Impact of Glucocorticoid‐Associated Stress‐Like Conditions on Aquaporin‐4 in Cultured Astrocytes and Its Modulation by Adenosine A 2A Receptors

ABSTRACT Astrocytes participate in brain clearance of extracellular proteins and metabolites, through the activity of the water channel aquaporin‐4 (AQP4), which can be deregulated in stress‐related disorders, impairing brain waste clearance. The present study investigates the impact of dexamethasone (Dexa), a synthetic glucocorticoid used as a simplified in vitro stress model, on astrocytic AQP4 and its modulation by adenosine A 2A receptors (A 2A R), which blockade reverses conditions related with maladaptive stress, such as anxiety and depression. The clearance of proteins in primary astrocytic cultures, assessed using 5 kDa FITC‐dextran and 45 kDa TRITC‐dextran uptake, was decreased by a 24 h exposure to 100 nM Dexa. The Dexa exposure decreased α‐syntrophin density, a protein‐targeting AQP4 to astrocytic processes, potentially affecting AQP4 location and, consequently, its activity. Accordingly, Dexa exposure decreased astrocytic water influx (assessed with calcein fluorescence), which paralleled the impairment of dextran clearance. The Dexa‐induced decrease in extracellular protein uptake was prevented by the AQP4 activator TGN‐073 and A 2A R antagonism with SCH58261, showing that the impairment of AQP4‐mediated protein clearance was controlled by A 2A R in this Dexa‐simplified in vitro stress model. Additionally, the effects of Dexa in AQP4 location and activity were prevented by SCH58261, confirming that A 2A R modulate AQP4 function. This conclusion was reinforced by the observed AQP4/A 2A R physical interaction in astrocytes. Overall, the data indicate that in vitro conditions related to stress affect the localisation of astrocytic AQP4 and its role in extracellular protein uptake, which was modulated by A 2A R. These findings unveil a novel therapeutic mechanism to prevent brain extracellular protein accumulation and associated neurological disorders by tinkering with AQP4 and A 2A R. image