biomolecules
ArticleIL-6-Mediated Upregulated miRNAs in Extracellular Vesicles
Derived from Lund Human Mesencephalic (LUHMES) Cells:
Effects on Astrocytes and Microglia
Kento Nishi 1,2 , Hiroto Izumi 1,3, * , Taisuke Tomonaga 3 , Chikage Nagano 1,2 , Yasuo Morimoto 1,3
and Seichi Horie 1,2
1

2

3

*

Citation: Nishi, K.; Izumi, H.;
Tomonaga, T.; Nagano, C.; Morimoto,Y.; Horie, S. IL-6-MediatedUpregulated miRNAs in ExtracellularCenter for Stress-Related Disease Control and Prevention, University of Occupational and Environmental
Health, Kitakyushu 807-8555, Japan
Department of Health Policy and Management, Institute of Industrial Ecological Sciences,
University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan
Department of Occupational Pneumology, Institute of Industrial Ecological Sciences,
University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan
Correspondence: h-izumi@med.uoeh-u.ac.jp

Abstract: Psychological stress plays a major role in depression, and interleukin-6 (IL-6) is elevatedduring depression and psychological stress. MicroRNAs (miRNAs) in extracellular vesicles (EVs),
including exosomes and microvesicles, suppress mRNA expression in other cells when endocytosed.
In this study, we analyzed the effect of IL-6 on EVs secreted by neural precursor cells. Cells fromthe human immortalized neural precursor cell line LUHMES were treated with IL-6. EVs werecollected using a nanofiltration method. We then analyzed the uptake of LUHMES-derived EVs
by astrocytes (ACs) and microglia (MG). Microarray analysis of miRNAs was performed using
EV-incorporated RNA and intracellular RNA from ACs and MG to search for increased numbers ofmiRNAs. We applied the miRNAs to ACs and MG, and examined the cells for suppressed mRNAs.
IL-6 increased several miRNAs in the EVs. Three of these miRNAs were originally low in ACs andMG (hsa-miR-135a-3p, hsa-miR-6790-3p, and hsa-miR-11399). In ACs and MG, hsa-miR-6790-3p andhsa-miR-11399 suppressed four mRNAs involved in nerve regeneration (NREP, KCTD12, LLPH, and
CTNND1). IL-6 altered the types of miRNAs in EVs derived from neural precursor cells, by which
mRNAs involved in nerve regeneration were decreased in ACs and MG. These findings provide new
insights into the involvement of IL-6 in stress and depression.

Vesicles Derived from Lund Human
Mesencephalic (LUHMES) Cells:
Effects on Astrocytes and Microglia.
Biomolecules 2023, 13, 718. https://

Keywords: IL-6; microRNA; extracellular vesicles; neural precursor cells; astrocyte; microglia;
stress; depression

doi.org/10.3390/biom13050718
Academic Editor: Nela Pivac
Received: 6 March 2023
Revised: 7 April 2023
Accepted: 19 April 2023
Published: 22 April 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
The global prevalence of depression and depressive symptoms has increased in recent
decades, with the prevalence of the disorder ranging from 20% to 25% in women and
7% to 12% in men [1,2]. The main diagnostic criteria for depression are based on the
Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, but these criteria areepisodic and tend to be subjective in their assessment [3]. In recent years, attention has been
focused on the theory that stress induces an inflammatory response that leads to changes
in the neural structure as part of the pathophysiology of depression [4]. The number of
glial cells in the prefrontal cortex is markedly reduced in depressed patients [5]. Amongglial cells, astrocytes (ACs) form contacts with many synapses and regulate neuronal
activity through the release of glial transmitters such as adenosine triphosphate [6]. In
postmortem tissues of depressed patients, gene expression is markedly decreased in ACs [7],and proinflammatory cytokines elicited by microglia (MG) have been implicated in thedevelopment of depression [8].

Biomolecules 2023, 13, 718. https://doi.org/10.3390/biom13050718

https://www.mdpi.com/journal/biomoleculesBiomolecules 2023, 13, 718

2 of 16

IL-6 is a type of proinflammatory cytokine that is prominently elevated during infectious diseases, trauma, and autoimmune diseases and is a marker of the inflammatory
response [9]. IL-6 is produced by brown adipose tissue in response to sympathetic nerve
stimulation [10], which is independent of adrenal corticosteroids and is therefore also
attracting attention as a stress biomarker [8,11]. It is also elevated during psychiatricdisorders such as depression [12,13].
Extracellular vesicles (EVs) are secreted (exosomes, microvesicles) or formed (apoptotic vesicles) from various cells and different EV types can be distinguished by their size,
content, and mechanism of production [14]. EVs contain proteins and nucleic acids, such as
microRNA (miRNA) and mRNA, and enter cells predominantly via clathrin-independentendocytosis and macropinocytosis [15–17]. This suggests that EVs play a role in the transmission of information to distant cells. miRNAs are noncoding RNAs of 20–25 nucleotidesin length that bind to mRNAs and suppress gene expression by degrading mRNAs or
inhibiting their translation [14]. Many studies have focused on the miRNAs present in
EVs and analyzed their qualitative and quantitative changes [18,19]. For example, astrocytes stimulated by IL-1β or TNF-α were reported to release EVs containing miRNAs(miR-125a-5p and miR-16-5p) that are involved in neuronal survival, differentiation, andneurite outgrowth [20]. This suggests that the miRNAs in EVs are involved in neuronal
function. As mentioned above, IL-6 is elevated during chronic stress and may affect theseneurons. However, it remains unclear how IL-6 affects EV secretion and the qualitativeand quantitative responses of EVs to it. In this study, we used a nanofiltration method toisolate EVs secreted from cultured neural precursor cells and analyzed the quantitative andqualitative responses of miRNAs to IL-6.
2. Materials and Methods
2.1. Cell Culture
An immortalized human dopaminergic neuronal precursor cell line (LUHMES cells),
immortalized human microglia (IHMG), and immortalized human astrocytes (IHACs)
were purchased from Applied Biological Materials Inc. (Richmond, BC, Canada). LUHMES
cells, IHACs, and IHMG were maintained in DMEM/F12 medium, Prigrow IV medium,and Prigrow III medium, respectively. All cells were cultured in medium with 10% heatinactivated fetal bovine serum (FBS) and 1% (v/w) penicillin/streptomycin, and weremaintained at 37 ◦ C with 5% CO2 . IL-6 (HZ-1019) was purchased from Proteintech Group,
Inc. (Rosemont, IL, USA). Cells were treated with IL-6 at 0.1 or 1 µg/mL and collected
24 h later.
2.2. Preparation of Nluc-Fused CD81 Plasmids and Their Expression
The construction of nanoluciferase (Nluc)-fused CD81 plasmids that co-express GFP
and puromycin was performed as described previously [21]. Nonproliferative lentivirus was
packaged using the System Biosciences lentivirus packaging system. Briefly, nanoluciferase(Nluc)-fused CD81 plasmids were mixed with pPACKH1 plasmid mix (pPACKH1-GAG,
pPACKh1-REV, pVSV-G) and transfected into COS1 cells using X-tremeGENE™ 9 DNA
Transfection Reagent (Roche Molecular Biochemicals, Penzberg, Germany). After 36 h,
the culture medium was filtered through a 0.45 µm syringe filter and added directly
to the LUHMES cells. Infected cells were cultured with 10 µg/mL puromycin (ant-pr;
InvivoGen, San Diego, CA, USA) for more than 2 weeks to establish CD81-Nluc-expressing
LUHMES cells.
2.3. Fractionation and Isolation of EVs in Cell Culture Medium Using Nanofiltration
Isolation of EVs using a syringe filter was performed as described previously [17]. Syringe nanofilters of 220 nm (SFPES013022N; Membrane Solutions), 100 nm (SFPES013010N;
Membrane Solutions), 50 nm (SF16008; TISCH Scientific), and 20 nm (ANPEL Laboratory
Technologies, Shanghai, China) were used in this study. Figure 1 shows a schematic ofsample preparation. F and C in front of the numbers indicate fresh medium and culture2.3. Fractionation and Isolation of EVs in Cell Culture Medium using Nanofiltration

