Content uploaded by Willy Jaya Suento
Author content
All content in this area was uploaded by Willy Jaya Suento on Feb 16, 2021
Content may be subject to copyright.
Journal of Neurochemistry. 2020;00:1–16. wileyonlinelibrary.com/journal/jnc
|
1© 2020 International Society for Neurochemistry
Received: 26 May 2020
|
Revised: 14 September 2020
|
Accepted: 20 O ctober 2020
DOI: 10.1111/jnc.15222
ORIGINAL ARTICLE
Prefrontal cortex miR-874-3p prevents lipopolysaccharide-
induced depression-like behavior through inhibition of
indoleamine 2,3-dioxygenase 1 expression in mice
Willy Jaya Suento1,2 | Kazuo Kunisawa3 | Bolati Wulaer1,4 | Aika Kosuge3 |
Tsubasa Iida3 | Suwako Fujigaki1 | Hidetsugu Fujigaki1 | Yasuko Yamamoto1 |
Andi Jayalangkara Tanra2 | Kuniaki Saito1,4,5 | Akihiro Mouri3,5 |
Toshitaka Nabeshima4,5
Willy Ja ya Suent o and Kazuo Kunis awa contribute d equally to this w ork.
Abbreviations: 1-MT, 1-methyl-l-try ptophan; 3-HA A, 3-hydrox y anthranilic a cid; 3-HK, 3-hyd roxykynur enine; AA , anthr anilic a cid; CNS, centr al ner vous system; FS T, forced swi mming
test; H IP, hippo campus; IDO1, i ndoleamine 2 ,3-dioxygen ase 1; IFN, inter feron; IL, inte rleukin; KA , kynureni c acid; KYN, k ynurenine; LP S, lipopolys accharide; MD D, major depres sive
disord er; miRN A, micr oRNA ; NC, miR-ne gative co ntrol; OF T, open-f ield tes t; PCNA , prol ifera ting cell nu clear ant igen; PFC , prefro ntal cor tex; QUIN, quino linic aci d; RRID, Rese arch
Resour ce Identifier ( see scic runch.org) ; TDO, tryptop han 2,3-diox ygenase; TLR4, to ll-like receptor 4 ; TNF, tumor n ecrosi s facto r; TRP, tryptopha n.
1Depar tment of Disease Control and
Prevention, Fujit a Health Univer sity
Graduate School of Health Science, Aichi,
Japan
2Depar tment of Psychiatry, Hasanuddin
University Faculty of Me dicine , South
Sulawesi, Indonesia
3Depar tment of Regulato ry Science
for Evaluation & Development of
Pharmaceutic als & Devices, Fujita Health
University Graduate School of Health
Science , Aichi, Japan
4Advanced Diagnostic Sys tem Research
Labor atory, Fujita Health Universit y
Graduate School of Health Science, Aichi,
Japan
5Japanese Drug O rganization of A ppropriate
Use and Res earch, Aichi, Japan
Correspondence
Akihiro Mouri, Depar tment of Regulatory
Science for Evaluation and D evelopment of
Pharmaceutic als and D evices, Fujita Health
University, Gra duate School of He alth
Science s, Aichi, 470-1192, Japan.
Email: mouri@fujita-hu.ac.jp
Funding information
Japan Society for the Promotion of
Science , Grant/Award Numb er: 17H0 4252,
17K01969, 18K15377, 18K19761 and
19K07490; Ministr y of Education, Culture,
Sports, Science and Technolog y of Japan
(MEXT); Education and Research Facility of
Animal Models fo r Human Diseases at the
Fujita Health Universit y
Abstract
Indoleamine 2,3-dioxygenase 1 (IDO1) is the first rate-limiting enzyme that metabo-
lizes tryptophan to the kynurenine pathway. Its activity is highly inducible by pro-
inflammatory cytokines and correlates with the severity of major depressive disorder
(MDD). MicroRNAs (miRNAs) are involved in gene regulation and the development
of neuropsychiatric disorde rs including MDD. However, the role of miRNAs in target-
ing IDO1 in the pathophysiology of MDD is still unknown. In this study, we investi-
gated the role of novel miRNAs in the regulation of IDO1 activity and its effect on
lipopolysaccharide (LPS)-induced depression-like behavior in mice. LPS up-regulated
miR-874-3p concomitantly with increase in IDO1 expression in the prefrontal cortex
(PFC), increase in immobility in the forced swimming test as depression-like behav-
ior and decrease in locomotor activity as sickness behavior without motor dysfunc-
tion. The miR-874-3p increased in both neuron and microglia after LPS. Its mimic
significantly suppressed LPS-induced IDO1 expression in the PFC. Infusion of IDO1
inhibitor (1-methyl-l-tryptophan) and miR-874-3p into PFC prevented an increase in
immobility in the forced swimming test, but did not decrease in locomotor activity
induced by LPS. These results suggest that miR-874-3p may play an important role
in preventing the LPS-induced depression-like behavior through inhibition of IDO1
expression. This may also serve as a novel potential target molecule for the treatment
of MDD.
KEYWORDS
depression, IDO1, lipopolysaccharide, miRNA-874-3p, prefrontal cortex
2
|
SUENTO ET al.
1 | Introduction
Major depressive disorder (MDD) is a complex mood disorder in-
fluenced by various genetic and environmental factors that affect
350 million people worldwide (Flint & Kendler, 2014). MDD is one
of the leading causes of morbidity, mortality, and burden of health
care utilization (Benton et al., 2007; Murray & Lopez, 1996; Otte
et al., 2016). Although many studies have tried to optimize the treat-
ment of MDD, about 40% of patients do not respond to the currently
available treatments (Dwivedi, 2014; Fava & Davidson, 1996). This
may be because of poor understanding of the pathophysiology of
MDD.
It is well established that there is a strong correlation between
inflammation and MDD. Multiple studies have shown that elevated
levels of pro-inflammatory cytokines—such as tumor necrosis fac-
tor (TNF)-α, interleukin (IL)-6, and IL-1β—are found in the blood
and postmortem brain of patients with MDD (Maes et al., 1997;
Zou et al., 2018). Furthermore, interferon (IFN)-α therapy has been
shown to induce depressive symptoms in patients with hepatitis C
(Murakami et al., 2016).
Tryptophan (TRP) is an essential amino acid that acts as a
precursor for the biosynthesis of the neurotransmitter sero-
tonin (Badawy, 2017). Diminishment of the serotonin pathway
has been proposed as a pathophysiological mechanism of MDD
(Oxenkrug, 2013), although the majority of available TRP is me-
tabolized through the kynurenine (KYN) pathway. Indoleamine-
2,3-dioxygenase 1 (IDO1) is one of the two enzymes (the other
is tryptophan 2,3-dioxygenase; TDO) metabolizing TRP through
the KYN pathway. IDO1 is induced by several pro-inflammator y
cytokines (e.g., TNF-α, IL-1 β, and IFN-α) and lipopolysaccharide
(LPS) (Fujigaki et al., 2017). Inflammatory conditions acceler-
ate the KYN pathway, thus increasing the levels of neuroactive
metabolites like 3-hydroxykynurenine (3-HK) and 3-hydroxy an-
thranilic acid (3-HAA) (Maes et al., 2011). These neuroactive me-
tabolites play an important role in MDD pathophysiology (Dantzer
et al., 2008; Fujigaki et al., 2017). Interestingly, increase in IDO1
activity has been positively correlated with scores for severe de-
pression (Br adley et al., 2015). Reduced cir cu lating TR P leve ls con-
comitant with increase in KYN metabolites have been reported in
patients with MDD (Fujigaki et al., 2017; Savit z, 2017). In support
of clinical studies, IDO1-mediated activation of the K YN pathway
by systemic LPS injection results in both anxiety and depres-
sion-like behavior in mice (Salazar et al., 2012). More importantly,
the IDO1 inhibitor, 1-methyl-l-tryptophan (1-MT), reverses LPS-
induced depression-like behavior (Lawson et al., 2013; O'Connor,
et al., 2009). These data strongly suggest that IDO1 may be a use-
ful therapeutic target for MDD.
MiRNAs are short RNA molecules with an average of 22 nu-
cleotides that are found in all eukaryotic cells (Bartel, 20 04). They
are post-transcriptional regulators that bind to complementary
sequences on target mRNA transcripts, usually resulting in trans-
lational repression or gene silencing. They thus play an important
role in the regulation of target mRNA expression (Cai et al., 2009;
Schratt, 2009). MiRNA s are highly expressed in neurons and mi-
croglia of the central nervous system (CNS) and modulate both
neuronal and immune functions (Brites & Fernandes, 2015). They
are also involved in the development of neuropsychiatric disorders
including MDD (Dwivedi, 2014; Karthikeyan et al., 2016; Mouillet-
Richard et al., 2012; O'Connor et al., 2012). Recent preclinical
studies have demonstrated that miRNA could have therapeutic
potential for various CNS disorders, such as cognitive dysfunction
(Tang et al., 2019) and MDD (Deng et al., 2019). However, it re-
mains unclear whether the miRNAs inhibiting IDO1 is useful ther-
apeutic target s for MDD.
In this study, we aimed to identify novel miRNAs that inhibit
IDO1 activity and investigate whether it ameliorates LPS-induced
depression-like behavior in mice.
2 | Materials and Methods
2.1 | Animals
Male C57BL/6J mice (8 weeks old; Research Resource Identifiers,
RRID:IMSR_JAX:000664) were purchased from Japan SLC Inc.
(n = 214). Only male mice were used to exclude any potential es-
trous cycle ef fects. This study was not pre-registered and no ran-
domization/blinding was performed. No exclusion criteria were
pre-determined, and no animals were excluded. Sample size for each
experiment was determined based on our previous studies with the
relevant type of experiment (Miwa et al., 2011; Mouri et al., 2018;
Wulaer et al., 2020). No sample size calculations were performed.
Mice were housed in a plastic cage and maintained on a 12-hr light/
dark cycle (lights on at 8:00 a.m.) with food and water ad libitum. All
experiments were carried out in accordance with the guidelines es-
tablished by the Japanese Pharmacological Society and the Institute
for Experimental Animals at Fujita Health University. The protocols
were approved by the Ethics Committee of Animal E xperiments at
the Institute for Experimental Animals at Fujita Health University
(Permit Number: AP16044).
2.2 | LPS injection
LPS (serotype E. coli 0127: B8, Cat# L3880; Sigma-Aldrich) was dis-
solved in phosphate-buffered saline (PBS). The mice were intraperi-
toneally (i.p.) injected with LPS (0.5 mg/kg) 24 hr before behavioral
testing. This dose was used according to previous publication shown
that LPS (0.5 mg/kg; i.p.) induced depression-like behavior in the
forced swimming test (FST) (Yamawaki et al., 2018).
