SIRT1-ZKSCAN3 Pathway Regulates Autophagy-Lysosomal Function Of Neurons In Parkinson’s Disease Model

Background and PurposeParkinson’s diseae(PD)is defined as a neurological disease which affects 2%3%of the population over 65 years old.It has been the second-most common neurodegenerative disease after Alzheimer’s disease and the root cause remains unclear.The use of pesticide and industrial products increases the incidence of PD.And the exposure of heavy metal in the occupational environment aggravates the development of PD.With increasing public concern in occupational and environmental health,the effects of exposure to pesticides and industrial products on PD are also valued.Up to now,PD is an incurable neurodegenerative disorder.With the increase of aging population in China,PD has become a serious public health problem in China.Over 60 autophagy-lysosomal genes are repressed by ZKSCAN3,a transcriptional factor which regulates autophagy.ZKSCAN3 involves in the regulation of autophagy in Parkinson’s disease,but the precise mechanism remains unclear.Acetylation is one of the post-translational modifications of autophagic related proteins,whether ZKSCAN3 is regulated by acetylation also remains unknown.In this study,we try to investigate whether ZKSCAN3 mediates autophagy-lysosomal function involves in the process of MPTP/MPP+induced Parkinson’s disease models.In addition,we try to reveal the mechanism of SIRT1ZKSCAN3 pathway and provide new ideas for treatment of PD.Methods1.Immunoblotting to detect protein level2.Detection of mitochondrial membrane potential by JC-1 staining3.Immunohistochemical staining(IHC)to quantify antigen4.Detection of ROS production by DCFH-DA staining5.Detection of ROS production by MitoSOX staining6.Detection of mitochondrial morphology by Mitotracker Red staining7.Detection of the interaction between proteins by co-immunoprecipitation(co-IP)in the cells8.Detection of lysosomal quantity by LysoTracker Red staining9.Measurement of cell death by PI staining10.Detection of cell apoptosis by Flow Cytometry11.Animal behavioral experiments:Grip test,Climbing pole test and Rotarod test12.Determination of mRNA levels by RT-PCR13.Assessment of cell viability by CCK8Results1.The increase of nuclear ZKSCAN3 protein level in ventral midbrain of mice and SN4741 cells upon MPTP/MPP+-treated.2.The protein level of LC3-Ⅱ increased and the protein level of p62 decreased under the treatment of MPTP/MPP+,which indicates the dysfunction of autophagy-lysosomal pathway.While the function of autophagy-lysosomal pathway could be reversed by ZKSCAN3 knockdown.3.MPP+induced mitochondrial damage in SN4741 cells which includes increased mitochondrial fragment,decreased mitochondrial membrane potential and excess reactive oxygen species.4.NAC promoted ZKSCAN3 to translocate out of the nucleus in MPP+-treated SN4741 cells along with the recovered autophagic function,mitochondrial function and lysosomal quantity.5.The deacetylation of SIRT1 was inhibited in MPP+-treated SN4741 cells.SRT2104,a SIRT1 activator,promoted ZKSCAN3 to translocate out of the nucleus through deacetylating ZKSCAN3.This attenuated the injury induced by MPP+ in SN4741 cells.6.The use of SRT2104 reversed damage of autophagy-lysosomal function and mitochondrial injury in MPP+-treated SN4741 cells.7.SRT2104 recovered autophagic function,attenuated dopaminergic death and relieved impairment of motor capacity of MPTP mice.Conclusion1.Autophagy-lysosomal function was inhibited in MPTP/MPP+PD models.SRT2104 protects dopaminergic neurons from MPTP/MPP+toxicity through ZKSCAN3.2.Increased nuclear ZKSCAN3 related to the decreased autophagy and it could be recovered through ZKSCAN3 knockdown.3.ZKSCAN3 was deacetylated by SIRT1 and translocated out of nucleus.And then ZKSCNA3 lost its capability of transcriptional repression.4.Clearance of excess ROS activated SIRT1 function and upregulated autophagy in MPP+-induced SN4741 cells to protect dopaminergic survival.