Biomolecules 2023, 13, 718

Isolation of EVs using a syringe filter was performed as described previously [17].
Syringe nanofilters of 220 nm (SFPES013022N; Membrane Solutions), 100 nm
16(SFPES013010N; Membrane Solutions), 50 nm (SF16008; TISCH Scientific), and 203 of
nm(ANPEL Laboratory Technologies, Shanghai, China) were used in this study. Figure 1
shows a schematic of sample preparation. F and C in front of the numbers indicate fresh
mediumand culture
supernatant,respectively.
in allwas
cellfilteredcultureat
media
was(F220)
filteredsupernatant,respectively.
FBS in all
cell cultureFBS
media
220 nmorat(F220)or 20 visible
nm (F20)
to removevisiblebodies
solids derived
and apoptotic
bodies20 220nm nm(F20)
to removesolidsand apoptotic
from theFBS. derived
In somefromthe FBS. F220
In someexperiments,
F220
mediumwasF20used,
while wasin others
experiments,
mediumwas used,while
in others
mediumused.F20
Themedium
methodwasused. Themethod ofpreparation
is describedin detaileach analysis.
As aof sample
preparation
is sample
described
in detail foreach analysis.
Asfora commontechnique,commontechnique,when passing
mediumthroughsyringefilterof of
100onenmdropor less,
when passing
the medium
throughthea syringe
filterof 100 anm
or less,a rateperasecondrate ofwas
onestrictly
drop persecond was strictly employed.
employed.

Figure 1.
1. Preparation
Preparation of
ofmedia
mediausedusedininthisthisexperimentexperimentand
andsamples
samplesusedusedininthetheassay.
assay. This
This researchresearch
Figuremainly consisted
consistedof
offourfourmethods
methods(A–D).
(A–D).(A)(A,B)
the sample
preparation
for for
ELISA
analysis,
mainlyandshow(B) showthe sample
preparation
ELISA
analysis,(C)forthat
for activity
Nluc activity
analysis,and(D)foranalysis.
RNA analysis.
F220
F20 indicate
fresh(C) thatNluc
analysis,and (D)thatforthat
RNA
F220 andF20andindicate
fresh medium
mediumpassed through220nm
andfilters,
20 nmrespectively.
filters, respectively.
C220 is medium
filteredat 220after
passed through
220 and 20
C220 is medium
filtered at
220 nmafternm
culture.
culture.
F220
>
C220
and
F20
>
C220
refer
to
samples
obtainedbyculturing
cells
in
F220
or
F20
F220 > C220 and F20 > C220 refer to samples obtained by culturing cells in F220 or F20 medium,medium, respectively, and subsequently filtering the cell medium using a 220 nm syringe filter.
respectively, and subsequently filtering the cell medium using a 220 nm syringe filter. Adjusted
Adjusted samples were used in the experiments as indicated by the numbers in each figure.
samples were used in the experiments as indicated by the numbers in each figure.

2.4.
2.4. ELISAELISA Analysis
Analysis with
withAnti-CD9,
Anti-CD9,Anti-CD63,
Anti-CD63,andandAnti-CD81
Anti-CD81Antibodies
Antibodies
The
The indicatedindicated numbers
numbers ofof LUHMESLUHMES cells,
cells,ACs,
ACs,andandMG
MGwerewereseeded
seededininaaplateplatedish.
dish.
The
IL-6
was
added
andandthe themediumwas wascollected
24 h later.
The
The next
nextday,
day,11oror1010pg/mL
pg/mL
IL-6
was
added
mediumcollected
24 h later.
collected
mediumwaswaspassedthrougha 220
nmnm
filterandthen
The collected
mediumpassedthrougha 220
filterandthenanalyzed
analyzedimmediately.
immediately.
Tetraspanins
inbothbothfresh
fresh
mediumculturesupernatantquantified
anTetraspanins in
mediumandandculturesupernatantwerewerequantified
using using
an ELISA
ELISA
kit(296-83701
forCD9,
290-83601
for
CD63,
and
292-83801
for
CD81;
Wako,
Osaka,
kit (296-83701 for CD9, 290-83601 for CD63, and 292-83801 for CD81; Wako, Osaka, Japan).
Japan).
A sample50 µL
or 100wasdependingused, depending
the amountof tetraA sample
volumevolumeof 50 orof100
wasµL
used,
on the onamountof tetraspanins
spaninsin the sample
the sensitivityof the antibody.
Reporterlysis buﬀer
was addedin the sample
and theandsensitivityof the antibody.
Reporterlysis buffer
was added
to the
cells
thein
dish
was transferred
to a tube.
ThisThis
tubetubewaswasvortexed
fortotheincells
theanddisheachand sample
each sample
was transferred
to a tube.
vortexed
20 s20andcentrifuged
at 10,000
× g gfor
was
for
s and
centrifuged
at 10,000×for22min.
min.AnAnappropriate
appropriate amountamount of supernatant wastransferred totoaa96-well
96-wellwhite
whiteplate
plateand
and
mixed
with
equal
volumeof CellTiter-Glotransferred
mixed
with
an an
equal
volumeof CellTiter-Glo
2.0
2.0
Cell
Viability
Assay(Promega,
Madison,WI,
USA)
reagent.
After
5
min,
ATP
activity
Cell Viability Assay (Promega, Madison, WI, USA) reagent. After 5 min, ATP activity was
was measured
a luminometer(Luminescencer
JNII RAB-2300;
ATTO,
Tokyo,
Japan).
measuredusingusing
a luminometer(Luminescencer
JNII RAB-2300;
ATTO,
Tokyo,
Japan).
The
The values
obtained
via ELISA
normalized
the valuesof cellular
ATP activity
valuesobtained
via ELISA
were werenormalizedusingusing
the valuesof cellular
ATP activity
as an
as an internal
standard.
internal
standard.
2.5. Western
WesternBlot
BlotAnalysis
Analysiswith
withImmunoprecipitation
Immunoprecipitation
2.5.
Each cell
cell line
linewaswassonicatedsonicatedininlysis
lysisbuﬀer
bufferconsisting
consistingofofmM
Tris-HCl
(pH
8.0),
Each
5050
mM
Tris-HCl
(pH
8.0),
1
1
mM
EDTA,
120
mM
NaCl,
0.5%
Nonidet
P-40,
and
0.5
mM
PMSF,
and
centrifugedatmM EDTA, 120 mM NaCl,
0.5% Nonidet P-40, and 0.5 mM PMSF, and centrifuged at
21,000× g for 10 min at 4 ◦ C. Proteins (1 mg) were immunoprecipitated with 5 µg of antiIL-6R antibody (ab222101, Lot GR34013 9-1; Abcam, Cambridge, UK) or rabbit IgG for 4 h
at 4 ◦ C. Immune complexes were isolated by incubation with 10 µL of Protein A/G PLUSAgarose (sc-2003; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 4 ◦ C. Protein
A/G-agarose pellets were washed twice with 1 mL of lysis buffer. Each sample was subjected to SDS-PAGE and then transferred to a PVDF membrane. IL-6R was detected using

