NSC 19280 No.

of Pages 11
31 October 2019

NEUROSCIENCE
1
RESEARCH ARTICLE
F. Yu et al. / Neuroscience xxx (2018) xxx–xxx

3
2 Differential Levels of Hippo Signaling in Selected Brain and Peripheral
4 Tissues in Streptozotocin-Induced Cognitive Dysfunction in Mice
5 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
6 Ailin Luo, c Fei Hua a* and Chun Yang c*
a
7 Department of Endocrinology, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
b
8 Department of Neurosurgery, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
c
9 Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
d
10 Department of Critical Care Medicine, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China
e
11 Department of Cardiology, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China

13
12 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 metabo-
lism 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 streptozo-
tocin for 5 consecutive days to mimic diabetic models, and then hierarchical cluster analysis was adopted to clas-
sify 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.
All rights reserved.

key words: Diabetes, Cognitive dysfunction, Hippo signaling, Streptozotocin.

14 INTRODUCTION Although macrovascular and microvascular complications 21

of diabetes are well recognized, there is a lack of atten- 22
15 It is estimated that there are approximately 435 million tions on diabetes-induced cognitive dysfunction (CD) 23
16 people living with diabetes worldwide and the number (Munshi, 2017). Meanwhile, it has been reported that 24
17 will reach over 642 million by 2040 (Ingelfinger et al., patients with diabetes during midlife have a higher inci- 25
18 2017; Ogurtsova et al., 2017). The rising prevalence of dence of 19% than healthy individuals to experience CD 26
19 diabetes all over the world has gained public concerns lar- (Rawlings et al., 2014). 27
20 gely ascribing to its relevant long-term complications. Both type 1 and type 2 diabetes are predisposing 28
factors for the onset of CD, ultimately leading to 29
dementia in animal models and clinical studies (Wong 30
*Corresponding authors.
et al., 2014; Li et al., 2017; Yuan et al., 2017). Alzheimer’s 31
E-mail addresses: czhuafei@vip.sina.com (F. Hua), yangchuntz@si-
na.com (C. Yang).
disease (AD) is clinically characterized by CD or even 32
1
These authors contributed equally to this study. dementia, which is highly related to abnormal accumula- 33
Abbreviations: Ab, Amyloid b; ACC, anterior cingulate cortex; AD, tion and deposition of amyloid plaques and neurofibrillary 34
Alzheimer’s disease; ANOVA, analysis of variance; CD, cognitive tangles in pathology (Ow et al., 2014; Lane et al., 2018). 35
dysfunction; CONT, control; LATS1, large tumor suppressors 1; mPFC,
medial prefrontal cortex; MST1, mammalian sterile 20-like protein Several lines of evidence have suggested that abnormal 36
kinases1; MWMT, Morris water maze test; NAc, nucleus accumbens; Ab deposition and impaired glucose regulation might both 37
N.S., not significant; p-YAP, phosphorylation of YAP; STZ, underlie the mechanisms of comorbidity in AD and 38
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.

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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31 October 2019

2 F. Yu et al. / Neuroscience xxx (2019) xxx–xxx

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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F. Yu et al. / Neuroscience xxx (2019) xxx–xxx 3

