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Archival Report
Angiotensin-Converting Enzyme Inhibitor Rapidly
Ameliorates Depressive-Type Behaviors via
Bradykinin-Dependent Activation of Mammalian
Target of Rapamycin Complex 1
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,
and Jian-Guo Chen
ABSTRACT
BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEIs) are widely prescribed antihypertensive agents.
Intriguingly, case reports and clinical trials have indicated that ACEIs, including captopril and lisinopril, may have a
rapid mood-elevating effect in certain patients, but few experimental studies have investigated their value as fast-
onset antidepressants.
METHODS: The present study consisted of a series of experiments using biochemical assays, immunohistochem-
istry, and behavioral techniques to examine the effect and mechanism of captopril on depressive-like behavior in 2
animal models, the chronic unpredictable stress model and the chronic social defeat stress model.
RESULTS: Captopril (19.5 or 39 mg/kg, intraperitoneal injection) exerted rapid antidepressant activity in mice treated
under the chronic unpredictable stress model and mice treated under the chronic social defeat stress model.
Pharmacokinetic analysis revealed that captopril crossed the blood-brain barrier and that lisinopril, another ACEI with
better blood-brain barrier permeability, exerted a faster and longer-lasting effect at a same molar equivalent dose.
This antidepressant effect seemed to be independent of the renin-angiotensin system, but dependent on the
bradykinin (BK) system, since the decreased BK detected in the stressed mice could be reversed by captopril.
The hypofunction of the downstream effector of BK, Cdc42 (cell division control protein 42) homolog, contributed
to the stress-induced loss of dendritic spines, which was rapidly reversed by captopril via activating the mTORC1
(mammalian target of rapamycin complex 1) pathway.
CONCLUSIONS: Our findings indicate that the BK-dependent activation of mTORC1 may represent a promising
mechanism underlying antidepressant pharmacology. Considering their affordability and availability, ACEIs may
emerge as a novel fast-onset antidepressant, especially for patients with comorbid depression and hypertension.
Keywords: Angiotensin-converting enzyme inhibitor, Bradykinin, Captopril, Cdc42, MDD, mTORC1
https://doi.org/10.1016/j.biopsych.2020.02.005
Major depressive disorder (MDD) is a significant contributor to
the global burden of disease and affects approximately 16% of
the world’s population at some point in their lives. The major
problems in the therapy of MDD are that only 40% to 70% of
patients with depression respond to drug treatment and that
the onset of the therapeutic effect is delayed. In recent years,
there has been substantial clinical and preclinical progress in
identifying fast-onset antidepressants, such as ketamine and
scopolamine (1–3), and the enantiomer S-ketamine has been
recently approved to be prescribed for treatment-resistant
depression by the U.S. Food and Drug Administration. How-
ever, the use of available fast-onset antidepressants is limited
owing to their risk of dependence and psychomimetic side
effects.
Some clinic studies have revealed a possible relationship
between cerebrovascular disease and occurrence or out-
comes of depression in later life (4–6). Angiotensin II (Ang II),
the most important component of renin-angiotensin system
(RAS), is assumed to stimulate the hyperactivity of the
hypothalamic-pituitary-adrenocortical axis via activation of AT
1
receptor (AT1R) in corticotropin-releasing factor neurons (7,8).
Clinical data indicate that RAS-acting agents, including AT1R
blocker (ARB) and angiotensin-converting enzyme (ACE) in-
hibitor (ACEI), reduce the risk of mood disorders compared
with the patients taking other antihypertensive drugs such as
calcium channel blockers and
b
-blockers (9). Intriguingly,
although no randomized controlled trial has assessed the ef-
fects, in the last 40 years, a succession of case reports and
ª2020 Society of Biological Psychiatry. 1
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clinical studies have reported that ACEIs, not ARBs, elicit
mood-elevating effects in certain hypertensive patients
(10–17), which are indicated by the time axis in Supplemental
Figure S1. In the early 1980s, several cases reported that
captopril might rapidly improve the patient’s mood within 1 to 2
days at the daily dosages ranging from 37.5 to 200 mg
(10–11,13). In 2005, mood benefits were observed in 9 hy-
pertensive patients with MDD, who were treated with another
ACEI, lisinopril (18). ACEIs are a group of widely prescribed
antihypertensive agents, but to date, very few experimental
studies have investigated their antidepressant value (19,20). In
most patients with hypertension, the daily dosage of captopril
is 12.5 to 150 mg (a single dosage is 12.5–50 mg, 1 to 3 times
per day). Thus, we screened the effects of captopril on
depressive-like behaviors in mice as following: 2.44, 4.88, 9.75,
19.5, and 39 mg/kg/day (the equivalent of a single dosage of
11.87, 23.74, 47.48, 94.96, or 189.92 mg in a human weighing
60 kg), using the body surface area normalization method
described by U.S. Food and Drug Administration draft guide-
lines (21) (human dosage = mice dosage 3Km mice/Km hu-
man, Km = kg/m
2
, Km mice = 3, Km human = 37). We found
that only a high dosage of captopril produced a fast-onset
therapeutic effect on depressive-like behaviors.
