Chaperone Mediated Autophagy Regulates The Antioxidative Stress Response Of Keap1-Nrf2 System In The Pathogenesis Of Parkinson’s Disease

Background and objectiveOxidative stress plays an important role in the pathogenesis of neurodegenerative diseases including Parkinson’s disease.Chaperone-mediated autophagy(CMA)is a selective degradation process for substrate proteins via lysosomes.In CMA,substrate proteins carrying special CMA-targeting motif(KFERQ-like motif)are bound with Hsc70(heat shock cognate protein 71),transferred from cytosol to the lysosomal CMA receptor LAMP2A(lysosome-associated membrane protein type 2A),and eventually translocated into lysosome lumen for degradation.And LAMP2 A is the rating-limited factor of CMA.CMA could be activated during mild oxidative stress,and alleviates DA neurons’ oxidative damage through degrading oxidative damaged protein,maintaining the transcriptional function of the nucleus and protecting the morphology of mitochondria.However,the mechanisms of CMA maintaining neuronal homeostasis under oxidative stress are far from clear.Nuclear factor erythroid 2-related factor 2(Nrf2)is a mastertranscriptional regulator for antioxidative response and cellular homeostasis maintenance through transcriptional upregulating plenty of antioxidative factors.Nrf2 is primarily regulated by Keap1-CULLIN3 E3 ligase complex.Keap1(Kelch-like ECH associated protein 1)serves as a substrate scaffold for CULLIN3-containing E3 ubiquitin ligase,which induces continually ubiquitination and degradation of Nrf2 by 26 S proteasome.The modification or protein level change of Keap1 could directly influence Nrf2’s stabilization and activity.Our previous mass spectrometry results revealed that Keap1 may be the substrate of CMA.Therefore,this study aims to confirm whether CMA can selectively degrade Keap1 to promote Nrf2 levels and functions,for elucidating the mechanism of CMA’s resistance to oxidative stress in neurons and CMA dysfunction in the pathogenesis of PDMethods1.Detecting the effect of CMA on Nrf2 and related mechanism.SN4741 cells,a mouse embryonic substantia nigra-derived cell line,were used as model cells,and transfected Flag-LAMP2 A to activate CMA.We detected Nrf2 protein level and m RNA of target genes by western blot and q PCR.Meanwhile,we detected the stability and ubiquitination level of Nrf2.2.Investigating whether Keap1 was degraded by CMA.SN4741 cells were used as model cells.Through serum-free treatment,LAMP2 A overexpression or LAMP2 A knockdown to detect Keap1 protein level.Detecting the interaction between CMA chaperone Hsc70 and Keap1 through Co-IP and GST pull down assays.Purifying lysosomes and detecting whether lysosomes can bind and uptake Keap1 in vitro.Searching and validating the KFERQ-like motif of Keap1.3.Exploring the relationship between CMA and Keap1-Nrf2 signaling pathway under oxidative stress.SN4741 cells were used as model cells,and neurotoxin,6-OHDA was used to treat cells to induce oxidative stress.CMA activity was inhibited by transfection with si-LAMP2 A,and the effects of 6-OHDA treatment on Keap1 and Nrf2 protein levels were detected by western blot.The expression level ofdownstream genes of Nrf2 was detected by q PCR,and the ROS and apoptosis levels were detected by ROS assay kit and TUNEL staining.Results and Conclusion1.Activating CMA though overexpressing LAMP2 A could increase Nrf2 level and promote Nrf2 nucleus translocation.Cycloheximide(CHX)was used to inhibit cellular protein synthesis function,then detecting the degradation rate of Nrf2.The results showed that activating CMA can significantly reduce the degradation rate of Nrf2.Keap1’s binding to Nrf2 leads to continuous ubiquitination degradation of Nrf2.Our Co-IP results showed that after upregulation of CMA activity,the binding of Keap1 to Nrf2 was significantly reduced,and the ubiquitination level of Nrf2 was significantly reduced.These results indicate that CMA can stabilize and up-regulate Nrf2 levels by inhibiting Keap1 mediated ubiquitination of Nrf2,and promote Nrf2’s nuclear translocation and transcriptional activity.2.Prolonged serum deprivation and LAMP2 A overexpression could significantly decrease the protein level of Keap1.We found Keap1 has interaction with Hsc70 through GST pull-down and Co-IP assays.We demonstrated that Keap1 could be degrade by CMA-activated lysosomes by lysosomal binding and uptake assay in vitro.We searched NELRL68-72 sequence as a KFERQ-like motif through “KRERQ finder”.We mutated NELRL to AALRL(NE/AA)and detected the interaction between NE/AA and Hsc70 through Co-IP,and the results showed that NE/AA mutant could significantly inhibit the binding between Keap1 and Hsc70.Taken together,our findings establish Keap1 as a CMA substrate.3.SN4741 cells were treated with 6-OHDA for inducing oxidative damage,and we found Nrf2 was upregulated for antioxidative response,accompanying with increased LAMP2 A and reduced Keap1.Using si-LAMP2 A to inhibit CMA could block the degradation of Keap1 during 6-OHDA treatment and subsequently disturb the upregulation of Nrf2.Notably,study reported that Keap1 could also be degraded via p62-dependent macroautophagy.When using si-p62 to inhibit macroautophagy,wefound although si-p62 could upregulate Keap1 in basal condition,it could not inhibit Keap1 degradation induced by 6-OHDA.These date indicates that Keap1 is primarily degraded by CMA to promote Nrf2 during oxidative stress.In 6-OHDA treatment,disturbing CMA also inhibit the expression of Nrf2’s target genes,resulting the ROS aggravation and increased cell death.These results indicate that CMA is necessary for the normal antioxidative stress function of Nrf2.4.Previous study reported that Nrf2 could transcriptional upregulate LAMP2 A for activating CMA.We found LAMP2 A level change was consistent with Nrf2 through Nrf2 overexpression or knockdown in SN4741 cells.To further confirm the feedback regulation mode of CMA→Keap1/Nrf2→LAMP2A(CMA),we designed a q PCR primer for mice’s LAMP2 A and we validated the primer only target to endogenous mouse LAMP2 A not exogenous LAMP2 A plasmid through PCR and DNA electrophoresis.q PCR results showed that exogenous LAMP2 A plasmid could promote the expression of endogenous LAMP2 A,and this effect was inhibited in Nrf2-knockdown cells.These results indicate the existence of a positive feedback regulatory pathway between CMA and Keap1/Nrf2 system.In conclusion,our results reveal a previously unrecognized mechanism underlying the antioxidative stress function of CMA and establish a positive feedback loop between CMA and Nrf2,which critically contributes to maintaining DA neurons’ redox homeostasis.Our findings suggest that the disruption of CMA-Keap1-Nrf2 signaling pathway could be the pathogenic mechanism of PD,and it could also be targeted as promising therapeutic approaches for PD treatment.