Angiotensin-converting enzyme inhibitor rapidly ameliorates depressive-type behaviors via bradykinin-dependent activation of mTORC1

Journal Pre-proof Angiotensin-converting enzyme inhibitor rapidly ameliorates depressive-type behaviors via bradykinin-dependent activation of mTORC1 Han Luo, Peng-Fei Wu, Yu Cao, Ming Jin, Tian-Tian Shen, Ji Wang, Jian-Geng Huang, Qian-Qian Han, Jin-Gang He, Si-Long Deng, Lan Ni, Zhuang-Li Hu, Li-Hong Long, Fang Wang, Jian-Guo Chen PII:

S0006-3223(20)30094-9

DOI:

https://doi.org/10.1016/j.biopsych.2020.02.005

Reference:

BPS 14125

To appear in:

Biological Psychiatry

Received Date: 26 October 2019 Revised Date:

22 January 2020

Accepted Date: 3 February 2020

Please cite this article as: Luo H., Wu P.-F., Cao Y., Jin M., Shen T.-T., Wang J., Huang J.-G., Han Q.Q., He J.-G., Deng S.-L., Ni L., Hu Z.-L., Long L.-H., Wang F. & Chen J.-G., Angiotensin-converting enzyme inhibitor rapidly ameliorates depressive-type behaviors via bradykinin-dependent activation of mTORC1, Biological Psychiatry (2020), doi: https://doi.org/10.1016/j.biopsych.2020.02.005. 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. © 2020 Published by Elsevier Inc on behalf of Society of Biological Psychiatry.

Luo H et al

1

Angiotensin-converting

enzyme

inhibitor

rapidly

ameliorates

2

depressive-type behaviors via bradykinin-dependent activation of

3

mTORC1

4

Han Luo1,*, Peng-Fei Wu1,2,3,4,*, Yu Cao1, Ming Jin6, Tian-Tian Shen1, Ji Wang1,

5

Jian-Geng Huang6, Qian-Qian Han1, Jin-Gang He1, Si-Long Deng1, Lan Ni1,

6

Zhuang-Li Hu1,2,3, Li-Hong Long1,2,3,4, Fang Wang1,2,3,4,5, † and Jian-Guo Chen1,2,3,4,5, †

7

1

8

Huazhong University of Science and Technology, Wuhan City, Hubei 430030, China

9

2

Department of Pharmacology, School of Basic Medicine, Tongji Medical College,

Key Laboratory of Neurological Diseases (HUST), Ministry of Education of China,

10

Wuhan, Wuhan City, Hubei 430030, China

11

3

12

of Hubei Province, Wuhan City, Hubei 430030, China

13

4

14

University of Science and Technology, Wuhan, 430030, China

15

5

The Collaborative-Innovation Center for Brain Science, Wuhan 430030, China

16

6

Department of Pharmaceutics, College of Pharmacy, Tongji Medical College,

17

Huazhong University of Science and Technology, Wuhan, China

18

Short title: ACEI may serve as fast-acting anti-depressants

19

*

The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation

Laboratory of Neuropsychiatric Diseases, The Institute of Brain Research, Huazhong

Equally contributed authors † Correspondence to: Dr. Jian-Guo Chen or Dr. Fang 1

Luo H et al

1

Wang.

Department of Pharmacology, Tongji Medical College, Huazhong University

2

of Science and Technology, 13 Hangkong Road, Wuhan, Hubei, China 430030

3

E-mail: [email protected] or [email protected]

4

Tel: +86-27-83692636; FAX: +86-27-83692608

5 6

Key Words: MDD, Angiotensin-converting enzyme inhibitor, Bradykinin, Cdc42,

7

mTORC1 , captopril

8 9

Word count of the abstract:

247

10

Word count of the main text: 3982

11

Number of figures: 6

12

Number of table(s): 0

13

Number of supplement(s): 8

14 15 16 17 18 19 20 21 22 23 24 25 26 27 2

Luo H et al

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Abstract

2

BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEIs) are widely

3

prescribed anti-hypertensive agents. Intriguingly, case reports and clinical trials have

4

indicated that ACEIs, including captopril and lisinopril, may have a rapid

5

mood-elevating effect in certain patients, but few experimental studies have

6

investigated their value as fast-onset antidepressants. METHODS: The present study

7

consisted of a series of experiments using biochemical assays, immunohistochemistry

8

and behavioral techniques to examine the effect and mechanism of captopril on

9

depressive-like behavior in two animal models, chronic unpredictable stress (CUS)

10

model and chronic social defeat stress (CSDS) model. RESULTS: Captopril (19.5 or

11

39 mg/kg, intraperitoneal injection) exerted rapid antidepressant activity in the

12

CUS-treated and CSDS-treated mice. Pharmacokinetic analysis revealed that captopril

13

crossed the blood brain barrier (BBB) and that lisinopril, another ACEI with better

14

BBB permeability, exerted a faster and longer-lasting effect at a same molar

15

equivalent dose. This antidepressant effect seemed to be independent of the

16

renin-angiotensin system, but dependent on bradykinin (BK) system, since the

17

decreased BK detected in the stressed mice could be reversed by captopril. The

18

hypofunction of the downstream effector of BK, cell division control protein 42

19

homolog, contributed to the stress-induced loss of dendritic spines, which was rapidly

20

reversed by captopril via activating the mammalian target of rapamycin complex 1

21

(mTORC1) pathway. CONCLUSIONS: Our findings indicate that the BK-dependent 3

Luo H et al

1

activation of mTORC1 may represent a promising mechanism underlying

2

antidepressant pharmacology. Considering their affordability and availability, ACEIs

3

may emerge as a novel fast-onset antidepressant, especially for patients with

4

comorbid depression and hypertension.

5

6

Introduction

7

Major depressive disorder (MDD) is a significant contributor to the global burden

8

of disease and affects approximately 16% of the world’s population at some point in

9

their lives. The major problems in the therapy of MDD are that only 40-70% of

10

patients with depression respond to drug treatment, and that the onset of the

11

therapeutic effect is in a delayed manner. In recent years, there has been substantial

12

clinical and preclinical progress in identifying fast-onset antidepressants, such as

13

ketamine and scopolamine (1-3), and the enantiomer S-ketamine has been recently

14

approved to be prescribed for treatment-resistant depression by the U.S. food and drug

15

administration (FDA). However, the use of available fast-onset antidepressants is

16

limited due to their risk of dependence and psychomimetic side effects.

17

Some

clinic

studies

have

revealed

a

possible

relationship

between

18

cerebrovascular disease and occurrence or outcomes of depression in later life (4-6).

