Intramembrane proteolysis of ephrin-B2 by γ-secretase regulates podosome dynamics and migration of microglia

Abstract

The Eph-ephrin system plays pivotal roles during development and adulthood in cell-cell interaction, cell adhesion and migration. It has previously been shown that ephrin-B1 and 2 can be cleaved intramembranously by the γ-secretase. γ-Secretase mediated processing of ephrin-B has furthermore been linked to activation of Src, a kinase crucial for focal adhesion and podosome phosphorylation. Due to the finding that many familial Alzheimer’s disease (FAD) mutations are located in the catalytic subunit of the γ-secretase, i.e. presenilin, the γ-secretase has in the past decades mainly been subject of investigation in early Alzheimer’s disease (EOAD) studies. Additionally, the positive correlation between occurrence of late onset Alzheimer’s disease (LOAD) and mutations in genes mainly expressed by microglia has drawn attention to these cells.<br /> In this study, the role of γ-secretase and ephrin signaling on downstream effectors, podosomal turnover and the migration of embryonic stem cell derived microglia (ESdM) was tested. We demonstrate extracellular shedding and subsequent intramembranous ephin-B2 cleavage by the γ-secretase upon EphB1 receptor binding in a microglial cell model. It was furthermore found that proteolytic release of the ephrin-B2 intracellular domain (ICD) stimulates Src and FAK activity.<br /> ESdM from presenilin double knock out (PSdKO) and wild type (WT) mice were transduced with either WT presenilin 1 (PS1 WT) or with non-functional presenilin containing an inactivating mutation (PS1 DN). A rescue cell type of the PSdKO PS1 DN cells was generated, which additionally expressed the ephrin-B2 ICD. Expression of non-functional presenilin 1 could not rescue phosphorylation of Src and FAK. Cell motility and podosomal morphology were also changed in these cells. More specifically, this genotype was associated with decreased motility and an enlargement of podosomal surface. Also, the present results indicate impaired cleavage of podosomal proteins like FAK, talin and vinculin in cells with inactive γ-secretase.<br /> Interestingly, ephrin-B2 ICD expression could fully rescue this phenotype, indicating that ephrin-B2 ICD mediated activation of Src and FAK modulates podosomal dynamics in microglial cells. Together, these results identify γ-secretase as well as ephrin-B2 as important regulators of microglial migration.