(2024) 21:200
Chae et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03180-x

Journal of Neuroinflammation

Open Access

RESEARCH

The dopamine analogue CA140 alleviates
AD pathology, neuroinflammation, and rescues
synaptic/cognitive functions by modulating
DRD1 signaling or directly binding to Abeta
Sehyun Chae1†, Hyun‑ju Lee2†, Ha‑Eun Lee3†, Jieun Kim2, Yoo Joo Jeong2,7, Yuxi Lin4, Hye Yun Kim3,
Geoffray Leriche5, Rachel S. Ehrlich5, Sascha Castro Lingl5, Min‑Duk Seo6, Young‑Ho Lee4,8,9, Jerry Yang5*,
Jae‑Ick Kim3* and Hyang‑Sook Hoe2,7,10*

Abstract
Background We recently reported that the dopamine (DA) analogue CA140 modulates neuroinflammatory
responses in lipopolysaccharide-injected wild-type (WT) mice and in 3-month-old 5xFAD mice, a model of Alzheimer’s disease (AD). However, the effects of CA140 on Aβ/tau pathology and synaptic/cognitive function and its
molecular mechanisms of action are unknown.
Methods To investigate the effects of CA140 on cognitive and synaptic function and AD pathology, 3-month-old WT
mice or 8-month-old (aged) 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 10,
14, or 17 days. Behavioral tests, ELISA, electrophysiology, RNA sequencing, real-time PCR, Golgi staining, immunofluorescence staining, and western blotting were conducted.
Results In aged 5xFAD mice, a model of AD pathology, CA140 treatment significantly reduced Aβ/tau fibrillation, Aβplaque number, tau hyperphosphorylation, and neuroinflammation by inhibiting NLRP3 activation. In addition, CA140
treatment downregulated the expression of cxcl10, a marker of AD-associated reactive astrocytes (RAs), and c1qa,
a marker of the interaction of RAs with disease-associated microglia (DAMs) in 5xFAD mice. CA140 treatment also suppressed the mRNA levels of s100β and cxcl10, markers of AD-associated RAs, in primary astrocytes from 5xFAD mice.
In primary microglial cells from 5xFAD mice, CA140 treatment increased the mRNA levels of markers of homeostatic
microglia (cx3cr1 and p2ry12) and decreased the mRNA levels of a marker of proliferative region-associated microglia (gpnmb) and a marker of lipid-droplet-accumulating microglia (cln3). Importantly, CA140 treatment rescued
scopolamine (SCO)-mediated deficits in long-term memory, dendritic spine number, and LTP impairment. In aged
5xFAD mice, these effects of CA140 treatment on cognitive/synaptic function and AD pathology were regulated†

Sehyun Chae, Hyun-ju Lee, Ha-Eun Lee contributed equally to this study.

*Correspondence:
Jerry Yang
jerryyang@ucsd.edu
Jae‑Ick Kim
jikim220@unist.ac.kr
Hyang‑Sook Hoesookhoe72@kbri.re.kr
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article orparts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To
view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Chae et al. Journal of Neuroinflammation(2024) 21:200

Page 2 of 34

by dopamine D1 receptor (DRD1)/Elk1 signaling. In primary hippocampal neurons and WT mice, CA140 treatment
promoted long-term memory and dendritic spine formation via effects on DRD1/CaMKIIα and/or ERK signaling.
Conclusions Our results indicate that CA140 improves neuronal/synaptic/cognitive function and ameliorates Aβ/tau
pathology and neuroinflammation by modulating DRD1 signaling in primary hippocampal neurons, primary astrocytes/microglia, WT mice, and aged 5xFAD mice.
Keywords CA140, Dopamine D1 receptor, Learning and memory, LTP, Aβ, Tau, Reactive gliosis

Background
Extracellular senile plaque (SP) deposition and intraneuronal neurofibrillary tangle (NFT) accumulation are thepredominant pathophysiological hallmarks of Alzheimer’s disease (AD) [1]. SPs are aggregates of misfolded
amyloid-β (Aβ), and NFTs are composed of abnormally
hyperphosphorylated microtubule-associated tau proteins [1]. The accumulation of SPs and NFTs begins in
the entorhinal cortex and hippocampus, brain regions
associated with memory processing, and the resulting
axonal deterioration, decreased dendritic spine density,
neuronal loss, and weakened synaptic strength lead tocognitive dysfunction [2]. Under pathological conditions,SPs accelerate the transition of homeostatic microglia todisease-associated microglia (DAMs), neurodegenerative
phenotype microglia (MGnDs), and lipid-droplet-accumulating microglia (LDAMs) and the transition of naïve
astrocytes to AD-associated reactive astrocytes (RAs)[3–5]. The interaction of DAMs/MGnDs with RAs exacerbates neuroinflammatory dynamics, a key mechanismof the pathoprogression of neurodegenerative diseases,
including AD. Therefore, small molecules that prevent
harmful interactions of Aβ and tau with the cellular environment and SP- or NFT-mediated neuroinflammation
may have therapeutic value for AD [6].
Dopamine (DA) receptors ­(DRD1-5) are distributed
throughout the hippocampus during hippocampal formation, and DA signaling modulates learning, memory,and dendritic spine formation [7–9]. Brain levels of DA
and its receptor DRD1/DRD2 are lower in AD patients
than in healthy controls, and supplementation with DA
precursors or agonists rescues synaptic and cognitive
function [10, 11]. In medium spiny neuronal cultures,
DA promotes dendritic spine formation via DRD1 andDRD2 and participates in dendritic spine enlargement
and structural plasticity [12]. Pharmacological activation of DRD1 enhances recognition memory in rats [13],and DRD1/5 activation in the rat hippocampus promotes
initial and novel memory consolidation [14]. DRDs
expressed on glial cells function in anti-inflammation by
downregulating microgliosis and astrocytic activation;accordingly, DA regulates neuroinflammation in neurodegenerative diseases [15]. For example, the DA precursor levodopa attenuates LPS- or α-synuclein-mediated

NLRP3 inflammasome activation in human microglial
cultures [16]. Collectively, these observations suggestthat DA and DRDs play important roles in synaptic andcognitive function and neuroinflammatory responses.
In a direct application of these findings, we recently
designed and synthesized the DA analogue CA140 byintroduction of a 2-(methylamino) benzoyl group to theamine in DA [17]. CA140 penetrates the blood–brain barrier (brain:plasma concentration ratio = 1.91 ± 0.22) andinhibits neuroinflammatory responses in LPS-injectedwild-type (WT) mice and in 3-month-old 5xFAD mice[17]. However, the effects of CA140 on synaptic/cognitive function and AD pathology under normal and pathological conditions have not been examined. In the present
study, we aimed to investigate these effects and theirmolecular mechanisms of action in vitro and in vivo. We
found that CA140 directly bound Aβ aggregates in vitro.
In aged 5xFAD mice, CA140 treatment decreased Aβ/tau
fibrillation; Aβ plaque number; tau hyperphosphorylation; microgliosis/astrogliosis; and the expression of theneuroinflammation molecular target NLRP3, a marker
of AD-associated reactive astrocytes (cxcl10), and amarker of the interaction of RAs with disease-associatedmicroglia (DAMs; c1qa). In primary astrocytes (PACs)
and primary microglial cells (PMCs) from 5xFAD mice,
CA140 treatment suppressed the mRNA levels of markers of AD-associated RAs (s100β and cxcl10), proliferative region-associated microglia (PAMs; gpnmb) andlipid-droplet-accumulating microglia (LDAMs; cln3) andincreased the mRNA levels of markers of homeostatic
microglia (cx3cr1 and p2ry12). More importantly, CA140
treatment attenuated memory impairment, dendritic
spine loss and LTP impairment in aged 5xFAD mice bymodulating DRD1/Elk1 signaling. We then investigatedthe effects of CA140 on synaptic/cognitive function undernormal conditions and found that CA140 treatment
improved synaptic and cognitive function through DRD1/
CaMKIIα and/or ERK signaling in primary hippocampal
neurons and WT mice. Taken together, our data indicatethat the dopamine analogue CA140 modulates synaptic/
cognitive function, AD pathologies, reactive gliosis, andneuroinflammatory responses through DRD1 signaling inprimary hippocampal neurons, PACs, PMCs, WT mice,and aged 5xFAD mice.

Chae et al. Journal of Neuroinflammation(2024) 21:200

Methods
Mouse studies

All in vivo experiments were performed in accordance with approved animal protocols and guidelines
established by the Korea Brain Research Institute(IACUC-2016-0013, IACUC-2018-0018) and UNIST(UNISTIACUC-20-18). Male C57BL6/N mice were used
as WT mice, and F1 generation 5xFAD transgenic (Tg)mice were used as the mouse model of AD.
Wild‑type mice

Male C57BL6/N mice (3 months old, 25–30 g) were purchased from Orient-Bio Company (Gyeonggi-do, South
Korea) and housed in a pathogen-free facility under a12-h light/dark cycle at an ambient temperature of 22 °C.
To investigate whether CA140 modulates cognitive/synaptic function and its molecular mechanisms of action
under normal conditions, WT mice were injected withvehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for
10, 14, or 17 days. CA140 was synthesized as described
previously [17], and all mice were randomly assigned totreatment groups. An independent researcher blinded tothe experiments quantified all in vivo results.
5xFAD mice (mouse model of AD)

To ensure the integrity of the genetic background [18], F1 generation 5xFAD transgenic (Tg)mice (stock #34848-JAX, B6Cg-Tg APPSwFlLon,
PSEN1*M146L*L286V6799Vas/Mmjax) were purchasedfrom Jackson Laboratory. Only male mice were used tominimize bias caused by hormonal fluctuations in female
AD mice. To examine the effects of CA140 on cognitiveand synaptic function and AD pathologies and its molecular mechanisms of action under pathological conditions, 8-month-old 5xFAD mice were injected daily withvehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) for 14 or
17 days. Behavioral experiments and tissue analysis were
subsequently conducted.
Cell lines and cAMP assay

To determine whether CA140 acts as a DRD1 agonist
or antagonist, we used HEK293 cells, which have hightransfection efficiency for plasmid DNA [19]. HEK293
cells were maintained in Opti-MEM® (Invitrogen) with
10% fetal bovine serum (FBS, Life Technologies, Inc.) in
a 5% C­ O2 incubator, seeded in 12-well plates (30 × ­104
cells/well), and transiently transfected with eGFP-tagged
DRD1 constructs or control constructs using FuGENE®
6 (Roche). Twenty-four hours after transfection, theHEK293 cells were washed with serum-free media andtreated with a DRD1 antagonist (LE300, 10 μM) or vehicle (DMSO) for 30 min. The HEK293 cells were thentreated with a DRD1 agonist (A77636, 5 μM), DA (10Page 3 of 34

or 100 μM), CA140 (10, 50, or 100 μM), or vehicle (1%DMSO) for 1 h, followed by resuspension in 200 μL of
0.1 M HCl buffer and incubation for 10 min at room temperature (RT). The cell lysates were centrifuged (600×g)
for 10 min at 4 °C to remove cellular debris. The supernatant was used to measure cAMP levels using the cAMP
complete ELISA kit according to the manufacturer’s recommendations (Enzo Life Sciences, Farmingdale, NY,
USA).
Primary neuronal culture, transfection,and immunocytochemistry (ICC)

Primary cortical neurons (PCNs) from embryonic day(E) 16 Swiss–Webster mice were prepared as previously described [20]. In addition, primary hippocampal
neurons (PHNs) from E19 Sprague–Dawley rats were
cultured at 150 cells/mm2 as previously described withminor modifications [21]. Briefly, the hippocampus was
dissected from the brain, washed in dissection dissociation medium (DDM), and dissociated with trypsin. The
PHNs were plated on poly-d-lysine-coated coverslips in
MEM containing 10% fetal bovine serum, 1 mM pyruvate, 2 mM l-alanyl-l-glutamine dipeptide, and 0.45%
glucose. Two hours after plating, the DDM was replacedwith neurobasal medium supplemented with B27, Ara-C(0.5 μM), and 2 mM l-alanyl-l-glutamine dipeptide.
Twice weekly, one-half of the medium was replaced withfresh maintenance medium. At DIV 13 or 20, the PHNs
were transfected with GFP using Lipofectamine 2000
(Invitrogen, Carlsbad, CA, USA).
For GFP transfection, 1 μL of Lipofectamine 2000 was
diluted in 25 μL of neurobasal medium and incubated at
RT for 5 min. GFP plasmid DNA (2 μg) was also diluted
in 25 μL of neurobasal medium and incubated at RT for
5 min. The diluted Lipofectamine 2000 and GFP DNA
were then mixed and incubated at RT for 30 min in thedark. Finally, the Lipofectamine 2000/GFP DNA mixture (50 μL) was added to each well containing PHNs.
Twenty-four hours after transfection, the PHNs weretreated with vehicle (1% DMSO) or CA140 (1 or 5 μM)
for 24 h to examine the effects of CA140 on synaptic
function. The neurons were then fixed for 10 min in 4%
paraformaldehyde (PFA) at RT or methanol at − 20 °C,
followed by immunostaining with antibodies against
spinogenesis-related molecules (i.e., DRD1, synaptophysin, PSD-95, p-ERK, and p-CaMKIIα) in GDB buffer(0.1% gelatin, 0.3% Triton X-100, 16 mM sodium phosphate pH 7.4, 450 mM NaCl) overnight. The cells werethen immunostained with Alexa Fluor 555- and 488-conjugated secondary antibodies for 2 h. Detailed information on the primary and secondary antibodies is provided
in Table 2. Images were acquired with an LSM 510 laser
scanning confocal microscope (Zeiss). Confocal z-stackChae et al. Journal of Neuroinflammation(2024) 21:200

Page 4 of 34

image stacks encompassing entire dendrite segments
were analyzed using MetaMorph software (Universal
Imaging Corporation, Downingtown, PA, USA).

Sciences) eluted with PBS buffer. The purified tau protein
was concentrated by ultrafiltration (Amicon, 10 kDa cutoff, Millipore).

Preparation of cultures of primary astrocytes and primary
microglia from 5xFAD mice

Estimating the binding of CA140 to aggregated Aβ42

To investigate the effect of CA140 on the reactive state of
glial cells in an in vitro model of AD, mixed glial cultures
were prepared from 5xFAD pups at postnatal days 1–2 aspreviously described with modifications [22, 23]. Briefly,
the pups were genotyped using genomic DNA extracted
from tail snips. Then, whole brains from 5xFAD pups
were filtered through 70-μm nylon mesh and cultured
in low-glucose DMEM with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The mixed glial cultures were maintained in a 5% C
­ O2 incubator at 37 °C for
3 weeks. To obtain PACs from 5xFAD mice, 75 T flasks
containing mixed glial cells were shaken at 250 rpm ona rotary shaker at RT overnight. The conditioned culture
medium was then discarded, and the cells were dissociated with 0.25% trypsin–EDTA and centrifuged at 2000
rpm for 10 min. After centrifugation, the pellet (PACs)
was collected and used for experiments. To isolate PMCs
from 5xFAD mice, mixed glial cells were treated withmild trypsin digestion (0.5 M EDTA, 1 M ­CaCl2, and
0.25% trypsin–EDTA in low-glucose DMEM with 1%
penicillin and streptomycin) for 40 min in a 37 °C incubator. After removing the astrocyte layer, the PMCs weredissociated with trypsin–EDTA (0.25%) and centrifuged
twice at 2000 rpm for 10 min. Finally, the pellet (PMCs)
was collected and used for experiments.
Aβ and tau inhibition assays
Expression and purification of 2N4R tau

Escherichia coli BL21-CodonPlus (DE3) cells were transformed with a pET29b plasmid encoding human tau(2N4R isoform, 441 residues) and grown at 37 ℃ in LB
medium containing kanamycin (50 mg/mL). Protein
expression was induced by adding 0.5 mM isopropyl β-dthiogalactopyranoside (IPTG) at an ­OD600 of 0.5, and thecells were further grown at 37 ℃ for 4 h. The cells were
harvested by centrifugation and disrupted by sonication
in lysis buffer (20 mM Tris–HCl, 500 mM NaCl, 1 mM
­MgCl2, 5 mM DTT, 1 mM EDTA, pH 6.8). The lysate wasboiled for 30 min and then centrifuged at 18,000 rpm for
50 min. The supernatant was diluted and loaded onto
a cation exchange column (HiTrap SP HP, GE Healthcare Life Sciences) equilibrated with 20 mM Tris–HCl(pH 6.8) containing 50 mM NaCl, 1 mM M
­ gCl2, 2 mM
DTT, 1 mM EDTA, and 0.2 mM PMSF. Tau proteins
were eluted with a salt gradient (100 mM to 1 M NaCl).
Further purification was performed on a HiLoad 16/600
Superdex 200 prep-grade column (GE Healthcare Life

Aggregated Aβ was prepared from commercially available monomers (GenScript USA, Inc.) using a previously reported protocol [24]. The binding of CA140 toaggregated Aβ was measured according to a previously
described assay [25]. Briefly, 300 µL of solution containing a specific concentration of CA140 in 1% DMSO in
Milli-Q water was incubated with or without 30 µg of
aggregated Aβ to give a final solution volume of 330 µL.
The incubations were performed in duplicate and allowed
to reach equilibrium overnight at RT. The next day, thesolution was centrifuged for 20 min at 16,000×g and 4 °C.
The supernatant was removed, and the pellet was resuspended in 330 µL of fresh 1% DMSO in Milli-Q water.
Three 100-µL aliquots of each solution were pipetted into
separate wells of a black 96-well microplate, and fluorescence was measured in a microplate reader (Molecular
Devices SpectraMax i3x Multi-Mode Microplate Reader)with an excitation wavelength of 330 nm and emissionof 450 nm. The observed fluorescence of CA140 in thepresence of Aβ was reported as the fluorescence intensity measured after subtracting the fluorescence intensityof samples containing the same amount of CA140 but noAβ. The averages of each run were plotted with the error
bars representing the standard deviation from the mean.
The data were fit to the one-site specific binding algorithm to determine Kd: Y = Bmax × X/(Kd + X), where X isthe concentration of CA140 (in µM), Y is the observed
fluorescence intensity, and Bmax is the maximal observable fluorescence upon binding of CA140 to aggregated
Aβ.
Preparation of solutions of Aβ42 and tau

A stock solution of monomeric Aβ42 was prepared aspreviously described [26]. Briefly, lyophilized Aβ42 purchased from Peptide Institute, Inc. (Osaka, Japan) was
dissolved in chilled 10 mM NaOH at ~ 200 μM and sonicated in a cold-water bath for 15 s. The peptide solution was ultracentrifuged for 1 h at 40,000 rpm and
4 °C to remove large precipitates. The concentration ofAβ42 monomers was determined by measuring the UV
absorbance at 280 nm using a molar extinction coefficient of 1490 ­M−1 ­cm−1. Buffer exchange of the stock
solution of 2N4R tau against 20 mM sodium phosphatebuffer (pH 7.5) was performed using PD-10 columns(GE Healthcare Life Sciences, Massachusetts, USA). The
concentration of tau was determined by measuring theabsorbance at 280 nm with a molar extinction coefficientof 7,575 ­M−1 ­cm−1.

