SGLT2 - Novel Molecular Mechanism of AQP4 Regulation - SUMMARY In ischemic stroke, outcomes are determined in part by acute brain swelling, which is an independent predictor of poor outcome, and which contributes significantly to mortality, especially with large strokes. Due to its role in water transport, aquaporin-4 (AQP4) is crucial in brain edema but mechanisms that regulate AQP4 post-ischemia are poorly understood. The Na+/glucose co-transporter 2 (SGLT2) transports glucose accompanied by Na+ into the cell. SGLT2 inhibitors of the gliflozin class are beneficial in models of stroke, but mechanisms are not defined, especially mechanisms related to Na+ influx (as opposed to glucose). In this project, we will study the role of SGLT2 in two aspects of AQP4 regulation that are critical for AQP4 function: (1) trafficking of AQP4 to the cell membrane, which promotes H2O influx that drives cell swelling, and which is known to be mediated in part by Ca2+/calmodulin (CaM); (2) “AQP4 dysregulation”, the disease-associated process whereby AQP4 is distributed away from endfeet to other parts of the cell membrane, and which has been postulated to be due to Ca2+- activated proteases acting on members of the dystrophin-associated protein (DAP) complex that anchors AQP4 to endfeet. We recently discovered that Na+ influx plays an unexpected role in post-ischemic astrocytes – Na+ influx causes the bidirectional co-transporter, Na+/Ca2+ exchanger 1 (NCX1) to reverse from its usual “Ca2+ extrusion” mode to a “Ca2+ entry” mode, resulting Ca2+ influx. We theorize that Na+ influx due to SGLT2 activates NCX1 in reverse Ca2+-entry mode, and that the rise in Ca2+ promotes both trafficking of AQP4 to the cell-surface and activates Ca2+-dependent proteases. Central to our theory are new preliminary data: (1) In post-ischemic brain slices, live cell Na+ imaging showed that SGLT2 activation raised astrocyte Na+ by about 6 mM, and Ca2+ imaging of astrocyte endfeet showed that SGLT2-induced Ca2+ influx was blocked equally by canagliflozin and the NCX1 inhibitor SEA0400. (2) Following middle cerebral artery occlusion / reperfusion (MCAo/R), inhibiting SGLT2 reduced AQP4 trafficking to the membrane and AQP4 dysregulation. (3) Following MCAo/R, inhibiting SGLT2 reduced the death of neurons, implicating SGLT2 in Na+-linked neuronal death that is mechanistically independent of glutamate excitotoxicity. This project has 3 Specific Aims (SA). In SA1, using live cell imaging of MCAo/R brain slices, we will characterize the roles of and interactions between SGLT2 and NCX1 in Na+ and Ca2+ influx, in AQP4 trafficking and swelling of astrocyte endfeet. In SA2, using mice of various genotypes including astrocyte-RiboTag, we will use qPCR, immunohistochemistry and immunoblot of isolated microvessels to study the role of SGLT2/NCX1 in AQP4 dysregulation. In SA3, using mature mice of both sexes and MCAo/R, we will study the effects of astrocyte-specific and neuron-specific deletion of SGLT2 on multiple stroke outcomes. A key goal of this project is to evaluate the gliflizin class of drugs for treating cerebral edema and stroke. These drugs are FDA approved, have proven to be safe with minimal hypoglycemia, and could be repurposed rapidly for treating patients with stroke.
