Antibodies and reagents
The antibodies used in the present study are listed in the Additional file 1: Table S1. TG-101348 (special JAK2 inhibitor, from MCE), JAK2 siRNA (sc-39099, from Santa Cruz Biotechnology), SP600125 (the inhibitor of JNK1, from Santa Cruz) and FR180204 (the inhibitor of ERK1, from Santa Cruz) were purchased. PcDNA3.0 vector coded wild type STAT1 (WT-STAT1) plasmid was the gift of Dr. Xiao-Yuan Li (Institute of Biomedical Sciences, Academia Sinica, Taiwan), and PcDNA3.0 vector coded wild type STAT3 (WT-STAT3) plasmid was purchased from Shangdong Vigene Bioscience Biotechnology, co., LTD. EGFP-N1 vector coded mutant full-length human tau (P301L-hTau) plasmid, WT-STAT1 plasmid was mutated to pseudoacetylated STAT1 plasmids (single or double lysine sites (410, 413) mutated to glutamine, K410Q-, K413Q-, K410/3Q-STAT1) or unacetylated STAT1 plasmids (single or double lysine site (410, 413) mutated to arginine, K410R-, K413R-, K410/3R-STAT1) coded into RFP-N1 vector by Shanghai Baicheng Biotechnology, co., LTD.
Male C57 mice were purchased from the Animal Center of Tongji Medical College, Huazhong University of Science and Technology. STAT3flox/flox mice (B6; 129S-STAT3tm1Mam/Mmjax) and Tg4510 mice (FVB-Fgf14Tg(tetO-MAPT*P301L)4510Kha/JlwsJ) were purchased from Jackson Laboratory. All mice were kept at 22 ± 2 ºC on 12 h light-dark cycles with ad libitum access to food and water. All animal experiments were performed according to the ‘Policies on the Use of Animals and Humans in Neuroscience Research’ revised and approved by the Society for Neuroscience (USA) in 1995, and the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. The Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology approved the study protocol.
Stereotaxic brain injection
Adeno-associated virus coded for human mutant full-length P301L-hTau (AAV-P301L) with the N-terminal fused with enhanced green fluorescent protein (eGFP) and the control AAV-eGFP, AAV-STAT3, AAV-K410/3R-STAT1 virus were purchased from OBio Biologic Technology Co., Ltd. AAV-cre was purchased from Shanghai Genechem Co.,Ltd. The titer of AAV-P301L was 1.3×1013 v.g./ml, that of AAV-STAT3 virus was 1.2×1013 v.g./ml, and the titer for AAV-K410/3R-STAT1 virus was 1.1×1013 v.g./ml. The titer of AAV-Cre or the control virus was 7.9×1012 v.g./ml. All AAV viruses were driven by CMV-promoter. The in vivo overexpression efficiency was measured by immunohistochemical staining and Western blotting 1 month later after injection of the virus into the hippocampal CA3 region of mice. After positioned in a stereotaxic instrument, 3 month-old C57 or STAT3flox/flox mice were bilaterally injected the virus into the hippocampal CA3 region (AP ±2.0, ML -1.5, DV -2.0) at a rate of 0.10 μl/min. The needle syringe was left in place for ~3 min before being withdrawn. The injection did not significantly change the normal activity or increase the death rate of the mice compared with the non-injected controls.
~1 month after brain infusion of the virus, Morris water maze (MWM) test was used to assess spatial learning and memory . For spatial learning, mice were trained to find a hidden platform in the water maze for 6 consecutive days, 4 trials per day with a 30 s interval from 14:00 to 20:00 pm. On each trial, an operator let the animal face the wall of the pool and started from one of the four quadrants. When the animal climbed on the platform, the trial ended. If the mice did not locate the platform within 60 s, operators guided them to the platform and stayed for 30 s. The spatial memory was tested 24 h after the last training. The longer a mouse stayed in the quadrant where the platform previously located, the higher the score for spatial memory. The swimming path and the latency to find the platform or times passing through the previous platform-located quadrant (during test phase) were recorded by a video camera. The camera was fixed to the ceiling, 1.5 m from the water surface, and connected to a digital-tracking device, which attached to an IBM computer.
The contextual fear conditioning test was performed according to our previously established procedure . Briefly, prior to experimentation, in order to adapt to the environment, the mouse was kept in the cage for 3 min. The animal then received training via 3 min unsignaled foot-shocks (one shock at the 1st min, three shocks at the 2nd min and 8 shocks at the 3rd min; 0.5 mA, 2-sec duration, and 1 min apart). Long-term memory (LTM) was tested respectively 24 h post-training by placing the animal back into the conditioning chamber for 3 min and measuring the freezing time.
