Antibodies and reagents
All primary antibodies used in this study were as follow: γH2A.X (Proteintech, Cat#10856-1-ap), S345 (Gene Tex, Cat#39233), ChK1 (Bioss, Cat ac01184523; Abclonal, Cat#a7653), S727-STAT3 (Affinity, Cat#af3294), STAT3 (Zenbio, Cat#385805), CIP2A (Cell Signaling Technology, Cat#14805), GluA1 (Cell Signaling Technology, Cat#13185), Synaptophysin (Abcam, Cat#ab32127), β-actin (Abcam, Cat#ab8226), Synapsin 1 (Millipore, Cat#S193), GAPDH (Proteintech, Cat#60004-1-Ig).
Chk1 plasmid and GFAP-ChK1-AAV were from Shanghai Genechem Co.,Ltd. (Shanghai, China). Neofect DNA transfection reagent was purchased from Neofect Biological Technology Co, Ltd (Beijing, China). Neurobasal, B27, DMEM-high and protein marker were from Invitrogen (Grand Island, NY, USA). DMEM/F12 and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, New York, USA). Serine/Threonine Phosphatase Assay kit was purchased from Promega Company (Madison, Wisconsin, USA). Human or rat or mouse Aβ40/Aβ42 ELISA kits were purchased from Elabscience Biotechnology (Wuhan, China). Nissl staining solution was from Beyotime biotechnology (Shanghai, China). Golgi staining solution was from GENMED SCIENTIFICS INC. USA.
Animals and AAV delivering
For animal experiments, Sprague-Dawley rats and C57BL/6J mice were from Zhejiang Vital river Experimental Animal Technology Co. LTD Tongxiang branch. 3xTg-AD mice carrying human mutated APP, PS1 and tau genes were from Jackson Lab. All the mice can take food and water freely in an air-conditioned room (22 ± 2°C, 12 hours light/dark cycle). The behavior tests were performed on their active hours.
For AAV injection, C57 mice were deeply anesthetized with isoflurane. AAV virus particles (2.0 μl at 0.4 μl/min) was injected into the lateral ventricle (interaural 3.58 mm, bregma 0.22 mm, depth 0.30 mm). After the injection, the mice were kept under standard laboratory conditions.
HEK 293-T cell culture and transfection
For HEK293-T cell culture, the cells were cultured in DMEM-high medium supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin at 37°C in the presence of 5% CO2.The cells were cultured to 50%-60% confluence in 6-well plates and changed to fresh medium, then transfected with relevant plasmids together with Neofect DNA transfection reagent (Neofect Biological Technology, Beijing, China). After 48 hours, cells and culture media were collected for further detections.
Primary astrocyte and neuron culture
Primary astrocyte culture and neuron culture were performed following the method described previously [13, 20]. At the end of treatments, cells were collected and lyzed in RIPA buffer for further biological detections or fixed with 4% paraformaldehyde for immunofluorescence imaging.
Western blotting and Co-IP
For Western blotting, brain tissue homogenates or cell lysates were boiled at 100°C for 5 min in the loading buffer (50 mM Tris-HCl, pH 7.6, 2% SDS, 10% glycerol, 10 mM DTT, and 0.2% bromphenol blue). The proteins were electrophoresed in 10% SDS-polyacrylamide gel and the separated proteins were transferred to nitrocellulose membranes (Amersham Biosciences). The membranes were then blocked with 5% nonfat milk dissolved in TBS-Tween-20 (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.2% Tween-20) for 1 h and probed with primary antibody at 4°C overnight. Then the blots were detected using secondary antibodies at room temperature for 1 h and visualized using the Odyssey Infrared Imaging System (LicorBiosciences, Lincoln, NE, USA). The protein bands were quantitatively analyzed by Image J software (Rawak Software, Inc. Germany).
To analyze protein-protein interactions, Co-IP experiments were performed. Cells were lysed using Pierce IP lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche). The cell lysates were centrifuged, and then, immunoprecipitated overnight at 4°C using the indicated primary antibodies followed by incubation with Dynabeads Protein G (Life Technologies) for 1 h The immunocomplexes were washed twice with IP lysis buffer before being resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with indicated antibodies.
Fluorescence imaging and confocal microscopy
For cultured neurons, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.5% Triton X-100 for 10 min, followed by incubation with 3% bovine serum albumin (BSA) to block nonspecific sites. For mouse brain slices, sectioned slices were incubated with 3% bovine serum albumin (BSA) to block nonspecific sites. After blocking, primary antibody incubation was performed overnight at 4°C. Alexa 488-conjugated secondary antibody (1:200, A-21206, Invitrogen) was used for fluorescence labeling. All the images were observed under the LSM710 confocal microscope (Zeiss, Germany).
For cultured astrocytes, the previous steps were as above. After blocking, primary antibodies incubation was performed overnight at 4°C. Alexa 488 and 543-conjugated secondary antibody (1:200, A-21206, A-10036, Invitrogen) were used for double fluorescence labeling. All the images were observed under the LSM710 confocal microscope (Zeiss, Germany).
