Studies Of The Impact Of GJC2 Mutations In Patients With PMLD On The Subcellular Localization Of Cx47 Protein

Objective:The purpose of this study was to reveal the effects of five kinds of GJC2 mutations found in Chinese PMLD patients on the change of subcellular localization of Cx47 protein in Oligodendrocyte.In order to confirm that,mutation of GJC2 will leading Cx47 accumulation in the endoplasmic reticulum,can not coupling with Cx43 normally,which can damage the function of oligodendrocytes and lead to the death of oligodendrocytes,this may be a pathogenic mechanism of PMLD.At the same time,it provides the theoretical basis and experimental basis for the future treatment of PMLD.Methods:On the basis of p EGFP-N1-GJC2 wild type plasmid,5 new mutations of GJC2 in PMLD patients were constructed by site directed mutagenesis: GJC2 c.201C>G(p.C67W),c.216 del Gins AA(p.P73 fs X106),c.217C>T(p.P73S),c.735C>A(p.C245X),c.973G>C(p.A325P),transfect the human oligodendrocyte MO3.13 by Lipofectamine2000,establish wild and mutant model of GJC2 gene,and using Er-tracker to mark the endoplasmic reticulum with,using DAPI mark nucleus with blue,in order to observe the subcellular localization of wild-type Cx47 protein and mutant Cx47 protein by confocal microscopy.Results:5 kinds of mutant plasmid and a wild type plasmid were constructed successful;Observed by confocal microscopy,the wild-type Cx47 protein mainly distributed in the cell membrane and its expression rate is about 94.8%;while the mutant Cx47 protein is mainly stored in the endoplasmic reticulum,the rates of storage in ER are different in different types of GJC2 mutation,C67 W,P73fs X106,P73 S,C245X,A325 P storage rates were 91.4%,81.4%,87.1%,88.3%,89.7%.Conclusion:Cx47 that encoded by five different mutations of GJC2,cannot folding in the endoplasmic reticulum correctly and transporting normally,which change theirs subcellular localization,and accumulation in the endoplasmic reticulum with different degree,cannot expression in the cell membrane normally,this disturbed the Cx47/Cx43 coupling and destroyed the function of oligodendrocyte,which suppressed the formation of normal myelin in central nervous system;A large number of abnormal Cx47 gathered in the endoplasmic reticulum will cause endoplasmic reticulum,trigger unfolded protein response and apoptosis,which may cause PMLD.This may be another Pathogenic mechanism of PMLD,while this theory need to be further proved by detect the expression level of molecular markers on the stress pathway.