| Objective:Alzheimer's disease (AD) is the most common type of dementia in clinical. AD is increasing in prevalence year by year, with no specific treatment. Studying the etiology of AD and pathogenesis of AD are still the major issue which needs to be settled. Increasing research suggests that mitochondrial defects play a large role in Alzheimer's disease (AD) pathogenesis. A growing number of studies have shown that mitochondrial dynamics plays an important role in AD pathogenesis recently.However, the research about the changes of mitochondrial dynamics in different stages of AD is still rare at present,This needs further studies.We compared APPsw/PS1dE9 transgenic mice at age months 3, 6, 9 and 12 old as an animal model of AD and age-matched C57BL/6 mice as controls. We dynamically observed the behavior, mitochondrial morphology and mitochondrial structural proteins in brain of APP/PS1 transgenic mice in different age groups. The purpose of this study was to compared changes in mitochondrial fission/fusion in different stages of the AD mouse brain and brains from age-matched controls to determine whether mitochondrial dysfunction in AD pathogenesis is age-related.Methods:1. Animals and Tissues : Heterozygou ? -amyloid PPswe/PS1dE9 double-transgenic mice (APP/PS1) with a C57BL/6 background and age-matched C57BL/6 control mice (C57) at age 3, 6, 9, and 12 months were used. All mice were males (n= 5 per group) and all were obtained from Beijing HFK Bioscience (Permit no. scxk 2014-0004). Mice were deeply euthanized by 10% chloral hydrate followed by cervical dislocation. Within 3 min, the brains were rapidly removed; one hemisphere was dissected for EM, and the other was used for Western blotting.2. Behavior Test : We used the modified Morris water maze test to assess learning and spatial memory ability of mice. The procedure consisted of 1-day adapting tests without a platform and 5-days hidden platform tests, plus a spatial probe trial 24 h after the last hidden platform test; 15 mice were used in each group. In the spatial acquisition tests, the longest time for a mouse to finding the platform in the water maze pool was 60 s. We will guide the mouse to the platform,if it did not find the platform within 60 s. The platform was located in the southwest quadrant of the water tank. Starting locations were semirandomly selected at the north, east,southeast, and northwest corners. We trained every mouse four times semirandomly selecting the starting locations per day. The time taken for a mouse to explore and arrive at the platform was recorded as the escape latency. After 24h of the last the hidden platform test had been completed, A single probe trial was performed to test memory retention, in which the platform was removed. The new start location was northeast and each mouse was allowed to swim for 60 s. The time spent traveling in the target quadrant (where the platform was previously located) were recorded and the times of mice swimming through the original platform location were counted to test whether they remembered the original position of the platform.3. In order to determine whether mitochondrial morphology is chamged as AD progresses,we used TEM to observe the ultrastructure of hippocampal CA1 area of C57 mice and APP/PS1 transgenic mice at different ages. Changes of mitochondrial morphology were determined. 15-20 randomly selected micrographs per group were obtained at 15,000 magnification and analyzed by blinded investigator. Images of mitochondria in cell bodies of 15 CA1 neurons were obtained. We take quantitative analysis of the total number of mitochondria?the number of abnormal Mitochondria and mitochondrial length in CA1 neuronal cell bodies from APPsw/PS1dE9 and C57 mice based on electron microscopy examination. Mitochondrial length in neurons was measured using Image J software.4. To determine whether mitochondrial structural proteins are changed in level as AD progresses, we compared the protein levels of DRP1,FIS 1,MFN2, and OP A1 in hippocampus tissues from APP/PS1 and C57 mice at ages 3, 6, 9, and 12 months.Results:1. The learning ability and spatial memory ability of APP/PS1 transgenic mice showed significant differences compared with age-matched C57 mice until 9 and 12 months.In learning experiment, over the 5 days of training, the escape latency of each group reduced gradually. The escape latency of APP/PS1 mice was longer than controls, and showed significant differences until 9 and 12 months compared with C57 mice. However, no statistical differences were detected at 3 and 6 months.In the exploratory experiment, the number of platform location crossings and the time spent in the target quadrant of APP/PS1 mice were consistently lower and shorter than those of the controls, which suggests an overall significance. However,the times crossing platform location of APP/PS1 mice at 12 months compared with C57 mice show only a significant difference out of eight comparisons.2. The changes in mitochondrial morphology started at age 3 months in APP/PS1 mouse models before the onset of memory decline. Mitochondrial morphology changes in APP/PS1 mice started at age 3 months compared with the length and shape of mitochondria in C57 brains at the same age. Several elongated mitochondria were identified, mitochondria gathered around the nucleus, and mitochondria were increased in number because of the increased formation of the mitochondrial split ring. A mitochondrion divides by developing a ring around the organelle that constricts to pinch it into two halves. However, mitochondrial split rings were rare and mitochondria were larger. Many fragmented mitochondria marked with disrupted membranes and without clear cristae appeared in APP/PS1 transgenic mice at the age of 6 months. Then. the number of mitochondria split rings increased significantly for many damaged degenerative and fragmented mitochondria in AD mouse brains at the age of 9 months. Finally, the 'medullary corpuscle'(an accumulation of small mitochondria and structurally damaged mitochondria with broken cristae) was found in neuropils in APP/PS1 transgenic mice at the age of 12 months. These mitochondria changes were rarely observed in C57 mouse tissues.3. Altered expression of mitochondrial fission or fusion proteins may lead to changes in mitochondrial Morphology. Levels of proteins related to fission (DRP1 and FIS1) and fusion (OPA1 and MFN2) were significantly increased in APP/PS1 mice at the age of 3 months compared with C57 mice. In contrast, levels of fission proteins DRP1 and FIS1 and mitochondrial inner-membrane fusion protein OPA1 were decreased at 6 months, but those of the mitochondrial outer-membrane fusion protein MFN2 were increased in APP/PS1 mice compared with C57 mice.Protein levels of DRP1, FIS1, OPA1, and MFN2 at 9 and 12 months were significantly increased compared with the controls (but not FIS1 at 9 months).Our data suggest that the expression of fission and fusion proteins is altered in APP/PS1 mice with age and changes in mitochondrial morphology in APP/PS1 mice may be caused by altered expression of key proteins involved in mitochondrial dynamics.Conclusion:In summary, our study show that mitochondrial dynamics changes differ in APP/PS1 mice with age as compared with controls and that mitochondrial morphology changes occur in APP/PS1 mice before the onset of memory decline.mitochondrial morphology and mitochondrial dynamics in AD pathogenesis are age related, which are significant in developing new treatment strategies by modifying abnormal mitochondrial dynamics in AD at different stages of disease progression.More importantly, we found that the progressive decline of memory appears later than mitochondrial dysfunction in APP/PS1 transgenic mice with disease progression, thus,abnormal mitochondrial dynamics may be early events in AD progression. These changes may provide us with new insight to study the mechanism of AD and could be used for early diagnosis and monitoring AD progression. However, appropriate manipulation of fission and fusion genes to ameliorate neurodegenerative processes will be a challenge because the requirements for fission and fusion differ between cell types and cellular compartments. |
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