Biomolecules 2023, 13, 718

4 of 16anti-IL-6R antibody (ab222101, 1/1000 dilution) in PBS-T (Takara, Tokyo, Japan) containing
1% BSA Fraction V (Rockland Immunochemicals, Limerick, PA, USA) overnight at 4 ◦ C.
Bound secondary anti-rabbit IgG, HRP-Linked Whole Ab Donkey (NA934; Cytiva Life
Sciences, Marlborough, MA, USA), was visualized using an enhanced chemiluminescencekit (GE Healthcare Bio-Sciences, Marlborough, MA, USA).
2.6. Uptake of EVs and Nluc Activity Assay
Samples for Nluc activity measurement were prepared using two methods.
(1) LUHMES cells expressing CD81 were digested with trypsin. After neutralization
with medium, cells were washed twice with PBS and then resuspended in PBS. Suspended
cells were passed through 220, 100, 50, and 20 nm syringe filters, and Nluc activity after
each filtration was measured. The cell suspension before passing through a filter andthe supernatant obtained by centrifugation (2000× g for 2 min) were also analyzed. The
measurements were performed by mixing 100 µL of each sample with 100 µL of NanoGlo Luciferase Assay System (Promega) reagent. Nluc activity was measured using aluminometer. (2) The culture supernatant of LUHMES cells expressing CD81-Nluc waspassed through a 220 nm syringe filter, and IHACs and IHMG were cultured in this medium
for the indicated times. Cells were washed twice with PBS and lysed in reporter lysis buffer.
After collection, the lysate was vortexed (20 s) and then centrifuged (12,000 rpm for 2 min).
The supernatant (100 µL) was mixed with Nano-Glo Luciferase Assay System reagent
and light intensity was measured using a luminometer. To prepare a background sample,
after adding the culture supernatant containing CD81, cells were immediately washedand lysed as described above. The supernatant (20 µL) was also mixed with CellTiter-Glo
2.0 Cell Viability Assay reagent and light intensity was measured with a luminometer.
The value of Nluc activity was normalized using the level of cellular ATP activity as aninternal standard.
2.7. Reverse Transcription and Quantitative Real-Time PCR Analysis (qRT-PCR)
The indicated numbers of LUHMES cells, ACs, and MG were seeded in a plate
dish. The next day, 1 or 10 pg/mL IL-6 was added and cells were collected 24 h later.
Total RNA including miRNA was purified with an miRNeasy Kit (217084; Qiagen, Venlo,
the Netherlands). qRT-PCR assay was then performed as described previously [22], forwhich the primer sets were as follows: Hs00233521_m1 for CD9, Hs01041238_g1 for CD63,
Hs01002167_m1 for CD81, Hs01075664_m1 for IL-6R, and Hs01060665_g1 for β-actin(Applied Biosystems, Foster City, CA, USA). Values were normalized to those of human
β-actin. The comparative cycle time method was used to quantify gene expression. All
samples were analyzed in duplicate in each experiment.
2.8. miRNA Extraction
The indicated numbers of LUHMES cells, ACs, and MG were seeded in a plate dish.
The next day, 1 or 10 pg/mL IL-6 was added and the medium was collected 24 h later.
miRNA extraction was performed as described previously [21]. Briefly, total RNA of EVs
captured with a 20 nm filter was eluted with ISOGEN (Nippon Gene, Tokyo, Japan) andpurified using a NucleoSpin miRNA Plasma Kit (Macherey-Nagel, Düren, Germany).
2.9. miRNA Transfection
ACs and MG were transfected with the following miRNAs: hsa-miR-135-3p, hsa-miR6790-3p, hsa-miR-11399, and control miRNA (Invitrogen, Carlsbad, CA, USA). Briefly, each
25 pmol miRNA was mixed with 7.5 µL Lipofectamine RNAiMAX (Invitrogen) in 100 µL
Opti-MEM. The miRNA complexes were applied to 2 × 105 cells. After 24 h, cells werecollected and miRNA microarray analysis was performed.