Chae et al. Journal of Neuroinflammation(2024) 21:200

Page 5 of 34

Thioflavin T (ThT) fluorescence assay

AAV‑DRD1 shRNA injection

The ThT fluorescence assay was used to monitor amyloid
fibrillation of Aβ42 and 2N4R tau at 37 °C. The following
sample solutions were prepared: (1) 5 μM Aβ42, 20 mM
sodium phosphate buffer (pH 7.5), and 5 μM ThT withand without 5, 25, 50, and 100 μM CA140; (2) 30 μM tau,
20 mM sodium phosphate buffer (pH 7.5), 1 mM TCEP,
30 μM heparin, and 5 μM ThT with and without 150 and
600 μM CA140. Each sample solution (100 μL) was pipetted in quadruplicate into wells of a low-binding 96-wellplate (Corning, Kennebunk, ME, USA), and the plate wasfirmly sealed. The kinetic profiles of Aβ42 amyloid aggregation were monitored under quiescent conditions, while
tau amyloid formation was observed under continuous
shaking. ThT fluorescence intensity was recorded in a
SpectraMax iD3 microplate reader (Molecular Devices
LLC., San Jose, CA, USA) using excitation and emission
wavelengths of 445 and 490 nm, respectively.

To examine whether CA140 regulates cognitive function
in a DRD1-dependent manner under normal conditions,
adeno-associated viral (AAV) vectors expressing shDRD1
under the control of the U6 promoter (AAV-eGFPU6-mDRD1a-shRNA, 1.0 × ­1012 genome copies [GC]/mL) and control AAV expressing GFP (AAV-eGFP-U6shRNA) were purchased from Vector Biolabs (Malvern,
PA, USA). Male WT mice (3 months old, 25–30 g) wereanesthetized with 2,2,2-tribromoethanol (Sigma Aldrich,
St. Louis, MO, USA, 2.5% v/v, 150 mg/kg, i.p.) and placedin a stereotaxic apparatus (Stoelting, Wood Dale, Illinois,
USA). Next, the AAVs were injected bilaterally into the
hippocampal CA1 region in a volume of 0.5 μL at a flowrate of 0.1 μL/min using a 10-μL Hamilton syringe and 26
G needle. The stereotaxic coordinates of the hippocampal CA1 injection site were − 1.7 mm anterior/posterior(AP), ± 1.4 mm medial/lateral (ML), and − 1.5 mm dorsal/ventral (DV) from the bregma. The surgery was performed 7 days before beginning the treatment regimen ofdaily administration of CA140 (30 mg/kg, i.p.) or vehicle(10% DMSO) for 14 days. The behavioral tests were conducted on days 15 to 17 after beginning the treatment
regimen.
To examine whether CA140 regulates cognitive function in a DRD1-dependent manner under pathologicalconditions, 8-month-old 5xFAD mice were injected with
AAV-eGFP-U6-shRNA or AAV-eGFP-U6-mDRD1ashRNA in the bilateral hippocampal CA1 region in
a volume of 1.0 μL at a flow rate of 0.3 μL/min. The
coordinates were − 2.0 mm (AP), ± 1.5 mm (ML), and− 1.55 mm (DV) from the bregma. The surgery was performed 21 days before beginning the treatment regimenof daily administration of CA140 (30 mg/kg, i.p.) or vehicle (10% DMSO) for 14 days. Behavioral tests were performed on days 15 to 17 after beginning the treatment
regimen.

Behavioral tests

To assess the effects of CA140 on cognitive function
under normal and pathological conditions, WT mice, amouse model of amnesia induced by scopolamine (SCO)
injection, and 5xFAD mice were treated with vehicle (10%DMSO) or CA140 (30 mg/kg, i.p.), and Y-maze and novel
object recognition tests were performed. The Y-maze
test evaluates short-term spatial memory. The Y-mazeconsisted of three arms (35 cm × 7 cm × 15 cm) at anglesof 120°. To conduct the test, a mouse was placed in one
arm of the maze and allowed to explore freely for 5 min.
Spontaneous alternations among the arms were recorded
and analyzed using a video camera connected to tracking
software (Ethovision XT, Noldus, Wageningen, Netherlands), and the percentage of alternations was calculatedby dividing the number of alternations by the number ofalternation triads. To analyze recognition and long-term
memory, the novel object recognition (NOR) test wasused. The apparatus for this test consisted of an openfield box (40 cm × 40 cm × 25 cm). First, each mouse
was placed in the apparatus with two identical objects
for 5 min as the training phase. Twenty-four hours later,
the retention testing phase was performed by placing themouse in the same apparatus with one of the previously
encountered objects and one novel object for 5 min. The
locations of the two objects in the arena were counterbalanced, and between trials, odor cues were eliminatedby carefully cleaning the apparatus and objects with 70%
ethanol. Each trial was recorded, and the exploration
time was manually quantified. Exploratory behavior was
defined as pointing of the mouse’s nose toward an object.
The novel object preference (%) was calculated fromthe exploration times of the novel and familiar objects:
Object preference [%] = ­(TNovel/[TFamiliar + ­TNovel]) × 100.

Golgi staining and morphological analysis of dendritic
spinesTo evaluate the effects of CA140 on dendritic spine formation in vivo, we conducted Golgi staining using an FD
Rapid GolgiStain Kit (FD Neurotechnologies, Ellicott
City, MD, USA) as described previously [27–29]. Briefly,
vehicle- or CA140-injected WT and 5xFAD mice were
submerged in Solutions A and B for 2 weeks in the dark
and then transferred to Solution C for 24 h. Solution C
was replaced after the first 24 h. Individual mouse brains
were sliced at a thickness of 150 µm using a VT1000S
Vibratome (Leica, Bannockburn, IL, USA). Dendritic images were acquired with an Axioplan 2 (Zeiss,
Oberkochen, Germany) under brightfield microscopy.
Dendritic spine density was determined by measuringChae et al. Journal of Neuroinflammation(2024) 21:200

pyramidal neurons in the cortex and/or hippocampal
CA1 region from − 1.70 mm to − 2.30 mm relative to thebregma using ImageJ (version 1.53a, National Institutesof Health, Bethesda, MD, USA).
mRNA sequencing

To investigate the molecular mechanisms (including geneprofiling) by which CA140 inhibits neuroinflammatory
responses in an AD mouse model, 3-month-old 5xFAD
mice were injected with vehicle (10% DMSO) or CA140(30 mg/kg, i.p.) daily for 14 days. Total RNA was thenextracted from the mouse hippocampus. The integrity ofthe total RNA was analyzed using an Agilent Technologies 2100 Bioanalyzer. The RNA integrity number (RIN)
values were > 8.8 for all samples. Poly(A) mRNA isolationfrom total RNA and subsequent fragmentation were performed using the Illumina TruSeq Stranded mRNA LT
Sample Prep Kit according to the manufacturer’s instructions. The adapter-ligated libraries were sequenced using
an Illumina NovaSeq 6000 (Macrogen, Korea). mRNAsequencing was performed for two independent replicates under each condition. Adapter sequences (TruSeq
universal and indexed adapters) were removed from theresulting read sequences for each sample using cutadapt
software (version 2.7; https://​cutad​apt.​readt​hedocs.​io/​
en/​stable/). The remaining reads were then aligned to
the Mus musculus reference genome (GRCm38) using
TopHat2 software (version 2.1.1) with default parameters[30]. After alignment, we counted the numbers of reads
mapped to the gene features (GTF file of GRCm38.91)
using HTSeq [31]. Read counts for the samples in eachcondition were then normalized using TMM (trimmed
mean of M-values) normalization in the edgeR package[32].
Identification of differentially expressed genes (DEGs)

Differential expression analysis was performed using the
Bioconductor package DESeq2 (version 1.38) with default
parameters [33]. DEGs were identified by adjusted p values < 0.05 and absolute ­log2-fold changes > 0.58 (1.5-foldchange). Gene set enrichment analysis of the DEGs was
performed using DAVID software [34]. Gene Ontology
biological processes (GOBPs) with p values < 0.05 wereselected as the processes enriched in DEGs. Cytoscape
software was used to reconstruct a network model inwhich the nodes were arranged based on the locations
and associations of the corresponding genes in the KEGG
pathway and WikiPathways databases [35, 36].
Real‑time quantitative PCR (qPCR)

To validate the results of mRNA sequencing, the same
mRNA sample used for mRNA sequencing was reverse
transcribed to cDNA using the Superscript cDNA

Page 6 of 34

Premix Kit II with oligo (dT) primers (GeNetBio, Chungman, Korea). In addition, to investigate the effects of
CA140 on the reactive state of glial cells in AD, mRNA
from CA140-treated PACs and PMCs (from 5xFAD mice)
was reverse transcribed to cDNA using the Superscript
cDNA Premix Kit II with oligo (dT) primers (GeNetBio,
Chungman, Korea). The synthesized cDNA was thenused as the template in real-time qPCR with Fast SYBRGreen Master Mix (Thermo Fisher Scientific, Waltham,
MA, USA) in a QuantStudio 5 Real-Time PCR System(Applied Biosystems, Thermo Fisher Scientific, Waltham,
MA, USA). The primer sequences for real-time qPCR are
given in Table 1. Cycle threshold (Ct) values were normalized to the value for gapdh, and the fold change wascalculated relative to the control.
Electrophysiology
Long‑term potentiation (LTP)/basal synaptic transmission

To determine the effects of CA140 on SCO-mediatedsuppression of LTP and basal synaptic transmission,
WT mice were injected (i.p.) daily with CA140 (30 mg/
kg, i.p.) or vehicle (10% DMSO) for 14 days and injectedwith SCO (1 mg/kg) or PBS daily on days 3 through 14. In
addition, to examine the effects of CA140 on hippocampal synaptic plasticity in an aged AD mouse model,
8-month-old 5xFAD mice were injected daily with CA140(30 mg/kg, i.p.) or vehicle (10% DMSO, i.p.) for 14 days.
Acute hippocampal slices (300 μm) were obtained using
a vibratome (VT1200S, Leica). The brain slices wereallowed to recover for 20 min at 34 °C and 40 min at
RT in artificial cerebrospinal fluid (ACSF) containing
125 mM NaCl, 2.5 mM KCl, 1.25 mM ­NaH2PO4, 25 mM­NaHCO3, 1 mM ­MgCl2, 2 mM ­CaCl2 and 15 mM glucose
oxygenated with 95% O­ 2 and 5% C
­ O2. After recovery, thebrain slices were transferred to a submerged recording
chamber perfused with ACSF at a rate of 2–3 mL/min at
30–31 °C.
To measure basal synaptic transmission, borosilicate
glass pipettes (2.5–4.0 MΩ) were filled with Cs+-based
low Cl− internal solution containing 135 mM ­CsMeSO3,
10 mM HEPES, 1 mM EGTA, 3.3 mM QX-314 chloride,
0.1 mM ­CaCl2, 4 mM Mg-ATP, 0.3 mM ­Na3-GTP, and
8 mM ­Na2-phosphocreatine (adjusted to pH 7.3 with
CsOH). For theta-burst stimulation (TBS) LTP, the glass
pipettes were filled with K­ +-based internal solution containing 135 mM K
­ MeSO3, 3 mM KCl, 10 mM HEPES,
1 mM EGTA, 0.1 mM C
­ aCl2, 8 mM N
­ a2-phosphocreatine,
4 mM Mg-ATP, and 0.3 mM N
­ a3-GTP (adjusted to pH
7.3 with KOH). The access resistance was 10–20 MΩ,and only neurons with a change in access resistance < 20%
were included in the analysis. Pyramidal neurons in CA1
were voltage-clamped at − 70 mV, and the Schaffer collateral (SC) pathway was stimulated every 20 s. ThreeChae et al. Journal of Neuroinflammation(2024) 21:200

Page 7 of 34

Table 1 Primers used in real-time qPCR

Table 1 (continued)

Gene

Gene

nlrp3
il-1β
iba-1
gfap
s100β
wwtr1
neat1
cxcl10chi3l1
serpina3n
nestinsynemindst
tspo

Sequence
Forward5′-TCC ACA ATT CTG ACC CAC AA-3′

Reverse5′-ACC TCA CAG AGG GTC ACC AC-3′

Forward5′-TTG ACG GAC CCC AAA AGA TG-3′

Reverse5′-AGG ACA GCC CAG GTC AAA G -3′

Forward5′-GGA CAG ACT GCC AGC CTA AG-3′

Reverse5′-GAC GGC AGA TCC TCA TCA TT-3′

Forward5′-GTT TCA TCT TGG AGC TTC TGC-3′

Reverse5′-GGA GGT GGA GAG GGA CAA C-3′

Forward5′-TGG TTG CCC TCA TTG ATG TCT-3′

Reverse5′-CTC GTT GTT GAT AAG CTC CTT CAG-3′

Forward5′-GTG AAG GCT TCT CGG TTG AG-3′

Reverse5′-ACG TCT TGC TTG CCT TGT CT-3′

Forward5′-TGG AGA TTG AAG GCG CAA GT-3′

Reverse5′-ACC ACA GAA GAG GAA GCA CG-3′

Forward5′-GCC GTC ATT TTC TGC CTC A-3′

Reverse5′-GCT TCC CTA TGG CCC TCA TT-3′

Forward5′-CAA GGA ACT GAA TGC GGA AT-3′

Reverse5′-CTG TGA TGG CCT GTG ATT TG-3′

Forward5′-CCC TGA GGA AGT GGA AGA AT-3′

Reverse5′-CCT GAT GCC CAG CTT TGA AA-3′

Forward5′-AGG AGA AGC AGG GTC TAC AGA G-3′

Reverse5′-AGT TCT CAG CCT CCA GCA GAG T-3′

Forward5′-GCC TTT GGA GGG TTA AGG AC-3′

Reverse5′-CTT AGA TAC CCC TGC CAC CA-3′

Forward5′-GCT CCC TGC TCA GAA AAC AC-3′

Reverse5′-CTG TGA ACG TGT GGA TGG TC-3′

Forward5′-GCT GTG GAT CTT TCC AGA ACA-3′

Sequence
Reverse5′-AGT TTC CAT TCC ACC ACC AG-3′

slc33a1

Forward5′-GAA AAC GTG GGC TAT GCT TC-3′

Reverse5′-CCT TCT TCC ACC AGC TTC AG-3′

snx17

Forward5′-CTT CGA ACC TCA CTG GAA GC-3′

Reverse5′-CAG GGG TCA AAG AGA ACA GC-3′

gpnmb

Forward5′-CCA ATT ACG TGG CTG GTC TT-3′

Reverse5′-CCC AAA TCA TTC CTG CAG TT-3′

igf1

Forward5′-CAC AAA CTC ACC ACC CTG TG-3′

Reverse5′-CTC CTG GAA ACC CAG AAC AA-3′

itgax

Forward5′- CCA AGA CAT CGT GTT CCT GAT T-3′

Reverse5′-ACA GCT TTA ACA AAG TCC AGC A-3′

clec7aForward5′-AGA CAC AGG GAG AAG GCA AA-3′

Reverse5′-CAG AAA AGG TGG GCA GAC TC-3′

lpl

Forward5′-GGG AGT TTG GCT CCA GAG TTT-3′

Reverse5′-TGT GTC TTC AGG GGT CCT TAG-3′

ctsd

Forward5′-GCT TCC GGT CTT TGA CAA CCT-3′

Reverse5′-CAC CAA GCA TTA GTT CTC CTC C-3′

cd44

Forward5′-ACT AGA TCC CTC CGT TTC ATC C-3′

Reverse5′-GGT TAC ATT CAA ATC GAT CTG CTG-3′

apoE

Forward5′-GGT TCG AGC CAA TAG TGG AA-3′

Reverse5′-TAT TAA GCA AGG GCC ACC AG-3′

spp1

Forward5′-AGC AAG AAA CTC TTC CAA GCA A-3′

Reverse5′-GTG AGA TTC GTC AGA TTC ATC CG-3′

trem2

Forward5′-CAG TTT CTC CTG CTG CTG AT-3′

Reverse5′-CAG TGC TTC AAG GCG TCA TA-3′

grn

Forward5′-CTG TAG TGC AGA TGG GAA ATC CTG CT -3′

Reverse5′-GTG GCA GAG TCA GGA CAT TCA AAC T-3′

gapdh

Forward5′-TGT GTC CGT CGT GGA TCT GA-3′

Reverse5′-CCT GCT​TCA​CCA CCT TCT TGA -3′

Reverse5′-ATG CCA AGA GGG TTT CTG C-3′

mao-b

Forward5′-GGC TGC TAC ACA ACC TAC TTC C-3′

Reverse5′-CAG TGT GAG GCT GTT TCA GTG-3′

hspb1

Forward5′-GCT CAC AGT GAA GAC CAA GG-3′

Reverse5′-GAA GCA CCG AGA GAT GTA GC-3′

cryab

Forward5′-AGG TGT TGG GAG ATG TGA TTG A-3′

Reverse5′-GGA TGA AGT AAT GGT GAG AGG GT-3′

fabp7

Forward5′-CAA GAA CAC AGA GAT CAA TTT CCA-3′

Reverse5′-CAT CCA ACC GAA CCA CAG A-3′

cr3

Forward5′-GCA GGA GTC GTA TGT GAG G-3′

Reverse5′-TTA CTG AGG TGG GGC GTC T-3′

c1qa

Forward5′-AAA GGC AAT CCA GGC AAT ATC A -3′

Reverse5′-TGG​ TTC ​TGG ​TAT ​GGA ​C TC ​TCC-3′

cx3cr1

Forward5′-ATT CTT CAT CAC CGT CAT CAG-3′

Reverse5′-ACT AAT GGT GAC ACC GTG CT-3′

p2ry12

Forward5′-GCT GCC TTG CTG AAG TCT CT-3′

Reverse5′-AGG TGG TAT TGG CTG AGG TG-3′

cd164

Forward5′-GGT TTC AGT TCC CAT GTG CT-3′

Reverse5′-CAA ATG AAA GCC CCA CAA CT-3′

Miniature excitatory postsynaptic currents (mEPSCs)

