- Email: [email protected]
The correlation between adiponectin and FGF9 in depression disorder
Journal Pre-proofs Research report The correlation between adiponectin and FGF9 in depression disorder Xiao-Qing Wang, Wei-Hui Li, Ya-Hui Tang, Li-Feng Wu, Gui-Rong Zeng, YuHong Wang, Ze-Neng Cheng, De-Jian Jiang PII: DOI: Reference:
S0006-8993(19)30650-X https://doi.org/10.1016/j.brainres.2019.146596 BRES 146596
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
14 September 2019 2 December 2019 7 December 2019
Please cite this article as: X-Q. Wang, W-H. Li, Y-H. Tang, L-F. Wu, G-R. Zeng, Y-H. Wang, Z-N. Cheng, D-J. Jiang, The correlation between adiponectin and FGF9 in depression disorder, Brain Research (2019), doi: https:// doi.org/10.1016/j.brainres.2019.146596
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
The correlation between adiponectin and FGF9 in depression disorder Xiao-Qing Wanga,b, Wei-Hui Lic, Ya-Hui Tangb, Li-Feng Wub, Gui-Rong Zengb, YuHong Wangd, Ze-Neng Chenga,*, De-Jian Jiangb, d,* (a. XiangYa Pharmacy School, Central South University, Changsha 410083; b.
Hunan Center for Safety Evaluation and Research of Drugs & Hunan Key
Laboratory for Pharmacodynamics and Safety Evaluation of new drugs, Changsha 410013; c.
Department of Psychiatry, The Second XiangYa Hospital of Central South
University, Changsha 410011; d.
Institute of
Innovation and Applied Research in Chinese Medicine, Hunan
University of Chinese Medicine, Changsha 410208 ) *Corresponding authors Prof. De-Jian Jiang, E-mail address: [email protected] Phone: 86 731 83285157 Fax: 86 731 83285166 Prof. Ze-Neng Cheng, E-mail address: [email protected] Phone: 86 13874879386
1. Introduction Depression is a debilitating mental disease, characterized by persistent low mood and anhedonia. With growing incidences in the recent years, major depression disorder (MDD) has become one of the most common psychiatric disorders. Stress represents a major environmental risk factor for depression, which can induce these imbalances of between some endogenous anti-depressive and pro-depressive factors resulting in individual vulnerability or resilience in affective disorders (Suppes et al., 2017). The
stress-induced depression was also evidenced in animal models with chronic unpredictable mild stress (CUMS). These core symptoms of depression, such as weight loss and decreased response to reward as well as lack of pleasure, which leading to less sweet liquid consumption, were exerted in CUMS-simulated animals (Li et al., 2019). Adiponectin (ADPN) is an adipocytokine secreted by adipocytes. Studies discovered that ADPN has the functions of anti-inflammation, enhancing insulin sensitivity, improving lipid metabolism and anti-hyperglycemia (Bednarska-Makaruk et al., 2017). Other studies have shown that there is a close relationship between ADPN and the occurrence and development of depression. For example, Cizza et al. (Cizza et al., 2010) and Lehto et al. (Lehto et al., 2009) found that the serum ADPN level of depressive patients was significantly lower than that of the control group. A metaanalysis in six studies with a total of 4,220 subjects indicates that patients with depression had a lower ADPN level when compared to healthy subjects in European groups (Hu et al., 2015). In some animal studies, the negative correlation between serum level of ADPN and depression-like behavior was also reported. Compared with wild type mice, the plasma level of ADPN in adiponectin gene knockout (Adipo-/-) mice was significantly lower and their susceptibility to depression-like behavior was increased (Liu et al., 2012). FSL rats, a genetically modified depression model, have the characteristics of ADPN signaling pathway disorder compared with normal SD rats (Wilhelm et al., 2013). Though it is widely thought as an important antidepressive factor, ADPN how to exert its protective effects on depression is not unclear yet. Histopathological studies of postmortem brain tissue reveal prominent pathology in MDD. Some studies reported decreases in the packing density or number of the
general, Nissl-stained populations of glial cells in subjects diagnosed with MDD as compared to non-psychiatric control subjects. The studies indicated that changes were observed in fronto-limbic brain regions including the dorsolateral prefrontal cortex (Rajkowska et al., 1999; Cotter et al., 2002; Torres-Platas et al., 2002), orbitofrontal cortex (Rajkowska et al., 1999), subgenual cortex (Ongür et al., 1998) anterior cingulate cortex (Cotter et al., 2001; Gittins et al., 2011) and amygdala (Bowley et al., 2002). Both gene expression profiling in postmortem human brain and studies using animal models have implicated the fibroblast growth factor (FGF) family in affect regulation and suggest a potential role in the pathophysiology of MDD (Bernard et al., 2011). As a factor functional opposite to FGF2, which widely reported as a antidepressive factor, FGF9 was recently thought to be a key mediator in MDD via FGF receptor 3 (FGFR3)-dependent pathway (Aurbach et al., 2015). The level of FGF9 were elevated in both patients and animals model with depression. Both endogenous and exogenous FGF9 promotes anxiety- and depression-like behavior (Aurbach et al., 2015). Conversely, localized blockade of endogenous FGF9 expression decreases anxiety behavior. FGF9 represents a novel target for treating affective disorders (Aurbach et al., 2015). There are a few studies to show a regulation each other between adipocytederived adiponectin and FGF family members such as FGF15/19/21, which share common signaling cascades and exert similar beneficial functions in alcoholic liver disease (Yang et al., 2019) and atherosclerosis (Kikai et al., 2018). Based on these literatures mentioned above, we hypothesized that there are certain interactions between ADPN and FGF9, and the disorder of ADPN-FGF9/FGFR3 pathway is involved in progression of depression. In the present study, we observed firstly the correlation between ADPN and FGF9 levels in plasma and (or) hippocampus tissues
in patents with depression and in different month old adipo-/- mice. Furthermore, the role of ADPN-FGF9/FGFR3 pathway in depression was studied using injection of either exogenous ADPN or antibody of FGF9 in CUMS-induced normal or adipo-/mice. 2
Results
2.1 Human study There was no significant difference in body mass index (BMI) between depression patients and healthy people (P ＞ 0.05). Serum levels of ADPN and FGF9 were detected. Compared with non-depressive subjects, the decreased levels of ADPN (Fig. 1A, P<0.01) and the significantly increased levels of FGF9 (Fig. 1B, P<0.05) were observed in depressive patients. Furthermore, the negative correlation of the ratio of ADPN to FGF9 and the total score of Hamilton Depression Scale (Fig. 1C, R2 = 0.91, P < 0.01) was observed in total investigated subjects. 2.2 The relationship between depressive-like behavioral and ADPN/FGF9 levels in serum and hippocampus tissues of different month-aged adiponectin gene knockout mice The depressive-like behavior was evaluated using SPT, NST and FST in mice. No differences were observed in SPT and NST among 3, 6 and 12 month old adipo-/mice groups compared with the wild type C57BL/6J mice groups (Fig. 2A, Fig. 2B, P ＞0.05). The immobility time in FST of 12-month old group was obviously increased than that of control group (Fig. 2C, P<0.05). After behavioral evaluation, the amounts of FGF9 and ADPN in serum and hippocampus tissues were determined by ELISA method in all groups. In the serum, the levels of ADPN significantly decreased in the 3, 9 and 12 month-aged groups, while the levels of FGF9 significantly increased in
the 9 and 12 month-aged groups (Fig. 2D, Fig. 2E , P<0.05). However, there were no obvious differences in the levels of ADPN and FGF9 in the 3, 9 and 12 month-aged groups in both C57BL/6J and Adipo-/- mice in hippocampus tissues (Fig. 2F, Fig. 2G , P>0.05). The pathological results showed that hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic in the 12 month Adipo-/- mice compared with the age-matched control group (Fig 3). 2.3 The role of ADPN/FGF9 in CUMS-induced depression in male ICR mice Depression is often accompanied by significant body weight loss. The body weight of ICR mice in each group were monitored weekly during all the studies. No differences in baseline body weight were observed among each group (at Week 0: P>0.05). From the first week to the fourth week, the body weight of mice in the CUMS group significantly decreased(P<0.01), compared with the control group. After the treatment of ADPN or Anti-FGF9, the body weight of mice significantly increased, compared with the model group(Fig 4A). As shown in Fig. 4B, sucrose consumption significantly decreased in the CUMS group compared with the control group after 4 weeks (p<0.01). The anti-FGF9 group showed significant improvement in sucrose consumption (p<0.05) compared with the CUMS group. The ADPN group showed an upward trend, but the difference was not significant. The results of the NFT were summarized in Fig. 4C. The CUMS group exhibited a longer latency to eat than the control group (p<0.01). The ADPN and the anti-FGF9 group produced an obvious decrease latency to eat compared with the model CUMS group (p<0.05 or p<0.01, respectively). Fig. 4D shows the performance of mice in the FST. The immobility time of the CUMS group obviously increased compared with the control group (P<0.01). The ADPN and the anti-FGF9 group significantly decreased immobility time in CUMS mice (P<0.01). After behavioral evaluation, the amounts of FGF9 and
ADPN were determined in serum of different groups by ELISA method. CUMS exposure significantly decreased the expressions of ADPN and increased the expressions of FGF9 in serum (Fig. 4E, Fig. 4F, p<0.01). Compared with the CUMS group, ADPN levels displayed a significant increase in the ADPN treated group (Fig. 4E, p<0.01) but no difference in the anti-FGF9 treated group(Fig. 4E, P＞0.05) after the injection of recombinant ADPN, while the FGF9 levels displayed a significant decrease in both ADPN and anti-FGF9 treated groups (Fig. 4F, p<0.01) after the injection of anti-FGF9. The contents of ADPN and FGF9 in the hippocampus tissues after treatments are shown in Fig. 4G and Fig. 4H. CUMS exposure significantly decreased expressions of ADPN and increased the expressions of FGF9 in hippocampus tissues (Fig. 4G, Fig. 4H, p<0.01)compared with the control group. Compared with the CUMS group, ADPN levels displayed no significant differences both in the ADPN and the anti-FGF9 treated groups (Fig. 4G, p ＞ 0.05), while the FGF9 levels displayed a significant decrease in both ADPN and anti-FGF9 treated groups( Fig. 4H, p<0.05 or p<0.01, respectively). The pathological results showed that hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic in the CUMS group. After the treatment of ADPN or anti-FGF9, pathological changes could be alleviated (Fig. 5). The expression levels of FGFR3 in the hippocampus tissues were observed via immunohistochemistry, which showed that the expression of FGFR3 in the hippocampus tissues in the CUMS group was lower, compared with the control group. After the treatment of ADPN or anti-FGF9, the expression of FGFR3 was up-regulated (Fig. 6A, Fig. 6B). 2.4 The effects of anti-FGF9 on CUMS-induced depression in male Adipo-/- mice The body weight of Adipo-/- mice in each group were monitored weekly during all the studies. No differences in baseline body weight were observed among each group (at
Week 0: P>0.05). The body weight of mice in the CUMS group from week 1 to week 4 significantly decreased (P<0.01), compared with the control group. After the treatment of Anti-FGF9, the body weight of mice significantly increased, compared with the model group(Fig 7A). The results of the SPT, NST and FST were summarized in Fig. 7B, 7C, 7D. Sucrose consumption significantly decreased while the latency to eat in NST and the immobility time in FST increased in CUMS mice, compared with the control group(P<0.01). After the injection of anti-FGF9, the immobility time in FST decreased significantly, sucrose consumption increased and the latency to eat decreased although the differences were not significant. After behavioral evaluation, the amounts of FGF9 and ADPN in serum and hippocampus tissues of different groups were determined by ELISA method. CUMS exposure significantly decreased the expressions of ADPN and increased the expressions of FGF9 in serum (Fig. 7E, Fig. 7F, p<0.01). Compared with the CUMS group, ADPN levels displayed no differences (Fig. 7E, p ＞ 0.05) but FGF9 levels displayed a significant decrease in the anti-FGF9 treated group(Fig. 7F, P＞0.05). The contents of ADPN and FGF9 in the hippocampus tissues after treatments are shown in Fig. 7G and Fig. 7H. CUMS exposure significantly decreased expressions of ADPN and increased the expressions of FGF9 in hippocampus tissues (Fig. 7G, Fig. 7H, p<0.05 or p<0.01, respectively) compared with the control group. Compared with the CUMS group, ADPN levels displayed no significant differences in the anti-FGF9 treated group (Fig. 7G, p＞0.05), while the FGF9 levels displayed a significant decrease in anti-FGF9 treated group( Fig. 7H, p<0.01). The pathological results showed that hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic in the CUMS group. After the treatment of anti-FGF9, pathological changes could be alleviated (Fig. 8). The expression levels of FGFR3 in the
hippocampus tissues were observed via immunohistochemistry, which showed a down-regulated expression of FGFR3 in hippocampus tissues in the CUMS group, compared with the control group. After the treatment of anti-FGF9, the expression of FGFR3 was up-regulated (Fig. 9A, Fig. 9B). 3 Discussion There are a negative link between adiponectin and FGF9 in depressive disorder This series of studies is the first, to our knowledge, to demonstrate an association and crosstalk between the adiponectin and FGF9 in depressive disorders. In a few studies, adiponectin and FGF9 were reported as anti-depressive factor and pro-depressive factor, respectively (Yau et al., 2015; Aurbach et al., 2015). Adiponectin, a pleiotropic adipocyte-secreted hormone, has insulin-sensitizing and neurotrophic properties. Several clinic studies showed a close relationship between lower plasma level of ADPN and the occurrence and development of depression. The negative relationship between serum level of adiponectin and depression-like behavior were (Formolo et al., 2019) also reported in some depressive animal model (Huang et al., 2019). Adiponectin can cross the blood-brain barrier and target multiple brain regions where the adiponectin receptors that adiponectin and
are
detected.
