Glucocorticoids regulate small extracellular vesicle (sEV) release via activation of nSMase2

Chronic stress, marked by prolonged elevation of glucocorticoid (GC) stress hormones, is a major risk factor for Alzheimer's disease (AD) and accelerates AD pathology in mouse models. A key mechanism contributing to AD progression is the release of small extracellular vesicles (sEVs) carrying pathogenic proteins (e.g., tau, amyloid-beta) between brain regions, but the role of GCs in sEV biogenesis and release is unknown. Using total internal reflection fluorescence (TIRF) microscopy and the pH-sensitive marker mCh-CD63-pHluorin to visualize sEV release, we show that GCs stimulate sEV secretion in a neuronal cell line. This process requires the GTPase Rab27a and the enzyme neutral sphingomyelinase 2 (nSMase2), which catalyzes ceramide production and drives sEV formation. We further demonstrate 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-induced mitochondrial damage, specifically mPTP opening, to nSMase2 activation and enhanced sEV release by neuronal cells.