Biomolecules 2023, 13, 718

5 of 16

2.10. Microarray Analysis
Microarray analysis of miRNAs has been described previously [17]. Briefly, a Human
miRNA Oligo chip-4 plex (TORAY, Tokyo, Japan) containing 2565 probe sets was used.
Microarray analysis of mRNAs has been described previously [23]. Briefly, a Human Oligo
chip 25k set (TORAY) containing 24,460 probe sets was used. Each microRNA and mRNA
was normalized using the global normalization method, which adjusted the median of thedetected signal intensity to 25.
2.11. Statistical Analysis
Statistical analysis was performed using JMP pro 15 (SAS Institute Inc., Cary, NC,
USA). The results were compared using Tukey’s range test. Data are expressed as themean ± SD. p < 0.05 was considered to indicate a statistically significant difference.
3. Results
3.1. Purification of EVs Using Nanofiltration
In our previous study, EVs fractionated using nanofiltration using a 50 nm syringe
filter were analyzed, with the following results being obtained [21]. The EVs were spherical,
as determined using transmission electron microscopy (TEM), with a diameter of approximately 170 nm, as determined using dynamic light scattering (DLS). Furthermore, theEVs contained CD9, CD63, CD81, and EpCAM proteins, as determined using Western
blotting, and contained miRNAs of less than 30 bp in length, as revealed by an Agilent
2100 Bioanalyzer. EVs captured with a 50 nm syringe filter could be collected by reverse
flow, but EVs captured with a 20 nm syringe filter could not. Therefore, we are unable toshow the analytical results for EVs captured with a 20 nm syringe filter, but expect similar
results to those captured with a 50 nm syringe filter.
In accordance with the method described in Figure 1, F220 > C220, F20 > C220, F220,
and F20 samples were prepared, and CD9, CD63, and CD81 protein levels in each sample
were measured (Figure 2A). CD9, CD63, and CD81 were all found in the F220 > C220
and F20 > C220 samples, whereas almost no expression was observed in the F220 and F20
samples. These results suggest that the antibody used in this study recognized humantetraspanins, but not bovine tetraspanins, or that there were very few EVs derived frombovine serum.
Next, we examined whether nanofiltration damaged EVs. CD81-Nluc-expressing
cells were suspended in PBS and Nluc activities in PBS passed through 220, 100, 50, and20 nm syringe filters were measured. Since Nluc is released outside the cells when the cells
are damaged during passage through the syringe filter, cell damage can be confirmed bymeasuring the Nluc activity of the sample after passage. As shown in Figure 2B, no Nluc
activity was observed in any of the PBS samples, even when the smallest filter (20 nm)
was used. These results suggest that the cells suffered little damage when passaged at a
rate of less than two drops per second, as described in the Methods section. Therefore,
it was considered that EVs, which are much smaller than cells, were hardly damaged bypressurization when passing through the filter.
3.2. The Expression of Tetraspanin Proteins and mRNAs
The expression of tetraspanin proteins in media of LUHMES cells, ACs, and MG
was measured using ELISA (Figure 3A), and the expression of each tetraspanin mRNAin cells was analyzed using real-time PCR (Figure 3B). There was no correlation between
tetraspanin protein expression in the medium and cellular mRNA expression in each cell.
These analyses confirmed that all cells expressed three tetraspanins.

Biomolecules 2023, 13, 718

Biomolecules 2023, 13, 718

In accordance with the method described in Figure 1, F220 > C220, F20 > C220, F220,
and F20 samples were prepared, and CD9, CD63, and CD81 protein levels in each sample
were measured (Figure 2A). CD9, CD63, and CD81 were all found in the F220 > C220 and
F20 > C220 samples, whereas almost no expression was observed in the F220 and F20 samples. These results suggest that the antibody used in this study recognized human tetra6 of 16
spanins, but not bovine tetraspanins, or that there were very few EVs derived from bovine
serum.

7 of 1720 nm syringe filters were measured. Since Nluc is released outside the cells when thecells are damaged during passage through the syringe filter, cell damage can be confirmedby measuring the Nluc activity of the sample after passage. As shown in Figure 2B, no
Nluc activity was observed in any of the PBS samples, even when the smallest filter (20nm) was used. These results suggest that the cells suﬀered little
damage when passaged
Figure 2.
2. Evaluation
Evaluation ofof EVsEVs usingusing nanofiltration. (A)A
1055 cells
cells were
were seeded
seeded in aa six−well
FigureA totaltotal ofof44×
10
six−wellata rate ofless than two
drops nanofiltration.
per second, as(A)described
in ×theMethods
section.inTherefore,
plate. The
The next
next day,day, cells were washed with PBS
PBS and supplemented with 1 mL of F220 or F20. After
After
plate.
it was considered that EVs, which are much smaller than cells, were hardly damaged by24
24 h,h, culture
culture medium
medium waswas collectedcollected andand passed
passed throughthrough aa 220 nm filter. Each
Each tetraspanin
tetraspanin in
in thethepressurization
when passing
through
the filter.
medium was measured
using ELISA
and eachvalue was normalized using the cellular ATP activity

medium was measured using ELISA and each value was normalized using the cellular ATP activityas
as an
an internalinternal standard.
standard. Data
Data were
were obtained
obtained fromfrom three
three independent
independent experiments
experiments (n(n == 3),3), ****pp<<0.01.
0.01.
4 LUHMES cells expressing CD81−Nluc
3.2.
The
ExpressionofTetraspanin
Proteins
and
mRNAs
Error
bars
represent
standard
deviation.
(B)
A
total
of
1
×
10
Error bars represent standard deviation. (B) A total of 1 × 104 LUHMES cells expressing CD81−Nlucwere The
prepared.
Luciferase
activity proteinsin flow−through(FT)
was measured
afterandfiltrationofexpressionof tetraspanin
in media
ofmeasured
LUHMES
cells,
ACs,MG
waswere prepared.
Luciferase
activity in flow
−through(FT) wasafter
filtrationof CD81−Nluc.
CD81−Nluc.
The
filter
pores
were
20,
50,
100
and
220
nm
in
diameter.
Cellsuspensions
were
measmeasured
using
ELISA
(Figuretheexpressionof each tetraspaninmRNA
in intactcells
The filter pores
were
20, 50,100 and3A),
220 andnm indiameter.
Cell suspensions
were measured
withured with intact cells prior to filtration. Cell suspensions were centrifuged at 800× g for 5 min andwasanalyzedusing
real-time
PCR(Figure
3B).
There
was
no
correlation
betweentetracells prior towas
filtration.
Cell suspensions
were
centrifuged
at 800independent× g for 5 minexperiments
and supernatantwassupernatantalso measured.
Data wereobtainedfrom three(n = 3),**
spaninproteinData
expression
in thefrom
mediumand cellularexperiments
mRNA expressionin< eachcell.
alsomeasured.
wereobtainedthree
independent
(n
=
3),
**
p
0.01.
Error
p < 0.01. Error bars represent standard deviation.
These
analysesconfirmedthat all cells expressed three tetraspanins.
bars represent
standard
deviation.

Next, we examined whether nanofiltration damaged EVs. CD81-Nluc-expressing
cells were suspended in PBS and Nluc activities in PBS passed through 220, 100, 50, andFigure 3.
3. Expression
Expression analysis
analysis ofof tetraspaninstetraspanins associatedassociated with
with EVs.
(A) A
A totaltotal of1055 cells
cells were
were
FigureEVs. (A)of 22 ×× 10
seededininaa12-well
12-wellplate.
plate.The
Thenext
nextday,
day,thethecells
cellswere
werewashedwashedwith
withPBS
PBSandandsupplemented
supplementedwith
with1
seededmL of F220. After 24 h, culture medium was collected and passed through a 220 nm filter. Eachtetraspanin in the medium was measured using ELISA and each value was normalized using thecellular ATP activity as an internal standard. Data were obtained from three independent experiments (n = 3), ** p < 0.01. Error bars represent standard deviation. (B) A total of 1 × 106 cells wereseeded into a 60 mm dish. The next day, the cells were washed with PBS and supplemented with 3
mL of F20. After 24 h, the cells were collected and total RNA was purified. The expression levels of

Biomolecules 2023, 13, 718

7 of 16

1 mL of F220. After 24 h, culture medium was collected and passed through a 220 nm filter. Eachtetraspanin in the medium was measured using ELISA and each value was normalized using thecellular ATP activity as an internal standard. Data were obtained from three independent experiments(n = 3), ** p < 0.01. Error bars represent standard deviation. (B) A total of 1 × 106 cells were seeded
into a 60 mm dish. The next day, the cells were washed with PBS and supplemented with 3 mL
of F20. After 24 h, the cells were collected and total RNA was purified. The expression levels ofeach tetraspanin in each cell type were analyzed using qRT-PCR. Data were obtained from three
independent experiments (n = 3), ** p < 0.01. Error bars represent standard deviation.