Emerging
the adiponectin receptor
agonist
evidence could
has
suggested
promote
adult
neurogenesis, dendritic and spine remodeling, and synaptic plasticity in the hippocampus, resulting in antidepressant effects in adult mice (Nicolas et al., 2018). FGF9 is recently reported as a novel negative regulatory factor of depressive disorder. Chronic social defeat stress decreased social interaction and body weight and was associated with increased hippocampal FGF9 expression in rodents (Aurbach et al., 2015). Chronic FGF9 administration increased both anxiety- and depression-like behavior. Conversely, knocking down FGF9 expression in the dentate gyrus
decreased anxiety-like behavior (Garrett et al., 2018). In our present study, several lines of evidence link the dysregulation of adiponectin-FGF9 pathway in depressive disorder. Compared with the subjects without depression, the lower level of adiponectin and the higher level of FGF9 in plasma were found in clinic patients with depression. These results were consistent with some previous reports (Aurbach et al., 2015; Garrett et al., 2018). Interestingly, we analysis the correlation plasma levels of between adiponectin and FGF9 in these subjects and a strong negative correlation between two factors was shown. Also, the negative correlation were observed in different month old Adipo-/- mice. In 3, 9 and 12 month-aged adipo-/- mice, the plasma level of FGF9 was gradually increased. Beside FST in 12 month old is significant increase compared with month-matched wild-type mice, other depressivelike action is trend to increase following age increase but not statistically significance. A similar result has been reported that compared with wild type mice, their susceptibility to depression-like behavior was increased in adipo-/- mice. It is possible reason that the susceptibility of aging adipo-/- mice with high FGF9 level responding to pro-depressive stimulus is increased. These results suggested a potential negative link between adiponectin and FGF9 in depressive disorder (Aurbach et al., 2015). ADPN is an endogenous negative regulator of FGF9 in depressive disorder To further investigate the regulatory relationship between ADPN and FGF9 in depressive disorder, the CUMS-induced depressive mice model were used. These mice were chronically treated with different kinds of unpredictable mild stress every day, which is better to mimic human high stress living environment. Treatment with CUMS significantly decreased the levels of ADPN and increase the levels of FGF9 in plasma and hippocampus in ICR mice. In CUMS-treated ICR mice, these depressivelike behavior were markedly improved by injection with either recombinant ADPN or
antibody to FGF9 into the lateral ventricle. These results suggested a potential role of adiponectin-FGF9 pathway in depression. To certify the regulatory link between the two factors, the levels of FGF9 in plasma and hippocampus were significantly decreased after the injection of either recombinant ADPN or anti-FGF9 in CUMSinduced ICR mice. Additionally, in adipo-/- mice, CUMS-induced depressive-like behavior were attenuated by injection of anti-FGF9 into the lateral ventricle. The protein expression of FGF receptor 3 (FGFR3), the main receptor of FGF9, was significantly down-regulated in hippocampus tissues of CUMS-induced animals, which could be attenuated by treatment with either recombinant ADPN or anti-FGF9. These results demonstrated that ADPN is negative regulatory factor of FGF9 in depressive mice. ADPN–FGF9 axis participates in various signaling cascades, including inflammation and nerve injury. Nonetheless, exactly how ADPN and FGF9 regulate each other and the underlying mechanisms of their concerted actions in depression are presently unknown. In summary, we demonstrated here that ADPN is a negative regulatory factor of FGF9 and the disregulation of ADPN-FGF9 pathway plays a key role in depressive disorder. 4. Methods and materials 4.1 Human study Depressive patients in the Department of Psychiatry, Xiangya Second Hospital, Central South University were selected as the depression group from September 2016 to December 2017. Body mass index(BMI) was determined before the study. The ICD-10 and Hamilton Depression Scale (HAMD) were used to assess depressive patients conformed to the scores. The total score of HAMD less than 8 were taken for non-depression, 8 to 16 for mild depression, 17 to 23 for moderate depression and 24
for severe depression. The patients arranged in the depression group were conformed to the following inclusion criteria: (1) more than 9 years of education; (2) conformed to the International Classification of Diseases and Related Health Problems (10th Revision, ICD-10); (3) the total score of HAMD was 8 to 16. Patients with the following traits were excluded: (1) taking other antipsychotics, antidepressants and drugs with influence on blood glucose and blood lipid 3 weeks before enrollment; (2) drinking, smoking and taking drugs; (3) family history of diabetes; (4) serious physical disease; (5) other mental history; (6) brain organic diseases; (7) serious suicidal tendencies. Blood samples from patients in depression and healthy group(n = 10 each) were collected. The supernatant was collected by centrifuge at 3000 rpm for 10 min and stored in refrigerator at - 80 C for examination. The contents of ADPN and FGF9 in serum were detected by ELISA using commercial kits (human FGF9 201712, human ADPN 201712, Bio-Swamp, China), respectively. 4.2 Animals studies 4.2.1 Animals Thirty male C57BL/6J mice (body weight: 16 - 18g) and forty ICR mice (body weight: 18 - 22g) were purchased from Hunan SJA Laboratory Animal Co. Ltd（Changsha, China ） . Healthy adipo-/- mice (n = 60, 16 - 18g) were obtained from Hunan Experimental Animal Center (Changsha, China). The mice housed in groups of five per cage, in an environmentally controlled condition i.e. 22-26oC with 12 h light and dark cycles. Mouse were given standard diet and water ad libitum. They were allowed to acclimatize for 7 days before model development. Experimental protocols were approved by the Ethics Committee of Drug safety evaluation research center of Hunan province and were in accordance with the National Institute of Health Guidelines for Laboratory Animals (NIH Publications NO.80-23, revised 1996).