39 diabetes (Akter et al., 2011; Shinohara et al., 2017). How- by Western blot analysis. Furthermore, correlations 83
40 ever, the exact pathogenesis and interactions of the two between escape latency of MWMT results and levels of 84
41 diseases have not yet been fully elucidated (Huang Hippo signaling in selected brain and peripheral tissues 85
42 et al., 2014). Therefore, studying the pathogenesis and were also performed to verify the causal linkage. 86
43 exploring potential therapies of AD by investigating the
44 mechanisms of CD caused by diabetes are greatly EXPERIMENTAL PROCEDURES 87
45 necessary.
46 The Hippo signaling was first discovered in Drosophila Animals 88
47 using genetic manipulation and now has been All experimental protocols and animal handling 89
48 successfully established and determined in mammals (Ji procedures were carried out in strict accordance with 90
49 et al., 2017). The major physiologic functions of Hippo sig- the recommendations in the Guide for the Care and Use 91
50 naling are to limit tissue growth and control organ size, as of Laboratory Animals, published by the National 92
51 well as to regulate metabolic homeostasis by modulating Institutes of Health (NIH Publications No. 80-23, revised 93
52 cellular proliferation, apoptosis and regeneration in 1996). This study was approved by the Experimental 94
53 (Gumbiner et al., 2014; Li et al., 2017; Ardestani et al., Animal Committee of Tongji Hospital, Tongji Medical 95
54 2018). In mammalian systems, Hippo signaling is com- College, Huazhong University of Science and 96
55 posed of mammalian sterile 20-like protein kinases1 and Technology (Wuhan, China). 97
56 2 (MST1/2), large tumor suppressors1 and 2 (LATS1/2), Eight-week-old healthy male C57BL/6J mice weighing 98
57 Yes-associated protein (YAP) and transcriptional coacti- 20–25 g were purchased from Beijing Vital River 99
58 vator with PDZ-binding motif (TAZ) (Wang et al., 2015; Laboratory Animal Technology (Beijing, China). A total 100
59 Meng et al., 2016). Once the Hippo pathway is activated, of 26 mice used in this study were fed with food and 101
60 upstream regulators directly initiate activation of MST1/2 water ad libitum, on a 12-h light/dark cycle schedule. 102
61 and LATS1/2, and then active LATS1/2 phosphorylating The laboratory conditions were maintained with a 103
62 YAP/TAZ at Serine 127, finally leading to YAP/TAZ cyto- consistent temperature at 22 °C ± 2 °C and a relative 104
63 plasmic retention and promoting their ubiquitin- humidity of 60% ± 5%. All mice were allowed to 105
64 proteasome degradation. By contrast, the inactivated acclimate for a week before experiments (Fig. 1A). 106
65 state of Hippo pathway would help dephosphorylated
66 YAP/TAZ to translocate into the nucleus, which is associ-
67 ated with neoplastic growth and occurrence of tumors MODELS OF TYPE 1 DIABETES MELLITUS 107

68 (Plouffe et al., 2015; Yu et al., 2015; Ardestani et al., A total of 20 mice were fasted for 12 h prior to treatment 108
69 2018). after 7 days accommodation to induce a model of type 1 109
70 Hyper-activation of MST1 may have a significant diabetes (Kong et al., 2018). Subsequently, a fresh solu- 110
71 impact on regulating proteolytic processing of the tion of 10 mg/ml STZ (Absin Bioscience Inc., Shanghai, 111
72 precursor of Ab (Jang et al., 2007; Tomiyama, 2010; China) that dissolved in 0.1 M sodium citrate buffer 112
73 Huang et al., 2012). Hence, excessive activation of Hippo (pH4.5) was prepared, and then mice were injected 113
74 signaling might contribute to the development of neurode- intraperitoneally with STZ at a dose of 55 mg/kg for 5 con- 114
75 generative diseases (Plouffe et al., 2015). Interestingly, secutive days as previously described (Li et al., 2016). In 115
76 recent findings have suggested that Hippo pathway has addition, mice in control group were injected with the 116
77 a bidirectional interaction with glucose metabolism same volume of sodium citrate buffer. Metabolic parame- 117
78 (Wang et al., 2015; Peng et al., 2017). Collectively, we ters including body weight, water intake and food intake 118
79 proposed that Hippo signaling pathway might play a vital were recorded once a week, while fasting blood glucose 119
80 role in the pathogenesis of diabetes-induced CD. was assessed every 2 weeks using a tail vein blood sam- 120
81 For this end, we determined different expressions of ple via OneTouchÒ Ultra blood glucose meter. Mice with 121
82 Hippo signaling in selected brain and peripheral tissues fasting blood glucose levels more than 11.1 mmol/L were 122

Table 1. Mean blood glucose.

Week 0 2 4 6 8

CONT (mmol/L) 7.05 6.717 7.2 6.167 6.183

CD (mmol/L) 6.75 20.399 23.049 26.07 25.101
Non-CD (mmol/L) 6.85 19.69 21.339 22.061 23.458
CD: cognitive dysfunction; CONT: control.

Table 2. Mean body weight.

