Background
Increasing evidence indicates a critical role of the immune system in neurodegenerative diseases such as Alzheimer’s disease (AD) [
1]. Abnormal glial activation in patients with neurodegenerative diseases may be a hallmark diagnostic feature of these diseases, particularly AD [
2]. Neuroinflammation or inflammation of the central nervous system (CNS) is mainly mediated by the activation of microglia [
1]. In addition to releasing various neurotrophic factors that support neuronal cell survival and neurotoxic factors, activated microglia release proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) [
3,
4]. The neuroinflammation induced by the release of these proinflammatory cytokines may eventually lead to neuronal cell death and synaptic dysfunction. Therefore, the elucidation of the regulation of glial activation and inactivation may provide a potential therapeutic strategy for treating neurodegenerative diseases.
Lipopolysaccharide (LPS) is a well-established stimulator that induces the activation of microglial cells and is widely used both in vivo and in vitro to induce neuroinflammation in animal models [
5,
6]. The interaction between LPS and Toll-like receptor 4 (TLR4) activates inflammation-associated transcription factors [
7] and the mitogen-activated protein kinase (MAPK) family [
8,
9], which comprises at least three components: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38 MAPK. In addition, the association between LPS and TLR4 stimulates the release of both immune-related cytotoxic factors, including iNOS and COX-2, and proinflammatory cytokines (TNF-α, IL-1β, and IL-6) [
10]. A chronic inflammatory response may be accompanied by amyloid beta (Aβ) production, and microglia have been identified near the Aβ plaques of AD patients [
11,
12]. Aβ accumulation triggers AD pathogenesis through two mechanisms: neuronal apoptosis and glia-mediated inflammation leading to cell death [
13]. Extracellular Aβ deposits in senile plaques trigger changes in glial reactivity and stimulate neuroinflammation. Thus, Aβ accumulation may lead to neuronal loss through the overproduction of reactive proinflammatory cytokines [
14,
15]. CA140 is a chemically stable small-molecule analog of dopamine (DA) and is synthesized by acylation of the amine in DA with N-methylisatoic anhydride, which reduces the propensity of DA to undergo self-polymerization [
16]. DA is a neurotransmitter that regulates a wide range of functions, including initiation of movement and learning and memory [
17]. DA binds to several DA receptors, which are present on nearly all immune cells [
18]. Activation of these receptors via DA or DA agonists modulates the activation, proliferation, and cytokine production of immune cells [
19]. We therefore speculated that CA140 may also exhibit biological activity against the neuroinflammatory response.
In the present study, we examined whether CA140 regulates the neuroinflammatory response in vitro and in vivo. We discovered that CA140 reduced proinflammatory responses in LPS-stimulated BV2 microglial cells, primary microglial cells, and primary astrocytes. In addition, CA140 inhibited LPS-induced neuroinflammatory responses by inhibiting the dopamine D1 receptor (D1R)/ERK/STAT3 signaling pathways. Moreover, CA140 significantly decreased the activation of microglia and astrocytes in wild-type mice as well as a mouse model of AD. Taken together, our results indicate that CA140 is a potential therapeutic agent for treating and/or preventing neuroinflammation-related diseases, including AD.
Methods
Cell lines and culture conditions
BV2 microglial cells (a generous gift of Dr. Kyung-Ho Suk) or HEK cells (a generous gift of Dr. Hyung-Jun Kim) were maintained in high-glucose DMEM (Invitrogen, Carlsbad, CA, USA) with 5 or 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) in a 5% CO2 incubator.
Mouse primary microglial and astrocyte cultures
Mouse primary microglial and astrocyte cultures were prepared from mixed glial cultures as previously described [
20]. Briefly, whole brains of post-natal 1-day-old C57BL/6 mice were chopped and mechanically disrupted using a 70-μm nylon mesh. The cells were seeded in 75 T culture flasks and grown in low-glucose DMEM supplemented with 10% FBS, 100 unit/mL penicillin, and 100 μg/mL streptomycin. The culture medium was changed after 7 days and every 3 days thereafter. After 14 days, mixed primary glial cells were obtained for use in subsequent experiments. To obtain mouse primary astrocytes, mixed glial cells were cultured with shaking at 250 rpm overnight. The next day, the culture medium was discarded, and the cells were washed three times with PBS. The cells were dissociated using trypsin-EDTA and collected by centrifugation at 1200 rpm for 10 min. Primary astrocytes were maintained in low-glucose DMEM supplemented with 10% FBS and penicillin-streptomycin. To obtain mouse primary microglial cells, mixed primary glial cells were incubated with trypsin solution (0.25% trypsin, 1 mM EDTA in Hank’s balanced salt solution) diluted 1:4 in serum-free DMEM media [
21]. After the mouse primary astrocyte layer was fully detached, low-glucose DMEM containing 10% FBS was added, the supernatant was aspirated, and the remaining primary microglial cells were used for experiments.
