The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles
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The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrPC). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20–50, 100–200, and 300–400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrPC. STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrPC-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1–PrPC signaling.
KeywordsExosomes Chaperones Prion protein STI1 Extracellular vesicles
We are very grateful to Dr. Wes Sundquist for the donation of VPS4A plasmids. We thank Leica Microsystems and Ms. Lianne Dale for help in experiments done in a demo Leica GSD unit. This investigation was supported by grants from FAPESP to VRM (2009/14027-2) and GNMH (2012/04370-4), PrioNet-Canada, Canadian Institutes of Health Research (CIHR), Canadian Foundation for Innovation and Ontario Research Fund to MAMP, CNPq and FAPERJ to RL, FAPESP fellowships to CA, MVSD, MR and BS, and a postdoctoral CNPq fellowship to IPC are gratefully acknowledged.
Conflict of interest
The authors declare that there are no conflicts of interest.
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