Autophagy proteins differentially regulate extracellular vesicles (EVs) formation and secretion in the placenta

Authors

• A Singh
• D Johnson
• J Williams
• J Kuhn
• CM Guardia

Abstract

Placental trophoblast cells release extracellular vesicles (EVs) into maternal circulation, as a mechanism of fetal-maternal communication during pregnancy. EVs contain structural proteins, like CD63, specific lipids, and cytosolic cargo of endosomal origin. In pregnancy disorders like preeclampsia, EV circulation increases, and monitoring their number and composition has the potential to become an effective, non-invasive diagnostic tool. However, placental EV biogenesis remains poorly understood. Recent studies suggest that autophagy may impact EV secretion. However, autophagy regulation of EV formation and cargo packing is one of the critical gaps in this field. Thus, we knocked out (KO) three autophagy genes involved in autophagosome formation (ATG9A, ATG2A, ATG2B) and an essential gene of the LC3-conjugation machinery (ATG7). We isolated and characterized the EVs from the supernatant of WT and KO cells and observed that in the absence of autophagy genes, there was a substantial increase in the secretion of EVs. Treating cells with Lysosomal inhibitor further increased EVs secretion, with ATG2A/B double KO showing a 2-fold enrichment. Omics analysis revealed significant changes in the proteome of EVs secreted from KO cells, with lipidomic analysis showing altered distributions of phospholipids, ceramides, and sphingomyelins in both KO cells and secreted EVs. These results provide the evidence for the critical role of autophagy proteins in MVB packaging and EV secretion.

Scientific Focus Area: Cell Biology

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