Role Of The PrP~C In Autophagy-lysosomal Pathway Of Astrocytes During Oxygen-glucose Deprivation And Re-oxygenation Injury

Background and purposeAstrocyte-mediated immune-inflammatory response after cerebral ischemia-reperfusion injury（CIRI）is one of the important pathophysiological mechanisms in which the autophagy-lysosomal pathway（ALP）is involved.The specific role played by ALP in CIRI is still controversial,and it is particularly important to explore in depth the dynamics of its various components.Cellular prion protein（PrP^C）is a cell surface membrane glycoprotein encoded by the Prnp gene,and its isoform(Scrapie isoform of prion protein,PrP^Sc)can lead to prion diseases.The group previously found that PrP^C overexpressing microglia were in an inflammation-suppressive state,which was related to their maintenance of ALP patency,but the specific molecular mechanism was not clear.Elucidating the role and regulatory mechanism of PrP^C in astrocyte ALP during CIRI may provide new ideas for more effective intervention in ischemic stroke.ContentPart I PrP^C is involved in regulating the autophagy lysosomal pathway and cellular inflammatory phenotypic transformation in astrocytes during OGD/R Methods1.Wild type（WT）,Prnp^-/-and Prnp-overexpressing neonatal mouse cerebral cortex astrocytes were isolated,cultured and purified in vitro,and the oxygen-glucose deprivation and re-oxygenation（OGD/R）model was constructed.Astrocytes were collected at 2h of cytosolic oxygen-glucose deprivation and 3h,6h,12h,24h,48h and72h of re-oxygenation,respectively,and the cells activity of was detected by CCK-8method.2.Western blotting（WB）was used to detect the expression levels of ALP-related proteins:autophagosomal markers LC3B-II/Ⅰ,autophagy substrates SQSTMI and ubiquitin,lysosomal membrane protein LAMP1,lysosomal enzyme proteins CTSD and CTSB,as well as LC3B-II/Ⅰin the presence or absence of the lysosomal inhibitor chloroquine were used to assess autophagy lysosomal pathway patency.The fluorescence spectrophotometric assay was used to detect lysosomal enzyme CTSD activity,and immunofluorescence（IF）was used to double-stain intracellular LAMP1and LC3B as well as LAMP1 and CTSD,respectively:LC3B⁺particles represent autophagosomes,LAMP1⁺particles represent lysosomes,LAMP1⁺LC3B⁺particles represent autolysosomes and LAMP1⁺CTSD⁺particles represent functional lysosomes.3.Flow cytometric bead array（CBA）was used to detect the levels of pro-inflammatory cytokines such as tumor necrosis factor（TNF）,interferon-gamma（INF-γ）,interleukin 6（IL-6）and anti-inflammatory factor IL-10 in the cell culture supernatants.Results1.Overexpressing PrP^C ameliorates OGD/R-induced impaired astrocyte activity.Compared to WT astrocytes,Prnp^-/-astrocytes activity decreased at 12-72h of re-oxygenation and Prnp overexpression astrocytes activity increased at 3-6h and 48-72h of re-oxygenation.2.Overexpressing PrP^C ameliorates lysosomal dysfunction and upregulates lysosomal numbers during the early（3-24h of re-oxygenation）and late（48-72h of re-oxygenation）stages after OGD/R,thereby maintaining ALP patency.Compared with the normal group,the OGD/R（3-24h）groups showed an increase in the number of autophagosomes,a significant increase or a tendency to increase in the expression level of SQSTMI,and a significant decrease in the expression level of pro-CTSD and m CTSB,the activity of CTSD,and the number of functionally active lysosomes,whereas there was no significant change in the number of lysosomes and the level of expression of LAMP1.It is suggested that autophagosome formation is increased in the early stages of OGD/R,but there is lysosomal dysfunction that leads to accumulation of autophagosomes and substrates.However,the OGD/R（48-72h） groups exhibited a still increased number of autophagosomes,a significant decrease in the expression level of ubiquitin,and a significant increase in the expression level of LAMP1 and the number of lysosomes,with the expression levels of pro-CTSD and m CTSB,CTSD activity and the number of functional lysosomes restored to the baseline level,suggesting that the formation of autophagosomes was increased in the late stage of OGD/R,but the rise in the number of lysosomes and the improvement of dysfunction resulted in the autophagic substrate being rapidly degraded.Further,chloroquine