Week 0 1 2 3 4 5 6 7 8

CONT (g) 23.47 24.23 23.57 24.08 24.28 24.12 24.6 24.42 24.6
CD (g) 23.82 21.43 19.94 20.74 21.10 20.93 20.85 20.43 20.23
Non-CD (g) 23.94 22.2 20.53 21.29 21.74 21.28 21.55 21.02 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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31 October 2019

4 F. Yu et al. / Neuroscience xxx (2019) xxx–xxx

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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31 October 2019

F. Yu et al. / Neuroscience xxx (2019) xxx–xxx 5

123 used in the following experiments (Zheng et al., 2018). (Millipore, Bedford, MA, USA). Bands were blocked with 171
124 Eight weeks later, mice were subjected to behavioral tests 5% BSA in TBST (0.1% Tween 20 in Tris-buffered 172
125 to evaluate cognitive function. saline) at room temperature for 1 h. Appropriate primary 173
antibodies were incubated at 4 °C overnight: rabbit anti- 174
MST1 (1:1000; Proteintech, Wuhan, China), rabbit anti- 175
126 Morris water maze test
LATS1 (1:1000; Absin Bioscience Inc., Shanghai, 176
127 Spatial information acquisition and memory retention China), rabbit anti-p-YAP (1:1000; Cell Signaling 177
128 were assessed by the Morris water maze test (MWMT) Technology, Danvers, MA, USA) and rabbit anti-YAP 178
129 after 8 weeks of the final STZ injection. A circular pool (1:1000; Proteintech, Wuhan, China). Afterwards, bands 179
130 (diameter: 120 cm; height: 50 cm) was filled with warm were washed with TBST and incubated with secondary 180
131 (23 ± 1 °C) opaque water which was contained with antibodies at room temperature for 1.5 h: horseradish 181
132 nontoxic titanium white-colored dye. In the target peroxidase-conjugated goat anti-rabbit IgG antibody 182
133 quadrant, a movable clear platform with 15 cm in (1:5000; Affinity, Cincinnati, OH, USA). Finally, these 183
134 diameter was submerged 0.5–1 cm below water surface. bands were detected by enhanced chemiluminescence 184
135 The MWMT was performed 4 trials per day for reagents (Abbkine, Wuhan, China) using the 185
136 consecutive 5 days to determine the ability of mice in ChemiDocXRS chemiluminescence imaging system 186
137 spatial memory as previously described (Zhan et al., (Bio-Rad, Hercules, CA, USA). 187
138 2018, 2019). During each trial, all the mice were trained
139 to find the hidden platform in 60 s on which they sat for Statistical analysis 188
140 15 s before being removed from the pool. If a mouse did
141 not find the platform within 60 s, it was gently guided to The data show as the mean ± standard error of the mean 189

142 the platform and allowed to remain there for 15 s. For all (SEM). Statistical analyses were performed using 190

143 training trials, time and distance taken to reach the plat- GraphPad Prism 7 (GraphPad Software, San Diego, 191

144 form were recorded. The less time it took a mouse to CA, USA). Kolmogorov–Smirnov test was performed to 192

145 reach the platform, the better its learning ability (Han test data normality, and that Levene test, Welch test or 193

146 et al., 2013). On the day after finishing training, a probe Brown-Forsythe test was used to test equality of 194

147 test was conducted immediately to evaluate memory variance. Data in this study were analyzed by one-way, 195

148 retention. The platform was removed from the pool and two-way analysis of variance (ANOVA) or Fisher’s exact 196

149 that mice were allowed to swim freely for 60 s. The num- test, followed by Tukey’s multiple comparisons test or 197

150 ber of platform crossing and time spent in each quadrant Sidak’s multiple comparisons test. In Hierarchical cluster 198

151 were recorded. analysis, the data were firstly standardized by z scores. 199
Then, MWMT results (Escape latency on day 5 and 200
platform crossing on the probe trial) were clustered via 201
152 Western blotting using Ward’s method and mice were classified as CD or 202

153 On day 68, all mice were anesthetized with 5% isoflurane Non-CD groups. A correlation analysis was conducted 203

154 and immediately sacrificed. The selected brain tissues using Pearson’s product-moment coefficient. The P- 204

155 and peripheral tissues of mice, including medial values of less than 0.05 were considered statistically 205

