Study On The Effect And Mechanism Of TFEB In Regulating Iron Metabolism-related Proteins And Ferroptosis

Parkinson’s disease(PD)is the second most common neurodegenerative disease,which is characterized bytremor,rigidity,bradykinesia and postural instability in clinic.More and more studies have proved that iron deposition caused by abnormal brain iron metabolism is an important risk factor for the nigral dopaminergic neurodegeneration in PD.Lysosomes are important cellular iron-storage organelles.Due to the acidic and reducing environment,lysosomal iron usually exists in the form of ferrous iron.When lysosome membrane is damaged,a large amount of ferrous iron will be released into the cytoplasm,which will cause Fenton reaction and cell death.Transcription Factor EB(TFEB)is an important factor that regulates lysosomal regeneration and autophagy.It has been confirmed that overexpression of TFEB can improve lysosomal function and then play a neuroprotective role in PD,which is suggested to be one of the effective strategies to PD prevention or treatment.The existing studies mainly focus on the lysosomal regeneration and clearing the pathological aggregated proteins by TFEB,however,it is still unclear whether TFEB can regulate iron metabolism and inhibit ferroptosis.Objective:In this study,we will explore the role and possible mechanism of TFEB on regulating iron metabolism and inhibiting ferroptosis.Method:In order to establish the mouse model with overexpression of TFEB in the substantia nigra,adeno-associated virus carrying tfeb(AAV-TFEB)was microinjected into the substantia nigra of C57 BL /6 mice according to the mouse brain atlas.Open field test,pole test and rotarod test were employed to evaluate the motor function of mice.Both immunofluorescence staining and western blot were employed to detect the lysosomal regeneration and change of iron metabolism related proteins.PC12 cells were transfected with lentivirus carrying tfeb to construct PC12 cell models with overexpression of TFEB(Lv-GFP cells and Lv-TFEB cells),and then cells were treated with ferroptosis inducer(Erastin)or ferric ammonium citrate(FAC)to construct ferroptosis cell models.2′,7′-Dichlorofluorescin diacetate probe coupled with fluorescence microscope were employed to mintor the change of cellular ROS.Western blot was employed to detect the changes of iron metabolism and ferroptosis-related proteins.Under the iron overload condition,the effect of TFEB overexpression on the cell membrane potential was detected by patch clamp.On the PC12 cells,the changes of ferroptosis related proteins after knockdown the TFEB by si RNA were detected by western blot.Results:1.Overexpression of TFEB in the substantia nigra of mice can promote the lysosomal regeneration.Compared with the Vehicle group mice,both the levels of lysosome-associated membrane protein 1(LAMP1)and cathepsin B(CTSB)in the substantia nigra of AAV-TFEB mice were significantly increased,suggesting that the overexpression of TFEB in the substantia nigra of mice can promote the lysosomal regeneration and enhance the degradation ability.2.Overexpression of TFEB in substantia nigra can enhance the motor function of mice.In the open field test,compared with the vehicle mice,the total movement distance of of AAV-TFEB mice was significantly increased,however,there is no significant change in the time spent in the central region between vehicle mice and Lv-TFEB mice,suggesting that overexpression of TFEB in the substantia nigra could enhance the motor function of mice,but does not cause anxiety-like behavior.In both the pole test and the rotarod test,there was no significant difference between these two groups of mice in the total climbing time and the time spent on the rod,suggesting that overexpression of TFEB in the substantia nigra does not affect the motor coordination ability of mice.3.Effect of TFEB overexpression on the nigral iron metabolism related proteins.(1)Compared with vehicle mice,the level of transferrin receptor 1(TfR1)in the substantia nigra of AAV-TFEB mice was significantly increased,however,the protein of divalent metal transporter 1(DMT1)has no significant change,suggesting that the overexpression of TFEB in the substantia nigra may increase the cellular and lysosomal iron uptake ability,but does not affect the iron release from lysosomes.