Rat primary microglial and astrocyte cultures
Rat primary mixed glial cells were cultured from the cerebral cortices of 1-day-old Sprague Dawley rats. Briefly, the cortices were triturated into single cells in high-glucose DMEM containing 10% FBS/penicillin-streptomycin solution (5000 units/mL penicillin, 5 mg/mL streptomycin, Corning, Mediatech Inc., Manassas, VA, USA) and plated into 75 T culture flasks (0.5 hemisphere/flask) for 2 weeks. To harvest rat primary microglial cells, the plate was shaken continuously at 120 rpm for 2 h to facilitate microglial detachment from the plate. The fluid medium was subsequently collected and centrifuged at 1500 rpm for 15 min, and the cell pellets were resuspended to plate 1 × 105 cells per well. The remaining cells in the flask were harvested using 0.1% trypsin to obtain rat primary astrocytes. These rat primary astrocytes and primary microglial cells were cultured in 12-well plates (35 mm) pre-coated with poly-D-lysine (Sigma).
Wild-type mice
All experiments were performed in accordance with the approved animal protocols and guidelines established by the Korea Brain Research Institute (IACUC-2016-0013). C57BL6/N mice were purchased from Orient-Bio Company. Male C57BL6/N mice (8 weeks, 25–30 g) were housed in a pathogen-free facility with 12 h of light and dark per day at an ambient temperature of 22 °C. To determine if pretreatment with CA140 alters LPS-induced neuroinflammation, wild-type mice were intraperitoneally (i.p.) administered CA140 (30 mg/kg) or vehicle (10% DMSO) daily for 3 days and subsequently injected with LPS (Sigma, Escherichia coli, 10 mg/kg, i.p.) or PBS. After 3 h, immunostaining was performed with anti-IbaI or anti-GFAP antibodies. To examine whether post-treatment with CA140 regulates LPS-induced neuroinflammatory responses, wild-type mice were injected with LPS (10 mg/kg/day, i.p.) or PBS, followed 30 min later by injection with CA140 (30 mg/kg, i.p., twice with an interval of 1 h, followed 30 min later by a third injection) or vehicle (10% DMSO, i.p.). Immunohistochemistry was then performed with anti-Iba-1 and anti-GFAP antibodies.
Familial AD (5xFAD) mice
F1 generation 5xFAD mice (stock number 008730, B6SJL-Tg APPSwFlLon, PSEN1*M146 L*L286V6799Vas/Mmjax) were purchased from The Jackson Laboratory. 5xFAD mice overexpress two mutant human proteins: APP (695) with KM670/671NL (Swedish), I716V (Florida), and V717I (London) FAD mutations and PS1 with M146 L and L286 V FAD mutations. To examine the effects of CA140 on the neuroinflammatory response in a mouse model of AD, 5xFAD mice were injected with CA140 (30 mg/kg, i.p.) or vehicle (10% DMSO, i.p.) daily for 2 weeks, and immunohistochemistry was conducted with anti-Iba-1 or anti-GFAP antibodies. The animal groups were randomized for all experiments. Data were analyzed in a semi-automated manner using ImageJ software and confirmed by an independent researcher who did not participate in the current experiments. Only male mice were used for this study because the pathology of 5xFAD female mice is more severe than that of male mice, leading to huge variations in in vivo experiments.