cln3

Forward5′-AAA CCA GAG CCA TGT GGA AC-3′

Reverse5′-GCT CCC AGC AGA ACA AAC TC-3′

Forward5′-CCT GAA TAA GCT TCC GGT GA-3′

To determine the effects of CA140 on mEPSCs in an
AD mouse model, 6-month-old 5xFAD mice were
injected with CA140 (30 mg/kg, i.p.) or vehicle (10%DMSO) daily for 14 days and sacrificed on the last day

npc2

successive excitatory postsynaptic currents (EPSCs) wereaveraged and expressed relative to the normalized baseline. TBS consisted of a burst of five pulses at 100 Hz
repeated 10 times at 5 Hz. Four consecutive trains ofTBS were delivered at 10-s inter-train intervals. TBS wasdelivered in current clamp mode. Recording signals were
filtered at 2 kHz (Multiclamp 700B, Molecular Devices)and digitized at 10 kHz (NI PCIe-6259, National Instruments). Recording data were acquired online using the
WinWCP program (Strathclyde software, http://​spider.​scien​ce.​strath.​ac.​uk/​sipbs/​softw​are_​ses.​htm) and analyzed offline using Clampfit 10.7 software (Molecular
Devices) and OriginPro 2017 (OriginLab).

Chae et al. Journal of Neuroinflammation(2024) 21:200

of injection. To measure mEPSCs, borosilicate glass
pipettes (2.5–4.0 MΩ) were filled with Cs+-based low
Cl− internal solution containing 135 mM C
­ sMeSO3,
10 mM HEPES, 1 mM EGTA, 3.3 mM QX-314 chloride, 0.1 mM C
­ aCl2, 4 mM Mg-ATP, 0.3 mM N
­ a3-GTP,
and 8 mM ­Na2-phosphocreatine (adjusted to pH 7.3
with CsOH). Pyramidal neurons in CA1 were voltageclamped at − 70 mV to measure mEPSCs. ACSF containing 500 nM tetrodotoxin and 25 μM D-AP5 was
perfused during the recording. The amplitude and frequency of the mEPSCs were analyzed with MiniAnalysis (Synaptosoft).
Immunofluorescence staining (IF)To evaluate the effects of CA140 on AD pathology, neuroinflammation, and synaptic function under pathological conditions, 8-month-old 5xFAD mice were injectedwith CA140 (30 mg/kg, i.p.) or vehicle (10% DMSO)
daily for 14 days. In addition, to examine whether CA140
modulates AD pathology and neuroinflammation in a
DRD1-dependent manner, 8-month-old 5xFAD mice
were injected with AAV-DRD1 shRNA or AAV-control
shRNA in the bilateral hippocampal CA1 region. Three
weeks after AAV injection, the mice were injected withvehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for
17 days. To assess the mechanism by which CA140 regulates cognitive/synaptic function under normal conditions, 3-month-old WT mice were injected with vehicle(10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days.
The treated mice were perfused/fixed in 4% paraformaldehyde overnight and then in 30% sucrose solution for
2 days. A cryostat (Leica CM1850, Wetzlar, Germany)
was used to obtain coronal slices with a thickness of
35 µm, which were then permeabilized at RT for 1 h in
PBS containing 0.2% Triton X-100 (PBST) and 10% normal goat serum. The brain sections were immunostainedfor 24–72 h at 4 °C with anti-Aβ17-24 (4G8), anti-TauThr212/
Ser214(AT100), anti-Iba-1, anti-GFAP, anti-NLRP3, antiIL-1β, anti-CXCL10, anti-C1QA, anti-GPB2, anti-CD68,
anti-CD206, anti-synaptophysin, anti-PSD-95, or antiDRD1 antibodies diluted in PBST. After incubation withthe primary antibody, the sections were washed withPBST three times and incubated with the secondary
antibody at RT for 2 h. Finally, the sections were washedthree times with PBS and mounted in antifade mounting
medium with DAPI (Vector Laboratories, Burlingame,
CA, USA). Images of the sections were acquired by fluorescence microscopy (DMi8, Leica Microsystems, Wetzlar, Germany) and analyzed by the software ImageJ(version 1.53a, National Institutes of Health, Bethesda,
MD, USA). Detailed information regarding the primaryand secondary antibodies is provided in Table 2.

Page 8 of 34

Enzyme‑linked immunosorbent assay (ELISA) for plasma
IL‑1β and COX‑2

To determine whether long-term CA140 treatment
induces peripheral inflammatory responses (adverse
effects), plasma IL-1β and COX-2 levels were measured
by ELISA. For this experiment, WT mice were injectedwith water (control for vehicle), 10% DMSO (vehicle),or CA140 (30 mg/kg, i.p.) daily for 17 days, and retroorbital blood was collected via a capillary tube. The
blood was centrifuged at 2000 rpm for 10 min at 4 °C,
and the plasma was collected and stored at − 80 °C until
analysis. IL-1β and COX-2 levels (100 µL of plasma)
were measured using IL-1β and COX-2 ELISA kits (IL1β ELISA kit: 88-7013-88, Invitrogen, Waltham, Massachusetts, USA; COX-2 ELISA kit: DYC4198, R&D
Systems, Minneapolis, MN, USA) according to the
manufacturer’s instructions and as previously described[28].
Western blot

To demonstrate the mechanism by which CA140
modulates synaptic and cognitive function under normal and pathological conditions, WT and 5xFAD mice
were injected with vehicle (10% DMSO, i.p.) or CA140(30 mg/kg, i.p.) daily for 17 days, and the hippocampus
and/or cortex was dissected. In addition, to investigate
the in vivo toxicity of CA140, the livers and kidneysof CA140-treated WT mice were collected. The brain,
liver, and kidney tissues were homogenized in RIPA
lysis buffer (Merck Millipore, Billerica, MA, USA) containing 1% protease and phosphatase inhibitor cocktail(Thermo Scientific, Waltham, MA, USA) for 1 h on ice.
The lysates were then centrifuged twice for 15 min at
20,000×g and 4 °C, and the supernatant was stored at− 80 °C until analysis. To separate brain proteins byelectrophoresis, 20 μg of protein was heated for 10
min at 100 °C and loaded onto an 8% SDS–polyacrylamide gel. To separate liver or kidney proteins by electrophoresis, 50 μg of protein was heated for 10 min at
100 °C and loaded onto a 10% SDS–polyacrylamide gel.
The separated proteins were then electrotransferred
to a PVDF membrane (Millipore, Bedford, MA, USA),
which was blocked with 5% skim milk in TBST andincubated overnight with anti-pCaMKIIα, anti-pERK,
anti-pELK-1, anti-caspase-3, anti-cleaved caspase-3,
or β-actin antibodies at 4 °C. Next, the membrane wasincubated with the horseradish peroxidase-conjugatedsecondary antibody for 1 h at RT. Finally, ECL solution(ATTO, Tokyo, Japan) was added for detection, andimages were acquired and analyzed with the software
Fusion Capt Advance (Vilber Lourmat, Eberhardzell,
Germany).

Chae et al. Journal of Neuroinflammation(2024) 21:200

Page 9 of 34

Table 2 Antibodies used in IF, ICC and WB
Primary antibodies
Immunogen

Host species

Dilution

Manufacturer

Catalog no

Application

4G8

Mouse

1:500

BioLegend

800,704

IF

AT100

Mouse

1:200

Invitrogen

MN1060IF

Iba-1

Rabbit

1:500

Wako

019-19741

IF

GFAP

Chicken

1:500

Millipore

AB5541

IF

NLRP3

Goat

1:100

Abcam

AB4207IF

IL-1β

Rabbit

1:100

Abcam

AB9722IFCXCL10

Goat

1:50

R&D

AF-466-NA

IF

C1QA

Rat

1:20

Abcam

AB11861

IF

GBP2

Rabbit

1:100

Abcam

AB203238

IF

CD68

Rat

1:100

BioradMCA1957GA

IF

CD206

Rat

1:100

Thermo12-2061-82

IF

Synaptophysin

Rabbit1:200

Invitrogen

MA5-14532

IF

Synaptophysin

Mouse

1:200

Sigma-Aldrich

S5768

ICC

PSD-95

Mouse

1:200

Neuromab

75028

IF, ICC

DRD1

Rat

1:200

Sigma Aldrich

D2944

IF

DRD1

Rabbit1:200

Invitrogen

AB20066

ICC

pCaMKIIα

Rabbit

1:200

Cell Signaling

12716

WB

pCaMKIIα

Rabbit

1:200

Abcam

AB5683ICC

pERK

Rabbit

1:2000Cell Signaling

9101

WB

pERK

Rabbit1:200

Invitrogen

368800ICC

p-ELK-1 Ser383

Mouse

1:1000Cell Signaling

9186

WB

GFP

Mouse

1:200

Novus

NB600-597

ICC

GFP

Rabbit1:200

Invitrogen

A11122ICC

Caspase-3

Rabbit

1:500

Abcam

AB32351

WB

C. caspase-3

Rabbit1:1000Cell Signaling

9664

WB

β-actinMouse

1:2000Santa Cruz

SC-47778

WB

Secondary antibodies
Antibody

Dilution

Manufacturer

Catalog no

Application

Goat anti-rabbit IgG, 555

1:200

Invitrogen

A21428IF, ICC

Goat anti-mouse IgG, 555

1:200

Invitrogen

A21422

IF, ICC

Goat anti-mouse IgG, 488

1:200

Invitrogen

A11001

IF, ICC

Donkey anti-goat, 555

1:200

Invitrogen

A21432IF

Goat anti-chicken IgG, 488

1:200

Abcam

A150169

IF

Goat anti-rat IgG, 555

1:200

Invitrogen

A21434IF, ICC

Goat anti-rabbit, HRP

1:5000Enzo

ADI-SAB-300-J

WB

Goat anti-mouse, HRP

1:5000Enzo

ADI-SAB-100-J

WB

Statistical analysis

Comparisons between two groups were performed
using unpaired two-tailed t-tests, whereas one-way
ANOVA or two-way repeated-measures ANOVA wasused for multiple comparisons. Tukey’s or Dunn’s

multiple-comparison test was used for post hoc analyses with significance at *p < 0.05, **p < 0.01, and***p < 0.001. All analyses were performed in GraphPad Prism 7 software. Data are presented as themean ± S.E.M. Details of the statistical analyses are provided in Table S1.

Chae et al. Journal of Neuroinflammation(2024) 21:200

Results
CA140 binds to aggregated Aβ and reduces Aβ/tau
amyloid formation and Aβ/tau pathology

Several studies have demonstrated that DA interacts withaggregated Aβ and modulates Aβ metabolism [37]. Thus,
we examined whether the DA analogue CA140 binds toaggregated Aβ and found that CA140 interacted withaggregated Aβ with a ­Kd of 7.8 ± 2.4 µM (Fig. 1A).
We investigated the influence of CA140 on Aβ42 amyloid formation by performing ThT fluorescence assays.
In the absence of CA140, the fluorescence intensity
increased after a lag time of ~ 0.8 h and plateaued at~ 2 h (Fig. 1B, C). In the presence of CA140, similar sigmoidal ThT kinetics were observed, and the intensityof ThT intensity at the saturation phase decreased withincreasing CA140 concentration (Fig. 1B, C). We quantified amyloid fibrils using the ThT fluorescence intensity
at the endpoint of the experiments (i.e., the final ThT
intensities at 3 h) and found that as the concentrationof CA140 increased from 0 to 100 μM (5 molar equivalents of CA140 with respect to the concentration ofAβ42), the final ThT intensity decreased from ~ 1.6 × ­106
(0 μM CA140) to ~ 1.3 × ­106 (25 μM CA140) to ~ 1.0 × ­106
(100 μM CA140) (Fig. 1C). These results confirmed that
CA140 inhibits nucleation-limited Aβ42 amyloid fibrillation in a dose-dependent manner.
To examine whether CA140 affects Aβ levels in vitro,
primary cortical neurons (PCNs) and APP-overexpressing CHO cells were treated with vehicle (1% DMSO) or
CA140 (1 or 5 μM) for 24 h, and Aβ levels were measured
by ELISA. Compared with vehicle, 5 µM CA140 significantly reduced mouse and human Aβ levels in PCNs and
APP-overexpressing CHO cells, respectively (Fig. 1D, E).
Next, to determine the effects of CA140 on Aβ plaquelevels in aged 5xFAD mice, 8-month-old 5xFAD mice(late-phase AD mouse model with severe Aβ accumulation) were injected with CA140 (30 mg/kg, i.p.) or vehicle (10% DMSO) daily for 14 days, and brain sections
were immunostained with an anti-Aβ17-24 (4G8) antibody recognizing Aβ plaques. We found that CA140
treatment significantly reduced the number of Aβplaques in the cortex and hippocampal DG region (butPage 10 of 34

not the hippocampal CA1 region) in 5xFAD mice compared with vehicle treatment (Fig. 1F–I).
To further investigate the effects of CA140 on Aβplaque number with or without formic acid, 8-monthold 5xFAD mice were injected with CA140 (30 mg/kg,
i.p.) or vehicle (10% DMSO) daily for 14 days, and brain
sections were pretreated with 70% formic acid before
immunostaining with an anti-Aβ17-24 (4G8) antibody.
Consistent with the findings in Fig. 1, CA140 treatment significantly reduced the number of Aβ plaquesin the cortex, and immunostaining was not affected by
pretreatment with formic acid (Supplementary Fig. 1).
Taken together, these data imply that CA140 interacts
with aggregated Aβ to reduce Aβ fibril formation, Aβlevels, and Aβ plaque number in vitro and/or in aged
5xFAD mice.
Since CA140 reduced Aβ fibrillation and Aβ plaquenumber, we further examined the effects of CA140 on
tau fibril formation using the ThT fluorescence assay.
In this study, we used 2N4R tau, a 441-amino-acid tau
protein that is the largest human tau isoform. In theabsence of CA140, the ThT fluorescence intensity of
30 μM tau increased rapidly after a lag phase of ~ 2 hand reached a plateau at ~ 4 h (Fig. 1J, K), indicating
nucleation-dependent tau fibril formation. Treatment
with CA140 (5 or 20 molar equivalents) decreased ThT
intensity (Fig. 1J, K), indicating that CA140 inhibits
2N4R tau fibril formation.
We then investigated whether CA140 affects tau
hyperphosphorylation in aged 5xFAD mice. Eightmonth-old 5xFAD mice were injected with vehicle (10%DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days,
and immunostaining of brain slices was performed
with an anti-AT100 antibody, which detects tau thatis hyperphosphorylated at Thr212/Ser214. Comparedwith vehicle treatment, CA140 treatment significantly
reduced tau hyperphosphorylation at Thr212/Ser214
in the hippocampal CA1 and DG regions (Fig. 1L, M).
These data indicate that CA140 inhibits tau fibril formation and downregulates tau phosphorylation in anaged AD mouse model.

