Curcumin Stimulates Axonal Transport Of Autophagy Via Enhancing The Expression Of Rab7

Objectives: Alzheimer’s disease(AD)is a degenerative disease in central nervous system.Autophagy is an important pathway for the degradation of protein in eukaryotic cells which is closely related to the development of AD.The accumulation of autophagosomes in the distal axon,which can’t be combined with lysosomes,not only affects the metabolism of Aβ,but also causes autophagy stress which results in the death of nerve cell.Rab7 is a small GTP enzyme that plays an important role in the maturation of autophagosomes and the integration of autophagosomes with lysosomes.Studies have shown that as a polyphenolic plant compounds,curcumin plays a neuroprotective role in AD,however,the mechanism remains unknown.Our study aims to verify if curcumin will reduce the deposition of Aβ so that perform a protective effect on AD in vitro.Then the influence of curcumin on autophagy and autophagy flux will be observed.Furthermore,the role of curcumin in axonal transport of autophagosomes will be studied.At last,whether the mechanism is related to the expression of Rab7 will be explored.In our study,we select a new sight to explore the mechanism of curcumin on AD so that provide a new target and idea on the treatment of AD.Methods: First of all,the cells were divided into 4 groups: WT group(N2a/WT cells),Control group(N2a/APP695 swe cells),Sham group(N2a/APP695 swe cells treated with DMSO at 5μM for 24h),Curcumin group(N2a/APP695 swe cells treated with DMSO at 5μM for 24h).Flow cytometry was used to detect the differences of cell apoptosis and mitochondrial membrane potential in each group.ELISA assay was performed to analyze the expression of Aβ in the cell culture mediums.The quantity changes of autophagosomes and autolysosomes in each group were observed by transmission electron microscopy and laser confocal microscopy.At the same time,the expression of LC3 II,SQSTM1,Beclin1,Atg7,Atg16L1,Dynein,KIF3 B,and Rab7 were detected by Western Blot.And the expression of Rab7 at m RNA level was determined by q RT-PCR.Then the cells were divided into 5 groups after transfection of pc DNA3.1,pc DNA3.1-Rab7,NC-si RNA and Rab7-si RNA respectively: Control group,pc DNA3.1 group,pc DNA3.1-Rab7 group,NC-si RNA group and Rab7-si RNA group.Flow cytometry was employed to determine the cell apoptosis and mitochondrial membrane potential.ELISA assay was used to examine the expression of Aβ in the cell culture mediums.Transmission electron microscopy and laser confocal microscopy were carried out to observe the number of autophagosomes and autolysosomes in each group.Western Blot was performed to detect the expression of LC3 II,SQSTM1,Dynein and KIF3 B proteins.Results: Compared with the Control group,the mitochondrial membrane potential(P=0.000),the number of autophagosomes and autolysosomes,the expression of LC3II(P=0.000),Beclin1(P=0.000),Atg7(P=0.000),Atg16L1(P=0.003),Dynein(P=0.000)and Rab7(P=0.000)proteins and the expression of Rab7 at m RNA level(P=0.000)were increased;however,the cell apoptosis(P=0.000),the expression of Aβ(P=0.000),the expression of SQSTM1(P=0.000)and KIF3B(P=0.000)at protein levels were decreased in curcumin treated group.After transfection of pc DNA3.1-Rab7 in N2a/APP695 swe cells,the mitochondrial membrane potential(P=0.000),the quantity of autophagosomes and autolysosomes,the expression of Dynein protein(P=0.000)and KIF3 B protein(P=0.000)were elevated;meanwhile the cell apoptosis(P=0.000),the expression of Aβ(P=0.000)in the cell culture mediums,the expression of SQSTM1 at protein level(P=0.000)were attenuated;and there was no change of the expression of LC3 II protein(P=0.865)compared to the Control group.After transfection of Rab7-si RNA in N2a/APP695 swe cells,the results were oppositely in comparison with N2a/APP695 swe cells were transfected with pc DNA3.1-Rab7.Conclusion: Curcumin attenuates the cell apoptosis,increases the mitochondrial membrane potential,decreases the production of Aβ and enhances the axon transport of autophagy to promote autophagy and autophagy flux.The mechanism may be related to the regulation of the expression of Rab7.