2.3 | Behavioral tests
All behavioral tests were performed between 10:00 a.m. and
6:00 p.m. To reduce the influence of the previous experiment, the
|
3
SUENTO ET al .
sequence of behavioral tests was arranged in order of stress level
from low (open field test: Open-field test [OFT ]) to high (forced
swimming test: FST). The rotarod test was performed separate
group of animals from the OFT and FST. Behavioral experiment s
were carried out in a sound-attenuated and air-regulated experi-
mental room, to which the mice were habituated for more than
2 hr.
2.4 | Open-field test
OFT was performed as a previous report (Yamada et al., 2000) with
minor modifications. The open field consisted of a square area with
gray walls (42 × 42 × 40 cm) set in a dark, sound-attenuated room.
The floor of the field was divided into nine identical squares with
a light (200 lux) positioned 100 cm above the center of the floor.
Each mouse was placed in one corner of the open field. The mice
were allowed to explore the environment freely for 5 minutes. The
time it took the mouse to move to another section was repor ted as
its starting latency. The time spent in the center or corner zone and
the total distance traveled were measured using an ANY-maze video
tracking system (Cat# 6000, Stoelting Co., Ltd.; RRID:SCR_014289).
The number of rearing events was also counted by images captured
on video.
2.5 | Forced swimming test
The FST was performed according to the method outlined in previ-
ous repor t (Murai et al., 2007). Each mouse was placed in a trans-
parent glass cylinder (20 × 15 cm) that was filled with water to a
depth of 13 cm, at a temperature of 22 °C, and illuminated by a
lamp placed above the apparatus (100 lux). The mouse was forced
to swim for 6 minutes. The duration of swimming was measured
usi ng SCANET-40 (Ca t# MV-40 , MELQU EST Co., Ltd .). The immobil-
ity time was calculated in the last 5 min as follows: Immobility time
(s) = total time − swimming time.
2.6 | Rotarod test
The rotarod test was performed according to the method out-
lined in previous report (Iida et al., 1999) with minor modifica-
tions. Motor functions of the mice were examined using the
rotarod test (Cat# 47650; Muromachi Kikai Co., Ltd.). The test
was performed by placing a mouse on a rotating treadmill drum
(3 cm diameter) with constant illumination of approximately
20 lux, and measuring how long the mouse was able to maintain
its balance on the treadmill. Four trials were conducted in which
the treadmill rotated at 12 rpm for a maximum of 120 seconds.
Each mouse’s mean latency to fall was calculated and used in
subsequent analysis. To prevent experimental bias, a blinded ob-
server calculated the latency.
2.7 | Sample collection
The mice were deeply anesthetized with isoflurane (1 ml/ml; Cat#
095-06573; Wako Pure Chemical Co.) and transcardially perfused
with ice-cold PBS at 2, 6, 12, and 24 hr after LPS injection. The
entire brain was quickly removed and chilled in ice-cold saline. The
prefrontal cortex (PFC) and hippocampus (HIP) were manually dis-
sected on ice-cold plates and then immediately frozen using dry
ice because these regions have been associated with the patho-
physiolog y and progression of MDD (McKinnon et al., 2009;
Treadway et al., 2015). Specifically, the enhanced inflammation,
oxidative stress, and neurotransmitter disturbances (e.g., seroto-
ni n , glutam a te, ga mma -am i nobu tyri c ac id) in PF C an d HIP faci l itat e
the MDD progression and contribute to further brain structural
decline as the disease advances (Holmes et al., 2018; Setiawan
et al., 2015). All samples were stored at −80°C until needed for
analysis.
2.8 | Quantitative real-time reverse transcription
PCR (qRT-PCR)
Total RNA was is olate d usin g a Nucleo Sp in® RNA kit (Cat# U0 955;
Takara) according to the manufacturer ’s instructions. All PCR
pri mers were purchased from Integ rated DNA Tech nologie s. Fir st-
strand cDNA was synthesized using the ReverTra Ace qPCR RT
kit (Cat# FSQ-101; Toyobo). For quantitative PCR, SsoAdvancedTM
Universal Probes Supermix (Cat# 1725281; Bio-Rad) was used and
sub ject ed to re al-ti me PCR quant if ic at ion us ing a StepOn eTM Real-
Tim e PCR System (C at# 4376357; Life Technologies). Quant it ative
PCR analysis was performed using a StepOne analyzer (Cat#
4461357; Life Technologies; RRID:SCR_014281). The PCR reac-
tion program consisted of 40 cycles of 95°C for 30 seconds and
60°C for 1 minute. To discriminate specific amplification from
non-specific amplification, melting curve analysis was performed
after each PCR reaction. β-actin was used as a housekeeping gene
to normalize all PCR data.
Quantitative PCR for miRNA was isolated using a NucleoSpin®
miRNA kit (Cat# U0971; Takara). All PCR primers were purchased
from Applied Biological Materials Inc.. First-strand cDNA was syn-
thesized using an miRNA cDNA synthesis kit (Applied Biological
Materials Inc.). For the quantitative PCR, SsoAdvancedTM Universal
SYBR® Green Supermix (Cat# 1725271; Bio-Rad) was used and then
subjected to real-time PCR quantification using a StepOneTM Real-
Time PCR System (Life Technologies). The PCR cycle was as follows:
95°C for 10 min, 40 cycles of 95°C for 15 seconds, and 60°C for
1 minute. A melting curve analysis was performed at the end of each
experiment to ver ify that a single product per primer pair was ampli-
fied. U6 small nuclear RNA (U6 snRNA) was used as a housekeeping
gene to normalize all PCR data.
Primers used in this study include: IDO1 (Mm.PT.58.2954917),
IL-1β (Mm.PT.58.41616450), IL-6 (Mm.PT.58.10005566), TNF-α
(Mm.PT.58.12575861), β-actin (Mm.PT.39.a.22214843), Mmu-miR-
4
|
SUENTO ET al.
203-3p (MPM00983), Mmu-miR-384-3p (MPM0 0461), Mmu-miR-
874-3p (MPM02237), Mmu-miR-381-3p (MPM01286), U6 snRNA
(MPM00002), and Universal 3′ miRNA reverse primer (MPH00000).
Representative RT-PCR product bands are shown in Figure S1.
2.9 | Western blotting analysis
Western blotting was performed as described previously
(Kunisawa et al., 2017). Tissues were homogenized in an ice-cold
homogenization buffer (pH 7.4; 50 mM Tris-HCl [pH 8.0] con-
taining 4 mM EGTA, 10 mM EDTA, 150 mM sodium dihydrogen
phosphate, and 1% protease inhibitor cocktail) (Cat# 162-26031;
Fujifilm Wako Pure Chemical Co.) by sonication. After 15 minutes
of centrifugation at 15,300 g at 4°C, the protein concentration in
the supernatant was determined with Quick StartTM Bradford 1x
dye reagent (Cat# 500 0205; Bio-Rad) and normalized to 2.0 μg/μL.
Respective protein samples were then electrophoresed on 10%
(w/v) SDS-PAGE and subsequently transferred onto a 0.22 mm
PVDF membrane (Cat# GVWP04700; Millipore). The membranes
were blocked with 5% skim milk in TBST for 60 minutes at room
temperature (24°C) and probed with a primary antibody at 4°C
overnight. Next, PVDF membranes were washed in TBST (three
times for 10 minutes) and incubated in the appropriate HRP-
conjugated secondary antibody for 2 hr at room temperature.
The PVDF membranes were then washed again in TBST (3 times
for 10 minutes) and reacted with Immobilon Forte Western HRP
substrate (Cat# WBLUF0100; Millipore). Immunoreactive bands
were visualized using ATTO LuminoGraphI (Cat# WSE-6100;
ATTO). The band intensi ties were analyzed using ImageJ soft ware .
The primar y antibodies used were rat anti-IDO1 (1:500; Cat# sc-
53978; Santa Cruz Biotechnology; RRID:AB_831071), mouse anti-
β-actin (1:1000; Cat# A5441; Sigma-Aldrich, RRID:AB_476744),
and mouse anti-TLR4 (1:1000; Cat# sc-293072; Santa Cruz
Biotechnology; RRID:AB_10611320). The secondary antibodies
were diluted at 1:2000 for horseradish peroxidase-linked anti-
rat or anti-mouse IgG (Cat# NA935 and NA931; GE Healthcare,
RRID:AB_772207 and AB_772210).
2.10 | Mouse tissue preparation
For histological analysis, mice were deeply anesthetized with iso-
flurane (1 ml/ml, Wako Pure Chemical Co.). Once reflex responses
had disappeared, mice were transcardially perfused with 4% para-
formaldehyde in phosphate-buffered saline (PBS). Brains were
post-fixed in 4% paraformaldehyde overnight at 4°C. The post-
fixed tissues were cr yoprotected overnight in PBS containing
20% sucrose, embedded in OCT compound (Cat# 45833; Sakura
Finetechnical Co.), and cut into 20 μm sections using a cryostat
(Cat# Leica CM3050, RRID:SCR_016844) for in situ hybridization
and immunohistochemistry.
2.11 | In situ hybridization
Digoxigenin (DIG)-labeled single-stranded riboprobes for miR-
874-3p were purchased from QIAGEN (Cat# YD00616271-BCG).
The protocol for in situ hybridization was previously described
(Kunisawa et al., 2018). Briefly, the 20 μm sections were treated
with proteinase K (40 μg/ml for 30 min at room temperature; Cat#
107393; Millipore) and hybridized overnight at 50°C with DIG-
labeled antisense riboprobes in a hybridization solution consisting
of 40% formamide, 20 mM Tris-HCl (pH 7.5), 600 mM NaCl, 1 mM
EDTA, 10% dextran sulfate, 200 μg/mL yeast tRNA, 1x Denhardt’s
solution, and 0.25% SDS. The sections were washed three times in
1× SSC (150 mM NaCl and 15 mM sodium citrate) containing 50%
formamide at 50°C, followed by 0.1 M maleic buffer (pH 7.5) con-
taining 0.1% Tween 20 and 0.15 M NaCl. The bound DIG-labeled
probe was detected by overnight incubation with anti-DIG antibody
conjugated with alkaline phosphatase (Cat# 11093274910; Roche).
The probe was developed in a solution containing 4-nitro-blue tetra-
zolium chloride (NBT; Cat# 11383213001; Roche) and 5-bromo-4-
chloro-3-indolyl phosphate (BCIP; Cat# 11383221001; Roche) in the
dark at room temperature.