156 prefrontal cortex (mPFC), anterior cingulate cortex significant. 206

157 (ACC), nucleus accumbens (NAc), striatum,

158 hippocampus, heart, liver, kidney, right anterior foot RESULTS 207
159 muscle and gut were dissected and collected. Samples
Comparisons of metabolic parameters and behaviors 208
160 were homogenized on ice in the presence of protease
among CONT, CD and Non-CD groups 209
161 and phosphatase inhibitors that mixed in RIPA buffer
162 (150 mM sodium chloride, Triton X-100, 0.5% sodium Mice were randomly selected to construct type 1 diabetes 210
163 deoxycholate, 0.1% sodium dodecyl sulfate, 50mMTris, models. Two months after final exposure of streptozotocin 211
164 pH 8.0) for 30 min, and then homogenates were at a dose of 55 mg/kg (Fig. 1A), a total of 14 diabetic 212
165 centrifuged at 12,000 rpm at 4 °C for 15 min. Protein models were successfully established versus age- 213
166 concentration in the supernatants was quantified via matched control mice by comparing body weight, water 214
167 BCA protein assay kit (Boster, Wuhan, China). Proteins intake, food intake and blood glucose (Fig. 1B–E). 215
168 were analyzed by 10% sodium dodecyl sulfate- Obviously, STZ-treated mice significantly decreased 216
169 polyacrylamide gel electrophoresis (SDS-PAGE), and body weight than mice in CONT group (Time: 217
170 transferred to polyvinylidene difluoride membranes F8,80 = 1.89, P = 0.0729; Group: F1,10 = 17.41, 218

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, 223

P < 0.001. Fig. 1C), food intake 224
(Time: F8,80 = 2.307, P < 0.05; 225
Group: F1,10 = 288.1, P < 0.001; 226
Interaction: F8,80 = 2.526, P < 0.05. 227
Fig. 1D) and blood glucose (Time: 228
F4,40 = 27.65, P < 0.001; Group: 229
F1,10 = 232.7, P < 0.001; 230
Interaction: F4,40 = 31.23, 231
P < 0.001. Fig. 1E) in STZ-treated 232
mice. According to hierarchical 233
clustering analysis of MWMT results, 234
14 mice confirmed as diabetic 235
models were divided into CD (n = 6) 236
and Non-CD (n = 8) groups 237
(Fig. 1F). A notable difference of 238
swimming traces in MWMT was 239
represented among the three groups 240
(Fig. 1G). Furthermore, there was a 241
significant increase in escape 242
latency (Time: F4,60 = 13.75, 243
P < 0.001; Group: F2,15 = 8.466, 244
P < 0.01; Interaction: F8,60 = 2.669, 245
P < 0.05. Fig. 1H) and path length 246
(Time: F4,60 = 11.17, P < 0.001; 247
Group: F2,15 = 2.209, P > 0.05; 248
Interaction: F8,60 = 3.085, P < 0.01. 249
Fig. 1I) in CD group than those of 250
CONT or Non-CD group on day 5. In 251
platform crossing, CD mice showed 252
a significant decrease than CONT or 253
Non-CD mice (F2,15 = 9.286, 254
P < 0.01. Fig. 1J). Additionally, mice 255
in CD group spent significant less 256
time in the target quadrant as 257
compared to CONT or Non-CD mice 258
(Time: F3,45 = 3.201, P < 0.05; 259
Group: F2,15 = 5.672, P < 0.05; 260
Interaction: F6,45 = 5.253, 261
P < 0.001. Fig. 1K). Interestingly, 262
we found that the mean blood 263
glucose in CD mice was higher than 264
that of Non-CD mice in 2–8 weeks 265
(Table 1). Besides, the mean body 266
weight in CD mice was slightly 267
decreased than that of Non-CD mice 268
(Table 2). It is therefore likely that 269
STZ-treated mice with a higher level 270
of blood glucose were more likely to 271
suffer from CD. 272
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 Differential levels of Hippo 273
ratio in striatum. (E) MST1, LATS1 and p-YAP/YAP ratio in hippocampus. ACC: anterior cingulate signaling in selected brain tissues 274
cortex; LATS1: large tumor suppressors 1; mPFC: medial prefrontal cortex; MST1: mammalian among CONT, CD and Non-CD 275
sterile 20-like protein kinases1; NAc: nucleus accumbens; p-YAP: phosphorylation of YAP; YAP:
groups 276
Yes-associated protein.
We performed Western blot analysis 277
to determine the levels of Hippo 278
219 P < 0.01; Interaction: F8,80 = 1.89, P < 0.001. Fig. 1B). signaling including MST1, LATS1, p- 279
220 Two weeks after final STZ exposure, there was a YAP and YAP in selected brain tissues of CONT, CD 280
221 palpable increase in water intake (Time: F8,80 = 16.29, and Non-CD mice (Fig. 2A–E). Compared with CONT 281
222 P < 0.001; Group: F1,10 = 127.2, P < 0.001; group, mice in CD group showed a significant increase 282