An increasing number of studies have demonstrated that
activation of mTOR (mammalian target of rapamycin) signaling
in the medial prefrontal cortex (mPFC) mediate the rapid anti-
depressant actions of ketamine (3,22). A recent report has
indicated that direct activation of mTOR signaling via a leucine
sensing pathway by the sestrin modulator NV-5138 mimics the
rapid antidepressant effects of ketamine (23). It has been re-
ported that ACE may block mTOR signaling pathway (24,25),
but very little is known about the downstream signaling
mechanism. ACE function comprises the production of Ang II
and the breakdown of bradykinin (BK). Ang II, the main product
of ACE, increases the mTOR activity (26,27), whereas BK, a
degraded substrate for ACE, also activates mTOR signaling
pathway via B
2
receptor (B2R) in various conditions (28–30).
We further determined that ACEI may work by activating the
mTORC1 (mTOR complex 1) pathway via a non-RAS mecha-
nism (i.e., a BK-dependent pathway) and identified B2R as a
novel therapeutic target for depression.
METHODS AND MATERIALS
Detailed materials and methods are available in Supplement.
Animals and Behavioral Experiments
Male C57BL/6J mice (7–8 weeks of age, 18–21 g) from Hunan
SJA Laboratory Animal (Changsha, Hunan, China) were used in
our study. All the procedures were conducted following the
Declaration of Helsinki and the Guide for Care and Use of
Laboratory Animals as adopted and promulgated by the Na-
tional Institutes of Health. All experiments were approved by
the Review Committee for the Use of Human or Animal Sub-
jects of Huazhong University of Science and Technology.
Chronic unpredictable stress (CUS) and chronic social defeat
stress (CSDS) were used to induce depressive-like behavior,
and all the behavioral tests were conducted as previously
described with slight modifications (31–33). Sample sizes were
determined according to those used in previous publications
from our group and other similar studies (31,34,35) and justi-
fied by the power analyses.
Experiments in Molecular Biology
Quantitative real-time polymerase chain reaction was per-
formed on the StepOnePlus Real-Time PCR System (Applied
Biosystems, Foster City, CA) to analyze the gene expression.
Western blotting was used to analyze the protein level. The
vectors contained a cytomegalovirus-driven EGFP (enhanced
green fluorescent protein) and oligonucleotides encoding
shRNAs (short hairpin RNAs) of B2R were purchased from
Shanghai Genechem Co., Ltd. (Shanghai, China). The assay of
Cdc42 (cell division control protein 42) homolog activity was
performed by analyzing GTP-Cdc42/Cdc42 ratio as previous
reports (36).
Statistics
Analysis was performed using GraphPad Prism 7.0 or SPSS
18.0 software (SPSS Inc., Chicago, IL), and p,.05 was
considered statistically significant. All values were expressed
as mean 6SEM. Each ncorresponded to a single mouse. If
technical replicates were performed, their mean was consid-
ered as one n. We tested the data normality using
Kolmogorov-Smirnov test of normality with the Dallal-
Wilkinson-Lillie corrected pvalue (GraphPad Prism 7.0), and
variances were compared by Bartlett statistics to decide
whether parametric tests were applicable. Statistical analyses
were performed using 1-way analysis of variance followed by
least significant difference multiple comparison tests or 2-way
analysis of variance followed by Bonferroni test to compare
means of 3 or more groups, 1-way repeated measures analysis
of variance to examine means of repeated measured data, and
unpaired 2-tailed Student’sttest to compare 2 groups. For the
nonnormal distributed data (data for AT1R in the mPFC and
data for CUS and CUS 1Captopril group), Mann-Whitney
tests were used.
RESULTS
Captopril Rapidly Reverses Chronic Stress-Induced
Depressive-like Behaviors in Mice
First, the effects of captopril on behavioral despair were
measured by immobility time in 2 behavioral tests, the tail
suspension test (TST) and forced swimming test (FST). A wide
range of clinically relevant dosages of captopril was chosen
(2.44, 4.88, 9.75, 19.5, and 39 mg/kg/day in mice). Intraperi-
toneal injection of captopril at dosages of 9.75, 19.5 and
39 mg/kg/day produced rapid antidepressant responses in the
FST and TST 24 hours after administration (Figure 1A, B).
However, captopril exhibited few influences on the open field
test (Supplemental Figure S2A–C), elevated plus maze test
(Supplemental Figure S2D–F), and novelty-suppressed feeding
test (Supplemental Figure S2G), indicating that captopril may
exert little effect on anxiety. At the same dosage (19.5 or
39 mg/kg, intraperitoneally), captopril exerted little effect on
water drinking (12 hours, Supplemental Figure S2H). The effect
of captopril in the FST was detected within 24 hours and lasted
for 7 days after one dosing (Supplemental Figure S2I). The
levels of captopril in the mPFC were measured using liquid
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chromatography–tandem mass spectrometry following the
peripheral administration of captopril (19.5 mg/kg, Figure 1C).