Synthesis of CA140
N-Methylisatoic anhydride (19.6 mg, 110 μmol) was added to 28.1 mg of 3,4-dimethoxyphenethylamine (155 μmol, 1.3 equiv) and 25 μL of triethylamine (339 μmol, 3 equiv) in dichloromethane (DCM). The reaction mixture was stirred for 2 h at room temperature and overnight at 20 °C. The mixture was warmed to room temperature, and a vacuum was subsequently applied to remove volatile organics. The amide product was purified by column chromatography (7% ethyl acetate in DCM; Rf = 0.27) to provide 31.5 mg of N-(3,4-dimethoxyphenethyl)-2-(methylamino)benzamide as a white solid (91% isolated yield). 1H-NMR (400 MHz, CDCl3) δ ppm = 7.30 (t, 1H, J = 7.7 Hz, Ar-H), 7.20 (d, 1H, J = 7.7 Hz, Ar-H), 6.82 (d, 1H, J = 8.1 Hz, Ar-H), 6.74 (m 2H, Ar-H), 6.55 (t, 1H, J = 7.5 Hz, Ar-H), 3.86 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.63 (t, 1H, J = 6.7 Hz, -HCH-), 3.61 (t, 1H, J = 6.7 Hz, -HCH-), 2.84 (s, 3H, CH3), 2.84 (t, 2H, J = 6.8 Hz, CH2). 13C-NMR (100 MHz, CDCl3) δ ppm = 169.6, 150.0, 148.9, 147.6, 132.7, 131.4, 127.0, 120.6, 115.4, 114.8, 111.9, 111.4, 111.3, 55.8, 55.7, 40.9, 35.1, 29.8.
N-(3,4-Dimethoxyphenethyl)-2-(methylamino)benzamide (9.2 mg, 29.3 μmol) was dissolved in 0.5 mL of anhydrous DCM, and 120 μL of 1 M BBr3 in DCM (120 μmol, 4 equiv) was added. The reaction was stirred overnight under an inert atmosphere. Excess methanol was added to the mixture, and volatile organics were removed in vacuo. The addition and removal of methanol was repeated at least three times to remove boric acid as methyl borate. The target compound, N-(3,4-dihydroxyphenethyl)-2-(methylamino)benzamide (CA140), was obtained in quantitative yield. 1H-NMR (400 MHz, CD3OD) δ ppm = 7.91 (d, 1H, J = 8.0 Hz, Ar-H), 7.89 (t, 1H, J = 7.6 Hz, Ar-H), 7.74 (m, 2H, J = 7.6 Hz, Ar-H), 6.67 (d 2H, J = 9.6 Hz Ar-H), 6.56 (d, 1H, J = 8.0 Hz, Ar-H), 3.60 (t, 2H, J = 7.1 Hz, CH2), 3.01 (s, 3H, NCH3), 2.79 (t, 1H, J = 7.0 Hz, CH2). LC-MS ESI positive mode m/z [M + H]+ = 287.11 (calculated = 287.13).
Brain-to-plasma ratio in ICR (Institute for Cancer Research) mice
ICR mice (n = 3) were dosed with CA140 dissolved in DMSO/Tween-80/saline (10:5:85%) via a single intravenous administration (10 mg/kg). Blood was collected by cardiac puncture at 5 min and then centrifuged to isolate plasma. The brain was collected at 5 min and homogenized in PBS after washing with fresh PBS. The concentrations of CA140 in the plasma and brain were determined by LC-MS/MS. The LC-MS/MS system comprised a Nexera XR HPLC system (Shimadzu Co., Kyoto, Japan) coupled to a TSQ Vantage triple quadrupole mass spectrometer equipped with Xcalibur version 1.1.1 (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Stability studies of CA140 and dopamine in vitro
To examine the stability of CA140 in vitro, samples were generated from stock solutions (30 mM in DMSO) of either dopamine (DA, as a control for CA140) or CA140 by dilution in 1 mL of preheated PBS buffer to yield final concentrations of 500 μM. The solutions were incubated at 37 °C in a thermoblock, and the concentration of CA140 or DA was followed over time in triplicate. Aliquots (50 μL) were taken at 0, 1, 2, 4, 6, 8, and 22 h and added to 200 μL of acetonitrile. The samples were mixed by vortexing for 30 s and then centrifuged at 4 °C for 15 min at 14,000 rpm. The clear supernatants were diluted in PBS (2-fold) and analyzed by HPLC at 254 and 280 nm for CA140 and DA, respectively.