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/
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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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8 F. Yu et al. / Neuroscience xxx (2019) xxx–xxx

those of CD group (Fig. 2A). In ACC, 288

the levels of MST1 (F2,15 = 6.032, 289
P < 0.05) and p-YAP/YAP ratio 290
(F2,15 = 9.253, P < 0.01), but not 291
LATS1 (F2,15 = 0.4474, P > 0.05), 292
in CD mice were both higher than 293
mice in CONT or Non-CD group 294
(Fig. 2B). However, the levels of all 295
proteins were not significantly 296
different in NAc among CONT, CD 297
and Non-CD groups (MST1: 298
F2,15 = 0.0669, P > 0.05; LATS1: 299
F2,15 = 0.5167, P > 0.05; p-YAP/ 300
YAP ratio: F2,15 = 0.7625, P > 0.05. 301
Fig. 2C). The levels of MST1 302
(F2,15 = 7.246, P < 0.01), LATS1 303
(F2,15 = 4.639, P < 0.05) and p- 304
YAP/YAP ratio (F2,15 = 3.272, 305
P = 0.0554) were significantly higher 306
in the striatum of CD mice compared 307
to CONT mice. Although the level of 308
MST1 was significantly increased in 309
CD group than Non-CD group, no 310
distinct difference was detected in 311
the levels of LATS1 and p-YAP/YAP 312
ratio (Fig. 2D). In addition, there was 313
a significant increase in the levels of 314
MST1 (F2,15 = 5.348, P < 0.05), 315
LATS1 (F2,15 = 5.61, P < 0.05) and 316
p-YAP/YAP ratio (F2,15 = 8.809, 317
P < 0.01) in the hippocampus of CD 318
mice as compared with CONT mice 319
(Fig. 2E). 320

Correlations between escape 321

latency and levels of Hippo 322
signaling in selected brain tissues 323

Here we speculated that the 324

incidence of CD in STZ-induced 325
diabetic mice might be related to the 326
alterations in Hippo signaling, 327
including MST1, LATS1 and p-YAP. 328
Given the important role of escape 329
latency in MWMT, correlations 330
between the escape latency and the 331
expression of these proteins were 332
analyzed (Fig. 3A–E). Consequently, 333
there were significant positive 334
correlations between the escape 335
Fig. 5. Correlations between escape latency and levels of Hippo signaling in peripheral tissues latency and the expression of LATS1 336
(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/ 337
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 338
in muscle. (E) MST1, LATS1 and p-YAP/YAP ratio in gut. LATS1: large tumor suppressors 1;
MST1: mammalian sterile 20-like protein kinases1; p-YAP: phosphorylation of YAP; YAP: Yes- mPFC (Fig. 3A), as well as MST1 339

associated protein. (r = 0.6683, P < 0.05) and p-YAP/ 340

YAP ratio (r = 0.611, P < 0.05) in 341
ACC (Fig. 3B). By contrast, there 342
283 in the levels of MST1 (F2,15 = 6.237, P < 0.05) and was no correlation between the 343
284 LATS1 (F2,15 = 5.706, P < 0.05), as well as in p-YAP/ escape latency and all proteins in NAc (MST1: 344
285 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; 345
286 levels of MST1, LATS1 and p-YAP/YAP ratio in the p-YAP/YAP ratio: r = 0.09736, P > 0.05. Fig. 3C). 346
287 mPFC of Non-CD group were significantly lower than Moreover, a positive correlation between the escape 347