4.2.2 Detections of depressive-like behavior and ADPN/FGF9 levels in different month old adipo-/- mice 30 male C57BL/6J mice and 30 male adipo-/- mice weighing 16-18 g aged 3-12 months were randomly assigned to six groups (n = 10 each group): 3 month old, 9 month old and 12 month old group, respectively. The depressive-like behavior was evaluated using sucrose preference test (SPT), novelty suppressed feeding test (NST) and forced swimming test (FST) according to previously reported methods (Liu et al., 2107; Pahwa et al., 2019; Fernandez et al., 2014). After behavioral detection, the mice were sacrificed by rapid decapitation, and the hippocampus were rapidly removed and frozen in liquid nitrogen and kept in −80℃ until assay. ADPN and FGF9 contents in serum and hippocampus tissue homogenate were detected by ELISA. Hippocampus tissues were examined by routine histopathology. 4.2.3 Effects of injection of recombinant ADPN or anti-FGF9 into lateral ventricle on CUMS-induced depression in male ICR mice 40 male ICR mice of 4 weeks weighing 18-22g were used in the study. Among which 10 mice were taken as the normal control group. The other mice receiving 4 weeks of continuous processing of CUMS were randomly divided into 3 groups: the CUMS group, the ADPN group and the anti-FGF9 group. After the treatment mentioned above, the mice of each group were injected into the lateral ventricle with different materials. The normal control group and the CUMS group were injected with the phosphate buffer solution (PBS) in the lateral ventricle, while the ADPN group and the anti-FGF9 group were injected with recombinant ADPN and anti-FGF9 in the lateral ventricle, respectively (Miyatake et al., 2015). The depressive-like behavior was evaluated using SPT, NST and FST after a night recovery. The contents of ADPN and FGF9 in serum and hippocampus were detected by ELISA. Hippocampus tissues
were examined to evaluate neural injury by routine HE staining and to check the protein expression and location of FGFR3, the main receptor of FGF9, by immunohistochemistry method. 4.2.4 Effects of injection of anti-FGF9 into lateral ventricle on CUMS-induced depression in adipo-/- mice 30 male adipo-/- mice of 3 months weighing 18-22g were used in the study. Among which 10 mice were taken as the normal control group. The other mice receiving 4 weeks of continuous processing of CUMS were randomly divided into 2 groups: the CUMS group and the anti-FGF9 group. After the treatment mentioned above, the mice of each group were injected into the lateral ventricle with different materials. The normal control group and the CUMS group were injected with the phosphate buffer solution (PBS) in the lateral ventricle, while the anti-FGF9 group were injected with anti-FGF9 in the lateral ventricle (Miyatake et al., 2015). The depressive-like behavior was evaluated using SPT, NST and FST after a night recovery. ADPN and FGF9 contents in serum and hippocampus tissue homogenate were detected by ELISA. Neural injury and protein expression of FGFR3 were detected by these methods mentioned above. 4.2.5
Chronic unpredictable mild stress procedure
The procedures of CUMS to induce depression-like behaviors were performed as described in Tab. 1 according the previous report with slight modifications (Szewczyk et al., 2019). Briefly, the CUMS protocol consisted of the sequential application of a variety of mild stress as follows: (1) forced swimming for ten minutes (25 ℃), (2) food deprivation for 12 hours, (3) water deprivation for 12 hours, (4) stroboscopic illumination (120 flashes/min) for 6 hour, (5) inversion light/dark cycle, (6) damp sawdust cage (200 ml water in 100 g saw dust bedding) for 12 hours, (7)
tilted cage for 12 hours. These stressors were randomly selected 2 types of stress each day and scheduled over a one-week period, and repeated throughout the 4-week experiment. Non-stressed animals were left undisturbed in their home cages except during housekeeping procedures such as cage cleaning. 4.2.6 Behavioral Testing 4.2.6.1 Sucrose preference test Sucrose preference test was carried out at the end of 4-week CUMS exposure. In brief, 64 hours before the test, mice were trained to adapt to 1% sucrose solution (w/v) as follows: two bottles of 1% sucrose solution were placed in each cage. After 24 hours, 1% sucrose in one bottle was replaced with tap water. After the adaptation for 24 hours, mice were deprived of food for another 8 hours. Sucrose preference test was conducted at 9:00 am, in which mice were housed in individual cages and were free to access to two bottles containing either 100 ml of sucrose solution (1%, w/v) or 100 ml of water, respectively. After 15 hours, the weights of consumed sucrose solution and tap water were recorded and the sucrose preference was calculated by the following formula: sucrose preference index = sucrose consumption/(tap water consumption+ sucrose consumption) × 100% (Liu et al., 2107). 4.2.6.2 Novelty suppressed feeding test The latency to begin eating is used as an index of depression-like/anxiety behavior in the novelty suppressed feeding test (Pahwa et al., 2019), because classical anxiolytic drugs as well as chronic antidepressants decrease this measure. The NFT was carried out as described as follows. Briefly, mice were adapted 10 min in the box (42 × 31 × 20 cm) and food deprived 24 hours prior to the test. A single piece of mouse chow was placed in the center of the box with white paper platform in the test. All mice were placed in the corner of the box and allowed to bite the chow freely for 5 min,
and the time until the first feeding episode was recorded. 4.2.6.3 Forced swimming test Forced swimming test was carried out after novelty suppressed feeding test. The test was performed according to the previous reported method (Fernandez et al., 2014; Song, et al., 2018; Gawali, et al., 2017). Mice were forced to swim in a clear plexiglas cylinder (20 cm high, 14 cm in diameter, filled with 10 cm of water at 24 - 26 ℃) placed in a cabinet. The motilities of mice were recorded with a camera and mice were considered immobile when they absence of any limb or body movement. The total duration of immobile time was recorded during the last 4 min of a single 6-min test session while initial 2 min was served as mice adaption. 4.2.7 Biochemical analysis 4.2.7.1 ELISA assays For the lysate preparation, the tissues were homogenized in RIPA lysis buffer and centrifuged 3000 rpm for 10 minutes at 4 ℃. Protein levels of FGF9 and ADPN in serum and hippocampus were measured using commercially available ELISA kits (Mouse FGF9 MU30936, Mouse ADPN MU30297, Bio-Swamp, China) according to the manufacturer’s instructions. All samples were measured in duplicate in the same assay. 4.2.7.2 Histology Histological evaluation was conducted to observe total infarct volume, necrosis, neurons morphology, glial cell, inflammation and apoptosis. At the end of experiment, mice were sacrificed and brain dissected out and fixed in 4% paraformaldehyde. Then, the tissues were embedded in paraffin wax, cut into 4-μm-thick sections, and stained by routine hematoxylin-eosin (HE) processing(Li et al., 2015; Wang et al., 2017). Six
sections per group were observed and evaluated by optical microscopy and a photographic system (Olympus, Tokyo, Japan). 4.2.7.3 Immunohistochemistry The hippocampus tissue samples were paraffin-embedded and cut into 5-µm sections. Following dewaxing with dimethylbenzene and an ethanol concentration gradient, the sections were rinsed three times (5 minutes each rinse) with PBS and then added with antigen repair solution, heated for 5 minutes at 95℃ for 2 cycles in a microwave oven. The container was removed from the microwave oven and cooled at room temperature for 15-20 minutes. The sections were washed with distilled water, blocked for 15 minutes with 0.5% periodate (Sinopharm Chemical ReagentCo., Ltd., Shanghai, China) and incubated with primary antibody (dilution, 1:500; cat. no. ab133644) at 37°C for 30 minutes and incubated with second antibody (Zhongshan Company, Wuhan, China) overnight in a wet box at 4°C. The tissue sections were then rinsed three times (5 minutes each rinse) and added with diaminobenzidine solution (Zhongshan Company, Wuhan, China) for 10 min at room temperature, stained by hematoxylin, differentiated for 5 sec with HCI + ethanol, washed for 20 min, hydrated with an ethanol concentration gradient, and mounted. The Image-Pro-Plus was used to analyze integral optical density (IOD) (Zhang et al., 2019). 4.3 Statistical Analysis All results were analyzed using the SPSS statistic 17.0 (SPSS Inc., Illinois, Chicago, USA). Multiple comparisons were made by using one-way or two-way ANOVA. Significance levels were considered at P < 0.05, values are displayed as mean ± standard deviation (SD). Liner regression for the ratio of ADPN to FGF9 and the total score of Hamilton Depression Scale was constructed. 5. References
1. Aurbach EL, Inui EG, Turner CA, Hagenauer MH, Prater KE, Li JZ, Absher D, Shah N, Blandino P Jr, Bunney WE, Myers RM, Barchas JD, Schatzberg AF, Watson SJ Jr, Akil H. Fibroblast growth factor 9 is a novel modulator of negative affect. Proc Natl Acad Sci U S A. 2015; 112:11953-8. 2. Bednarska-Makaruk M, Graban A, Wiśniewska A, Łojkowska W, Bochyńska A, Gugała-Iwaniuk M, Sławińska K, Ługowska A, Ryglewicz D, Wehr H. Association of adiponectin, leptin and resistin with inflammatory markers and obe sity in dementia. Biogerontology. 2017; 18: 561-580. 3. Bowley MP, Drevets WC, Ongür D, Price JL. Low glial numbers in the amygdala in major depressive disorder. Biol Psychiatry. 2002; 52:404-12. 4. Bernard
R, Kerman
IA, Thompson
RC, Jones
EG, Bunney
WE, Barchas
JD, Schatzberg AF, Myers RM, Akil H, Watson SJ. Altered expression of glutamate signaling, growth factor, and glia genes in the locus coeruleus of patients with major depression. Mol Psychiatry. 2011; 16: 634-646. 5. Cotter D, Mackay D, Chana G, Beasley C, Landau S, Everall IP. Reduced neuronal size and glial cell density in area 9 of the dorsolateral prefrontal cortex in subjects with major depressive disorder. Cereb Cortex. 2002; 12:386-94. 6. Cotter D, Mackay D, Landau S, Kerwin R, Everall I. Reduced glial cell density and neuronal size in the anterior cingulate cortex in major depressive disorder. Arch Gen Psychiatry. 2001; 58:545-53. 7. Cizza G, Nguyen VT, Eskandari F, Duan Z, Wright EC, Reynolds JC, Ahima RS, Blackman MR, POWER Study Group. Low 24-hour adiponectin and high nocturnal leptin concentrations in a case-control study of community-dwelling premenopausal women with major depressive disorder: the Premenopausal,
Osteopenia/Osteoporosis, Women, Alendronate, Depression (POWER) study. J Clin Psychiatry. 2010; 71:1079-87. 8. Formolo DA, Lee TH, Yau SY2. Increasing Adiponergic System Activity as a Potential Treatment for Depressive Disorders.
Mol
Neurobiol.
2019;
doi:
10.1007/s12035-019-01644-3. 9. Fernandez JW, Grizzell JA, Philpot RM, Wecker L. Postpartum depression in rats: differences in swim test immobility, sucrose preference and nurturing behaviors. Behavioural brain research . 2014; 272: 75-82. 10. Gawali NB, Bulani VD, Gursahani MS, Deshpande PS, Kothavade PS, Juvekar AR2. Agmatine attenuates chronic unpredictable mild stress-induced anxiety, depression-like behaviours and cognitive impairment by modulating nitrergic signalling pathway. Brain Res. 2017; 1663:66-77. 11. Garrett L, Becker L, Rozman J, Puk O, Stoeger T, Yildirim AÖ2, Bohla A, Eickelberg O, Hans W, Prehn C, Adamski J, Klopstock T, Rácz I, Zimmer A, Klingenspor M, Fuchs H, Gailus-Durner V, Wurst W, Hrabě de Angelis M, Graw J, Hölter SM. Fgf9 Y162C Mutation Alters Information Processing and Social Memory in Mice. Mol Neurobiol. 2018; 55:4580-4595. 12. Gittins RA, Harrison PJ. A morphometric study of glia and neurons in the anterior cingulate cortex in mood disorder. J Affect Disord. 2011; 133:328-32. 13. Huang C, Kogure M, Tomata Y, Sugawara Y, Hozawa A, Momma H, Tsuji I, Nagatomi R2. Association of serum adiponectin levels and body mass index with worsening dep
ressivesymptoms in elderly individuals:
a
10-year
longitudinal study. Aging Ment Health. 2019; 18: 1-7. 14. Hu Y, Dong X, Chen J. Adiponectin and depression: A meta-analysis. Biomed Rep. 2015; 3: 38-42.
15. Kikai M, Yamada H, Wakana N, Terada K, Yamamoto K, Wada N, Motoyama S, Saburi M, Sugimoto T, Irie D, Kato T, Kawahito H, Ogata T, Matoba S. Adrenergic receptor-mediated activation of FGF-21-adiponectin axis exerts atheroprotectiveeffects
in
in brown adipose tissue-transplanted apoE-/- mice.
Biochem Biophys Res Commun. 2018; 497: 1097-1103. 16. Li H, Buisman-Pijlman FTA, Nunez-Salces M, Christie S, Frisby CL, Inserra A, Hatzinikolas
G, Lewis
MD, Kritas
S, Wong
ML, Page
AJ.
Chronic stress induces hypersensitivity of murine gastric vagal afferents. Neurogastroenterol Motil. 2019; 26: e13669. 17. Liu J, Guo M, Zhang D, Cheng SY, Liu M, Ding J, Scherer PE, Liu F, Lu XY.Adiponectin is critical in determining susceptibility to depressive behaviors an d has antidepressant-like activity. Proc Natl Acad Sci USA. 2012; 109: 12248-53. 18. Lehto
SM, Huotari
H, Honkalampi
A, Niskanen
K, Ruotsalainen
L, Tolmunen
H, Herzig
T, Koivumaa-Honkanen
KH, Viinamäki
H, Hintikka
J.