was applied to block autophagosome and lysosome formation and inhibit lysosomal degradation,and the expression level of LC3II/I was examined in the normal,OGD/R（6h）and OGD/R（48h）groups,respectively,and the results showed that,compared with the normal group,the degree of elevation of LC3II/I after chloroquine treatment was significantly reduced in the OGD/R（6h）group,but restored to the baseline level in the OGD/R（48h）group,suggesting that ALP is blocked in the early stage of OGD/R and ameliorated in the late stage of OGD/R.In Prnp overexpressing astrocytes,the number of autophagosomes and the expression level of insoluble SQSTM1 were significantly decreased during OGD/R（3-72h）,while the expression levels of CTSD,m CTSB,LAMP,the number of lysosomes,the activity of CTSD,and the number of functionally active lysosomes were either elevated or increased,compared with that of the Normal group;the extent of LC3B-II/Ⅰelevation after chloroquine treatment was not significantly changed in the OGD/R（6h）group but was significantly increased in the OGD/R（48h）group.It is suggested that overexpression of PrP^C ameliorates ALP in the early and late stages of OGD/R by up-regulating lysosome number and enhancing lysosomal function,leading to autophagosomal and substrate degradation.In Prnp^-/-astrocytes,the number of autophagosomes,LC3B-II/Ⅰand the expression level of insoluble SQSTM1were significantly increased during OGD/R（3-72h）,the expression level of CTSD and m CTSB,the activity of CTSD and the number of functional lysosomes were significantly decreased,and there was no significant change in the LAMP1 and the number of lysosomes;the degree of elevation of LC3B-II/Ⅰafter chloroquine treatment was significantly decreased in both the OGD/R（6h）and OGD/R（48h）groups,and the Prnp knockdown resulted in the blockade of ALP in late stage of OGD/R by inhibiting the up-regulation of the number of lysosomes and improvement of their functions.3.Overexpressing PrP^C contributed to the conversion of astrocytes from a pro-inflammatory phenotype to an anti-inflammatory phenotype during OGD/R.Compared with WT astrocytes,the levels of cytokine IL-10 in the supernatants of Prnp^-/-astrocytes were significantly decreased at OGD/R（72h）,and the levels of cytokines TNF and INF-γwere significantly increased at OGD/R（24-48h）.On the contrary,the levels of cytokine IL-10 in the supernatants of Prnp-overexpressing astrocytes were significantly increased at OGD/R（3-72h）,the levels of the levels of cytokines TNF and INF-γwere significantly decreased at OGD/R（3-72h）,and cytokine IL-6 was significantly decreased at OGD/R（6-12h）.Part II The Ca²⁺/PPP3 pathway affects the autophagy lysosomal pathway and astrocyte inflammatory phenotypic transformation by regulating TFEB nuclear translocation at different times of OGD/RMethods1.WT astrocytes were cultured in vitro,and WB technique was used to detect the total expression level of TFEB during OGD/R in WT astrocytes and the detection of TFEB nuclear translocation by IF technique,quantification of the extent of TFEB nuclear translocation by Mandela co-localization system.Further（1）Evaluation of the pathway related to positive regulation upstream of TFEB:intracellular Ca²⁺ concentration was detected by Fluo-4 AM calcium fluorescent probe,PPP3 expression level was detected by WB method,and PPP3 enzyme activity was detected by fluorescence spectrophotometry.（2）Evaluation of the negative upstream regulatory pathway of TFEB:the expression levels of p-m TOR and m TOR were detected by WB method.Finally,the effects of the upstream regulatory mechanism of TFEB on its nuclear translocation were evaluated by applying the PPP3 inhibitor Cyclosporin A,the m TOR activity inhibitor Rapamycin,and the m TOR activity activator MHY1485,respectively.2.The small interfering RNA（si RNA）was applied to down-regulate the expression of TFEB.Next,WB method was used to compare the ALP-related protein levels of the normal group and the OGD/R（48h）group,including the expression levels of LC3B-Ⅱ/Ⅰ,SQSTMI,ubiquitin,LAMP1,CTSD and CTSB;CCK-8 technology to detect cell activity;CBA technology to detect the levels of factors such as TNF,INF-γ,IL-6,and IL-10 in the supernatant of WT astrocytes culture.3.WT astrocytes were cultured in vitro to up-regulate the expression level