Prions are protein-based infectious agents that autocatalytically convert the cellular prion protein PrPC to its pathological isoform PrPSc Subsequent aggregation and accumulation of PrPSc in nervous tissues causes several invariably fatal neurodegenerative diseases in humans and animals. Prions can infect recipient cells when packaged into endosome-derived nanoparticles called exosomes, which are present in biological fluids such as blood, urine, and saliva. Autophagy is a basic cellular degradation and recycling machinery that also affects exosomal processing, but whether autophagy controls release of prions in exosomes is unclear.
Researchers at the University of Calgary investigated the effect of autophagy modulation on exosomal release of prions and how this interplay affects cellular prion infection. Exosomes isolated from cultured murine central neuronal cells (CAD5) and peripheral neuronal cells (N2a) contained prions as shown by immunoblotting for PrPSc, prion-conversion activity, and cell culture infection. They observed that autophagy stimulation with the mTOR inhibitor rapamycin strongly inhibited exosomal prion release. In contrast, inhibition of autophagy by wortmannin or CRISPR/Cas9-mediated knockout of the autophagy protein Atg5 (autophagy-related 5) greatly increased the release of exosomes and exosome-associated prions. The researchers also show that a difference in exosomal prion release between CAD5 and N2a cells is related to differences at the level of basal autophagy. Taken together, these results indicate that autophagy modulation can control lateral transfer of prions by interfering with their exosomal release. The researchers describe a novel role of autophagy in the prion life cycle, an understanding that may provide useful targets for containing prion diseases.
Characterization of exosomes isolated from CAD5/ScCAD5 neuronal cells
A, representative TEM of exosomes isolated from CAD5 culture medium reveals a homogenous population of vesicles of 100 nm in diameter characteristic for exosomes (some denoted by black arrows). Scale bar, 100 nm. B, immunoblot of ScCAD5 cell lysate and exosome preparations probed for total PrP (−PK) and PrPSc (+PK) (anti-PrP mAb 4H11). Actin was used as loading control. Flotillin-1 (Flot-1) was used as an exosome marker. C, RT-QuIC of CAD5 exosome, ScCAD5 exosome, ScCAD5 cell lysate, 10% brain homogenate from terminally prion-sick mice (22L) or left unseeded (negative control). The average increase of thioflavin-T fluorescence of replicate wells is plotted as a function of time. The y axis represents RFU, and the x axis represents time (h). D, immunoblot analysis of ScCAD5 cell lysate and exosomes isolated from ScCAD5 culture medium. Exosome preparation is positive for exosome markers Alix, HSC70, Tsg-101, flotillin-1, CD63, and CD9 and negative for mitochondrial marker Bcl2, Golgi marker GM130, and nuclear marker nucleoporin p62. Actin was used loading control. E, ScCAD5 exosome pellet loaded on the top of a continuous sucrose gradient and ultracentrifuged. Fractions were analyzed by Western blotting and probed for HSC70, flotillin-1, and mAb 4H11 to detect PrP/PrPSc. Lanes 9 and 10 represent cell lysate before and after PK digestion, respectively.