ associated protein. YAP ratio (r = 0.611, P < 0.05) in ACC (Fig. 3B). By contrast, there in the levels of MST1 (F2,15 = 6.237, P < 0.05) and was no correlation between the LATS1 (F2,15 = 5.706, P < 0.05), as well as in p-YAP/ escape latency and all proteins in NAc (MST1: YAP ratio (F2,15 = 9.449, P < 0.01). By contrast, the r = 0.08041, P > 0.05; LATS1: r = 0.3086, P > 0.05; levels of MST1, LATS1 and p-YAP/YAP ratio in the p-YAP/YAP ratio: r = 0.09736, P > 0.05. Fig. 3C). mPFC of Non-CD group were significantly lower than Moreover, a positive correlation between the escape
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latency and the level of MST1 (r = 0.6314, P < 0.05. Fig. 3D) was exhibited in striatum, as well as the ratio of p-YAP/YAP in the hippocampus (r = 0.6351, P < 0.05. Fig. 3E).
Differential levels of Hippo signaling in peripheral tissue among CONT, CD and Non-CD groups In peripheral tissues (heart, liver, kidney, skeletal muscle and gut), Western blot analysis was also adopted to evaluate the levels of MST1, LATS1 and p-YAP/YAP ratio (Fig. 4A–E). Obviously, a significant decrease in the expression of LATS1 (F2,15 = 1.003, P < 0.05) was noted in liver of CD group, but not in CONT and NonCD groups (Fig. 4B). The levels of LATS1 (F2,15 = 14.17, P < 0.001) and p-YAP/YAP ratio (F2,15 = 12.21, P < 0.001) were lower in kidney of CD mice than those in CONT, but no statistical difference in the ratio of p-YAP/YAP between CD and Non-CD groups. Interestingly, the expression of p-YAP/YAP ratio in Non-CD group was significantly lower than CONT group, associated with a downward trend towards the LATS1 expression in Non-CD group than CONT group (Fig. 4C). In skeletal muscle, there was a significant decrease in the levels of LATS1 (F2,15 = 3.469, P = 0.0578) and p-YAP/YAP ratio (F2,15 = 9.385, P < 0.01) in CD mice as compared with CONT mice, while the ratio of p-YAP/YAP was lower in Non-CD mice than that of CONT mice (Fig. 4D). As to the Hippo signaling in gut, mice in CD group showed a significant decrease in the levels of MST1 (F2,15 = 4.796, P < 0.05) and LATS1 (F2,15 = 6.251, P < 0.05) than CONT group, as well as in the ratio of p-YAP/YAP (F2,15 = 3.876, P < 0.05. Fig. 4E). Nevertheless, there were no significant changes in the levels of Hippo signaling in heart tissue (MST1: F2,15 = 0.1847, P > 0.05; LATS1: F2,15 = 0.003881, P > 0.05; p-YAP/ YAP ratio: F2,15 = 0.5127, P > 0.05. Fig. 4A).
Correlations between escape latency and levels of Hippo signaling in peripheral tissues We performed correlation analysis between escape latency of MWMT and levels of Hippo signaling in peripheral tissues. Similarly, correlations between the escape latency and the expressions of MST1, LATS1 and p-YAP/YAP ratio were analyzed (Fig. 5A–E). The results showed a significant positive correlation between the escape latency and the level of LATS1 in liver (r = 0.7414, P < 0.01. Fig. 5B), kidney (r = 0.6248, P < 0.05. Fig. 5C) and gut tissues (r = 0.7122, P < 0.01. Fig. 5E). However, there were no significant correlations between the escape latency and the levels of Hippo signaling in heart (MST1: r = 0.1291, P > 0.05; LATS1: r = 0.114, P > 0.05; p-YAP/YAP ratio: r = 0.0529, P > 0.05. Fig. 5A) and muscle tissues (MST1: r = 0.09339, P > 0.05; LATS1: r = 0.174, P > 0.05; p-YAP/YAP ratio: r = 0.4758, P > 0.05. Fig. 5D).