19

Angiotensin II (Ang II), the most important component of renin-angiotensin-system

20

(RAS),

is

assumed

to

stimulate

the

hyperactivity

of

the 4

Luo H et al

1

hypothalamic-pituitary-adrenocortical axis via activation of Ang II type 1 receptor

2

(AT1R) in corticotropin-releasing-hormone neurons (7-8). Clinical data indicate that

3

RAS-acting agents, including AT1R blocker (ARB) and angiotensin-converting

4

enzyme (ACE) inhibitor, reduce the risk of mood disorders compared with the

5

patients taking other antihypertensive drugs such as calcium channel blockers and

6

β-blockers (9). Intriguingly, although no randomized controlled trial has assessed the

7

effects, in the last forty years, a succession of case reports and clinical studies have

8

reported that ACEIs, not ARBs, elicit mood-elevating effects in certain hypertensive

9

patients (10-17), which were indicated by the time axis in Fig.S1. In the early 1980s,

10

several cases reported that captopril might rapidly improve the patient's mood within

11

1-2 days at the daily dosages ranging from 37.5 - 200 mg (10-11, 13). In 2005, mood

12

benefits were observed in 9 hypertensive patients with MDD, who were treated with

13

another ACEI, lisinopril (18). ACEIs are a group of widely prescribed

14

anti-hypertensive agents, but to date, very few experimental studies have investigated

15

their antidepressant value (19-20). In most hypertensive patients, the daily dosage of

16

captopril is 12.5 - 150 mg (a single dosage is 12.5 - 50 mg, one to three times per day).

17

Thus, we screened the effects of captopril on depressive-like behaviors in mice as

18

following: 2.44, 4.88, 9.75, 19.5 and 39 mg/kg/day (the equivalent of a single dosage

19

of 11.87, 23.74, 47.48, 94.96 or 189.92 mg in a human weighing 60 kg), using the

20

body surface area normalization method described by FDA draft guidelines (21,

21

human dosage = mice dosage × Km mice /Km human, Km mice = 3, Km human = 5

Luo H et al

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37). We found that only high dosage of captopril produced a fast-onset therapeutic

2

effect on depressive-like behaviors.

3

An increasing number of studies have demonstrated that activation of mammalian

4

target of rapamycin (mTOR) signaling in the medial prefrontal cortex (mPFC)

5

mediate the rapid antidepressant actions of ketamine (3, 22). A recent report has

6

indicated that direct activation of mTOR signaling via a leucine sensing pathway by

7

the sestrin modulator NV-5138 mimics the rapid antidepressant effects of ketamine

8

(23). It has been reported that ACE may block mTOR signaling pathway (24-25), but

9

very little is known about the downstream signaling mechanism. ACE function

10

comprises the production of Ang II and the breakdown of bradykinin (BK). Ang II,

11

the main product of ACE, increases the mTOR activity (26-27), whereas BK, a

12

degraded substrate for ACE, also activates mTOR signaling pathway via B2 receptor

13

(B2R) in various conditions (28-30). We further determined that ACEI may work by

14

activating the mammalian target of rapamycin complex 1 (mTORC1) pathway via a

15

non-RAS mechanism, i.e., a BK-dependent pathway, and identified B2R as a novel

16

therapeutic target for depression.

17

18

Materials and Methods

19

Detailed Materials and Methods are available in Supplementary Materials and

20

Methods.

6

Luo H et al

1

2

Animals and behavioral experiments. Male C57BL/6J mice (7-8 weeks of age,

3

18-21 g) from Hunan SJA Laboratory Animal (Changsha, Hunan, China) were used in

4

our study. All the procedures were conducted following the Declaration of Helsinki

5

and the Guide for Care and Use of Laboratory Animals as adopted and promulgated

6

by the National Institutes of Health. All experiments were approved by the Review

7

Committee for the Use of Human or Animal Subjects of Huazhong University of

8

Science and Technology. Chronic unpredictable stress (CUS) and chronic social

9

defeat stress (CSDS) were used to induce depressive-like behavior and all the

10

behavioral tests were conducted as previously described with slight modifications

11

(31-33). Sample sizes were determined according to those used in previous

12

publications from our group and others similar studies (31, 34-35) and justified by the

13

power analyses.

14

15

Experiments in molecular biology. Quantitative real-time PCR (qPCR) was

16

performed on the StepOnePlusTM Real-Time PCR System (Applied Biosystems,

17

Foster City, CA) to analyze the gene expression. Western blotting was used to analyze

18

the protein level. The vectors contained a cytomegalovirus-driven enhanced green

19

fluorescent protein (EGFP) and oligonucleotides encoding short hairpin RNAs

20

(shRNAs) of B2R were purchased from Shanghai Genechem Co., Ltd. (Shanghai,

21

China). The assay of cell division control protein 42 homolog (Cdc42) activity was 7

Luo H et al

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performed by analyzing GTP-Cdc42/Cdc42 ratio as previous reports (36).

2

3

Statistics. Analysis was performed using Graphpad Prism 7.0 or SPSS 18.0 software

4

(SPSS Inc., Chicago, IL, USA) and p < 0.05 was considered statistically significant.

5

All values were expressed as mean ± S.E.M. Each “n” corresponded to a single mouse.

6

If technical replicates were performed, their mean was considered as one “n”. We

7

tested the data normality using Kolmogorov-Smirnov test of normality with the

8

Dallal-Wilkinson-Lillie corrected P value (GraphPad Prism 7.0), and variances were

9

compared by Bartlett statistics to decide whether parametric tests were applicable.

10

Statistical analyses were performed using one-way ANOVA followed by LSD

11

multiple comparison tests or two-way ANOVA followed by Bonferroni test to

12

compare means of three or more groups, one-way repeated measures ANOVA to

13

examine means of repeated measured data and unpaired two-tailed Student’s t-test to

14

compare two groups. For the non-normal distributed data (data for AT1R in the mPFC

15

in Fig.3A, data for CUS and CUS + Captopril group in Fig.3B), Mann-Whitney test

16

were used.

17

18

Results

19

Captopril rapidly reverses chronic stress-induced depressive-like behaviors in

20

mice. 8

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First, the effects of captopril on behavioral despair were measured by immobility

2

time in two behavioral tests, the tail suspension test (TST) and forced swimming test

3

(FST). A wide range of clinically relevant dosages of captopril were chosen (2.44,

4

4.88, 9.75, 19.5, and 39 mg/kg/day in mice). Intraperitoneal injection of captopril at

5

dosages of 9.75, 19.5 and 39 mg/kg/day produced rapid antidepressant responses in

6

the FST and TST 24 h after administration (Fig. 1A, B). However, captopril exhibited

7

few influences on the open field test (OFT, Fig. S2A-C), elevated plus maze (EPM,

8

Fig. S2D-F) and the novelty-suppressed feeding test (NSFT, Fig. S2G), indicating that

9

captopril may exert little effect on anxiety. At the same dosage (19.5 or 39 mg/kg, i.p),

10

captopril exerted little effect on water drinking (12 h, Fig. S2H). The effect of

11

captopril in the FST was detected within 24 h and lasted for 7 days after one dosing

12

(Fig. S2I). The levels of captopril in the mPFC were measured using liquid

13

chromatography-tandem mass spectrometry (LC-MS/MS) following the peripheral

14

administration of captopril (19.5 mg/kg, Fig. 1C). The plasma concentrations of

15

captopril reached peak levels (184.06 ± 20.25 ng/ml) at 0.5 h after administration. In

16

this experimental condition, the detection limit of captopril concentration was 23.01

17

nM (5 ng/ml). A modest concentration of captopril was detected in the mPFC tissue

18

(24.6 ± 2.82 ng/ml) 0.5 h after administration, indicating that captopril can enter the

19

blood brain barrier (BBB) at high dosage.

