Cellular Prion Protein PrP~c Affects Microglia Phenotypic Transformation By Regulating Its Autophagy During Glucose Oxygen Deprivation/Reperfusion Injury

Background:Stroke is the second leading cause of death worldwide,with ischemic strokes being the most common.Microglia,as a key cellular effector of CNS response to I/R,can protect neurons by regulating microglia activation status,which is a promising therapeutic concept for CNS diseases.PrPc is a membrane glycoprotein that is centrally expressed in the central nervous system.Autophagy is a lysosome-dependent protein degradation pathway.Recent studies have found that both PrPc and autophagy can exert neuroprotective effects by regulating microglia activation status.However,so far,we are still unclear whether there is a link between the two?This is the scientific question to be addressed in this project.Methods:We selected several newborn mammary mice,including wild type,PrP~c knockout,and PrPc overexpression.Firstly,we isolated,cultured and purified neonatal mouse cerebral cortex microglia and constructed a glyoxylation deprivation/reperfusion injury(OGD/R)model.than,we collected normal group and OGD(2h)/R(24h,48h,72h)microglia and cell culture supernatants,respectively.Secondly,we examined the phenotypic differentiation of microglia and the release of pro-and anti-inflammatory factors in different genotypes and treatment groups by flow cytometry and CBA techniques to observe the association between PrP~c and microglia phenotypic transformation after OGD/R.At the same time,the association between PrPc and microglia autophagy after OGD/R was also observed by immunofluorescence and Western Blotingt techniques to detect autophagic changes in microglia of different genotypes and treatment groups.Finally,we observed microglia performance transformation after adding autophagy inhibitors.In order to study the link between PrP~c,autophagy and microglia performance transformation.Results:We found that after OGD/R treatment,microglia autophagy was activated,microglia phenotype was predominantly M1 phenotype,and autophagy occurred before phenotypic transformation.When we inhibited the activation of microglia autophagy after OGD/R treatment,the microglia phenotype was mainly transformed to M1 phenotype.WB tests showed that after OGD/R treatment,LC3B expression level increased,P62 expression level decreased,LAMP1expression level did not increase,and autophagy was activated,most obviously with OGD(2h)/R(24h).CBA and flow tests showed that M1-type microglia and IL-6 increased significantly after OGD/R treatment,most significantly with OGD(2h)/R(48h),while M2-type microglia and IL-10 were not significantly elevated.When we inhibited microglia autophagy activation after OGD/R treatment with autophagy inhibitor 3-MA or Bafilomycin A1,microglia phenotype was mainly transformed to M1 phenotype.We found that PrP~C promoted microglia autophagy activation and promoted microglia conversion to M2 type after OGD/R treatment.WB tests showed that in the PRNP overexpression group,LC3B expression level increased,P62 expression level decreased,LAMP1 expression level increased,after OGD/R treatment,and autophagy was activated;in PRNP knockdown group,LC3B expression level was decreased,P62 expression level was increased,LAMP1 expression level was decreased,and autophagy was inhibited.CBA and flow cytometry tests showed that after OGD/R treatment,PRNP overexpression group,the proportion of M2 type microglia and IL-10 were predominantly increased;in the PRNP knockdown group,the proportion of M1-type microglia and IL-6 were predominantly elevated.We found that after OGD/R,PrP~C could induce microglia to convert mainly to M2 type by promoting microglia autophagy activation.CBA and flow tests showed that when we inhibited autophagy activation in TGA type microglia after OGD/R treatment with autophagy inhibitor Bafilomycin A1,the conversion of TGA type microglia to M2 type was inhibitedConclusions:We propose that PrP~C induces microglial cell conversion to M2 type by promoting microglial cell autophagy activation during OGD/R injury.