Autophagy Controls Exosomal Release of Prions and Lateral Prion Infection
Bibliographic record
Abstract
Prion diseases are fatal transmissible neurodegenerative disorders that affect both human and animals. They are characterized by conformational conversion of the normal cellular prion protein (PrP c ) into a disease associated pathological isoform (PrP Sc ). It has been shown that PrP Sc can infect recipient cells when it is packaged into endosomal derived nanoparticles called exosomes. Exosomes are present in all biological fluids such as blood, urine, milk, and saliva. Exosome secretion is under the influence of the autophagic system, a basic cellular degradation machinery. Autophagy stimulation can inhibit exosomal release, whereas inhibition of autophagy seems to enhance exosomal release. In this paradigm, our work investigates the effect of autophagy modulation on exosomal release of prions and how this interplay impacts prion infection. We successfully isolated and characterized exosomes from a prion‐infected neuronal cell line (ScN2a) using differential ultracentrifugation technique. The isolated exosomes contained PrP Sc as shown by immunoblot. Treatment of ScN2a cells with the neutral sphingomyelinase inhibitor GW4869 resulted in a block of the exosomal release and reduced PrP Sc levels in exosomes. When we stimulated autophagy in ScN2a cells using rapamycin, a well‐known autophagy stimulator that inhibits the mammalian target of Rapamycin (mTOR) pathway, we observed a strong inhibition of exosomal release and decreased levels of PrP Sc in exosomes compared to vehicle treated cells. To further assess the impact of autophagy on exosomal prion release, we knocked‐out Atg5 (a main player in the autophagic machinery) in N2a cells using the CRISPR‐Cas9 system. CRISPR‐Cas9 is the clustered, regularly interspaced, short palindromic repeats (CRISPR)‐associated protein (Cas) system which involves RNA–guided site specific DNA double strand cleavages. Non‐homologous end joining is used by cells for repair of double strand breaks and may result in functional gene knock‐out by introduction of insertions and deletions. Using this technology we generated various N2a cell clones with functional knockouts in different exons of the Atg5 gene, as verified by sequencing and immunoblot analysis. Upon stably infecting these Atg5 knock‐out cells with prions, we found a highly increased release of exosomes and exosome‐associated PrP Sc compared to wild type cells. Taken together, our data shows that autophagy modulation can control lateral prion infection by interfering with exosomal release of PrP Sc . Our present work correlates these findings to outcomes of prion infection in recipient cells and in mouse animal models. Overall, our study describes a novel interplay of basic cellular machinery which affects the live cycle of prions. This new understanding will result in novel targets for therapy against prion diseases and protein misfolding disorders. Support or Funding Information The Natural Sciences and Engineering Research Council of Canada (NSERC) Alberta Prion Research Institute (APRI) National Institute of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) Alberta Innovates Health Solutions (AIHS)
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".