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The Adeno-associated virus (AAV) was provided by Shanghai Jima Pharmaceutical Technology Co., LTD (Qingdao, China). The brain stereoscopic locator was purchased from Anhui Zhenghua Biological Instrument Equipment Co., LTD (Anhui, China). The Morris Maze was purchased from Shanghai Xin Soft Information Technology Co., LTD (Shanghai, China). Commercial assay kits for total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). Detailed information on the primary antibodies used is presented in Table 1. Horseradish-peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG were purchased from Proteintech (Wuhan, China).
Antibody Producers Catalogue
numberSource Dilution APP Cell Signaling Technology 15126S Rabbit 1:1,000 Aβ40 Cell Signaling Technology 12990S Rabbit 1:1,000 Aβ42 Cell Signaling Technology 14974s Rabbit 1:1,000 p-Tau Cell Signaling Technology 23214S Rabbit 1:1,000 Tau Cell Signaling Technology 46687S Rabbit 1:1,000 Ox-DJ1 Emd Millipore Corporation MABN1773 Rat 1:10,000 DJ1 Abcam ab18257 Rabbit 1:1,000 Nrf2 Cell Signaling Technology 12721S Rabbit 1:1,000 Keap1 Cell Signaling Technology 8047S Rabbit 1:1,000 HO-1 Cell Signaling Technology 86806S Rabbit 1:1,000 p62/SQSTM1 Cell Signaling Technology 23214S Rabbit 1:1,000 LC3 Proteintech 14600-1-AP Rabbit 1:5,000 Beclin1 Servicebio GB11228 Rabbit 1:4,000 p-AMPK Abclonal AP883 Rabbit 1:2,000 AMPK Proteintech 18167-1-AP Rabbit 1:2,000 P-mTOR Abcam ab109268 Rabbit 1:10,000 mTOR Abcam ab32028 Rabbit 1:5,000 Caspase3 Proteintech 19677-1-AP Rabbit 1:2,000 Bax Proteintech 50599-2-Ig Rabbit 1:10,000 Bcl-2 Proteintech 26593-1-AP Rabbit 1:2,000 PCNA Cell Signaling Technology 13110S Rabbit 1:1,000 β-actin Proteintech 20536-1-AP Rabbit 1:5,000 GAPDH Abcam ab181602 Rabbit 1:10,000 Table 1. Detailed information of primary antibodies used
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Forty male 7-month-old SPF APP/PS1 mice were selected and purchased from Kavenberg Model Animal Research Co., LTD (Suzhou, China). The animals were raised in the barrier environment of the Experimental Animal Center, School of Public Health, Zhengzhou University. The experimental mice were housed in cages with free access to water and food, and had light/dark cycling of 12 h. The ambient temperature was 20–25 °C and the humidity was 50%–60%. After one week of adaptive feeding, the 40 APP/PS1 mice were randomly divided into four equal groups. Overexpression or knockdown of AAV was injected into the hippocampus of two groups of mice through brain localization surgery to construct AD model mice with DJ1 overexpression or knockdown. The four groups were: AD model control group (MC), AAV vector control group (NC), DJ1 up-regulation group (DJ1+), and DJ1 knockdown group (DJ1−). After 21 days of feeding, behavioral tests were performed, followed by anesthesia and euthanasia, and samples were collected. The protocol was reviewed and approved by the Life Science Ethics Committee of Zhengzhou University (ethical approval number zzuGZR2018-03).
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AAV is a single-stranded linear DNA virus that is a safe, durable, efficient, and highly specific gene-manipulation tool. To date, AAV-based gene vectors have been used in a large number of clinical trials for gene therapy[26]. AAV has multiple serotypes (1-9, Rh10), among which AAV-9 is capable of vectorial translocation and long-term expression in the hippocampus[27]. We used the brain localization injection technique to inject AAV9-DJ1 into the bilateral hippocampal regions of the mice. During localization, a locating needle was used to locate and drill holes 2 mm behind the fontanelle and approximately 2 mm beside the sagittal suture, and then 2 µL of AAV-DJ1 was injected into each hippocampus.
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The Morris water maze (MWM) behavioral study was performed on APP/PS1 mice in each group three weeks after brain localization injection. MWM was performed according to previous reports with some modifications[28]. Before the formal water maze experiment began, the APP/PS1 mice were acclimatized for two days. In the hidden platform training, the mice were trained for four days and were given a limit of 90 s to find the platform that was submerged 1 cm below the water surface. The mice performed 4 trials per day at 15 min intervals. On the last day of the space exploration test, the platform was removed, and the number of times the mice crossed the platform region for 1 min was recorded. All data were recorded using a computer program.
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All APP/PS1 mice were fasted overnight, weighed, and euthanized using sodium pentobarbital (40 mg/kg). Blood samples collected by cardiac aspiration were centrifuged at 3,000 rpm at 4 °C for 15 min to separate the upper serum. Samples were stored at –80 °C[29] before further biochemical analysis. For histological studies, the brains of mice were removed and immediately divided into two halves on ice. One half was fixed in 4% paraformaldehyde for morphological identification, and hippocampal tissue from the other half was collected and stored at –80 °C for subsequent experiments.
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AD is a progressive neurological dysfunction disease and studies have found that as the disease progresses, the brains of patients with AD experience brain cell death and atrophy compared to the brains of healthy individuals[30]. The brain index is a simple and effective index for evaluating brain atrophy and was calculated as: Brain index = brain weight (g)/rat total body weight (g).