20

Next, the mice that exposed to CUS were used to evaluate the effects of captopril.

21

CUS mice displayed anhedonia, a core symptom of depression measured by the SPT, 9

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1

increased despair behaviors and a reduction in body weight (Fig. S3A-E). Notably, the

2

CUS-induced reduction in sucrose preference was reported to be reversed by daily

3

treatment with tricyclic antidepressants for 2 to 3 weeks (37-38); however, it was

4

rapidly reversed by a single dosage (19.5 or 39 mg/kg, Fig. 1D) of captopril within 24

5

h. This effect was not due to the effect of angiotensin on drinking behavior because

6

the CUS mice treated with captopril (0, 19.5 or 39 mg/kg, i.p) displayed similar total

7

fluid consumption in the SPT (n = 8-12, Fig. S3F). Moreover, the increased sucrose

8

preference was observed at 1, 3 and 7 days after interperitoneal injection of captopril

9

(19.5 mg/kg), indicating that the antidepressant effects of captopril on anhedonia can

10

last for at least 1 week (Fig. 1E). These results consistently demonstrate that captopril

11

elicits fast and sustained antidepressant effects in mice.

12

We also employed another animal model of depression, CSDS, to evaluate the

13

effect of captopril on social deficits. We found that the social index increased at 24 h

14

after intraperitoneal injection of captopril (19.5 mg/kg, Fig. 1F), indicating that

15

captopril improved the social interaction in the socially defeated mice. The effects of

16

a single dosage of captopril on blood pressure and locomotor activity in CUS mice

17

were very limited 24 h after administration (Fig. S4), indicating that its rapid

18

antidepressant activity may not be associated with the alterations in blood pressure.

19

20

Other RAS-acting agents are insufficient to elicit rapid antidepressant effects. 10

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The Ang II reducing effect largely mediates the cardiovascular effects of ACEI.

2

Thus, we investigated whether other RAS-acting agents mimicked the rapid

3

antidepressant activity of ACEI. First, we employed a direct renin inhibitor (DRI),

4

aliskiren, to mimic the Ang II-reducing effect of captopril (Fig. 2A). Aliskiren (10

5

mg/kg, 50 mg/kg) induced a significant reduction in Ang II levels 24 h after

6

administration (Fig. S5A), but failed to exert significant antidepressant effects in the

7

CUS-treated mice (Fig. 2B), indicating that Ang II-reducing effect may be insufficient

8

to elicit rapid antidepressant effects. Then, valsartan, a specific and widely used ARB,

9

was applied to mimic the captopril-mediated hypofunction of AT1R. A previous

10

report found that pretreatment with losartan (20 mg/kg), another ARB, reduced the

11

duration of immobility in the FST of normal normal male CD mice (39). However, in

12

the CUS mice, the intraperitoneal injection of valsartan (30 or 60 mg/kg) elicited no

13

observed rapid antidepressant effects (Fig. 2C), which was consistent with a very

14

recent report performed on CUS-treated rats (40). We also administered an Ang II

15

neutralizing antibody (nAb), aliskiren and valsartan directly into the mPFC, again,

16

and they did not exert rapid antidepressant effects (Fig. 2D and Fig. S5B), suggesting

17

that down-regulation of Ang II function in either the peripheral or central, may be

18

insufficient to produce rapid antidepressant effects in the stressed mice.

19

Then, we asked whether other ACEIs have similar antidepressant effects and

20

compared the efficacy/sustainability of lisinopril, a long-lasting ACEI that can pass

21

through the BBB (41), with that of captopril. At a molar equivalent dosage to that of 11

Luo H et al

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captopril (19.5 mg/kg), the duration of the antidepressant effect of lisinopril (39.6

2

mg/kg) was much longer than that of captopril in non-stressed mice (Fig. 2E). In the

3

stressed mice, lisinopril (39.6 mg/kg) exerted a much faster antidepressant effect

4

(within two hours) than that of captopril (Fig. 2F).

5

6

Reactivation of the BK system mediates the rapid antidepressant effects of

7

ACEI.

8

We observed that the concentration of Ang II, as well as the mRNA levels of

9

angiotensinogen (AGT), ACE, AT1R and Ang II type 2 receptor (AT2R), exhibited

10

few changes in the mPFC, hippocampus and nucleus accumbens (NAc) of the

11

CUS-treated mice (Fig. 3A). As a vasodilator nonapeptide that is degraded by ACE,

12

BK is another important molecular mediator underlying the cardiovascular effects of

13

ACEI as a non-RAS mechanism. Interestingly, the levels of BK were significantly

14

decreased in the mPFC and plasma of stressed mice (Fig. 3B-D), an effect that was

15

reversed by captopril (19.5 mg/kg, i.p.). To assess the relevance of BK levels to MDD,

16

we analyzed the BK concentration in the plasma of human subjects who were

17

diagnosed with MDD. Notably, a similar change in BK levels was observed in the

18

plasma of depressed patients (Fig. 3E), as determined by ELISA. BK exerts its effects

19

via two different receptor subtypes: B1 receptor (B1R) and B2R. The level of B2R,

20

not B1R in the mPFC of CUS mice was up-regulated (Fig. 3F). The altered expression 12

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of B2R may confer a compensatory mechanism of BK deficits induced by CUS.

2

Previous reports have indicated that when administered in the central nervous

3

system (CNS), BK leads to initial rapid excitation (42) and hyperalgesia (43). Thus,

4

we asked whether the BK system mediated the rapid antidepressant effect of captopril.

5

To address this issue, BK (50 ng/per side) was bilaterally infused into the mPFC of

6

stressed mice to mimic the BK-potentiating property of captopril. We found that the

7

local administration of BK in the mPFC rapidly reversed depressive-like behaviors in

8

the stressed mice (Fig. 3G). Next, we explored whether the inhibition of BK function

9

was associated with depressive-like behaviors. Repeated administration of HOE140

10

(65.226 µg/kg per day, i.p), a blocker of B2R, but not DALBK (99.818 µg/kg per day,

11

i.p), a blocker of B1R, for 7 days significantly increased the immobility time in the

12

FST of mice (Fig. 3H), suggesting that hypofunction of B2R may contribute to the

13

pathophysiology of depression. Together, these results demonstrate that B2R

14

signaling may play a key role in the antidepressant activity of captopril.

15

To further confirm the role of B2R in the antidepressant mechanism of captopril,

16

we employed both pharmacological and genetic approaches in this study. Intra-mPFC

17

infusion of HOE140 (100 nM, 1 µl per side), but not DALBK (100 nM, 1 µl per side),

18

completely blocked captopril-induced reduction in the immobility time of TST and

19

FST (Fig. 4A). We further examined the influence of HOE140 on the antidepressant

20

effect of captopril in the CUS-treated mice. Intra-mPFC injection of HOE140

21

abolished the behavioral responses to captopril in stressed mice (Fig. 4B). To directly 13

Luo H et al

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explore the role of B2R in the effects of captopril, we used a lentivirus that expressed

2

short hairpin RNAs to knockdown B2R expression. As shown in Fig.S6, the

3

lentivirus-guided EGFP expression was predominantly located in the mPFC and the

4

B2R protein level was significantly down-regulated by LV-B2R siRNA. We found

5

that captopril failed to exert an antidepressant effect in the mice with lentivirus-guided

6

knockdown of B2R (Fig.S6 and Fig. 4C). These results demonstrate that BK-B2R

7

signaling determines the antidepressant effect of captopril.