The plasma concentrations of captopril reached peak levels
(184.06 620.25 ng/mL) at 0.5 hours after administration. In
this experimental condition, the detection limit of captopril
concentration was 23.01 nM (5 ng/mL). A modest concentra-
tion of captopril was detected in the mPFC tissue (24.6 62.82
ng/mL) 0.5 hours after administration, indicating that captopril
can enter the blood-brain barrier at high dosage.
Next, the mice exposed to CUS were used to evaluate the
effects of captopril. CUS mice displayed anhedonia, a core
symptom of depression measured by the sucrose preference
test (SPT); increased despair behaviors; and a reduction in
body weight (Supplemental Figure S3A–E). Notably, the CUS-
induced reduction in sucrose preference was reported to be
reversed by daily treatment with tricyclic antidepressants for 2
to 3 weeks (37,38); however, it was rapidly reversed by a single
dosage (19.5 or 39 mg/kg, Figure 1D) of captopril within 24
hours. This effect was not due to the effect of angiotensin on
drinking behavior because the CUS mice treated with captopril
(0, 19.5, or 39 mg/kg, intraperitoneally) displayed similar total
fluid consumption in the SPT (n=8–12, Supplemental
Figure S3F). Moreover, the increased sucrose preference was
observed at 1, 3, and 7 days after interperitoneal injection of
captopril (19.5 mg/kg), indicating that the antidepressant ef-
fects of captopril on anhedonia can last for at least 1 week
(Figure 1E). These results consistently demonstrate that
captopril elicits fast and sustained antidepressant effects in
mice.
We also employed another animal model of depression,
CSDS, to evaluate the effect of captopril on social deficits.
We found that the social index increased at 24 hours after
intraperitoneal injection of captopril (19.5 mg/kg, Figure 1F),
indicating that captopril improved the social interaction in the
socially defeated mice. The effects of a single dosage of
captopril on blood pressure and locomotor activity in CUS
Figure 1. Captopril rapidly ameliorates CUS and CSDS-induced depressive-type behaviors in mice. (A, B) Single intraperitoneal injection of captopril (9.75,
19.5, or 39 mg/kg) significantly reduced the immobility time in the TST (A) and FST (B) (n=6–20, **p,.01). (C) Time-concentration curve of captopril in the
plasma, mPFC, and CSF of mice (19.5 mg/kg, intraperitoneally, 30 minutes: n=7–11, 1 hour: n=7–11, 3 hours: n=6–8, 6 hours: n=5–7, 12 hours: n=3–8).
(D) The effect of captopril (9.75, 19.5, or 39 mg/kg, intraperitoneally) on sucrose preference and immobility time in TST and FST of CUS-treated mice (n=7–16,
*p,.05, **p,.01). (E) Sucrose preference was measured in the CUS-treated mice at 1 day (n=6–11), 3 days (n=16–24), and 7 days (n=17–24) after
captopril injection (19.5 mg/kg, intraperitoneally, *p,.05, **p,.01 vs. CON,
##
p,.01 vs. CUS). (F) Social index in the interaction zone of CSDS-treated mice
was measured in social interaction test 24 hours after captopril administration (19.5 mg/kg, intraperitoneally, n=8–13, **p,.01). Data are expressed as mean
6SEM. CON, control; CSDS, chronic social defeat stress; CSF, cerebrospinal fluid; CUS, chronic unpredictable stress; FST, forced swimming test; i.p.,
intraperitoneally; mPFC, medial prefrontal cortex; TST, tail suspension test.
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mice were very limited 24 hours after administration
(Supplemental Figure S4), indicating that its rapid antidepres-
sant activity may not be associated with the alterations in
blood pressure.
Other RAS-Acting Agents Are Insufficient to Elicit
Rapid Antidepressant Effects
The Ang II–reducing effect largely mediates the cardiovascular
effects of ACEI. Thus, we investigated whether other RAS-
acting agents mimicked the rapid antidepressant activity of
ACEI. First, we employed a direct renin inhibitor, aliskiren, to
mimic the Ang II–reducing effect of captopril (Figure 2A). Alis-
kiren (10 mg/kg, 50 mg/kg) induced a significant reduction in
Ang II levels 24 hours after administration (Supplemental
FigureS5A) but failed to exert significant antidepressant ef-
fects in the CUS-treated mice (Figure 2B), indicating that Ang II–
reducing effect may be insufficient to elicit rapid antidepressant
effects. Then, valsartan, a specific and widely used ARB, was
applied to mimic the captopril-mediated hypofunction of AT1R.
A previous report found that pretreatment with losartan (20 mg/
kg), another ARB, reduced the duration of immobility in the FST
of normal male CD mice (39). However, in the CUS mice, the
intraperitoneal injection of valsartan (30 or 60 mg/kg) elicited no
observed rapid antidepressant effects (Figure 2C), which was
consistent with a very recent report performed on CUS-treated
rats (40). We also administered an Ang II–neutralizing antibody,
aliskiren, and valsartan directly into the mPFC, again, and they
did not exert rapid antidepressant effects (Figure 2D and
Supplemental Figure5B), suggesting that downregulation of
Ang II function in either the peripheral or central nervous sys-
tems may be insufficient to produce rapid antidepressant effects
in the stressed mice.
