Chemicals and reagents. A selective p38α/β MAPKs inhibitor NJK14047 was synthesized by a previously reported procedure (>97%, HPLC) [23, 25]. Dulbecco’s modified Eagle’s medium (DMEM), Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from GE healthcare HyClone™. Cell culture flasks and plates were purchased from SPL (70075, 30006, 30024, 30048, 30096). Lipopolysaccharide (LPS) from Escherichia coli serotype O55:B5 (L6529, ≥500,000 EU/mg), Methylthiazolyldiphenyl-tetrazolium bromide (MTT), Thioflavin S (T1892) and DPX mounting medium (06522) were obtained from Sigma-Aldrich. Neurobasal medium (21103-049), B27 supplements (17504-044), RIPA buffer (89901), protease and phosphatase inhibitor cocktail (78445) and Griess reagent (G7921) were obtained from ThermoFisher Scientific. Fluorescence mounting medium (S3023) was purchased from Dako. TUNEL (G3250) assay kits were obtained from Promega. Information about antibodies used in this study is listed in Table.1.
Animals and treatment. 5XFAD mice on B6/SJL background (34840-JAX, Tg6799) and wild type B6/SJL mice from Jackson Laboratory were bred and maintained in an individual ventilated cage, 12 h light/dark cycle and 22 oC condition. 5XFAD mice were divided into two experimental groups (treatment group and vehicle group) using the block randomization method. Similar to previous studies [26, 27], treatment group was treated with NJK14047 at 2.5 mg/kg every other day from 6-month-old to 9-month-old by intraperitoneal injection. Vehicle group and its littermate wild-type mice were treated with the same volume of vehicles by intraperitoneal injection. NJK14047 was dissolved in pure DMSO at 25 mg/ml which was used for 40X stock solution. For working solution, the aliquot was diluted in PBS and filtered using 0.25 µm syringe filter. The body weights of the mice did not significantly differ among groups and any conspicuous side effects (weight loss, anorexia, convulsion or death) were not observed. All mice used in this study were sacrificed at 9-month-old. Both sexes of mice were used in Morris water maze test and Aβ1-42 ELSIA and there was no sex difference in therapeutic effects. To minimize the sex difference on the degree of amyloidopathy, the protein and RNA samples were acquired from male mice and histological sections were obtained from female mice. All experiments were approved by the Kyung Hee University Institutional Animal Care and Use Committee (IACUC, KHUASP(SE)-17-126-1).
Behavioral test. The Morris water maze task was used to assess spatial memory performance. The water maze was a white circular PVC tank (130-cm diameter, 40-cm height) in which water was filled to 31.5 cm in depth (23 ± 1 °C). The submerged target platform was a 10 cm diameter circular zone 1.5 cm below the surface of the water. Titanium dioxide (TiO2) was dispersed in the water to camouflage the target platform. The position of the platform was varied from mouse to mouse and distributed equally between experimental groups. One day before training, mice were habituated to the maze and swimming in the absence of cues. During the training period, mice were subjected to four trials per day. The start position of each trial was equally distributed. In each trial, the mouse was given 60 s to find the target platform in the presence of cues around the maze. The spent time before finding the platform was recorded as the latency of each trial. If the mouse did not find the platform within 60 s, it was guided to the platform and allowed to stay on the platform for 10 s. After a total 10 days of training, a probe test was performed (day 11). The mouse was subject to a trial in the absence of the platform for 60 s. The start position was standardized to the opposite direction of the area in which the platform had existed. All trials were recorded using a camera and analyzed by a free tracking software tool, Toxtrac [28]. The software, the user manual, and the documentation are available at https://toxtrac.sourceforge.io.
Immunoblotting. Immunoblotting analysis was performed as previously described [29]. In brief, mice were sacrificed after the behavioral test. Cortex and hippocampus were quickly isolated and stored at −80 °C until use. The brain tissues were homogenized in 10X volume of lysis buffer (RIPA buffer including 1 % 100X protease inhibitor) and centrifuged at 13,000 rpm for 20 min. The supernatant was collected; concentrations were measured using the Bradford method (Bio-Rad, 5000006). Equal amounts of protein samples (~50 µg) were fractionated by SDS-PAGE, then transferred to PVDF membranes. The membranes were blocked with 5 % skim milk and probed with primary antibodies at 4 °C overnight. After several washes in TBS-T, the membranes were probed with corresponding secondary antibodies conjugated with HRP. The immunoblot signals were developed with an enhanced chemiluminescence (ECL, Bio-Rad, 1705061) detection system. The band intensity was quantified using image J densitometry (NIH, Bethesda, MD, USA) and the data were normalized with respective β-actin level.