3.3. Uptake of LUHMES-Derived EVs by Astrocytes (ACs) and Microglia (MG)EVs derived from cells are taken up by other cells and their contents, including
proteins and RNAs, are transferred into the cells and function there. Culture medium of
LUHMES (neural progenitor) cells expressing CD81-Nluc was added to ACs and MG, andthe uptake of EVs containing CD81-Nluc in each cell was examined. Before uptake analysis,
we examined whether the expression of each endogenous tetraspanin was altered by theexpression of CD81-Nluc. As shown in Figure 4A, the expression of each tetraspanin in
the EVs was not significantly different after expression of the CD81-Nluc protein. Thissuggested that the amount of CD81 in EVs was controlled, and EVs expressing CD81-Nlucwere expected to be taken up similarly to normal EVs. Note that the Nluc activity shown in
Figure 4B and 4C does not indicate simply the degree of uptake, but rather the combinationof uptake and degradation/recycling of EVs. Time course analysis revealed that Nluc
activity in both ACs and MG peaked at 4 h and was stable after 8 h (Figure 4B). These
results suggest that uptake and decomposition after 8 h were in equilibrium. Because wefound stable Nluc activity after 8 h, the uptake of EVs derived from LUHMES cells wascompared between ACs and MG at 8 h. As shown in Figure 4C, EV uptake by ACs washigher than that by MG. This suggests that ACs receive more information from LUHMES
cells via EVs than MG do.
3.4. Effect of IL-6 on EV Secretion and Uptake
IL-6 is well known to be induced by stress. We thus first evaluated the expression
level of IL-6R in LUHMES cells, ACs, and MG. Since no antibody specific to the IL-6R
protein was available, immunoprecipitation was performed with an anti-IL-6 antibody
and Western blotting was performed with the same antibody. As shown in Figure 5A, weobserved three signals and found that the middle major signal and upper weak signal were
derived from IL-6R and a lower signal was nonspecific. All three cell types expressed theIL-6R protein. To compare IL-6R expression among the cell types, mRNA was quantified
by qRT-PCR. IL-6R mRNA expression was highest in MG and lowest in LUHMES cells.
Next, we analyzed whether IL-6 affected the secretion of LUHMES-cell-derived EVs andtheir uptake into ACs and MG. The concentration of IL-6 selected for administration inthis experiment was based on a meta-analysis [12]. We considered that 0 pg/mL was nota physiological state because healthy people also have about 1 pg/mL IL-6 (up to about
5 pg/mL). Meanwhile, IL-6 tends to be higher in depressed individuals than in healthy
subjects, but it was expected that it would rarely exceed 10 pg/mL. Based on these results,
1 pg/mL IL-6 was set for the control group. As shown in Figure 5B,C, the expression oftetraspanin proteins in EVs from LUHMES cells and the cellular expression of tetraspanin
mRNA in LUHMES cells were not affected by IL-6. Additionally, analysis of EV uptake
showed no changes of Nluc activity in ACs and MG (Figure 5D). These results suggest that
IL-6 is not involved in the degree of EV secretion and uptake.
3.5. IL-6 Affects the Amounts of miRNAs in EVs
Because EV secretion and uptake were not affected by IL-6, we focused on the expression ratio of each miRNA in the EVs. LUHMES cells were treated with 1 or 10 pg/mL
IL-6 and then EVs in the culture supernatant were fractioned using nanofiltration. Total
RNAs were eluted from the filters, and microarray analysis was performed. We found that

Biomolecules 2023, 13, 718

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Biomolecules 2023, 13, 718

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were expected to be taken up similarly to normal EVs. Note that the Nluc activity shownin Figure 4B and 4C does not indicate simply the degree of uptake, but rather the combinationmiRNAsof uptakeandincreased
degradation/recyclingof identify
EVs. Timecourse analysis
that
many
were
or decreased. To
significant
miRNAs,revealed
we selected
Nluc activity
infollowing
both ACs criteria:
and MG(1)peakedat 4 h andwas stable after 8 EVs
h (Figure
4B). Thesethem
using theexpression
in LUHMES-derived
was >100
whenresultssuggestthat applied;
uptake anddecomposition
after
8 hpg/mL
were inwas
equilibrium.
we
10
pg/mL
IL-6 was(2) expressionof IL-6at 10
more thanBecause
twice thatat
1 pg/mL;
expression
ACs
and
MG
was <100.
miRNAsidentified,foundstable(3)Nlucactivity in
after
8 h,
the
uptakeof EVsFour
derivedfromwere
LUHMES
cells andwas
threemiRNAs(hsa-miR-135a-3p,
hsa-miR-6790-3p,
hsa-miR-11399)
in EVstreatedcompared
betweenACs and MG at
8 h. As shown inand
Figure4C, EV uptake
by ACs
was
with
10
pg/mL
IL-6
were
significantly
increasedcompared
with
those
in
EVs
treatedwithhigher than that by MG. This suggests that ACs receive more information from LUHMES
1cellspg/mL(Table1).MGInterestingly,
these miRNAs were not very high in ACs or MG.
via EVsthando.