2.12 | Immunohistochemistry
Immunofluorescence staining was performed as described previ-
ously (Kunisawa et al., 2018). Cryosections were immunostained
with a mouse anti-IDO1 antibody (1:500; Cat# MABF850; Millipore),
mouse anti-PCNA antibody (1:1000; Cat# MS-106-P; Thermo;
RRID:AB_64275), mouse anti-GFAP antibody (1:1000; Cat#
SAB5201104; Sigma-Aldrich; RRID:AB_2827276), rabbit anti-NeuN
antibody (1:1000; Cat# ab177487; Abcam; RRID: AB_2532109), rab-
bit anti-Iba1 antibody (1:500; Cat# 019-19741; Wako Pure Chemical
Co.; RRID:AB_83950 4), and rat anti-F4/80 antibody (1:500; Cat#
ab6640; Abcam; RRID:AB_1140040). The coronal sections between
1.42 and 2.10 mm from bregma (Paxinos & Franklin, 2004) were
heated in a microwave in 10 mM citrate buffer (pH 6.0) up to 90°C for
5 minutes. After washing with PBS containing 0.3% Triton-X (PBST),
sections were blocked with 5% fetal bovine serum (Cat# 174012,
Nichirei Biscience Inc.) in PBST for 2 hr and then incubated with pri-
mary antibodies in PBST at 4°C overnight. After washing with PBST,
the sec tions were incubated with secondary antibodies (1:2000;
Alexa488-conjugated goat anti-mouse IgG and Alexa568-conjugated
goat anti-rabbit IgG; Cat# A28175 and A11011; Molecular Probes;
RRID:AB_2536161 and AB_143157) and Hoechst 33342 (0.1 µg /ml;
Cat# 346-07951; Dojindo; RRID:KU039) for 3 hr at room tempera-
tu r e. Se cti o ns we r e then ri nse d with PBST, mou nted and cove r ed wi t h
glass coverslips, and then visualized under a Zeiss L SM-710FSX100
confocal laser microscope (Olympus; RRID:SCR_018063). The im-
munohistochemical controls were performed as described above
except for the omission of the primar y antibodies. No positive im-
munostained cells were found in any of the controls.
|
5
SUENTO ET al .
Sections used for 3,3’-diaminobenzidine (DAB) staining were
blocked with 5% fetal bovine serum (Cat# 174012; Nichirei Biscience
Inc.) in PBST for 1 hr, then incubated with mouse anti-NeuN an-
tibody (1:1000; Cat# ab177487; Millipore; RRID:AB_2532109)
and rabbit anti-Iba1 antibody (1:500; Cat# 019-19741; Wako Pure
Chemical Co., RRID:AB_839504) at 4°C overnight. After washing
with PBST, the sections were incubated with secondary antibod-
ies (1:400; biotinylated goat anti-mouse IgG and biotinylated goat
anti-rabbit IgG; Cat# RPN1001 and RPN1004, Vector Laboratories,
RRID:AB_1062579 and AB_1062582) for 1 hr at room tempera-
ture. The sections were then incubated with avidin-biotin complex
(ABC) solution (horseradish peroxidase-streptavidin-biotin complex,
Vectastain ABC kit; Cat# PK-6100; Vector Laboratories) for 1 hr at
room temperature. The HRP signals were detected by DAB solution
with 0.03% H2O2.
The number and density of cells positive for immunoreactivities
were analyzed using ImageJ software. The average of at least three
slices in each mouse was calculated in a 360 μm × 260 μm prelimbic
area of the PFC and used for statistical analysis.
2.13 | Surgery and intracranial injections
The surgery was performed as described previously (Mouri
et al., 2012). An miR-negative control (miR-NC; Cat# A07001) and
miR-874-3p mimic (Cat# A03001) were purchased from GenePharma
co. Mice were anesthetized with a mixture of anesthetic, muscle
relaxant, analgesic and sedative such as medetomidine (0.3 mg/
kg; Domitor®; Nippon Zenyaku Kogyo), butorphanol (5.0 mg/
kg; Vetorphale®; Meiji Seika Pharma), and midazolam (4.0 mg/kg;
Midazolam Sandoz®; Sandoz) to minimize the pain and reversed
by atipamezole (0.15 mg/kg; Antisedan®; Nippon Zenyaku Kogyo)
after completed the surger y. Stainless steel guide cannulas (9.7 mm,
0.4 mm inner diameter, 0.5 mm outer diameter; Cat# AG-6; Eicom)
were bilaterally implanted into the PFC. The coordinates were
+1.7 mm anterior and ± 0.5 mm lateral from the bregma, at a depth
of 1.5 mm from the skull. The guide cannulas were fixed using dental
cement (Cat# 204310196; Shofu Inc.). To seal the top of the guide
cannula and prevent tissue entry into the cannula, a dummy cannula
(0.3 mm in diameter; Cat# AD-6; Eicom) was lef t in place throughout
the experiment. Three days after recovery from surgery, mice were
injected with sterile endotoxin-free PBS (0.5 μl/site) or 1-methyl-l-
tryptophan (1-MT: 1 μg/μL, 0.5 μl/site; C at# 4 47439; Sigma-Aldrich)
for 1-MT experiment, and miR-NC (100 nM, 0.5 μL/site), or miR-
874-3p mimic (100 nM, 0.5 μL/site) for miR-mimic experiment bilat-
erally using a 27-ga ug e in fu si on needle (1.0 mm lo ng er than the guide
cannulas; Cat# 80300; Plastics One) connected to a 10 μl Hamilton
microsyringe at a rate of 0.1 μl/min. After the injection, the needle
wa s left in pl ace fo r an add i tio nal 3 min be f ore wi thdr awa l. It is know n
that miRNA mimics have high stability in vivo and can be applied for
microinjection (Krutzfeldt et al., 2005; Rupaimoole & Slack, 2017).
The OFT and FST were performed 24 hr after the infusion.
2.14 | Measurement of KYN
metabolites
The KYN metabolites were measured as described previously
(Tashiro et al., 2017). For TRP, KYN, kynurenic acid (KA), anthranilic
acid (AA), and 3-HAA, the PFC was weighed and homogenized
(1:3, w/v) in 10% perchloric acid (Cat# 162-00715; Fujifilm Wako
Pure Chemical Co.). After mixing, the precipitated proteins were
removed by centrifugation (13,000 rpm, 15 min). Fifty microliters
of the resulting supernatant were subjected to high-per formance
liquid chr omatograp hy (HPLC ; C at # SPD -M 30A; SH IMADZU ) anal-
ysis. TRP, KYN, KA, A A, and 3-HAA were isocratically eluted from
a reverse phase column (TSKgel ODS-100V, 3 μm, 4.6 mm (ID) ×
15 cm (L); Cat# 0021829; Tosoh Co.) using a mobile phase contain-
ing 10 mM sodium acetate and 2% acetonitrile (pH adjusted to 4.5
with acetic acid) at a flow rate of 0.8 mL/min. TRP and KYN were
detected by ultra-violet and spectrophotometric apparatus (TRP
[UV wavelength, 280 nm], KYN (UV wavelength, 365 nm); Cat#
SPD-M30A; SHIMADZU). AA, 3-HA A, and KA were detected by
a fluorescent detector (A A and 3-HAA [excitation wavelength:
320 nm, emission wavelength: 420 nm], K A [excitation wave-
length: 344 nm, emission wavelength: 40 4 nm]; Cat# RF-20A;
SHIMADZU).
For 3-HK, the PFC was weighed and homogenized (1:3, w/v)
in 10% perchloric acid (Fujifilm Wako Pure Chemical Co.). After
mixing, the precipitated proteins were removed by centrifugation
(13,000 rpm, 15 min). Twenty microliters of the resulting super-
natant was applied to a C18 octadecylsilyl (ODS) silica-gel column
(2.1 mm × 150 mm; Cat# SC-50DS; Eicom) using a mobile phase
consisting of 5% acetonitrile, 0.9% trimethylamine, 0.59% phos-
phoric acid, 0.27 mM EDTA, 8.9 mM sodium heptane sulfonic acid,
and a flow rate of 0.5 mL/min. 3-HK was detected by an electro-
chemically detector (oxidation potential: +0.55 V; Cat# ECD-300;
Eicom).
2.15 | Data analyses
All statistical analyses were performed using GraphPad Prism 6
Software (GraphPad Software Inc.). Significance was assessed
using t tests, or in the case of multiple comparisons, an analysis
of variance (ANOVA) with repeated measures. The Tukey–Kramer
test was used for post hoc analyses when F ratios were significant.
An assessment of the normality of the data prior to the statisti-
cal comparisons has not been carried out; however, this type of
analysis was resistant to deviations from the assumptions of the
traditional ordinary-least-squares ANOVA, and are robust to outli-
ers, thus being insensitive to distributional assumptions (such as
no rma lit y) (H ube r, 1996). Data were not as s ess ed fo r nor mal ity, and
no test for outliers was conducted. The criterion for a significant
difference was p < .05 for all statistical evaluations. All data are
expressed as mean ± SEM.
6
|
SUENTO ET al.
3 | Results
3.1 | LPS triggers the development of depression-
like and sickness behaviors
We induced acute depression-like symptom in mouse with intra-
peritoneal injection of LPS as described by Yamawaki et al. (2018).
Mice were subjected to behavioral tests to investigate whether
LPS-induced depression-like behavior 24 hr af ter injection. LPS sig-
nificantly increased mouse immobility during the FST (Figure 1a;
Student’s t test, t = 3.050, df = 18, **p < .01). Since LPS induces
sickness behavior, such as a decrease in body weight and locomo-
tor activity (Dantzer et al., 2008), mice were weighed before and
24 hr after the LPS injection. LPS significantly decreased body
weight in mice compared to mice in the PBS group (Figure 1b;
two-way ANOVA followed by Tukey’s multiple comparison test, time
[F(1,3 6) = 3.564, p = .0671], treatment [F(1,36) = 35.01, **p < .01] ,
time × treatment [F(1,36) = 40.89, **p < .01]). During the OFT, LPS
significantly decreased locomotor activity (Figure 1c; Student’s t
test, t = 5.594, df = 18, **p < .01) and the number of rearing be-
haviors (Figure 1d; Student’s t test, t = 4.373, df = 18, **p < .01).