Serum adiponectin and resistin levels in major depressive disorder. Acta Psychiatr Scand. 2009; 121: 209-15. 19. Liu YM, Hu CY, Shen JD, Wu SH, Li YC, Yi LT. Elevation of synaptic protein is associated with the antidepressant-like effects of ferulic acid in a chronic model of depression. Physiol Behav. 2017; 169: 184-188. 20. Li Q, Yang D, Wang J, Liu L, Feng G, Li J, Liao J, Wei Y, Li Z. Reduced amount of olfactory receptor neurons in the rat model of depression. Neurosci Lett. 2015; 603:48-54. 21. Leng L, Zhuang K, Liu Z, Huang C, Gao Y, Chen G, Lin H, Hu Y, Wu D, Shi M, Xie W, Sun H, Shao Z, Li H, Zhang K, Mo W, Huang TY, Xue M, Yuan
Z, Zhang X, Bu G, Xu H, Xu Q, Zhang J. Menin Deficiency Leads to Depressivelike Behaviors in Mice by Modulating Astrocyte-Mediated Neuroinflammation. 22. Nicolas S, Debayle D, Béchade C, Maroteaux L, Gay AS, Bayer P, Heurteaux C, Guyon A, Chabry J. Adiporon, an adiponectin receptor agonist acts as an antidepressant and metabolic regulator in a mouse model of depression. Transl Psychiatry. 2018; 8: 159. 23. Ongür D, Drevets WC, Price JL. Glial reduction in the subgenual prefrontal cortex in mood disorders. Proc Natl Acad Sci USA. 1998; 95:13290-5. 24. Pahwa
P, Goel
RK.
Antidepressant-like effect of
a
standardized
hydroethanolic extract of Asparagus adscendens in mice. Indian J Pharmacol. 2019; 51: 98–108. 25. Rajkowska
G, Miguel-Hidalgo
JJ, Wei
J, Dilley
G, Pittman
SD, Meltzer
HY, Overholser JC, Roth BL, Stockmeier CA. Morphometric evidence for neuronal and glial prefrontal cell pathology in major depression. Biol Psychiatry. 1999; 45:1085-98. 26. Szewczyk B, Pochwat B, Muszyńska B, Opoka W, Krakowska A, Rafało-Ulińska A, Friedland K, Nowak G. Antidepressant-like activity of hyperforin and changes in BDNF and zinc levels in mice exposed to chronic unpredictable mild stress. Behav Brain Res. 2019; 372: 112045. 27. Song Y, Sun R, Ji Z, Li X, Fu Q, Ma S. Perilla aldehyde attenuates CUMSinduced depressive-like behaviors via regulating TXNIP/TRX/NLRP3 pathway in rats. Life Sci. 2018; 206:117-124. 28. Suppes T, Ostacher M. Mixed features in major depressive disorder: diagnoses and treatments. CNS. 2017; 22: 155-160.
29. Torres-Platas D, Mackay D, Chana G, Beasley C, Landau S, Everall IP. Reduced neuronal size and glial cell density in area 9 of the dorsolateral prefrontal cortex in subjects with major depressive disorder. Cereb Cortex. 2002; 12:386-94. 30. Wilhelm
CJ, Choi
D, Huckans
M, Manthe
L, Loftis
JM.
Adipocytokine signaling is altered in Flinders sensitive line rats, and adiponectin correlates in humans with some symptoms of depression. Pharmacol Biochem Behav. 2013; 3: 643-51. 31. Wang W, Shi W, Qian H, Deng X, Wang T, Li W. Stellate ganglion block attenuates chronic stress induced depression in rats. PLoS One. 2017; 12: e0183995. 32. Yau SY, Li A, Xu A, So KF. Fat cell-secreted adiponectin mediates physical exercise-induced
hippocampal
neurogenesis:
an
alternative anti-
depressive treatment?. Neural Regen Res. 2015; 10: 7-9. 33. Yang Z, Kusumanchi P, Ross RA, Heathers L, Chandler K, Oshodi A, Thoudam T, Li F, Wang L, Liangpunsakul S. Serum Metabolomic Profiling Identifies Key Metabolic Signatures Associated With Pathogenesis of Alcoholic Liver Disease in Humans. Hepatol Commun . 2019; 3: 542-557. 34. Zhang H, Zhuo C, Zhou D, Zhang F, Chen M, Xu S, Chen Z. Association between the
expression
of
carbonic
anhydrase II
and
clinicopathological features of hepatocellular carcinoma. Oncol Lett. 2019; 17: 5721-5728.
Figure lengends Fig. 1 Serum levels of ADPN and FGF9 in healthy and depressive adults There was no significant difference in body mass index (BMI) between depression patients and healthy people. Serum levels of ADPN and FGF9 were detected. Compared with non-depressive subjects, the decreased levels of ADPN (Fig. 1B, P<0.01) and the significantly increased levels of FGF9 (Fig. 1C, P<0.05) were observed in depressive patients. The data are presented as the mean ± SD, with n=10 for each group. *P<0.05 and **P<0.01 compared to the control group. Furthermore, the negative correlation of the ratio of ADPN to FGF9 and the total score of Hamilton
Depression Scale (Fig. 1D, R2 = 0.91, P < 0.01) was observed in total investigated subjects. Fig. 2 The depressive-like behavior and ADPN/FGF9 levels in serum and hippocampus tissues in different month-aged adiponectin gene knockout mice.
Sucrose preference in the SPT (A), latency to eat in the NST (B) and immobility time in the FST (C) were observed. The levels of ADPN(D) and FGF9(E) in serum and the levels of ADPN(F) and FGF9(G) in hippocampus tissues were detected. The data are presented as the mean ± SD, with n=10 for each group. *P<0.05 and **P<0.01 compared to the control group. Fig. 3 Representative histological sections displaying of hippocampus in different month-aged normal C57BL/6J mice and adiponectin gene knockout mice (magnification, ×100). a-c: normal C57BL/6J mice aged 3, 9, 12 month. d-f: Adipo-/- mice aged 3, 9, 12 month. Hippocampus cells in the 12 month aged Adipo-/- mice were sparsely arranged and disordered, and some vertebral cells were necrotic.
Fig. 4 The effects of ADPN and anti-FGF9 injection in lateral ventricle on the depressive-like behavior and ADPN/FGF9 levels in serum and hippocampus tissues in CUMS-induced male ICR mice. The changing trend of body weight during 4 weeks of treatment(A). Sucrose preference in the SPT (B), latency to eat in the NST (C) and immobility time in the FST (D) were observed. The levels of ADPN(E) and FGF9(F) in serum and the levels of ADPN(G) and FGF9(H) in hippocampus tissues were detected. The data are presented as the mean ± SD, with n=10 for each group. *P<0.05 and **P<0.01
compared to the control group, +P<0.05 and
++P<0.01
compared to the CUMS model
group. Fig. 5 Representative histological sections displaying of hippocampus tissues in CUMS-induced male ICR mice after injection of ADPN or anti-FGF9 in lateral ventricle (magnification, ×100). a: Control group, b: CUMS model group, c: ADPN treated group, d: anti-FGF9 treated group. Hippocampus cells of the normal control group were closely arranged and the vertebral body cells were normal. But in the CUMS group, hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic. After the treatment of ADPN or anti-FGF9, pathological changes could be alleviated. Fig. 6 Expression of FGFR3 in hippocampus tissues samples (magnification, ×100). a: Control group, b: CUMS model group, c: ADPN treated group, d: anti-FGF9 treated group. The expression of FGFR3 in the hippocampus tissues was detected using immunohistochemistry. The expression of FGFR3 was up-regulated after the treatment of ADPN or anti-FGF9. *P<0.05 and **P<0.01 compared to the control group, +P<0.05 and ++P<0.01 compared to the CUMS model group. Fig. 7 The effects of anti-FGF9 injection in lateral ventricle on the depressive-like behavior and ADPN/FGF9 levels in serum and hippocampus tissues in CUMS-induced male Adipo-/-
mice. The changing trend of body weight during 4 weeks of treatment(A). Sucrose preference in the SPT (B), latency to eat in the NST (C) and immobility time in the FST (D) were observed. The levels of ADPN(E) and FGF9(F) in serum and the levels of ADPN(G) and FGF9(H) in hippocampus tissues were detected. The data are presented as the mean ± SD, with n=10 for each group. *P<0.05 and **P<0.01
compared to the control group, +P<0.05 and
++P<0.01
compared to the CUMS model
group. Fig. 8 Representative histological sections displaying of hippocampus tissues in CUMS-induced male Adipo-/- mice after injection of anti-FGF9 in lateral ventricle (magnification, ×100). a: Control group, b: CUMS model group, c: anti-FGF9 treated group. Hippocampus cells of the normal control group were closely arranged and the vertebral body cells were normal. But in the CUMS group, hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic. After the treatment of anti-FGF9, pathological changes could be alleviated. Fig. 9 Expression of FGFR3 in hippocampus tissues samples (magnification, ×100). a: Control group, b: CUMS model group, c: anti-FGF9 treated group. The expression of FGFR3 in the hippocampus tissues was detected using immunohistochemistry. The expression of FGFR3 was up-regulated after the treatment of anti-FGF9. *P<0.05 and **P<0.01 compared to the control group, +P<0.05 and CUMS model group.
++P<0.01
compared to the
Figures and Table A
B
C
D
Fig 1
A
B
C
D
Fig. 2A- D
E
F
G
Fig. 2E- G
Fig. 3
A
B
C
D
Fig. 4A- D
E
F
G
H
Fig. 4E-H
Fig. 5
A
B
Fig. 6
A
B
C
D
Fig. 7A-D
E
F
G
H
Fig. 7E-H
Fig. 8
A
B
Fig. 9
Author Contributions 1. Designed the work that led to the submission: Xiao-Qing Wang, Wei-Hui Li, YaHui Tang, Gui-Rong Zeng, Yu-Hong Wang, Ze-Neng Cheng, De-Jian Jiang 2.
Performed the animal experiments: Xiao-Qing Wang , Ya-hui Tang, Li-Feng Wu, Gui-Rong Zeng
3. Acquired human data: Wei-Hui Li, Ya-Hui Tang 4. Drafted and revised the manuscript: Xiao-Qing Wang, Ze-Neng Cheng, De-Jian Jiang 5. Helped perform the analysis with constructive discussions: Wei-Hui Li, Gui-Rong Zengb, Yu-Hong Wang, Ze-Neng Cheng, De-Jian Jiang 6. Approved the final version: Xiao-Qing Wang, Wei-Hui Li, Ya-Hui Tang, Li-Feng Wu, Gui-Rong Zeng, Yu-Hong Wang, Ze-Neng Cheng, De-Jian Jiang.
