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

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.