However, there was no change in either index of anxiety in LPS
group compared to PBS group: star ting latency (Figure 1e; Student’s
t test, t = 0.6089, df = 18, p = .55) or time spent in the center or
corner zone (Figure 1f; Student’s t test, center [t = 1.189, df = 18,
p = .25], corner [t = 0.3833, df = 18, p = .76]). A rotarod test was
performed to investigate whether these LPS-induced behavioral
changes were because of motor dysfunction. There was no dif-
ference observed in the duration of maintained balance between
the LPS and PBS groups (Figure 1g; two-way ANOVA followed by
FIGURE 1 LPS triggers the development of depression-like and sickness behavior. (a) The FST was performed 24 hr after LPS injection
(Student’sttest, **p < .01;n = 8–12 mice in each group). (b) Body weight was measured before and 24 hr after LPS injection (two-way ANOVA
followed by Tukey’s multiple comparison test, time [p > .05], treatment [**p < .01], time × treatment [**p < .01];n = 8–12 mice in each group).
(c–f) The OFT was performed 24 hr after LPS injection (n = 8–12 mice in each group). All data were analyzed with Student ’sttest showing
distance traveled (c,**p < .01), rearing counts (d, **p < .01), starting latency (e,p > .05), and time spent in the center and corner of the zone
(f, center [p > .05], corner [p > .05];n = 8–12 mice in each group). (g) Rotarod test was per formed 24 hr after LPS injection (two-way ANOVA
followed by Tukey’s multiple comparison test, session [**p < .01], treatment [p > .05], session × treatment [p > .05];n = 8–10 mice in each
group). All data are expressed as mean ± SEM
(a) (b) (c)
(e)(d) (f)
(g)
|
7
SUENTO ET al .
Tukey’s multiple comparison test, session [F(3,64) = 4.9 29, **p < .01],
treatment [F(1,6 4) = 0.04569, p = .8314], session × treatment
[F(3,64) = 0. 5570, p = .6 453]). These results suggest that acute LPS
injection induces depression-like behavior and sickness behavior in
mice without inducing anxiety or motor dysfunction.
3.2 | LPS up-regulates IDO1 expression in the PFC,
but not in the HIP
IDO1 is essential for LPS-induced depression-like behavior in mice
(O'Connor, et al., 2009). The increase in pro-inflammator y cytokines
in the brain contributes to IDO1 up-regulation (Jo et al., 2015). The
increased expression of pro-inflammatory cytokines such as IL-1β
and TNF-α in the PFC and HIP were observed after LPS injection
(Figures S2a,b,d,e; Student’s t test, IL-1β in PFC [t = 11.32, df = 18,
**p < .01], TNF-α in PFC [t = 9.363, df = 18, *p < .01], IL-1β in HIP
[t = 13.96, df = 18, **p < .01], TNF-α in HIP [t = 15.24, df = 18 ,
**p < .01]), but that of IL-6 was significantly decreased only in
the PFC by qRT-PCR (Figures S2c,f; Student’s t test, IL-6 in PFC
[t = 2.752, df = 18, *p < .05], IL-6 in HIP [t = 1.356, df = 18, p = .19]).
To further investigate whether LPS-induced depression-like behav-
ior is related to up-regulation of IDO1 in the brain, the expression
of IDO1 mRNA and protein was examined in the mouse PFC and
HIP by qRT-PCR and western blotting, respectively. Tissue samples
were collected at 2, 6, 12, and 24 hr after LPS injection (Figure 2a).
The mRNA level of IDO1 was up-regulated in the PFC between 6 hr
and 24 hr after LPS injec tion (Figure 2b; two-way ANOVA followed
by Tukey’s multiple comparison test, F(3,55) = 15.34, **p < .01).
IDO1 mRNA levels in the HIP were up-regulated only 6 hr after LPS
FIGURE 2 LPS up-regulates IDO1 expression in the PFC, but not in the HIP. (a) Experimental time course. (b, c) The mRNA levels of
IDO1 were measured in the PFC (b) and HIP (c) at 2, 6, 12, and 24 hr after LPS injection by qRT-PCR (two-way ANOVA followed by Tukey’s
multiple comparison test, PFC [**p < .01], HIP [**p < .01];n = 7–10 mice in each group). (d, e) The protein levels of IDO1 were measured in the
PFC (d) and HIP (e) at 2, 6, 12, and 24 hr after LPS injection by western blotting (two-way ANOVA followed by Tukey’s multiple comparison
test, PFC [**p < .01], HIP [p > .05];n = 7–10 mice in each group). Data are normalized to the expression levels in the PBS group. (f) Schematic
illustration showing the PFC section that was included in this analysis (Red dotted lines). (g, h) Representative images of double staining with
anti-IDO1 (green) and anti-NeuN (g, red) or anti-Iba1 (h, red) in the PFC 24 hr after LPS injection. Scale bar: 100 μm. (i) IDO1 signal intensity
and (j) NeuN-positive cells were measured in the PFC (Student’sttest, IDO1 signal intensity [*p < .05], NeuN-positive cells [p > .05];n = 4–5
mice in each group). (k) The number of IDO1/NeuN double-positive cells was quantified in the PFC (Student’sttest,p > .05;n = 4–5 mice
in each group). (l) IDO1 signal intensit y and (m) Iba1-positive cells were measured in the PFC (Student’sttest, IDO1 [*p < .05], Iba1-positive
cells [**p < .0 1];n = 4–5 mice in each group). (n) The number of IDO1/Iba1 double-positive cells was quantified in the PFC (Student’sttest,
*p < .05;n = 4–5 mice in each group). The data are expressed as mean ± SEM. Cg, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic
cortex
(a)
(b) (c)
(d) (e)
(f) (g) (h)
(i) (j) (k) (l) (m) (n)
8
|
SUENTO ET al.
injection (Figure 2c; two-way ANOVA followed by Tukey’s multiple
comparison test, F(3,49) = 5.197, **p < .01). Moreover, the IDO1
protein level in the PFC was up-regulated 24 hr after LPS injection
(Figure 2d; two-way ANOVA followed by Tukey’s multiple compari-
son test, F(3,49) = 3.880, **p < .01). Howev er, the IDO1 prote in level
in the HIP was the same at all timepoints for the LPS and PBS groups
(Figure 2e; two-way ANOVA followed by Tukey’s multiple compari-
son test, F(3,48) = 0.1675, p = .9178). To confirm this result, the lo-
calization of IDO1 was determined by double immunostaining with
IDO1 and NeuN (neuronal marker) or Iba1 (microglia marker) in the
PFC (Figure 2f–h). Quantification data showed that IDO1 intensity
was significantly higher in the IDO1/NeuN-stained samples in the
FIGURE 3 LPS transiently increases the expression of miR-874-3p in the PFC, and its over-expression down-regulates IDO1 protein level.
(a) Predicted regulatory miRNAs to IDO1. The left middle panel represents the intersection of the three databases. The numbers in the panel
indicate candidate miRNAs in each database. Four miRNAs that have a high probability of regulating IDO1 expression are shown in the right
box. (b) Sequence alignment of the miR-203-3p, miR-384-3p, miR-874-3p, and miR-381-3p base-pairing site in the 3′ UTR of IDO1 mRNAs
in mice. (c-e) The expression levels of miR-203-3p (c), miR-384-3p (d), and miR-874-3p (e) of the PFC were analyzed by qRT-PCR at 2, 6, 12,
and 24 hr after LPS injection (two-way ANOVA followed by Tukey’s multiple comparison test, miR-203-3p [p > .05], miR-384-3p [p > .05],
miR-874-3p [**p < .01];n = 5–8 mice in each group). (f) Representative images ofin situhybridization for miR-874-3p (blue) in the PFC 6 hr
after LPS injection. Scale bar: 100 μm. (g) Double staining byin situhybridization for miR-874-3p (blue) and immunostaining with anti-NeuN
(brown) or anti-Iba1 (brown) in the PFC 6 hr after LPS injection. Arrowheads indicate double-positive cells for both miR-874-3p and NeuN
or Iba1. Scale bar: 50 μm. (h) The percentage of miR-874-3p-positive cells expressing neuron or microglia marker was quantified in the PFC
(Student’sttest, NeuN [**p < .01], Iba1 [*p < .05];n = 3 mice in each group). (i) The experimental time course. (j) The protein level of IDO1
were examined in the PFC 24 hr after LPS injection followed by miR-874-3p mimic microinjection by western blotting (two-way ANOVA
followed by Tukey’s multiple comparison test, treatment [ p > .05], microinjection [p > .05], treatment with microinjection [**p < .01];n = 6–7
mice in each group). The data are expressed as mean ± SEM. NC, miR-negative control
(a)
(b)
(c) (e) (f)
(g) (h)
(i) (j) (k)
|
9
SUENTO ET al .
LPS group (Figure 2i; Student’s t test, t = 3.023, df = 6, *p < .05).
No differences were observed in the number of NeuN-positive
cells (Figure 2j; Student’s t test, t = 0.5335, df = 6, p = .6129) or
IDO1/NeuN-double-positive cells between the LPS and PBS groups
(Figure 2k; Student’s t test, t = 1.964, df = 6, p = .0972). Similarly,
the intensity of IDO1 was higher in the IDO1/Iba1-stained sam-
ples in the LPS-group (Figure 2l; Student’s t test, t = 3.115, df = 7,
*p < .05). The numbers of Iba1-positive cells (Figure 2m; Student’s t
test, t = 4.856, df = 7, **p < .01) and IDO1/Iba1-do ub le-positive cells
(Figure 2n; Student’s t tes t , t = 2.716 df = 7, *p < .05) wer e also si gnif-
icantly increased in the LPS group. Quantification of Iba1 and PCNA
(proliferation cell marker)-double-positive cells was significantly
higher in the LPS-group (Figure S3a,d; Student’s t test, t = 18. 51,
df = 7, **p < .01). Co-localization of LPS-induced Iba1-positive cells
with GFAP (astrocyte marker) and F4/80 (macrophage marker)-posi-
tive cells was not obser ved (Figure S3b,c). This suggested that these
Iba1-positive cells reflect the microglial proliferation rather than the
migration of macrophages and other cell types. These results pro-
posed that LPS-induced depression-like behavior is likely related to
up-regulation of IDO1 in both the neurons and microglia of the PFC.