of TFEB using plasmid transfection with Tfeb technology,and WB methods were used to compare the changes in the levels of ALP-related proteins in the normal group and the OGD/R（6h）group,including the expression levels of LC3B-Ⅱ/Ⅰ,SQSTMI,ubiquitin,LAMP1,CTSD and CTSB.Results1.The Ca²⁺/PPP3 pathway occupies an important role in the regulation of TFEB nuclear translocation during OGD/R.1 Total TFEB expression level and its nuclear translocation increased in WT astrocytes after OGD/R earlier than the improvement of ALP function during 48-72h of re-oxygenation.WB results showed that compared with the normal group,the total TFEB expression level in WT astrocytes showed a decrease in the ultra-early stage（3-6h of re-oxygenation）,a gradual increase in the early stage（12-24h of re- oxygenation）,and a return to the baseline level in the late stage（48-72h of re-oxygenation）during OGD/R.The IF results showed that in WT astrocytes of the normal group,TFEB staining was weak and diffuse,with a small amount of expression in the nucleus;at the ultra-early stage,there was a tendency of perinuclear aggregation of TFEB,while the Mander overlap coefficient（the ratio of intra-nuclear to total TFEB）was significantly decreased;at 12-48h of re-oxygenation,perinuclear aggregation of TFEB was obvious,which was manifested by the increase of the average optical density,and the Mander overlap coefficient was significantly increased;at 72h of re-oxygenation,the perinuclear aggregation of TFEB was gradually diminished,and the Mandela’s overlap coefficient was gradually restored to the baseline level.2 The Ca²⁺/PPP3 pathway occupies an important role in TFEB nuclear translocation during OGD/R.The results showed that,compared with the normal group,the intracellular Ca²⁺level decreased at 6h of re-oxygenation,increased at 12-48h of re-oxygenation,and returned to the baseline level at 72h of re-oxygenation in WT astrocytes during OGD/R treatment.The expression level of PPP3 protein did not change significantly,while PPP3 enzyme activity increased significantly at 12-48h of re-oxygenation.Cyclosporin A was further applied to inhibit PPP3 activity,and the IF results showed that the nuclear translocation of TFEB at 12-48h of re-oxygenation was significantly reduced compared with that of the untreated groups.3 Changes in the m TOR pathway during OGD/R.The results showed that compared with the normal group,the ratio of p-m TOR to m TOR did not change significantly at 3-6h of re-oxygenation but increased significantly at 12-72 of re-oxygenation in WT astrocytes during OGD/R treatment.The theoretical inhibition of TFEB nuclear translocation by m TOR is not consistent with the actual observation of TFEB nuclear translocation at 12-48h of re-oxygenation.2.TFEB was involved in regulating ALP affecting astrocytic activity and cellular inflammatory phenotypic transformation during OGD/R.Compared with solvent control group,WB results showed that the expression levels of LC3B-II/Ⅰand ubiquitin were significantly increased,and the expression levels of LAMP1,CTSD,and m CTSB were significantly decreased in OGD/R（48h）si-Tfeb group.CCK-8 results showed a significant decrease in cell activity in the OGD/R（48h）si-Tfeb group.CBA results showed a decrease in IL-10 levels and a significant increase in TNF and IL-6 levels in the OGD/R（48h）si-Tfeb group.3.Up-regulation of TFEB expression did not improve early ALP blockade during OGD/R treatment.Compared with the solvent control group,the WB results showed no significant changes in the expression levels of LC3BII/Ⅰ,LAMP1,CTSD and m CTSB in the OGD/R（6h）Tfeb group.Part III PrP^C maintains ALP patency during OGD/R and induces astrocyte transformation from a pro-inflammatory to an anti-inflammatory phenotype by regulating TFEB nuclear translocationMethods1.IF technique was used to detect TFEB nuclear translocation during OGD/R in Prnp^-/-and Prnp overexpressing astrocytes.WB technique was used to detect total TFEB expression.2.Prnp overexpressing astrocytes were cultured in vitro,and the si RNA was applied to down-regulate the expression of TFEB.WB methods were used to compare the changes in the levels of ALP-related proteins in the normal,OGD/R（6h）and OGD/R（48h）groups,including the expression levels of LC3B-Ⅱ/Ⅰ,SQSTMI,ubiquitin,LAMP1,CTSD and CTSB;CCK-8 