The new object recognition test was performed according to a published procedure . A 5-min habituation without objects was conducted. After one hour, an object familiarization phase with two of Object A for 5 min was conducted. A further 5-min testing period with one of Object A and Object B, which were randomly assigned as A and B, was conducted after one hour later. A blinded investigator scored object recognition times. The novel object recognition ratio was calculated as: [(time novel object)/(time novel object +time familiar object)]. Object exploration was defined as active investigation of an object within ~2 cm or less of its nose.
A Leica VT1000S vibratome (Milton Keynes, UK) was used to cut horizontal brain slices (400 μm) containing the dorsal hippocampus at 4 º C in artificial cerebrospinal fluid (aCSF), which consisted of: 126 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 24 mM NaHCO3, 2 mM MgSO4, 2 mM CaCl2 and 10 mM glucose (pH 7.4; 305 mOsm), and saturated with carbogen (95% O2 and 5% CO2). Immediately after slicing, sections were transferred and maintained in an interface chamber continuously perfused with aCSF, and the slices were equilibrated at least for 30 min prior to recording at room temperature.
For extracellular recordings, slices were placed in the interface recording chamber at 32 º C and the perfusion rate was normally 3 ml/min, while maintaining a thin ﬁlm of aCSF covering the slice to make sure applied substances could diffuse into the area recorded. Field potentials were amplified with Neurolog AC-coupled NL 104 preamplifiers (Digitimer Ltd, Welwyn, UK). The excitatory postsynaptic potential (fEPSP) was recorded by a 0.1-MΩ tungsten monopolar electrode from the dendritic layer of the stratum radiatum of the CA3 ﬁeld following electrical stimulation of the mossy-fiber pathway. The electrical pulses were delivered using a bipolar platinum/iridium electrode (25 mm wire-diameter, at an inter-wire distance of 100 mm, World Precision Instruments, USA). The fEPSP was quantiﬁed by 30 % of the maximum slope of its rising phase. We used theta-burst stimulation (TBS), which consisted of 4 pulses at 100 Hz, repeated 3 times with a 200-ms interval, to induct long-term potentiation (LTP). LTP magnitude was expressed as the mean percentage of baseline fEPSP initial slope [37, 38].
HEK293 (human embryonic kidney293) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with fetal bovine serum (10%, v/v) and penicillin/streptomycin (1%), in a humidified atmosphere containing 5% CO2 at 37 ºC. The cells grew in plates for 24 h, and then the indicated plasmid(s) were transfected into the cells using Lipofectamine2000 according to the instructions of manufacturers.
For primary neuron cultures, 18-day embryonic (E18) hippocampus of rat was seeded on 6-well plates at 30,000-40,000 cells per well, which coated with Poly-D-Lysine/Laminin (Bioscience) in neurobasal medium (Invitrogen) supplemented with B27 (2%)/ glutamine (0.5 mM) / glutamate (25 mM). Half the culture medium was changed every 2 days with neurobasal medium supplemented with B27 (2%) and glutamine (0.5 mM). All cultures were kept at 37 º C in a humidified 5% CO2-containing atmosphere. After cultured for 7 to 17 div, more than 90% of the cells were neurons, which was verified by positive staining for the neuronal specific marker MAP2 (dendritic marker, Millipore).
Preparation of nuclear fractionation
According to instructions of the manufacturer, the nuclear extracts were prepared using the nuclear extraction kit (Signosis, Inc., Sunnyvale, CA, USA). Briefly, Buffer I working reagent was added to the culture dish and rocked at 200 rpm on a shaking platform at 4 º C for 10 min. The HEK293 cells were collected and centrifuged at 12,000 rpm at 4 ºC for 5 min. The supernatant was discarded, and the pellets were re-suspended by adding Buffer II working reagent. For tissues, the hippocampal CA3 areas (location of viral infected) were rapidly cut into small pieces, added Buffer I working reagent, and homogenized at 4 ºC until a single cell suspension was observed microscopically. After spinning at 500 g at 4 ºC for 5 min, and the supernatant was removed, the cell pellets were re-suspended in Buffer I working reagent, and the preparation rocked at 200 rpm on a shaking platform at 4 ºC for 10 min. The cells were then centrifuged at 10,000 rpm at 4 ºC for 5 min, and the pellets were re-suspended by adding Buffer II working reagents. Lastly, the cell lysate was shaken at 200 rpm on a platform at 4 ºC for two hours. After centrifugation at 12,000 rpm at 4 ºC for 5 min, the supernatant (nuclear extract) was collected and stored at -80 ºC until use.