3.3 | LPS transiently increases the expression of
miR-874-3p in the PFC, and its over-expression down-
regulates IDO1 protein level
MiRNAs regulate protein expression by targeting mRNA and may
serve as a potent ial therapeutic target (O'Connor et al., 2012). To in-
vestigate whether miRNAs are involved in the regulation of IDO1 ex-
pression, three databases were employed to determine the candidate
miRNAs. A total of 85 potential regulatory miRNAs were predicted
fro m the miRmap, 65 from Target Sc an, and four from the micr oR NA.
org database. Four miRNAs (miR-203-3p, miR-384-3p, miR-874-3p,
and miR-381-3p) were chosen for their high exact probabilit y by
Venn diagram analysis (Figure 3a). The interactions of IDO1 mRNA
at the 3′ UTR are shown in Figure 3b. qRT-PCR was per formed to
confirm which miRNAs are important for LPS-induced up-regulation
of IDO1 in the PFC. Among the miRNAs, miR-381-3p was not de-
tectable in either the LPS or PBS groups. The expression levels of
miR-203-3p and miR-384-3p did not differ at any timepoint between
the LPS and PBS groups (Figures 3c,d; two-way ANOVA followed
by Tukey’s multiple comparison test, miR-203-3p [F(3,37) = 0.735,
p = .5378], miR-384-3p [F(3,46) = 0.6642, p = .5783]). Importantly,
the expression level of miR-874-3p was significantly up-regulated
6 hr after LPS injection (Figure 3e; two-way ANOVA followed by
Tukey’s multiple comparison test, F(3,40) = 10.30, **p < .01). The
increase in miR-874-3p expression after LPS injection was further
confirmed by in situ hybridization (Figure 3f). The increase in miR-
874-3p was observed in both NeuN- and Iba1-positive cells after LPS
injection by double labeling of miR-874-3p and NeuN or Iba1 by in
situ hybridization and immunostaining, respectively (Figures 3g,h;
Student’s t test, NeuN [t = 10.69, df = 4, **p < .01], Iba1 [t = 4.594,
df = 4, *p < .05]). Altogether, miR-874-3p may play an important role
in LPS-induced up-regulation of IDO1 in the PFC. To further confirm
its role in the regulation of IDO1 levels, mice were injected with LPS
(0.5 mg/kg) and then immediately infused miR-NC or miR-874-3p
mimic into the PFC (Figure 3i and Figure S4a). MiR-874-3p prevented
the increase in IDO1 mRNA and protein levels in the PFC 24 hr after
LPS injection as seen by qRT-PCR and western blotting, respec-
tively (Figure S3j and S4b; two-way ANOVA followed by Tukey’s
multiple comparison test, IDO1 mRNA (treatment [F(1,26) = 28.33,
**p < .01], microinjection [F(1,26) = 8.624, **p < .01], treatment
with microinjection [F(1, 26) = 4.474, *p < .05]; IDO1 protein (treat-
ment [F(1, 21) = 1.445, p = .2427], microinjection [F(1,21) = 2.664,
p = .1176], tr eat men t wit h mic roi nje cti on [F(1,21) = 8.0 67, **p < .01]).
Toll-like receptor 4 (TLR4) is known as an LPS receptor and induces
pro-inflammatory cytokines (De Paola et al., 2012). To examine the
potential off-target effect s of miR-874-3p, we measured the expres-
sion of TLR4 and pro-inflammatory cytokines. MiR-874-3p failed
to af fect the expression of TLR4, IL-1β, TNF-α, and IL-6 in the PFC
(Figure S5; two-way ANOVA followed by Tukey’s multiple compari-
son test, TLR4 (treatment [F(1,28) = 0.1314, p = .7197], microinjec-
tion [F(1,28) = 0.8784, p = .3566], treatment with microinjection
[F(1,28) = 0.3011, p = .3011]), IL-1β (treatment [F(1,26) = 18.12,
**p < .01], microinjection [F(1, 26) = 0. 2110 , p = .6498], treatment
with microinjection [F(1, 26) = 6.915, *p < .05]), TNF-α (treat-
ment [F(1,26) = 31.06, **p < .01], microinjection [F(1,26) = 2 . 8 47,
p = .1035], treatment with microinjection [F(1,26) = 0.0000677,
p = .9935]), IL-6 (treatment [F(1,26) = 0.06351, p = .8030], microin-
jection [F(1,26) = 0 .198 9, p = .6593], treatment with microinjection
[F(1,26) = 2.806, p = .1059]). These results suggest that miR-874-3p
regulates IDO1 protein levels after LPS injection.
3.4 | Microinjection of 1-MT or miR-874-3p mimic
into the PFC improves LPS-induced depression-
like behavior
It has been suggested that miRNAs are novel targets of thera-
peutic strategies for depression-like behavior in animal models
(Deng et al., 2019; Lopizzo et al., 2019; O'Connor et al., 2012).
Therefore, we specu late d that th e inhibition of IDO1 in th e PF C by
miR-874-3p mimic might have a preventive effect on LPS-induced
depression-like behavior in mice. First, we infused 1-MT, a potent
IDO1 antagonist, into the PFC of the LPS group and subjected the
mouse to OFT and FST 24 hr after the LPS injection (Figure 4a).
Intra-PFC infusion of 1-MT prevented the increase in immobility
in the FST of the LPS group (Figure 4b; two-way ANOVA followed
by Tukey’s multiple comparison test, treatment [F(1,35) = 3 .42 9,
p = .0725], microinjection [F(1, 35) = 7. 136, p = .0114], treat-
ment with microinjection [F(1,35) = 4.415, *p < .05]). However,
1-MT failed to prevent the decrease in distance traveled
(Figure 4c; two-way ANOVA followed by Tukey’s multiple com-
parison test, treatment [F(1,35) = 15.26, **p < .01], microinjec-
tion [F(1,35) = 1.1 3 7, p = .2935], treatment with microinjection
[F(1,35) = 0.04056, *p < .05]) and number of rearing behaviors in
10
|
SUENTO ET al.
the OF T (Fig ur e 4d; two-w ay ANOVA followe d by Tukey’s mu lt iple
comparison test, treatment [F(1,35) = 64.95, **p < .01], microin-
jection [F(1,35) = 3.230, p = .0809], treatment with microinjec-
tion [F(1,35) = 0.0002297, p = .9880]). In a separate experiment,
the increase in IDO1 level was inhibited by over-expression of
mi R-8 74 -3p in th e PFC of the LP S gro u p (Fig ure 3j ). In a sim ila r way,
the miR-874-3p mimic was infused into the PFC of the LPS group
and conducted OF T and FST 24 hr later (Figure 4e). Interestingly,
the increase in immobility in the FST of the LPS group was also
prevented by the miR-874-3p mimic infusion (Figure 4f; two-way
FIGURE 4 Microinjection of 1-MT or miR-874-3p mimic into the PFC improves LPS-induced depression-like behavior. (a) The 1-MT
microinjection time course. (b) The FST was performed 24 hr after LPS injection followed by 1-MT microinjection into the PFC (two-
way ANOVA followed by Tukey’s multiple comparison test, treatment [ p > .05], microinjection [p > .05], treatment with microinjection
[*p < .05];n = 7–13 mice in each group). (c-d) The OFT was per formed 24 hr after LPS injection followed by 1-MT microinjection into
PFC (two-way ANOVA followed by Tukey’s multiple comparison test showing distance traveled (c) (treatment [**p < .01], microinjection
[p > .05], treatment with microinjection [*p < .05];n = 7–13 mice in each group) and rearing (d) (treatment [**p < .01], microinjection [p > .05],
treatment with microinjection [p > .05];n = 7–13 mice in each group). (e) The miR-874-3p microinjec tion time course. (f) The FST was
performed 24 hr after LPS injection followed by miR-874-3p mimic microinjection in the PFC (two-way ANOVA followed by Tukey’s multiple
comparison test, treatment [*p < .05], microinjection [*p < .05], treatment with microinjection [*p < .05];n = 6–10 mice in each group). (g-h)
The OFT was performed 24 hr after LPS injection followed by miR-874-3p mimic microinjection into the PFC (two-way ANOVA followed by
Tukey’s multiple comparison test showing distance traveled (g) (treatment [**p < .01], microinjection [ p > .05], treatment with microinjection
[p > .05];n = 6–10 mice each) and rearing (h) (treatment [**p < .01], microinjection [*p < .05], treatment with microinjection [p > .05];n = 6 -10
mice in each group). The data are expressed as mean ± SEM. 1-MT, 1-methyl-l-tryptophan; NC, miR-negative control
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
|
11
SUENTO ET al .
FIGURE 5 Microinjection of miR-874-3p mimic into the PFC improves the levels of 3-HA A. (a) Schematic of KYN metabolism. (b-f)
The concentrations of TRP (b), KYN (c), 3-HK (d), 3-HAA (e) and A A (f) were measured 24 hr after LPS injection followed by miR-874-3p
mimic microinjection into PFC 4by HPLC (two-way ANOVA followed by Tukey’s multiple comparison test, TRP (treatment [**p < .01],
microinjection [p > .05], treatment with microinjection [p > .05]), KYN (treatment [**p < .01], microinjection [p > .05], treatment with
microinjection [p > .05]), 3-HK (treatment [**p < .01], microinjection [p > .05], treatment with microinjection [p > .05], 3-HAA (treatment
[**p < .01], microinjection [**p < .01], treatment with microinjection [**p < .01], AA (treatment [**p < .01], microinjection [p > .05], treatment
with microinjection [p > .05]);n = 8 mice in each group). The data are expressed as mean ± SEM. TRP, tryptophan; KYN, kynurenine;
3-HK, 3-hydroxyk ynurenine; K A, k ynurenic acid; A A, anthranilic acid; 3-HAA, 3-hydroxy anthranilic acid; NAD+, nicotinamide adenine
dinucleotide; NC, miR-negative control
(a) (b)
(c) (d)
(e) (f)
12
|
SUENTO ET al.
ANOVA followed by Tukey’s multiple comparison test, treat-
ment [F(1, 34) = 6.246, *p < .05], microinjection [F(1, 34) = 7.14 6,
*p < .05], treatment with microinjection [F(1,34) = 4 .176,
*p < .05]). In contrast, similar to the results of 1-MT, the miR-
874-3p mimic infusion failed to prevent the decrease in the dis-
tance traveled (Figure 4g; two-way ANOVA followed by Tukey’s
multiple comparison test, treatment [F(1,32) = 33.22, **p < . 01],
microinjection [F(1,32) = 0.004486, p = .9470], treatment with
microinjection [F(1,32) = 0.1119, p = .7401]) and number of rear-
ing behaviors (Figure 4h; two-way ANOVA followed by Tukey’s
multiple comparison, treatment [F(1,32) = 50.80, **p < .01], mi-
croinjection [F(1,32) = 4.879, *p < .05], treatment with microin-
jection [F(1,32) = 0.0005185, p = .9820] in the OFT. There was
no difference obser ved in the time spent of the center or cor-
ner zone after 1-MT and miR-874-3p mimic infusion (Figure S6;
two-way ANOVA followed by Tukey’s multiple comparison test,
center with 1-MT (treatment [F(1,35) = 1.994, p = .1668], micro-
injection [F(1, 35) = 6.214, *p < .05], treatment × microinjection
[F(1,35) = 0.0003981, p = .9842]), corner with 1-MT (treatment
[F(1,32) = 2. 597, p = .1169], microinjection [F(1,32) = 0. 30 40,
p = .5852], treatment × microinjection [F(1,32) = 0 .6193 ,
p = .4371], center with miR-873-3p (treatment [F(1,32) = 1.295,
p = .2635], microinjection [F(1,32) = 0.5918 , p = .5918], treat-
ment × microinjection [F(1,32) = 0.08959, p = .7666]), corner
with miR-873-3p (treatment [F(1,35) = 1.721, p = .1981], micro-
injection [F(1,35) = 3.445, p = .0719], treatment × microinjection
[F(1,35) = 0.01696, p = .8971]). Collectively, these data suggest
that microinjection of miR-874-3p into the PFC prevents LPS-
induced depression-like behavior, but not sickness behavior,
through suppression of IDO1 activity.