5 cells were seeded
FigureFigure 4.
4. UptakeUptake ofof LUHMES-derived
LUHMES-derivedEVs
EVsby
byACs
ACsandandMG.
MG.(A)(A)AAtotal
totalofof22×× 10
105 cells
were seededinin aa 12-well
12-well plate.
plate. The
The next
next day,day, thethe cells
cells were
were washedwashed with
with PBS
PBS andand supplemented
supplemented with
with 11 mL
mL of
of
F20.
F20. After
After 2424 h,h, culture
culture medium
medium waswas collectedcollected andand passed
passed throughthrough aa 220
220 nmnm filter.
filter. Each
Each tetraspanin
tetraspanininin thethe media
media ofof CD81-Nluc
CD81-Nluc transgenictransgenic LUHMESLUHMES andand wild-typewild-type LUHMESLUHMES cells
cells was
was measured
measured using
using
ELISA. These
Thesevalues
valueswere
werenormalized
normalizedusingcellularactivityof eachcell. Data
obELISA.
using
thethecellular
ATPATP
activityof eachcell. Data
were wereobtainedtainedfromthree
independent
experiments
(n
=
3),
*
p
<
0.05,
**
p
<
0.01.
Error
bars
represent
standfrom three independent experiments (n = 3). Error bars represent standard deviation. (B) A total
wereseededa 12-welland cultured
withmediumard2 deviation.
A totalof 2into
× 10a5 cellsof× 105 cells(B)wereseeded12-wellplate
andintocultured
withplate
mediumcontaining
LUHMEScontaining LUHMES-derived CD81-Nluc expressed in EVs for the indicated time. The backgroundderived CD81-Nluc expressed in EVs for the indicated time. The background was subtracted fromwas subtracted from each value of Nluc activity and the result was normalized using cellular ATP
each value of Nluc activity and the result was normalized using cellular ATP activity as an internal
activity as an internal standard. The value after 8 h was set to 1 for each cell. Data were obtained
standard.
value after experiments
8 h was set to(n1 =forDataobtainedfrom represent
three independent
from threeThe
independent3),each* p <cell.
0.05,** pwere< 0.01.
Error bars
standard
experiments= 3). Error
bars represent CD81-Nlucstandard deviation.
Uptakeof LUHMES-derived
deviation. (C)(nUptakeof LUHMES-derived
expressed(C)in EVsin eachcell was quantified
CD81-Nluc
expressed in from
EVs incell ofwasquantified
8h
after administrationfrom the resultsof
8 h after administration
theeachresults(B).
Data wereobtainedfrom three independent
experi(B).
Dataexperiments(n = 3),* p < 0.05, ** p < 0.01. Error bars
ments
(n were= 3), *obtained
p < 0.05, from** p <three
0.01. independent
Error bars represent
standard
deviation.
represent standard deviation.

Biomolecules
Biomolecules2023,
2023,13,
13,718718

10 9ofof17
16

Figure5.5.Eﬀects
Effectsof
ofIL-6IL-6onontetraspanin
tetraspaninexpression
expressionand
anduptakeuptakeofof EVs.
EVs.(A)(A)IL-6R
IL-6Rexpression
expressionin
ineacheach
Figurecell
cellwaswasanalyzed
analyzedusing
usingWestern
Westernblotting
blottingand
andqRT-PCR.
qRT-PCR. Each
Each cell
cell lysate
lysate waswas immunoprecipitatedimmunoprecipitatedwithwith anti-IL-6R
anti-IL-6Rororanti-IgG
anti-IgGantibodies
antibodiesand
andimmunoblotted
immunoblottedwith
withanti-IL-6R
anti-IL-6R((left,middle)((left,middle)panels).
panels).
Expression
levels
of
IL-6R
in
each
cell
were
analyzed
using
qRT-PCR((right)
panel).
DataData
werewereobExpression levels of IL-6R in each cell were analyzed using qRT-PCR ((right) panel).
tained from three independent experiments (n = 3), * p < 0.05, ** p < 0.01. Error bars represent standobtained from three independent experiments (n = 3), ** p < 0.01. Error bars represent standard
ard deviation. (B) A total of 2 ×5 105 cells were seeded into a 12-well plate. The next day, the cells
deviation. (B) A total of 2 × 10 cells were seeded into a 12-well plate. The next day, the cells were
were washed with PBS and supplemented with 1 mL of F20 with 1 or 10 pg/mL IL-6. After 24 h,washedwith PBS
andsupplemented
withthrough1 mL of aF20
or 10Eachpg/mL
IL-6. After
24 h,cultureculture medium
wascollected
and passed
220withnm 1filter.
tetraspaninin themediummediumwas collected
and andpassedthrougha 220
nm filter. Eachtheactivity
mediumwasmeasuredusing ELISA
eachvalue wasnormalizedusingtetraspanin
the cellularin
ATP
aswas
an
measuredusing ELISA
and eachvaluefromwas threenormalizedusing thecellular ATP
an internalinternal
standard.
Data wereobtained
independent
experiments(n =activity3), ** pas< 0.01.
Error
standard.
Data
were obtainedfromindependent
experiments(n = 3). Error
bars represent
bars
represent
standard
deviation.
(C)three
The expression
levelsof each tetraspanin
in LUHMES
cells
treatedwith
1 or 10 pg/mL
were analyzed
qRT-PCR.
Datainwere
obtainedthreeindestandard
deviation.
(C) TheIL-6
expression
levels ofby
eachtetraspanin
LUHMES
cellsfromtreatedwith
1 orpendent
experiments(n = 3), **byp qRT-PCR.
< 0.01. Error
bars
represent
standard
deviation.
(D) Aexperiments
total of 2 ×
10 pg/mL
IL-6 were analyzed
Data
were
obtainedfrom three
independent
5
were bars
seededinto a standard
12-well plateand cultured
withofmedium
containing
LUHMES-derived
10(n =cells3). Error
represent
deviation.
(D) A total
2 × 105 cells
were seeded
into a 12-well
CD81-Nluc expressed in EVs with 1 or 10 pg/mL IL-6 for 8 h. The background was subtracted fromplate and cultured with medium containing LUHMES-derived CD81-Nluc expressed in EVs witheach value of Nluc activity and the result was normalized using cellular ATP activity as an internal
1 or 10 pg/mL IL-6 for 8 h. The background was subtracted from each value of Nluc activity and thestandard. Data were obtained from three independent experiments (n = 3), ** p < 0.01. Error bars
result wasstandard
normalizedusing cellular ATP activity as an internal standard. Data were obtained from
represent
deviation.
three independent experiments (n = 3). Error bars represent standard deviation.

3.5. IL-6 Aﬀects the Amounts of miRNAs in EVs

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Table 1. Expression of miRNAs in EVs and in cells.
miRNA Expression in EVs

miRNA IDs
hsa-miR-135a-3p
hsa-miR-11399
hsa-miR-6790-3p

miRNA Expression in Cells

1 pg/mL IL-6

10 pg/mL IL-6

Ratio *

p-Value **

ACs

MG

120.4 (±25.1)
242.3 (±81.4)
176.4 (±89.3)

284.5 (±39.5)
538.8 (±151.4)
383.5 (±29.0)

2.4
2.2
2.2

0.04950.04950.049517.4 (±6.4)15.9 (±5.5)
26.5 (±9.3)

48.0 (±5.2)
32.6 (±3.1)
67.3 (±4.6)* miRNA expression of 10 pg/mL IL-6/miRNA expression of 1 pg/mL IL-6. Mean (± standard deviation), n = 3.
** Wilcoxon signed-rank test.