Then, we asked whether other ACEIs have similar antide-
pressant effects and compared the efficacy/sustainability of
lisinopril, a long-lasting ACEI that can pass through the blood-
brain barrier (41), with that of captopril. At a molar equivalent
dosage to that of captopril (19.5 mg/kg), the duration of the
antidepressant effect of lisinopril (39.6 mg/kg) was much
longer than that of captopril in nonstressed mice (Figure 2E). In
the stressed mice, lisinopril (39.6 mg/kg) exerted a much faster
antidepressant effect (within 2 hours) than that of captopril
(Figure 2F).
Figure 2. Pharmacological regulators of angiotensin, such as direct renin inhibitor, ARB, and Ang II nAb, cannot mimic the rapid antidepressant activity of
ACEI. (A) Schematic showing pharmacology of direct renin inhibitor, ACEI, ARB, and Ang II nAb. (B) Alis kiren (10 and 50 mg/kg, intraperitoneally) did not
display rapid antidepressant activity in CUS-treated mice (n=9–13). (C) Valsartan (30 or 60 mg/kg, intraperitoneally) did not mimic the rapid antidepressant
activity of captopril in the CUS-treated mice (n=9–16). (D) Direct downregulation of central Ang II levels by local infusion with Ang II nAb (1
m
g/
m
L per side) did
not mimic the rapid antidepressant activity of captopril (n=7–9). (E) Lisinopril (39.6 mg/kg, at the same molar equivalent dose of 19.5 mg/kg captopril), a long-
lasting ACEI, exerted similar rapid antidepressant activities to captopril in the FST (n=8,*p,.05, **p,.01 vs. vehicle). (F) Lisinopril (39.6 mg/kg) exerted a
faster and longer antidepressant effect than that of captopril in the CUS-treated mice. Sucrose preference at 2 hours, 24 hours, 7 days after systemic injection
of lisinopril (39.6 mg/kg) and captopril (19.5 mg/kg) in the CUS-treated mice (n=7–16, **p,.01 vs. CON,
##
p,.01 vs. CUS). Data are expressed as mean 6
SEM. ACEI, angiotensin-converting enzyme inhibitor; AGT, angiotensinogen; Ang, angiotensin; ARB, angiotensin II receptor blocker; AT1R, AT
1
receptor; CON,
control; CUS, chronic unpredictable stress; FST, forced swimming test; IgG, immunoglobulin G; nAb, neutralizing antibody; TST, tail suspension test.
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Reactivation of the BK System Mediates the Rapid
Antidepressant Effects of ACEI
We observed that the concentration of Ang II, as well as the
messenger RNA (mRNA) levels of angiotensinogen, ACE,
AT1R, and AT
2
receptor (AT2R), exhibited few changes in the
mPFC, hippocampus, and nucleus accumbens of the CUS-
treated mice (Figure 3A). As a vasodilator nonapeptide that is
degraded by ACE, BK is another important molecular mediator
underlying the cardiovascular effects of ACEI as a non-RAS
mechanism. Interestingly, the levels of BK were significantly
decreased in the mPFC and plasma of stressed mice
(Figure 3B–D), an effect that was reversed by captopril (19.5
mg/kg, intraperitoneally). To assess the relevance of BK levels
to MDD, we analyzed the BK concentration in the plasma of
human subjects who were diagnosed with MDD. Notably, a
similar change in BK levels was observed in the plasma of
depressed patients (Figure 3E), as determined by enzyme-
linked immunosorbent assay. BK exerts its effects via two
different receptor subtypes: B
1
receptor (B1R) and B2R. The
level of B2R, not B1R, in the mPFC of CUS mice was upre-
gulated (Figure 3F). The altered expression of B2R may confer
a compensatory mechanism of BK deficits induced by CUS.
Previous reports have indicated that when administered in
the central nervous system, BK leads to initial rapid excitation
(42) and hyperalgesia (43). Thus, we asked whether the BK
system mediated the rapid antidepressant effect of captopril.
To address this issue, BK (50 ng/per side) was bilaterally
infused into the mPFC of stressed mice to mimic the BK-
potentiating property of captopril. We found that the local
administration of BK in the mPFC rapidly reversed depressive-
like behaviors in the stressed mice (Figure 3G). Next, we
explored whether the inhibition of BK function was associated
with depressive-like behaviors. Repeated administration of
HOE140 (65.226
m
g/kg per day, intraperitoneally), a blocker of
B2R, but not DALBK (99.818
m
g/kg per day, intraperitoneally), a
blocker of B1R, for 7 days significantly increased the immobility
time in the FST of mice (Figure 3H), suggesting that hypofunction
of B2R may contribute to the pathophysiology of depression.
