UBC Theses and Dissertations
Dystroglycan-dependent modulation of aquaporin-4 distribution : a new target to prevent brain edema Noel, Geoffroy Pierre Jean-Claude
Aquaporin-4 (AQP4) constitutes the principal water channel in the brain and is mainly clustered at the perivascular astrocyte endfeet. This polarized distribution is of major importance because it enhances water fluxes thereby modulating brain swelling in different pathophysiological conditions. Evidence points to a role of the dystroglycan (DG) complex in the localization of AQP4. To investigate in vivo the role of extracellular matrix (ECM) ligand-binding to glycosylated sites on DG in the polarized distribution of AQP4, I used the Largemyd mouse that presents defective O-glycosylation of DG and found a loss of AQP4 at astrocyte endfeet. Using a mixture of ECM molecules present at the perivascular basal lamina, I found that DG clustering is regulated by laminin in astrocyte cultures. Furthermore, I show that laminin induces a reduction in AQP4-mediated water transport. Subsequently, the analysis of cell surface compartmentalization of AQP4 showed that it depends on both cholesterol and DG. Further experiments revealed an interdependent regulation between laminin binding to DG and lipid raft reorganization. I next investigated the signaling events that may be involved in the coclustering of AQP4 and DG in astrocytes. An increase in tyrosine phosphorylation was observed at 3h in laminin-treated astrocytes and this was concomitant to the maximum laminin-induced clustering of lipid rafts and AQP4. I identified the protein-serine kinase C delta (PKCdelta) as one of the main kinases exhibiting an increase in tyrosine phosphorylation upon laminin treatment. The inhibition of PKCdelta showed that it is involved not only in the regulation of the laminin-induced clustering of AQP4 but also in AQP4-mediated water transport in astrocytes. Given the crucial role of AQP4 distribution in brain edema, I finally focused on the identification of drugs modulating the laminin-dependent AQP4 clustering which may prevent brain edema. By screening a chemical library, I identified 6 drugs and found that chloranil prevents AQP4 clustering by activating metalloproteinases that cleave DG. These findings revealed the molecular mechanisms regulating the laminin-induced and DG-dependent clustering of AQP4 at astrocyte endfeet and provide a tool to identify modulators of AQP4 clustering that will be tested in models of brain edema.
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