| Alzheimer’s disease (AD) is a neurodegenerative disease with the clinical presentation of progressive cognitive deterioration. The amyloid plaque accumulation, neurofibrillary tangle and neuronal loss are the three typical pathological characters. Recent researches found the existence of intracellular A β, which together with mitochondrial dysfunction have become the hot spots in AD.ABAD locates in mitochondrial matrix. It catalyzes the oxidative or reductive reactions of alcohol, aldehyde, ketone and hydroxysteroid. A β can bind ABAD and change its structure, which leads to oxidative stress and cell death. In A β-free situation, ABAD protects the cell from stress damages.Conditional ABAD knockout mouse is a good model to observe the role of ABAD in AD pathogenesis. Now we had successfully built this kind of model with ABAD gene knockout in neuron, so we investigated its basic characters and its mitochondrial function in neuron. Moreover, we built the stable SK-N-SH cell lines overexpressing wtABAD and three ABAD point mutants to further investigate the role of ABAD in mitochondrial function.Section1The characteristics and mitochondrial functions of ABAD knockout miceObject:The mouse with conditional ABAD gene knockout in neuron is a new animal model for mechani sm research in Alzheimer’s disease. Because it was newly built, we here settled its genotyping methods, the basic characteristics and its possible reasons for mitochondrial dysfunction in neurons as well. Methods:CREB PCR and FLOX PCR were applied to identify the genotyping of ABAD knockout mice. Western blot and fluorescence immunostaining was used to measure the quantity of ABAD and other related protein such as CypD. TMRM and MitoSOX immunostaining were used to observe the mitochondrial membrane potential and the existence of reactive oxygen species from mitochondria. ATP assay was applied to measure the concentration of ATP in mice brains and TUNEL assay to stain the apoptosis cells to investigate cell death in mice brains. We used ImageJ from NIH to quantify the intensity of Western blot bands and the colors representing a specific protein. And student t test was used to compare the differences between wild type group and ABAD knockout group.Results:The genotyping of ABAD knockout mice is CREB(+) and FLOX Δ neo. The ABAD expression in neo-cortex and hippocampus of ABAD knockout mice was low with the intensity of0.81±0.129vs.0.29±0.031, P=0.00098,1.74±0.144vs.1.00±0.160, P=0.01894respectively. MHB staining showed the MHB intensity in ABAD knockout mice increased significantly in neo-cortex (11.14±0.609vs.6.92±0.354, P=0.018). However, there was no difference in hippocampus between ABAD knockout mice and wt mice (P=0.48). The intensity of TMRM staining decreased in entorhinal cortex in ABAD ko mice brains (1.00±0.081vs.0.75±0.049, P=0.036), but not in motorsensory cortex (P=0.754).The intensity of MitoSOX was higher in entorhinal cortex in ABAD ko mice brains (1.00±0.062vs.1.37±0.085, P=0.004), while no difference was seen in motorsensory cortex between two groups (P=0.336).As for the concentration of ATP in brain tissue, there was no difference between two groups though the value of ABAD ko mice was higher (wt mice2.54±0.282, ABAD ko mice2.11±0.210, P=0.264).TUNEL staining found the existence of TUNEL positive neurons in the neo-cortex and hippocampus in ABAD knockout mice. Moreover, there was neuronal loss in entorhinal cortex (neuron count/total cells count in entorhinal cortex was0.298±0.069in ABAD ko mice,0.67±0.050in wt mice, P=0.032). There was no significantly positive TUNEL staining and neuronal loss in ABAD ko mice of3-5months. Moreover, the intensity of CypD, nrf2, SODII, GFAP increased significantly in brain of ABAD knockout while COXIV, PSD95, Synaptophysin decreased. Conclusion:The genotyping of ABAD knockout mice is CREB(+) plus FLOX Δneo. ABAD expression was found lower in cortex and hippocampus in ABAD knockout mice. Its substrate, MHB, accumulated in the corresponding areas, which might lead to mitochondrial dysfunction and oxidative reaction with diminishing membrane potential and increasing ROS, which were the reasons of neuron apoptosis. The high level of CypD in the brains of ABAD ko mice might be another reason for mitochondrial dysfunction. And the increased GFAP, SODII and Nrf2identified by western blot might be related to the reactivity of astrocytes to protect neuron from oxidative stress. The decreased expression of PSD95and synaptophysin might be related to the diminishing synaptic function.Section2The role of ABAD and3mutants in mitochondrial oxidative stressObject:MHBDD is caused by point mutants in ABAD gene. People took the accumulation of MHB in brain as the cause of MHBDD. Recently, one investigation presumed other ways affecting mitochondrial function besides MHB accumulation. Here we built the stable cell lines overexpressing the wtABAD and3ABAD mutants to observe the role of ABAD in mitochondrial oxidative stress.Methods:The stable cell lines of SK-N-SH overexpressing wtABAD and3mutABAD were built. Western blot was used to measure the quantity of ABAD in each clone while AEC staining was used to observe the purity and figure of the clone. When the proper clones had been selected, MTT assay and ATP assay were applied to measure the MTT reduction or ATP concentration of the cells treated by hydrogen peroxide in different concentrations. And the differences between these groups were carried out by student t test.Results:The stable cell lines of SK-N-SH overexpressing wtABAD and3mutABAD were built. Treated by hydrogen peroxide with the concentration of25uM,50uM,100uM for24hours or25uM,50uM for48hours, the wtABAD cell line had an increased MTT value(P<0.05). Among the wtABAD clones expressing different concentration of ABAD, protective effect was shown against the oxidative stress induced by the hydrogen peroxide. In the mutABAD clones, treated by hydrogen peroxide with the concentration of100uM for24hours, the mutABAD cell line carrying D86G had a decreased MTT value (R130C:0.80±0.033vs.0.73±0.059, P=0.340; Q165H:0.80±0.033vs.0.89±0.050, P=0.162, D86G:0.80±0.033vs.0.48±0.011, P=0.011). Treated by hydrogen peroxide with the concentration of250uM for24hours, the mutABAD cell line carrying R130C and D86G had decreased MTT values while Q165H had an increased value(D86G:0.50±0.076vs.0.08±0.002, P=0.001; R130C:0.50±0.076vs.0.15±0.021, P=0.001, Q165H:0.50±0.076vs.0.69±0.019, P=0.030). The same results were seen in ATP concentration in wtABAD cell lines treated by hydrogen peroxide of different concentration (100uM:0.90±0.086vs.1.18±0.079, P=0.036;200uM:0.66±0.071vs.1.07±0.092, P=0.006). As for the3mutants, no differences were found in ATP concentration comparing with the control, which were both treated with hydrogen peroxide in different concentrations.Conclusion:wtABAD transfected SK cell had a cell protective effect against the oxidative stress induced by hydrogen peroxide. The MTT value showed that R130C and D86G transfected cell lines might have a hazardous effect while Q165H transfected cell line might have a protective effect towards the oxidative stress caused by hydrogen peroxide, while ATP concentration in3mutABAD groups remained the same as that in control group. Further experiments should be done to explore the other mechanisms of the mitochondrial dysfunction in ABAD mutants. |
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