| Objectives: Autophagy is a conserved lysosomal degradation pathway in cells that is essential for maintaining cell homeostasis and adapting to external stimuli.However,due to the polarization of neuron cells,the formation and degradation of autophages have the characteristics of spatial separation,resulting in axons easy to accumulate and degrade proteins and damaged organelles.This can easily lead to many neurodegenerative diseases such as Alzheimer's disease(AD).In the early stage of AD,autophagosomes can be observed to accumulate at the axon terminals,resulting in autophagy stress.In neurons,Dynein drives macromolecular substances to move retrograde along microtubules,which plays a key role in the transport of substances.The expression of the Dynein intermediate chain DIC reduced in the brains of AD patients,Dynein-mediated transport was impaired in the brains of AD mice,and autophagosomes accumulated in large numbers along dystrophic neurites.However,the mechanism of autophagosome transport in AD brain is not yet clear,and the specific role of Dynein in axonal transport of autophagosomes is poorly understood.Therefore,the purpose of this study was to dynamically observe the changes of cognitive function,A ? deposition,autophagy and axonal transport related proteins in APP/PS1 double transgenic mice.The effects of DIC on cognitive function,A? deposition,autophagy and axonal transport related proteins in AD model mice were further analyzed by overexpression and interference with DIC.Methods: APP/PS1 double transgenic mice were propagated and divided into Non Tg(C57BL/6J mice-6 months old),Tg-3M(APPswe/ PSEN1dE9 mice-3 months old),Tg-6M(APPswe/PSEN1dE9 mice-6 months old),Tg-9M(APPswe/PSEN1dE9 mice-9 months old),Tg-12M(APPswe/PSEN1dE9 mice-12 months old)carried out the first part of the experiment.Firstly,the water maze test was used to detect the cognitive level of mice.Immunohistochemistry was used to detect the deposition of insoluble A? in hippocampus of mice.Transmission electron microscopy was used observe autophagy in hippocampal region,and Western Blot was carried to detecte the protein expression levels of LC3 and P62.The protein expression levels of LAMP2 and cathepsin D were measured,too.Furthermore,the protein expression of Kinesin,Dynein protein and axonal transport-related proteins P150,P50,Rab7 and ORP1 L were also detected.On this basis,DIC interference / overexpression adeno-associated virus was used to inject Tg mice,and then the second part of the experiment was carried out.After verifying the efficiency of virus interference,the cognitive level of mice was detected by water maze test,the insoluble A ? deposition in hippocampus of mice was detected by immunohistochemistry,and the protein expression of LC3,P62,LAMP2 and cathepsin D was detected by Western Blot.Furthermore,the protein expression of Kinesin,Dynein protein and axonal transport-associated proteins P150,P50 and Rab7 were detected.Result: The average escape latency of APP/PS1 mice was higher than that of Non Tg mice,and increased with age.The number of mice crossing the platform in space exploration experiments showed a decreasing trend in APP/PS1 mice;Immunohistochemistry showed that there was A? deposition in the hippocampus of Non Tg mice,but began to deposit in the hippocampus of Tg-3M mice and increased gradually with the increase of age.There was no obvious autophagy in hippocampal region of Non Tg mice and APP/PS1-3M mice observed by electron microscope.With the increase of age,the autophagy in the hippocampus of APP/PS1 mice showed a stacking state,and the protein levels of LC3? and p62 in APP/PS1 mice were higher than those in Non Tg mice.The levels of DIC and KIF5 B in APP/PS1 mice were higher than those in Non Tg mice,but decreased with age.The expressions of P150,P50,Rab7,ORP1 L and LAMP2 in hippocampus of APP/PS1 mice were higher than those of NonTg,but with the increase of age showed a first increase and then downward trend;The expression of cathepsin D gradually decreased in Tg group,indicating that lysosomal function was impaired.Then,AD mice were subjected to DIC interference or overexpression,and the Part ? experiment was carried out.It was found that the interference of DIC expression aggravated the cognitive impairment of AD mice and promoted the deposition of A?.Overexpression of DIC improved the recognition of AD mice.Western Blot showed that the change of LC3 was not statistically significant,but the increase of p62 after interfering with DIC indicated that autophagy degradation was hindered,and p62 decreased after overexpression of DIC,indicating that overexpression of DIC promoted autophagy degradation.The protein levels of DIC,P150,P50,LAMP2 and CTSD decreased and the expression of driving protein KIF5 B increased.after overexpression of DIC,the protein levels of DIC,P150,P50,LAMP2 and CTSD increased significantly,while the expression of driving protein KIF5 B decreased,while the expression of Rab7 increased after interfering with DIC,after interfering with DIC.The increase of Rab7 level was more obvious after overexpression of DIC.Conclusion: The learning and cognitive impairment and insoluble A ? deposition in AD transgenic mice increased gradually with the increase of age.And autophagic accumulation increased in the hippocampus of AD mice,the expression of axonal transport related proteins decreased,and lysosome function was impaired.DIC can promote the expression of axonal transport related proteins,improve lysosome function,reduce the accumulation of autophages,and reduce the deposition of insoluble A ?,thus improving the cognitive impairment of AD mice.It can be seen that,Dynein plays an important role in alleviating autophagy accumulation in AD,and is expected to provide a new idea for the treatment of AD. |
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