ChK1 and PP2A activity and ELISA assay
ChK1 and PP2A activity was measured according to the protocols provided by the manufacturers (ChK1, GMS50155.2 GENMEDSCIENTIFICS INC. U.S.A ; PP2A, V2460 kit; Promega). The culture medium or brain tissue homogenates which were lyzed in RIPA buffer were collected. The levels of IL-6, TNF-α, Aβ40 and Aβ42 were detected by ELISA following the construction offered by the assay kit manufacturer (Elabscience Biotechnology, Wuhan, China).
Behavior Tests
Novel objective recognition test(NORT)
The mice were habituated to the arenas (50 cm × 50 cm ×50 cm wooden container which was from Techman Software Co., Ltd., Chengdu, China) for 5 min without objects 24 h prior to the test. Arenas were cleaned with 70% ethanol between each habituation period. The day after the mice re-entered the arenas from the same starting point and were granted 5 minutes to familiarize themselves with the A object and B object. One hour after the familiarization period, B object was replaced with C object, and the mice were granted 5 minutes to explore both objects. After 24 h, C object was replaced with D object, and the mice were granted 5 minutes to explore both objects. All the behavior was recorded by a camera above the arena. The recognition index was calculated by TA/(TA+TB), TB/(TA+TB), TC/(TA+TC),TD/(TA+TD). The discrimination index was calculated by (TC-TA)/(TA+TC), (TD-TA)/(TA+TD). TA, TB, TC, TD were respectively the time mice exploring the object A, B, C and D.
Object location test(OLT)
The animals were habituated to the container that is same as novel objective recognition test. During the training session at the 6th day, the mice freely explored the floor of the box that contained two different objects. To get a measure of the OLT, one object was moved 45 cm to a new position, the total time of exploration of the familiar and novel object localization was measured 24 and 48 hours after the training. The objects and boxes were cleaned with ethanol after every training or test.
Morris water maze test
Spatial learning and memory were detected by Morris Water Maze (Techman Software Co., Ltd., Chengdu, China). Briefly, a circular arena (120 cm×50 cm) was filled with water (23 ± 2 °C). An escape platform (10×10×15 cm) was placed into the pool, 1.5 cm below the water surface. The water was made opaque by the addition of a white titanium dioxide. The test room contained several permanent extra-maze cues such as posters, a flag or other objects on the walls. A video-tracking camera above the center of the pool surface monitored the trajectory of the mice. Learning consisted of six consecutive daily acquisition sessions, each of them consisting of four trials, with a maximum trial duration of 60 s. Latency time (s) to find the hidden platform were recorded during each trial of each learning session. If the mice found the platform within the maximum trial time allowed, it was left on the platform for 20 s. If the mice did not find the platform within the time limit, it was gently placed on the platform for a 20 s period. Probe tests were carried out 1 or 2 days after acquisition.
Fear conditioning test
The fear conditioning test paradigm was performed following the methods previously described. In brief, the test was conducted in a conditioning chamber (33 cm* 33 cm*33 cm) equipped with white board walls, a transparent front door, a speaker, and a grid floor (Techman Software Co., Ltd., Chengdu, China). On day 1, mice were placed into the conditioning chamber and allowed free exploration for 2 minutes before the delivery of the conditioned stimulus (CS) tone (20 seconds, 80 dB, 2000 Hz) paired with a foot shock unconditioned stimulus (US; 2 seconds, 0.95 mA) through a grid floor at the end of the tone. A total of five CS-US pairs with a 60-s intertrial interval (ITI) were presented to each animal in the training stage. The mouse was removed from the chamber 1 minute after the last foot shock and placed back in its home cage. The contextual fear conditioning stage started 24 hours after the training phase, when the animal was put back inside the conditioning chamber for 5 minutes. The animal's freezing responses to the environmental context were recorded. The animal was placed back into the same chamber with different contextual cues, including blue wall, smooth plastic floor, and alcohol drops condition for 5 minutes, and the animal's freezing responses to the altered context were recorded. The tone fear conditioning stage started 24 hours after the different contextual stage. After 2 minutes of free exploration, the mouse was exposed to the exact same 3-CStones with 20-s ITI in the training stage without the foot shock, and its freezing responses to the tones were recorded.
Nissl staining
Thirty micrometers of coronal sections were mounted on gelatin-coated slides. Then, the sections were incubated in Cresyl violet for 3 minutes at room temperature, following with dehydration through 50%, 75%, 95%, and 100% alcohol, cleaning in xylene, and cover slipped with neutral balsam. Images were observed using light microscope.
Golgi staining
The mice were anesthetized and perfused intracardially with 400 ml normal saline containing 0.5% sodium nitrite, followed with 400 ml 4% formaldehyde and the Golgi dye solution containing 5% chloral hydrate, 4% formaldehyde, and 5% potassium dichromate. After perfusion, the brains were dissected into 5 mm × 5 mm sections and transferred to a vial containing Golgi dye solution for 3 days in dark, then immersed in solution containing 1% silver nitrate for another 3 days. The brains were serially sectioned into 100 μm thick slices using a vibrating microtome (Leica, VT1000S, Germany). Images were observed under the microscope (Nikon, Tokyo, Japan).
Statistical analysis
Data are expressed as mean ± SEM and analyzed using GraphPad Prism 8 statistical software (USA, GraphPad Software). The one-way analysis of variance (ANOVA) procedure followed by Tukey was used to determine the differences among groups. For the comparison between two groups, student-t test was used. The significance was set at P < 0.05. All results shown correspond to individual representative experiments.