DISCUSSION
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Currently, a sharp increase in the prevalence of diabetesinduced CD has gained widespread attentions owing to its ambiguous pathogenesis and a lack of effective therapeutic strategies (Dik et al., 2007; Tanaka et al., 2019). In the present study, we observed the crucial role of Hippo signaling in the pathogenesis and progress of CD induced by diabetes. Differential expressions of Hippo signaling in selected brain and peripheral tissues, including increased levels of MST1 or p-YAP/YAP ratio in mPFC, ACC, striatum and hippocampus, while decreased levels of LATS1 in liver, kidney, skeletal muscle and gut tissues in diabetes-induced CD, implying a clue in the effective treatment of AD, dementia or other symptoms with CD. Accumulating studies have revealed that both T1DM and T2DM are high risk factors in the development of AD, accompanied by attenuated performance on multiple aspects of cognitive function (Karan et al., 2012). It is well recognized that individuals with T1DM are prone to have mild to moderate CD compared with non-diabetic controls (Li et al., 2017). In addition, abnormalities in glucose metabolism related to impaired insulin signaling in AD might suggest that diabetes-induced CD and neurodegenerative diseases may share a common underlying pathologic mechanism (Banks et al., 2012). We here successfully constructed type 1 diabetic rodent models with an intraperitoneal injection of STZ at a dose of 55 mg/kg for 5 consecutive days as previously described (Li et al., 2016). During the eight weeks of observation, STZ-treated mice significantly increased fluid intake, food intake and blood glucose levels, while decreased body weight, which is consistent with the classic features of type 1 diabetes in the clinic. Several lines of evidence support that the core kinase in Hippo signaling, MST1, is highly associated with the neuronal cell death, and that it is defined as an apoptosis-promoting kinase (Li et al., 2018a,b). On the other hand, the downstream mediator of Hippo pathway, YAP, is widely expressed in human brain tumors and promotes glioblastoma growth. Therefore, YAP is proven to play a critical role in the normal human brain development (Orr et al., 2011). To further confirm the change of Hippo signaling in the central nervous system, we detected the expressions of MST1, LATS1 and p-YAP/AYP ratio in selected brain tissues among the groups. We here demonstrated that the levels of both MST1 and p-YAP/ YAP ratio were increased significantly in mPFC, ACC, striatum and hippocampus of diabetes-induced CD mice, but not Non-CD or control mice. We also found positive correlations between escape latency of MWMT results and levels of MST1 or p-YAP/YAP ratio in mPFC, ACC, striatum and hippocampus tissues. These interesting results provided a novel perspective to study the pathogenesis and further develop therapeutic strategies of CD caused by diabetes. Peripheral tissues, such as skeletal muscle and liver, are the emerging key roles in regulating the intake and utilization of blood glucose (Yamanaka et al., 2007). Severe lesions in peripheral tissues are tightly associated with the development of insulin resistance, and then trigger the
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onset of diabetes (Sugimoto et al., 2016). Although few studies about Hippo signaling pathway in peripheral tissues of CD rodents has been reported, this pathway is established to regulate peripheral insulin pathway and maintain glucose homeostasis by mediating the distinct expression of MST1, LATS1 or YAP (Iglesias et al., 2017). An in-vivo study shows that exceedingly active YAP could reduce plasma glucose levels via suppressing gluconeogenic gene expression and increase the size of liver (Hu et al., 2017). Given the fact that diabetic cardiomyopathy and diabetic nephropathy are two major pathological changes (Li et al., 2012), along with the strong association between gut-brain axis and cognitive impairment in our previous studies (Zhan et al., 2018, 2019), and the key role of energy synthesis and metabolism in skeletal muscle and liver tissues, we therefore enrolled peripheral tissues, including kidney, heart, liver, muscle, and gut to study Hippo signaling pathway in this study. Interestingly, we determined decreased expression of LATS1 in liver, kidney, skeletal muscle and gut tissues of mice in CD group, as well as p-Yap/YAP ratio. Positive correlations between escape latency and the level of LATS1 in liver, muscle and gut tissues were also exhibited. Intriguingly, we observed that lower level of p-YAP/ YAP ratio in kidney and muscle tissues of Non-CD mice than the controls. This finding is consistent with the role of Hippo signaling in glucose metabolism by mediating the down regulation of YAP (Hu et al., 2017). Collectively, different levels of Hippo signaling pathway in selected brain and peripheral tissues might have a causal linkage in the occurrence and progression of CD induced by diabetes. There are several limitations in this study. First, largesize number of mice is needed to diminish the discrepancy among the groups. Second, we did not adopt inhibitors of Hippo signaling, or interference plasmid with lentivirus vectors into mice to knock down the expression of Hippo signaling. Considering the pivotal role of Hippo signaling in diabetes-induced CD, further studies are required. In conclusion, our results suggest that alterations of Hippo signaling in selected brain and peripheral tissues may contribute to the incidence of CD caused by diabetes. Hence, therapeutic interventions improving Hippo signaling might be beneficial to the treatment of AD and other disorders characterized by cognitive function, which provides a new insight into the investigations of neurodegenerative diseases in the future.
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ACKNOWLEDGMENTS
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We thank the Beijing Genomics Institute for providing assistance with the data analysis of 16S rRNA sequencing.
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DECLARATION OF COMPETING INTEREST
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All the authors declare no conflicts of interest.
FUNDING
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This study was supported by grants from the National Natural Science Foundation of China (to A.L., 81974160, 81771159 and 81571047; to C.Y., 81974171 and 81703482), and was partially supported by the Program of Bureau of Science and Technology Foundation of Changzhou (to B.Z., CJ20159022; to L. Y., CJ20160030) and Major Science and Technology Projects of Changzhou Municipal Committee of Health and Family Planning (to B.Z., ZD201505; to L.Y., ZD201407) and Changzhou High-Level Medical Talents Training Project (to F.H., 2016ZCL J020).
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(Received 12 May 2019, Accepted 12 September 2019) (Available online xxxx)
Please cite this article in press as: Yu F et al. Differential Levels of Hippo Signaling in Selected Brain and Peripheral Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice. Neuroscience (2019), https://doi.org/ 10.1016/j.neuroscience.2019.09.018
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