(See figure on next page.)
Fig. 1 CA140 reduces Aβ/tau aggregate formation, Aβ levels, and AD pathology in vitro and in vivo. A Graph of fluorescence intensity
versus concentration of CA140 in the presence of aggregated Aβ42 (average fluorescence measurements from three independent experiments).
B Real-time monitoring of the inhibitory effects of CA140 on amyloid formation. C Quantification of the ThT intensity of Aβ42 at the final time
point. D, E Aβ levels in primary cortical neurons and APP-overexpressing CHO cells. F–I Eight-month-old 5xFAD mice were injected with vehicle(10% DMSO) or CA140 (30 mg/kg) daily for 14 days, and immunofluorescence (IF) staining of brain slices was conducted with an anti-Aβ17-24(4G8) antibody (n = 16 brain slices from 4 mice/group). J, K Quantification of the ThT intensity of 2N4R full-length tau at the final time point. L,
M 8-month-old 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and IF staining of brain slices
was conducted with an anti-TauThr212/Ser214 (AT100) antibody (Veh, n = 18 brain slices from 4 mice; CA140, n = 20 brain slices from 4 mice). Scalebar = 200 μm. *p < 0.05, **p < 0.01

Chae et al. Journal of Neuroinflammation

Fig. 1 (See legend on previous page.)(2024) 21:200

Page 11 of 34

Chae et al. Journal of Neuroinflammation(2024) 21:200

CA140 downregulates genes involvedin neuroinflammation in a mouse model of AD

We previously reported that CA140 has anti-inflammatory effects on LPS-evoked neuroinflammation in
WT mice and in 3-month-old 5xFAD mice (a model ofthe early phase of AD in which the neuroinflammatory
response occurs) [17]. To extend these findings, we performed RNA sequencing to investigate the molecularmechanism by which CA140 inhibits neuroinflammatory
responses in 3-month-old 5xFAD mice. Three-month-old
5xFAD mice were injected with vehicle (10% DMSO) or
CA140 (30 mg/kg, i.p.) daily for 14 days. The hippocampus was dissected, and RNA sequencing was performed.
By comparing mRNA expression profiles, we identified
822 differentially expressed genes (DEGs), including 183
upregulated and 639 downregulated genes, in CA140injected 5xFAD mice compared with vehicle-injected
5xFAD mice (Fig. 2A, B and Table S2). Notably, CA140
treatment downregulated several glial cell-specificmarker genes in 3-month-old 5xFAD mice (Fig. 2B): iba1
for microglia, gfap for astrocytes, cxcl10 for AD-associated RAs, trem2 and grn for MGnDs, clec7a and itgax for
DAMs, and c1qa and cr3 for interactions between RAs
and DAMs (Fig. 2A, B, E and Table S2). In addition, we
found that CA140 treatment significantly reduced themRNA levels of cd68 (a marker of M1 microglia) and
gbp2 (a marker of A1 astrocytes) in this mouse model ofthe early phase of AD (Fig. 2B, E).
To further examine the cellular processes affected by
CA140, we performed enrichment analysis of the up- or
downregulated DEGs using DAVID software [34]. The
DEGs that were downregulated by CA140 treatment
in 5xFAD mice were strongly associated with pathways
related to the inflammatory response (cytokine production, TLR, RIG-like/NOD, NF-κB, and Nlrp3 inflammasome signaling pathways) and glial cell activation/
proliferation (Fig. 2C, E and Table S3A). By contrast,
the DEGs that were upregulated by CA140 treatment
in 5xFAD mice were mainly associated with pathways

Page 12 of 34

related to synaptic function in the hippocampus (learning or memory, cognition, synaptic signaling, and cAMP/
calcium signaling pathways) (Fig. 2D and Table S3B).
To validate the RNA sequencing results, we performed
real-time PCR and confirmed that representative inflammatory response-related molecules (nlrp3 and il1β) andglial cell markers (iba-1 and gfap) were downregulated in
CA140-injected 5xFAD mice (Fig. 2F).
To visualize the collective actions of CA140 in thismouse model of AD, we constructed a network model
describing the interactions among the downregulated
inflammatory response-related genes based on interaction information in the KEGG pathway database and
WikiPathways [35, 36]. The network model suggested
that CA140 downregulates TLR1/4-dependent signaling, which is followed by inhibition of the downstream
NF-κB and IRF5/7 signaling pathways that produce proinflammatory cytokines/chemokines and Nlrp3 inflammasome-mediated cytokine production (Fig. 2G). These
data suggest that CA140 reduces neuroinflammatory
responses through NLRP3 in 3-month-old 5xFAD mice.
CA140 downregulates microglial/astrocyte activationin 8‑month‑old 5xFAD mice

Neuroinflammation is closely associated with Aβ/tau
pathology in AD mouse models and patients with AD andis predominantly regulated by glial cells [38, 39]. Since
CA140 downregulated neuroinflammatory responses ina mouse model of the early phase of AD (3-month-old
5xFAD mice), we examined whether CA140 regulates
neuroinflammatory-associated glial activation in an aged
AD mouse model. Eight-month-old 5xFAD mice were
injected with CA140 (30 mg/kg, i.p.) or vehicle (10%DMSO) daily for 14 days, and IF staining was conductedwith anti-Iba-1 (a microglial marker) and anti-GFAP(an astrocyte marker) antibodies. We found that CA140
treatment significantly diminished Iba-1 fluorescenceintensity and the number of Iba-1-positive cells in the
hippocampal CA1 region but not in the hippocampal(See figure on next page.)
Fig. 2 Identification of genes affected by CA140 in 3-month-old 5xFAD mice. A Heatmap of 183 upregulated and 639 downregulated genes
in CA140-injected 5xFAD mice compared with vehicle-injected 5xFAD mice. B Volcano plot showing differentially expressed genes (DEGs)
in CA140-treated 5xFAD mice. The X- and Y-axes present the ­log2-fold-change and –log10 (p value), respectively. Red and green dots represent
upregulated and downregulated genes, respectively. Gray dots represent genes without significant differences in expression. C, D Gene ontology
biological processes (GOBPs) represented by the downregulated (C) and upregulated (D) genes. The dotted line indicates the p value cutoff
used. The number of genes in each biological process is indicated in parentheses. E DEGs involved in the inflammatory response, glial cell
activation/proliferation, and Nlrp3 inflammasome signaling. The color bar shows the z-score gradient. F Relative mRNA levels of the indicatedgenes in CA140- or vehicle-treated 5xFAD mice were analyzed by real-time PCR (n = 2 mice/group). G Network model describing the interactions
among inflammatory response-related signaling pathways. The node colors represent downregulation (green) and no change (yellow)of the corresponding genes in CA140-injected 5xFAD mice. Nodes are arranged and connected according to the activation (arrows) informationin the KEGG pathway and WikiPathways databases. Solid and dotted lines denote direct and indirect interactions, respectively. “+p”, phosphorylation.
“+u”, ubiquitination. *p < 0.05, **p < 0.01

Chae et al. Journal of Neuroinflammation

Fig. 2 (See legend on previous page.)(2024) 21:200

Page 13 of 34

Chae et al. Journal of Neuroinflammation(2024) 21:200

Page 14 of 34

Fig. 3 CA140 injection regulates microglial activation and astrocytic morphology in 8-month-old 5xFAD mice. A–H Eight-month-old 5xFAD mice
were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and immunofluorescence staining of brain slices was conductedwith anti-Iba-1 and anti-GFAP antibodies (Iba-1: Veh, n = 23–24 brain slices from 4 mice; CA140, n = 17 brain slices from 4 mice; GFAP: Veh, n = 24
brain slices from 4 mice; CA140, n = 17 brain slices from 4 mice). Scale bar = 200 μm. *p < 0.05, **p < 0.01

Chae et al. Journal of Neuroinflammation(2024) 21:200

DG region (Fig. 3A–D). In addition, CA140 treatment
only significantly decreased the number of GFAPpositive cells in the hippocampus and had no effect on
GFAP fluorescence intensity or the GFAP-positive area
(Fig. 3E–H). These results demonstrate that CA140 also
suppresses neuroinflammatory responses in an aged AD
mouse model and that this suppression occurs via thedownregulation of microglial and astrocyte activation.
CA140 downregulates neuroinflammatory responses
and AD‑associated reactive gliosis in 8‑month‑old 5xFAD
mice

A recent study found that the induction of neuroinflammation by proinflammatory cytokines, including IL-1β,
is activated by NLRP3 inflammasome formation in thebrain in 5xFAD mice [40]. Our real-time PCR analysis demonstrated that CA140 treatment significantly
reduced nlrp3 and il-1β mRNA levels in the brain in
3-month-old 5xFAD mice (Fig. 2). We therefore examined whether CA140 modulates NLRP3 expression to
downregulate neuroinflammatory responses in an aged
AD mouse model. Eight-month-old 5xFAD mice were
injected with CA140 (30 mg/kg, i.p.) or vehicle (10%DMSO) daily for 14 days, and IF staining was conductedwith anti-NLRP3 and anti-IL-1β antibodies. Comparedwith vehicle, CA140 significantly reduced NLRP3 levels in the hippocampus in 8-month-old 5xFAD mice
(Fig. 4A, C). In addition, CA140 treatment significantly
reduced the levels of the proinflammatory cytokine IL-1β
in the hippocampus in 5xFAD mice (Fig. 4B, D).
Since we observed that CA140 treatment diminishedmicroglial/astrocyte activation and proinflammatory
cytokine IL-1β and NLRP3 levels in a mouse model ofAD, we assessed the effects of CA140 on neuroinflammatory dynamics and the functional states of neuroinflammatory responses in vivo. For this experiment,
8-month-old 5xFAD mice were injected with CA140(30 mg/kg, i.p.) or vehicle (10% DMSO) daily for 14 days,and IF staining was conducted with anti-CXCL10 andanti-C1QA antibodies. We found that CA140 treatment significantly reduced the expression of CXCL10(a marker of RAs) and C1QA (a marker of interactions
between RAs and DAMs) in the hippocampus in 5xFAD
mice (Fig. 4E–H). We also examined the effects of CA140
on M1/M2 and A1/A2 polarization states in this aged
AD mouse model and found that CA140 treatment did
not alter the mRNA expression of GBP2 (a marker of
A1 astrocytes), CD68 (a marker of M1 microglia), andCD206 (a marker of M2 microglia) in 5xFAD mice (Supplementary Fig. 2).
Taken together, these results reveal that CA140 inhibits neuroinflammatory responses by suppressing NLRP3Page 15 of 34

activation and reactive gliosis in an aged AD mousemodel.
CA140 decreases reactive gliosis in primary astrocytes
and primary microglia from 5xFAD mice

PACs and PMCs were used to investigate the effect ofCA140 on a broad spectrum of astroglial/microglial reactive states, including AD-associated RAs [41, 42], homeostatic microglia, MGnDs, DAMs, PAMs, and LDAMs[43, 44]. For this experiment, mixed glial cells preparedfrom whole brains of postnatal day 1–2 5xFAD pups
were cultured for 3 weeks. Then, PACs and PMCs wereisolated and treated with vehicle (1% DMSO) or CA140(5 μM) for 24 h, and real-time PCR was conducted. We
found that CA140 treatment significantly decreased themRNA levels of s100β (a cell signaling marker of ADassociated RAs) and cxcl10 (a secreted protein marker ofAD-associated RAs) in PACs from 5xFAD mice (Fig. 5A,
B). However, CA140 treatment did not alter the mRNAlevels of metabolism, cytoskeleton, or chaperone markers of AD-associated RAs in PACs from 5xFAD mice
(Fig. 5C–E). In addition, CA140 significantly reduced themRNA levels of cr3 and c1qa (markers of the interactionsof RAs and DAMs) in PACs from 5xFAD mice (Fig. 5F).
These data suggest that CA140 alters neuroinflammatory
responses by modulating the expression of cell signaling
and secreted protein markers of AD-associated RAs andmarkers of the interactions of RAs and DAMs in PACs
from 5xFAD mice (Fig. 5G).
In PMCs from 5xFAD mice, CA140 treatment significantly increased the mRNA levels of cx3cr1 and p2ry12(markers of homeostatic microglia) (Fig. 5H). Interestingly, in PMCs from 5xFAD mice, CA140 treatment significantly downregulated the mRNA levels of a marker
of PAMs, gpnmb whose level was also increased in the
MGnDs (Fig. 5I) [45]. However, CA140 treatment did notmodulate the mRNA levels of markers of DAMs (itgax,
clec7a, lpl, cstd, and cd44) and MGnDs (apoE, spp1,
trem2, and grn) in PMCs from 5xFAD mice (Fig. 5J, K).
In addition, CA140 decreased the mRNA levels of cln3 (amarker of LDAMs) and cr3 and c1qa (markers of interactions of RAs and DAMs) in PMCs from 5xFAD mice
(Fig. 5L–N). Collectively, these experiments show that
CA140 downregulates reactive gliosis during the neuroinflammatory dynamics of AD pathophysiology andupregulates homeostatic microglial function in PACs
and/or PMCs.
CA140 prevents scopolamine (SCO)‑induced impairments
in long‑term memory, dendritic spine number, and LTP
in WT mice

To determine whether CA140 regulates cognitive andsynaptic function under pathological conditions, weChae et al. Journal of Neuroinflammation(2024) 21:200

Page 16 of 34

Fig. 4 CA140 administration reduces NLRP3, IL-1β levels and reactive gliosis in 8-month-old 5xFAD mice. A–H Eight-month-old 5xFAD mice were
injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and immunofluorescence staining of brain slices was performed
with anti-NLRP3 (A, B), anti-IL-1β (C, D), anti-CXCL10 (E, F) and anti-C1QA (G, H) antibodies (n = 16–23 brain slices from 4 mice). Scale bar = 200 μm.
*p < 0.05, **p < 0.01, ***p < 0.001

Chae et al. Journal of Neuroinflammation(2024) 21:200

used a model of amnesia induced by scopolamine (SCO),
a muscarinic acetylcholine receptor antagonist that is
involved in hippocampal LTP reduction and memory
impairment [46, 47]. WT mice were injected with CA140(30 mg/kg, i.p.) or vehicle (10% DMSO) daily for 14 days.
On days 3 to 14, the mice were also injected with SCO(1 mg/kg, i.p.) or PBS daily. Behavior experiments were
performed on days 12 to 14. Interestingly, compared withvehicle treatment, SCO treatment significantly impaired
long-term memory but had no effect on short-term
spatial memory in WT mice (Fig. 6A, B). Importantly,
CA140 treatment rescued SCO-induced long-term memory deficits in WT mice but did not affect short-term
memory (Fig. 6A, B).
To demonstrate the molecular mechanisms by which
CA140 improves cognitive function, the effects of CA140
on dendritic spine formation, basal transmission, and
LPT in SCO-injected WT mice were analyzed. First, todetermine whether CA140 modulates dendritic spine
formation in SCO-treated WT mice, Golgi staining was
performed. Dendritic spine density in the hippocampal
basal shaft (BS), cortical apical oblique (AO) and cortical
BS region was significantly reduced in SCO-treated WT
mice compared with vehicle-treated WT mice (Fig. 6C–
E). CA140 treatment restored hippocampal and cortical dendritic spine numbers in SCO-treated WT mice
(Fig. 6C–E), indicating that CA140 rescues SCO-mediated dendritic spine loss in WT mice.
Second, to examine the potential recovery effect ofCA140 treatment on hippocampal LTP, WT mice were
injected with vehicle (10% DMSO) or CA140 (30 mg/kg,
i.p.) daily for 14 days and PBS or SCO (1 mg/kg, i.p.) daily
on days 3 to 14. Subsequent measurement of basal synaptic transmission in the hippocampus (Fig. 6F–H) andinput–output curve analysis showed that basal synaptic transmission at the SC-CA1 pathway was unchanged
in SCO-treated WT mice compared to vehicle-treated
WT mice. Administration of CA140 to SCO-treated
WT mice also had no effect on basal synaptic transmission (Fig. 6F–H). However, consistent with previous
findings [32, 33], SCO treatment suppressed LTP induction at SC-CA1 synapses in WT mice (Fig. 6I–M). More
importantly, CA140 treatment fully reversed LTP attenuation in SCO-treated WT mice (Fig. 6I–M). These results

Page 17 of 34

demonstrate that CA140 significantly reduces the inhibition of LTP by SCO in vivo.
CA140 restores long‑term memory, dendritic spine
number, LTP, and synaptic function in aged 5xFAD mice

Given the positive effects of CA140 on cognitive function, dendritic spine number, and LTP in the SCOmediated amnesia mouse model, we further investigatedthe effects of CA140 on synaptic/cognitive function
in an aged AD mouse model. Eight-month-old 5xFAD
mice were injected with vehicle (10% DMSO) or CA140(30 mg/kg, i.p.) daily for 17 days, and Y-maze and NOR
tests were conducted on days 15 to 17. Strikingly, CA140
treatment increased the object preference (%) in the
NOR test but not alternation triplets (%) in the Y-maze
test compared with vehicle treatment (Fig. 7A, B). These
results indicate that CA140 improves long-term memory
deficits in aged 5xFAD mice.
To further explore the memory-improving effects of
CA140 in 8-month-old 5xFAD mice, we assessed whether
CA140 modulates dendritic spine formation, which is
associated with cognitive function [48]. In 8-month-old
5xFAD mice, CA140 treatment significantly increased
dendritic spine number in the hippocampal AO and BS
region (Fig. 7C, D) and cortical AO region but not in thecortical BS (Supplementary Fig. 3). These data indicatethat CA140 improves long-term memory and dendritic
spine formation in aged 5xFAD mice.
Cognitive function is closely associated with hippocampal synaptic strength, and 5xFAD mice that are
older than 6 months exhibit memory dysfunction andattenuated LTP compared with WT mice [49, 50]. We
therefore examined the effect of CA140 on LTP induction in 8-month-old 5xFAD mice and found that CA140
tended to increase LTP, although not significantly(Fig. 7E–G). Taken together, these results demonstratethat CA140 ameliorates impairments in cognitive dysfunction, dendritic spine density, and LTP in an aged AD
mouse model.
To further investigate the effects of CA140 on synaptic function in 5xFAD mice, 6-month-old 5xFAD mice
were injected with vehicle (10% DMSO) or CA140 (30mg/kg, i.p. for consecutive 14 days); then, mEPSCs
were monitored. We found that CA140 treatment(See figure on next page.)
Fig. 5 CA140 treatment regulates reactive gliosis in primary astrocytes (PACs) and primary microglia (PMC) from 5xFAD mice. A–F PACs from 5xFAD
mice were treated with vehicle (1% DMSO) or CA140 (5 μM) for 24 h, and the relative mRNA levels of the indicated genes were analyzed by real-time
PCR (n = 12–15/group). G Summary illustration of the regulatory effect of CA140 on reactive astrogliosis in PACs from 5xFAD mice. H–M PMCs
from 5xFAD mice were treated with vehicle (1% DMSO) or CA140 (5 μM) for 24 h, and the relative mRNA levels of the indicated genes were analyzed
by real-time PCR (n = 5–6/group). N Summary illustration of the regulatory effect of CA140 on microglial reactive state in PMCs from 5xFAD mice.
*p < 0.05, **p < 0.01, ***p < 0.001