Antibodies and inhibitors
The following primary antibodies were used throughout this study: rat anti-mouse CD11b (1:400, Abcam), rabbit anti-F-actin (1:1000, Abcam), rabbit anti-COX-2 (1:1000, Abcam), rabbit anti-IL-1β (1:200, Abcam), rabbit anti-GFAP (1:5000, Neuromics), rabbit anti-Iba-1 (1:1000, Wako), goat anti-Iba-1 (1:500, Wako), rabbit anti-AKT (1:1000, Santa Cruz), rabbit anti-p-AKT (Ser473, Thr308) (1:1000, Cell Signaling), rabbit anti-ERK (1:1000, Santa Cruz), rabbit anti-p-ERK (Thr42/44) (1:1000, Cell Signaling), rabbit anti-STAT3 (1:1000, Cell Signaling), rabbit anti-p-STAT3 (Ser727, Abcam), mouse anti-PCNA (1:1000, Santa Cruz), rabbit anti-D2R (1:1000, Abcam), and rabbit anti-D1R (1:1000, Millipore) antibodies. We used the following small molecules: D1R antagonists (LE300, 10 μM, Sigma-Aldrich; SCH23390, 30 μM, Tocris), D1R agonist (A77636 hydrochloride, 10 nM, Tocris), D2R antagonist (eticlopride hydrochloride, 100 nM, Sigma-Aldrich), a STAT3 inhibitor (S3I-201, 50 μM, Sigma-Aldrich), and an ERK inhibitor (PD98059, 10 μM, Millipore).
MTT assay
BV2 microglial cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-di
phenyltetrazolium bromide (MTT) assay. BV2 microglial cells were seeded in 96-well plates and treated with various concentrations of CA140 (1–50 μM) or vehicle (1% DMSO) for 24 h in the absence of FBS. The cells were subsequently treated with 0.5 mg/mL MTT and incubated for 3 h at 37 °C in a 5% CO
2 incubator. The absorbance was read at 580 nm.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from cells using TRIzol (Invitrogen) following the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using a Superscript cDNA Premix Kit II with oligoDT (GeNetBio, Korea), and RT-PCR was performed using Prime Taq Premix (GeNetBio, Korea). RT-PCR products were separated by electrophoresis on 1.5% agarose gels with Eco Dye (1:5000, Korea) and photographed. Images were analyzed using ImageJ (NIH) and Fusion (Korea).
Immunocytochemistry
BV2 microglial cells were fixed in ice-cold methanol for 8 min, washed three times with 1 × PBS, and incubated with CD11b and COX-2 or CD11b and IL-1β antibodies in GDB buffer (0.1% gelatin, 0.3% Triton X-100, 16 mM sodium phosphate pH 7.4, and 450 mM NaCl) overnight at 4 °C. The next day, the cells were washed three times with 1 × PBS and incubated with the following secondary antibodies for 1 h at room temperature: Alexa Fluor 488 and Alexa Fluor 555 (1:200, Molecular Probes, USA). Images were obtained on a single plane using a confocal microscope (Nikon, Japan) and analyzed using ImageJ software.
Immunohistochemistry and immunofluorescence
Animals were perfused and fixed with 4% paraformaldehyde (PFA) solution, and brain tissues were flash-frozen and dissected using a cryostat (35-mm-thick sections). Each brain section was processed for immunofluorescence or immunohistochemical staining. For immunofluorescence staining, sections were rinsed in PBS and incubated with rabbit anti-Iba-1 (1:1000, Wako, Japan) for microglia or rabbit anti-GFAP (1:5000, Neuromics) for astrocytes. Antibodies were diluted in 0.5% bovine serum albumin (BSA) and incubated at 4 °C overnight. The following day, tissues were rinsed with 0.5% BSA and incubated with Alexa Fluor 555-conjugated anti-rabbit IgG (1:200, Molecular Probes) for 1 h at room temperature. The tissues were subsequently mounted on a gelatin-coated cover glass and covered with DAPI-containing mounting solution (Vector Laboratories). Images of the stained tissues were captured using confocal microscopy (TI-RCP, Nikon).