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Mouse brains were fixed in 4% paraformaldehyde for 48 h, followed by graded dehydration in ethanol, xylene removal, and paraffin-embedding. Paraffin sections (10 μm thick) were soaked in hematoxylin stain for 5 min, washed with water, and then fractioned and liquefied. Slides were washed with water and then with performing anti-blue. The sections were dehydrated with gradient alcohol and soaked in an eosin staining solution for 5 min. The stained sections were soaked in anhydrous ethanol and xylene and then sealed with neutral resin. The stained images were acquired and analyzed using an inverted microscope.
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Paraffin sections were soaked in Congo Red A solution overnight, washed with water for 2 min, and fractioned with Congo Red B solution. The differentiated sections were placed in Congo Red C solution for 1 min, washed with water, and stained with anti-blue. The treated sections were dehydrated with anhydrous ethanol and xylene and finally sealed with neutral gum. The stained images were acquired and analyzed using an inverted microscope. The number of age spots was quantified using ImageJ software.
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Paraffin sections were fixed in 4% paraformaldehyde for 30 min and washed 3 times with perbenzoic acid (PBA) for 5 min each. After antigen repair, the sections were washed three times with PBA for 5 min. The sections were blocked with BSA in a circle around the tissue for 30 min and incubated overnight at 4 °C with the primary antibody. The corresponding secondary antibody was added to cover the tissue and incubated for 50 min at room temperature in the dark. Sections were double-stained with DAPI for cell nuclei and sealed with a fluorescent quencher. The stained images were acquired and analyzed using a fluorescent microscope. The intensity of the positive product fluorescence images was quantified using ImageJ software.
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T-SOD, MDA, T-AOC, and GSH-PX are important indicators of the level of oxidative damage in the body. T-SOD plays a crucial role in the balance between oxidation and antioxidant activity in the body, and can scavenge superoxide anion free radicals, thus protecting the body from damage. MDA level often reflects the degree of lipid peroxidation in the body and indirectly reflects the degree of tissue and cell damage. GSH-PX is an important peroxidase enzyme widely present in the body, and its level can reflect the strength of the body’s antioxidant capacity. T-AOC can reflect the strength of the antioxidant capacity of the body’s defense system. These four indicators were measured in the hippocampus and serum of APP/PS1 mice, and the assays were performed strictly according to the manufacturer’s instructions.
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First, the ipsilateral hippocampal tissue of each group of APP/PS1 mice was weighed using an electronic analytical balance, and protein extract was added at a ratio of 1:10. The protein extract solution was prepared with RIPA lysate, phenyl methane sulfonyl fluoride (PMSF), and phosphatase inhibitor at a ratio of 98:1:1. The tissues were completely ground in an ice and water bath. The supernatant was extracted after centrifugation, and tissue protein content was quantified using the BCA protein assay. Intranuclear Nrf2 protein was extracted using an Intranuclear Protein Extraction Kit (Cell Signaling Technology). The tissue protein solution was mixed with 5 × sodium dodecyl sulfate (SDS) loading buffer at 95 °C and boiled for 5 min to denature the protein. Equal amounts of protein samples (30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes. Subsequently, the membranes were blocked in 5% skim milk for 2 h at room temperature, after which they were incubated at room temperature for 1.5 h with the following primary antibodies: APP (1:10,000), p-Tau (1:50,000), DJ-1 (1:1,000), Nrf2 (1:2,000), AMPK (1:10,000), mTOR (1:10,000), Bax (1:10,000), Caspase3 (1:2,000), B cell lymphoma-2 (Bcl-2) (1:2,000), and β-actin (1:5,000) (details of primary antibodies are listed in Table 1). The membranes were incubated with an HRP-coupled secondary antibody (1:10,000) for 1.5 h at room temperature. Finally, the proteins were visualized using an enhanced chemiluminescence kit (Biosharp), and the density values of the protein bands were quantified using ImageJ software. Most of the proteins were normalized to β-actin as a control, whereas their phosphorylated and oxidized proteins were normalized to the corresponding total proteins.
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All experiments were conducted in triplicate, and the experimental data were analyzed using SPSS 25.0 software (SPSS, Chicago, IL, USA). Measures that conform to a normal distribution are expressed as mean ± standard deviation (M ± SD). One-way ANOVA was used for comparisons between groups, and significance was calculated using the LSD test. If the data did not conform to a normal distribution or the variance was not uniform, the Kruskal–Wallis rank-sum test was used. The data from each group of the MWM training period were processed with repeated ANOVA measures and simple effects analysis and are expressed as mean ± standard error (M ± SE). All test levels were bilateral (α = 0.05).
DJ1 Ameliorates AD-like Pathology in the Hippocampus of APP/PS1 Mice
doi: 10.3967/bes2023.133
- Received Date: 2022-11-22
- Accepted Date: 2023-03-13
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Key words:
- Alzheimer’s disease /
- DJ1 /
- NRF2/HO-1 /
- Oxidative stress /
- AMPK/mTOR /
- Autophagy /
- Apoptosis.
Abstract:
The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study.
&These authors contributed equally to this work.
Citation: | PENG Yang Yang, LI Meng Xin, LI Wen Jie, XUE Yuan, MIAO Yu Fan, WANG Yu Lin, FAN Xiao Chen, TANG Lu Lu, SONG Han Lu, ZHANG Qian, LI Xing. DJ1 Ameliorates AD-like Pathology in the Hippocampus of APP/PS1 Mice[J]. Biomedical and Environmental Sciences, 2023, 36(11): 1028-1044. doi: 10.3967/bes2023.133 |