Chae et al. Journal of Neuroinflammation

Fig. 5 (See legend on previous page.)(2024) 21:200

Page 18 of 34

Chae et al. Journal of Neuroinflammation(2024) 21:200

markedly increased the frequency but not the amplitudeof mEPSCs in 6-month-old 5xFAD mice (Fig. 7H–J).
These data indicate that CA140 treatment may facilitatepresynaptic function or increase the number of synapses
to alter synaptic and cognitive function in aged 5xFAD
mice (Fig. 7H–J).
We then examined whether CA140 modulates presynaptic and postsynaptic protein expression to rescue cognitive and synaptic function in aged 5xFAD mice. For thisexperiment, eight-month-old 5xFAD mice were injectedwith vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) dailyfor 14 days, and IF staining was conducted with anti-synaptophysin and anti-PSD-95 antibodies. We found that
CA140 treatment significantly increased synaptophysin
expression in the hippocampal CA1 region in 8-monthold 5xFAD mice (Fig. 7K–L). However, CA140 treatment
did not alter PSD-95 expression in the hippocampal CA1
region in 8-month-old 5xFAD mice (Fig. 7M, N). These
data indicate that CA140 improves presynaptic performance in aged 5xFAD mice to restore cognitive and synaptic function.
CA140 improves cognitive function through DRD1
signaling in 8‑month‑old 5xFAD mice

We previously demonstrated that the DA analogue
CA140 suppresses the LPS-evoked inflammatory
response via modulating DRD1 in BV2 microglial cells[17]. Here, we followed up on this finding by testingwhether CA140 acts as an agonist or antagonist of DRD1.
HEK293 cells were transfected with DRD1 plasmid
DNA for 48 h, pretreated with a LE300 (10 μM, DRD1
antagonist) or vehicle (1% DMSO) for 30 min, and finallytreated with A77636 (5 μM, DRD1 agonist), DA (10 or
100 μM), or CA140 (10, 50, or 100 μM) for 1 h. Subsequent cAMP ELISA analysis showed that treatment with
A77636, DA, or CA140 significantly increased cAMP levels compared with vehicle treatment (Fig. 8A). However,pretreatment with LE300 followed by CA140 treatment

Page 19 of 34(100 μM) did not enhance cAMP levels, suggesting that
CA140 increases cAMP levels by acting as a DRD1 agonist (Fig. 8A).
Next, we investigated whether CA140 regulates cognitive function in a DRD1-dependent manner underpathological conditions. Eight-month-old 5xFAD mice
were injected with AAV-DRD1 shRNA or AAV-control
shRNA in the bilateral hippocampal CA1 regions. Three
weeks after AAV injection, the mice were injected withvehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) dailyfor 14 days, followed by IF staining of brain slices withan anti-DRD1 antibody. We found that treatment with
DRD1 shRNA and vehicle significantly reduced DRD1
levels compared with treatment with control shRNAand vehicle, confirming successful DRD1 shRNA infection in the hippocampal CA1 region in aged 5xFAD
mice (Fig. 8B, C). Cognitive behavioral tests (Y-maze andNOR) were then performed. Consistent with the findings presented in Fig. 7A, neither treatment with control
shRNA and CA140 nor treatment with DRD1 shRNA
and CA140 affected short-term memory compared withtreatment with control shRNA and vehicle (Fig. 8D).
However, treatment with control shRNA and CA140
improved long-term memory compared with treatment
with control shRNA and vehicle (Fig. 8E). More importantly, treatment with DRD1 shRNA and CA140 did not
improve long-term memory compared with treatment
with DRD1 shRNA and vehicle (Fig. 8E). These data indicate that CA140 enhances cognitive function throughDRD1 in an aged AD mouse model.
We further investigated the molecular mechanisms
by which CA140 improves cognitive function underpathological conditions. Because CaMKIIα/ERK/ELK-1
signaling is closely associated with dendritic spine formation and synaptic plasticity [51–53], we examined
whether CA140 modulates downstream CaMKIIα/ERK/
ELK-1 signaling in a DRD1-dependent manner in aged
5xFAD mice. For this experiment, 8-month-old 5xFAD(See figure on next page.)
Fig. 6 CA140 treatment reverses scopolamine (SCO)-induced impairments in long-term memory, dendritic spine number, and LTP in wild-type(WT) mice. A, B WT mice were injected daily with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) for 14 days. On days 3–14, the mice werealso injected daily with PBS or SCO (1 mg/kg, i.p.). Y-maze and NOR tests were performed on days 12 and 14, respectively (n = 8 mice/group).
C–E Representative AO and BS dendrites from the hippocampal CA1 region and cortical layer V region of mice treated with CA140 and/or SCO(hippocampus AO: Veh, n = 77 neurons from 8 mice; SCO, n = 76 neurons from 8 mice; SCO + CA140, n = 76 neurons from 8 mice; hippocampus
BS: Veh, n = 76 neurons from 8 mice; SCO, n = 77 neurons from 8 mice; SCO + CA140, n = 76 neurons from 8 mice; cortex AO and BS: n = 24 neurons
from 4 mice/group). Scale bar = 10 μm. F WT mice were injected daily with CA140 (30 mg/kg, i.p.) or vehicle (10% DMSO) for 14 days and werealso injected with SCO (1 mg/kg, i.p.) or PBS on days 3–14. G Representative excitatory postsynaptic current (EPSC) traces from the vehicle, SCO,
and SCO + CA140 treatment groups. H Input–output curves from the vehicle, SCO, and SCO + CA140 treatment groups (Veh, n = 18 cells from 5
mice; SCO, n = 17 cells from 5 mice; SCO + CA140, n = 14 cells from 5 mice). I Representative EPSC traces before and after LTP induction in the vehicle,
SCO, and SCO + CA140 treatment groups (1: before LTP; 2: after LTP). J, K Effects of SCO on LTP induction in the presence/absence of CA140. L
Overlay of the two graphs in J, K. M Summary statistics for LTP induction (last 5 min: Veh, n = 20 cells from 8 mice; SCO, n = 22 cells from 8 mice;
SCO + CA140, n = 22 cells from 7 mice). *p < 0.05, **p < 0.01, ***p < 0.001

Chae et al. Journal of Neuroinflammation

Fig. 6 (See legend on previous page.)(2024) 21:200

Page 20 of 34

Chae et al. Journal of Neuroinflammation(2024) 21:200

mice were injected with control shRNA or DRD1 shRNAvirus into the hippocampal CA1 region. Three weeks
after virus injection, the mice were injected with vehicle(10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 17 days.
Subsequent western blotting of hippocampal slices
showed that treatment with control shRNA and CA140
did not alter p-CaMKIIα and p-ERK levels comparedwith treatment with control shRNA and vehicle-treated
5xFAD mice (Fig. 8F, G). In addition, treatment with
DRD1 shRNA and CA140 did not change p-CaMKIIαand p-ERK levels compared with treatment with DRD1shRNA and vehicle-treated 5xFAD mice (Fig. 8F, G). By
contrast, treatment with control shRNA and CA140 suppressed p-ELK1 levels compared with treatment with
control shRNA and vehicle (Fig. 8H), but treatment with
DRD1 shRNA and CA140 did not significantly decreased
p-ELK1 levels compared with treatment with DRD1shRNA and vehicle-treated 5xFAD mice (Fig. 8H). These
data indicate that the regulatory effect of CA140 on cognitive function is partially regulated by DRD1/Elk-1 signaling in aged 5xFAD mice.
CA140 downregulates Aβ pathology via DRD1 signalingin 8‑month‑old 5xFAD mice

Since CA140 improved long-term recognition memory through DRD1 signaling under pathological conditions, we next examined whether CA140 attenuatesAβ pathology in a DRD1-dependent manner in anaged AD mouse model. Eight-month-old 5xFAD mice
were injected with AAV-DRD1 shRNA or AAV-control
shRNA in the bilateral hippocampal CA1 region. Three
weeks after AAV injection, the mice were administeredvehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for
14 days. IF staining of brain slices was then performed
with anti-Aβ17-24 (4G8) antibody. Treatment with control shRNA and CA140 significantly reduced Aβ plaquenumber in 5xFAD mice compared with treatment with
control shRNA and vehicle (Fig. 8I, J). More importantly,
treatment with DRD1 shRNA and CA140 did not affect

Page 21 of 34

Aβ plaque number in 5xFAD mice compared with treatment with DRD1 shRNA and vehicle (Fig. 8I, J). These
data indicate that CA140 reduces Aβ pathology by modulating DRD1 signaling under pathological conditions.
CA140 enhances long‑term memory and dendritic spine
number in WT mice

To examine whether CA140 modulates cognitive andsynaptic function under normal conditions, WT mice
were injected with vehicle (10% DMSO) or CA140(30 mg/kg) daily for 7 days and then subjected to Y-mazeand novel object recognition (NOR) tests on days 8 to 10.
CA140 treatment significantly enhanced the long-term
memory but not short-term memory of WT mice compared with vehicle treatment (Fig. 9A, B), indicating that
CA140 affects cognitive function in WT mice.
We then investigated whether vehicle (10% DMSO)
treatment has specific or non-specific effects on memory
function in WT mice. WT mice were injected with water(control for vehicle), 10% DMSO (vehicle), or CA140(30 mg/kg in 10% DMSO, i.p.) daily for 17 days, and cognitive function was analyzed on days 15 to 17. We foundthat vehicle treatment did not alter memory function in
WT mice compared to water treatment (Supplementary Fig. 4A, B). However, CA140 treatment significantly
increased long-term memory in WT mice compared tovehicle treatment (Supplementary Fig. 4A, B). These datasuggest that the apparent effects of CA140 treatment on
cognitive function in WT mice are not due to effects ofvehicle (10% DMSO).
After the behavioral tests, the systemic toxicity of
CA140 treatment in WT mice was assessed by western
blotting analysis of cleaved caspase-3 expression. CA140
administration did not induce cleaved caspase-3 expression in the liver and kidney, indicating that CA140 does
not have in vivo toxicity (Supplementary Fig. 4C, D).
Peripheral inflammation is directly or indirectly associated with adverse effects [54]; thus, we examined whether
CA140 treatment influences peripheral proinflammatory(See figure on next page.)
Fig. 7 CA140 improves long-term memory and dendritic spine number in 5xFAD mice. A, B Y-maze and NOR tests of 8-month-old 5xFAD mice
injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 17 days (n = 10 mice/group). C, D Representative hippocampal AO and BS
dendrites of 8-month-old 5xFAD mice injected with vehicle (10% DMSO) or 30 mg/kg CA140 daily for 14 days (AO: Veh, n = 39 neurons from 5 mice;
CA140, n = 35 neurons from 5 mice; BS: Veh, n = 35 neurons from 5 mice; CA140, n = 37 neurons from 5 mice). Scale bar = 10 μm. E Measurementof LTP in ex vivo hippocampal slices from 8-month-old 5xFAD mice injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days.
Representative excitatory postsynaptic current (EPSC) traces before and after long-term potentiation (LTP) induction are shown (1: before LTP; 2:
after LTP). F Effects of CA140 on LTP induction in 8-month-old 5xFAD mice. G Summary statistics for LTP induction (last 5 min: Veh, n = 26 cells from 7
mice; CA140, n = 23 cells from 7 mice). H Representative traces of mEPSCs of 6-month-old 5xFAD mice injected with vehicle (10% DMSO) or CA140(30 mg/kg, i.p.) daily for 14 days. I, J Summary graphs of mEPSC amplitude and frequency in the vehicle and CA140-treated groups (Veh, n = 19 cells
from 4 mice; CA140, n = 24 cells from 5 mice). K–N Eight-month-old 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.)
daily for 14 days, and immunofluorescence staining was conducted with anti-synaptophysin (K, L) or anti-PSD-95 antibodies (M, N) (n = 16 brainslices from 4 mice/group). Scale bar = 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001

Chae et al. Journal of Neuroinflammation

Fig. 7 (See legend on previous page.)(2024) 21:200

Page 22 of 34

Chae et al. Journal of Neuroinflammation(2024) 21:200

responses under normal conditions. WT mice were
injected with water (for comparison with the effects ofvehicle on proinflammatory cytokine levels), vehicle(10% DMSO), or CA140 (30 mg/kg in 10% DMSO, i.p.)
daily for 17 days, and plasma levels of the proinflammatory cytokines IL-1β and COX-2 were measured. CA140
treatment did not alter plasma IL-1β and COX-2 levels in
WT mice compared with water or vehicle (10% DMSO)
treatment (Supplementary Fig. 4E, F). These data indicatethat chronic administration of CA140 does not induce
adverse effects in WT mice.
Finally, to determine whether CA140 improves longterm memory under normal conditions by enhancing
dendritic spine density, WT mice were injected withvehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) dailyfor 10 days, and Golgi staining was performed to measure dendritic spine number in the hippocampus andcortex. Compared with vehicle treatment, CA140 treatment increased dendritic spine number in the hippocampus but not in the cortex in WT mice (Supplementary
Fig. 5A-C). These observations suggest that CA140 selectively promotes dendritic spine formation in the hippocampus in WT mice.
DRD1 is essential for the effects of CA140 on cognitive
function and dendritic spine density in WT mice

For comparison with the effects of CA140 on cognitive/
synaptic function via DRD1 under pathological conditions (Fig. 8), we investigated whether CA140 works with
DRD1 to improve long-term memory under normal conditions. WT mice were injected with AAV-DRD1 shRNA
or AAV-control shRNA in the bilateral hippocampal CA1
region. One week after AAV injection, the mice were
injected with vehicle (10% DMSO) or CA140 (30 mg/kg,
i.p.) daily for 17 days. IF staining and behavior experiments (Y-maze and NOR) were conducted on days 15
to 17. We found that DRD1 shRNA treatment reduced
DRD1 immunoreactivity in the hippocampal CA1 region

Page 23 of 34

in WT mice, confirming successful transduction of AAVDRD1 shRNA (Fig. 9C).
Consistent with our findings in Fig. 8, treatment with
control shRNA and CA140 improved long-term memory
but not short-term memory in WT mice compared withtreatment with control shRNA and vehicle (Fig. 9D, E).
Treatment with DRD1 shRNA and vehicle did not alter
short- and long-term memory in WT mice comparedwith treatment with control shRNA and vehicle (Fig. 9D,
E). Importantly, the rescue of long-term memory by
CA140 was lost when WT mice were treated with DRD1shRNA and CA140 (Fig. 9D, E). These findings indicatethat CA140 enhances cognitive function through DRD1
under normal conditions.
To demonstrate the molecular mechanisms by which
CA140 promotes cognitive function under normal conditions, WT mice were injected with vehicle (10% DMSO)or CA140 (30 mg/kg, i.p.) daily for 17 days. The hippocampus and cortex were dissected, and western blotting was conducted with p-ELK-1, anti-p-CaMKIIα, or
anti-p-ERK antibodies. Compared with vehicle treatment, CA140 treatment did not alter pELK-1 levelsin the hippocampus and cortex in WT mice (Fig. 9F,
G). In addition, a trend of increased p-CaMKIIα levels
was observed in the hippocampus but not the cortex
in CA140-treated WT mice (Fig. 9H and Supplementary Fig. 6A). Unexpectedly, CA140 treatment did notmodulate p-ERK levels in the hippocampus and cortex
in WT mice (Fig. 9I and Supplementary Fig. 6B). These
data indicate that CA140 treatment upregulates DRD1/
CaMKIIα signaling in WT mice.
CA140 increases dendritic spine formation in primaryhippocampal neurons

To determine whether CA140 affects dendritic spine formation in vitro, primary hippocampal neurons (PHNs)
were transfected with GFP plasmid DNA for 24 h (tovisualize dendritic spines) and subsequently treatedwith CA140 (1 or 5 μM) or vehicle (1% DMSO) for 24 h.

