| Objective:Alzheimer's disease(AD) is an urgent problem in the aging societyand there is no effective treatment method. Recent studies have confirmed that apoptosis induced by mitochondrial damage is involved in the pathogenesis of AD. It is very important to remove the damaged mitochondria in time. Mitophagy is a process of the selective removal of damaged or abnormal mitochondria to protect the cell, which is a hot research topic in recent years. The APPsw/PS1dE9 double transgenic mice and 20E2 cell (HEK293 cell with APPsw) were used to be the model of Alzheimer's disease. We dynamically observed the behavior, mitochondrial structure, autophagy and mitophagy in brain of APP/PS1 double transgenic mice in different age groups. Mitochondrial structure, autophagy and mitophagy of HEK293 and 20E2 cell in vitro were also recorded. This will provide us a useful reference for studying mitophagy in AD.Methods:1. We used the APPsw/PS1dE9 double transgenic mice as AD model. The mice were killed at 3,6,9,12 month.8-9 month-old C57BL/6 mice were control. All mice were male (n= 15 per group).20E2 cells (APPsw stably expressing HEK293 cells) were used to be the cell model of Alzheimer's disease, HEK293s cell were control.2. Learning and memory ability was evaluated by Morris water maze. The whole process included 1 day adaptive phase without platform,5 days learning phase with hiding platform, and 1 day exploratory phase after 24h of the last learning phase. At learning phase, the longest time of finding the hidden platform was 60s. If a mouse find the platform in the specified time, then stay on the platform for 15s.If a mouse failed to arrive in time, it would be guided to the platform and stay on the platform for 15s in order to remember the location. Every mouse was trained 4 times per day. The platform was located at SW. Start locations were randomly divided at N, E, SE, NW. The mouse explored and arrived at the platform was described as latent time. After 24h of the last learning experiment, the platform was removed to perform exploratory experiment to test memory ability. And NE was the new start location. The experiment time was 60s.The times crossing the point where the platform had been located were measured.The latency, times crossing the point where the platform had been located and times in target quadrant were recorded to evaluate the learning and memory ability. The data of Morris water maze used analysis of variance (ANOVA). The comparison of mean used LSD post analysis.3. Transmission electron micro was used to observe the ultrastructure in hippocampus CA3 zone of mice, and the ultrastructure in HEK293 cells and 20E2 cells.The change of mitochondrial morphology was determined.4. Autophagy related protein LC3-?/LC3-? and p62 in hippocampus and cells were detected by Western blotting to determine the dynamic change of autophagy in ADanimal model and the changes of autophagy in cell model.5. Mitophagy related protein PINK1 and Parkin in hippocampus and cells were detected by Western blotting to determine the dynamic change of mitophagy in AD animal model and the changes of mitophagy in cell model.Results:1. The cognition of 6 month-old APP/PS1 double transgenic mice was changed. In learning experiment, the latent time was longer than control in 6 month group. With the increase of age group, the latent time was increasingly longer, which was more obvious at 5th day. In the exploratory experiment, the time and times of getting old goal location of 6 month-old APP/PS1 double transgenic mice were longer than control, and the gap was enhanced with the increase of age group. And their learning and memory ability were remarkably reduced, which was more obvious with the increase of age.The learning and memory ability of 6 month-old APP/PS1 double transgenic mice showed a significant decrease, which was more obvious with the increase of age.2. Mitochondrial changes were observed in APPsw/PS1dE9 double transgenic mice brain tissue and 20E2 cells. In normal C57 mouse brain, the mitochondrial membrane structure was complete, and the mitochondrial ridge was clear. The brain tissue in APP/PS1 double transgenic mice had many damaged degenerative mitochondrion and intramedullary lesions with many autophagic vacuoles.most of the mitochondria in the hippocampus of 3 month-old APP/PS1 mice had a complete structure, and some mitochondria showed increased volume, loose matrix and disorder of the ridge. Structural abnormalities of the mitochondria in the 6 month-old APP/PS1 mice were greater. There were a large number of damaged degenerative mitochondrions in 9 month-old APP/PS1 mice hippocampus, double membrane dissolved, the cristae damage, and even extensive degradation of mitochondrial pyknosis, variable depth matrix and in axons accumulation formation of myeloid bodies. A large number of autophagy vacuoles and medullary bodies were formed in the hippocampus of 12 month-old APP/PS1 mice. And these changes were greater with the increase of age.20E2 cell had mitochondrial swelling, disappeared cristae, vacuolus, compared to HEK293 control.The changes of mitochondrial morphology in AD animal and cell model were suggested.And this morphological change can be observedbefore the symptoms of cognitive dysfunction.3. Autophagy in 6 month-old APP/PS1 double transgenic mice and 20E2 cells was increased. In Western blot results, autophagy markers LC3-?/LC3-? were up-regulated and p62 were down-regulated in 6 month-old APP/PS1 double transgenic mice and 20E2 cells, compared to control. But, there was no significantly difference between 6 and 9,12 month-old groups. For 3 month-old group, there was no significantly difference between transgenic mice and control.4. Mitophagy associated protein in APP/PS1 double transgenic mice and 20E2 cells was increased. In Western blot results, PINKl and Parkin were up-regulated in APP/PS1 double transgenic mice and 20E2 cells, compared to control. And there was no significantly difference among different age groups. In AD animal and cell models,the PINK1/Parkin induced mitophagy was enhanced. In animal experiments, increased mitophagy can be observed before the symptoms of cognitive dysfunction.Conclusion:In summary, we dynamically observed the behavior, mitochondrial structure, autophagy and mitophagy in brain of APP/PS1 double transgenic mice in different age groups. Mitochondrial structure, autophagy and mitophagy of HEK293 and 20E2 cell in vitro were also recorded. In our results, the mitochondrion was changed significantly in APP/PS1 double transgenic mice and 20E2 cell.Autophagy associated protein,LC3-?/LC3-? was up-regulated and p62 was down-regulated. Mitophagy associated protein PINK1 and PARKIN were up-regulated. So, we believe that the the PIN1l/Parkin induced mitophagy was enhanced in the AD animal and cell model, and plays a protective role in the neurons. The change of morphology and up-regulation of mitophagy in AD animals has occurred before the symptoms of cognitive dysfunction, which is an early event of AD pathology. |
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