Animals

Adult healthy TauTKO rats (constructed by our laboratory) and wild-type SD rats (WT rats, purchased from Huafukang Biotechnology company, Beijing, China) weighed between 250 and 300 g (male and female unlimited). The experiment was divided into two groups: the TauTKO group and the WT group. Each group of rats was randomly selected.

All rats were raised in an SPF environment where the temperature was about 25 °C, the air humidity was kept at 50%, and the light and dark cycle were 12 h/d. All the experimental animals were examined by the experimental Animal Ethics Committee of the Institute of Radiology, Chinese Academy of Medical Sciences.

Sample collection

Rats were anesthetized for euthanasia and sacrificed with 10% chloral hydrate (0.3 mL/100 g). The cerebral cortex was removed rapidly and snap-frozen into liquid nitrogen before storage at -80 °C until needed.

TMT labeled quantitative proteomicsSample preparation

Three cortical samples from each group were taken for proteomic analysis. Then, 400µL of 8 M urea was added to the sample, and the protease inhibitor was added at 10% of the lysate. After centrifuging at 14000 g for 20 min, the supernatant was collected. The protein concentration was determined by the Bradford method.

Then 100 µg of protein was extracted from each sample for reduction. A 100mM solution of dithiotreitol (DTT) was added and incubated at 30 °C for 1 h, then alkylated with sufficient iodoacetamide (IAM) for 1 h at room temperature. Add 25 mM ammonium bicarbonate buffer and dilute 4 times. Trypsin (trypsin: protein = 1:50) was added and incubated overnight at 37℃. Finally, high urea was removed by C18 filter cartridge, and the samples were dried by vacuum centrifugation.

TMT labeling

The TMT reagent was incubated to room temperature. Add 41 μL (0.8 mg/ tube) anhydrous ethanol to TMT reagent and mix. Add 41 μL TMT reagent to 100 μg digested sample. Then, the reaction was terminated by shaking, centrifugation and incubation at room temperature for 1 h and in 5% quenching agent (8 μL) for 15 min. The samples were freeze-dried and preserved.

Peptide separation by high pH reversed-phase HPLC

Gradient elution was performed on mobile phases A (2% acetonitrile, pH adjusted to 10.0 with ammonium hydroxide) and B (98% acetonitrile, pH adjusted to 10.0 with ammonium hydroxide). The lyophilized powder was dissolved in solution A and centrifuged at 14000 g at room temperature for 20 min. Firstly, RIGOL L-3000 double gradient high performance liquid chromatography (HPLC, Rigol Technologie, Co. LTD, China) was used with the Agela Durshell-C18 column (4.6 × 250 mm i.d., 5 μm, 100 Å). The polypeptide was isolated. The column temperature is set at 45 °C. Solvent gradient: 5% B, 0 min; B5-8%; B. 30 min; B. 27 min; 32–95%-B, 6 min; 95–5%, 4 min. The eluent was detected under UV at 214 nm. Every minute, one tube was collected and fused into 10 fractions. All fractions were dried under a vacuum.

LC–MS/MS analysis

The dry polypeptide was dissolved in 10μL A solution and centrifuged at 14000 RPM for 20 min. The 1 µg of supernatant was injected into a nano-UPLC-MS/MS system consisting of a Nanoflow HPLC system (Easy-NLC 1000 system, ThermoFisher Scientific) and an Orbitrap Fusion Lumos Mass Spectrometer (ThermoFisher Scientific). Each sample was injected into the column and eluted in gradients from 6 to 38 % solution B in 70 min, from 38 to 100 % B in 1 min and 100 % B in 10 min at 200 nL/min (Solvent A, 100 % H2O; Solvent B, 80 % acetonitrile; both containing 0.1% (v/v) formic acid). The ion source of the mass spectrometer operates nanospray flex (NSI), the spraying voltage is 2.3 kV and the ion transport capillary temperature is 320 °C.  A precursor MS1 scan (m/z 300–1400) was acquired in the Orbitrap at the nominal resolution setting of 120,000 (200 m / z )with an automatic gain control (AGC) target of 5E5 and a maximum ion injection time of 100 ms. The highest rich top 40 data-dependent mode was analyzed by higher energy selection and dispersed collision dissociation (HCD) and MS/MS. MS/MS spectra were collected in the Orbitrap (50,000 resolution) with an AGC target of 2.5E4 and a maximum ion injection time of 86 ms. The peptide fragmentation collision energy was set to 32%. The dynamic discharge resistance range is set to 18s.

