PEG-ceramide Nanomicelles Induce Autophagy And Degrade Tau In N2a Cells

Alzheimer's disease(AD)is a neurodegenerative disease that occurs in elder people and characterized by memory loss,behavioral abnormalities,and learning dysfunction.Its main pathological mechanism is the aggregation of amyloid-beta protein and phosphorylation of Tau protein.Many other factors contribute to the pathological progression of AD in a complex way.Autophagy is a cellular,physiological process clearing structural or functional abnormal organelles or protein polymers.In neurodegenerative diseases,the ability of autophagy decreases,leading to deposition or misfolded proteins that cannot be effectively eliminated,resulting in clinical symptoms.At present,there are many studies showing that some small molecule can induce autophagy to clear cell over-expressed proteins.While,in the central nervous system,the existence of blood-brain barrier makes most molecules unable to penetrate into brain.Nanomedicine provides a new strategy for brain targeted therapy,while some nanomaterials can induce the occurrence of autophagy.In the past research,we have prepared a stable and low-toxic nano-carrier,PEG-ceramide nanomicelle.There is no relevant research to explore the possibility of its potential treatment of AD.Based on this,we assume this novel nanomaterial may clear the overexpressed Tau protein by inducing cell autophagy.In the first part of this experiment,we optimized the formulation by the film dispersion method to obtain stable blank PEG-ceramide nanomicelles and determine the particle size.Secondly,we established the culture method of N2 a cells,and constructed in vitro nervous system model.The cytotoxicity of PEG-ceramide nanomicelles was investigated by CCK8 method,then we selected the safe concentration.In the second part,we used Western Blot to detect the effect of PEG-ceramide nanomicelles on the content of autophagy-associated protein LC3 and p62 in N2 a cells,to determine its effect on autophagy.The results showed that the ratio of LC3-II/LC3-I increased and the expression level of p62 decreased,suggesting autophagy activation.Secondly,we used the autophagic flow detection method to infect cells with the virus expressing mRFP-GFP-LC3 fusion protein,and labeled and traced LC3,observed the fluorescence expression of the cells by confocal microscopy.The results showed that the autophagosomes and autophagic lysosome spots of N2 a cells treated with PEG-ceramide nanomicelles increased significantly,showing an enhanced trend of autophagic flow.In the third part of this experiment,we transfected N2 a cells to express WT-Tau and P301L-Tau which are two type of human Tau protein,and then used Western Blot to detect the effect of PEG-ceramide nanomicelles on intracellular LC3 and Tau protein levels.The results showed that PEG-ceramide nanomicelles could increase the ratio of LC3-II/I,activate autophagy,and down-regulate the content of WT-Tau and P301L-Tau in cells.Based on the above experiments,we have initially confirmed that PEG-ceramide nanomicelles can enhance autophagy and autophagic flow in N2 a cells,and degrade both human WT-Tau and P301L-Tau proteins level in N2 a cells by regulating autophagy.We believe that this nanoparticle can be used in diagnosis and treatment of AD in the future.