Figure 3. Deficits in the BK signaling contributes to the CUS-induced depressive-like behaviors in mice. (A) CUS did not affect the central expression of the
RAS. The level of Ang II, ACE, AT1R, and AT2R were detected in the mPFC, hippocampus, and NAc of the stressed mice (n=4–11). (B-D) The BK level
significantly decreased in the mPFC, which was reversed by captopril (B, C,n=9–13), and in the plasma of CUS mice, which was also reversed by captopril
(C, D).(E) BK levels in the plasma of depressive patients were lower than those of healthy subjects (n=7–12). (F) The expression of B1R and B2R in the mPFC
of CUS mice (n=7–9). (G) Infusion of BK (50 ng/
m
L per side) into the mPFC rapidly reversed CUS-induced depressive-like behaviors in the sucrose preference
test and TST (n=10–13). (H) Successive administration of HOE140 (65.226
m
g/kg per day, intraperitoneally, 7 days), but not DALBK (99.818
m
g/kg per day,
intraperitoneally, 7 days), significantly increased the immobility time in the FST (n= 10). Data are expressed as mean 6SEM. *p,.05, **p,.01. ACE,
angiotensin-converting enzyme; Ang II, angiotensin II; AT1R, AT
1
receptor; AT2R, AT
2
receptor; B1R, B
1
receptor; B2R, B
2
receptor; BK, bradykinin; CON,
control; CUS, chronic unpredictable stress; FST, forced swimming test; mPFC, medial prefrontal cortex; NAc, nucleus accumbens; RAS, renin-angiotensin
system; TST, tail suspension test.
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Together, these results demonstrate that B2R signaling may play
a key role in the antidepressant activity of captopril.
To further confirm the role of B2R in the antidepressant
mechanism of captopril, we employed both pharmacological
and genetic approaches in this study. Intra-mPFC infusion of
HOE140 (100 nM, 1
m
L per side), but not DALBK (100 nM, 1
m
L
per side), completely blocked captopril-induced reduction in
the immobility time of TST and FST (Figure 4A). We further
examined the influence of HOE140 on the antidepressant ef-
fect of captopril in the CUS-treated mice. Intra-mPFC injection
of HOE140 abolished the behavioral responses to captopril in
stressed mice (Figure 4B). To directly explore the role of B2R in
the effects of captopril, we used a lentivirus (LV) that expressed
shRNAs to knockdown B2R expression. As shown in
Supplemental Figure S6, the lentivirus-guided EGFP expres-
sion was predominantly located in the mPFC and the B2R
protein level was significantly downregulated by LV-B2R small
interfering RNA (siRNA). We found that captopril failed to exert
an antidepressant effect in the mice with lentivirus-guided
knockdown of B2R (Supplemental Figure S6 and Figure 4C).
Figure 4. Both pharmacological and genetic blockade of B2R abolished the antidepressant activity of captopril. (A) Local bilateral infusion of HOE140, not
DALBK, abolished captopril’s effect on despair behavior in the TST and FST (n=8–11). (B) HOE140 abolished captopril’s effect on the CUS-induced
depressive-like behaviors (n=12–15) in the SPT, TST, and FST. (C) The stressed mice were stereotaxically injected with GFP-tagging LV-B2R-shRNA
or scrambled shRNA. B2R knockdown abolished captopril’s effect on the CUS-induced depressive-like behaviors in the SPT, TST, and FST (n=12–15).
Data are expressed as mean 6SEM. *p,.05, **p,.01. B2R, B
2
receptor; CON, control; CUS, chronic unpredictable stress; FST, forced swimming test; GFP,
green fluorescent protein; i.p., intraperitoneally; LV, lentivirus; shRNA, short hairpin RNA; SPT, sucrose preference test; TST, tail suspension test.
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These results demonstrate that BK-B2R signaling determines
the antidepressant effect of captopril.
BK-Stimulated Cdc42 Activity Confers the
Antidepressant Effects of Captopril via Activation of
the mTORC1 Pathway
B2R is widely distributed in the central nervous system (44),
and a previous study has revealed that BK depolarizes motor
neurons by postsynaptic activation of B2R (45). We hypothe-
sized that B2R may initiate the antidepressant activity via
postsynaptic action. BK is a powerful stimulator of Cdc42, a
critical Rho GTPase protein (46,47). Growing evidence sug-
gests that Cdc42 controls rapid presynaptic maturation to
facilitate synaptogenesis (48) and also contributes to post-
synaptic maturation (49). Furthermore, Cdc42 regulates the
activation of the mTORC1 signaling pathway (50,51), which is
critical in the synaptic mechanisms underlying rapid-acting
antidepressants. Thus, we asked whether the Cdc42-mTOR
signaling pathway contributes to the antidepressant activity
of captopril.
First, we found that the activity of Cdc42 was reduced
significantly in the mPFC of CUS-treated mice, and captopril
rapidly rescued the decrease in Cdc42 activity in the mPFC
(Figure 5A). As shown in Figure 5B and C, the intra-mPFC
infusion of a selective Cdc42 inhibitor ML141 (8.15 mg/
m
L/
side) blocked the effect of captopril on despair behaviors in the
nonstressed mice (Figure 5B), and the preinfusion of ML141
into the mPFC abolished the antidepressant activity of
captopril in the stressed mice (Figure 5C). These results sug-
gest that Cdc42 plays a role in the mechanism in the antide-
pressant activity of captopril.