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/
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348 latency and the level of MST1 (r = 0.6314, P < 0.05. DISCUSSION 403
349 Fig. 3D) was exhibited in striatum, as well as the ratio of
350 p-YAP/YAP in the hippocampus (r = 0.6351, P < 0.05. Currently, a sharp increase in the prevalence of diabetes- 404

351 Fig. 3E). induced CD has gained widespread attentions owing to its 405
ambiguous pathogenesis and a lack of effective 406
therapeutic strategies (Dik et al., 2007; Tanaka et al., 407
2019). In the present study, we observed the crucial role 408
352 Differential levels of Hippo signaling in peripheral of Hippo signaling in the pathogenesis and progress of 409
353 tissue among CONT, CD and Non-CD groups CD induced by diabetes. Differential expressions of Hippo 410
signaling in selected brain and peripheral tissues, includ- 411
354 In peripheral tissues (heart, liver, kidney, skeletal muscle ing increased levels of MST1 or p-YAP/YAP ratio in 412
355 and gut), Western blot analysis was also adopted to mPFC, ACC, striatum and hippocampus, while decreased 413
356 evaluate the levels of MST1, LATS1 and p-YAP/YAP levels of LATS1 in liver, kidney, skeletal muscle and gut 414
357 ratio (Fig. 4A–E). Obviously, a significant decrease in tissues in diabetes-induced CD, implying a clue in the 415
358 the expression of LATS1 (F2,15 = 1.003, P < 0.05) was effective treatment of AD, dementia or other symptoms 416
359 noted in liver of CD group, but not in CONT and Non- with CD. 417
360 CD groups (Fig. 4B). The levels of LATS1 Accumulating studies have revealed that both T1DM 418
361 (F2,15 = 14.17, P < 0.001) and p-YAP/YAP ratio and T2DM are high risk factors in the development of 419
362 (F2,15 = 12.21, P < 0.001) were lower in kidney of CD AD, accompanied by attenuated performance on 420
363 mice than those in CONT, but no statistical difference in multiple aspects of cognitive function (Karan et al., 421
364 the ratio of p-YAP/YAP between CD and Non-CD 2012). It is well recognized that individuals with T1DM 422
365 groups. Interestingly, the expression of p-YAP/YAP ratio are prone to have mild to moderate CD compared with 423
366 in Non-CD group was significantly lower than CONT non-diabetic controls (Li et al., 2017). In addition, abnor- 424
367 group, associated with a downward trend towards the malities in glucose metabolism related to impaired insulin 425
368 LATS1 expression in Non-CD group than CONT group signaling in AD might suggest that diabetes-induced CD 426
369 (Fig. 4C). In skeletal muscle, there was a significant and neurodegenerative diseases may share a common 427
370 decrease in the levels of LATS1 (F2,15 = 3.469, underlying pathologic mechanism (Banks et al., 2012). 428
371 P = 0.0578) and p-YAP/YAP ratio (F2,15 = 9.385, We here successfully constructed type 1 diabetic rodent 429
372 P < 0.01) in CD mice as compared with CONT mice, models with an intraperitoneal injection of STZ at a dose 430
373 while the ratio of p-YAP/YAP was lower in Non-CD mice of 55 mg/kg for 5 consecutive days as previously 431
374 than that of CONT mice (Fig. 4D). As to the Hippo described (Li et al., 2016). During the eight weeks of 432
375 signaling in gut, mice in CD group showed a significant observation, STZ-treated mice significantly increased 433
376 decrease in the levels of MST1 (F2,15 = 4.796, fluid intake, food intake and blood glucose levels, while 434
377 P < 0.05) and LATS1 (F2,15 = 6.251, P < 0.05) than decreased body weight, which is consistent with the clas- 435
378 CONT group, as well as in the ratio of p-YAP/YAP sic features of type 1 diabetes in the clinic. 436
379 (F2,15 = 3.876, P < 0.05. Fig. 4E). Nevertheless, there Several lines of evidence support that the core kinase 437
380 were no significant changes in the levels of Hippo in Hippo signaling, MST1, is highly associated with the 438
381 signaling in heart tissue (MST1: F2,15 = 0.1847, neuronal cell death, and that it is defined as an 439
382 P > 0.05; LATS1: F2,15 = 0.003881, P > 0.05; p-YAP/ apoptosis-promoting kinase (Li et al., 2018a,b). On the 440
383 YAP ratio: F2,15 = 0.5127, P > 0.05. Fig. 4A). other hand, the downstream mediator of Hippo pathway, 441
YAP, is widely expressed in human brain tumors and pro- 442
motes glioblastoma growth. Therefore, YAP is proven to 443
play a critical role in the normal human brain development 444
384 Correlations between escape latency and levels of
(Orr et al., 2011). To further confirm the change of Hippo 445
385 Hippo signaling in peripheral tissues
signaling in the central nervous system, we detected the 446
386 We performed correlation analysis between escape expressions of MST1, LATS1 and p-YAP/AYP ratio in 447
387 latency of MWMT and levels of Hippo signaling in selected brain tissues among the groups. We here 448
388 peripheral tissues. Similarly, correlations between the demonstrated that the levels of both MST1 and p-YAP/ 449
389 escape latency and the expressions of MST1, LATS1 YAP ratio were increased significantly in mPFC, ACC, 450
390 and p-YAP/YAP ratio were analyzed (Fig. 5A–E). The striatum and hippocampus of diabetes-induced CD mice, 451
391 results showed a significant positive correlation between but not Non-CD or control mice. We also found positive 452
392 the escape latency and the level of LATS1 in liver correlations between escape latency of MWMT results 453
393 (r = 0.7414, P < 0.01. Fig. 5B), kidney (r = 0.6248, and levels of MST1 or p-YAP/YAP ratio in mPFC, ACC, 454
394 P < 0.05. Fig. 5C) and gut tissues (r = 0.7122, striatum and hippocampus tissues. These interesting 455
395 P < 0.01. Fig. 5E). However, there were no significant results provided a novel perspective to study the patho- 456
396 correlations between the escape latency and the levels genesis and further develop therapeutic strategies of 457
397 of Hippo signaling in heart (MST1: r = 0.1291, CD caused by diabetes. 458
398 P > 0.05; LATS1: r = 0.114, P > 0.05; p-YAP/YAP Peripheral tissues, such as skeletal muscle and liver, 459
399 ratio: r = 0.0529, P > 0.05. Fig. 5A) and muscle tissues are the emerging key roles in regulating the intake and 460
400 (MST1: r = 0.09339, P > 0.05; LATS1: r = 0.174, utilization of blood glucose (Yamanaka et al., 2007). Sev- 461
401 P > 0.05; p-YAP/YAP ratio: r = 0.4758, P > 0.05. ere lesions in peripheral tissues are tightly associated with 462
402 Fig. 5D). the development of insulin resistance, and then trigger the 463