HIGHLIGHTS
There are strong negative correlation between the ratio of ADPN to FGF9 and the total score of Hamilton Depression Scale in clinic patients
The injection of recombinant ADPN or FGF9 antibody into lateral ventricle could significantly attenuate the depressive-like symptoms and decrease FGF9 level in CUMS-induced depressive mice
The susceptibility of aging adipo-/- mice with high FGF9 level responding to prodepressive stimulus was increased in adipo-/- mice, which could be attenuated by the injection of recombinant ADPN or FGF9 antibody into lateral ventricle
ADPN maybe a key negative regulator of FGF9/FGFR3 in depressive disorder
S0006-8993(19)30650-X https://doi.org/10.1016/j.brainres.2019.146596 BRES 146596
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
14 September 2019 2 December 2019 7 December 2019
Please cite this article as: X-Q. Wang, W-H. Li, Y-H. Tang, L-F. Wu, G-R. Zeng, Y-H. Wang, Z-N. Cheng, D-J. Jiang, The correlation between adiponectin and FGF9 in depression disorder, Brain Research (2019), doi: https:// doi.org/10.1016/j.brainres.2019.146596
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
The correlation between adiponectin and FGF9 in depression disorder Xiao-Qing Wanga,b, Wei-Hui Lic, Ya-Hui Tangb, Li-Feng Wub, Gui-Rong Zengb, YuHong Wangd, Ze-Neng Chenga,*, De-Jian Jiangb, d,* (a. XiangYa Pharmacy School, Central South University, Changsha 410083; b.
Hunan Center for Safety Evaluation and Research of Drugs & Hunan Key
Laboratory for Pharmacodynamics and Safety Evaluation of new drugs, Changsha 410013; c.
Department of Psychiatry, The Second XiangYa Hospital of Central South
University, Changsha 410011; d.
Institute of
Innovation and Applied Research in Chinese Medicine, Hunan
University of Chinese Medicine, Changsha 410208 ) *Corresponding authors Prof. De-Jian Jiang, E-mail address: [email protected] Phone: 86 731 83285157 Fax: 86 731 83285166 Prof. Ze-Neng Cheng, E-mail address: [email protected] Phone: 86 13874879386
1. Introduction Depression is a debilitating mental disease, characterized by persistent low mood and anhedonia. With growing incidences in the recent years, major depression disorder (MDD) has become one of the most common psychiatric disorders. Stress represents a major environmental risk factor for depression, which can induce these imbalances of between some endogenous anti-depressive and pro-depressive factors resulting in individual vulnerability or resilience in affective disorders (Suppes et al., 2017). The
stress-induced depression was also evidenced in animal models with chronic unpredictable mild stress (CUMS). These core symptoms of depression, such as weight loss and decreased response to reward as well as lack of pleasure, which leading to less sweet liquid consumption, were exerted in CUMS-simulated animals (Li et al., 2019). Adiponectin (ADPN) is an adipocytokine secreted by adipocytes. Studies discovered that ADPN has the functions of anti-inflammation, enhancing insulin sensitivity, improving lipid metabolism and anti-hyperglycemia (Bednarska-Makaruk et al., 2017). Other studies have shown that there is a close relationship between ADPN and the occurrence and development of depression. For example, Cizza et al. (Cizza et al., 2010) and Lehto et al. (Lehto et al., 2009) found that the serum ADPN level of depressive patients was significantly lower than that of the control group. A metaanalysis in six studies with a total of 4,220 subjects indicates that patients with depression had a lower ADPN level when compared to healthy subjects in European groups (Hu et al., 2015). In some animal studies, the negative correlation between serum level of ADPN and depression-like behavior was also reported. Compared with wild type mice, the plasma level of ADPN in adiponectin gene knockout (Adipo-/-) mice was significantly lower and their susceptibility to depression-like behavior was increased (Liu et al., 2012). FSL rats, a genetically modified depression model, have the characteristics of ADPN signaling pathway disorder compared with normal SD rats (Wilhelm et al., 2013). Though it is widely thought as an important antidepressive factor, ADPN how to exert its protective effects on depression is not unclear yet. Histopathological studies of postmortem brain tissue reveal prominent pathology in MDD. Some studies reported decreases in the packing density or number of the
general, Nissl-stained populations of glial cells in subjects diagnosed with MDD as compared to non-psychiatric control subjects. The studies indicated that changes were observed in fronto-limbic brain regions including the dorsolateral prefrontal cortex (Rajkowska et al., 1999; Cotter et al., 2002; Torres-Platas et al., 2002), orbitofrontal cortex (Rajkowska et al., 1999), subgenual cortex (Ongür et al., 1998) anterior cingulate cortex (Cotter et al., 2001; Gittins et al., 2011) and amygdala (Bowley et al., 2002). Both gene expression profiling in postmortem human brain and studies using animal models have implicated the fibroblast growth factor (FGF) family in affect regulation and suggest a potential role in the pathophysiology of MDD (Bernard et al., 2011). As a factor functional opposite to FGF2, which widely reported as a antidepressive factor, FGF9 was recently thought to be a key mediator in MDD via FGF receptor 3 (FGFR3)-dependent pathway (Aurbach et al., 2015). The level of FGF9 were elevated in both patients and animals model with depression. Both endogenous and exogenous FGF9 promotes anxiety- and depression-like behavior (Aurbach et al., 2015). Conversely, localized blockade of endogenous FGF9 expression decreases anxiety behavior. FGF9 represents a novel target for treating affective disorders (Aurbach et al., 2015). There are a few studies to show a regulation each other between adipocytederived adiponectin and FGF family members such as FGF15/19/21, which share common signaling cascades and exert similar beneficial functions in alcoholic liver disease (Yang et al., 2019) and atherosclerosis (Kikai et al., 2018). Based on these literatures mentioned above, we hypothesized that there are certain interactions between ADPN and FGF9, and the disorder of ADPN-FGF9/FGFR3 pathway is involved in progression of depression. In the present study, we observed firstly the correlation between ADPN and FGF9 levels in plasma and (or) hippocampus tissues
in patents with depression and in different month old adipo-/- mice. Furthermore, the role of ADPN-FGF9/FGFR3 pathway in depression was studied using injection of either exogenous ADPN or antibody of FGF9 in CUMS-induced normal or adipo-/mice. 2
Results
2.1 Human study There was no significant difference in body mass index (BMI) between depression patients and healthy people (P ＞ 0.05). Serum levels of ADPN and FGF9 were detected. Compared with non-depressive subjects, the decreased levels of ADPN (Fig. 1A, P<0.01) and the significantly increased levels of FGF9 (Fig. 1B, P<0.05) were observed in depressive patients. Furthermore, the negative correlation of the ratio of ADPN to FGF9 and the total score of Hamilton Depression Scale (Fig. 1C, R2 = 0.91, P < 0.01) was observed in total investigated subjects. 2.2 The relationship between depressive-like behavioral and ADPN/FGF9 levels in serum and hippocampus tissues of different month-aged adiponectin gene knockout mice The depressive-like behavior was evaluated using SPT, NST and FST in mice. No differences were observed in SPT and NST among 3, 6 and 12 month old adipo-/mice groups compared with the wild type C57BL/6J mice groups (Fig. 2A, Fig. 2B, P ＞0.05). The immobility time in FST of 12-month old group was obviously increased than that of control group (Fig. 2C, P<0.05). After behavioral evaluation, the amounts of FGF9 and ADPN in serum and hippocampus tissues were determined by ELISA method in all groups. In the serum, the levels of ADPN significantly decreased in the 3, 9 and 12 month-aged groups, while the levels of FGF9 significantly increased in
the 9 and 12 month-aged groups (Fig. 2D, Fig. 2E , P<0.05). However, there were no obvious differences in the levels of ADPN and FGF9 in the 3, 9 and 12 month-aged groups in both C57BL/6J and Adipo-/- mice in hippocampus tissues (Fig. 2F, Fig. 2G , P>0.05). The pathological results showed that hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic in the 12 month Adipo-/- mice compared with the age-matched control group (Fig 3). 2.3 The role of ADPN/FGF9 in CUMS-induced depression in male ICR mice Depression is often accompanied by significant body weight loss. The body weight of ICR mice in each group were monitored weekly during all the studies. No differences in baseline body weight were observed among each group (at Week 0: P>0.05). From the first week to the fourth week, the body weight of mice in the CUMS group significantly decreased(P<0.01), compared with the control group. After the treatment of ADPN or Anti-FGF9, the body weight of mice significantly increased, compared with the model group(Fig 4A). As shown in Fig. 4B, sucrose consumption significantly decreased in the CUMS group compared with the control group after 4 weeks (p<0.01). The anti-FGF9 group showed significant improvement in sucrose consumption (p<0.05) compared with the CUMS group. The ADPN group showed an upward trend, but the difference was not significant. The results of the NFT were summarized in Fig. 4C. The CUMS group exhibited a longer latency to eat than the control group (p<0.01). The ADPN and the anti-FGF9 group produced an obvious decrease latency to eat compared with the model CUMS group (p<0.05 or p<0.01, respectively). Fig. 4D shows the performance of mice in the FST. The immobility time of the CUMS group obviously increased compared with the control group (P<0.01). The ADPN and the anti-FGF9 group significantly decreased immobility time in CUMS mice (P<0.01). After behavioral evaluation, the amounts of FGF9 and
ADPN were determined in serum of different groups by ELISA method. CUMS exposure significantly decreased the expressions of ADPN and increased the expressions of FGF9 in serum (Fig. 4E, Fig. 4F, p<0.01). Compared with the CUMS group, ADPN levels displayed a significant increase in the ADPN treated group (Fig. 4E, p<0.01) but no difference in the anti-FGF9 treated group(Fig. 4E, P＞0.05) after the injection of recombinant ADPN, while the FGF9 levels displayed a significant decrease in both ADPN and anti-FGF9 treated groups (Fig. 4F, p<0.01) after the injection of anti-FGF9. The contents of ADPN and FGF9 in the hippocampus tissues after treatments are shown in Fig. 4G and Fig. 4H. CUMS exposure significantly decreased expressions of ADPN and increased the expressions of FGF9 in hippocampus tissues (Fig. 4G, Fig. 4H, p<0.01)compared with the control group. Compared with the CUMS group, ADPN levels displayed no significant differences both in the ADPN and the anti-FGF9 treated groups (Fig. 4G, p ＞ 0.05), while the FGF9 levels displayed a significant decrease in both ADPN and anti-FGF9 treated groups( Fig. 4H, p<0.05 or p<0.01, respectively). The pathological results showed that hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic in the CUMS group. After the treatment of ADPN or anti-FGF9, pathological changes could be alleviated (Fig. 5). The expression levels of FGFR3 in the hippocampus tissues were observed via immunohistochemistry, which showed that the expression of FGFR3 in the hippocampus tissues in the CUMS group was lower, compared with the control group. After the treatment of ADPN or anti-FGF9, the expression of FGFR3 was up-regulated (Fig. 6A, Fig. 6B). 2.4 The effects of anti-FGF9 on CUMS-induced depression in male Adipo-/- mice The body weight of Adipo-/- mice in each group were monitored weekly during all the studies. No differences in baseline body weight were observed among each group (at
Week 0: P>0.05). The body weight of mice in the CUMS group from week 1 to week 4 significantly decreased (P<0.01), compared with the control group. After the treatment of Anti-FGF9, the body weight of mice significantly increased, compared with the model group(Fig 7A). The results of the SPT, NST and FST were summarized in Fig. 7B, 7C, 7D. Sucrose consumption significantly decreased while the latency to eat in NST and the immobility time in FST increased in CUMS mice, compared with the control group(P<0.01). After the injection of anti-FGF9, the immobility time in FST decreased significantly, sucrose consumption increased and the latency to eat decreased although the differences were not significant. After behavioral evaluation, the amounts of FGF9 and ADPN in serum and hippocampus tissues of different groups were determined by ELISA method. CUMS exposure significantly decreased the expressions of ADPN and increased the expressions of FGF9 in serum (Fig. 7E, Fig. 7F, p<0.01). Compared with the CUMS group, ADPN levels displayed no differences (Fig. 7E, p ＞ 0.05) but FGF9 levels displayed a significant decrease in the anti-FGF9 treated group(Fig. 7F, P＞0.05). The contents of ADPN and FGF9 in the hippocampus tissues after treatments are shown in Fig. 7G and Fig. 7H. CUMS exposure significantly decreased expressions of ADPN and increased the expressions of FGF9 in hippocampus tissues (Fig. 7G, Fig. 7H, p<0.05 or p<0.01, respectively) compared with the control group. Compared with the CUMS group, ADPN levels displayed no significant differences in the anti-FGF9 treated group (Fig. 7G, p＞0.05), while the FGF9 levels displayed a significant decrease in anti-FGF9 treated group( Fig. 7H, p<0.01). The pathological results showed that hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic in the CUMS group. After the treatment of anti-FGF9, pathological changes could be alleviated (Fig. 8). The expression levels of FGFR3 in the
hippocampus tissues were observed via immunohistochemistry, which showed a down-regulated expression of FGFR3 in hippocampus tissues in the CUMS group, compared with the control group. After the treatment of anti-FGF9, the expression of FGFR3 was up-regulated (Fig. 9A, Fig. 9B). 3 Discussion There are a negative link between adiponectin and FGF9 in depressive disorder This series of studies is the first, to our knowledge, to demonstrate an association and crosstalk between the adiponectin and FGF9 in depressive disorders. In a few studies, adiponectin and FGF9 were reported as anti-depressive factor and pro-depressive factor, respectively (Yau et al., 2015; Aurbach et al., 2015). Adiponectin, a pleiotropic adipocyte-secreted hormone, has insulin-sensitizing and neurotrophic properties. Several clinic studies showed a close relationship between lower plasma level of ADPN and the occurrence and development of depression. The negative relationship between serum level of adiponectin and depression-like behavior were (Formolo et al., 2019) also reported in some depressive animal model (Huang et al., 2019). Adiponectin can cross the blood-brain barrier and target multiple brain regions where the adiponectin receptors that adiponectin and
are
detected.
