2.1 Materials
Kunming mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. (BA#: 2021 − 208, Changsha, China). All the peptides were synthesized by Jill Biochem. Co., Ltd., Shanghai, China. AG99 was provided by Wuhan Costan Biotech. Co., Ltd., Wuhan, China, and is a tyrosine phosphorylation inhibitor that competes with ATP to bind tyrosine. Recombinant human Akt1 (ab226416) was purchased from Abcam Pic. (Cambridge, UK).
2.2 YP Peptide Morphology Observation by TEM
The aqueous solution of YP peptide was dropped on a copper mesh. After drying, the samples were dyed with 1% phosphotungstic acid solution for 1 hour and then dyed with 0.2% uranium acetate solution for 1 hour. When dried, it was washed with distilled water 3 times. After drying again, the peptide was observed by a transmission electron microscope (H7670, Hitachi, Japan), and the morphology was photographed.
2.3 YP Phosphorylation Identification
Fifty microliters of 1 mg/ml YP peptide and NP peptide saline solutions were individually incubated with 10 µl of 1 mg/ml ATP. Three hours later, along with the YP and NP saline solution, they were individually loaded tointo a mass spectrometer. If complexes formed, they could be identified from the spectrum peaks. All the above incubated solutions and the peptide monomers were also assayed by SDS-PAGE. If there was phosphorylation, it was seen in the lane in the Coomassie stained polyacrylamide gel.
Other ATP-incubated YP or NP peptides were dialyzed to remove the unconjugated ATP and then freeze-dried for further usage.
2.4 Establishment of the Dementia Senile Mouse Model, Drug Intervention and Water Maze Test
D-galactose can be reduced to galactol and produce free radicals in the body. The radicals attack the lipid, protein and mtRNA of cell mitochondria, thus causing aging and disease. D-galactose-induced mice exhibit symptoms similar to those of natural aging mice, such as memory and immunity decreases, therefore, they have been commonly used as senile dementia models in antiaging experiments[14]. In our experiment, 52 two-month-old Kunming mice were used as subjects. Except for the normal control, each mouse was injected with 1 mg/ml galactose twice every 3 days (200 µl each time). After 50 days, all the mice were divided into the normal control group (briefed as “CTL”, tail vein-injected with 200 µl saline once every 3 days), senile group (briefed as “SN”, the dementia senescent mice were tail vein-injected with 200 µl saline once every 3 days), dementiaed senescent YP group (briefed as “SN + YP”, the dementia senescent mice were tail vein-injected with 200 µl 1 mg/ml saline dissolved YP peptide once every 3 days), dementia senescent NP group (briefed as “SN + NP”, the dementia senescent mice were tail vein-injected with 200 µl 1 mg/ml saline dissolved NP peptide (5-repeats “HEDA” amino acid residues consisted oligomer once every 3 days) and dementia senescent YP inhibition group (briefed as “SN + AG99 + YP”, the dementia senescent mice were tail vein-injected with 200 µl 1 mg/ml saline dissolved AG99 (100 µg/ml) inhibited YP peptide once every 3 days). One month later, the intelligence of mice in each group was analyzed in a 1.5 diameter water maze (MWM). The distance and the time taken to return the platform were used to index the intelligence of the mice. After the MWM, the mice were sacrificed according to the Animal Welfare Ethics. The brains were removed. Part of each brain was fixed for 24 h at 4°C with 30% sucrose − 4% paraformaldehyde solution and then frozen sectioned (15 µm thick, CM1950, Leica Inc., Germany) for neuron necrosis assays, histone methylation immunofluorescence analysis, and iron staining. Some remaining brain was cool lysed for WB, proteomics and phosphorylated proteomics analysis and free radical measurement, and the rest was frozen for ATAC sequencing and ChIP sequencing.
2.5 Brain Iron Staining and Iron Content Determination
Each brain slice was immersed in 100 µl 10% potassium ferricyanide aqueous solution for 30 minutes, washed with PBS 3 times and then sealed. The brain slices were observed under a microscope (BX53, Olympus, Japan). The areas with more iron will be dyed brown.
The fresh brain tissues were ground with lysate individually, and the protein concentration of each sample was measured with a micro ultraviolet spectrophotometer (Nano drop 2000, Thermofish Inc., Germany). After adjusting to equal protein concentrations, the iron content in each sample was determined using an iron content detection kit (BA1711, Shanghai Biol. Tech. Co Ltd., China).
2.6 Brain Slice Iron Distribution Mapping
The brains of demented senile and normal mice brain sections (refer to Fig. 34 [15]) were placed under a X-ray fluorescence work station (Institute of High Energy Physics, Chinese Academy of Science, Beijing, China) for iron content detection using an automatic step-ping platform. The iron content in each scanned point was contour mapped using MATLAB software.
2.7 Determination of Free Radicals in the Brain
Ten microliters of 1 mg/ml FeSO4 aqueous solution was added to each 50 µl protein concentration brain lysis sample. After 10 min of incubation, 10 µl of 0.1 mg/ml ascorbic acid was dripped onto the previous solution, and after another 10 min of incubation, 10 µl of 0.1 mg/ml pyridine aqueous solution was added. Then, the sample was submitted to a microplate reader (Multiskan FC, Thermofish, Germany). The light absorption value was used to index the concentration of radicals in each sample.
