| Objective:Establishing the primary neuronal model of Alzheimer disease through human ApoE3 and ApoE4 gene replaced mice.Detecting the expression of Sirtuin 3（Sirt3）in different neurons,and the effect of ApoE4 on mitochondrial function.To assess the effects of Sirt3 changes on mitochondrial function,cell metabolism in primary neurons from ApoE3 and ApoE4 mice.Methods:1. Cortices were dissected from Day 0 pups of transgenic mice that carry either human ApoE4 or ApoE3.Tissue were digest in trypsin and primary neurons were cultured in neuron culture medium（Neurobasal supplemented with L-glutamine,Penicillin-Streptomycin and B27 supplement）。the cells were placed into anaerobic incubator with 5%CO2and maintained at 37°C.2. The exogenous mouse Sirt3 cDNA sequence was subcloned into the Lenti-CMV-GFP vector to overexpress Sirt3.A short sequence of 19nucleotides was constructed and Sirt3 site 764 was targeted to Omics Link small hairpin RNA（sh RNA）expression clones to knock down Sirt3expression,The sh RNA vector,Lenti-Sirt3 vector,and a control vector were packaged into Lenti-Virus transfection system.After the cortical neurons matured in petri dish for 7 days,we added transfecting viral particles into ApoE3 and ApoE4 neuron,and puromycin was used to screen the untransfected cells.Successfully transfected cells express green fluorescence.3. The expression of Sirt3 in primary neurons from ApoE4 and ApoE3transgenic mice were detected by Western blot.4. Oxygen consumption and ATP levels were evaluated with Mito Xpress Xtra-oxygen consumption assay and Luminescent ATP Detection Assay Kit,respectively.5. All data were presented as mean±SEM.The comparison between multiple groups was performed by ANOVA.The data were processed by Graph Pad Prism 5 statistical analysis software.P<0.05 was considered as statistically significant.Results:1.Sirt3 levels in ApoE4 mice neuron were decreased compared to that in ApoE3 mice neurons.2.Compared with ApoE3 neurons,amplitude of oxygen consumption curves was reduced and the ATP level was decreased in ApoE4 neurons,the slope of oxygen consumption curves was higher.3.Compared with ApoE4 neurons,The amplitude of oxygen consumption curves and ATP level was reduced in ApoE4 neurons and ApoE3 neurons with Sirt3 knockdown,the slope of oxygen consumption curves was higher in ApoE4 neurons with Sirt3 knock down.whereas them were improved in ApoE4 neurons with Sirt3 overexpression.Conclusions:1.Decreased Sirt3 expression and impaired mitochondrial function in ApoE4 neurons.2.ApoE4 damage mitochondrial function through Sirt3. |