The data analysis

LC–MS/MS raw data were searched and quantitative analyzed from protein sequence database (Uniprot_RAT_2020_08). The mass spectrometry analysis of TMT was completed by Orbitrap Fusion mass spectrometry, and the original mass spectrometry files generated were processed by Proteome Discoverer 2.4. The searched parameters are set as follows: enzyme:trypsin; static modification: carbamidomethyl (C); dynamic modification: Oxidation(M), acetyl (protein N-terminal); species: rat; precursor ion mass tolerance: ± 15 ppm; Fragment ion mass tolerance: ± 0.5 Da; max missed cleavages: 2. the differentially expressed proteins (DEPs) were satisfied the following conditions: average ratio-fold change > 1.2, as well as p-value < 0.05.

Bioinformatics analysis

A global heat map was exerted to exhibit DEPs. The Gene Ontology (GO, which includes biological process (BP), cellular component (CC), and molecular function (MF)) annotation of the identified proteins was derived from the UniProt database. The KEGG database (https://www.genome.jp/kegg/) was used to perform the enrichment analysis of pathways. A study called STRING (https://www.string-db.org) was conducted to create protein–protein interaction (PPI) networks, which were used to identify interactions.

Western blot

The cerebral cortices of TauTKO rats and WT rats were collected and total proteins were extracted using efficient radioimmunoprecipitation assay (RIPA) lysate kit (solarbio, R0010, China). The protein concentration was determined using the BCA kit (solarbio, PC0020, China). 40 μg proteins were loaded and separated on 10% polyacrylamide SDS-PAGE gel and then were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk in TBST solution for 2 h at room temperature. Then, membranes were incubated with the rabbit anti-TauT(1:1000, Santcruz biotechnology, USA), mouse anti-GAPDH (1:10000, Proteintech, 60004–1-lg, USA) overnight at 4 °C. After being washed for 3 times with TBST (10 min each time), membranes were incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG secondary antibody (1:10000, ZB-2305, bs-10966R, China) and goat anti-rabbit IgG secondary antibody (1:10000, ZSGB-BIO, ZB-2301, China) for 90 min at room temperature. Then they were washed for 3 times with TBST (10 min each time). Membranes were incubated with Enhanced chemiluminescence (ECL)reagent (Merck Millipore, Germany). protein expression was analyzed with a gel imaging system (Fusion FX7, ViIbert Lourmat, France). The relative expression of the target protein was the ratio of the gray value of the target protein band to that of the internal reference protein band.

Immunofluorescence

After PBS perfusion, the brain tissue of rats were fixed with 4% paraformaldehyde for 24 h, dehydrated with 30% sucrose to the bottom, embedded with OCT glue, quickly frozen with liquid nitrogen and preserved at -80 ℃.The frozen slicer was used to cut into 6 micron slices, fixed with 4% paraformaldehyde for 15 min, washed with PBS for 3 times, 3 min each, and 0.5% Triton for 30 min, washed with PBS for 3 times, 3 min each, and sealed with goat serum for 30 min. rabbit anti-Annexin6 (1:100, Proteintech, 12542–1-AP, USA) and mouse anti-Pik3r2 (1:100, Proteintech, 67644–1-Ig, USA)) primary antibodies were incubated overnight, washed with PBS 3 times for 3 min each, and goat anti-rabbit(1:50, Proteintech, SA00003-1, USA)) and goat anti-mouse(1:50, Proteintech, SA00003-2, USA)) secondary antibodies were incubated at 37° for 1 h, washed with PBS 3 times for 3 min each, stained with DAPI and sealed.The results were observed under a confocal microscope (LSM 800, Zeiss, Germany), and 5 fields at high magnification (200 ×) were randomly selected for observation.

Statistics

All data were expressed as mean ± standard deviation (SD). Statistical analysis was conducted by SPSS software (version 17.0). Results were compared with an independent t-test. A p-value of 0.05 was considered statistically significant.