2.8 Necrotic Neuron Detection in the Brain
A TUNEL Kit (terminal deoxynucleotidyl transfer mediated nick end labeling, Beyotime, China) was used to stain dead cells in tissues. After death, the DNA of the cell is broken, and the 3'-OH terminal can be bound by fluorescein. In the experiment, each brain slice was covered with TUNEL reagent and incubated for 30 minutes. After 3 washes with PBS, the FITC-labeled antibody was added dropwise and incubated in the dark for 2 hours. After washing again with PBS 3 times, the slices were sealed and observed under a fluorescence microscope (BX53, Olympus, Japan). The bright green dots in the brain section were the dead cells. More fluorescent spots indicated more dead cells.
2.9 Western Blot (WB) for Protein Expression.
Brain tissues were ground on ice with RIPA (Radio Immunoprecipitation Assay) lysis buffer. Half an hour later, the lysates were centrifuged for 10 minutes at 12000 rpm. The supernatant of each sample was taken to determine the concentration, and then 5× loading buffer was added and boiled for 5 minutes. Then, SDS‒PAGE electrophoresis was performed for equal amounts of protein from each sample. After electrophoresis, the proteins of each sample were transferred to a PVDF membrane. The PVDF membrane was covered with antibody solution and incubated overnight. The next day, after 3 washes with PBS, the proteins of each sample on the PVDF membrane waswere incubated with HRP-labeled secondary antibodies. Two hours later, the PVDF membrane was washed 3 times with PBS. Then, the proteins on the membrane were visualized with an ECL chemiluminescence kit and developed (ChemiDoc-XRS+, Bio-Rad Inc., USA). Stronger fluorescence indicated more protein in the lane on the PVDF membrane.
2.10 Proteomics Determine
Brain lysates at equal concentration volume ratios were digested into peptides by proteases, and then subjected to mass spectrometry. By comparing the signals with a protein spectrometry database, we can determine the protein types and abundances in each sample and understand the effects of aging and YP on the expression of proteins in the mouse brain.
Accordingly, the proteins in each brain lysate with equal concentration volume ratios were labeled with different isotopes of TiO2 and then digested into peptide segments with proteases. The peptide segment solutions were loaded into the HPLC column, and eluted by adjusting the pH. The eluents were detected by mass spectrometry. The peaks were compared with a phosphorylated proteome database to identify the phosphorylation of the proteins.
2.11 Brain Histone Methylation Immunofluorescence
Brain slices on slides were soaked in 1% H2O2 solution for 10 min for endogenous peroxidase inactivation and then washed 3 times with PBS and blocked with 1% BSA. Half an hour, a methylated H3K27 rabbit anti-mouse antibody was added for overnight incubation at 4 ℃. The next day, the unbound antibody on the slices was washed away using PBS, and slices were covered with rhodanmine-labeled secondary antibody and incubated for another 2 hours at room temperature in the dark. Next, after washing away the unbound secondary antibody, the slices were sealed with DAPI-coated cover glass, and then were observed and photographed with a fluorescence microscope (OLYMPUS, BX53, Japan). The red bright spots were the sites of methylated H3K27s, which were located in the nucleus. Larger spots indicated more methylated H3K27.
2.12 ATAC Sequence (Assay for Targeting Accessible-Chromatin sequencing) for Gene Accessibility
Briefly, each fresh brain was cut and digested for 30 min with trypsin. Then, 10 ml PBS was added for 5 min of 1000 rpm centrifugation. When the supernatant was discarded, 10 ml DMEM containing 1% formaldehyde was added to the cell sediment, which was incubated at room temperature for 10 minutes with shaking, and then 1 ml 2.5 M glycine solution was added to stop the crosslinking. After shaking again at room temperature for 5 min, the reaction was terminated on ice for 15 min. Next, the cell suspension was centrifuged for 10 min at 1000 rpm at 4 ℃, and the precipitate was transferred to a 1.5 ml EP tube for nuclear extraction with basal cell lysate. Tn5 transposase was added to cut the duplex DNA, and the DNA fragments were labeled, purified, amplified by PCR and sequenced (HiSeq 2000, Illumina, USA).
2.12 ChIP Sequence-Based Assay on Histone H3K27 Methylation
For the ChIP (chromatin immunoprecipitation) sequence, brain digestion, fixation, nuclear extraction, and DNA and protein cross-linking processing were the same as those described above for ATAC SEQ. After the cross-linking of DNA and protein, the chromosomes were ultrasound broken, and then the DNA-H3K27me complex was pulled down by H3K27me antibody-conjugated magnetic beads. After washing away the nonbinding proteins, the proteins attached to DNA were digested, and the remaining DNA iswas collected and purified for library and sequencing.
2.13 Data Processing
All data were presented as the means ± SEMs of three or more independent experiments, and p < 0.05 in the t-tests was considered as statistically significant.