Glucocorticoids regulate small extracellular vesicle release via activation of nSMase2.

Glucocorticoid (GC) steroid hormones mediate the stress response, which requires dynamic communication between brain regions to enable an organism to react to and overcome perceived stressors. While many effects of GCs on the brain have been well-studied, little is known about whether and how GCs regulate brain cell-cell communication through the release of small extracellular vesicles (sEVs), nanoparticles that carry bioactive molecules between cells. Here, we use total internal reflection fluorescence (TIRF) microscopy and the pH-sensitive sEV marker mCh-CD63-pHluorin to visualize sEV release in a neuronal cell line. We find that GCs stimulate sEV secretion and that this process requires the GTPase Rab27a and the enzyme neutral sphingomyelinase 2 (nSMase2), which catalyzes ceramide production and drives sEV formation. We further show that GCs promote sEV release by activating nSMase2 downstream of mitochondrial reactive oxygen species production and opening of the mitochondrial permeability transition pore (mPTP). These findings link GC impacts on mitochondria, specifically via mPTP opening, to nSMase2 activation and enhanced sEV release by neuronal cells.