Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles

Abstract

Background information. TSEs (transmissible spongiform encephalopathies) are neurodegenerative disorders affecting humans and animals. PrPSc, a conformationally altered isoform of the normal prion protein (PrPC), is thought to be the pathogenic agent. However, the biochemical composition of the prion agent is still matter of debate. The potential transmission risk of the prion agent through biological fluids has been shown, but the development of competitive diagnostic tests and treatment for TSEs requires a more comprehensive knowledge of the agent and the cellular mechanisms by which it is disseminated. With this aim, we initiated characterization of the prion agent and the pathways by which it can be propagated using the cellular model system neuroblastoma (N2a). Results. The present study shows that N2a cells infected with scrapie release the prion agent into the cell culture medium in association with exosome‐like structures and viral particles of endogenous origin. We found that both prion proteins and scrapie infectivity are mainly associated with exosome‐like structures that contain viral envelope glycoprotein and nucleic acids, such as RNAs. Conclusions. The dissemination of prions in N2a cell culture is mediated through the exosomal pathway.

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

“…In agreement with this result, mouse 4 showed no clinical signs of TSE and tested negative for PrP TSE in the brain by Western blotting. The calculated LD 50 value for the Mo-vCJD brain homogenate, used for inoculation, was estimated to be 10 Ϫ6. 3 .…”

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins

“…The exosome biogenesis pathway can be "hijacked" by pathogens like the human immunodefi ciency virus (HIV), by proteins like prions involved in Creutzfeld-Jacob disease ( 10 ), and by the amyloid precursor protein (APP) of Alzheimer's disease ( 11,12 ).…”

“…Culture volume was doubled every day in the spinner bottles to maintain a cell density of around 0.25 × 10 6 cells/ml for good cell viability until about 10 9 cells were produced overall. The cells were spun down, washed with DMEM medium, and concentrated to 10 8 cells in 10 ml of DMEM without FCS to avoid contamination by any microvesicles that might be present in the fetal calf serum. Exosomes were recovered following 20 min cell stimulation by ionomycin (1 µM fi nal) and purifi ed by differential centrifugations as reported previously ( 16 ).…”

Protein Targeting to Exosomes/Microvesicles by Plasma Membrane Anchors

“…immune signaling (2), development (12,13) and in human disease (e.g. cancer (14 -16), amyloidopathies (17)(18)(19), and viral infections (4, 7, 20 -22)). …”