3.5 | Microinjection of miR-874-3p mimic into the
PFC improves the levels of 3-HAA
IDO1-mediated activation of KYN pathway is related to depressive-
like symptoms in mice (O'Connor, et al., 2009). To further investigate
the effects of miR-874-3p on the KYN pathway, we infused an miR-
874-3p mimic into the PFC of the LPS group and measured the con-
centrations of KYN metabolites (TRP, KYN, 3-HK , KA, 3-HAA , and
AA; Figure 5a) in the PFC by HPLC. Among the KYN metabolites, KA
was not detectable in either the PBS or LPS groups. Compared to the
PBS group, the levels of KYN, 3-HK , and 3-HA A but not TRP or A A
were significantly increased in the LPS group (Figure 5b–f; two-way
ANOVA followed by Tukey’s multiple comparison test, TRP (treat-
ment [F(1,28) = 9.573, **p < .01], microinjection [F(1,28) = 0.6610,
p = .4231], treatment with microinjection [F(1,28) = 2.298,
p = .1407]), KYN (treatment [F(1,28) = 70.45, **p < .01], microin-
jection [F(1,28) = 0. 2079, p = .6519], treatment with microinjection
[F(1,28) = 0.6644, p = .4214]), 3-HK (treatment [F(1,28) = 112.0,
**p < .01], microinjection [F(1,28) = 0.1279, p = .7233], treatment
with microinjection [F(1,28) = 1.128, p = .2973], 3-HA A (treatment
[F(1,28) = 121 .9, **p < .01], microinjection [F(1,28) = 22.87, **p < .01] ,
treatment with microinjection [F(1,28) = 28.59, **p < .01], AA (treat-
ment [F(1,28) = 10.22 , **p < .01], microinjection [F(1,28) = 0.2287,
p = .6362], treatment with microinjection [F(1,28) = 0.2287,
p = .6362])). The miR-874-3p mimic potently reduced 3-HAA levels
(Figure 5e), whereas K YN and 3-HK remained unchanged compared
to miR-NC gr oup (Figur e 5c, d). Thes e re sul t s su g ge st th at th e ben efi-
cial effect of miR-874-3p on LPS-induced depression-like behavior
may be associated with reduced 3-HAA levels.
4 | Discussion
In this study, we showed that LPS-induced depression-like behav-
ior was related to increase in IDO1 expression in the PFC. Our data
confirmed that IDO1 is an important regulator of depression-like
behavior induced by LPS, since pharmacological inhibition of IDO1
by 1-MT protected mice from LPS-induced depression-like behav-
ior. Furthermore, microinjection of miR-874-3p into the PFC also
reversed depression-like behavior through a decrease in IDO1 pro-
tein levels. The limitation of our study is because of the nature of
the acute inflammatory stimulus that caused inflammation-induced
depression. LPS-induced depression-like behavior recapitulates the
main features of inflammation-induced depression but lacks the
chronicity that is characteristic of MDD. In fact, the expression of
miR-874-3p was changed in the mice exposed to chronic social de-
feated stress (unpublished data). Although further validation using
other depression models is needed, this is the first report to provide
new insight for miRNA-874-3p as a potential therapeutic target for
MDD.
In a previous study, mice injected with LPS were originally de-
veloped as an animal model for sickness behavior characterized by a
decrease in body weight, food intake, and exploratory activity. This
behavior resulted from the production of pro-inflammatory cyto-
kines in the periphery and brain (Dantzer et al., 2008). We found
that the direct inhibition of IDO1 in the PFC suppressed the increase
in immobility in the FST, but not the decrease in open-field activity,
in the LPS group. These data suggest that IDO1 selectively related
to inflammation-induced depression-like behavior, but not anxiety
or sickness behavior. This finding is consistent with a previous study
that showed a different pathogenesis of sickness and depression-like
behavior af ter LPS injection (O'Connor, et al., 2009).
Previous reports have indicated that change in miRNA levels in
the bra in may si gnificant ly contribute to the development of depres-
sion in animal models (Deng et al., 2019; Lo Iacono et al., 2019; Lou
et al., 2019). Using Venn diagram analysis from three databases, we
identified miR-203-3p, miR-384-3p, miR-874-3p, and miR-381-3p for
the regulator of IDO1 expression at the 3′ UTR. Among them, miR-
874-3p was significantly up-regulated after LPS injection. However,
there are no reports on the role of miR-874-3p in the pathogenesis
of MDD. In some cancers, miR-874-3p can act as a tumor suppres-
sor (Han et al., 2016). Importantly, other studies have indicated that
up-regulated miR-874-3p increases proliferation and suppresses
apoptosis of neurons by inhibiting an apoptotic pathway in cerebral
|
13
SUENTO ET al .
isch emia mo del s (Ji an g et al ., 2019). ID O1 is el evate d in mice wit h ce-
rebral ischemia and correlates with increased risk of death (Jackman
et al., 2011). IDO1-derived KYN metabolites can promote apoptosis
in some types of cells in vitro and in vivo (Morita et al., 2001). We
found that IDO1 was up-regulated in the PFC after LPS injection.
Microinjection of miR-874-3p into the PFC down-regulates LPS-
induced IDO1 expression and prevents the LPS-induced increase in
immobility in the FST. These results suggest that miR-874-3p also
exerted protective roles against LPS-induced depression-like behav-
ior by suppressing an apoptotic pathway via IDO1 inhibition.
It is well known that LPS-induced inflammatory response in the
CNS is depend on TLR4 (Singhal & Baune, 2017; Walter et al., 2007).
TLR4 is expressed in both microglia and neuron (De Paola
et al., 2012) and involved in the proliferation and differentiation of
the cells (Leitner et al., 2019; Rolls et al., 2007). We demonstrated
that an increase in Iba1-positive cells after LPS injection reflects the
microglial proliferation rather than the migration of macrophage and
other cell types. Our results suggest that the microglial proliferation
was increased because of TLR4 ac tivation in the LPS-treated mice.
Furthermore, we found that IDO1 and miR-874-3p express not only
in microglia but also in neuron after LPS injection. Although the pre-
cise molecular mechanisms are not yet clear, it is possible that IDO1,
localized to the neurons, contributes to the development of depres-
sion-like behavior. Further studies on IDO1 deficiency in a specific
cell subset will provide important insights into the role of IDO1 in the
pathogenesis of MDD.
During inflammation, IDO1 and TDO are the major enzymes
that metabolizes TRP in the KYN pathway (Fujigaki et al., 2017).
The increase in IDO1 enzymatic activity is correlated with inflam-
mation-associated depression (Dantzer et al., 2008; Murakami
et al., 2016). We demonstrated that microinjection of 1-MT into PFC
could partially reverse LPS-induced depressive-like states in mice.
The behavioral effects of 1-MT could not be explained by the in-
hibition of the TDO as 1-MT has been shown to specifically act on
IDO1 (O'Connor, et al., 2009). These findings suggest that IDO1 ac-
tivation is an important feature of depressive-like states induced by
inflammatory processes. Preclinical, in vitro, and postmortem data
have demonstrated that elevation of K YN metabolites levels is also
associated with neuronal damage and/or suppression of neurogene-
sis (Fischer et al., 2015). We demonstrated that LPS disturbed KYN
metabolites in the PFC. LPS did not affect the TRP and AA levels;
meanwhile, KYN, 3-HK, and 3-HAA were increased in LSP-injected
mice. Of note, miR-874-3p strongly inhibits LPS-induced 3-HAA,
whereas no difference was observed in KYN and 3-HK levels. It has
been recognized that 3-HAA generates the potent oxidative spe-
cies superoxide (O2
–), hydroxyl radical (HO-) and hydrogen peroxide
(H2O2), which frequently contribute to increased apoptosis (Smith
et al., 2009). In line with these data, 3-HAA induced neuronal cell
death with apoptotic features following generation of ROS in primary
neurons (Stone & Darlington, 2002). Recent studies have identified
neuronal apoptosis and reduced volume of frontal cortex in patients
with MDD (Lucassen et al., 2001; McKernan et al., 2009; Shelton
et al., 2011). Although additional experiments were necessary to
fully understand the role of 3-HAA and in LPS-induced behavioral
changes, 3-HAA may be responsible for LPS-induced depression-like
behavior. Aside from IDO1, these data suggested that other rate lim-
iting enzymes for downstream KYN met abolites were also responsi-
ble for LPS-induced depression-like behavior. In fact, LPS increased
the expression of kynrenine-3-monoox y-genase (KMO), and IDO1
was observed in the rats after systemic administration (Connor
et al., 2008).
In summary, our data indicated two findings. First, LPS-induced
depression-like behavior is related to an increase in IDO1 expres-
sion in the PFC. Second, IDO1 inhibition by miR-874-3p or IDO1
antagonist via intra-PFC infusions can reverse depression-like be-
havior in mice. Our data implicate IDO1 in the PFC as an import-
ant component of LPS-induced depression-like behavior. Moreover,
miR-874-3p exerted antidepressant-like effects by down-regu-
lating IDO1 in the PFC. These findings suggest that miR-874-3p
may suppress the pathophysiology of MDD by inhibiting IDO1 ex-
pression. The beneficial effects of miR-874-3p were might be the
consequence of suppressed LPS-induced 3-HAA levels. Our study
provides the first insight into miR-874-3p as a novel potential thera-
peutic target for MDD.
ACKNOWLEDGMENTS
No financial or non-financial interests in relation to the work de-
scribed in this manuscript is declared by the authors. This work
was supported by Grants-in-Aid for Scientific Research from the
Japan Society for the Promotion of Science (17H04252, 17K01969,
18K15377, 18K19761, and 19K07490) and by the Private University
Research Branding Project from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (MEXT). This work was
supported by a grant from the Education and Research Facilit y of
Animal Models for Human Diseases at the Fujita Health Universit y.
