Aquaporin-4 regulation by NCX1 in post-ischemic brain swelling

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Brain edema and brain swelling are major complications of ischemic stroke. Two molecular pathways, aquaporin- 4 (AQP4), and SUR1-TRPM4, have been linked to edema. Water transport via AQP4 is governed principally by the rate-limiting number of AQP4 channels present in the plasmalemmal membrane, and by the transmembrane osmotic gradient. It was recently reported that in cultured cells, surface localization of AQP4 is regulated by Ca2+/calmodulin (CAM), with CAM binding causing a conformational change in the carboxyl terminus of AQP4 that drives cell-surface localization. Although this seminal discovery hints at a potentially important way to manipulate AQP4 to influence brain swelling, a major gap in knowledge is the mechanism of dynamic regulation of surface localization of AQP4 in ischemia in vivo. We hypothesize that in post-ischemic perivascular astrocyte endfeet, Na+ influx via SUR1-TRPM4 shifts Na+/Ca2+ exchanger 1 (NCX1) into “Ca2+ entry mode” (CaEnt-NCX1), which extrudes Na+ and causes Ca2+ influx, and that CaEnt-NCX1-mediated Ca2+ influx activates CAM to drive surface localization of AQP4 in endfeet that promotes endfoot swelling, blood-brain barrier (BBB) dysfunction and brain swelling. Central to our theory are new findings and new preliminary data: In mouse brain tissues after middle cerebral artery occlusion (MCAo), both SUR1-TRPM4 (a “Na+ channel” activated by ATP depletion) and NCX1 are upregulated in perivascular astrocyte endfeet. In post-MCAo brain slices, preliminary data with Ca2+ imaging of perivascular endfeet show that activation of SUR1-TRPM4 causes an increase in Ca2+ in endfeet that is blocked by inhibitors of SUR1 and by inhibitors of CaEnt-NCX1. Also central to our theory is our recent development of a method to measure the surface localization of AQP4 in brain slices, an important development that provides a novel tool for studying post-ischemic brain swelling. In post-MCAo brain slices, preliminary data indicate that surface localization of AQP4 is increased by ischemia, and the increase is blocked by pretreatment with CaEnt-NCX1 inhibition. In this project, we will confirm and expand on these preliminary data by pursuing the following specific aims: In Specific Aim 1, making use of a novel mouse model we developed (Slc8a1fl/fl;+GFAP-cre/ERT2 mouse), we will characterize the role of astrocyte NCX1 in post-ischemic BBB dysfunction and brain swelling. In Specific Aim 2, making use of our unique method to quantify surface localization of AQP4 in brain slices, we will characterize the role of astrocyte NCX1 in post-MCAo AQP4 surface localization. In Specific Aim 3, using PC::G5-tdT mice with transgene expression (GCaMP5G and tdTomato) regulated by pGFAP-cre/ERT2, we will characterize the role of astrocyte NCX1 in post-MCAo perivascular endfoot swelling and Ca2+ signaling. This project, which is focused on astrocyte endfoot NCX1 and AQP4, advances a novel theory that unifies the SUR1-TRPM4 and AQP4 edema pathways by incorporating an intermediate element – NCX1 – in a novel molecular mechanism of brain edema. We anticipate that the science emerging from this project will be highly impactful, and will have great translational potential.