Cholesterol deficiency directs autophagy-dependent secretion of extracellular vesicles.

Extracellular vesicle (EV) secretion is an important, though not fully understood, intercellular communication process. Lipid metabolism has been shown to regulate EV activity, though the impact of specific lipid classes is unclear. Through analysis of small EVs (sEVs), we observe aberrant increases in sEV release within genetic models of cholesterol biosynthesis disorders, where cellular cholesterol is diminished. Inhibition of cholesterol synthesis at multiple synthetic steps mimics genetic models in terms of cholesterol reduction and sEVs secreted. Further analyses of sEVs from cholesterol-depleted cells revealed structural deficits and altered surface marker expression, though these sEVs were also more easily internalized by recipient cells. Transmission electron microscopy of cells with impaired cholesterol biosynthesis demonstrated multivesicular and multilamellar structures potentially associated with autophagic defects. We further found autophagic vesicles being redirected toward late endosomes at the expense of autophagolysosomes. Through CRISPR-mediated inhibition of autophagosome formation, we mechanistically determined that release of sEVs after cholesterol depletion is autophagy dependent. We conclude that cholesterol imbalance initiates autophagosome-dependent secretion of sEVs, which may have pathological relevance in diseases of cholesterol disequilibrium.