Functional, structural and metabolic consequences of ionic dysbalance in stroke

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Ischemic stroke is a leading cause of death and disability worldwide. There is a pressing need to better understand and therapeutically leverage the molecular and cellular changes contributing to stroke pathogenesis. Spreading depolarization (SD) waves represent a particularly relevant disease mechanism during acute stroke. These waves, which are triggered in the hypoxic area by an extracellular overload of glutamate and potassium, can propagate through the ischemic area and into healthy tissue. We and others have shown that SDs are accompanied by strong elevations of the intracellular calcium concentration in astrocytes and neurons, which can increase cellular damage by triggering glutamate release. Based on our data from the first funding period, we will investigate several pathways relevant for calcium and sodium dysbalance and glutamate release during ischemic SDs. First, we will investigate the role of the glutamate transporter GLT1 in the post-acute phase after stroke, based on our preliminary data that astroglial GLT1 is downregulated after transient focal ischemia. We will determine the consequences of this downregulation for calcium and sodium signaling as well as extracellular glutamate, using in vivo two-photon microscopy of fluorescent reporters, and whether pharmacological upregulation of GLT1 with ceftriaxone can counteract these effects. Second, we will investigate the role of astroglial cell swelling mediated by volume- regulated anion channels (VRAC) in glutamate release during ischemic SDs, using mice in which the VRAC subunit SWELL1 has been specifically deleted in astrocytes. Third, we will further investigate preliminary data that the frequency of calcium microdomains in astrocytes is increased after ischemic SDs. Specifically, we will determine if these signals co-localize with mitochondria, and what the roles of mitochondrial sodium-calcium exchanger are in these calcium changes. Moreover, we will also investigate the consequences of these changes for glutamate release and mitochondrial morphology and energy metabolism. Finally, we will build on previous work that transient receptor vanilloid 4 (TRPV4) channels contribute to calcium influx into astrocytes and neurons and subsequent extracellular glutamate accumulation during ischemic SDs, to investigate the role of TRPV4 in sodium changes during SDs. In all of these projects, we will also determine SD threshold, infarct volume and motor outcome. Together, our experiments will identify novel pathways relevant for calcium and sodium overload and glutamate release during SDs after stroke, and may lead to the development of new translational treatment strategies to attenuate these deleterious events.