Reagents-
PGA1 and biotin-PGA1 (bio-PGA1) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Streptavidin-HRP (str-HRP) and streptavidin-resin (str-resin) were purchased from GenScript Biotech Corp. (Piscataway, NJ, USA). Antibodies against β-actin, p-tauThr181, p-tauSer202, p-tauSer404, tau and HRP-labeled secondary antibodies were obtained from Cell Signalling Technology (Danvers, MA, USA). Antibody specific for PPP2R1A was purchased from Abcam (Shanghai, China). All primers were synthesized by GENEWIZ, Inc. (Suzhou, Jiangsu, China). OA, an inhibitor of PP2A phosphatases, was got from Santa Cruz Biotechnology (Dallas, TX, USA). All reagents used for the quantitative real-time PCR (qRT-PCR) and SDS-PAGE experiments were purchased from Bio-Rad Laboratories (Hercules, CA, USA), and all other reagents were purchased from Thermo Fisher Scientific/Invitrogen (Shenyang, Liaoning, China), unless specified otherwise.
Tg Mice and treatments-
TauP301S Tg mice (Stock No. 008169) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA), as a model of AD. Genotyping was performed at 1 month after birth. Mice were housed in a standard environment. 3-month-old Tg mice were intranasally instilled with PGA1 (2.5 mg/kg/day) for 6 months. The learning ability of the mice were determined by Morris water maze test or nest construction assay. After behavior assay, the mice were anesthetized, perfused and fixed before collecting the brains for further physiological and biochemical test.
Morris water maze test-
The mice were trained and tested in a Morris water maze after 6-month treatment with PGA1. Briefly, the mice were pretrained in a circular water maze with a visible platform for 2 d. The mice were then allowed to swim freely in a hidden platform pool by adding milk for 6 d. On the last day, the platform was removed and the times that the mice passed through the memorized platform were recorded.
Nest construction assay-
The mice were housed in cages with corncob bedding for 1 w before the nest construction assay. 2 h before the formal experiments, 8 pieces of paper (5 × 5 cm2) were put into the cage for nesting. The nest score were determined by the 4-point system: 1, no biting/tearing and random dispersion of the paper; 2, no biting/tearing of the paper, with paper gathered in a corner/side of the cage; 3, moderate biting/tearing of the paper, with gathering in a corner/side of the cage; and 4, extensive biting/tearing of the paper, with gathering in a corner/side of the cage (30).
Intracerebroventricular Injection-
5 µl of vehicle (DMSO), PGA1 (1 µg/µl) and OA (2 ng/µl) was intracerebroventricular injected (i.c.v.) to the mice. Briefly, the mice were fixed on stereotaxic instrument after anesthesia. The coordinate from the bregma was adjusted to mediolateral, − 1.0 mm; anteroposterior, − 0.22 mm; and dorsoventral, − 2.8 mm. 5 µl solution was slowly injected to the ventricles of the mice. After 48 h, the mice were anesthetized, perfused and fixed before collecting the brains for further physiological and biochemical test.
Cell Culture-
Mouse neuroblastoma (n) 2a cells and human embryonic kidney (HEK) 293T cells were grown in 37 °C with 5% CO2 on 6 cm tissue culture dishes in DMEM and 10% FBS medium. In a separate set of experiments, cells were grown in serum-free medium for an additional 12 h before incubation with 20 nM OA in the absence or presence of 10 µM PGA1.
qRT-PCR-
qRT-PCR assays were performed with the MiniOpticon Real Time PCR detection system (Bio-Rad Laboratories) using total RNA and the Go Taq One-step Real-Time PCR kit with SYBR green (Promega, Madison, WI). Forward and reverse primers for mouse PPP2R1A were 5′-GGACGTTCAGCTTCGTCTCA-3′ and 5′-CAGCAGACAGTGCACATACT-3′, respectively. The gene expression levels were normalized to GAPDH. The forward and reverse primers were 5′-GCTCATGACCACAGTCCATGCCAT-3′ and 5′-TACTTGGCAGGTTTCTCCAGGCGG-3′, respectively.