We thank our lab members for the helpful discussions. The authors
declare no conflict of interest.
All experiments were conducted in compliance with the ARRIVE
guidelines.
AUTHORS’ CONTRIBUTIONS
WJS devised the project and the main conceptual ideas, participated
in all experiments, and wrote the manuscript. KK supervised the
work and wrote the manu script. BW, AK , TI , an d SF assisted with ex-
pe r ime nts. HF, YY, AJT, an d KS co n tri b ute d to the manus crip t dis cus-
sion. AM and TN super vised the work and finalized the manuscript.
ORCID
Kazuo Kunisawa https://orcid.org/0000-0002-4786-9681
Akihiro Mouri https://orcid.org/0000-0003-3833-4041
REFERENCES
Badawy, A. A . (2017). Kynurenine Pathway of Tryptophan Metabolism:
Regulatory and Functional Aspects. International Journal of
Tryptophan Research,10, 1178646917691938.
14
|
SUENTO ET al.
Bartel, D. P. (200 4). MicroRNAs: genomic s, biogenesis, mechanism, and
function. Cell, 116, 281–297.
Benton, T., Staab, J., & Evans, D. L . (2007). Medical co-morbidit y in de-
pressive disorders. Annals of Clinical Psychiatry, 19, 289–303.
Bradley, K. A., Case, J. A., Khan, O., Ricart, T., Hanna, A., Alonso, C . M.,
& Gabbay, V. (2015). The role of the kynurenine pathway in suicidal-
ity in adolescent major depressive disorder. Psychiatry Research, 227,
206–212.
Brites, D., & Fernandes, A. (2015). Neuroinflammation and depression:
Microglia activation, extracellular microvesicles and microRNA dys-
regulation. Frontiers in Cellular Neuroscience, 9, 476.
Cai, Y., Yu, X., Hu, S., & Yu, J. (2009). A brief review on the mechanisms
of miRNA regulation. Genomics Proteomics Bioinformatics, 7, 14 7–1 54.
Connor, T. J., Starr, N., O'Sullivan, J. B., & Harkin, A. (2008). Induction
of indolamine 2,3-dioxygenase and kynurenine 3-monooxygenase in
rat brain followin g a sys tem ic inflammatory challen ge: a role fo r IFN-
gamma? Neuroscience Letters, 441, 29–34.
Dantzer, R., O'Connor, J. C., Freund, G. G., Johnson, R. W., & Kelley, K.
W. (2008). From inflammation to sickness and depression: when the
immune system subjugates the brain. Nature Reviews Neuroscience,
9, 46–56.
De Paola, M., Mariani, A., Bigini, P., Peviani, M., Ferrara, G., Molteni,
M., Gemma, S., Veglianese, P., Castellaneta, V., Boldrin, V., Rossetti,
C., Chiabrando, C., Forloni, G., Mennini, T., & Fanelli, R . (2012).
Neuroprotective effec ts of toll-like receptor 4 antagonism in spinal
cord cultures and in a mouse model of motor neuron degeneration.
Molecular Medicine, 18, 971–981.
Deng, Z. F., Zheng, H. L., Chen, J. G., Luo, Y., Xu, J. F., Zhao, G., Lu, J. J.,
Li, H. H., Gao, S. Q., Zhan g, D. Z., & Zh u, L . Q. (2019). miR-214-3p tar-
gets beta-catenin to regulate depressive-like behaviors induced by
chronic social defeat stress in mice. Cerebral Cortex, 29, 1509–1519.
Dwivedi, Y. (2014). Emerging role of microRNAs in major depressive dis-
order: diagnosis and therapeutic implications. Dialogues in Clinical
Neuroscience, 16, 43–61.
Fava, M., & Davidson, K . G. (1996). Def inition and epidemiology of treat-
ment-resistant depression. Psychiatric Clinics of North America, 19,
179 –200.
Fischer, C. W., Eskelund, A., Budac, D. P., Tillmann, S., Liebenberg, N.,
Elfving, B., & Wegener, G. (2015). Inter feron-alpha treatment induces
depression-like behaviour accompanied by elevated hippocampal quin-
olinic acid levels in rats. Behavioural Brain Research, 293, 166–172.
Flint, J., & Kendler, K. S. (2014). The genetics of major depression. Neuron,
81, 484–503.
Fujigaki, H., Yamamoto, Y., & Saito, K. (2017). L-Tryptophan-kynurenine
pathway enzymes are therapeutic target for neuropsychiatric dis-
eases: Focus on cell type differences. Neuropharmacology, 11 2,
26 4–274 .
Han, J., Liu, Z., Wang, N., & Pan, W. (2016). MicroRNA-874 inhibits
growth, induces apoptosis and reverses chemoresistance in col-
orectal cancer by targeting X-linked inhibitor of apoptosis protein.
Oncology Reports, 36, 542–550.
Holmes, S. E., Hinz, R., Conen, S., Gregory, C. J., Matthews, J. C., Anton-
Rodriguez, J. M., Gerhard, A ., & Talbot, P. S. (2018). Elevated translo-
cator protein in anterior cingulate in major depression and a role for
inflammation in suicidal thinking: A positron emission tomography
stud y. Biological Psychiatry, 83, 61–69.
Huber, P. J. (1996) Robust statistic al procedures, Vol. 68: CBMS-NSF
regional conference series in applied mathematic s. Society for
Industrial and Applied Mathematics, .
Iida, R., Yamada, K., Mamiya, T., Saito, K ., Seishima, M., & Nabeshima, T.
(1999). Characterization of learning and memory deficits in C57BL/6
mice infected with LP-BM5, a murine model of AIDS. Journal of
Neuroimmunology, 95, 65–72.
Jackman, K. A., Brait, V. H., Wang, Y., Maghzal, G. J., Ball, H. J., McKenzie,
G., De Silva, T. M., Stocker, R., & Sobey, C. G. (2011). Vascular
expression, activity and function of indoleamine 2,3-dioxygen-
ase-1 following cerebral ischaemia-reperfusion in mice. Naunyn-
Schmiedeberg's Archives of Pharmacology, 383, 471–481.
Jiang, D., Sun, X., Wang, S., & Man, H. (2019). Upregulation of miR-874-3p
decreases cerebral ischemia/reperfusion injury by directly targeting
BMF and BCL2L13. Biomedicine & Pharmacotherapy, 117, 108941.
Jo, W. K., Zhang, Y., Emrich, H. M., & Dietrich, D. E. (2015). Glia in the
cytokine-mediated onset of depression: fine tuning the immune re-
sponse. Frontiers in Cellular Neuroscience, 9, 268.
Karthikeyan, A., Patnala, R., Jadhav, S. P., Eng-Ang, L., & Dheen, S. T.
(2016). MicroRNAs: Key players in microglia and astroc yte mediated
inflammation in CNS pathologies. Current Medicinal Chemistry, 23,
3528–3546.
Krutzfeldt, J., Rajewsky, N., Braich, R ., Rajeev, K. G ., Tuschl, T.,
Manoharan, M., & Stoffel, M. (2005). Silencing of microRNAs in vivo
with 'antagomirs'. Nature, 438, 685–689.
Kunisawa, K., Kido, K., Nakashima, N., Matsukura, T., Nabeshima, T.,
& Hiramat su, M. (2017). Betaine attenuates memory impairment
after water-immersion restr aint stress and is regulated by the
GABAergic neuronal system in the hippoc ampus. European Journal of
Pharmacology, 796, 122–130.
Kunisawa, K., Shimizu, T., Kushima, I., Aleksic, B., Mori, D., Osanai, Y.,
Kobayashi, K., Taylor, A. M., Bhat, M. A., Hayashi, A., Baba, H., Ozaki,
N., & Ikenaka, K. (2018). Dysregulation of schizophrenia-related
aquaporin 3 through disruption of paranode influences neuronal via-
bility. Journal of Neurochemistry, 147, 395–408.
Lawson, M. A., Parrott, J. M., McCusker, R. H., Dant zer, R., Kelley, K. W.,
& O'C onnor, J. C. (2013). Intrac erebrovent ricul ar admini st ratio n of li-
popolysaccharide induces indoleamine-2,3-dioxygenase-dependent
depression-like behaviors. Journal of Neuroinflammation, 10, 87.
Leitner, G. R., Wenzel, T. J., Marshall, N., Gates, E. J., & Klegeris, A. (2019).
Targeting toll-like receptor 4 to modulate neuroinflammation in cen-
tral nervous system disorders. Expert Opinion on Therapeutic Targets,
23, 865–882.
Lo Iacono, L., Ielpo, D., Accoto, A., Di Segni, M., Babicola, L ., D’Addario,
S. L., Ferlazzo, F., Pascucci, T., Ventura, R., & Andolina, D. (2019).
MicroRNA-34a regulates the depression-like behavior in mice
by modulating the expression of target genes in the dorsal raphe.
Molecular Neurobiology, 57, 823–836.
Lopizzo, N., Zonca, V., Cattane, N., Pariante, C. M., & Cattaneo, A.
(2019). miRNAs in depression vulnerability and resilience: Novel
target s for preventive strategies. Journal of Neural Transmission,
126, 1241–1258.
Lou, D., Wang, J., & Wang, X. (2019). miR-124 ameliorates depressive-like
behavior by targeting STAT3 to regulate microglial activation.
Molecular and Cellular Probes, 48, 101470.
Lucassen, P. J., Muller, M. B., Holsboer, F., Bauer, J., Holtrop, A., Wouda, J.,
Hoogendijk, W. J., De Kloet, E. R., & Swaab, D. F. (2001). Hippocampal
apoptosis in major depression is a minor event and absent from sub-
areas at risk for glucocorticoid overexposure. The Americ an Journal of
Pathology, 158, 453–468.
Maes, M., Bosmans, E., De Jongh, R., Kenis, G., Vandoolaeghe, E., &
Neels, H. (1997). Increased serum IL-6 and IL-1 receptor antagonist
concentrations in major depression and treatment resistant depres-
sion. Cytokine, 9, 853–858.
Maes, M., Leonard, B. E., Myint, A. M., Kubera, M., & Verkerk, R. (2011).
The new '5-HT' hypothesis of depression: cell-mediated immune ac-
tivation induces indoleamine 2,3-dioxygenase, which leads to lower
plasma tryptophan and an increased synthesis of detrimental tryp-
tophan catabolites (TRYCATs), both of which contribute to the onset
of depression. Progress in Neuro-Psychopharmacolog y and Biological
Psychiatry, 35, 702–721.
McKernan, D. P., Dinan, T. G., & Cryan, J. F. (2009). "Killing the Blues": A
role for cellular suicide (apoptosis) in depression and the antidepres-
sant response? Progress in Neurobiology, 88, 246–263.
|
15
SUENTO ET al .