For immunohistochemistry, sections were permeabilized for 1 h in PBS with 0.2% Triton X-100 and 1% BSA at room temperature. The sections were then incubated with primary antibodies at 4 °C overnight. The next day, the tissues were washed three times with 0.5% BSA and incubated with biotin-conjugated anti-rabbit antibody (1:400, Vector Laboratories) for 1 h at room temperature. After rinsing with 0.5% BSA, the sections were incubated for 1 h at room temperature in avidin-biotin complex solution (Vector Laboratories, Burlingame, CA), followed by rinsing three times in 0.1 M phosphate buffer (PB). The signal was detected by incubating the sections in 0.5 mg/mL 3,3′-diaminobenzidine (DAB, Sigma-Aldrich) in 0.1 M PB containing 0.003% H2O2. The sections were rinsed in 0.1 M PB and mounted on gelatin-coated slides, and images were obtained under a bright-field microscope (Leica).
Enzyme-linked immunosorbent assay (ELISA)
To measure the effects of pre- or post-treatment with CA140 on IL-1β, an enzyme-linked immunosorbent assay (ELISA) was performed. Briefly, BV2 microglial cells were treated with LPS (100 ng/mL) or PBS for 30 min, followed by treatment with CA140 (10 μM) or vehicle (1% DMSO). IL-1β ELISA was then performed using the conditioned medium. Mouse IL-1β ELISA kits (ELISA development reagents; R&D Systems, Minneapolis, MN) were used according to the manufacturer’s recommendations. Recombinant mouse IL-1β protein (R&D Systems) was used as a standard. The absorbance of the samples was measured at 450 nm using a microplate reader (BMG Labtech, Offenburg, Germany).
Griess assay
To examine the effects of CA140 on nitrite (NO) production, the Griess assay was performed. BV2 microglial cells were incubated with CA140 (10 μM) or vehicle (1% DMSO) for 30 min, followed by treatment with LPS (100 ng/mL) or PBS for 23.5 h. The conditioned medium was mixed with Griess reagent (0.1% N-(1-naphthyl)ethylenediamine dihydrochloride and 1% sulfanilamide in 2% phosphoric acid) in 96-well plates and incubated at room temperature for 5 min. The absorbance was measured at 540 nm using a microplate reader, and the level of nitrite was analyzed against a standard curve of sodium nitrite.
Western blotting
Cells were lysed using RIPA buffer containing protease and phosphatase inhibitor tablets (Roche, USA). Western blot analysis was performed as previously described [
22]. Images were analyzed using Fusion software or ImageJ.
Cytosolic and nuclear fractionation
BV2 microglial cells were lysed in cytosolic fractionation buffer (10 mM HEPES pH 8.0, 1.5 mM MgCl
2, 10 mM KCl, 0.5 mM DTT, 300 mM sucrose, 0.1% NP-40, and 0.5 mM PMSF). After 5 min, the cell lysates were centrifuged at 10,000 rpm at 4 °C for 1 min, and the supernatant was stored as the cytosolic fraction. The pellet was lysed in nuclear fractionation buffer (10 mM HEPES pH 8.0, 20% glycerol, 100 mM KCl, 100 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF) on ice for 15 min, followed by centrifugation at 10,000 rpm at 4 °C for 15 min. The cytosolic and nuclear fractions were analyzed by western blot as previously described [
23].
Statistical analyses
All data were analyzed using a two-tailed T test or ANOVA with GraphPad Prism 4 software. Post hoc analyses were performed using Tukey’s multiple comparison test with significance set at p < 0.05. Data are presented as the mean ± S.E.M. (*p < 0.05, **p < 0.01, ***p < 0.001).
Discussion
Increasing evidence is highlighting the critical role of the immune system in neurodegenerative diseases such as AD. Unchecked glial activation and neuroinflammation may represent hallmark diagnostic features of neurodegenerative diseases. However, research to explicate the mechanisms underlying neuroinflammation has been limited.
Several recent studies have demonstrated that microglia and astrocytes release proinflammatory cytokines, leading to neuronal cell death and synaptic dysfunction in neurodegenerative diseases, including AD [
35,
36]. The release of these cytokines may be induced by LPS in vivo and in vitro via Toll-like receptors [
37,
38]. McGeer et al. determined that neuroinflammation stimulated by a single intraperitoneal injection of LPS lasted 10 months in the mouse brain and eventually led to neurodegeneration. Therefore, the identification of agents that reduce proinflammatory cytokine levels may represent a promising strategy for developing drugs to treat neurodegenerative diseases.