Preparation of insoluble tau
Insoluble tau aggregates were isolated from the virus infected-hippocampal tissue by a modification of a published procedure . Brain tissues were homogenized in lysis buffer (in mM): Tris-HCl 10, NaCl 150, NaF 20, Na3VO4 1, EGTA 2, Triton X-100 0.5%, and SDS 0.1% with protease inhibitor mixture and centrifuged at 13,000 × g for 20 min. The resulting supernatant was designated as the soluble tau fraction. The pellet was resuspended in 1% SDS buffer with 10 times ultrasonication and designated as insoluble aggregated tau.
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate equal amounts of protein, and the separated proteins were transferred onto nitrocellulose membranes. For analysis of STAT3 dimerization, cell lysates were incubated with 1 mM DSS, blocked with 0.5 mM NH4OH for 20 min, and then, used for Western blotting . The membranes were blocked in non-fat milk (5%) at 20 ºC for 1 h and then incubated with primary antibody (Additional file 1: Table S1) at 4 ºC overnight. Blots were then incubated with IRDye 800CW-conjugated affinity-purified anti-mouse IgG (Rockland) or IRDye 800CW anti-rabbit IgG secondary antibody (Rockland) at 20 ºC for 1 h. Odyssey Infrared Imaging System (Licor Biosciences, Lincoln, NE, USA) was used to visualize immunoreactive bands.
Reverse transcription and real-time quantitative PCR
According to manufacturer’s instruction (TaKaRa, Dalian, China), reverse transcription and real-time quantitative PCR were carried out. The PCR system contained MgCl2 (3 mM), forward and reverse primers (0.5 μM), SYBR Green PCR master mixes (2 μl) and cDNA (2 μl), and the standards for each gene. A Rotor Gene 300 Real-time Cycler (Corbett Research, Sydney, Australia) was used to assay the samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the housekeeping gene which was not changed by the treatments, was used to normalize the expression level of the interest gene. PCR primers employed in the present study are as follow: Mmu-GluA1 forward and reverse primers, 5’-CAATGACCGCTATGAGGG-3’ and 5’- AAGGACTGAAACGGCTGA-3’; mmu-GluA2 forward and reverse primers, 5’- GTGTCGCCCATCGAAAGTG-3’ and 5’-AGTAGGCATACTTCCCTTTGGAT-3’; mmu-synapsin1 (Syn1) forward and reverse primers, 5’- AGGACGAGGTGAAAGC-3’ and 5’-TCAGTCGGAGAAGAGG-3’; mmu-synaptotagmin1 (Syt1) forward and reverse primers, 5’- CCATAGCCATAGTTGC-3’ and 5’-GTTTCAGCATCGTCAT-3’; mmu-GluN1 forward and reverse primers, 5’- GTCCACCAGACTAAAGA-3’ and 5’- TCCCATCATTCCGT-3’; mmu-GluN2A forward and reverse primers, 5’- CTTTTGAGGACGCC-3’ and 5’- AAATGAGACCCGATG-3’; mmu-GluN2B forward and reverse primers, 5’- GGCTGACTGGCTACG-3’ and 5’- CTTGGGCTCAGGGAT-3’; mmu-GAPDH forward primer 5’-GGAGCGAGATCCCTCCAAAAT-3’ and reverse primer 5’-GGCTGTTGTCATACTTCTCATGG-3’.
Electrophoresis mobility shift assay (EMSA)
The non-radioactive EMSA-STAT3 Kit was purchased from Signosis (Sunnyvale, CA, USA). EMSA was performed according the instruction of the manufacturer. Briefly, a biotinized oligonucleotide probe which containing a STAT3 binding site, were incubated with the samples, and then, the samples were separated on a non-denaturing polyacrylamide gel and transferred to nylon membranes. UV crosslinking was used to immobilize the transferred oligonucleotides. In order to detect the oligonucleotides, Streptavidin-HRP was added to the membrane, and the blots were developed by ECL according to the instructions of manufacturers. Excess amounts of unlabeled cold probe which containing STAT3 binding site, was used to perform a competition experiment.
Luciferase reporter assay
Activity of the transcription factors (TFs) was analyzed with the specific luciferase reporter vector pSTAT3-Luc (Signosis). This vector contains a cis-element (DNA binding sequence), a minimal promoter, and a firefly luciferase gene. The activated transcription factors binds to the cis-element and transactivates expression of the luciferase gene correlating with the measured luciferase enzyme activity. Therefore, the luciferase activity in this assay represents activation of the transcription factor. Briefly, HEK293 cells were transfected with P301L-hTau plasmid or its empty vector control in combination with pSTAT3-Luc reporter construct and pRL-TK for 48 h. Then the cells were washed and lysed in 100 μl of the 1×CCLR (Promega). Luciferase activity was measured according to the manufacturer’s instruction (Promega). The activity of TF (i.e. firefly luciferase) was normalized to transfection efficiency by using Renilla luciferase activity (pRL-TK).