Emerging
the adiponectin receptor
agonist
evidence could
has
suggested
promote
adult
neurogenesis, dendritic and spine remodeling, and synaptic plasticity in the hippocampus, resulting in antidepressant effects in adult mice (Nicolas et al., 2018). FGF9 is recently reported as a novel negative regulatory factor of depressive disorder. Chronic social defeat stress decreased social interaction and body weight and was associated with increased hippocampal FGF9 expression in rodents (Aurbach et al., 2015). Chronic FGF9 administration increased both anxiety- and depression-like behavior. Conversely, knocking down FGF9 expression in the dentate gyrus
decreased anxiety-like behavior (Garrett et al., 2018). In our present study, several lines of evidence link the dysregulation of adiponectin-FGF9 pathway in depressive disorder. Compared with the subjects without depression, the lower level of adiponectin and the higher level of FGF9 in plasma were found in clinic patients with depression. These results were consistent with some previous reports (Aurbach et al., 2015; Garrett et al., 2018). Interestingly, we analysis the correlation plasma levels of between adiponectin and FGF9 in these subjects and a strong negative correlation between two factors was shown. Also, the negative correlation were observed in different month old Adipo-/- mice. In 3, 9 and 12 month-aged adipo-/- mice, the plasma level of FGF9 was gradually increased. Beside FST in 12 month old is significant increase compared with month-matched wild-type mice, other depressivelike action is trend to increase following age increase but not statistically significance. A similar result has been reported that compared with wild type mice, their susceptibility to depression-like behavior was increased in adipo-/- mice. It is possible reason that the susceptibility of aging adipo-/- mice with high FGF9 level responding to pro-depressive stimulus is increased. These results suggested a potential negative link between adiponectin and FGF9 in depressive disorder (Aurbach et al., 2015). ADPN is an endogenous negative regulator of FGF9 in depressive disorder To further investigate the regulatory relationship between ADPN and FGF9 in depressive disorder, the CUMS-induced depressive mice model were used. These mice were chronically treated with different kinds of unpredictable mild stress every day, which is better to mimic human high stress living environment. Treatment with CUMS significantly decreased the levels of ADPN and increase the levels of FGF9 in plasma and hippocampus in ICR mice. In CUMS-treated ICR mice, these depressivelike behavior were markedly improved by injection with either recombinant ADPN or
antibody to FGF9 into the lateral ventricle. These results suggested a potential role of adiponectin-FGF9 pathway in depression. To certify the regulatory link between the two factors, the levels of FGF9 in plasma and hippocampus were significantly decreased after the injection of either recombinant ADPN or anti-FGF9 in CUMSinduced ICR mice. Additionally, in adipo-/- mice, CUMS-induced depressive-like behavior were attenuated by injection of anti-FGF9 into the lateral ventricle. The protein expression of FGF receptor 3 (FGFR3), the main receptor of FGF9, was significantly down-regulated in hippocampus tissues of CUMS-induced animals, which could be attenuated by treatment with either recombinant ADPN or anti-FGF9. These results demonstrated that ADPN is negative regulatory factor of FGF9 in depressive mice. ADPN–FGF9 axis participates in various signaling cascades, including inflammation and nerve injury. Nonetheless, exactly how ADPN and FGF9 regulate each other and the underlying mechanisms of their concerted actions in depression are presently unknown. In summary, we demonstrated here that ADPN is a negative regulatory factor of FGF9 and the disregulation of ADPN-FGF9 pathway plays a key role in depressive disorder. 4. Methods and materials 4.1 Human study Depressive patients in the Department of Psychiatry, Xiangya Second Hospital, Central South University were selected as the depression group from September 2016 to December 2017. Body mass index(BMI) was determined before the study. The ICD-10 and Hamilton Depression Scale (HAMD) were used to assess depressive patients conformed to the scores. The total score of HAMD less than 8 were taken for non-depression, 8 to 16 for mild depression, 17 to 23 for moderate depression and 24
for severe depression. The patients arranged in the depression group were conformed to the following inclusion criteria: (1) more than 9 years of education; (2) conformed to the International Classification of Diseases and Related Health Problems (10th Revision, ICD-10); (3) the total score of HAMD was 8 to 16. Patients with the following traits were excluded: (1) taking other antipsychotics, antidepressants and drugs with influence on blood glucose and blood lipid 3 weeks before enrollment; (2) drinking, smoking and taking drugs; (3) family history of diabetes; (4) serious physical disease; (5) other mental history; (6) brain organic diseases; (7) serious suicidal tendencies. Blood samples from patients in depression and healthy group(n = 10 each) were collected. The supernatant was collected by centrifuge at 3000 rpm for 10 min and stored in refrigerator at - 80 C for examination. The contents of ADPN and FGF9 in serum were detected by ELISA using commercial kits (human FGF9 201712, human ADPN 201712, Bio-Swamp, China), respectively. 4.2 Animals studies 4.2.1 Animals Thirty male C57BL/6J mice (body weight: 16 - 18g) and forty ICR mice (body weight: 18 - 22g) were purchased from Hunan SJA Laboratory Animal Co. Ltd（Changsha, China ） . Healthy adipo-/- mice (n = 60, 16 - 18g) were obtained from Hunan Experimental Animal Center (Changsha, China). The mice housed in groups of five per cage, in an environmentally controlled condition i.e. 22-26oC with 12 h light and dark cycles. Mouse were given standard diet and water ad libitum. They were allowed to acclimatize for 7 days before model development. Experimental protocols were approved by the Ethics Committee of Drug safety evaluation research center of Hunan province and were in accordance with the National Institute of Health Guidelines for Laboratory Animals (NIH Publications NO.80-23, revised 1996).