Western Blot Analysis-
Tissues or cells were lysed with RIPA buffer (Thermo Fisher Scientific, Shenyang, Liaoning, China). The protein concentration of the cell lysates was determined with a BCA protein assay kit (Thermo Fisher Scientific, Shenyang, Liaoning, China). The total cell lysates (20 µg) were subjected to SDS-PAGE, transferred to a membrane, and probed with a rabbit polyclonal antibody to PPP2R1A (1:2000,1 µg/µl), rabbit monoclonal antibody to p-tauThr181, p-tauSer202, p-tauSer404 or tau (1:2000, 1 µg/µL) as the primary antibodies. Each membrane was probed with only one antibody. β-actin served as a loading control.
Immunofluorescence-
n2a cells were cultivated on cover slips coated with poly-D-lysine (Merck/Sigma-Aldrich, Shanghai, China) in 6-well plates (Corning Incorporated, NY, USA). After treatment with10 µM PGA1or bio-PGA1 for 2 h, cells were washed with PBS (-), fixed with ice-cold methanol, and rehydrated with PBS (-). The cells were then blocked by a blocking buffer containing 2% BSA and 4% goat serum (Merck/Sigma-Aldrich, Shanghai, China). The slides were then stained with 1:200 diluted iFluor™ Streptavidin-555 (Biolegend, San Diego, CA, USA) for 1 h at room temperature. After extensively washing with PBS (-), the cells were counterstained with DAPI (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). The slides were finally mounted with fluorescent mounting medium (Dako Corporation, Carpinteria, CA, USA) and observed under confocal microscopy (Leica, Beijing, China).
Cloning procedures-
Sequences encoding PPP2R1A, PPP2R1AC377A and PPP2R1AC390A were ligated to the pBobi vector with 3 × flag-tags at amino-terminus using ligation-independent cloning method. Forward and reverse primers for mouse PPP2R1A were 5′-tagagaattcggatccatggcagctgccgacggtgacga-3′ and 5′-gcttccatggctcgagggcaagagagagaacagtca-3′; for mouse PPP2R1AC377A were 5′-tggctcagctgaaggatgaggctcctgaagtccgactgaatat-3′ and 5′-atattcagtcggacttcaggagcctcatccttcagctgagcca-3′; for mouse PPP2R1AC390A were 5′-atatcatctccaacctggatgctgtgaacgaggtgattggcat-3′ and 5′-atgccaatcacctcgttcacagcatccaggttggagatgatat-3′. Sequences encoding PPP2R1A, PPP2R1AC377A, PPP2R1AC390A, PPP2R2A and PPP2CA were ligated to the pET-28a-c (+) vector with 6 × His-tags at carboxy-terminal. These plasmids were used to produce and purify the recombinant proteins by transfecting BL21 E.coli (Agilent Technologies, Beijing, China). Forward and reverse primers for mouse PPP2R1A were 5′-CAGCAAATGGGTCGCGGATCCatggcagctgccgacggtga-3′ and 5′-CTCGAGTGCGGCCGCAAGCTTggcaagagagagaacagtca-3′; for mouse PPP2R2A were 5′-CAGCAAATGGGTCGCGGATCCatggcaggagctggaggagg-3′ and 5′-CTCGAGTGCGGCCGCAAGCTTattcactttgtcttgaaata-3′; for mouse PPP2CA were 5′-CAGCAAATGGGTCGCGGATCCatggacgagaagttgttcac-3′ and 5′-CTCGAGTGCGGCCGCAAGCTTcaggaagtagtctggggtac-3′. His-tagged proteins were induced expression by 1 mM isopropyl thio-β-D-galactoside (IPTG, Merck/Sigma-Aldrich, Shanghai, China) for 12 h at 25 °C. Glutathioneagarose (Merck/Sigma-Aldrich, Shanghai, China) and Ni-NTA agarose (Qiagen, Shanghai, China) were used to purify the recombinant proteins as previously described (31). His-tagged proteins were used for mass spectrum (MS) or in vitro PP2A activity assay.