technology was used to detect cell activity;CBA technology was used to detect the levels of factors such as TNF,INF-γ,IL-6,and IL-10 in the supernatant of cell culture.Results1.overexpression of PrP^C prolonged and promoted OGD/R-induced TFEB nuclear translocation.Compared to the normal group,Prnp^-/-astrocytes OGD/R treatment resulted in a significant decrease in total TFEB expression levels from 3-24h of re-oxygenation,and TFEB nuclear translocation was only observed as perinuclear aggregation in 12h images of reoxygenation,as evidenced by an increase in the mean optical density and a significant increase in the Mandela’s overlap coefficient.Prnp overexpressing astrocytes OGD/R treatment resulted in a decrease in total TFEB expression levels at 3-6h of re-oxygenation and a significant increase at12h and 48-72h of re-oxygenation,TFEB nuclear translocation was observed in perinuclear aggregates in both re-oxygenation 3-72h images,as evidenced by an increase in mean optical density and a significant increase in Mandela’s overlap coefficient.Compared to WT astrocytes,TFEB nuclear translocation in Prnp overexpressing astrocytes was significantly increased by 3-72h of re-oxygenation and significantly decreased by 24-48h of re-oxygenation in Prnp^-/-astrocytes.2.Overexpression of PrP^C maintains ALP patency during OGD/R and affects astrocyte inflammatory phenotypic transformation by regulating TFEB nuclear translocation:Compared with solvent control,WB results showed that LC3B-II/Ⅰand ubiquitin expression levels were significantly increased,pro-CTSD and m CTSB expression levels were significantly decreased,and LAMP1 and m CTSD were not significantly altered in the OGD/R（6h）si-Tfeb group,while in the OGD/R（48h）si-Tfeb group,LC3B-II/Ⅰand ubiquitin expression levels were significantly increased,and LAMP1,CTSD and m CTSB expression levels were significantly decreased.CCK-8 results showed a significant decrease in cell activity in both OGD/R（6h）and OGD/R（48h）si-Tfeb groups.CBA results showed decreased levels of IL-10 and significantly increased levels of TNF and IL-6 in both OGD/R（6h）and OGD/R（48h）si-Tfeb groups.Part IV PrP^C promotes TFEB nuclear translocation through the Ca²⁺/PPP3pathwayMethodsWT,Prnp^-/-and Prnp overexpressing astrocytes were isolated and cultured in vitro for OGD/R modeling;intracellular Ca²⁺concentration was detected by Fluo-4AM calcium fluorescent probe.WB and fluorescence spectrophotometry were used to detect the protein content and activity of PPP3 in the cells,respectively;Cyclosporin A was applied to inhibit the activity of PPP3 in the Prnp overexpression group of astrocytes after OGD/R,and the subsequent nuclear translocation of TFEB was observed.ResultsCompared with WT astrocytes,PPP3 activity in the Prnp overexpressing cell group was significantly increased at 3-6h and 24-48h of re-oxygenation,and intracellular Ca²⁺concentration was increased at 3-12h and 48h-72h of re-oxygenation,and decreased at 24h of reoxygenation,while PPP3 activity in the Prnp^-/-cell group was significantly decreased at 12h and 48-72h of re-oxygenation,and intracellular Ca²⁺concentration decreased at 3h and 12-72h of re-oxygenation.Application of Cyclosporin significantly reversed OGD/R（3-72）TFEB nuclear translocation in Prnp overexpressing astrocytes.Conclusions1.In the early stage of OGD/R,the ALP of astrocytes was blocked,and the ALP blockage was relieved in the late stage;overexpression of PrP^C maintained the ALP patency of astrocytes in the different stages of OGD/R by up-regulating the number of lysosomes and improving the lysosomal function,which improved the impaired cellular activity induced by OGD/R,and facilitated the transformation of the pro-inflammatory to the anti-inflammatory phenotypes of astrocytes.2.TFEB is involved in the regulation of ALP patency in late OGD/R and affects the transformation of the inflammatory phenotype of astrocytes,and the Ca²⁺/PPP3pathway plays an important role in the regulation of nuclear translocation of TFEB;however,up-regulation of TFEB expression did not improve the ALP blockage in early OGD/R in astrocytes.3.Overexpression of PrP^C maintains ALP patency and promotes the transformation of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype at different stages of OGD/R through the Ca²⁺/PPP3/TFEB pathway.