4.2.2 Detections of depressive-like behavior and ADPN/FGF9 levels in different month old adipo-/- mice 30 male C57BL/6J mice and 30 male adipo-/- mice weighing 16-18 g aged 3-12 months were randomly assigned to six groups (n = 10 each group): 3 month old, 9 month old and 12 month old group, respectively. The depressive-like behavior was evaluated using sucrose preference test (SPT), novelty suppressed feeding test (NST) and forced swimming test (FST) according to previously reported methods (Liu et al., 2107; Pahwa et al., 2019; Fernandez et al., 2014). After behavioral detection, the mice were sacrificed by rapid decapitation, and the hippocampus were rapidly removed and frozen in liquid nitrogen and kept in −80℃ until assay. ADPN and FGF9 contents in serum and hippocampus tissue homogenate were detected by ELISA. Hippocampus tissues were examined by routine histopathology. 4.2.3 Effects of injection of recombinant ADPN or anti-FGF9 into lateral ventricle on CUMS-induced depression in male ICR mice 40 male ICR mice of 4 weeks weighing 18-22g were used in the study. Among which 10 mice were taken as the normal control group. The other mice receiving 4 weeks of continuous processing of CUMS were randomly divided into 3 groups: the CUMS group, the ADPN group and the anti-FGF9 group. After the treatment mentioned above, the mice of each group were injected into the lateral ventricle with different materials. The normal control group and the CUMS group were injected with the phosphate buffer solution (PBS) in the lateral ventricle, while the ADPN group and the anti-FGF9 group were injected with recombinant ADPN and anti-FGF9 in the lateral ventricle, respectively (Miyatake et al., 2015). The depressive-like behavior was evaluated using SPT, NST and FST after a night recovery. The contents of ADPN and FGF9 in serum and hippocampus were detected by ELISA. Hippocampus tissues
were examined to evaluate neural injury by routine HE staining and to check the protein expression and location of FGFR3, the main receptor of FGF9, by immunohistochemistry method. 4.2.4 Effects of injection of anti-FGF9 into lateral ventricle on CUMS-induced depression in adipo-/- mice 30 male adipo-/- mice of 3 months weighing 18-22g were used in the study. Among which 10 mice were taken as the normal control group. The other mice receiving 4 weeks of continuous processing of CUMS were randomly divided into 2 groups: the CUMS group and the anti-FGF9 group. After the treatment mentioned above, the mice of each group were injected into the lateral ventricle with different materials. The normal control group and the CUMS group were injected with the phosphate buffer solution (PBS) in the lateral ventricle, while the anti-FGF9 group were injected with anti-FGF9 in the lateral ventricle (Miyatake et al., 2015). The depressive-like behavior was evaluated using SPT, NST and FST after a night recovery. ADPN and FGF9 contents in serum and hippocampus tissue homogenate were detected by ELISA. Neural injury and protein expression of FGFR3 were detected by these methods mentioned above. 4.2.5
Chronic unpredictable mild stress procedure
The procedures of CUMS to induce depression-like behaviors were performed as described in Tab. 1 according the previous report with slight modifications (Szewczyk et al., 2019). Briefly, the CUMS protocol consisted of the sequential application of a variety of mild stress as follows: (1) forced swimming for ten minutes (25 ℃), (2) food deprivation for 12 hours, (3) water deprivation for 12 hours, (4) stroboscopic illumination (120 flashes/min) for 6 hour, (5) inversion light/dark cycle, (6) damp sawdust cage (200 ml water in 100 g saw dust bedding) for 12 hours, (7)
tilted cage for 12 hours. These stressors were randomly selected 2 types of stress each day and scheduled over a one-week period, and repeated throughout the 4-week experiment. Non-stressed animals were left undisturbed in their home cages except during housekeeping procedures such as cage cleaning. 4.2.6 Behavioral Testing 4.2.6.1 Sucrose preference test Sucrose preference test was carried out at the end of 4-week CUMS exposure. In brief, 64 hours before the test, mice were trained to adapt to 1% sucrose solution (w/v) as follows: two bottles of 1% sucrose solution were placed in each cage. After 24 hours, 1% sucrose in one bottle was replaced with tap water. After the adaptation for 24 hours, mice were deprived of food for another 8 hours. Sucrose preference test was conducted at 9:00 am, in which mice were housed in individual cages and were free to access to two bottles containing either 100 ml of sucrose solution (1%, w/v) or 100 ml of water, respectively. After 15 hours, the weights of consumed sucrose solution and tap water were recorded and the sucrose preference was calculated by the following formula: sucrose preference index = sucrose consumption/(tap water consumption+ sucrose consumption) × 100% (Liu et al., 2107). 4.2.6.2 Novelty suppressed feeding test The latency to begin eating is used as an index of depression-like/anxiety behavior in the novelty suppressed feeding test (Pahwa et al., 2019), because classical anxiolytic drugs as well as chronic antidepressants decrease this measure. The NFT was carried out as described as follows. Briefly, mice were adapted 10 min in the box (42 × 31 × 20 cm) and food deprived 24 hours prior to the test. A single piece of mouse chow was placed in the center of the box with white paper platform in the test. All mice were placed in the corner of the box and allowed to bite the chow freely for 5 min,
and the time until the first feeding episode was recorded. 4.2.6.3 Forced swimming test Forced swimming test was carried out after novelty suppressed feeding test. The test was performed according to the previous reported method (Fernandez et al., 2014; Song, et al., 2018; Gawali, et al., 2017). Mice were forced to swim in a clear plexiglas cylinder (20 cm high, 14 cm in diameter, filled with 10 cm of water at 24 - 26 ℃) placed in a cabinet. The motilities of mice were recorded with a camera and mice were considered immobile when they absence of any limb or body movement. The total duration of immobile time was recorded during the last 4 min of a single 6-min test session while initial 2 min was served as mice adaption. 4.2.7 Biochemical analysis 4.2.7.1 ELISA assays For the lysate preparation, the tissues were homogenized in RIPA lysis buffer and centrifuged 3000 rpm for 10 minutes at 4 ℃. Protein levels of FGF9 and ADPN in serum and hippocampus were measured using commercially available ELISA kits (Mouse FGF9 MU30936, Mouse ADPN MU30297, Bio-Swamp, China) according to the manufacturer’s instructions. All samples were measured in duplicate in the same assay. 4.2.7.2 Histology Histological evaluation was conducted to observe total infarct volume, necrosis, neurons morphology, glial cell, inflammation and apoptosis. At the end of experiment, mice were sacrificed and brain dissected out and fixed in 4% paraformaldehyde. Then, the tissues were embedded in paraffin wax, cut into 4-μm-thick sections, and stained by routine hematoxylin-eosin (HE) processing(Li et al., 2015; Wang et al., 2017). Six
sections per group were observed and evaluated by optical microscopy and a photographic system (Olympus, Tokyo, Japan). 4.2.7.3 Immunohistochemistry The hippocampus tissue samples were paraffin-embedded and cut into 5-µm sections. Following dewaxing with dimethylbenzene and an ethanol concentration gradient, the sections were rinsed three times (5 minutes each rinse) with PBS and then added with antigen repair solution, heated for 5 minutes at 95℃ for 2 cycles in a microwave oven. The container was removed from the microwave oven and cooled at room temperature for 15-20 minutes. The sections were washed with distilled water, blocked for 15 minutes with 0.5% periodate (Sinopharm Chemical ReagentCo., Ltd., Shanghai, China) and incubated with primary antibody (dilution, 1:500; cat. no. ab133644) at 37°C for 30 minutes and incubated with second antibody (Zhongshan Company, Wuhan, China) overnight in a wet box at 4°C. The tissue sections were then rinsed three times (5 minutes each rinse) and added with diaminobenzidine solution (Zhongshan Company, Wuhan, China) for 10 min at room temperature, stained by hematoxylin, differentiated for 5 sec with HCI + ethanol, washed for 20 min, hydrated with an ethanol concentration gradient, and mounted. The Image-Pro-Plus was used to analyze integral optical density (IOD) (Zhang et al., 2019). 4.3 Statistical Analysis All results were analyzed using the SPSS statistic 17.0 (SPSS Inc., Illinois, Chicago, USA). Multiple comparisons were made by using one-way or two-way ANOVA. Significance levels were considered at P < 0.05, values are displayed as mean ± standard deviation (SD). Liner regression for the ratio of ADPN to FGF9 and the total score of Hamilton Depression Scale was constructed. 5. References
1. Aurbach EL, Inui EG, Turner CA, Hagenauer MH, Prater KE, Li JZ, Absher D, Shah N, Blandino P Jr, Bunney WE, Myers RM, Barchas JD, Schatzberg AF, Watson SJ Jr, Akil H. Fibroblast growth factor 9 is a novel modulator of negative affect. Proc Natl Acad Sci U S A. 2015; 112:11953-8. 2. Bednarska-Makaruk M, Graban A, Wiśniewska A, Łojkowska W, Bochyńska A, Gugała-Iwaniuk M, Sławińska K, Ługowska A, Ryglewicz D, Wehr H. Association of adiponectin, leptin and resistin with inflammatory markers and obe sity in dementia. Biogerontology. 2017; 18: 561-580. 3. Bowley MP, Drevets WC, Ongür D, Price JL. Low glial numbers in the amygdala in major depressive disorder. Biol Psychiatry. 2002; 52:404-12. 4. Bernard
R, Kerman
IA, Thompson
RC, Jones
EG, Bunney
WE, Barchas
JD, Schatzberg AF, Myers RM, Akil H, Watson SJ. Altered expression of glutamate signaling, growth factor, and glia genes in the locus coeruleus of patients with major depression. Mol Psychiatry. 2011; 16: 634-646. 5. Cotter D, Mackay D, Chana G, Beasley C, Landau S, Everall IP. Reduced neuronal size and glial cell density in area 9 of the dorsolateral prefrontal cortex in subjects with major depressive disorder. Cereb Cortex. 2002; 12:386-94. 6. Cotter D, Mackay D, Landau S, Kerwin R, Everall I. Reduced glial cell density and neuronal size in the anterior cingulate cortex in major depressive disorder. Arch Gen Psychiatry. 2001; 58:545-53. 7. Cizza G, Nguyen VT, Eskandari F, Duan Z, Wright EC, Reynolds JC, Ahima RS, Blackman MR, POWER Study Group. Low 24-hour adiponectin and high nocturnal leptin concentrations in a case-control study of community-dwelling premenopausal women with major depressive disorder: the Premenopausal,
Osteopenia/Osteoporosis, Women, Alendronate, Depression (POWER) study. J Clin Psychiatry. 2010; 71:1079-87. 8. Formolo DA, Lee TH, Yau SY2. Increasing Adiponergic System Activity as a Potential Treatment for Depressive Disorders.
Mol
Neurobiol.
2019;
doi:
10.1007/s12035-019-01644-3. 9. Fernandez JW, Grizzell JA, Philpot RM, Wecker L. Postpartum depression in rats: differences in swim test immobility, sucrose preference and nurturing behaviors. Behavioural brain research . 2014; 272: 75-82. 10. Gawali NB, Bulani VD, Gursahani MS, Deshpande PS, Kothavade PS, Juvekar AR2. Agmatine attenuates chronic unpredictable mild stress-induced anxiety, depression-like behaviours and cognitive impairment by modulating nitrergic signalling pathway. Brain Res. 2017; 1663:66-77. 11. Garrett L, Becker L, Rozman J, Puk O, Stoeger T, Yildirim AÖ2, Bohla A, Eickelberg O, Hans W, Prehn C, Adamski J, Klopstock T, Rácz I, Zimmer A, Klingenspor M, Fuchs H, Gailus-Durner V, Wurst W, Hrabě de Angelis M, Graw J, Hölter SM. Fgf9 Y162C Mutation Alters Information Processing and Social Memory in Mice. Mol Neurobiol. 2018; 55:4580-4595. 12. Gittins RA, Harrison PJ. A morphometric study of glia and neurons in the anterior cingulate cortex in mood disorder. J Affect Disord. 2011; 133:328-32. 13. Huang C, Kogure M, Tomata Y, Sugawara Y, Hozawa A, Momma H, Tsuji I, Nagatomi R2. Association of serum adiponectin levels and body mass index with worsening dep
ressivesymptoms in elderly individuals:
a
10-year
longitudinal study. Aging Ment Health. 2019; 18: 1-7. 14. Hu Y, Dong X, Chen J. Adiponectin and depression: A meta-analysis. Biomed Rep. 2015; 3: 38-42.
15. Kikai M, Yamada H, Wakana N, Terada K, Yamamoto K, Wada N, Motoyama S, Saburi M, Sugimoto T, Irie D, Kato T, Kawahito H, Ogata T, Matoba S. Adrenergic receptor-mediated activation of FGF-21-adiponectin axis exerts atheroprotectiveeffects
in
in brown adipose tissue-transplanted apoE-/- mice.
Biochem Biophys Res Commun. 2018; 497: 1097-1103. 16. Li H, Buisman-Pijlman FTA, Nunez-Salces M, Christie S, Frisby CL, Inserra A, Hatzinikolas
G, Lewis
MD, Kritas
S, Wong
ML, Page
AJ.
Chronic stress induces hypersensitivity of murine gastric vagal afferents. Neurogastroenterol Motil. 2019; 26: e13669. 17. Liu J, Guo M, Zhang D, Cheng SY, Liu M, Ding J, Scherer PE, Liu F, Lu XY.Adiponectin is critical in determining susceptibility to depressive behaviors an d has antidepressant-like activity. Proc Natl Acad Sci USA. 2012; 109: 12248-53. 18. Lehto
SM, Huotari
H, Honkalampi
A, Niskanen
K, Ruotsalainen
L, Tolmunen
H, Herzig
T, Koivumaa-Honkanen
KH, Viinamäki
H, Hintikka
J.