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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10 F. Yu et al. / Neuroscience xxx (2019) xxx–xxx

464 onset of diabetes (Sugimoto et al., 2016). Although few FUNDING 519
465 studies about Hippo signaling pathway in peripheral tis-
466 sues of CD rodents has been reported, this pathway is This study was supported by grants from the National 520

467 established to regulate peripheral insulin pathway and Natural Science Foundation of China (to A.L., 521

468 maintain glucose homeostasis by mediating the distinct 81974160, 81771159 and 81571047; to C.Y., 81974171 522

469 expression of MST1, LATS1 or YAP (Iglesias et al., and 81703482), and was partially supported by the 523

470 2017). An in-vivo study shows that exceedingly active Program of Bureau of Science and Technology 524

471 YAP could reduce plasma glucose levels via suppressing Foundation of Changzhou (to B.Z., CJ20159022; to L. 525

472 gluconeogenic gene expression and increase the size of Y., CJ20160030) and Major Science and Technology 526

473 liver (Hu et al., 2017). Given the fact that diabetic car- Projects of Changzhou Municipal Committee of Health 527

474 diomyopathy and diabetic nephropathy are two major and Family Planning (to B.Z., ZD201505; to L.Y., 528

475 pathological changes (Li et al., 2012), along with the ZD201407) and Changzhou High-Level Medical Talents 529

476 strong association between gut-brain axis and cognitive Training Project (to F.H., 2016ZCL J020). 530

477 impairment in our previous studies (Zhan et al., 2018,

478 2019), and the key role of energy synthesis and metabo- REFERENCES 531
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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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664 (Received 12 May 2019, Accepted 12 September 2019)
665 (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