8

9 10

BK-stimulated Cdc42 activity confers the antidepressant effects of captopril via activation of the mTORC1 pathway.

11

B2R is widely distributed in the CNS (44) and a previous study has revealed that

12

BK depolarizes motor neurons by postsynaptic activation of B2R (45). We

13

hypothesized that B2R may initiate the antidepressant activity via postsynaptic action.

14

BK is a powerful stimulator of Cdc42, a critical Rho GTPase protein (46-47).

15

Growing evidence suggests that Cdc42 controls rapid presynaptic maturation to

16

facilitate synaptogenesis (48) and also contributes to postsynaptic maturation (49).

17

Furthermore, Cdc42 regulates the activation of the mTORC1 signaling pathway

18

(50-51), which is critical in the synaptic mechanisms underlying rapid-acting

19

antidepressants. Thus, we asked whether the Cdc42-mTOR signaling pathway

20

contributes to the antidepressant activity of captopril. 14

Luo H et al

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First, we found that the activity of Cdc42 was reduced significantly in the mPFC

2

of CUS-treated mice, and captopril rapidly rescued the decrease in Cdc42 activity in

3

the mPFC (Fig. 5A). As shown in Fig. 5B & C, the intra-mPFC infusion of a selective

4

Cdc42 inhibitor ML141 (8.15 mg/µl/side) blocked the effect of captopril on despair

5

behaviors in the non-stressed mice (Fig. 5B), and the pre-infusion of ML141 into the

6

mPFC abolished the antidepressant activity of captopril in the stressed mice (Fig. 5C).

7

These results suggest that Cdc42 plays a role in the mechanism in the antidepressant

8

activity of captopril.

9

We next examined the effect of captopril on the mTORC1 activity. The

10

phosphorylated forms of mTORC1 (p-mTOR) and the key downstream target of

11

mTORC1, p70S6 kinase (p-p70S6K) represent the activation of mTORC1 signaling.

12

We found that captopril activated mTORC1 in the non-stressed mice, moreover, the

13

intra-mPFC infusion of ML141 prevented the captopril-induced activation of

14

mTORC1 (Fig. 5D). In the CUS-treated mice, captopril reversed the stress-induced

15

inhibition of mTORC1 activity (Fig. 5E, p-mTOR and p-p70S6K) and induced an

16

increased level of BDNF (Fig. 5E). The genetic knockdown of B2R abolished the

17

captopril-induced activation of mTORC1 and BDNF synthesis in the stressed mice

18

(Fig. 5E). We next assessed whether increased mTOR activity is sufficient to mediate

19

the rapid antidepressant activity of captopril. Accordingly, a selective mTORC1

20

inhibitor, rapamycin, was infused into the mPFC of CUS mice. We found that the

21

infusion of rapamycin into the mPFC abolished captopril-elicited rapid antidepressant 15

Luo H et al

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activity (Fig. 5F).

2

To further address the relationship between BK and mTORC1 activity, we

3

observed a direct action of BK on mTORC1 activity in primary cultured neurons.

4

Incubation of BK (10 nM) increased phosphorylation of p70S6K and BDNF level in

5

primary cultured neurons of mPFC (Fig. S7A-B). Additionally, in vivo, we found that

6

rapamycin blocked BK-elicited rapid antidepressant responses (Fig. S7C). Thus,

7

captopril may exert antidepressant effects through the BK-B2R-Cdc42-mTORC1

8

signaling pathway.

9

10

Captopril reverses CUS-induced synaptic loss and stimulates synaptogenesis.

11

Considering that Cdc42 activity can regulate dendritic spine plasticity, we asked

12

whether the reactivation of Cdc42 function can alleviate the CUS-induced synaptic

13

loss. Using confocal microscopy, the dendritic spine density in the mPFC of stressed

14

mice was measured 24 h after captopril administration. As previously reported (52),

15

dendritic spines were classified by functional subtype: long thin, mushroom and

16

stubby. In our study, the CUS exposure significantly reduced the number and density

17

of dendritic spines, especially long thin spines, in the mPFC of mice. Moreover,

18

captopril rapidly rescued the synaptic loss observed in the mPFC of CUS-treated mice

19

by increasing the spine density and number of long thin and mushroom dendritic

20

spines (Fig. 6A-B). The morphological changes were abolished by the intra-mPFC 16

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infusion of ML141 (Fig. 6A-B), indicating that Cdc42-dependent synaptogenesis

2

mediates the effect of captopril on CUS-induced synaptic loss. Furthermore, captopril

3

significantly increased the levels of key synaptic proteins, including GluA1 and

4

PSD95, in the mPFC of CUS mice (Fig. 6C), which strengthens our hypothesis that

5

captopril exerts antidepressant effects by increasing synapse numbers. Captopril also

6

increased the levels of synaptic proteins, including GluA1 and PSD95 in the

7

hippocampus of CUS mice (Fig. 6D), suggesting that in addition to the mPFC, other

8

brain areas may be involved in the effect of captopril.

9

10

Discussion

11

In the present study, we demonstrated that ACEIs produced a rapid and

12

long-lasting reversal of chronic stress-induced depressive-like behaviors by

13

potentiating the BK-B2R-Cdc42-mTORC1 signaling pathway (Fig. 6E). Our study

14

proposed a new property of ACEI, an important class of RAS-acting agents. As

15

widely used anti-hypertensive agents, the clinical antidepressant value of RAS-acting

16

agents has not been established. Our results indicated that RAS-acting agents

17

including DRI, ARB and captopril at a single clinic dosage, did not exert a rapid

18

antidepressant activity in CUS-treated rodents. Interestingly, our data indicated that

19

the BK action, an acute physiological outcome that was often limited by the clinical

20

dosage regimen to avoid side effects, mediated the antidepressant effects of ACEIs at 17

Luo H et al

1

high dosages. An alternative interpretation for the overlook of the ACEI effect may be

2

that captopril initiated a rapid but transient action for a few days, like ketamine, which

3

was approved for anesthetic in 1970, but its antidepressant value has been established

4

until recent years.

5

BK plays a key role in the pharmacological effect of captopril (53). A notable

6

finding of the present study is that the altered BK system may contribute to the

7

development of depressive-like behaviors. Decreased levels of BK in the blood and

8

mPFC were observed in mice exposed to CUS. The administration of BK in the

9

mPFC rapidly rescued CUS-induced behavior deficits. Consistently, the levels of BK

10

were also decreased in the plasma of MDD patients. It should be noted that the

11

differences in the gender composition of human samples may induce bias. We

12

analyzed the bradykinin level between wemen and men, and no differences were

13

found, both in the MDD patients and healthy volunteers. However, considering the

14

sex ratio in control population does not mirror the ratio in the experimental group, and

15

all the experiments were performed on the male mice, the role of bradykinin in

16

women depressive patients should be further evaluated.