We next examined the effect of captopril on the mTORC1
activity. The phosphorylated forms of mTORC1 and p70S6K,
the key downstream target of mTORC1, represent the activa-
tion of mTORC1 signaling. We found that captopril activated
mTORC1 in the nonstressed mice; moreover, the intra-mPFC
infusion of ML141 prevented the captopril-induced activation
of mTORC1 (Figure 5D). In the CUS-treated mice, captopril
reversed the stress-induced inhibition of mTORC1 activity
(Figure 5E, phosphorylated mTOR and phosphorylated
p70S6K) and induced an increased level of BDNF (brain-
Figure 5. Bradykinin-stimulated Cdc42 activity confers the antidepressant effects of captopril via activating the mTORC1 pathway. (A) Captopril reversed
CUS-induced defect in Cdc42 activity, which was assayed by detecting the GTP-bound Cdc42 (n=9–11). (B) Local bilateral infusion of ML141 blocked the
effect of captopril on immobility in TST and FST (n=8–9). (C) Local bilateral infusion of ML141 blocked the effect of captopril on CUS-induced depressive-like
behaviors (n=9–11). (D) Captopril activated the mTORC1 pathway and preinfusion of ML141 into the mPFC blocked captopril’s effect on p-mTOR (n= 4).
(E) Captopril (19.5 mg/kg, intraperitoneally) reversed the CUS-induced decrease in p-mTOR, p-p70S6K, and BDNF levels in the mPFC, which was abolished by
LV-B2R-shRNA (1
m
L per side, n=4–7). (F) Preinjection of rapamycin into mPFC blocked the effect of captopril (19.5 mg/kg) on sucrose preference test and
FST in CUS mice (n=7–11). Data are expressed as mean 6SEM. *p,.05, **p,.01.B2R, B
2
receptor; BDNF, brain-derived neurotrophic factor; CON, control;
CUS, chronic unpredictable stress; FST, forced swimming test; LV, lentivirus; mPFC, medial prefrontal cortex; mTORC1, mammalian target of rapamycin
complex 1; p-mTOR, phosphorylated mTORC1; p-p70S6K, phosphorylated p70S6 kinase; shRNA, short hairpin RNA; TST, tail suspension test.
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derived neurotrophic factor) (Figure 5E). The genetic knock-
down of B2R abolished the captopril-induced activation of
mTORC1 and BDNF synthesis in the stressed mice (Figure 5E).
We next assessed whether increased mTOR activity is suffi-
cient to mediate the rapid antidepressant activity of captopril.
Accordingly, a selective mTORC1 inhibitor, rapamycin, was
infused into the mPFC of CUS mice. We found that the infusion
of rapamycin into the mPFC abolished captopril-elicited rapid
antidepressant activity (Figure 5F).
To further address the relationship between BK and
mTORC1 activity, we observed a direct action of BK on
mTORC1 activity in primary cultured neurons. Incubation of BK
(10 nM) increased phosphorylation of p70S6K and BDNF level in
primary cultured neurons of mPFC (Supplemental Figure S7A,
B). Additionally, in vivo, we found that rapamycin blocked BK-
elicited rapid antidepressant responses (Supplemental
Figure S7C). Thus, captopril may exert antidepressant effects
through the BK-B2R-Cdc42-mTORC1 signaling pathway.
Captopril Reverses CUS-Induced Synaptic Loss and
Stimulates Synaptogenesis
Considering that Cdc42 activity can regulate dendritic spine
plasticity, we asked whether the reactivation of Cdc42 function
can alleviate the CUS-induced synaptic loss. Using confocal
microscopy, the dendritic spine density in the mPFC of
stressed mice was measured 24 hours after captopril admin-
istration. As previously reported (52), dendritic spines were
classified by functional subtype: long thin, mushroom, and
stubby. In our study, the CUS exposure significantly reduced
the number and density of dendritic spines, especially long thin
spines, in the mPFC of mice. Moreover, captopril rapidly
rescued the synaptic loss observed in the mPFC of CUS-
treated mice by increasing the spine density and number of
long thin and mushroom dendritic spines (Figure 6A, B). The
morphological changes were abolished by the intra-mPFC
infusion of ML141 (Figure 6A, B), indicating that Cdc42-
dependent synaptogenesis mediates the effect of captopril
on CUS-induced synaptic loss. Furthermore, captopril signifi-
cantly increased the levels of key synaptic proteins, including
GluA1 and PSD95 (postsynaptic density protein 95), in the
mPFC of CUS mice (Figure 6C), which strengthens our hy-
pothesis that captopril exerts antidepressant effects by
increasing synapse numbers. Captopril also increased the
levels of synaptic proteins, including GluA1 and PSD95 in
the hippocampus of CUS mice (Figure 6D), suggesting that in
addition to the mPFC, other brain areas may be involved in the
effect of captopril.
DISCUSSION
In the present study, we demonstrated that ACEIs produced a
rapid and long-lasting reversal of chronic stress-induced
depressive-like behaviors by potentiating the BK-B2R-
Cdc42-mTORC1 signaling pathway (Figure 6E). Our study
proposed a new property of ACEI, an important class of RAS-
acting agents. As widely used antihypertensive agents, the
clinical antidepressant value of RAS-acting agents has not
been established. Our results indicated that RAS-acting agents
including direct renin inhibitor, ARB, and captopril at a single
clinic dosage did not exert a rapid antidepressant activity in
CUS-treated rodents. Interestingly, our data indicated that the
BK action, an acute physiological outcome that was often
limited by the clinical dosage regimen to avoid side effects,
mediated the antidepressant effects of ACEIs at high dosages.
