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). 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), and protease/phosphatase inhibitor cocktail (78445) 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 with 12 h light/dark cycles at 22oC. 5XFAD mice were divided into two experimental groups (treatment vs. 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 the age of six months to nine months by intraperitoneal injection. Vehicle group and its littermate wild-type mice were treated with the same volume of vehicle by intraperitoneal injection. NJK14047 was dissolved in pure DMSO at 25 mg/ml which was used as a 40X stock solution. For working solution, an aliquot of the 40X stock solution 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 nine months of age. Both sexes of mice were used in Morris water maze test and Aβ1-42 ELSIA without any effect of sex on therapeutic effects. To minimize the sex difference on the degree of amyloidopathy, the protein and RNA samples were obtained from male mice, and histological sections 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 located 1.5 cm below the surface of the water. Titanium dioxide (TiO2) was dispersed in water to camouflage the target platform. The position of the platform was varied for each mouse with equal distribution 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, and 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 time spent finding the platform was recorded as the latency of each trial. If a 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 subjected to a trial in the absence of the platform for 60 s. The start position was standardized to the opposite direction of the area where the platform was located. 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. The 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 containing 1% 100X protease inhibitor) and centrifuged at 13,000 rpm for 20 min. The supernatant was collected and the concentrations were measured using the Bradford method (Bio-Rad, 5000006). Equal amounts of protein samples (~50 µg) were fractionated by SDS-PAGE and 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 detection system (ECL, Bio-Rad, 1705061). The band intensity was quantified using Image J densitometry (NIH, Bethesda, MD, USA), normalized with respect to the β-actin level.
Tissue preparation and immunofluorescence. The mice were anesthetized with intraperitoneal injection of 2.5% Avertin (2,2,2-tribromoethanol) and immediately perfused through the heart with PBS followed by 4% paraformaldehyde in PBS. Brains were excised, post-fixed in 4% paraformaldehyde at 4°C overnight, and incubated in 30% sucrose at 4°C until reaching equilibrium. The brains were embedded in 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; and then incubated with primary antibodies at 4°C overnight. After incubation with primary antibodies, the brain 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, and 100% EtOH for 5 min, respectively; afterward, coverslip was applied over fluorescence mounting medium.
Aβ ELISA. An enzyme-linked immunosorbent assay (ELISA) for human Aβ1-42 was performed using fluorescence‐based ELISA kits (Invitrogen) and appropriate Aβ standards in compliance with 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 at pH 8.0. Homogenates were mixed at room temperature for 4 h and then, diluted in PBS containing 5% BSA, 0.03% Tween 20, and protease inhibitor cocktail.
Quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted from the mouse cortex and hippocampus with a Hybrid-R total RNA purification kit (GeneAll®, 305-101) in accordance with the manufacturer’s instructions. The NanoDrop™-2000c (ThermoFisher Scientific) was used to assess the concentration and purity of the RNA samples. cDNA was synthesized 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 measured data were normalized to the GAPDH level using 2-ΔΔCT method and expressed as the fold change with respect to the mean of the control group.
Fluoro-jade B staining. To detect 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. Afterward, the brain sections were incubated with 0.06% potassium permanganate for 10 min, followed by a 2-min wash with distilled water. The sections were then 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 either 1 μM or 10 μM NJK14047. After 22 h of LPS stimulation, all media were changed to fresh neurobasal medium. The cells were incubated in neurobasal media 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, and then used to culture primary neurons within 24 h.
Primary cells and treatment. Primary mouse cortical microglia and astrocytes were prepared according to the commonly used protocols as previously described [32, 33]. Briefly, mouse cortical mixed glia was 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 three days. After five to seven days, the flasks were vigorously tapped, and the supernatant was collected and centrifuged at 3,000 rpm for 10 min to obtain microglia. 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 the cells were found to be positive for ionized calcium-binding adapter molecule 1 (Iba-1) which is a marker of microglia. Afterward, the flasks were tapped, and the supernatant was discarded; 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 to harvest astrocytes. Approximately 98% of the cells were positive for glial fibrillary acidic protein (GFAP) which is a marker of astrocytes. 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 media were changed to fresh neurobasal medium to incubate the cells for another 24 h. The conditioned neurobasal medium was obtained and centrifuged at 1,500 rpm for 5 min. The supernatant was collected stored at 4°C, and then treated to primary neurons within 24 h.
Primary mouse cortical neurons were prepared as previously described [34]. In brief, mouse cortical neurons were obtained from E17 C56BL/6J mouse embryos. The neurons were seeded on poly-L-lysine pre-coated 48-well plates or 24-well coverslips for the MTT and the TUNEL assay, respectively. Neurobasal medium containing 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. The MTT assay is generally used to assess the cell viability by measuring the metabolic activity of the cells [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 medium conditioned with BV2, C8-D1A, primary cultured microglia, and primary cultured astrocytes, respectively, for 24 h. The media were replaced with neurobasal media containing 10% MTT. Absorbance at 570 nm corrected with 690 nm values were measured by a microplate reader. The measured cell viability was expressed as a percentage relative to the control mean.
TUNEL assay. Apoptotic DNA fragmentation was detected using the TUNEL method [36]. To measure the apoptotic cell death of primary neuronal cells, mouse 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 in each conditioned medium for 24 h. After incubation, the cells were subjected to TUNEL staining in accordance with the manufacturer’s instructions. Apoptotic cells were detected as localized bright green cells (positive cells) in a blue background using an Olympus BX51 microscope. The extent of apoptosis was quantified 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 areas area (2 left; 2 right) and four hippocampus areas area (2 left; 2 right) were imaged in one brain slice. For each mouse, thioflavin S-positive area, 6E10-positive area, and Fluoro-jade B-positive cells were quantified with four to five brain slices (Bregma −1.30 to −2.70, 300 µm apart) using Image J software. The number of Iba-1- and GFAP-positive cells were quantified using Cell Profiler software [37]. To quantify the number of plaques associated microglia, Iba-1-positive cells within the 20 µm range of plaques were manually counted. A total of 70-75 plaques (> 10 µm) from five mice per group were measured [26].
Statistical analysis. In all in vivo studies, n represents the number of animals used in the corresponding experiment. For in vitro studies, n is the number of independent experiments. The group size for each experiment was based on our previous results [38]. The operators responsible for the experimental procedure and data analysis were blinded and unaware of group allocation throughout the experiments. All data were analyzed using SPSS version 25 (IBM corporation, NY, USA) with statistical significance defined as a P-value less than 0.05. Parametric tests such as ANOVA were used when the data satisfied the null hypothesis of the Levene’s test. The Tukey’s post-hoc test was performed if the P-value was < 0.05 in one-way ANOVA. In case of qRT-PCR and the MTT assay, the Kruskal-Wallis test followed by Dunn’s post-hoc multiple comparisons was used to compare among the experimental groups. Latency of Morris water maze task was analyzed using the generalized estimating equation (GEE) method. Descriptive statistics were used to summarize the data using mean ± standard error of the mean (SEM). All graphs were constructed using Graph Pad Prism 5.0 software (Graph Pad software Inc., CA, USA).