Serum adiponectin and resistin levels in major depressive disorder. Acta Psychiatr Scand. 2009; 121: 209-15. 19. Liu YM, Hu CY, Shen JD, Wu SH, Li YC, Yi LT. Elevation of synaptic protein is associated with the antidepressant-like effects of ferulic acid in a chronic model of depression. Physiol Behav. 2017; 169: 184-188. 20. Li Q, Yang D, Wang J, Liu L, Feng G, Li J, Liao J, Wei Y, Li Z. Reduced amount of olfactory receptor neurons in the rat model of depression. Neurosci Lett. 2015; 603:48-54. 21. Leng L, Zhuang K, Liu Z, Huang C, Gao Y, Chen G, Lin H, Hu Y, Wu D, Shi M, Xie W, Sun H, Shao Z, Li H, Zhang K, Mo W, Huang TY, Xue M, Yuan
Z, Zhang X, Bu G, Xu H, Xu Q, Zhang J. Menin Deficiency Leads to Depressivelike Behaviors in Mice by Modulating Astrocyte-Mediated Neuroinflammation. 22. Nicolas S, Debayle D, Béchade C, Maroteaux L, Gay AS, Bayer P, Heurteaux C, Guyon A, Chabry J. Adiporon, an adiponectin receptor agonist acts as an antidepressant and metabolic regulator in a mouse model of depression. Transl Psychiatry. 2018; 8: 159. 23. Ongür D, Drevets WC, Price JL. Glial reduction in the subgenual prefrontal cortex in mood disorders. Proc Natl Acad Sci USA. 1998; 95:13290-5. 24. Pahwa
P, Goel
RK.
Antidepressant-like effect of
a
standardized
hydroethanolic extract of Asparagus adscendens in mice. Indian J Pharmacol. 2019; 51: 98–108. 25. Rajkowska
G, Miguel-Hidalgo
JJ, Wei
J, Dilley
G, Pittman
SD, Meltzer
HY, Overholser JC, Roth BL, Stockmeier CA. Morphometric evidence for neuronal and glial prefrontal cell pathology in major depression. Biol Psychiatry. 1999; 45:1085-98. 26. Szewczyk B, Pochwat B, Muszyńska B, Opoka W, Krakowska A, Rafało-Ulińska A, Friedland K, Nowak G. Antidepressant-like activity of hyperforin and changes in BDNF and zinc levels in mice exposed to chronic unpredictable mild stress. Behav Brain Res. 2019; 372: 112045. 27. Song Y, Sun R, Ji Z, Li X, Fu Q, Ma S. Perilla aldehyde attenuates CUMSinduced depressive-like behaviors via regulating TXNIP/TRX/NLRP3 pathway in rats. Life Sci. 2018; 206:117-124. 28. Suppes T, Ostacher M. Mixed features in major depressive disorder: diagnoses and treatments. CNS. 2017; 22: 155-160.
29. Torres-Platas D, Mackay D, Chana G, Beasley C, Landau S, Everall IP. Reduced neuronal size and glial cell density in area 9 of the dorsolateral prefrontal cortex in subjects with major depressive disorder. Cereb Cortex. 2002; 12:386-94. 30. Wilhelm
CJ, Choi
D, Huckans
M, Manthe
L, Loftis
JM.
Adipocytokine signaling is altered in Flinders sensitive line rats, and adiponectin correlates in humans with some symptoms of depression. Pharmacol Biochem Behav. 2013; 3: 643-51. 31. Wang W, Shi W, Qian H, Deng X, Wang T, Li W. Stellate ganglion block attenuates chronic stress induced depression in rats. PLoS One. 2017; 12: e0183995. 32. Yau SY, Li A, Xu A, So KF. Fat cell-secreted adiponectin mediates physical exercise-induced
hippocampal
neurogenesis:
an
alternative anti-
depressive treatment?. Neural Regen Res. 2015; 10: 7-9. 33. Yang Z, Kusumanchi P, Ross RA, Heathers L, Chandler K, Oshodi A, Thoudam T, Li F, Wang L, Liangpunsakul S. Serum Metabolomic Profiling Identifies Key Metabolic Signatures Associated With Pathogenesis of Alcoholic Liver Disease in Humans. Hepatol Commun . 2019; 3: 542-557. 34. Zhang H, Zhuo C, Zhou D, Zhang F, Chen M, Xu S, Chen Z. Association between the
expression
of
carbonic
anhydrase II
and
clinicopathological features of hepatocellular carcinoma. Oncol Lett. 2019; 17: 5721-5728.
Figure lengends Fig. 1 Serum levels of ADPN and FGF9 in healthy and depressive adults There was no significant difference in body mass index (BMI) between depression patients and healthy people. Serum levels of ADPN and FGF9 were detected. Compared with non-depressive subjects, the decreased levels of ADPN (Fig. 1B, P<0.01) and the significantly increased levels of FGF9 (Fig. 1C, P<0.05) were observed in depressive patients. The data are presented as the mean ± SD, with n=10 for each group. *P<0.05 and **P<0.01 compared to the control group. Furthermore, the negative correlation of the ratio of ADPN to FGF9 and the total score of Hamilton
Depression Scale (Fig. 1D, R2 = 0.91, P < 0.01) was observed in total investigated subjects. Fig. 2 The depressive-like behavior and ADPN/FGF9 levels in serum and hippocampus tissues in different month-aged adiponectin gene knockout mice.
Sucrose preference in the SPT (A), latency to eat in the NST (B) and immobility time in the FST (C) were observed. The levels of ADPN(D) and FGF9(E) in serum and the levels of ADPN(F) and FGF9(G) in hippocampus tissues were detected. The data are presented as the mean ± SD, with n=10 for each group. *P<0.05 and **P<0.01 compared to the control group. Fig. 3 Representative histological sections displaying of hippocampus in different month-aged normal C57BL/6J mice and adiponectin gene knockout mice (magnification, ×100). a-c: normal C57BL/6J mice aged 3, 9, 12 month. d-f: Adipo-/- mice aged 3, 9, 12 month. Hippocampus cells in the 12 month aged Adipo-/- mice were sparsely arranged and disordered, and some vertebral cells were necrotic.
Fig. 4 The effects of ADPN and anti-FGF9 injection in lateral ventricle on the depressive-like behavior and ADPN/FGF9 levels in serum and hippocampus tissues in CUMS-induced male ICR mice. The changing trend of body weight during 4 weeks of treatment(A). Sucrose preference in the SPT (B), latency to eat in the NST (C) and immobility time in the FST (D) were observed. The levels of ADPN(E) and FGF9(F) in serum and the levels of ADPN(G) and FGF9(H) in hippocampus tissues were detected. The data are presented as the mean ± SD, with n=10 for each group. *P<0.05 and **P<0.01
compared to the control group, +P<0.05 and
++P<0.01
compared to the CUMS model
group. Fig. 5 Representative histological sections displaying of hippocampus tissues in CUMS-induced male ICR mice after injection of ADPN or anti-FGF9 in lateral ventricle (magnification, ×100). a: Control group, b: CUMS model group, c: ADPN treated group, d: anti-FGF9 treated group. Hippocampus cells of the normal control group were closely arranged and the vertebral body cells were normal. But in the CUMS group, hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic. After the treatment of ADPN or anti-FGF9, pathological changes could be alleviated. Fig. 6 Expression of FGFR3 in hippocampus tissues samples (magnification, ×100). a: Control group, b: CUMS model group, c: ADPN treated group, d: anti-FGF9 treated group. The expression of FGFR3 in the hippocampus tissues was detected using immunohistochemistry. The expression of FGFR3 was up-regulated after the treatment of ADPN or anti-FGF9. *P<0.05 and **P<0.01 compared to the control group, +P<0.05 and ++P<0.01 compared to the CUMS model group. Fig. 7 The effects of anti-FGF9 injection in lateral ventricle on the depressive-like behavior and ADPN/FGF9 levels in serum and hippocampus tissues in CUMS-induced male Adipo-/-
mice. The changing trend of body weight during 4 weeks of treatment(A). Sucrose preference in the SPT (B), latency to eat in the NST (C) and immobility time in the FST (D) were observed. The levels of ADPN(E) and FGF9(F) in serum and the levels of ADPN(G) and FGF9(H) in hippocampus tissues were detected. The data are presented as the mean ± SD, with n=10 for each group. *P<0.05 and **P<0.01
compared to the control group, +P<0.05 and
++P<0.01
compared to the CUMS model
group. Fig. 8 Representative histological sections displaying of hippocampus tissues in CUMS-induced male Adipo-/- mice after injection of anti-FGF9 in lateral ventricle (magnification, ×100). a: Control group, b: CUMS model group, c: anti-FGF9 treated group. Hippocampus cells of the normal control group were closely arranged and the vertebral body cells were normal. But in the CUMS group, hippocampus cells were sparsely arranged and disordered, and some vertebral cells were necrotic. After the treatment of anti-FGF9, pathological changes could be alleviated. Fig. 9 Expression of FGFR3 in hippocampus tissues samples (magnification, ×100). a: Control group, b: CUMS model group, c: anti-FGF9 treated group. The expression of FGFR3 in the hippocampus tissues was detected using immunohistochemistry. The expression of FGFR3 was up-regulated after the treatment of anti-FGF9. *P<0.05 and **P<0.01 compared to the control group, +P<0.05 and CUMS model group.
++P<0.01
compared to the
Figures and Table A
B
C
D
Fig 1
A
B
C
D
Fig. 2A- D
E
F
G
Fig. 2E- G
Fig. 3
A
B
C
D
Fig. 4A- D
E
F
G
H
Fig. 4E-H
Fig. 5
A
B
Fig. 6
A
B
C
D
Fig. 7A-D
E
F
G
H
Fig. 7E-H
Fig. 8
A
B
Fig. 9
Author Contributions 1. Designed the work that led to the submission: Xiao-Qing Wang, Wei-Hui Li, YaHui Tang, Gui-Rong Zeng, Yu-Hong Wang, Ze-Neng Cheng, De-Jian Jiang 2.
Performed the animal experiments: Xiao-Qing Wang , Ya-hui Tang, Li-Feng Wu, Gui-Rong Zeng
3. Acquired human data: Wei-Hui Li, Ya-Hui Tang 4. Drafted and revised the manuscript: Xiao-Qing Wang, Ze-Neng Cheng, De-Jian Jiang 5. Helped perform the analysis with constructive discussions: Wei-Hui Li, Gui-Rong Zengb, Yu-Hong Wang, Ze-Neng Cheng, De-Jian Jiang 6. Approved the final version: Xiao-Qing Wang, Wei-Hui Li, Ya-Hui Tang, Li-Feng Wu, Gui-Rong Zeng, Yu-Hong Wang, Ze-Neng Cheng, De-Jian Jiang.
HIGHLIGHTS
There are strong negative correlation between the ratio of ADPN to FGF9 and the total score of Hamilton Depression Scale in clinic patients
The injection of recombinant ADPN or FGF9 antibody into lateral ventricle could significantly attenuate the depressive-like symptoms and decrease FGF9 level in CUMS-induced depressive mice
The susceptibility of aging adipo-/- mice with high FGF9 level responding to prodepressive stimulus was increased in adipo-/- mice, which could be attenuated by the injection of recombinant ADPN or FGF9 antibody into lateral ventricle
ADPN maybe a key negative regulator of FGF9/FGFR3 in depressive disorder