17

Increased B2R levels in the mPFC of stressed mice were observed, and these

18

increased levels may be a compensatory mechanism in response to chronic

19

stress-induced deficits in BK signaling. We found that repeated administration of the

20

B2R blocker HOE140 (65.226 µg/kg per day, i.p) for 7 days increased FST

21

immobility time (seen in Fig.3H). However, in the Fig.4A, intra-mPFC infusion of 18

Luo H et al

1

HOE140 (100 nM, 1 µl per side, once) did not affect the immobility time after 24

2

hours, indicating that a long-term blockade, not acute blockade of B2R, may generate

3

despair behaviors. We hypothesized that chronic blockade of B2R in the PFC would

4

also have increased immobility, and this point was supported by our data that the

5

genetic knockdown of B2R in the mPFC increased FST immobility time in control

6

mice (Fig.4C). Both pharmacological and genetic approaches revealed that B2R, not

7

B1R, mediated the antidepressant effects of BK and captopril. Considering that a

8

neuroprotective role of B2R has been revealed (54), our findings raise the possibility

9

that BK-potentiating peptides or drugs may emerge as a new class of antidepressants.

10

Notably, BK-induced cough and hyperalgesia may affect treatment compliance,

11

however, it could be prevented by a rational drug regimen or a peripheral blocker of

12

BK receptors.

13

Previous studies have reported that BK potentiates synaptic transmission via

14

activating both presynaptic and postsynaptic B2Rs (45, 55). As a G-protein-coupled

15

receptor, B2R strongly stimulates Cdc42 activation (46-47). It is generally believed

16

that the Cdc42 signaling pathway plays a key role in the structural plasticity of

17

dendritic spines, dendritic morphogenesis, synaptic maturation and axon guidance

18

(56). MDD has been linked to aberrant dendritic spine and synapse development (57).

19

Until now, very little is known about the role of Cdc42 in depression. In our study,

20

we found that CUS induced a robust defect in Cdc42 activation in the mPFC, which

21

could be rescued by captopril. Meanwhile, captopril restored CUS-induced spine loss. 19

Luo H et al

1

Both the behavior and morphologic effects of captopril were abolished by the Cdc42

2

inhibitor ML141, suggesting that a Cdc42-dependent mechanism may be critical to

3

the effect of captopril.

4

In the recent decade, ketamine and scopolamine have been developed as

5

rapid-acting antidepressants that can improve depressive symptoms within hours or

6

days in patients. The promotion of mTORC1 has been recognized as a common

7

signaling pathway that mediates rapid-acting antidepressant effects. A recent report

8

indicated that direct activation of mTORC1 via a leucine sensing pathway by

9

NV-5138 mimicked the rapid antidepressant effects of ketamine without affecting

10

glutamate receptors (23), and Navitor Pharmaceuticals has commenced Phase I

11

Clinical Evaluation of NV-5138 in patients with treatment-resistant depression.

12

Similarly, we found that captopril activated the mTORC1 pathway via a

13

BK-dependent pathway. BK incubation directly activated the mTOR signaling in the

14

cultured neurons (Fig. S7A-B), which may work through a BK-B2R-Cdc42-mTORC1

15

signaling pathway. Previous study has reported that, in an in vitro study,

16

overexpression of ACE reduced the level of p70S6K, whereas captopril increased the

17

levels of p70S6K (24), which was in consistent with our observations. We found that

18

captopril promoted synaptogenesis, which may be due to that captopril and BK,

19

similar to other rapid-acting antidepressants, facilitated the mTORC1-dependent

20

synaptogenesis.

21

ACEIs are widely used antihypertensive agents, and some studies also indicated 20

Luo H et al

1

ACEIs might be beneficial for psychiatric diseases (58). Considering the affordability

2

and availability of ACEIs, long-lasting ACEIs that can cross the BBB may be used as

3

new rapid-onset antidepressants. The pharmacodynamics and toxicity of ACEIs have

4

been well studied. Considering the fetal toxicity, and the increase of neonatal

5

morbidity and death, the use of ACEI for depression during pregnancy should be

6

limited. In patients with comorbid depression and diabetes or impaired renal function,

7

ACEI should be used with caution. A further large-scale, randomized, controlled

8

clinical study should be performed to evaluate the antidepressant effect of ACEIs.

9

10

Acknowledgments

11

This work was supported by grants from the Foundation for Innovative Research

12

Groups of NSFC (No. 81721005 to J.G.C. and F. W.), National Natural Science

13

Foundation of China (No. 81773712 to P.F.W., No. 81471377 and No. 81671438 to

14

F.W., No. 81473198 and No. 81673414 to J.G.C.), PCSIRT (No. IRT13016) to J.G.C.,

15

the Program for HUST Academic Frontier Youth Team and Integrated Innovative

16

Team for Major Human Diseases Program of Tongji Medical College, HUST, to F. W.

17

18

Author contributions

19

H.L. performed most molecular and behavioral experiments, stereotaxic surgeries and

21

Luo H et al

1

analyzed data. Peng-Fei Wu designed the experiments, performed molecular

2

experiments, helped methodology and analyzed data. Y.C., Q.Q.H. and S.L.D.

3

contributed to animal experiments and stereotaxic surgeries. M.J and J.G. H

4

performed LC-MS/MS analysis of captopril. T.T.S. performed CSDS model and

5

contributed to behavioral experiments. J.W. and L.N. contributed to confocal

6

microscopy experiments. J.G.H. contributed to measure plasma BK content. Z.L.H.

7

and L.H.L. provided the technique supports. F.W. and J.G.C. supervised the project,

8

designed the experiments, revised the manuscript and supported funding acquisition.

9

P.F.W., H.L., F.W. and J.G.C. wrote the paper with contributions from all of the other

10

authors.

11

12

Financial Disclosures: The authors report no biomedical financial interests or

13

potential conflicts of interest.

14 15

Reference

16

1.

17

models. Mol Psychiatr. 22:（5（-（（5.

18

2.

19

synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science.

20

329:959-9（4.

Ramaker MJ, Dulawa SC (2017–: Identifying fast-onset antidepressants using rodent

Li N, Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M, et al. (2010–: mTOR-dependent

22

Luo H et al

Voleti B, Navarria A, Liu RJ, Banasr M, Li N, Terwilliger R, et al. (2013–: Scopolamine

1

3.

2

rapidly increases mammalian target of rapamycin complex 1 signaling, synaptogenesis, and

3

antidepressant behavioral responses. Biol Psychiatry. 74:742-749.

4

4.

5

evolution of depressive symptoms in the Cardiovascular Health Study. Stroke, 33: 1（3（-1（44

6

5.

7

Carotid artery stiffness and incident depressive symptoms: The Paris Prospective Study III.

8

Biol Psychiatry, 85: 498-505.

9

（.

