The Relationship Between Ferroptosis And Microtubule-associated Protein Tau Aggregation And The Accosiated Mechanisms

Ferroptosis is a non-apoptotic form of programmed cell death,which is characterized by the iron-dependent accumulation of lipid peroxides.Research indicates that excessive accumulation of iron and peroxidation damage of neurons in brain regions like the hippocampus are closely related to tau-related neurodegenerative diseases such as Alzheimer’s disease(AD).However,the relationship between ferroptosis of neurons and pathological accumulation of tau protein has not been reported.Based on the background,this study was designed to establish ferroptotic cell death model on Mouse Neuroblastoma N2a cells by treating with ferroptosis inducer erastin.In order to research the relationship between ferroptotic cell death and abnormal aggregating of microtubule-related proteins tau.Then the possible mechanism was elucidated by detecting the changes in the content of microtubule-associated protein tau,the degree of phosphorylation modification,intracellular ubiquitin homeostasis,20S proteasome activity and the ratio of autophagy activity marker LC3-II/I.In this experiment,N2a cells were treated with 10μM ferroptosis inducer erastin,10 μM erastin plus 2 μM ferroptosis inhibitor ferrostatin-1 for 6 hours and 12 hours respectively.After that Cell Counting Kit-8(CCK-8)was used to detect the cells activity;using Phase Contrast Microscopy to observe the changes of cells morphology and number;the content of Reactive Oxygen Species(ROS)in N2a cells was detected by DCFH-DA;GPX Assay Kits was used to detect the activity of Glutathione peroxidase(GPX);Iron Assay Kit was used to detect the level of iron;Proteasome Assay Kit was used to detect the proteasome viability;the degree of tau protein aggregation was detected by Immunofluorescence double-labeling technology;the degree of phosphorylation modification of tau,the content of tau protein and the autophagy activity marker LC3-Ⅱ/Ⅰ were detected by Western Blotting technology.The experimental results are as follows:(1)Compared with the control group,After N2a cells were treated with erastin for 6 hours and 12 hours,the cell activity was significantly declined(p<0.05),the number of cells was reduced,and the neurites were significantly shortened,the intracellular GPX activity was significantly decreased(p<0.001),the content of ROS and iron were significantly increased(p<0.001),the above cellular damaging changes can be significantly reversed by ferrostatin-1.These results suggest that erastin induces ferroptotic cell death in N2a cells.(2)Compared with control group,after N2a cells were treated with erastin for 6 hours and 12 hours,there was an obvious aggresome of tau protein in the nucleus,the cellular damaging changes can be significantly reversed by ferrostatin-1.These results suggest that neuronal ferroptotic cell death can formation of tau protein aggregates.(3)Compared with control group,After N2a cells were treated with erastin for 6 hours and 12 hours,the intracellular total tau protein content was significantly increased(p<0.01),the tau-Ser-396 and tau-Thr-231 sites were hyperphosphorylated(p<0.001),the intracellular level of Mono-Ub was decreased(p<0.001),Poly-Ub was increased(p<0.01),the 20S proteasome activity was decreased(p<0.001),and the ratio of autophagy activity marker LC3-Ⅱ/Ⅰ was increased(p<0.05),the above cellular damaging changes can be significantly reversed by ferrostatin-1.To sum up,in the process of erastin induced ferroptotic cell death in N2a cells,microtubule-associated protein tau pathological aggregation.The pathological changes of tau are related to the intracellular total tau protein content,excessive phosphorylated modification,activity inhibition of the Ubiquitin-Proteasome System(UPS),and the compensatory activation of the Autophagy-Lysosome System(ALS)can not degerate the accumulation of tau protein effectively.This study reveals the relationship between ferroptotic cell death and abnormal accumulation of tau protein in neurons,and provides a new research idea for the pathological research of tau protein-related neurodegenerative diseases.