An alternative interpretation for the overlook of the ACEI effect
may be that captopril initiated a rapid but transient action for a
few days, like ketamine, which was approved for anesthetic in
1970, but its antidepressant value has not been established
until recent years.
BK plays a key role in the pharmacological effect of
captopril (53). A notable finding of the present study is that the
altered BK system may contribute to the development of
depressive-like behaviors. Decreased levels of BK in the blood
and mPFC were observed in mice exposed to CUS. The
administration of BK in the mPFC rapidly rescued CUS-
induced behavior deficits. Consistently, the levels of BK were
also decreased in the plasma of patients with MDD. It should
be noted that the differences in the sex ratio of patients may
induce bias. We analyzed the BK level between women and
men and found no differences, both in the patients with MDD
and in healthy volunteers. However, considering that the sex
ratio in the control population does not mirror the ratio in the
experimental group and that all the experiments were per-
formed on the male mice, the role of BK in female patients with
MDD should be further evaluated.
Increased B2R levels in the mPFC of stressed mice were
observed, and these increased levels may be a compensatory
mechanism in response to chronic stress-induced deficits in
BK signaling. We found that repeated administration of the
B2R blocker HOE140 (65.226
m
g/kg per day, intraperitoneally)
for 7 days increased FST immobility time (seen in Figure 3H).
However, as seen in Figure 4A, intra-mPFC infusion of HOE140
(100 nM, 1
m
L per side, once) did not affect the immobility time
after 24 hours, indicating that a long-term blockade, not acute
blockade, of B2R may generate despair behaviors. We hy-
pothesized that chronic blockade of B2R in the PFC would
also have increased immobility, and this point was supported
by our data that the genetic knockdown of B2R in the mPFC
increased FST immobility time in control mice (Figure 4C). Both
pharmacological and genetic approaches revealed that B2R
not B1R, mediated the antidepressant effects of BK and
captopril. Considering that a neuroprotective role of B2R has
been revealed (54), our findings raise the possibility that BK-
potentiating peptides or drugs may emerge as a new class
of antidepressants. Notably, BK-induced cough and hyper-
algesia may affect treatment compliance; however, they could
be prevented by a rational drug regimen or a peripheral blocker
of BK receptors.
Previous studies have reported that BK potentiates synaptic
transmission via activating both presynaptic and postsynaptic
B2Rs (45,55). As a G-protein-coupled receptor, B2R strongly
stimulates Cdc42 activation (46–47). It is generally believed
that the Cdc42 signaling pathway plays a key role in the
structural plasticity of dendritic spines, dendritic morphogen-
esis, synaptic maturation, and axon guidance (56). MDD has
been linked to aberrant dendritic spine and synapse develop-
ment (57). Until now, very little is known about the role of
Cdc42 in depression. In our study, we found that CUS induced
a robust defect in Cdc42 activation in the mPFC, which could
be rescued by captopril. Meanwhile, captopril restored CUS-
ACEI May Serve as Fast-Acting Antidepressants
8Biological Psychiatry --, 2020; -:-–-www.sobp.org/journal
Biological
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induced spine loss. Both the behavior and morphologic effects
of captopril were abolished by the Cdc42 inhibitor ML141,
suggesting that a Cdc42-dependent mechanism may be crit-
ical to the effect of captopril.
In the recent decade, ketamine and scopolamine have been
developed as rapid-acting antidepressants that can improve
depressive symptoms within hours or days in patients. The
promotion of mTORC1 has been recognized as a common
signaling pathway that mediates rapid-acting antidepressant
effects. A recent report indicated that direct activation of
mTORC1 via a leucine sensing pathway by NV-5138 mimicked
the rapid antidepressant effects of ketamine without affecting
glutamate receptors (23), and Navitor Pharmaceuticals has
commenced phase I clinical evaluation of NV-5138 in patients
with treatment-resistant depression. Similarly, we found that
captopril activated the mTORC1 pathway via a BK-dependent
pathway. BK incubation directly activated the mTOR signaling
in the cultured neurons (Supplemental Figure S7A, B), which
may work through a BK-B2R-Cdc42-mTORC1 signaling
pathway. A previous in vitro study has reported that
Figure 6. Captopril reverses the CUS-induced synaptic loss and stimulates synaptogenesis. (A) Mice were bilaterally injected with vehicle or ML141 into the
mPFC. After 30 minutes, mice were intraperitoneally injected with vehicle or captopril, and the spine density was observed after 24 hours using a confocal
microscope. Representative 3-D reconstructing image of dendritic spines (A) and quantification of average dendritic spine density (B,n=6–12) are shown.