Steffens DC, Krishnan KR, Crump C, Burke GL. (2002–: Cerebrovascular disease and

van Sloten TT, Boutouyrie P, Tafflet M, Offredo L, Thomas F, Guibout C, et al. (2019–:

Steffens DC (2019–: Vascular Depression: Is an Old Research Construct Finally Ready for

10

Clinical Prime Time? Biol Psychiatry, 85:441-442.

11

7.

12

corticotropin-releasing-hormone neurons by angiotensin II. Neuroendocrinology. （1:437-444.

13

8.

14

Polymorphisms in the angiotensin-converting enzyme gene are associated with unipolar

15

depression, ACE activity and hypercortisolism. Mol Psychiatry. 11:1003-1015.

16

9.

17

Monotherapy With Major Antihypertensive Drug Classes and Risk of Hospital Admissions for

18

Mood Disorders. Hypertension. （8:1132-1138.

19

10. Zubenko GS, Nixon RA (1984–: Mood-elevating effect of captopril in depressed patients.

Aguilera G, Young WS, Kiss A, Bathia A (1995–: Direct regulation of hypothalamic

Baghai TC, Binder EB, Schule C, Salyakina D, Eser D, Lucae S, et al. (200（–:

Boal AH, Smith DJ, McCallum L, Muir S, Touyz RM, Dominiczak AF, et al. (201（–:

23

Luo H et al

1

Am J Psychiatry. 141:110-111.

2

11. Deicken RF (198（–: Captopril treatment of depression. Biol Psychiatry. 21:1425-1428.

3

12. Croog SH, Levine S, Testa MA, Brown B, Bulpitt CJ, Jenkins CD, et al. (198（–: The

4

effects of antihypertensive therapy on the quality of life. N Engl J Med. 314:1（57-1（（4.

5

13. Germain L, Chouinard G (1988–: Treatment of recurrent unipolar major depression with

6

captopril. Biol Psychiatry. 23:（37-（41.

7

14. Cohen BM, Zubenko GS (1988–: Captopril in the treatment of recurrent major depression.

8

J Clin Psychopharmacol. 8:143-144.

9

15. Vuckovic A, Cohen BM, Zubenko GS (1991–: The use of captopril in treatment-resistant

10

depression: an open trial. J Clin Psychopharmacol. 11:395-39（.

11

1（. Michalsen A, Wenzel RR, Mayer C, Broer M, Philipp T, Dobos GJ (2001–: [Quality of life

12

and psychosocial factors during treatment with antihypertensive drugs. A comparison of

13

captopril and quinapril in geriatric patients]. Herz. 2（:4（8-47（.

14

17. Braszko JJ, Karwowska-Polecka W, Halicka D, Gard PR (2003–: Captopril and enalapril

15

improve cognition and depressed mood in hypertensive patients. J Basic Clin Physiol

16

Pharmacol. 14:323-343.

17

18. Hertzman M, Adler LW, Arling B, Kern M (2005–: Lisinopril may augment antidepressant

18

response. J Clin Psychopharmacol. 25:（18-（20.

19

19. Giardina WJ, Ebert DM (1989–: Positive effects of captopril in the behavioral despair swim 24

Luo H et al

1

test. Biol Psychiatry. 25:（97-702.

2

20. Martin P, Massol J, Puech AJ (1990–: Captopril as an antidepressant? Effects on the

3

learned helplessness paradigm in rats. Biological psychiatry. 27:9（8-974.

4

21. Nair A, Morsy MA, Jacob S (2018–: Dose translation between laboratory animals and

5

human in preclinical and clinical phases of drug development. Drug Develop Res. 79:373-382.

6

22. Li N, Liu RJ, Dwyer JM, Banasr M, Lee B, Son H, et al. (2011–: Glutamate

7

N-methyl-D-aspartate receptor antagonists rapidly reverse behavioral and synaptic deficits

8

caused by chronic stress exposure. Biol Psychiatry. （9:754-7（1.

9

23. Kato T, Pothula S, Liu RJ, Duman CH, Terwilliger R, Vlasuk GP, et al. (2019–: Sestrin

10

modulator NV-5138 produces rapid antidepressant effects via direct mTORC1 activation. J

11

Clin Invest. 129:2542-2554.

12

24. Mori S, Tokuyama K (2007–: ACE activity affects myogenic differentiation via mTOR

13

signaling. Biochem Biophys Res Commun. 3（3:597-（02.

14

25. Chen LJ, Xu YL, Song B, Yu HM, Oudit GY, Xu R, et al. (201（–: Angiotensin-converting

15

enzyme 2 ameliorates renal fibrosis by blocking the activation of mTOR/ERK signaling in

16

apolipoprotein E-deficient mice. Peptides. 79:49-57.

17

2（. Hafizi S, Wang X, Chester AH, Yacoub MH, Proud CG (2004–: ANG II activates effectors

18

of mTOR via PI3-K signaling in human coronary smooth muscle cells. Am J Physiol Heart Circ

19

Physiol. 287:H1232-1238.

25

Luo H et al

1

27. Wang RH, He JP, Su ML, Luo J, Xu M, Du XD, et al. (2013–: The orphan receptor TR3

2

participates in angiotensin II-induced cardiac hypertrophy by controlling mTOR signalling.

3

Embo Mol Med. 5:137-148.

4

28. Fu C, Cao Y, Li B, Xu R, Sun Y, Yao Y (2019–: Bradykinin protects cardiac c-kit positive

5

cells from high-glucose-induced senescence through B2 receptor signaling pathway. J Cell

6

Biochem.

7

29. Liu YP, Lu ZY, Cui M, Yang Q, Tang YP, Dong Q (201（–: Tissue kallikrein protects

8

SH-SY5Y neuronal cells against oxygen and glucose deprivation-induced injury through

9

bradykinin B2 receptor-dependent regulation of autophagy induction. J Neurochem.

10

139:208-220.

11

30. Yu HS, Wang SW, Chang AC, Tai HC, Yeh HI, Lin YM, et al. (2014–: Bradykinin promotes

12

vascular endothelial growth factor expression and increases angiogenesis in human prostate

13

cancer cells. Biochem Pharmacol. 87:243-253.

14

31. Li MX, Zheng HL, Luo Y, He JG, Wang W, Han J, et al. (2018–: Gene deficiency and

15

pharmacological inhibition of caspase-1 confers resilience to chronic social defeat stress via

16

regulating the stability of surface AMPARs. Mol Psychiatry. 23:55（-5（8.

17

32.

18

Protein 150 and Protein Kinase A Complex in the Basolateral Amygdala Contributes to

19

Depressive-like Behaviors Induced by Chronic Restraint Stress. Biol Psychiatry. 8（:131-142.

20

33. Fogaca MV, Campos AC, Coelho LD, Duman RS, Guimaraes FS (2018–: The anxiolytic

Zhou HY, He JG, Hu ZL, Xue SG, Xu JF, Cui QQ, et al. (2019–: A-Kinase Anchoring

26

Luo H et al

1

effects of cannabidiol in chronically stressed mice are mediated by the endocannabinoid

2

system: Role of neurogenesis and dendritic remodeling. Neuropharmacology. 135:22-33.