(See figure on next page.)
Fig. 8 CA140 alleviates cognitive function/Aβ pathology through DRD1 signaling in an aged AD mouse model. A HEK293 cells were transfectedwith DRD1 plasmid DNA, pretreated with a DRD1 antagonist (LE300, 10 μM) or vehicle (1% DMSO) for 30 min, and treated with A77636 (5 μM),
dopamine (10 or 100 μM), or CA140 (10, 50, or 100 μM) for 1 h. cAMP levels were then measured by ELISA (n = 3/group). B–E Eight-month-old
5xFAD mice were injected with AAV-DRD1 shRNA or AAV-control shRNA in the bilateral hippocampal CA1 region. Three weeks after AAV injection,
the mice were administered vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and immunofluorescence staining of brain slices
was performed with an anti-DRD1 antibody (n = 16 brain slices from 4 mice/group). Y-maze and NOR tests were performed on days 15–17 (n = 8–9
mice/group) F–H 8-month-old 5xFAD mice were injected with AAV-DRD1 shRNA or AAV-control shRNA in the bilateral hippocampal CA1 region.
Three weeks after AAV injection, the mice were administered vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 17 days, and western blottingof brain lysates was performed with anti-p-Elk1, anti-p-ERK, anti-p-CaMKIIα, or β-actin antibodies (n = 6–8 mice/group). I, J Eight-month-old 5xFAD
mice were injected with AAV-DRD1shRNA or AAV-control shRNA in the bilateral hippocampal CA1 region. Three weeks after AAV injection, the mice
were administered vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and immunofluorescence staining of brain slices was performed
with anti-Aβ17-24 (4G8) antibody (n = 16 brain slices from 4 mice/group). Scale bar = 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001

Chae et al. Journal of Neuroinflammation

Fig. 8 (See legend on previous page.)(2024) 21:200

Page 24 of 34

Chae et al. Journal of Neuroinflammation(2024) 21:200

Page 25 of 34

Fig. 9 CA140 modulates DRD1 signaling to alter cognitive and synaptic function in wild-type (WT) mice. A, B WT mice were injected with vehicle(10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 10 days, and Y-maze and novel object recognition (NOR) tests were conducted (n = 10 mice/group).
C–E Y-maze and NOR tests of WT mice injected with AAV-Control shRNA or AAV-DRD1 shRNA, followed by daily injections of CA140 (30 mg/kg, i.p.)
or vehicle (1% DMSO) for 17 days (n = 4 mice/group). F–I WT mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 17 days,
and western blotting of brain lysates was conducted with anti-p-ELK-1, anti-p-CaMKIIα, or anti-p-ERK antibodies (n = 8 mice/group). *p < 0.05,**p < 0.01, ***p < 0.001

Dendritic spine number was then measured. At 14 daysin vitro (DIV14), which corresponds to the peak of synaptogenesis, dendritic spine number was significantly

higher in PHNs treated with 1 or 5 μM CA140 than in
PHNs treated with vehicle (Fig. 10A, B). In matureneurons (DIV21), treatment with 1 or 5 μM CA140

Chae et al. Journal of Neuroinflammation(2024) 21:200

Page 26 of 34

Fig. 10 CA140 enhances dendritic spine formation by regulating functional synapses in primary hippocampal neurons (PHNs). A, C Dendritic
spine density was measured in GFP-transfected PHNs treated with vehicle (1% DMSO) or CA140 (1 or 5 μM) for 24 h on DIV14 and DIV21. B, D
Quantification of data from A and C (DIV14: Veh, n = 30; 1 μM CA140, n = 47; 5 μM CA140, n = 48; DIV21: Veh, n = 161; 1 μM CA140, n = 60; 5 μM
CA140, n = 50). E–H PHNs were transfected with GFP for 24 h, treated with vehicle (1% DMSO) or CA140 (5 μM) for 24 h, and immunostainedwith anti-synaptophysin (E) or anti-PSD-95 (G) antibodies (synaptophysin intensity: Veh, n = 11; CA140, n = 12; synaptophysin puncta number: Veh,n = 55; CA140, n = 42; PSD-95 intensity: Veh, n = 16; CA140, n = 16; PSD-95 puncta number: Veh, n = 15; CA140, n = 14). Scale bar = 20 μm. **p < 0.01,***p < 0.001

significantly increased dendritic spine number comparedwith vehicle treatment (Fig. 10C, D). These data indicatethat CA140 enhances dendritic spine formation in bothdeveloping and mature PHNs.
We then evaluated whether the enhancement in dendritic spine number was correlated with increased excitatory functional synapses in vitro. For this experiment,
PHNs were transfected with GFP plasmid DNA for 24 h,treated with vehicle (1% DMSO) or CA140 (5 µM) for
24 h, and immunostained with anti-synaptophysin or
anti-PSD-95 antibodies. We found that CA140 treatment
significantly enhanced synaptophysin and PSD-95 puncta
numbers in PHNs without altering the total levels ofsynaptophysin and PSD-95 (Fig. 10E–H), suggesting that
CA140 increases functional synapses during the mature
stages to alter dendritic spine density in PHNs.
CA140 promotes dendritic spine formation through DRD1/
CaMKIIα/ERK signaling in primary hippocampal neurons

To examine the effects of the DA analogue CA140 on
DRD1 levels in vitro, PHNs were transfected with GFP
for 24 h and then treated with vehicle (1% DMSO) or
CA140 (5 μM) for 24 h. Immunocytochemistry with ananti-DRD1 antibody showed that CA140 treatment significantly increased DRD1 levels in PHNs compared withvehicle treatment (Fig. 11A, B).

Chae et al. Journal of Neuroinflammation(2024) 21:200

Next, to determine whether DRD1 is required for the
positive effects of CA140 on dendritic spine formationin vitro, PHNs were transfected with GFP for 24 h,
pretreated with the DRD1 antagonist LE300 (10 μM)
or vehicle (1% DMSO) for 1 h, and treated with vehicle(1% DMSO) or CA140 (5 μM) for 23 h. Measurementof dendritic spine number revealed that treatment
with LE300 and CA140 eliminated the positive effectsof CA140 on dendritic spine formation in PHNs, indicating that CA140 improves dendritic spinogenesis in
a DRD1-dependent manner in vitro (Fig. 11C, D).
We then investigated the molecular mechanisms bywhich CA140 promotes synaptic/cognitive function
under normal conditions by assessing the levels of proteins associated with CaMKIIα/ERK signaling, which
influences synaptic plasticity and cognitive function
by promoting dendritic spine density [55–57]. For
this experiment, GFP plasmid-transfected PHNs weretreated with vehicle (1% DMSO) or CA140 (5 μM) for
24 h, and immunostaining was conducted with anti-pCaMKIIα or p-ERK antibodies. We found that CA140
treatment significantly increased the phosphorylation of CaMKIIα and ERK in PHNs (Fig. 11E–H). By
contrast, treatment with the DRD1 antagonist LE300(10 μM) significantly reduced CaMKIIα and ERK
phosphorylation in PHNs (Fig. 11I–L).
To determine whether CA140 alters dendritic spine
number via CaMKIIα and/or ERK signaling in vitro,
PHNs were transfected with GFP for 24 h and pretreated with the CaMKIIα inhibitor KN93 (10 μM),
the ERK inhibitor PD98059 (10 μM), or vehicle (1%DMSO) for 1 h. Then, the PHNs were treated with
CA140 (5 μM) or vehicle (1% DMSO) for 23 h, anddendritic spine number was measured. Strikingly, pretreatment with KN93 or PD98059 abolished the positive effects of CA140 on dendritic spine number in
PHNs (Fig. 11M–P). These data indicate that CA140
modulates DRD1/CaMKIIα/ERK signaling to promote
synaptic function in PHNs.

Page 27 of 34

Discussion
Dopamine (DA) release from the predominant DA pathways, namely, the mesocorticolimbic and nigrostriatal
pathways, and even from the minor locus coeruleus todorsal hippocampus pathway is associated with cognitive
function and synaptic plasticity [58–62]. Thus, understanding the effects of DA on learning and memory, synaptic strength, and dendritic spinogenesis is critical fordeveloping therapeutic options for the prevention and/or treatment of memory-related diseases, including AD
and Parkinson’s disease (PD). We previously reported
that the DA analogue CA140 reduces neuroinflammatory
responses in WT and 3-month-old 5xFAD mice [17]. In
the present study, we investigated the effects of CA140
on AD pathology and synaptic and cognitive function
and its molecular mechanisms of action under normal and pathological conditions. We found that CA140
directly interacted with aggregated Aβ and decreased Aβpathology and tau hyperphosphorylation in aged 5xFAD
mice. CA140 also mitigated reactive gliosis during theneuroinflammatory dynamics of AD pathophysiology in
5xFAD mice and in PACs and PMCs from 5xFAD mice.
In addition, CA140 rescued impairments in long-term
memory, mEPSCs, LTP and dendritic spinogenesis ina SCO-mediated model of amnesia in WT mice and in
8-month-old 5xFAD mice. Moreover, CA140 ameliorated
AD pathology (including Aβ deposition, tau hyperphosphorylation, and microgliosis) and rescued long-term
memory in partial regulated by DRD1/Elk1 signaling inaged 5xFAD mice. Under normal conditions, CA140 promoted synaptic and cognitive function through effects on
DRD1/CaMKIIα and/or ERK signaling in WT mice and
PHNs. Collectively, our results suggest that CA140 regulates AD pathology, neuroinflammatory responses, andsynaptic and cognitive function via DRD1 signaling in
WT mice and aged 5xFAD mice.
DA and its derivatives suppress Aβ aggregation andinhibit fibrillation of Aβ40 monomers [63, 64]. In addition, DA and its metabolites stabilize neurotoxic Aβ oligomers and reduce Aβ-induced toxicity in vitro [37, 63].
There is also evidence that DA receptors are involved(See figure on next page.)
Fig. 11 CA140 promotes dendritic spine formation through DRD1/CaMKII/ERK signaling in primary hippocampal neurons (PHNs). A, B DRD1 levels
in GFP-transfected PHNs treated with vehicle (1% DMSO) or CA140 (5 μM) for 24 h were measured by immunostaining with anti-DRD1 antibodies(Veh, n = 152; CA140, n = 155). C, D Dendritic spine number in GFP-transfected PHNs pretreated with DRD1 inhibitor (LE300, 10 μM) or vehicle (1%DMSO) for 1 h and treated with CA140 (5 μM) or vehicle (1% DMSO) for 23 h (Veh, n = 79; CA140, n = 54; LE300, n = 72; LE300 + CA140, n = 62). E–H
Immunostaining of p-CaMKIIα or p-ERK in GFP-transfected PHNs treated with CA140 (5 μM) or vehicle (1% DMSO) for 24 h (p-CaMKIIα: Veh, n = 93;
CA140, n = 98; p-ERK: Veh, n = 29; CA140, n = 28). I–L Immunostaining of p-CaMKIIα or p-ERK in GFP-transfected PHNs treated with LE300 (10 μM)
or vehicle (1% DMSO) for 24 h (p-CaMKIIα: Veh, n = 99; LE300, n = 85; p-ERK: Veh, n = 78; LE300, n = 93). Scale bar = 10 μm. M–P GFP-transfected PHNs
were pretreated with vehicle (1% DMSO), CaMKIIα inhibitor (10 μM), or ERK inhibitor (PD98059) for 1 h and treated with CA140 (5 μM) or vehicle (1%DMSO) for 23 h. Then, dendritic spine number was measured (KN93: Veh, n = 37; CA140, n = 41; KN93 + Veh, n = 36; KN93 + CA140, n = 33; PD98059:
Veh, n = 49; CA140, n = 45; PD98059 + Veh, n = 24; PD98059 + CA140, n = 34). Scale bar = 20 μm. *p < 0.05, **p < 0.01, ***p < 0.001

Chae et al. Journal of Neuroinflammation

Fig. 11 (See legend on previous page.)(2024) 21:200

Page 28 of 34

Chae et al. Journal of Neuroinflammation(2024) 21:200

in Aβ aggregation and tauopathy. For example, a DRD1
agonist, A68930 alleviates Aβ-mediated neuropathology, including memory deficits and neuroinflammatory
responses, in Aβ-treated WT mice [65]. Moreover, aDRD1 agonist blocks the interaction of Aβ with growth
hormone secretagogue receptor 1α (GHSR1α), which is
involved in hippocampal plasticity [66]. Overall, theseobservations indicate that DA and its receptor DRD1
might suppress Aβ pathogenic processes. In the present
study, we found that the DA analogue CA140 directly
bound aggregated Aβ and inhibited Aβ/Tau fibrillation(Fig. 1). Moreover, we discovered that CA140 reduced Aβlevels in vitro and attenuated Aβ plaque deposition andtau hyperphosphorylation in aged 5xFAD mice (Fig. 1).
Based on these results, we hypothesized that the DA analogue CA140 modulates AD pathologies through effects
on its receptor DRD1. To test this hypothesis, we examined the effects of CA140 on AD pathologies in an aged
AD mouse model. We found that CA140 did not reduce
Aβ plaque deposition in 5xFAD mice in which DRD1
was knocked down (Fig. 8). Our results and the literature
indicate that CA140 binds directly/indirectly to DRD1
and downregulates AD pathologies under pathologicalconditions. It is also possible that CA140 directly interacts with Aβ to alter AD pathologies via bidirectional
pathways, which we will address in a future study.
Our mRNA-seq analysis identified molecular signatures associated with the actions of CA140 in earlyphase AD mice. CA140 treatment altered the mRNA
expression of 822 genes involved in various cellular
processes, including 40 genes involved in inflammatory
response-related cellular processes (Fig. 2, Tables S2 andS3). Among these 40 genes, we focused on the effects of
CA140 on neuroinflammation, including NLRP3 inflammasome activation, a key regulator of innate immunity,and IL-1β secretion from microglia [67]. Recent studies have shown that DA administration inhibits NLRP3
inflammasome activation and IL-1β secretion in primary human microglia [16, 68]. In the present study, weinvestigated the effect of CA140 on reactive gliosis invarious stages and models of AD (early/late-phase AD
mouse models and PACs and PMCs from 5xFAD mice)at both the mRNA and protein levels. We found that in
3-month-old 5xFAD mice, CA140 treatment significantly
downregulated the mRNA levels of reactive gliosis markers, including trem2/grn (MGnDs), clec7a/itgax (DAMs),
cxcl10 (AD-associated RAs), and cr3/c1qa (interactionsof RAs and DAMs), and the proinflammatory mediatornlrp3/il-1β (Fig. 2). In addition, in 8-month-old 5xFAD
mice, CA140 treatment significantly reduced neuroinflammatory dynamics, including the protein expressionof markers of AD-associated reactive gliosis (CXCL10),NLRP3/IL-1β, and a marker of interactions of RAs andPage 29 of 34

DAMs (C1QA) (Figs. 3, 4). Furthermore, in PACs or
PMCs from 5xFAD mice, CA140 downregulated cellsignaling and secreted protein (s100β, cxcl10) markers of
RAs and markers of PAMs (gpnmb), LDAMs (cln3), andinteractions of RAs and DAMs (cr3, c1qa) and upregulated homeostatic microglial markers (cx3cr1, p2ry12)(Fig. 5).
Our findings raise the following question: why did
CA140 downregulate MGnD and DAM markers inaged 5xFAD mice but not in PMCs from 5xFAD mice?
In 5xFAD mice, AD pathology begins to develop at 2–3
months of age, but the primary glial cells were isolated
from P1–P2 5xFAD pups, which may not have been
old enough to reflect the disease-associated state. PAM
populations have been identified in developing microglia(P7 AD mouse pups), and the expression of the C1 cluster of PAM markers (gpnmb, igf1, spp1, clec7a) was also
upregulated in MGnDs [45]. The present study foundthat in PMCs from P1–2 5xFAD pups, CA140 treatment downregulated a PAM marker gpnmb whose level
was increased in MGnD implying that CA140 can mitigate AD-like pathologies even in the developing state. A
future study will address whether CA140 modulates the
populations of AD-mediated reactive microglia (MGnDs,
DAMs, LDAMs) in PMCs from early- and late-phase AD
mice.
CA140 treatment also affected the expression of genes
in the Toll-like receptor signaling pathway. TLR2/4
expression is increased on glial cells surrounding Aβ proteins [69–71]. Moreover, TLR4 polymorphisms are protective against AD, and cytokine secretion is reduced in amouse model of AD in which TLR4 is knocked out, leading to increased glial cell activation in the brain [72–74].
Additional analyses of the genes that are differentiallyexpressed following CA140 treatment will help elucidate
their roles in neuroinflammation under normal and pathological conditions.
Scopolamine (SCO) induces cognitive decline byantagonizing muscarinic cholinergic receptors (mAChR)
and is widely used in models of cognitive decline (e.g.,dementia and amnesia) [75–78]. Interestingly, in the present study, we observed that SCO treatment decreased
the number of cortical dendritic spines but not the number of hippocampal AO dendritic spines (Fig. 6). What isthe basis of this region-specific antagonistic effect of SCO
on dendritic spine formation in the brains of WT mice?
Because SCO is a mAChR antagonist, differences in the
distribution of mAChR between hippocampal AO/BS
and cortical AO/BS may explain this discrepancy in thedecrease in dendritic spine number. Specifically, mAChR
immunoreactivity is observed in apical dendrites, basal
dendrites, and soma of cortical layer V pyramidal cells[79], whereas in the hippocampal CA1 region, mAChR

Chae et al. Journal of Neuroinflammation(2024) 21:200

immunoreactivity is predominantly distributed in basal
dendrites and soma of pyramidal cells and is largely
absent from naïve apical dendrites [79]. On the basis ofprevious reports and our findings, we speculate that theantagonistic effect of SCO on dendritic spine formationis enhanced in strongly mAChR-immunoreactive regions(cortical AO/BS and hippocampal BS) compared withweakly mAChR-immunoreactive regions (hippocampal AO). The influence of the differential distribution ofmAChR on the effect of SCO on the activation of cholinergic signaling to regulate dendritic spine formation will
be investigated in a subsequent study.
SCO reduces DA turnover in the hippocampus andfrontal cortex, and stimulation of DA receptors rescues
SCO-induced amnesia [80]. Interestingly, a DRD1 agonist
improves SCO-induced cognitive decline by increasing
the secretion of acetylcholine, an essential neurotransmitter for synaptic plasticity [81]. In line with previous
reports, we found that CA140 treatment rescued SCOmediated memory deficits and dendritic spine loss in
WT mice (Fig. 6). These results suggest that CA140
affects DA metabolism and/or acetylcholine secretion to
alleviate SCO-induced memory impairment. Strikingly,
we found that CA140 treatment completely recovered
SCO-induced LTP impairments at hippocampal SC-CA1
synapses (Fig. 6). Given that LTP impairments in the hippocampus can be rescued by activation of cholinergic
signaling [82, 83] or DA receptor agonists [84], our findings imply that CA140 might modulate hippocampal
LTP by interacting with dopaminergic and/or cholinergic signaling. In a future study, we will examine whether
CA140 modulates the cholinergic system to regulate cognitive/synaptic function in SCO-treated WT mice.
DA receptors (­DRD1-5) are distributed throughout the
hippocampus [9], implicating the DA system in regulating learning and memory and dendritic spine formation[8, 85]. Specifically, DA promotes dendritic spine formation via DRD1 and DRD2 and participates in dendritic spine enlargement and structural plasticity [12].
In addition, experiments in mouse models have shownthat manipulating the activities of DRD1 and/or DRD5
affects recognition memory enhancement and hippocampal memory consolidation [13]. Furthermore, dorsal hippocampal DA originating from the locus coeruleus
enhances spatial memory via DRD1/5 [58]. Here, weinvestigated the effects of CA140 on synaptic/cognitive
function and its molecular mechanisms of action underpathological conditions and found that CA140 acted asa DRD1 agonist to rescue long-term memory/dendritic
spine loss and improve synaptophysin expression in aged
5xFAD mice (Figs. 7,8). We next determined whether
CA140 regulates synaptic and cognitive function in aged
AD mice via Ras-CaMKIIa-ELK1 signaling, which isPage 30 of 34

involved in Aβ-mediated synaptic dysfunction [53, 86].
Phosphorylation of ELK1 initiates neuronal death byenabling the translocation of ELK1 from dendrites to the
nucleus and thus is involved in the progression of neurodegenerative diseases (e.g., AD and PD) [87, 88]. Importantly, we demonstrated that treatment with control
shRNA and treatment downregulated pELK-1 expression in aged 5xFAD mice, whereas treatment with DRD1shRNA and CA140 did not significantly decrease pELK-1
expression compared with treatment with DRD1 shRNAand vehicle (Fig. 9). These data indicate that DRD1/
ELK-1 might be involved in the positive effects of CA140
on cognitive function in 8-month-old 5xFAD mice. Of
course, it is possible that CA140 also directly interacts
with Aβ (e.g., via bidirectional pathways involving DRD1
and Aβ) or DRD2 (which is involved in cortical synaptic pruning [89]) to alter synaptic/cognitive function inaged AD mice. Further study will address whether CA140
cooperate with Aβ or DRD2 to regulate AD pathologies
and will investigate other possibilities and underlying
mechanisms in AD mice model. Interestingly, mRNAseq analysis revealed that CA140 treatment upregulated
the expression of ngf, tac1, and egr1, which are involved
in synaptic function and learning and memory [90–92],
in 3-month-old 5xFAD mice (Fig. 2). In animal models
and patients with AD, Tac1 expression is downregulated
in the hippocampus, making tac1 a potential target genefor the treatment of cognitive memory impairments [92].
In animal models of memory deficits, NGF is involved
in improving hippocampal/cortical synaptic formation,cholinergic transmission, and spatial memory [91, 93].
egr1 mRNA and EGR1 protein levels are reduced in thecortex and hippocampus in late-phase AD animal models [90, 94]. It is possible that CA140 improves cognitiveand synaptic function by modulating ngf, tac1, and egr1
expression in 5xFAD mice. Further studies will reveal the
specific mechanisms by which CA140 enhances synaptic
plasticity and memory in mouse models of AD (including
young vs. aged AD models).
Under pathological conditions, CA140 treatment
alleviated synaptic/cognitive impairments and AD
pathologies in partial regulation of DRD1/ELK-1 signaling in aged 5xFAD mice (Fig. 8). We therefore investigated the effects of CA140 on synaptic and cognitive
function under normal conditions and whether theseeffects also involved with DRD1/ELK-1 signaling. We
found that CA140 treatment improved cognitive andsynaptic function via DRD1 signaling in WT mice
(Fig. 9). CA140 treatment did not affect ELK-1 signaling
but showed tendency to increase CaMKIIα signaling in
WT mice (Fig. 9), suggesting that CA140 improves synaptic and cognitive function through DRD1/CaMKIIαbut not ELK-1 under normal conditions. Why does