Tissue preparation and immunofluorescence. The mice were anesthetized with intraperitoneal injection of 2.5 % Avertin (2, 2, 2-tribromoethanol) and immediately cardiac perfused with PBS followed by 4 % paraformaldehyde in PBS. Brains were excised and post-fixed in 4 % paraformaldehyde at 4 °C overnight and incubated in 30 % sucrose at 4 °C until they had equilibrated. The brains were embedded into O.C.T. compound blocks at −80 °C. Sequential 30-µm coronal sections were obtained with a cryostat (CM30 50S; Leica). Every tenth section (300 µm apart) of the brain (Bregma −1.30 to −2.70 mm) was used for immunohistochemistry. Free-floating brain sections were rinsed in PBS, blocked for 1 h in 2 % normal goat serum, 2 % BSA and 0.4 % Triton-X100, then incubated with primary antibodies at 4 °C overnight. After incubation with primary antibodies, sections were washed with PBS and incubated for 2 h with corresponding secondary antibodies conjugated with Alexa-Fluorescence.
Thioflavin S staining. To stain fibrillary Aβ, the brain sections were incubated with 0.1 % Thioflavin S in 50 % EtOH for 10 min. Brain sections were sequentially washed with 50 % EtOH, 70 % EtOH, 100 % EtOH for 5 min respectively and coversliped with fluorescence mounting medium.
Aβ ELISA. An enzyme linked immunosorbent assay (ELISA) for human Aβ1-42 was performed using fluorescent‐based ELISA kits (Invitrogen) and appropriate Aβ standards, according to the manufacturer's protocol. The hippocampus and frontal cortex from one hemisphere were homogenized in 10X volume of guanidine buffer with a final concentration of 50 mM Tris and 5 M guanidine HCl, pH 8.0. Homogenates were mixed at room temperature for 4 hours. After mixing, homogenates were diluted in PBS containing 5 % BSA, 0.03 % Tween 20, and protease inhibitor cocktail.
Quantitative real time polymerase chain reaction (qRT-PCR). Total RNA extraction from mouse cortex and hippocampus was performed with a Hybrid-R total RNA purification kit (GeneAll®, 305-101), according to the manufacturer’s instructions. The concentration and purity of the RNA samples were assessed with a NanoDrop™-2000c (ThermoFisher Scientific). cDNA synthesis was performed using TOPscript RT DryMIX (Enzynomics, RT200), according to the manufacturer’s instructions. cDNA samples were subjected to qRT-PCR using SYBR Green Mix (Enzynomics, RT500) and a CFX Connect real time PCR system (Bio-Rad). The qRT-PCR protocol was as follows: first holding stage at 95 °C for 3 min, followed by a cycling stage at 95 °C for 10 s, 55 °C for 10 s, 72 °C for 30 s (30 cycles total), and finally a holding stage at 95 °C for 10 s. The information about primers used in this study is listed in Table.2. The data were normalized with respective GAPDH level using 2-ΔΔCT method and shown as fold of control mean.
Fluoro-jade B staining. To stain late-stage degenerating neuronal cells, Fluoro-jade B staining was performed [30, 31]. Brain sections were incubated with 1 % NaOH in 80 % EtOH for 5 min, followed by sequential washes with 70 % EtOH and distilled water. Then the brain sections were incubated with 0.06 % potassium permanganate for 10 min. After a 2-min wash with distilled water, the sections were incubated in 0.0004 % Fluoro-jade B (Millipore, AG310) plus 0.01 % acetic acid solution. After three washes with distilled water, the sections were fully dried and coverslipped with DPX mounting medium.
Cell lines and treatment. The BV2 (RRID: CVCL_0182) murine microglia cell line and the C8-D1A (RRID: CVCL_6379) murine astrocyte cell line were cultured in DMEM with 10 % FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5 % CO2. Cells were seeded on 6-well plates at 5 105 cells/well and stimulated with 500 ng/ml LPS after 2 h of pre-treatment with 1 μM or 10 μM NJK14047. After 22 h of LPS stimulation, all medium was changed to fresh neurobasal medium. The cells were incubated in neurobasal medium for another 24 h. The conditioned neurobasal medium was obtained and centrifuged at 1,500 rpm for 5 min to remove the remaining cells. The supernatant was collected and stored at 4 °C, then used to culture primary neurons within 24 h.
