?-Amyloid Induces Mitophagy Through Fatty Acid Transporter,Leading To Ferroptosis Of Pericytes,and Its Mechanism Of Blood-Brain Barrier Damage

Part One A?1-40 leads to the destruction of the blood-brain barrier by up-regulating the expression level of CD36 in pericytes.Purpose:Pericytes are an essential part of the neurovascular unit and blood-brain barrier(BBB),acting as the BBB gatekeeper.The production and deposition of excessive A?in the brain further lead to the death of pericytes and the destruction of the BBB.CD36 is a multifunctional glycoprotein of the innate immune receptor involved in A?transport.Knockout of CD36 expression increased the number of pericytes and induced partial normal pericytes.Can offset the cerebrovascular dysfunction caused in APP/PS1 mice.However,the molecular mechanism and its role in destroying pericytes and the blood-brain barrier are still unclear.This study explores the effect and significance of A?1-40 pericyte CD36 on the destruction of the BBB and further explores the specific signal transduction mechanism.Methods:At the experimental animal level,the experimental group is 7mo and 9mo APP/PS1 double transgenic mice,and the control group is wild-type mice(WT)of the same strain,age,and sex-matched.We used PET/CT to measure the blood-brain barrier.A?spots were stained by immunohistochemistry,PDGFR?(pericytes),CD36,GLUT1(endothelial cells),A?were stained by immunofluorescence.Protein levels in CD36,LRP1,NG2,and PDGFR?were detected by western blot.At the cell level,the primary pericytes were extracted and co-cultured with the endothelial cell(b End.3 cell line)to establish the Transwell in vitro contact BBB model.The compactness of the BBB model was tested by transendothelial impedance(TEER).The BBB permeability was measured by the permeability of FITC-dextran and FITC-A?1-40 with a fluorescent microplate reader.We incubated pericytes and Hylite Fluor^TM 555-A?1-40(red fluorescence)for one hour.Then,we observed the pericytes endocytosis of A?1-40 and co-localized with CD36 by immunofluorescence.The lentiviral transfection method knocks down the expression of CD36 and detects the inhibition efficiency at the protein level and transcription level.When measuring the effect of A?1-40on BBB of pericytes,we add A?1-40 100n M and 1u M to the lower chamber of Transwell for 6 hours after the established BBB is tested by TEER and permeability.Experimental groups:(1)Control group(CTR group):cells are cultured typically,and the medium is exchanged.(2)DMSO group:After the cells were in the in vitro blood-brain barrier model for 6 days,1u M DMSO at the same concentration as A?1-40 was added to the lower chamber of Transwell as a solvent control group.(3)A?1-40 group:Two dose of A?1-40100n M and 1u M were added to the lower section of Transwell,and the follow-up test was performed 6 hours after treatment.(4)Si-CD36 group:Use RNA interference technology to down-regulate pericyte CD36 and then establish an in vitro blood-brain barrier model.The treatment is the same as the A?1-40 group to verify the role of CD36 in the pericyte blood-brain barrier.(5)si-NC group:As a negative control for si-CD36,the BBB in vitro was established after the carrier control was turned out.Follow-up experiments were the same as the si-CD36 group.Western blot and RT-q PCR were used to observe the changes in the expression of CD36 and LRP1.Result:1)The BBB of APP/PS1 mice was damaged in 6mo and 9mo,and the pericytes decreased,and the expression of CD36 decreased.2)APP/PS1 6mo mouse A?plaques are formed,and pericytes express CD36 and co-localize with A?.3)In vitro experiments verified that pericytes endocytosed A?1-40 by CD36,and pericytes highly expressed CD36 after treatment with A?1-40.4)A?1-40 increases the permeability of BBB and down-regulates the expression of CD36 in pericytes to improve the permeability of BBB.Conclusion:PDGFR?,CD36,and?-amyloid protein are co-localized,and A?1-40 damage the BBB by up-regulating the expression level of CD36 in pericytes.After si CD36,the compact type increases,and the permeability decreases.Part two A?1-40 promotes mitophagy through the CD36/PINK1/Parkin pathway and induce ferroptosis of pericytesPurpose: After pericytes endocytose A?1-40 into the cells via CD36,the degradation of A?1-40 is imbalanced in the cells to explore the effect of A?1-40 on pericytes,to detect changes in pathway proteins,and to explore the signal transduction mechanism of its impact.And further,explore the role and significance of CD36 molecules in A?1-40 damage to pericytes.Methods: This experiment uses primary brain pericytes as the main research object.A?1-40 100 n M and 1u M were used to stimulate pericytes cultured in vitro,and CCK-8 was used to observe its effect on proliferation.And observe the changes of mitochondrial reactive oxygen species and mitochondrial membrane potential by flow cytometry.The autophagy agonist CCCP,autophagy inhibitor CQ,and Mdivi-1 were used to verify its effect on mitochondrial autophagy.The discussion on ferroptosis was verified with the ferroptosis agonist Erastin and the inhibitor FER-1.The knockdown of CD36 uses lentiviral transfection technology,and an empty vector is used as a control.Experimental grouping: 1).CTR group 2).DMSO group 3).A?1-40(100n M or 1u M)group 4).CCCP group 5).CQ group 6).CQ + A?1-40 1u M group 7).Mdivi-1 Group 8).Mdivi-1 + A?1-40 1u M group 9).Erastin group 10).FER-1 group 11).FER-1 + A?1-40 1u M group 12).si-NC group 13).si CD36 group 14).si-NC + A?1-40 1u M group 15).si CD36+A?1-40 1u M group.Autophagy flow changes were observed by immunofluorescence staining of mitochondrial matrix protein HSP60,autophagosomal protein LC3.Lysosomal tracer and mitochondrial changes autophagosome and mitochondrial damage were observed by the electron microscope.The apoptosis rate was observed by flow cytometry.The kit method measures the changes in ATP,GSH-PX,and iron ion levels,and the active lipid oxygen and Fe2+ levels are measured by a fluorescent microplate reader.Fluorescent probes DHE and ROS measure the changes of reactive oxygen species.Western blot detection of CD36,HSP60,Tim23,LC3II/LC3 I,BCL2,BAX,caspase3(P35),and cleaved-caspase3(P17),GPX4,x CT,Ferritin,NOX1,S65,BNIP3,NIX,PINK1,Parkin,and other protein levels Variety.RT-q PCR detects the changes of HSP60 and Tim23 at the transcription level.Result: 1)A?1-40 inhibits pericyte proliferation,causes mitochondrial damage,and promotes mitophagy.2)Treatment of pericytes with a small dose of A?1-40 for 6 hours did not lead to apoptosis,but pericyte oxidative stress increased.3)A?1-40 leads to ferroptosis dependent on mitophagy in pericytes.4)A?1-40 induces mitophagy in pericytes through the CD36/PINK1/Parkin pathway.Conclusion: A?1-40 induces pericyte mitophagy-dependent ferroptosis through CD36/ PINK1/Parkin pathway.