Scale bar = 5
m
m. (C, D) Captopril restored the CUS-induced decrease in synaptic proteins, including GluA1 and PSD95 in the mPFC (C,n= 4) and hip-
pocampus (D,n=4).(E) A pharmacology model for the fast-acting antidepressant activity of ACEIs. ACEIs enter the central nervous system, potentially inhibit
the ACE activity and increase central BK level, following by an activation of B2R-Cdc42-mTORC1-dependent synaptogenesis. Data are expressed as mean 6
SEM. *p,.05, **p,.01. ACE, angiotensin-converting enzyme; ACEI, ACE inhibitors; B2R, B
2
receptor; BDNF, brain-derived neurotrophic factor; BK, bra-
dykinin; CON, control; CUS, chronic unpredictable stress; GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; i.p.,
intraperitoneally; LV, lentivirus; mPFC, medial prefrontal cortex; mTORC1, mammalian target of rapamycin complex 1; PSD95, postsynaptic density protein 95.
ACEI May Serve as Fast-Acting Antidepressants
Biological Psychiatry --, 2020; -:-–-www.sobp.org/journal 9
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overexpression of ACE reduced the level of p70S6K, whereas
captopril increased the levels of p70S6K (24), which was
inconsistent with our observations. We found that captopril
promoted synaptogenesis, which may be because captopril
and BK, similar to other rapid-acting antidepressants, facili-
tated the mTORC1-dependent synaptogenesis.
ACEIs are widely used antihypertensive agents, and some
studies also indicated ACEIs might be beneficial for psychiatric
diseases (58). Considering the affordability and availability of
ACEIs, long-lasting ACEIs that can cross the blood-brain
barrier may be used as new rapid-onset antidepressants. The
pharmacodynamics and toxicity of ACEIs have been well
studied. Considering the fetal toxicity and the increase of
neonatal morbidity and death, the use of ACEI for depression
during pregnancy should be limited. In patients with comorbid
depression and diabetes or impaired renal function, ACEI
should be used with caution. A further large-scale, random-
ized, controlled clinical study should be performed to evaluate
the antidepressant effect of ACEIs.
ACKNOWLEDGMENTS AND DISCLOSURES
This work was supported by grants from the Foundation for Innovative
Research Groups of NSFC (Grant No. 81721005 [to J-GC and FW]), National
Natural Science Foundation of China (Grant No. 81773712 to [P-FW], Grant
Nos. 81471377 and 81671438 [to FW], Grant Nos. 81473198 and 81673414
[to J-GC]), Program for Changjiang Scholars and Innovative Research Team
in University (Grant No. IRT13016 [to J-GC]), the Program for Academic
Frontier Youth Team and Integrated Innovative Team for Major Human
Diseases Program of Tongji Medical College, Huazhong University of Sci-
ence and Technology (to FW).
HL performed most molecular and behavioral experiments, stereotaxic
surgeries, and analyzed data. P-FW designed the experiments, performed
molecular experiments, helped with methodology, and analyzed data. YC,
Q-QH, and S-LD contributed to animal experiments and stereotaxic sur-
geries. MJ and J-GH performed liquid chromatography–tandem mass
spectrometry analysis of captopril. T-TS performed CSDS model and
contributed to behavioral experiments. JW and LN contributed to confocal
microscopy experiments. J-GH contributed to measure plasma BK content.
Z-LH and L-HL provided the technique supports. FW and J-GC supervised
the project, designed the experiments, revised the manuscript, and sup-
ported funding acquisition. P-FW, HL, FW, and J-GC wrote the paper with
contributions from all of the other authors.
The authors report no biomedical financial interests or potential conflicts
of interest.
ARTICLE INFORMATION
From the Department of Pharmacology (HL, P-FW, YC, T-TS, JW, Q-QH,
J-GH, S-LD, LN, Z-LH, L-HL, FW, J-GC), School of Basic Medicine;
Department of Pharmaceutics (MJ, J-GH), College of Pharmacy, Tongji
Medical College, Huazhong University of Science and Technology; Key
Laboratory of Neurological Diseases (HUST) (P-FW, Z-LH, L-HL, FW, J-GC),
Ministry of Education of China; The Key Laboratory for Drug Target Re-
searches and Pharmacodynamic Evaluation of Hubei Province (P-FW, Z-LH,
L-HL, FW, J-GC); Laboratory of Neuropsychiatric Diseases (P-FW, L-HL,
FW, J-GC), The Institute of Brain Research, Huazhong University of Science
and Technology; and The Collaborative-Innovation Center for Brain Science
(FW, J-GC), Wuhan, Hubei, China.
HL and P-FW contributed equally to this work.
Address correspondence to Jian-Guo Chen, M.D., Ph.D., or Fang Wang,
M.D., Ph.D., Department of Pharmacology, Tongji Medical College, Huaz-
hong University of Science and Technology, 13 Hangkong Road, Wuhan,
Hubei, China 430030. E-mail: wangfangtj0322@163.com or chenj@mails.
tjmu.edu.cn or wangfangtj0322@163.com.
Received Oct 26, 2019; revised Jan 22, 2020; accepted Feb 3, 2020.
Supplementary material cited in this article is available online at https://
doi.org/10.1016/j.biopsych.2020.02.005.
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