Primary cells and treatment. Primary mouse cortical microglia and astrocytes were prepared according to public protocols [32, 33]. Briefly, mouse cortical mixed glia were obtained from P1 to P4 C57BL/6J mouse pups and seeded on poly-L-lysine pre-coated 75T flasks at 1.5 107 cells/flask. Culture medium was changed every 3 days. After 5–7 days, to obtain microglia, the flasks were vigorously tapped and the supernatant which contained the microglia was collected and centrifuged at 3,000 rpm for 10 min. The cell pellet was dispersed in new medium and seeded on poly-L-lysine pre-coated 6-well plates at 5 105 cells/well. After 2 h of cell seeding, the culture medium was replaced with fresh medium to remove the oligodendrocytes. Approximately 96 % of cells were found to be positive for a marker of microglia, ionized calcium-binding adapter molecule 1 (Iba-1). After flask tapping and supernatant discarding, to obtain astrocytes, the rest of the cells in the flask were rinsed with PBS and seeded onto poly-L-lysine pre-coated 6-well plates at 5 105 cells/well. Approximately 98 % of cells were positive for a marker of astrocytes, glial fibrillary acidic protein (GFAP). Prepared microglia and astrocytes were stimulated with 50 ng/ml LPS after 2 h of pre-treatment with 10 μM NJK14047. After 22 h of LPS stimulation, all medium was changed to fresh neurobasal medium, in which the cells were incubated for another 24 h. The conditioned neurobasal medium was obtained and centrifuged at 1,500 rpm for 5 min. The supernatant was collected and stored at 4 °C, then used to primary neurons within 24 h.
Primary mice cortical neurons were prepared as previously described [34]. In brief, mouse cortical neurons were obtained from E17 C56BL/6J mouse embryos and seeded on poly-L-lysine pre-coated 48-well plates, for the MTT assay, or 24-well coverslips, for the TUNEL assay. Neurobasal medium including 2 % B27 supplement, 2 mM L-glutamine (Welgene, LS 002-01) and 1 % P/S was used to culture neurons. Primary neurons were used in experiments at 10 days in vitro (DIV).
Cytotoxicity assay. MTT assay measures the metabolic activity of the cells so it is generally used to assess the cell viability [35]. To assess the cytotoxicity of glia-conditioned medium, mouse cortical primary neurons were seeded on poly-L-lysine pre-coated 48-well plates at 1 105 cells/well. On DIV10, neurons were incubated in the BV2, C8-D1A, primary cultured microglia and primary cultured astrocyte conditioned medium for 24 h. Media were exchanged for neurobasal media including 10 % MTT. Absorbance at 570 nm corrected with 690 nm values were measured by a microplate reader. The data were normalized to the control group and shown as % of control mean.
Nitric oxide concentration measurement. To measure the Nitric oxide (NO) concentration in the conditioned medium obtained from each cell line, conditioned medium was mixed with Griess reagent at 1:1 ratio. Absorbance at 550 nm values were measured by a microplate reader.
TUNEL assay. TUNEL is a widely used method to detecting apoptotic DNA fragmentation [36]. To measure the apoptotic cell death of primary neuronal cells, mice cortical primary neurons were seeded on poly-L-lysine pre-coated 24-well cover glasses at 1.5 105 cells/well. On DIV10, neurons were incubated with each conditioned medium for 24 h. After incubation, the cells were subjected to TUNEL staining in according to the manufacturer’s instructions. Apoptotic cells were detected as localized bright green cells (positive cells) in a blue background by using an Olympus BX51 microscope. The quantification of apoptosis was performed using Image J software.
Confocal microscopy and image analysis. All stained brain sections were imaged by confocal microscopy. Z-stacked images were acquired at 1.5 µm intervals (total 15 optical slices). Four cortex area (2 left; 2 right) and four hippocampus area (2 left; 2 right) were imaged in one brain slice. Four to five brain slices (Bregma −1.30 to −2.70 mm, 300 µm apart) were used to quantify one mouse. Thioflavin S positive area, 6E10 positive area and Fluoro-jade B positive cells were quantified using Image J software. The number of Iba-1 positive cells and GFAP positive cells were quantified using Cell Profiler software [37]. To quantify the number of plaque associated microglia, Iba-1 positive cells within 20 µm range of plaques were manually counted. Total 70-75 plaques (> 10 µm) from five mouse per group were measured [26].
Statistical analysis. In all in vivo studies, the ‘n’ number means the number of animals used in the statistical analysis. Regarding in vitro studies, the ‘n’ number means the number of independent experiments. The group sizes for each experiment was based on our previous results [38]. The operators responsible for experimental procedure and data analysis were blinded and unaware of group allocation throughout the experiments. All data were analyzed using SPSS ver.25 (IBM corporation, NY, USA) and P-values less than 0.05 were considered statistically significant. Parametric tests were used when the data satisfied with the null hypothesis of Levene’s test. Tukey’s post-hoc test were used if the p-value < 0.05 in one-way ANOVA. In case of qRT-PCR and MTT assay, Kruskal-Wallis test followed by Dunn’s multiple comparison test was used. Latency of Morris water maze task was analyzed using generalized estimating equation (GEE) analysis. To analyze the group differences of each day, one-way ANOVA was used satisfying the assumption of Levene’s test followed by Tukey’s post-hoc test. The general data were expressed as mean ± standard error of the mean (SEM) and all graphs were drawn using Graph Pad Prism 5.0 software (Graph Pad software Inc., CA, USA).