OATP1A2 Mediates The Transport Of Aβ_1-42 And Its Mechanism Of Action In Alzheimer’s Disease

Research BackgroundAlzheimer’s Disease（AD）is a neurodegenerative disease of unknown etiology.At present,many studies believe that the imbalance between the clearance and accumulation of Aβin the brain of AD patients is the main cause of AD disease.It is found that drug transporters play a very important role in the in vivo transport of drugs,endogenous substances and the occurrence and development of many diseases.Are these transporters also related to the occurrence and development of the imbalance between Aβclearance and accumulation?OATP1A2 is the most abundant subfamily of amphiphilic substrates in the family of organic anion transporting polypeptides（OATPs）,and its high expression on both sides of endothelial cells in the brain suggests its role in the delivery of drugs and neuroactive peptides to brain tissue Does it play a crucial role in mediating the production and accumulation of Aβin the central system,resulting in an imbalance between the clearance and accumulation of Aβ,leading to the occurrence and development of AD disease?This will be a bold and innovative scientific hypothesisResearch ObjectiveThe present study was designed to investigate the uptake of Aβ_1-42in HEK293T,OATP1A2-HEK293T transgenic cells and human astrocytes,and to confirm whether OATP1A2 is involved in the transport of Aβ_1-42at the cellular level,as well as the expression and transport of Aβ_1-42by OATP1A2 in neuronal cells closely related to AD.This study investigates the transport properties of the OATP1A2 transporter for Aβ_1-42and its potential role in AD disease from the perspective of drug uptake transporters,with a view to elucidating the mechanism of imbalance between Aβprotein clearance and accumulation in vivo,and providing new targets and therapeutic directions for the treatment of AD disease.Research methods（1）Using lentiviral vector technology to construct HEK293T transgenic cell model stably expressing NC and OATP1A2;（2）After subculturing the overexpressed transgenic cells,RT-qPCR,flow cytometry and Western blot were used to detect the expression of target gene m RNA and protein in the transgenic cells;（3）Aβ_1-42of 0μM,0.4μM,1μM and 2.5μM series concentration（CK,low,medium and high concentration）was added to HEK293T cells,OATP1A2-HEK293T cells and human astrocytes and incubated for 24h and 48h respectively,72h,then collect and process the cells.The expression of Aβ_1-42in cell lysate after centrifugation was detected by flow cytometry and Western blot;（4）The transport and transport properties of OATP1A2 mediated Aβ_1-42in cells were verified by immunofluorescence experiments..Research Result（1）The OATP1A2-HEK293T transgenic cell model was successfully established in this study,and the m RNA and protein expression levels of the target gene OATP1A2 in the overexpression transgenic cells were significantly higher than those in the control cells;（2）The Aβ_1-42protein in NC-HEK293T cells,OATP1A2-HEK293T cells and human astrocytes increased with the prolongation of time（24-72h）and the addition of Aβ_1-42concentration（0.4-2.5μM）The levels gradually increased,indicating that the uptake of Aβ_1-42by these cells was time-and concentration-dependent;（3）The results of Western Blot and flow cytometry showed the same trend of Aβ_1-42protein uptake,that is,the uptake of Aβ_1-42by OATP1A2-HEK293T cells was significantly higher than that in NC-HEK293T control cells,especially at 48h and72h of culture,the uptake of Aβ_1-42by OATP1A2-HEK293T was about twice that of the NC-HEK293T group,and its immunofluorescence intensity was also significantly higher than that of the NC-HEK293T cell control group;（4）Aβ_1-42protein uptake in OATP1A2-HEK293T cells was higher than that in human astrocytes at the same time and at the same concentration,while the intensity of immunofluorescence was comparable to that in human astrocytes.Research Conclusion（1）The OATP1A2 transporter is responsible for the transport of Aβ_1-42,suggesting that OATP1A2 may be an important carrier of Aβ_1-42from blood to brain tissue and that it is involved in mediating the accumulation of Aβ_1-42in cells.（2）Human astrocytes show OATP1A2 expression and transport of Aβ_1-42,suggesting that OATP1A2 expression in neuronal cells has an impact on AD pathology.（3）OATP1A2 is involved in the transport of Aβ_1-42protein,suggesting that OATP1A2 mediates the production and accumulation of Aβin the central system,resulting in an imbalance between the clearance and accumulation of Aβ,leading to the development of AD disease.