OPTN Regulates Autophagy And Eliminates Abnormal TAU Protein Through PI3K/AKT/mTOR Pathway To Relieve Neurotoxicity

Objective Obtained recombinant adeno-associated virus capable of overexpressing OPTN and TAU-P301-L genes,infecting 293 T cells in vitro and Kunming mice in vivo,and to explore the effects of OPTN and TAU-P301-L proteins on The effects of apoptosis and AKT / PI3 K / m TOR pathways provide new ideas for the treatment of Alzheimer's disease(AD).Methods A plasmid containing the target gene was extracted from E.coli containing the target gene,and the target gene was obtained by PCR amplification.The target gene OPTN was constructed into the p AAV-IRES-Zs Green1 vector,and the target gene TAU-P301L-V5 was constructed into p AAV-CMV-mkate-IRES vector.Plasmid-positive clones were identified and sequenced.Recombinant plasmids p AAV-IRES-Zs Green1-AAV9-OPTN and p AAV-CMV-mkate-IRES-AAV9-Tau-P301L-V5 were co-transfected with packaging plasmid p AAV-RC9 and helper plasmid p Helper into 293 T cells through LipofiterTM 2000 to obtain AAV9 virus.Using the same method,the corresponding green and red AAV9 control viruses were constructed without using the gene of interest.The q PCR method was used to determine the titer of recombinant adeno-associated virus vector.The virus vector infected 293 T cells for virus effectiveness and safety identification.Western blotting and RT-PCR were used for protein expression identification,The effect of OPTN protein and TAU-P301-L protein on the viability of 293 T cells was detected by MTT.q PCR was used to detect TAU,OPTN,AKT,PI3 K and m TOR m RNA,and Western blotting was used to detect OPTN,TAU,p TAU,caspase 3,AKT,m TOR,PI3 K and LC3 proteins.The pathological diffusion of TAU-P301-L protein and the colocalization of OPTN protein and TAU protein were observed by fluorescence microscope.Results The sequencing results showed that the p AAV-IRES-Zs Green1-AAV9-OPTN and p AAV-CMV-mkate-IRES-AAV9-Tau-P301L-V5 adeno-associated virus vectors were successfully constructed.The virus titer measured by q PCR was 1.0 × 1012 vg / m L.The transfection efficiency of vector-infected 293 T cells was 33.6% and 20.5%.Western blot and RT-PCR showed protein overexpression.MTT results showed that the OPTN group rescued the apoptosis of the TAU-P301-L group.OPTN protein and TAU protein have achieved high colocalization in mice.The cargo protein receptor OPTN can enhance autophagy by reducing the activity of the AKT / PI3 K / m TOR pathway.Conclusion Adeno-associated virus over-expressing OPTN and TAU-P301-L genes was successfully constructed.OPTN protein can reduce the cytotoxicity of TAU-P301-L protein.The cargo protein receptor OPTN reduces the apoptosis caused by TAU protein cells,clears the accumulation of abnormal protein TAU,relieves neurotoxicity,and provides new possibilities for the treatment of AD.