3

34.

4

ventral tegmental area regulate behavioral responses to chronic stress. Elife. 7: e32420.

5

35.

6

Antidepressant effects of Fibroblast Growth Factor-2 in behavioral and cellular models of

7

depression. Biol Psychiatry. 72: 258 2（5.

8

3（. Benard V, Bohl BP, Bokoch GM (1999–: Characterization of Rac and Cdc42 activation in

9

chemoattractant-stimulated human neutrophils using a novel assay for active GTPases.

Zhong P, Vickstrom CR, Liu X, Hu Y, Yu L, Yu HG, et al. 2018

: HCN2 channels in the

Elsayed M, Banasr M, Duric V, Fournier NM, Licznerski P, Duman RS (2012–:

10

Journal of Biological Chemistry. 274:13198-13204.

11

37. Willner P, Towell A, Sampson D, Sophokleous S, Muscat R (1987–: Reduction of sucrose

12

preference by chronic unpredictable mild stress, and its restoration by a tricyclic

13

antidepressant. Psychopharmacology (Berl–. 93:358-3（4.

14

38. Navarria A, Wohleb ES, Voleti B, Ota KT, Dutheil S, Lepack AE, et al. (2015–: Rapid

15

antidepressant actions of scopolamine: Role of medial prefrontal cortex and M1-subtype

16

muscarinic acetylcholine receptors. Neurobiol Dis. 82:254-2（1.

17

39. Gard PR, Mandy A, Sutcliffe MA (1999–: Evidence of a possible role of altered angiotensin

18

function in the treatment, but not etiology, of depression. Biol Psychiatry. 45: 1030-4.

19

40. Costa-Ferreira M, Morais-Silva G, Gomes-de-Souza L, Marin MT, Crestani CC (2019–:

27

Luo H et al

1

The AT1 Receptor Antagonist Losartan Does Not Affect Depressive-Like State and Memory

2

Impairment Evoked by Chronic Stressors in Rats. Front Pharmacol. 10.

3

41. Tan J, Wang JM, Leenen FH (2005–: Inhibition of brain angiotensin-converting enzyme by

4

peripheral administration of trandolapril versus lisinopril in Wistar rats. Am J Hypertens.

5

18:158-1（4.

6

42. Okada Y, Tuchiya Y, Yagyu M, Kozawa S, Kariya K (1977–: Synthesis of bradykinin

7

fragments and their effect on pentobarbital sleeping time in mouse. Neuropharmacology.

8

1（:381-383.

9

43. Dalmolin GD, Silva CR, Belle NAV, Rubin MA, Mello CF, Calixto JB, et al. (2007–:

10

Bradykinin into amygdala induces thermal hyperalgesia in rats. Neuropeptides. 41:2（3-270.

11

44. Viel TA, Lima Caetano A, Nasello AG, Lancelotti CL, Nunes VA, Araujo MS, et al. (2008–:

12

Increases of kinin B1 and B2 receptors binding sites after brain infusion of amyloid-beta 1-40

13

peptide in rats. Neurobiol Aging. 29:1805-1814.

14

45. Bouhadfane M, Kaszas A, Rozsa B, Harris-Warrick RM, Vinay L, Brocard F (2015–:

15

Sensitization of neonatal rat lumbar motoneuron by the inflammatory pain mediator bradykinin.

16

Elife. 4:e0（195.

17

4（. Kozma R, Ahmed S, Best A, Lim L (1995–: The Ras-related protein Cdc42Hs and

18

bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3

19

fibroblasts. Mol Cell Biol. 15:1942-1952.

28

Luo H et al

1

47. Sakurada K, Kato H, Nagumo H, Hiraoka H, Furuya K, Ikuhara T, et al. (2002–: Synapsin I

2

is phosphorylated at Ser（03 by p21-activated kinases (PAKs– in vitro and in PC12 cells

3

stimulated with bradykinin. The Journal of biological chemistry. 277:45473-45479.

4

48. Shen W, Wu B, Zhang Z, Dou Y, Rao ZR, Chen YR, et al. (200（–: Activity-induced rapid

5

synaptic maturation mediated by presynaptic cdc42 signaling. Neuron. 50:401-414.

6

49. Choi J, Ko J, Racz B, Burette A, Lee JR, Kim S, et al. (2005–: Regulation of dendritic spine

7

morphogenesis by insulin receptor substrate 53, a downstream effector of Rac1 and Cdc42

8

small GTPases. J Neurosci. 25:8（9-879.

9

50. Fang Y, Park IH, Wu AL, Du G, Huang P, Frohman MA, et al. (2003–: PLD1 regulates

10

mTOR signaling and mediates Cdc42 activation of S（K1. Curr Biol. 13:2037-2044.

11

51. Endo M, Antonyak MA, Cerione RA (2009–: Cdc42-mTOR signaling pathway controls

12

Hes5 and Pax（ expression in retinoic acid-dependent neural differentiation. J Biol Chem.

13

284:5107-5118.

14

52. Radley JJ, Anderson RM, Hamilton BA, Alcock JA, Romig-Martin SA (2013–: Chronic

15

stress-induced alterations of dendritic spine subtypes predict functional decrements in an

16

hypothalamo-pituitary-adrenal-inhibitory prefrontal circuit. J Neurosci. 33:14379-14391.

17

53. Siltari A, Korpela R, Vapaatalo H (201（–: Bradykinin -induced vasodilatation: Role of age,

18

ACE1-inhibitory peptide, mas- and bradykinin receptors. Peptides. 85:4（-55.

19

54. Caetano AL, Dong-Creste KE, Amaral FA, Monteiro-Silva KC, Pesquero JB, Araujo MS,

29

Luo H et al

1

et al. (2015–: Kinin B2 receptor can play a neuroprotective role in Alzheimer's disease.

2

Neuropeptides. 53:51-（2.

3

55. Zhou JR, Shirasaki T, Soeda F, Takahama K (2014–: Cholinergic EPSCs and their

4

potentiation by bradykinin in single paratracheal ganglion neurons attached with presynaptic

5

boutons. J Neurophysiol. 112:933-941.

6

5（. Hedrick NG, Harward SC, Hall CE, Murakoshi H, McNamara JO, Yasuda R (201（–: Rho

7

GTPase complementation underlies BDNF-dependent homo- and heterosynaptic plasticity.

8

Nature. 538:104-108.

9

57. Kang HJ, Voleti B, Hajszan T, Rajkowska G, Stockmeier CA, Licznerski P, et al. (2012–:

10

Decreased expression of synapse-related genes and loss of synapses in major depressive

11

disorder. Nat Med. 18:1413-1417.

12

58.

13

renin-angiotensin pathway in posttraumatic stress disorder: angiotensin-converting enzyme

14

inhibitors and angiotensin receptor blockers are associated with fewer traumatic stress

15

symptoms. J Clin Psychiatry 73:849-55.