3.6. Target Analysis of Hsa-miR
Hsa-miRs in EVs derived from LUHMES cells were increased by IL-6. This result
suggests that these miRNAs were increased in ACs and MG after uptake of EVs. Next, we
introduced these miRNAs into ACs and MG via lipofection, and then microarray analysis
of cellular mRNA was performed to identify target genes. The conditions for selection
were as follows: (1) mRNA expression in the control group (introduction of ctrl miRNA) of>100; (2) mRNA expression at the time of introduction of each miRNA/mRNA expression
at the time of introduction of ctrl miRNA of <0.5. The number of mRNAs that met theabove criteria is shown in Table 2. mRNAs with expressions that were <0.5 for the same
miRNAs in both of the two cell types are shown in Table 3. As shown in Tables 2 and 3, thenumber of mRNAs decreased by hsa-miR-135-3p was the lowest. Additionally, mRNAs
with expressions that were <0.5 for different miRNAs in the same cell type are shown in
Table 4. Four mRNAs, NREP and RPEL1 in ACs, and CTNND1 and KCTD12 in MG, werecommonly suppressed by two miRNAs. mRNAs that were simultaneously suppressed bythree miRNAs were not observed. We identified mRNAs involved in the nervous systemusing the data in Tables 3 and 4, and searched for the target sequence of each miRNA. The
database from which the nucleotide sequences and target sites were retrieved is shown in
Table 5.
Table 2. Number of mRNAs for which expression was less than 1/2 due to transfection of miRNAs.
Cell

Transfected miRNA

Number of mRNAs

ACs

hsa-miR-135-3p
hsa-miR-6790-3p
hsa-miR-11399

2
15
547

MG

hsa-miR-135-3p
hsa-miR-6790-3p
hsa-miR-11399

13
34
205

Commonhsa-miR-135-3p
hsa-miR-6790-3p
hsa-miR-11399

1
10
47

Table 3. mRNAs for which expression was less than 1/2 in both ACs and MG due to transfectionof miRNAs.
Transfected miRNAs

mRNAs Expression Less than 1/2

hsa-miR-135-3p

UBE2D3hsa-miR-6790-3p

MINOS1-NBL1
PTS
RAB6A
RPE
SET

MRPL11
RPEL1
LLPH
IMP4
EI24

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Table 3. Cont.
Transfected miRNAs

mRNAs Expression Less than 1/2

hsa-miR-135-3p

UBE2D3hsa-miR-11399

ACTR3
CDKN1A
CENPF
NCKAP1
SUPT16H
MRFAP1L1CREB1CTNND1
ARL6IP6
ZNF836
ZNF721EFNB2RHOBTB3
PUM2
SF3B1
BAMBI

TOR1AIP1
AMD1
GATC
ANXA1
IPO5
LIMS1
FAM45AZNF117LARSZFR
POLR2BANLN
ZFAND6
NDFIP2NOL8
EEF1A1P31

BCCIP
SERINC1SOAT1
TOP2A
HIST2H2AA4
CHCHD7
HIST1H2BG
HIST1H2BH
CASP3MARVELD1
WDR75DGKZUSO1PRPF4B
HIST3H2A
TTC37
G3BP2

Table 4. Common mRNAs reduced by less than 1/2 following transfection of different miRNAs incells of the same species.
Gene

hsa-miR-135-3p

hsa-miR-6790-3p

hsa-miR-11399-3p

NREP of ACs
RPEL1 of ACs
CTNND1 of MG
KCTD12 of MG

NE
NE
NE
NE

↓
↓
↓
↓

↓
↓
↓
↓↓

↓: less than 1/2, ↓↓: less than 1/10, NE: no effect.

Table 5. Comparison of alignment using Target Scan or Emboss water.
miRNA Name

hsa-miR-135a-3p

hsa-miR-11399

hsa-miR-6790-3p

miRNA
sequence

UAUAGGGAUUGGAGCCGUGGCGUUCAGGUCUGG
GGCUGAAACCUCGACCUCGGCGACCCCUCACU

NREP(Target Scan)

none

none

UGUAUGGGUAUUGAUGAGGUCAU

NREP(Emboss water)

none

GAUGCUGCC
ACAGGACCUGAGUGUAUGGGUAUUGAUGAGGUCKCTD12(Target Scan)

none

none

CUCCAAGCGCC
GUAGGAGGUCAC

KCTD12(Emboss water)none

GGUUGCAGCUCCUGAGUGCAGCGCGGCUUCCUG

GCCCGGGGC
CGCCGCCCUCG

LLPH(Target Scan)

none

none

GAGGCAGGUGG
AUCACGAGGUCA

LLPH(Emboss water)

none

none

UGAGGCAGAUGG
AUCACCUGAGGUCCTNND1(Target Scan)

none

none

none

CTNND1(Emboss water)

none

GGCUCCGCCCCUUACCUUCAAGAGAGGAGUGAAGCUCGCCGGAAAC

Underlined bases indicate miRNA target alignment. Below the mRNA is the name of the database from which themiRNA target was retrieved.