In this study, we synthesized a novel analog of dopamine, CA140 that can penetrate the blood-brain barrier. We determined that 10 μM CA140 was effective for lowering LPS-induced proinflammatory cytokine levels in BV2 microglial cells regardless of the timing of treatment (Fig.
2, Additional file
1: Figure S1). However, post-treatment with 5 μM CA140 only reduced the mRNA levels of LPS-induced IL-1β and not those of other proinflammatory cytokines. Our findings imply that an appropriate concentration of CA140 may be efficiently employed to both reduce and prevent neuroinflammatory responses in LPS-stimulated BV2 microglial cells. In addition, we observed that pretreatment with CA140 significantly reduced LPS-induced proinflammatory cytokine levels in rat primary microglia and primary astrocytes under high-glucose conditions (Additional file
1: Figure S6). However, post-treatment with CA140 only affected the LPS-stimulated proinflammatory response in rat primary microglial cells and not primary astrocytes under high-glucose conditions (Additional file
1: Figure S6). Why do pre- and post-treatment with CA140 have different effects on LPS-induced proinflammatory responses? Several recent studies have reported that high glucose levels induce primary glial cell activation [
25‐
28]. Thus, we conducted additional experiments to assess the anti-inflammatory effects of CA140 on primary glial cells under low-glucose conditions. Pre- or post-treatment with CA140 significantly reduced LPS-stimulated proinflammatory cytokine levels in mouse primary microglial cells under low-glucose conditions (Fig.
3a–f, Additional file
1: Figure S7). In addition, pretreatment with CA140 significantly reduced LPS-induced proinflammatory cytokine levels in mouse primary astrocytes (Additional file
1: Figure S7). Interestingly, post-treatment with CA140 only reduced LPS-induced iNOS mRNA levels in mouse primary astrocytes under low-glucose conditions (Fig.
3). These data suggest that pre- or post-treatment with CA140 may have different effects depending on cell type and culture conditions (e.g., low vs high glucose).
The physiological functions of the catecholaminergic neurotransmitter DA, which range from voluntary movement and reward to hormonal regulation and hypertension, are mediated by G-protein-coupled DA receptors (D1, D2, D3, D4, and D5) [
39,
40]. DA receptors have also been identified as important factors for controlling immunity in the CNS [
41]. Importantly, D1R and D2R are expressed in rodent and human microglia from brains damaged by stroke or neurodegeneration [
41‐
43]. Here, we observed that D1R and D2R were expressed in BV2 microglial cells and upregulated by LPS treatment (Additional file
1: Figure S8). Previous studies and our results may imply that the upregulation of DA receptors in microglia contributes to neuroinflammation in pathological conditions. Spiperone, a D1/D2R antagonist, inhibits DA-induced chemotaxis in cultured human microglia [
44]. Pretreatment with SCH23390, an antagonist of D1R, suppresses NO production by microglia in LPS-injected mice [
45]. Consistent with these findings, pretreatment with LE300 or SCH23390, antagonists of D1R, significantly suppressed COX-2 and IL-1β mRNA levels in LPS-stimulated BV2 microglial cells (Fig.
4). A68930, an agonist of D1R, inhibits the production of proinflammatory cytokines in mice [
46], and pretreatment with SKF83959, an atypical D1R agonist, reduces proinflammatory cytokine levels in LPS-stimulated BV2 microglia [
47]. However, in the present study, pretreatment with A77636, a selective agonist of D1R, did not reduce LPS-induced proinflammatory cytokine levels (Fig.
5). More importantly, treatment with A77636, LPS, and CA140 significantly suppressed LPS-stimulated IL-1β mRNA levels compared with treatment with A77636 and LPS, suggesting that CA140 regulates D1R to alter the LPS-induced neuroinflammatory response (Fig.
5).