Transfection-
The retroviral vectors encoding the sequences of PPP2R1A, PPP2R1AC377A and PPP2R1AC390A were cotransfected to HEK293T cells with the lentivirus packaging vectors pLP/VSVG, pRSV-Rev and pMDLg/pRRE by a calcium phosphate transfection kit (AMOGENE, Fujian, China). The flag-tagged proteins were used for pull-down (PD) or immunoprecipitation (IP) assay. For knocking down the expression of PPP2R1A, siRNA was transfected to n2a cells by Lipofectamine 2000 (Thermo Fisher Scientific/Invitrogen, Shenyang, Liaoning, China) according to the manufacturer’s protocol. The oligonucleotide sequence of siRNA is 5'-GCACUCACCUUCCGAUCUA-3'.
Pull down assay-
The proteins were extracted from cells or brains, which were further incubated with str-resin. Briefly, 100 µg of protein were incubated with str-resin in the buffer containing 1% NP-40 and 0.1% SDS. The unbound proteins were removed by extensive washes with 0.1 mM EDTA, 50 mM Tris-HCl (pH 7.6), 50 mM NaF, 0.1 mM EGTA, 0.1 mM β-mercaptoethanol, 1% NP-40 and 0.1% SDS. The proteins bound on str-resin were collected by centrifugation after boiling for 10 min. The supernatant was used for western blots. The bio-PGA1 conjugated proteins were visualized with a mouse monoclonal antibody against flag-tag (1:2000, 1 µg/µl) or str-HRP (1:5000, 1 µg/µl).
Immunoprecipitation-
PPP2R1A with or without the mutation of Cys377Ala or Cys390Ala was immunoprecipitated with anti-flag antibody using protein A/G agarose (Thermo Fisher Scientific/Invitrogen, Shenyang, Liaoning, China). The proteins bounded on A/G agarose bounded were collected by centrifugation after boiling 10 min. The supernatant was used for western blots. The bio-PGA1 and PPP2R1A were visualized by a str-HRP (1:5000, 1 µg/µl) or mouse monoclonal antibody against flag-tag (1:2000, 1 µg/µl).
Mass spectrum-
1 µg His-tagged PPP2R1A was incubated with 1 µM PGA1 at 37 °C for 1 h. The reacting solution was centrifuged for collecting supernatant, which was then subjected to HPLC-MS-MS analysis. As previously described (32), unassigned ions or those with a charge < 1 + or > 7 + were rejected by MS/MS. Raw data was processed by Thermo Proteome Discoverer (version 2.1) against the SwissProt proteome database using an extracted FASTA file specified for “mouse” taxonomy. The searches were carried out with the maximum 10 ppm and 0.02 Da error tolerances for the survey scan and MS/MS analysis, respectively. The protein identifications were based on at least six amino acids in length and < 1% of the false discovery rate (FDR). On the basis of this analysis, enriched pathways were further identified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) or gene ontology (GO) analysis.
PP2A activity assay-
The cells or tissues were lysed in the lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100. 40 nM PPP2CA was incubated with 50 nM PPP2R1A in TBS with or without mutation of PPP2R1A in the absence or presence of 10 µM PGA1 for 30 min at 37 °C. For determining the activity of PP2A, 60 mM Thr phosphopeptide (KRpTIRR, Merck/Millipore, Shanghai, China) was added to the solution and incubated at 37 °C for 25 min. The free phosphate was measured colorimetrically by malachite green kit (Cayman) according to manufacturer’s instructions.
Animal Committee-
All animals were handled according to the guidelines for the care and use of medical laboratory animals (Ministry of Health, Peoples Republic of China, 1998) and the guide lines of the laboratory animal ethical standards of Northeastern University.
Statistical Analysis-
All data are presented as the means ± S.E. of independent experiments. The statistical significance of the differences between the means was determined using Student’s t test or one-way analysis of variance, where appropriate. If the means were significantly different, multiple pair wise comparisons were performed using Tukey’s post hoc test.