Biomolecules 2023, 13, 718

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4. Discussion
There are various methods to collect EVs, but none have been optimized. EVs werefirst collected using ultracentrifugation and subsequently using gel filtration and capture
methods using antibodies [24]. In this study, EVs were collected via a microfiltration
method using 220 and 20 nm syringe filters (termed nanofiltration). The advantage ofthis method is that particles of uniform size are collected, but the sample is contaminatedby microvesicles of 100–220 nm in size. Additionally, there are concerns that filter pores
may be blocked by EVs, and that EVs may be destroyed by the membrane pressure [25,26].
Therefore, we examined cell destruction after passing through the filter, and confirmed thatcells were not destroyed at a rate of two drops or less per second. Because EVs are smaller
than cells, they are less likely to be destroyed when they pass through a syringe filter. We
also believe that only approximately 5 min to pass 10 mL of medium through a 13 mm
syringe filter of 50 nm in size is very useful for analysis. EVs trapped in a syringe filter canbe easily eluted using phenol or detergent, but intact EVs cannot be recovered.
IL-6 is a proinflammatory cytokine that is increased by stress and involved in the development of depression [4]. Because there are reports that dysregulation of the dopamine
system is involved in the pathophysiology of depression [27–29], in this study we investigated how IL-6 affects dopaminergic neuronal cells (LUHMES), astrocytes, and microglia.
In particular, we applied IL-6 to neural precursor cells and analyzed quantitative andqualitative changes in EVs released by these cells as well as uptake of the EVs by astrocytes
and microglia. First, we analyzed the intracellular proteins CD9, CD63, and CD81 andtheir mRNAs in EVs under normal culture conditions. The proteins and mRNAs of thesetetraspanins were not correlated. It was suggested that the expression of each tetraspanin
and its localization to EVs differs depending on the cell.
Tetraspain fused with a sensitive luciferase such as Nluc is used for in vitro and in vivo
assays [30]. In this study, nanoluciferase-fused CD81 was expressed in EVs and analyzed
for uptake by other cells. Intracellular uptake of EVs released by neural precursor cells
peaked at 4 h and reached an equilibrium after 8 h in all cell types. Additionally, astrocytes
had the highest uptake, and microglia showed a similar uptake to neural precursor cells.
In vivo analysis has shown that retinal ganglion cells and microglia exhibit maximum
fluorescence at 14 and 7 days, respectively, after administration of fluorescently labeled
EVs in rats [31]. Therefore, several days may be required for EVs to reach cells in animals.
Our results suggest that EVs released by neighboring cells are taken up within hours. It isthought that the difference in the uptake degree depending on the cell type is influenced
by the combination of adhesion molecules expressed on the EV membrane and the plasma
membrane by each cell type. For example, Horibe et al. reported that EV endocytosis
depends on the expression level of related adhesion molecules [32]. This report suggeststhat the mechanism of EV uptake via endocytosis depends on the recipient cell type, whichis consistent with the difference in EV uptake between astrocytes and microglia.
In this study, we found that IL-6 does not affect EV secretion or uptake. TNFα andIL-1β modify the miRNA cargo of astrocyte-shed EVs [33] and IL-6 stimulation resultsin high production of miR-455-3p by human umbilical cord mesenchymal stem cells [34].
Therefore, we focused on miRNAs among the molecules contained in EVs and comprehensively analyzed neural-precursor-cell-derived exosomal miRNAs of which the expression
was increased or decreased by IL-6. Although many miRNAs were increased or decreased,
we identified three miRNAs (hsa-miR-135a-3p, hsa-miR-6790-3p, and hsa-miR-11399) thatwere significantly increased dose-dependently by IL-6. Unfortunately, we did not findany studies of these miRNAs in relation to depression or psychological stress. Interestingly, there is a report that hsa-miR-135a-5p is weakly expressed in depressed patients [35]and that depressed patients with high expression levels of miR-135a-5p respond better toantidepressant treatment and are more likely to remain in remission [36].
We found that the expression levels of many genes were increased or decreased
by transfection with each miRNA. Among them, NREP mRNA was decreased by bothhsa-miR-6790-3p and hsa-miR-11399. In the Target Scan ver. 8.0 database, NREP is directlyBiomolecules 2023, 13, 718

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targeted by hsa-miR-6790-3p, but not hsa-miR-11399. Using Emboss water to match locations from each sequence (https://www.ebi.ac.uk/Tools/psa/emboss_water/) accessedon 16 August 2022, revealed that hsa-miR-11399 might directly target NREP with a highidentity of 75%. These results suggest that the NREP gene might be targeted by bothhsa-miR-6790-3p and hsa-miR-11399. NREP, which is also known as P311, promotes axonal
regeneration [37] and cellular differentiation [38], and ectopic expression of NREP augments the motility of glioma cells [39]. These findings suggest that IL-6-related miRNAs in
EVs may suppress the neuronal function of ACs.
In the present study, three miRNAs were transfected into MG and KCTD12 wasfound to be reduced by both hsa-miR-6790-3p and hsa-miR-11399. KCTD12 is a directtarget of hsa-miR-6790-3p (Target Scan ver. 8.0). However, hsa-miR-11399 is not in theTarget Scan database. Searching Emboss water revealed 48% homology between KCTD12and hsa-miR-11399. KCTD12 is an important auxiliary subunit in GABAB receptor signaling [40]. KCTD12 expression in peripheral blood mononuclear cells decreases underchronic stress [41]. Conversely, KCTD12 expression in the circulating CD11b+ macrophagesof bipolar disorder patients is significantly higher as a percentage than that in healthy
controls [42]. Furthermore, a well-validated mouse model of chronic social duet stressrevealed an increase of KCTD12 expression in the dentate gyrus, a subregion of the hippocampus [43]. Thus, no consensus has been reached regarding KCTD12 expression during
stress. Although KCTD expression is widely spread over many areas of the brain, its
distribution has been reported to vary by brain region and intracellular location. We thinkthat this may explain why KCTD expression exhibits diverse responses to stress. [44,45].
We identified several mRNAs that were commonly decreased in ACs and MG by eachmiRNA (Table 3). Among them, LLPH is a direct target of hsa-miR-6790-3p (Target Scan ver.
8.0). LLPH has been predicted to be involved in dendrite extension and positive regulationof dendritic spine development. In mice, LLP is strongly expressed in the developing nervous system and its application to cultured neurons promotes dendrite outgrowth [46]. It
is possible that the suppression of LLPH inhibits neuronal dendrite outgrowth in situationsof elevated IL-6, i.e., chronic neuroinflammation, as was the condition in this study. This
supports the idea that an altered neurostructure is part of the etiology of depression [4].
CTNND1 was commonly downregulated in ACs and MG by hsa-miR-11399 andby hsa-miR-6790-3p in MG. In Target Scan, CTNND1 is listed for hsa-miR-6790-3p andhsa-miR-11399, but in the Emboss water analysis, hsa-miR-6790-3p and hsa-miR-11399 exhibited 53.8% and 65.0% identity with CTNND1, respectively. CTNND1 encodes p120-catenin.
p120-catenin promotes reorganization of the actin cytoskeleton during the formation ofcell adhesions [47]. Interestingly, wild-type astrocytes have a mesenchymal phenotype(i.e., E-cadherin negative and highly motile) and CTNND1 phosphorylation regulates theirmesenchymal–epithelial transition [48]. These data suggest that decreased expression of
CTNND1 impairs the MET function.
5. Conclusions
This study utilized nanofiltration to capture EVs in a culture medium. Nanofiltration
has the advantage of easily capturing EVs, making it useful for analyzing proteins andnucleic acids in EVs. However, since the EVs captured by the filter cannot be collected in
an intact condition, it is difficult to analyze their in vitro or in vivo functions. Our findings
suggest that the uptake of IL-6-derived EVs secreted from neural precursor cells by ACs andMG may suppress the expression of genes involved in neural regeneration, indicating thathigh levels of IL-6, associated with depression, contribute to decreased neuronal functions
via miRNAs in EVs.

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Author Contributions: Conceptualization, K.N. and H.I.; methodology, H.I.; validation, K.N. and
H.I.; formal analysis, K.N. and H.I.; investigation, K.N. and H.I.; resources, H.I.; data curation, K.N.
and H.I.; writing—original draft preparation, K.N.; writing—review and editing, K.N. and H.I.;
visualization, K.N.; supervision, H.I., T.T., C.N., Y.M. and S.H.; project administration, K.N. and H.I.;
funding acquisition, H.I., Y.M. and S.H. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank Ai Baba, Hiromi Morishita and Yukiko Yoshiura (Center
for Stress-related Disease Control and Prevention, University of Occupational and Environmental
Health, Japan) for their assistance.
Conflicts of Interest: The authors declare no conflict of interest.

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