(2)Compared with Vehicle mice,the levels of both ferritin light chain(FTL)and ferritin heavy chain(FTH)in the substantia nigra of AAV-TFEB mice were significantly increased,suggesting that overexpression of TFEB in the substantia nigra might enhance the iron storage capacity of cells.4.Overexpression of TFEB does not affect the level of ferritinophagy selective receptor nuclear receptor coactivator 4(NCOA4).After overexpression of TFEB in the substantia nigra of mice or in the PC12 cells,there is no significant change in the protein level of the ferritinophagy selective receptor NCOA4,suggesting that overexpression of TFEB may not affect the process of NCOA4-mediated ferritinophagy.5.Mechanism of TFEB in regulating ferroptosis.(1)PC12 cells with overexpression of TFEB(no fluorescence)were constructed,and cells were treated with ferroptosis inducer Erastin(10 μM)for 24 h.The level of LDH in the Lv-TFEB group was significantly lower than that in the Lv-Flag group,suggesting that high expression of TFEB can alleviate Erstin induced cytotoxicity(2)PC12 cells with overexpression of TFEB were treated with ferroptosis inducer Erastin(10 μM)for 24 h,and western blot results revealed that the level of TfR1 protein in the Lv-TFEB group was significantly higher than that in the Lv-GFP group,however,there is no significant change in the level of DMT1,suggesting that overexpression of TFEB still have the ability to increase the lysosomal iron uptake even in the present of Erastin.(3)PC12 cells with overexpression of TFEB were treated with FAC(100 μM)for 24 h,and western blot results revealed that FAC significant decreased the level of TfR1 protein in both the Lv-GFP and Lv-TFEB cells.However,compared with the cells in the Lv-GFP+FAC group,the level of TfR1 protein in the cells in Lv-TFEB+FAC group was still significantly higher,suggesting that under the iron overload condition,overexpression of TFEB could slow down the decline of TfR1 protein level.(4)PC12 cells with overexpression of TFEB were treated with Erastin or FAC for 24 h,western blot results revealed that there is no significant change in the protein level of NCOA4,suggesting that overexpression of TFEB may not affect the NCOA4-mediated ferritinophagy in the above two cell models.(5)In current clamp mode,both Lv-GFP and Lv-TFEB cells have no spontaneous and evoked firing.The average resting membrane potential in 5 recorded cells were-19.39 mV(Lv-GFP cells)or-21.06 mV(Lv-TFEB cells).After application of FAC(100 μM)to the external solution for 10 minutes,the membrane potential of Lv-GFP cells were significantly decreased to-28.53 mV(Figure 2E,2F),however,there was no significant change in Lv-TFEB cells,suggesting that the effect of TFEB on maintaining the cellular electrical activity under the iron overload condition.(6)PC12 cells with overexpression of TFEB(no fluorescence)were constructed,and cells were treated with ferroptosis inducer Erastin(10 μM)for 24 h.The level of ROS in the Lv-Flag group was significantly higher than that in the Lv-TFEB group,suggesting that overexpression of TFEB could alleviate the increase of ROS level induced by Erastin.(7)Compared with Vehicle mice,the protein levels of xCT and glutathione peroxidase 4(GPX4)in substantia nigra of AAV-TFEB mice were significantly increased.Meanwhile,in the PC12 cells,the protein level of GPX4 was significantly increased after overexpression of TFEB,however,the level of GPX4 was significantly decreased after knockdown of TFEB.These results suggest that TFEB may inhibit ferroptosis by regulating the xCT-GPX4 pathway.Conclusion:Taken together,overexpression of TFEB in substantia nigra can enhance motor function of mice,up-regulate the levels of iron metabolism-related proteins,including TfR1 、 FTL and FTH.However,overexpression of TFEB does not affect level of ferritinophagy selective receptor NCOA4.In the PC12 cells,TFEB may(1)increase the lysosomal iron storage capacity and stabilize the cell membrane potential by up-regulating TfR1,(2)regulate the xCT-GPX4 pathway and reduce the intracellular ROS level,thereby inhibit ferroptosis by reducing the accumulation of both cellular iron and lipid peroxide.These results in this study will not only provide a theoretical basis for enriching TFEB functions,but also provide a new perspective and strategie for PD prevention or treatment.