Chae et al. Journal of Neuroinflammation(2024) 21:200

the DA analogue CA140 differentially regulate downstream signaling under normal and pathological conditions? DRD1 expression is reduced in patients with AD(pathological conditions) compared with healthy controls (normal conditions) [10], which may affect DRD1mediated downstream signaling under pathologicalconditions. The reduced expression of DRD1 underpathological conditions may underlie the differential
effects of the DRD1–CA140 association under pathological and normal conditions. The specific mechanism
by which CA140 differentially modulates DRD1-mediated downstream signaling under normal and pathological conditions will be explored in a future study.
In WT mice, the DA-DRD1 interaction regulates hippocampal synaptic plasticity via Gαq-CaMKIIα signaling [95]. In addition, the RAS/CaMKIIα/ERK pathway
is a critical mediator of dendritic spine formation,neuronal plasticity, and cognitive function throughregulation of the α-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid receptor (AMPAR) andN-methyl-D-aspartate receptor (NMDAR) [96–101]. In
the present study, we explored the molecular mechanisms by which CA140 affects synaptic function andmolecular targeting/signaling under normal conditionsin PHNs. We found that CA140 increased CaMKIIα/ERK phosphorylation in PHNs, whereas a DRD1 antagonist (LE300) decreased CaMKIIα/ERK phosphorylation (Fig. 11). Notably, inhibition of CaMKIIα or ERK
eliminated the CA140-induced increase in dendritic
spine number in PHNs (Fig. 11). Why do the effectsof CA140 on downstream CaMKIIα and ERK signaling differ between PHNs and WT mice? Comparedwith PHNs, whole brain lysates contain multiple typesof cells, including microglia, neurons, and astrocytes;
these additional cells may influence the effects of
CA140 on p-CaMKIIα and p-ERK expression in WT
mice. In a future study, we will systematically investigate the effects of CA140 on CaMKIIα/ERK signaling
in glial cells (i.e., microglia vs. astrocytes) in vitro andin vivo and compare them with the effects of CA140
on CaMKIIα/ERK signaling in PHNs. Our data suggestthat CA140 promotes synaptic function by modulating
DRD1/CaMKIIα and/or ERK signaling in PHNs and/or
WT mice. Collectively, our findings suggest that CA140
enhances synaptic function and long-term memory byactivating DRD1 signaling under normal and pathological conditions. However, it is possible that CA140
affects other synaptic and cognitive function-related
signaling networks via bi- and/or multidirectional
pathways. Additional studies are needed to unveil the
specific mechanisms of action by which CA140 differentially regulates cognitive and synaptic function underpathological and normal conditions.

Page 31 of 34

Conclusions
This study demonstrates that the DA analogue CA140
has novel therapeutic effects on Aβ/tau pathology andsynaptic/cognitive function under normal and pathological conditions. In aged 5xFAD mice, CA140 treatment
alleviates Aβ accumulation, tau phosphorylation, anddeficits in long-term memory, dendritic spine formation,
synaptic function and LTP through DRD1. In addition,
CA140 ameliorates reactive gliosis in 5xFAD mice, PACs
and PMCs. In WT mice and PHNs, CA140 treatment
promotes synaptic and cognitive function via DRD1/
CaMKIIα and/or ERK signaling. Collectively, these findings suggest that CA140 is a multitarget therapeutic drugfor neurodegenerative diseases, including AD.
Abbreviations
AD	Alzheimer’s disease
CaMKII	Calcium–calmodulin-dependent protein kinase IIDA	Dopamine
DRD1	Dopamine receptor D1
ERK	Extracellular signal-regulated kinase
LTP	Long-term potentiationmEPSC	Miniature excitatory postsynaptic current
NLRP3	Nucleotide-binding domain, leucine-rich–containing family, pyrin
domain–containing-3
SCO	Scopolamine
TLR	Toll-like receptor
PHN	Primary hippocampal neuron

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03180-x.
Supplementary file 1.
Supplementary file 2.
Supplementary file 3.
Acknowledgements
Confocal microscopy (Nikon, T-RCP) data were acquired at the Advanced
Neural Imaging Center at the Korea Brain Research Institute (KBRI). We thankprevious and current neurodegenerative diseases lab members, specificallyDr. Jin Han Nam, Dr. Youngpyo Nam, Dr. Hee-Jeong Choi, HyunHee Park forediting, technical assistance/support for in vitro and in vivo work, valuablecomments, and behavior studies in our manuscript. In addition, we thank Dr.
Daniel Pak, Dr. Jeongyeon Kim. and Dr. Misun Kang’s technical assistance/support for in vitro and in vivo work.
Author contributions
J.Y., J. I. K., and H.S.H. conceived and participated in the design of the study.
S.C., H.J.L., H.E.L., J.Y., Y. L., M.D.S., Y.H.L., J.I.K., and H.S.H. wrote the manuscript.
S.C. analyzed the RNA sequencing data. H.J.L., J.K., Y. J., Y.L., S.Y.S., H.P., G.L., R.S.E,
S.C.L. performed in vitro and vivo experiments, quantification of data, andhistological analysis. H.E.L conducted LTP experiments. H.J.L., H.E.L., and H.Y.K
performed the experiments, quantification of data, and statistical analysis during the revision. All authors read and approved the final manuscript.
Funding
This work was supported by the Basic Science Research Program throughthe National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (Grants 2019R1A2B5B01070108 to H.S.H. and
2021R1A4A1031644, 2021M3A9G8022960, 2022M325E8017907, and2023R1A2C1006489 to J.I.K, RS-2023-00253215 to S.C.) and the KBRI basic
research program through KBRI funded by the Ministry of Science, ICT, andChae et al. Journal of Neuroinflammation(2024) 21:200

Future Planning (Grants 24-BR-02-03, 23-BR-02-12, 24-BR-03-07, 24-BR-05-02,
24-BR-03-01, and 24-BR-03-05 to H.S.H., 21-BR-02-12 to J.I.K., and 23-BR-01-02
to S.C.). This work was also supported by the National Institute on Aging at the
National Institutes of Health (award number R01AG053577 to G.L., R.S.E, S.C.L.
and J.Y.). In addition, this work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (Grant NRF2022R1A2C1011793 to Y.-H.L.), the KBSI fund (grant A439200 to Y.-H.L.) andthe Korea Ministry of Science and ICT’s Special Account for Regional Balanced
Development for Commercialization supervised by the NIPA (National IT
Industry Promotion Agency) to support AI-based digital medical devices forneurodevelopmental disorders (H0301-24-1001 to H.S.H.).
Availability of data and materials
All data generated and/or analyzed during this study are included in thisarticle and the supplementary information files.

Declarations
Ethical approval and consent to participate
All in vivo experiments were performed in accordance with approved animal
protocols and guidelines established by the Korea Brain Research Institute(IACUC-2016–0013, IACUC-2018-0018).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Neurovascular Unit, Korea Brain Research Institute (KBRI), 61, Cheomdan‑ro,
Dong‑gu, Daegu 41068, Republic of Korea. 2 Neurodegenerative Unit, Korea
Brain Research Institute (KBRI), 61, Cheomdan‑ro, Dong‑gu, Daegu 41068,
Republic of Korea. 3 Department of Biological Sciences, Ulsan National
Institute of Science and Technology (UNIST), 50 UNIST‑Gil, Ulsan 44919,
Republic of Korea. 4 Biopharmaceutical Research Center, Korea Basic Science
Institute (KBSI), Ochang, ChungBuk 28119, Republic of Korea. 5 Departmentof Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA
92093‑0358, USA. 6 College of Pharmacy and Department of Molecular Scienceand Technology, Ajou University, Suwon, Gyeonggi 16499, Republic of Korea.
7
Department of Brain & Cognitive Sciences, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu 42988, Republic of Korea. 8 Bio‑Analytical
Science, University of Science and Technology (UST), Daejeon 34113, Republicof Korea. 9 Department of Systems Biotechnology, Chung-Ang University,
Gyeonggi 17546, Republic of Korea. 10 Department of Neural Development
and Disease, Korea Brain Research Institute (KBRI), 61 Cheomdan‑ro, Dong‑gu,
Daegu 41068, Republic of Korea.
Received: 10 July 2023 Accepted: 17 July 2024

References
1. DeTure MA, Dickson DW. The neuropathological diagnosis of Alzheimer’s disease. Mol Neurodegener. 2019;14(1):32.
2. DeKosky ST, Scheff SW. Synapse loss in frontal cortex biopsies in
Alzheimer’s disease: correlation with cognitive severity. Ann Neurol.
1990;27(5):457–64.
3. Sobue A, Komine O, Yamanaka K. Neuroinflammation in Alzheimer’s
disease: microglial signature and their relevance to disease. Inflamm
Regen. 2023;43(1):26.
4. Muzio L, Viotti A, Martino G. Microglia in neuroinflammation and neurodegeneration: from understanding to therapy. Front Neurosci. 2021;15:742065.
5. Perez-Nievas BG, Serrano-Pozo A. Deciphering the astrocyte reaction in
Alzheimer’s disease. Front Aging Neurosci. 2018;10:114.
6. Selkoe DJ. Resolving controversies on the path to Alzheimer’s therapeutics. Nat Med. 2011;17(9):1060–5.

Page 32 of 34

7. Kramar CP, Chefer VI, Wise RA, Medina JH, Barbano MF. Dopamine in thedorsal hippocampus impairs the late consolidation of cocaine-associated memory. Neuropsychopharmacology. 2014;39(7):1645–53.
8. Yagishita S, Hayashi-Takagi A, Ellis-Davies GC, Urakubo H, Ishii S, Kasai
H. A critical time window for dopamine actions on the structural
plasticity of dendritic spines. Science. 2014;345(6204):1616–20.
9. Rezaei M, Sadeghian A, Roohi N, Shojaei A, Mirnajafi-Zadeh J. Epilepsy and dopaminergic system. Physiol Pharmacol. 2017;21(1):1–14.
10. Pan X, Kaminga AC, Wen SW, Wu X, Acheampong K, Liu A. Dopamineand dopamine receptors in Alzheimer’s disease: a systematic review
and network meta-analysis. Front Aging Neurosci. 2019;11:175.
11. Dubovyk V, Manahan-Vaughan D. Gradient of expression of dopamine D2 receptors along the dorso-ventral axis of the hippocampus.
Front Synaptic Neurosci. 2019;11:28.
12. Fasano C, Bourque MJ, Lapointe G, Leo D, Thibault D, Haber M,
Kortleven C, Desgroseillers L, Murai KK, Trudeau LE. Dopamine facilitates dendritic spine formation by cultured striatal medium spiny
neurons through both D1 and D2 dopamine receptors. Neuropharmacology. 2013;67:432–43.
13. de Lima MN, Presti-Torres J, Dornelles A, Scalco FS, Roesler R, Garcia
VA, Schroder N. Modulatory influence of dopamine receptors on
consolidation of object recognition memory. Neurobiol Learn Mem.
2011;95(3):305–10.
14. Lemon N, Manahan-Vaughan D. Dopamine D1/D5 receptors
gate the acquisition of novel information through hippocampal
long-term potentiation and long-term depression. J Neurosci.
2006;26(29):7723–9.
15. Xia QP, Cheng ZY, He L. The modulatory role of dopamine receptors
in brain neuroinflammation. Int Immunopharmacol. 2019;76: 105908.
16. Pike AF, Longhena F, Faustini G, van Eik JM, Gombert I, Herrebout
MAC, Fayed M, Sandre M, Varanita T, Teunissen CE, et al. Dopamine
signaling modulates microglial NLRP3 inflammasome activation:
implications for Parkinson’s disease. J Neuroinflamm. 2022;19(1):50.
17. Lee JY, Nam JH, Nam Y, Nam HY, Yoon G, Ko E, Kim SB, Bautista MR,
Capule CC, Koyanagi T, et al. The small molecule CA140 inhibits theneuroinflammatory response in wild-type mice and a mouse modelof AD. J Neuroinflamm. 2018;15(1):286.
18. Na D, Zhang J, Beaulac HJ, Piekna-Przybylska D, Nicklas PR, Kiernan
AE, White PM. Increased central auditory gain in 5xFAD Alzheimer’s
disease mice as an early biomarker candidate for Alzheimer’s diseasediagnosis. Front Neurosci. 2023;17:1106570.
19. Abaandou L, Quan D, Shiloach J. Affecting HEK293 cell growth andproduction performance by modifying the expression of specificgenes. Cells. 2021;10(7):1667.
20. Hoe HS, Freeman J, Rebeck GW. Apolipoprotein E decreases tau
kinases and phospho-tau levels in primary neurons. Mol Neurodegener. 2006;1:18.
21. Pak DT, Yang S, Rudolph-Correia S, Kim E, Sheng M. Regulation ofdendritic spine morphology by SPAR, a PSD-95-associated RapGAP.
Neuron. 2001;31(2):289–303.
22. van Gijsel-Bonnello M, Baranger K, Benech P, Rivera S, Khrestchatisky
M, de Reggi M, Gharib B. Metabolic changes and inflammation in
cultured astrocytes from the 5xFAD mouse model of Alzheimer’s
disease: alleviation by pantethine. PLoS ONE. 2017;12(4): e0175369.
23. Ryu KY, Lee HJ, Woo H, Kang RJ, Han KM, Park H, Lee SM, Lee JY, Jeong
YJ, Nam HW, et al. Dasatinib regulates LPS-induced microglial andastrocytic neuroinflammatory responses by inhibiting AKT/STAT3
signaling. J Neuroinflamm. 2019;16(1):190.
24. Ehrlich RS, Shiao AL, Li M, Teppang KL, Jeoung KY, Theodorakis EA,
Yang J. Exploring the effect of aliphatic substituents on aryl cyano
amides on enhancement of fluorescence upon binding to amyloidbeta aggregates. ACS Chem Neurosci. 2021;12(15):2946–52.
25. Capule CC, Brown C, Olsen JS, Dewhurst S, Yang J. Oligovalentamyloid-binding agents reduce SEVI-mediated enhancement of
HIV-1 infection. J Am Chem Soc. 2012;134(2):905–8.
26. Roche J, Shen Y, Lee JH, Ying J, Bax A. Monomeric Abeta(1–40) and
Abeta(1–42) peptides in solution adopt very similar ramachandran
map distributions that closely resemble random coil. Biochemistry.
2016;55(5):762–75.
27. Nam Y, Joo B, Lee JY, Han KM, Ryu KY, Koh YH, Kim J, Koo JW, We
YM, Hoe HS. ALWPs improve cognitive function and regulate Abeta

Chae et al. Journal of Neuroinflammation

28.
29.

30.
31.
32.
33.
34.

35.
36.
37.
38.
39.
40.

41.

42.
43.

44.
45.

46.

47.

48.

