APP/PS1/LC3三转基因小鼠的构建及其自噬流水平研究

Autophagy flux in the APP/PS1/LC3 triple-transgenic mouse model of Alzheimer’s disease Chen Jingfei，He Guiqiong，Long Zhimin

摘要:

目的：研究APP/PS1/LC3三转基因小鼠的构建及自噬流情况。方法：将CAG-mRFP-eGFP-LC3转基因自噬流模型小鼠与APP/PS1双转基因阿尔茨海默病（Alzheimer’s Disease，AD）模型小鼠交配繁殖，对产下的子代进行基因分型鉴定，选出同时含有 CAG-mRFP-eGFP-LC3基因和APP/PS1的小鼠为APP/PS1/LC3三转基因小鼠，Morris水迷宫实验检测小鼠行为学变化。取6月龄APP/PS1/LC3三转基因小鼠和同窝CAG-mRFP-eGFP-LC3 单转基因小鼠各6只，断头取脑后在透射电镜及荧光显微镜下观察自噬流的变化，采用免疫组织化学法检测老年斑的形成，用Western blot检测自噬标志物。结果：基因分型证实APP/PS1/LC3三转基因小鼠构建成功。Morris水迷宫显示，6月龄APP/PS1/LC3三转基因小鼠找到平台的平均潜伏期和路程均明显增加（F=87.096，P=0.000；F=41.583，P=0.000），穿越平台的次数明显减少（2.000±0.707 vs. 4.800±0.800，t=2.622，P=0.031）。透射电镜下观察，6月龄APP/PS1/LC3三转基因小鼠脑神经元内出现更多的自噬小泡。荧光显微镜下观察，与同窝CAG-mRFP-eGFP-LC3单转基因小鼠比较，6月龄APP/PS1/LC3三转基因小鼠海马区自噬体及自噬溶酶体数量均明显增高（5.894±0.742 vs. 14.820±3.350，t=0.017，P=0.000；1.204±0.420 vs. 1.840±0.559，t=3.156，P=0.005），大脑皮质内自噬体数量亦明显升高（1.943±0.415 vs. 10.030±4.382，t=6.364，P=0.000），但自噬溶酶体数量比较，差异未见统计学意义（0.562±0.207 vs. 0.686±0.195，t=0.156，P=0.878）。免疫组化染色结果显6月龄APP/PS1/LC3三转基因大脑皮质及海马区域出现明显老年斑，而同窝CAG-mRFP-eGFP-LC3单转基因小鼠脑内未见老年斑。Western blot结果显示：与同窝CAG-mRFP-eGFP-LC3单转基因小鼠比较，APP/PS1/LC3三转基因小鼠脑内APP蛋白明显升高（0.294±0.070 vs. 0.690±0.275，t=3.423，P=0.007），自噬相关蛋白LC3、 Beclin1、P62水平均增高（0.241±0.004 vs. 0.534±0.019，t=37.170，P=0.000；0.479±0.020 vs. 1.180±0.255，t=6.820，P=0.000；0.188±0.007 vs. 0.356±0.021，t=18.850，P=0.000），但溶酶体膜蛋白LAMP1表达水平降低（1.450±0.065 vs. 0.773±0.043，t=8.705，P=0.000）。结论：APP/PS1/LC3三转基因小鼠自噬被激活，但自噬溶酶体降解受阻，是研究AD自噬流水平的理想动物模型。

Abstract:

Objective：To establish a new APP/PS1/LC3 triple-transgenic mice model and to study its autophagy flux activities. Methods：APP/PS1 double-transgenic Alzheimer’s disease（AD） model mouse was breed with CAG-mRFP-eGFP-LC3 transgenic autophagy flux model mouse，then their filial generations were identified by genotyping. Triple-transgenic mice expressing both CAG-mRFP-eGFP-LC3 gene and APP/PS1 gene were selected. Six APP/PS1/LC3 triple-transgenic mice and six CAG-mRFP-eGFP-LC3 transgenic mice were involved in this study，all in their sixth month. The Morris water maze was employed to test learning and memory ability. The activities of autophagy flux were observed under fluorescence microscope and transmission electron microscopy（TEM）. Immunohistochemical staining was used to detect the formation of senile plaques；autophagy related proteins were measured by Western blot. Results：The genotyping confirmed that the APP/PS1/LC3 triple-transgenic mouse model was succ-essfully established. Morris water maze test showed that，compared with those of CAG-mRFP-eGFP-LC3 transgenic mice，the escape latency and path length of APP/PS1/LC3 triple-transgenic mice were significantly increased（F=87.096，P=0.000；F=41.583，P=0.000），and the passing times were decreased（2.000±0.707 vs. 4.800±0.800，t=2.622，P=0.031）. TEM revealed more autophagosomes in APP/PS1/LC3 triple-transgenic mice neurons than in CAG-mRFP-eGFP-LC3 transgenic mice. Compared with those of sixth month CAG-mRFP-eGFP-LC3 transgenic mice，both autophagosomes and autolysosomes were increased in the same age triple-transgenic mice（5.894±0.742 vs. 14.820±3.350，t=0.017，P=0.000；1.204±0.420 vs. 1.840±0.559，t=3.156，P=0.005） in hippocampus area. Autophagosomes were significantly elevated in the cortex region of APP/PS1/LC3 triple-transgenic mice（1.943±0.415 vs. 10.030±4.382，t=6.364，P=0.000），but showed no difference in autolysosome number（0.562±0.207 vs. 0.686±0.195，t=0.156，P=0.878）. The staining showed that sixth month triple-transgenic mice had appar－ently senile plaques deposition in brains，while the CAG-mRFP-eGFP-LC3 transgenic mice had none. Western blot showed that，compared with that of brood CAG-mRFP-eGFP-LC3 transgenic mice，APP protein in the brain of APP/PS1/LC3 triple-transgenic mice increased significantly（0.294±0.070 vs. 0.690±0.275，t=3.423，P=0.007）. The proteins related with autophagy including LC3，Beclin1，P62 also increased in brains of APP/PS1/LC3 triple-transgenic mice（0.241±0.004 vs. 0.534±0.019，t=37.170，P=0.000；0.479±0.020 vs. 1.180±0.255，t=6.820，P=0.000；0.188±0.007 vs. 0.356±0.021，t=18.850，P=0.000），but lysosome membrane pro－tein LMAP1 decreased in APP/PS1/LC3 triple-transgenic mice，compared with brood CAG-mRFP-eGFP-LC3 transgenic mice （1.450±0.065 vs. 0.773±0.043，t=8.705，P=0.000）. Conclusion：Autophagy is induced in APP/PS1/LC3 triple-transgenic mice，and the degradation of autolysosomes is blocked，which is an ideal mice model for researching autophagy flux function in AD.