McKinnon, M. C ., Yucel, K., Nazarov, A., & MacQueen, G. M. (2009). A
meta-analysis examining clinical predictors of hippocampal volume
in patients with major depressive disorder. Journal of Psychiatry &
Neuroscience, 34, 41–54.
Miwa, M., Tsuboi, M., Noguchi, Y., Enokishima, A., Nabeshima, T., &
Hiramatsu, M. (2011). Effects of betaine on lipopolysaccharide-in-
duced memory impairment in mice and the involvement of GABA
transporter 2. Journal of Neuroinflammation, 8, 153.
Morita, T., Saito, K ., Takemura, M., Maekawa, N., Fujigaki, S., Fujii,
H., Wada, H ., Takeuchi, S., Noma, A., & Seishima, M. (2001).
3-Hydroxyanthranilic acid, an L-tryptophan metabolite, induces
apoptosis in monocyte-derived cells stimulated by interfer-
on-gamma. Annals of Clinical Biochemistry, 38, 242–251.
Mouillet-Richard, S., Baudry, A., Launay, J. M., & Kellermann, O. (2012).
MicroRNAs and depression. Neurobiology of Disease, 46, 272–278.
Mouri, A., Sasaki, A., Watanabe, K., Sogawa, C., Kitayama, S., Mamiya, T.,
Miyamoto, Y., Yamada, K., Noda, Y., & Nabeshima, T. (2012). MAGE-D1
regulates expression of depression-like behavior through serotonin
transporter ubiquitylation. Journal of Neuroscience, 32, 4562–4580.
Mouri, A ., Ukai, M., Uchida, M., Hasegawa, S., Taniguchi, M., Ito, T., Hida,
H., Yoshimi, A., Yamada, K., Kunimoto, S., Ozaki, N., Nabeshima, T.,
& Noda, Y. (2018). Juvenile social defeat stress exposure persistently
impairs social behaviors and neurogenesis. Neuropharmacology, 133,
23 –37.
Murai, R., Noda, Y., Matsui, K., Kamei, H., Mouri, A., Matsuba, K., Nitta,
A., Furukawa, H., & Nabeshima, T. (2007). Hypofunctional glutama-
tergic neurotransmission in the prefrontal cortex is involved in the
emotional deficit induced by repeated treatment with phencycli-
dine in mice: implications for abnormalities of glutamate release and
NMDA-CaMKII signaling. Behavioural Brain Research, 180, 152–160.
Murakami, Y., Ishibashi, T., Tomita, E., Imamura, Y., Tashiro, T.,
Watcharanurak, K., Nishikawa, M., Takahashi, Y., Takakura, Y.,
Mitani, S ., & Fujigaki, H. (2016). Depressive symptoms as a side ef-
fect of Interferon-alpha therapy induced by induction of indoleamine
2,3-dioxygenase 1. Scientific Reports, 6, 29920.
Murray, C. J., & Lopez, A. D. (1996). Evidence-based health policy–les-
sons from the global burden of disease study. Science, 2 74, 740–743.
O'Connor, J. C., L awson, M. A., Andre, C., Briley, E. M., Szegedi, S. S.,
Lestage, J., Castanon, N., Herkenham, M., Dantzer, R., & Kelley, K. W.
(2009). Induction of IDO by bacille Calmette- Guerin is responsible
for development of murine depressive-like behavior. The Journal of
Immunology, 182, 3202–3212.
O'Connor, J. C., Lawson, M. A ., Andre, C., Moreau, M., Lestage,
J., Castanon, N., Kelley, K. W., & Dant zer, R. (2009).
Lipopolysaccharide-induced depressive-like behavior is mediated
by indoleamine 2,3-dioxygenase activation in mice. Molecular
Psychiatry, 14, 511–522.
O'Connor, R. M., Dinan, T. G., & Cryan, J. F. (2012). Little things on which
happiness depends: microRNAs as novel therapeutic targets for
the treatment of anxiety and depression. Molecular Psychiatry, 17,
359–376 .
Otte, C., Gold, S. M., Penninx, B. W., Pariante, C. M., Etkin, A ., Fava, M.,
Mohr, D. C., & Schat zberg, A. F. (2016). Major depressive disorder.
Nature Reviews Disease Primer s, 2, 16065.
Oxenkrug, G. (2013). Serotonin-kynurenine hypothesis of depression:
historical overview and recent developments. Current Drug Targets,
14, 514–521.
Paxinos, G., & Franklin, K. B. J. (2004). The mouse brain in stereotaxic coor-
dinate s. Elsevier Academic Press.
Rolls, A., Shechter, R., London, A., Ziv, Y., Ronen, A., Levy, R., & Schwar tz,
M. (20 07 ). Toll -l ike re ce pt or s mod ul at e ad ul t hi ppo ca mpal ne ur og en -
esis. Nature Cell Biology, 9, 1081–1088 .
Rupaimoole, R., & Slack, F. J. (2017). MicroRNA therapeutics: towards a
new era for the management of cancer and other diseases. Nature
Reviews Drug Discovery, 16, 203–222.
Salazar, A., Gonzalez-Rivera, B. L., Redus, L., Parrott, J. M., & O'Connor,
J. C. (2012). Indoleamine 2,3-dioxygenase mediates anhedonia and
anxiety-like behaviors caused by peripheral lipopolysaccharide im-
mune challenge. Hormones and Behavior, 62, 202–209.
Savitz, J. (2017). Role of kynurenine metabolism pathway activation in
major depressive disorders. Current Topics in Behavioral Neurosciences,
31, 249–267.
Schrat t, G. (2009). Fine-tuning neural gene expression with microRNAs.
Current Opinion in Neurobiology, 19, 2 13–219.
Setiawan, E., Wilson, A. A., Mizrahi, R., Rusjan, P. M., Miler, L., Rajkowska,
G., Suridjan, I., Kennedy, J. L., Rekkas, P. V., Houle, S., & Meyer, J.
H. (2015). Role of transloc ator protein densit y, a marker of neuroin-
flammation, in the brain during major depressive episodes. JAMA
Psychiatry, 72, 268–275.
Shelton, R. C ., Claiborne, J., Sidoryk-Wegrzynowicz, M., Reddy, R.,
Aschner, M., Lewis, D. A., & Mirnics, K. (2011). Altered expression
of genes involved in inflammation and apoptosis in frontal cortex in
major depression. Molecular Psychiatry, 16, 751–762.
Singhal, G., & Baune, B. T. (2017). Microglia: An interface between
the loss of neuroplasticity and depression. Frontiers in Cellular
Neuroscience, 11, 270.
Smith, A. J., Smith, R. A., & Stone, T. W. (2009). 5-Hydroxyanthranilic
acid, a tr yptophan metabolite, generates oxidative stress and neuro-
nal death via p38 activation in cultured cerebellar granule neurones.
Neurotoxicity Research, 15, 303–310.
Stone, T. W., & Darlington, L. G. (2002). Endogenous kynurenines as
target s for drug discovery and development. Nature Reviews Drug
Discover y, 1, 609–620.
Tang, C. Z., Zhang, D. F., Yang, J. T., Liu, Q. H., Wang, Y. R., & Wang, W. S.
(2019). Overexpression of microRNA-301b accelerates hippocampal
microglia activation and cognitive impairment in mice with depres-
sive-like behavior through the NF-kappaB signaling pathway. Cell
Death & Disease, 10, 316.
Tashiro, T., Murakami, Y., Mouri, A., Imamura, Y., Nabeshima, T.,
Yamamoto, Y., & Saito, K. (2017). Kynurenine 3-monooxygenase is
implicated in antidepressants-responsive depressive-like behaviors
and monoaminergic dysfunctions. Behavioral Brain Research, 317,
279–285 .
Treadway, M. T., Waskom, M. L., Dillon, D. G., Holmes, A . J., Park, M. T.
M., Chakravarty, M. M., Dutra, S. J., Polli, F. E., Iosifescu, D. V., Fava,
M., Gabrieli, J. D. E., & Pizzagalli, D. A. (2015). Illness progression,
recent stress, and morphometr y of hippocampal subfields and me-
dial prefrontal cortex in major depression. Biological Psychiatry, 77,
28 5–2 94.
Walter, S., Letiembre, M., Liu, Y., Heine, H., Penke, B., Hao, W., Bode,
B., Manietta, N., Walter, J., Schulz-Schüffer, W., & Fassbender,
K. (2007). Role of the toll-like receptor 4 in neuroinflammation
in Alzheimer's disease. Cellular Physiology and Biochemistry, 20,
947–95 6 .
Wulaer, B., Kunisawa, K., Hada, K., Jaya Suento, W., Kubota, H., Iida, T.,
Kosuge, A., Nagai, T., Yamada, K., Nitta, A., Yamamoto, Y., Saito, K.,
Mouri, A., & Nabeshima, T. (2020). Shati/Nat8l deficiency disrupts
adult neurogenesis and causes attentional impairment through do-
paminergic neuronal dysfunction in the dentate gyrus. Journal of
Neurochemistry, ht tp s://doi.o rg /10.1111 /jnc.150 22
Yamada, K., Iida, R., Miyamoto, Y., Saito, K., Sekikawa, K., Seishima,
M., & Nabeshima, T. (2000). Neurobehavioral alterations in mice
with a targeted deletion of the tumor necrosis factor-alpha gene:
implications for emotional behavior. Journal of Neuroimmunology,
111, 131–138.
Yamawaki, Y., Yoshioka, N., Nozaki, K., Nozaki, K., Ito, H., Oda, K., Harada,
K., Shirawachi, S., Asano, S., Aizawa, H., Yamawaki, S., Kanematsu, T.,
& Akagi, H. (2018). Sodium butyrate abolishes lipopolysaccharide-in-
duced depression-like behaviors and hippocampal microglial activa-
tion in mice. Brain Research, 16 80, 13–38.
16
|
SUENTO ET al.
Zou, W., Feng, R., & Yang, Y. (2018). Changes in the serum levels of in-
flammatory cytokines in antidepressant drug-naive patients with
major depression. PLoS One, 13, e0197267.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Suento WJ, Kunisawa K, Wulaer B, et al.
Prefrontal cortex miR-874-3p prevents lipopolysaccharide-
induced depression-like behavior through inhibition of
indoleamine 2,3-dioxygenase 1 expression in mice. J Neurochem.
2020;00:1–16. https://doi.org/10.1111/jnc.15222
A preview of this full-text is provided by Wiley.
Content available from Journal of Neurochemistry
This content is subject to copyright. Terms and conditions apply.