With respect to the effects of D2R on neuroinflammation, the D2R agonist pramipexole increases nitrites in cultured primary microglia [
42]. In addition, sulpiride, an antagonist of D2R, reduces LPS-induced TNF-α and NO production [
45]. In astrocytes, D2R contributes to the suppression of neuroinflammation; however, microglial D2R is not involved in neuroinflammation according to studies of D2R-deficient mice or an ischemic mouse model [
48,
49]. In our study, the D2R antagonist eticlopride hydrochloride (EH) did not alter LPS-stimulated proinflammatory cytokine levels in BV2 microglial cells (Additional file
1: Figure S9). This discrepancy may be a result of differences in the details of the experimental procedures, such as treatment duration (i.e., 6 h compared with 24 h), the effective dose of antagonist or agonist, and/or pre- or post-treatment with a DA receptor antagonist. Based on the existing literature and our current findings, we suggest that CA140 may directly or indirectly interact with D1R and thereby regulate neuroinflammatory responses. However, we do not exclude other possibilities; for example, CA140 may regulate other neuroinflammation-related receptors (e.g., TLR4, other DA receptors) to modulate neuroinflammatory responses. Additional studies are required to fully dissect the molecular mechanisms involved in the CA140/DA receptors-induced neuroinflammatory response in vivo.
Activation of TLR receptors via LPS turns on downstream signaling cascades, such as MAP kinases, including ERK and AKT signaling in microglia and astrocytes [
10,
50,
51]. Therefore, inhibiting the MAP kinase signaling pathway has been suggested as a potential target for therapeutic drugs for anti-inflammation. Moreover, MAP kinase has been suggested as a downstream effector of both D1R and D2R stimulation [
52,
53]. Treatment with the D1R agonist SKF 38393 and the D2R agonist quinpirole activates ERK signaling in primary cultured striatal neurons [
54], and in cultured neuroblastoma cells, treatment with the D1R agonist SKF 38393 results in oxidative stress and cytotoxicity via ERK activation [
55]. Interestingly, our results indicated that pre- or post-treatment with CA140 significantly suppressed LPS-stimulated ERK signaling in BV2 microglial cells (Fig.
6, Additional file
1: Figure S10). In addition, we found that CA140 further reduced proinflammatory cytokine levels when combined with an ERK inhibitor, which suggests that CA140 alters LPS-induced ERK phosphorylation to modify the neuroinflammatory response.
STAT3, a member of the STAT family, is a transcription factor that plays a critical role in regulating microglial activation and inflammatory responses [
56,
57]. STAT3 levels in microglia are enhanced in brain injury and a neurodegenerative disease model [
58,
59]. Thus, we examined whether CA140 alters the nuclear localization of STAT3 to regulate the neuroinflammatory response and found that pre- or post-treatment with CA140 reduced cytosolic and nuclear p-STAT3 levels in LPS-stimulated BV2 microglial cells (Fig.
7, Additional file
1: Figure S11). Taken together, our data suggest that CA140 may alter neuroinflammation by regulating the ERK/STAT3 signaling pathway.
Systemic injection of LPS in wild-type mice significantly induces astrocyte and microglial activation and proinflammatory cytokine expression [
60‐
62]. Moreover, a single injection of LPS induces robust expression of IL-1β and TNF-α mRNA in various brain regions of wild-type mice [
63]. Intracerebral LPS injection in rats induces inflammatory responses and β-secretase-1 (BACE1) in the cortex and hippocampus, with axonal and dendritic pathologies similar to those present in AD [
64,
65]. Other studies have also demonstrated that LPS treatment exacerbates the accumulation of amyloid beta and tau pathology in a mouse model of AD [
59,
66,
67]. Interestingly, a recent study has shown that both SCH23390, a D1R antagonist, and sulpiride, a D2R antagonist, suppress proinflammatory cytokine levels in LPS-injected mice [
45]. DA released by electroacupuncture reduces proinflammatory cytokine levels through D1R in LPS-injected mice [
68]. In addition, the regulation of catecholamines by pharmacological agents, such as methylphenidate, enhances neuroinflammatory responses and microgliosis in 5xFAD mice, a transgenic AD mouse model [
32,
69]. In the present study, our novel drug CA140, which is structurally related to DA, also substantially reduced astrocyte and microglial activation as well as proinflammatory cytokine levels in LPS-injected wild-type mice and 5xFAD mice (Fig.
8–
10, Additional file
1: Figure S12). Taken together, our results suggest that CA140 may serve as a therapeutic agent for the prevention/treatment of neuroinflammation-related diseases, including AD.