To generate luciferase reporter plasmids of GluN1, GluN2A or GluN2B promoter, after copied from the mouse genomic DNA, PCR fragments were subcloned into pGL3 basic luciferase expression vector (Promega, Madison, WI) between the BglII and NcoI sites. The GeneTailor system (Invitrogen) was used to introduce mutation of the pGL3-GluN1/GluN2A/GluN2B luciferase plasmid. Luciferase reporter plasmids were transfected into HEK293 cells by Lipofectamine Plus (Invitrogen) according to the manufacturer’s instructions. To assay the luciferase activity, pGL3-construct, P301L-hTau and pRL-TK plasmid were co-transfected into HEK293 cells, after the cells were seeded into 24-well plates one day prior to transfection. 24 h later, cells were harvested and lysed with Passive Lysis Buffer (100 μl). 20 μl cell extracts were used for luciferase activity assay by a Lumat LB9507 luminometer (Berthold) and the Dual Luciferase Reporter (DLR) assay system (Promega).
Chromatin immunoprecipitation (ChIP) assay
The DNA and protein were cross-linked with formaldehyde (1%) for 10 min, and washed 3 times and scraped into cold PBS with protease inhibitors. After centrifugation, the cell pellet was re-suspended in buffer (in mM): 20 HEPES, pH 7.9, 420 NaCl, 1.5 MgCl2, and 0.2 EDTA, with 25 % glycerol and protease inhibitors, incubated for 20 min on ice, and centrifuged. The pellet (nucleus) was re-suspended in breaking buffer (in mM): 50 Tris-HCl, pH 8.0, 1 EDTA, 150 NaCl with 1 % SDS, 2 % Triton X-100 and protease inhibitors, and sonicated for 5~10 s, and Triton buffer (in mM): 50 Tris-HCl, pH 8.0, 1 EDTA, 150 NaCl, with 0.1% Triton X-100 was added. An aliquot was reserved as the input, and the remainder was divided to immunoprecipitate with control mouse IgG (Millipore,) or STAT3 (Abcam) antibody and incubated with protein G beads. After washed 3 times in Triton buffer, the samples were added with SDS buffer consisted of 62.5 mM Tris-HCl, pH 6.8, 200 mM NaCl, 2 % SDS, 10 mM DTT, 2 μl of proteinase K (40 mg/ml), and then, samples were vortexed. To reverse cross-linking, samples were incubated at 65 ºC overnight. After isolated using phenol/chloroform extraction, DAN was re-suspended in distilled H2O. Primers used for ChIP PCR were as following: GluN1 forward and reverse primer, 5’- TAGCATTGGCATTGACCC-3’, 5’-GCTGGTGCGGTGATGTGA-3’; GluN2A forward and reverse primer, 5’-TCGGCTTGGACTGATACGTG-3’, 5’- AGGATAGACTGCCCCTGCAC-3’; GluN2B forward and reverse primer, 5’- TCTCCACCGTGCTGATGT-3’, 5’-CTCTCCGAGTCTACCTGTTC-3’. PCR products were analyzed by 2% agarose gel electrophoresis.
In brief, mice were terminated using chloral hydrate (1 g/kg) and perfused through aorta with physiological saline (100 ml) followed by phosphate buffer containing 4 % paraformaldehyde (400 ml). After removed and postfixed in perfusate overnight, brains were cut into sections (30 μm) using a vibratome (Leica, Nussloch, Germany; S100, TPI), and then, sections were collected consecutively in PBS for immunohistochemistry. Free floating sections or FTDP-17 brain sections were blocked with 0.3% H2O2 in absolute ethanol for 30 min at room temperature, and incubated with bovine serum albumin (BSA) for another 30 min to block nonspecific sites. Primary antibodies were used to incubate with sections at 4 ºC overnight. Immunoreaction was developed and visualized with diaminobenzidine (brown color) by Histostain TM-SP kits. FTDP-17 brain sections were counterstained with hematoxylin, and mouse or human sections dehydrated through a graded ethanol series, mounted on glass slides, and sealed with glass coverslips. For each primary antibody, 3-5 consecutive sections from each brain were used. A microscope (Olympus BX60, Tokyo, Japan) was used to observe the images.
The human brain tissues used in the present study were provided by Dr. K Ye of the Emory University School of Medicine, USA (Additional file 1: Table S2). The study was approved by the Biospecimen Committee. Informed consent was obtained from the subjects.
All data were collected and analyzed in a blinded manner. Data were expressed as mean ± SD or mean ± SEM. Statistical analysis was performed using Mann-Whitney test (two-group comparison), one-way ANOVA or two-way repeated measures ANOVA followed by Bonferroni’s post hoc test with SPSS 12.0 statistical software (SPSS Inc. Chicago, IL, USA). A p-value of < 0.05 was considered as statistical significance in all experiments.