Autophagy regulates exosomal release of prions in neuronal cells

Abstract

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. Our work 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. We 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. We 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, our results indicate that autophagy modulation can control lateral transfer of prions by interfering with their exosomal release. We describe a novel role of autophagy in the prion life cycle, an understanding that may provide useful targets for containing prion diseases.

Exosomes: Revisiting their role as “garbage bags”

“…For example, depletion of the essential autophagy protein Atg5 was shown to decrease the amount of exosomes released during basal autophagy . Conversely, autophagy blockage was shown to increase exosomal secretion of PrPsc and α‐synuclein, two proteins involved in neurological disorders . Atg12‐Atg3 has also been shown to control exosome biogenesis through its interaction with Alix .…”

“…Similarly, exosomes released by infected cells were demonstrated to contain the abnormally folded prion GPI‐protein scrapie (PrPsc), and could contribute to intercellular membrane exchange and the spread of prions . PrPsc release through exosomes is increased when autophagy is blocked …”

“…44 Conversely, autophagy blockage was shown to increase exosomal secretion of PrPsc and α-synuclein, two proteins involved in neurological disorders. 52,53 Atg12-Atg3 has also been shown to control exosome biogenesis through its interaction with Alix. 54 Conversely, ESCRT components have been shown to be involved in autophagy.…”

New insight into the role of extracellular vesicles in kidney disease

“…Under conditions that stimulate autophagy, MVBs are directed to the autophagic pathway that consequently inhibits exosome release . In neuronal cells, autophagy stimulation with the mTOR inhibitor rapamycin strongly inhibited exosomal prion release . When uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because they can neutralize lysosomal pH.…”

Exosome release and cargo in Down syndrome

“…Enhanced exosome secretion is thought to be a compensatory mechanism in response to cellular stressors. Recent molecular studies suggest that specific molecular checkpoints, such as ATG5, ATG7, and CD63, interface with the control of exosomal release (Abdulrahman, Abdelaziz, & Schatzl, 2018;Uddin et al, 2018). Tetraspanins, a family of transmembrane proteins (e.g., CD63 and CD81) are among the most abundant surface proteins on exosomes (Booth et al, 2006;Raposo & Stoorvogel, 2013) and are often used to quantify total exosome secretion.…”