Khoury NM, Marvar PJ, Gillespie CF, Wingo A, Schwartz A, Bradley B, et al. (2012– The

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Figure Legends

19

30

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1

Fig. 1 Captopril rapidly ameliorates CUS and CSDS-induced depressive-type

2

behaviors in mice. (A-B) Single intraperitoneal injection of captopril (9.75, 19.5 or

3

39 mg/kg) significantly reduced the immobility time in the TST (A) and FST (B) (n =

4

6-20, **p<0.01). (C) Time-concentration curve of captopril in the plasma, mPFC and

5

cerebrospinal fluid (CSF) of mice (19.5 mg/kg, i.p., 30 min: n=7-11, 1 h: n=7-11, 3 h:

6

n=6-8, 6 h: n=5-7, 12 h: n=3-8). (D) The effect of captopril (9.75, 19.5 or 39 mg/kg,

7

i.p) on sucrose preference and immobility time in TST and FST of CUS-treated mice

8

(n=7-16, *p<0.05, **p<0.01). (E) Sucrose preference was measured in the

9

CUS-treated mice at 1 d (n=6-11), 3 d (n=16-24) and 7 d (n=17-24) after captopril

10

injection (19.5 mg/kg, i.p., *p<0.05, **p<0.01 vs CON, ##p<0.01 vs CUS). (F) Social

11

index in the interaction zone of CSDS-treated mice was measured in social interaction

12

test 24 h after captopril administration (19.5 mg/kg, i.p., n=8-13, **p<0.01). Data are

13

expressed as mean ± S.E.M.

14

15

Fig. 2 Pharmacological regulators of angiotensin, such as DRI, ARB and Ang II

16

nAb, can not mimic the rapid antidepressant activity of ACEI. (A) Schematic

17

showing pharmacology of DRI, ACEI, ARB and Ang II nAb. (B) Aliskiren (10 and

18

50 mg/kg, i.p.) did not display rapid antidepressant activity in CUS-treated mice

19

(n=9-13). (C) Valsartan (30 or 60 mg/kg, i.p.) did not mimic the rapid antidepressant

20

activity of captopril in the CUS-treated mice (n=9-16). (D) Direct down-regulation of

21

central Ang II levels by locally infusion with Ang II nAb (1 µg/µl per side) did not 31

Luo H et al

1

mimic the rapid antidepressant activity of captopril (n=7-9). (E) Lisinopril (39.6

2

mg/kg, at the same molar equivalent dose of 19.5 mg/kg captopril), a long-lasting

3

ACEI, exerted similar rapid antidepressant activities to captopril in the FST (n=8,

4

**p<0.01 vs vehicle). (F) Lisinopril (39.6 mg/kg) exerted a faster and longer

5

antidepressant effect than that of captopril in the CUS-treated mice. Sucrose

6

preference at 2 h, 24 h, 7 d after systemic injection of lisinopril (39.6 mg/kg) and

7

captopril (19.5 mg/kg) in the CUS-treated mice (n=7-16, **p<0.01 vs CON, ##p<0.01

8

vs CUS). Data are expressed as mean ± S.E.M.

9

10

Fig. 3 Deficits in the BK signaling contributes to the CUS-induced

11

depressive-like behaviors in mice. (A) CUS did not affect the central expression of

12

RAS system. The level of Ang II, ACE, AT1R and AT2R were detected in the mPFC,

13

hippocampus, and NAc of the stressed mice (n=4-11). (B-D) The BK level

14

significantly decreased in the mPFC, which was reversed by captopril (B-C, n=9-13),

15

and in the plasma of CUS mice, which was also reversed by captopril (C-D). (E) BK

16

levels in the plasma of depressive patients were lower than those of healthy subjects

17

(n=7-12). (F) The expression of B1R and B2R in the mPFC of CUS mice (n=7-9). (G)

18

Infusion of BK (50 ng/µl per side) into the mPFC rapidly reversed CUS-induced

19

depressive-like behaviors in the SPT and TST (n=10-13). (H) Successive

20

administration of HOE140 (65.226 µg/kg per day, i.p, 7 days), but not DALBK

21

(99.818 µg/kg per day, i.p, 7 days) significantly increased the immobility time in the 32

Luo H et al

1

FST (n=10). Data are expressed as mean ± S.E.M. *p<0.05, **p<0.01.

2

3

Fig. 4 Both pharmacological and genetic blockade of B2R abolish the

4

antidepressant activity of captopril. (A) Locally bilateral infusion of HOE140, not

5

DALBK, abolished captopril’s effect on despair behavior in the TST and FST

6

(n=8-11). (B) HOE140 abolished captopril’s effect on the CUS-induced

7

depressive-like behaviors (n=12-15) in the SPT, TST and FST. (C) The stressed mice

8

were stereotaxically injected with GFP-tagging LV-B2R-shRNA or scrambled

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shRNA. B2R knockdown abolished captopril’s effect on the CUS-induced

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depressive-like behaviors in the SPT, TST and FST (n=12-15). Data are expressed as

11

mean ± S.E.M. *p<0.05, **p < 0.01.

12

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Fig. 5 BK-stimulated Cdc42 activity confers the antidepressant effects of

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captopril via activating the mTORC1 pathway. (A) Captopril reversed

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CUS-induced defect in Cdc42 activity, which was assayed by detecting the

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GTP-bound Cdc42 (n=9-11). (B) Locally bilateral infusion of ML141 blocked the

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effect of captopril on immobility in TST and FST (n=8-9). (C) Locally bilateral

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infusion of ML141 blocked the effect of captopril on CUS-induced depressive-like

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behaviors (n = 9-11). (D) Captopril activated the mTORC1 pathway and pre-infusion

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of ML141 into the mPFC blocked captopril’s effect on p-mTOR (n=4). **p<0.01. (E) 33

Luo H et al

1

Captopril (19.5 mg/kg, i.p) reversed the CUS-induced decrease in p-mTOR,

2

p-p70S6K and BDNF levels in the mPFC, which was abolished by LV-B2R-shRNA

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(1 µl per side, n=4-7). (F) Pre-injection of rapamycin into mPFC blocked the effect of

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captopril (19.5 mg/kg) on SPT and FST in CUS mice (n=7-11). Data are expressed as

5

mean ± S.E.M. *p<0.05, **p<0.01.

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Fig. 6 Captopril reverses the CUS-induced synaptic loss and stimulates

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synaptogenesis. (A) Mice were bilaterally injected with vehicle or ML141 into the

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mPFC. After 30 min, mice were intraperitoneally injected with vehicle or captopril,

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and the spine density was observed after 24 h using a confocal microscope.

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Representative 3D reconstructing image of dendritic spines (A) and quantification of

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average dendritic spine density (B, n=6-12) were shown. Scale bar: 5 µm. (C-D)

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Captopril restored the CUS-induced decrease in synaptic proteins, including GluA1

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and PSD95 in the mPFC (C, n=4) and hippocampus (D, n=4). (E) A pharmacology

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model for the fast-acting antidepressant activity of ACEIs. ACEIs enter the central

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nervous system, potentially inhibit the ACE activity and increase central BK level,

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following by an activation of B2R-Cdc42-mTORC1-dependent synaptogenesis. Data

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are expressed as mean ± S.E.M. *p<0.05, **p<0.01.

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