(2024) 21:200

plaque and tau hyperphosphorylation in a mouse model of Alzheimer’s disease. Front Mol Neurosci. 2019;12:192.
Lee HJ, Hoe HS. Inhibition of CDK4/6 regulates AD pathology, neuroinflammation and cognitive function through DYRK1A/STAT3 signaling.
Pharmacol Res. 2023;190: 106725.
Lee HJ, Jeon SG, Kim J, Kang RJ, Kim SM, Han KM, Park H, Kim KT, Sung
YM, Nam HY, et al. Ibrutinib modulates Abeta/tau pathology, neuroinflammation, and cognitive function in mouse models of Alzheimer’s
disease. Aging Cell. 2021;20(3): e13332.
Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions
with RNA-Seq. Bioinformatics. 2009;25(9):1105–11.
Anders S, Pyl PT, Huber W. HTSeq–a Python framework to work withhigh-throughput sequencing data. Bioinformatics. 2015;31(2):166–9.
Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010;11(3):R25.
Love MI, Huber W, Anders S. Moderated estimation of fold change
and dispersion for RNA-seq data with DESeq2. Genome Biol.
2014;15(12):550.
Sherman BT, Hao M, Qiu J, Jiao X, Baseler MW, Lane HC, Imamichi T,
Chang W. DAVID: a web server for functional enrichment analysis andfunctional annotation of gene lists (2021 update). Nucleic Acids Res.
2022;50(W1):W216–21.
Kanehisa M, Furumichi M, Sato Y, Ishiguro-Watanabe M, Tanabe M.
KEGG: integrating viruses and cellular organisms. Nucleic Acids Res.
2021;49(D1):D545–51.
Martens M, Ammar A, Riutta A, Waagmeester A, Slenter DN, Hanspers K,
Miller RA, Digles D, Lopes EN, Ehrhart F, et al. WikiPathways: connecting
communities. Nucleic Acids Res. 2021;49(D1):D613–21.
Cataldi R, Chia S, Pisani K, Ruggeri FS, Xu CK, Sneideris T, Perni M, Sarwat
S, Joshi P, Kumita JR, et al. A dopamine metabolite stabilizes neurotoxic
amyloid-beta oligomers. Commun Biol. 2021;4(1):19.
Kinney JW, Bemiller SM, Murtishaw AS, Leisgang AM, Salazar AM, Lamb
BT. Inflammation as a central mechanism in Alzheimer’s disease. Alzheimers Dement. 2018;4:575–90.
Bernaus A, Blanco S, Sevilla A. Glia crosstalk in neuroinflammatory
diseases. Front Cell Neurosci. 2020;14:209.
Lin W, Li Z, Liang G, Zhou R, Zheng X, Tao R, Huo Q, Su C, Li M, Xu N,
et al. TNEA therapy promotes the autophagic degradation of NLRP3
inflammasome in a transgenic mouse model of Alzheimer’s disease via
TFEB/TFE3 activation. J Neuroinflamm. 2023;20(1):21.
Escartin C, Galea E, Lakatos A, O’Callaghan JP, Petzold GC, Serrano-Pozo
A, Steinhauser C, Volterra A, Carmignoto G, Agarwal A, et al. Reactive
astrocyte nomenclature, definitions, and future directions. Nat Neurosci.
2021;24(3):312–25.
Yu W, Li Y, Zhong F, Deng Z, Wu J, Yu W, Lu Y. Disease-associatedneurotoxic astrocyte markers in Alzheimer disease based on integrative
single-nucleus RNA sequencing. Cell Mol Neurobiol. 2024;44(1):20.
Jo KW, Lee D, Cha DG, Oh E, Choi YH, Kim S, Park ES, Kim JK, Kim
KT. Gossypetin ameliorates 5xFAD spatial learning and memory
through enhanced phagocytosis against Abeta. Alzheimers Res Ther.
2022;14(1):158.
Paolicelli RC, Sierra A, Stevens B, Tremblay ME, Aguzzi A, Ajami B, Amit
I, Audinat E, Bechmann I, Bennett M, et al. Microglia states and nomenclature: a field at its crossroads. Neuron. 2022;110(21):3458–83.
Li Q, Cheng Z, Zhou L, Darmanis S, Neff NF, Okamoto J, Gulati G,
Bennett ML, Sun LO, Clarke LE, et al. Developmental heterogeneity of
microglia and brain myeloid cells revealed by deep single-cell RNA
sequencing. Neuron. 2019;101(2):207–23.
Lee JS, Kim HG, Lee HW, Han JM, Lee SK, Kim DW, Saravanakumar A, Son
CG. Hippocampal memory enhancing activity of pine needle extract
against scopolamine-induced amnesia in a mouse model. Sci Rep.
2015;5:9651.
Lee HJ, Woo H, Lee HE, Jeon H, Ryu KY, Nam JH, Jeon SG, Park H, Lee JS,
Han KM, et al. The novel DYRK1A inhibitor KVN93 regulates cognitive
function, amyloid-beta pathology, and neuroinflammation. Free Radic
Biol Med. 2020;160:575–95.
Dorostkar MM, Zou C, Blazquez-Llorca L, Herms J. Analyzing dendritic
spine pathology in Alzheimer’s disease: problems and opportunities.
Acta Neuropathol. 2015;130(1):1–19.

Page 33 of 34

49. Zhang M, Zhong L, Han X, Xiong G, Xu D, Zhang S, Cheng H, Chiu K, Xu
Y. Brain and retinal abnormalities in the 5xFAD mouse model of Alzheimer’s disease at early stages. Front Neurosci. 2021;15: 681831.
50. Forner S, Kawauchi S, Balderrama-Gutierrez G, Kramar EA, Matheos DP,
Phan J, Javonillo DI, Tran KM, Hingco E, da Cunha C, et al. Systematic
phenotyping and characterization of the 5xFAD mouse model of
Alzheimer’s disease. Sci Data. 2021;8(1):270.
51. Jourdain P, Fukunaga K, Muller D. Calcium/calmodulin-dependent
protein kinase II contributes to activity-dependent filopodia growthand spine formation. J Neurosci. 2003;23(33):10645–9.
52. Ohta KI, Suzuki S, Warita K, Kaji T, Kusaka T, Miki T. Prolonged maternal
separation attenuates BDNF-ERK signaling correlated with spine formation in the hippocampus during early brain development. J Neurochem. 2017;141(2):179–94.
53. Szatmari EM, Oliveira AF, Sumner EJ, Yasuda R. Centaurin-alpha1Ras-Elk-1 signaling at mitochondria mediates beta-amyloid-induced
synaptic dysfunction. J Neurosci. 2013;33(12):5367–74.
54. Jones MR, Urits I, Wolf J, Corrigan D, Colburn L, Peterson E, Williamson A,
Viswanath O. Drug-induced peripheral neuropathy: a narrative review.
Curr Clin Pharmacol. 2020;15(1):38–48.
55. Arendt T, Gartner U, Seeger G, Barmashenko G, Palm K, Mittmann
T, Yan L, Hummeke M, Behrbohm J, Bruckner MK, et al. Neuronal
activation of Ras regulates synaptic connectivity. Eur J Neurosci.
2004;19(11):2953–66.
56. Zhu JJ, Qin Y, Zhao M, Van Aelst L, Malinow R. Ras and Rap control AMPA
receptor trafficking during synaptic plasticity. Cell. 2002;110(4):443–55.
57. Wu L, D’Amico A, Hochrein H, O’Keeffe M, Shortman K, Lucas K. Development of thymic and splenic dendritic cell populations from different
hemopoietic precursors. Blood. 2001;98(12):3376–82.
58. Kempadoo KA, Mosharov EV, Choi SJ, Sulzer D, Kandel ER. Dopamine release from the locus coeruleus to the dorsal hippocampus
promotes spatial learning and memory. Proc Natl Acad Sci USA.
2016;113(51):14835–40.
59. Calabresi P, Picconi B, Tozzi A, Di Filippo M. Dopamine-mediatedregulation of corticostriatal synaptic plasticity. Trends Neurosci.
2007;30(5):211–9.
60. Denenberg VH, Kim DS, Palmiter RD. The role of dopamine in learning,memory, and performance of a water escape task. Behav Brain Res.
2004;148(1–2):73–8.
61. Jay TM. Dopamine: a potential substrate for synaptic plasticity andmemory mechanisms. Prog Neurobiol. 2003;69(6):375–90.
62. Takeuchi T, Duszkiewicz AJ, Sonneborn A, Spooner PA, Yamasaki M,
Watanabe M, Smith CC, Fernandez G, Deisseroth K, Greene RW, et al.
Locus coeruleus and dopaminergic consolidation of everyday memory.
Nature. 2016;537(7620):357–62.
63. Nam E, Derrick JS, Lee S, Kang J, Han J, Lee SJC, Chung SW, Lim MH. regulatory activities of dopamine and its derivatives toward metal-free andmetal-induced amyloid-beta aggregation, oxidative stress, and inflammation in Alzheimer’s disease. ACS Chem Neurosci. 2018;9(11):2655–66.
64. Li J, Zhu M, Manning-Bog AB, Di Monte DA, Fink AL. Dopamine andL-dopa disaggregate amyloid fibrils: implications for Parkinson’s andAlzheimer’s disease. FASEB J. 2004;18(9):962–4.
65. Cheng ZY, Xia QP, Hu YH, Wang C, He L. Dopamine D1 receptor agonist
A-68930 ameliorates Abeta(1–42)-induced cognitive impairment andneuroinflammation in mice. Int Immunopharmacol. 2020;88: 106963.
66. Tian J, Guo L, Sui S, Driskill C, Phensy A, Wang Q, Gauba E, Zigman
JM, Swerdlow RH, Kroener S, et al. Disrupted hippocampal growth
hormone secretagogue receptor 1alpha interaction with dopamine
receptor D1 plays a role in Alzheimer’s disease. Sci Transl Med. 2019.
https://​doi.​org/​10.​1126/​scitr​anslm​ed.​aav62​78.
67. Latz E, Xiao TS, Stutz A. Activation and regulation of the inflammasomes. Nat Rev Immunol. 2013;13(6):397–411.
68. Yan Y, Jiang W, Liu L, Wang X, Ding C, Tian Z, Zhou R. Dopamine controls
systemic inflammation through inhibition of NLRP3 inflammasome.
Cell. 2015;160(1–2):62–73.
69. Letiembre M, Liu Y, Walter S, Hao W, Pfander T, Wrede A, SchulzSchaeffer W, Fassbender K. Screening of innate immune receptors
in neurodegenerative diseases: a similar pattern. Neurobiol Aging.
2009;30(5):759–68.

Chae et al. Journal of Neuroinflammation(2024) 21:200

70. Frank S, Copanaki E, Burbach GJ, Muller UC, Deller T. Differential regulation of toll-like receptor mRNAs in amyloid plaque-associated brain
tissue of aged APP23 transgenic mice. Neurosci Lett. 2009;453(1):41–4.
71. Walter S, Letiembre M, Liu Y, Heine H, Penke B, Hao W, Bode B, Manietta
N, Walter J, Schulz-Schuffer W, et al. Role of the toll-like receptor 4
in neuroinflammation in Alzheimer’s disease. Cell Physiol Biochem.
2007;20(6):947–56.
72. Minoretti P, Gazzaruso C, Vito CD, Emanuele E, Bianchi M, Coen E, Reino
M, Geroldi D. Effect of the functional toll-like receptor 4 Asp299Gly
polymorphism on susceptibility to late-onset Alzheimer’s disease.
Neurosci Lett. 2006;391(3):147–9.
73. Jin JJ, Kim HD, Maxwell JA, Li L, Fukuchi K. Toll-like receptor 4-dependent upregulation of cytokines in a transgenic mouse model of Alzheimer’s disease. J Neuroinflamm. 2008;5:23.
74. Tahara K, Kim HD, Jin JJ, Maxwell JA, Li L, Fukuchi K. Role of toll-likereceptor signalling in Abeta uptake and clearance. Brain. 2006;129(Pt11):3006–19.
75. Preston GC, Brazell C, Ward C, Broks P, Traub M, Stahl SM. The scopolamine model of dementia: determination of central cholinomimetic
effects of physostigmine on cognition and biochemical markers inman. J Psychopharmacol. 1988;2(2):67–79.
76. Stone WS, Croul CE, Gold PE. Attenuation of scopolamine-induced
amnesia in mice. Psychopharmacology. 1988;96(3):417–20.
77. Ebert U, Kirch W. Scopolamine model of dementia: electroencephalogram findings and cognitive performance. Eur J Clin Invest.
1998;28(11):944–9.
78. Bajo R, Pusil S, Lopez ME, Canuet L, Pereda E, Osipova D, Maestu F,
Pekkonen E. Scopolamine effects on functional brain connectivity: apharmacological model of Alzheimer’s disease. Sci Rep. 2015;5:9748.
79. van der Zee EA, Luiten PG. Muscarinic acetylcholine receptors in the
hippocampus, neocortex and amygdala: a review of immunocytochemical localization in relation to learning and memory. Prog Neurobiol. 1999;58(5):409–71.
80. Memo M, Missale C, Trivelli L, Spano PF. Acute scopolamine treatment
decreases dopamine metabolism in rat hippocampus and frontal
cortex. Eur J Pharmacol. 1988;149(3):367–70.
81. Piri M, Rostampour M, Nasehi M, Zarrindast MR. Blockade of thedorsal hippocampal dopamine D1 receptors inhibits the scopolamine-induced state-dependent learning in rats. Neuroscience.
2013;252:460–7.
82. Drever BD, Riedel G, Platt B. The cholinergic system and hippocampal
plasticity. Behav Brain Res. 2011;221(2):505–14.
83. Chen J, Nakamura M, Kawamura T, Takahashi T, Nakahara D. Roles ofpedunculopontine tegmental cholinergic receptors in brain stimulation
reward in the rat. Psychopharmacology. 2006;184(3–4):514–22.
84. Yuan Xiang P, Janc O, Grochowska KM, Kreutz MR, Reymann KG. Dopamine agonists rescue Abeta-induced LTP impairment by Src-familytyrosine kinases. Neurobiol Aging. 2016;40:98–102.
85. Kramar CP, Barbano MF, Medina JH. Dopamine D1/D5 receptors in thedorsal hippocampus are required for the acquisition and expressionof a single trial cocaine-associated memory. Neurobiol Learn Mem.
2014;116:172–80.
86. Tong L, Balazs R, Thornton PL, Cotman CW. Beta-amyloid peptide
at sublethal concentrations downregulates brain-derived neurotrophic factor functions in cultured cortical neurons. J Neurosci.
2004;24(30):6799–809.
87. Sharma A, Callahan LM, Sul JY, Kim TK, Barrett L, Kim M, Powers JM,
Federoff H, Eberwine J. A neurotoxic phosphoform of Elk-1 associateswith inclusions from multiple neurodegenerative diseases. PLoS ONE.
2010;5(2): e9002.
88. Barrett LE, Sul JY, Takano H, Van Bockstaele EJ, Haydon PG, Eberwine JH.
Region-directed phototransfection reveals the functional significanceof a dendritically synthesized transcription factor. Nat Methods.
2006;3(6):455–60.
89. Zhang YQ, Lin WP, Huang LP, Zhao B, Zhang CC, Yin DM. Dopamine D2
receptor regulates cortical synaptic pruning in rodents. Nat Commun.
2021;12(1):6444.
90. Hu YT, Chen XL, Huang SH, Zhu QB, Yu SY, Shen Y, Sluiter A, Verhaagen
J, Zhao J, Swaab D, et al. Early growth response-1 regulates acetylcholinesterase and its relation with the course of Alzheimer’s disease. Brain
Pathol. 2019;29(4):502–12.

Page 34 of 34

91. Eu WZ, Chen YJ, Chen WT, Wu KY, Tsai CY, Cheng SJ, Carter RN, Huang
GJ. The effect of nerve growth factor on supporting spatial memory
depends upon hippocampal cholinergic innervation. Transl Psychiatry.
2021;11(1):162.
92. Liu YJ, Liu TT, Jiang LH, Liu Q, Ma ZL, Xia TJ, Gu XP. Identification of hub
genes associated with cognition in the hippocampus of Alzheimer’s
Disease. Bioengineered. 2021;12(2):9598–609.
93. Garofalo L, Ribeiro-da-Silva A, Cuello AC. Nerve growth factor-induced
synaptogenesis and hypertrophy of cortical cholinergic terminals. Proc
Natl Acad Sci USA. 1992;89(7):2639–43.
94. Koldamova R, Schug J, Lefterova M, Cronican AA, Fitz NF, Davenport
FA, Carter A, Castranio EL, Lefterov I. Genome-wide approaches reveal
EGR1-controlled regulatory networks associated with neurodegeneration. Neurobiol Dis. 2014;63:107–14.
95. Kern A, Mavrikaki M, Ullrich C, Albarran-Zeckler R, Brantley AF, Smith RG.
Hippocampal dopamine/DRD1 signaling dependent on the ghrelin
receptor. Cell. 2015;163(5):1176–90.
96. Barcomb K, Buard I, Coultrap SJ, Kulbe JR, O’Leary H, Benke TA, Bayer KU.
Autonomous CaMKII requires further stimulation by Ca2+/calmodulinfor enhancing synaptic strength. FASEB J. 2014;28(8):3810–9.
97. Pi HJ, Otmakhov N, El Gaamouch F, Lemelin D, De Koninck P, Lisman
J. CaMKII control of spine size and synaptic strength: role of phosphorylation states and nonenzymatic action. Proc Natl Acad Sci USA.
2010;107(32):14437–42.
98. Ryu HH, Lee YS. Cell type-specific roles of RAS-MAPK signaling inlearning and memory: implications in neurodevelopmental disorders.
Neurobiol Learn Mem. 2016;135:13–21.
99. Cornelia Koeberle S, Tanaka S, Kuriu T, Iwasaki H, Koeberle A, Schulz A,
Helbing DL, Yamagata Y, Morrison H, Okabe S. Developmental stagedependent regulation of spine formation by calcium-calmodulindependent protein kinase IIalpha and Rap1. Sci Rep. 2017;7(1):13409.
100. Wayman GA, Lee YS, Tokumitsu H, Silva AJ, Soderling TR. Calmodulinkinases: modulators of neuronal development and plasticity. Neuron.
2008;59(6):914–31.
101. Lee YS, Silva AJ. The molecular and cellular biology of enhanced cognition. Nat Rev Neurosci. 2009;10(2):126–40.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.