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ABSTRACTIt is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation.In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.

The prion protein is infamous for its involvement in a group of neurodegenerative diseases known as Transmissible Spongiform Encephalopathies. In the longstanding quest to decipher the physiological function of its cellular isoform, PrPC, the discovery of its participation to the self‐renewal of hematopoietic and neural stem cells has cast a new spotlight on its potential role in stem cell biology. However, still little is known on the cellular and molecular mechanisms at play. Here, by combining in vitro and in vivo murine models of PrPCdepletion, we establish that PrPCdeficiency severely affects the Notch pathway, which plays a major role in neural stem cell maintenance. We document that the absence of PrPCin a neuroepithelial cell line or in primary neurospheres is associated with drastically reduced expression of Notch ligands and receptors, resulting in decreased levels of Notch target genes. Similar alterations of the Notch pathway are recovered in the neuroepithelium of Prnp−/−embryos during a developmental window encompassing neural tube closure. In addition, in line with Notch defects, our data show that the absence of PrPCresults in altered expression of Nestin and Olig2 as well as N‐cadherin distribution. We further provide evidence that PrPCcontrols the expression of the epidermal growth factor receptor (EGFR) downstream from Notch. Finally, we unveil a negative feedback action of EGFR on both Notch and PrPC. As a whole, our study delineates a molecular scenario through which PrPCtakes part to the self‐renewal of neural stem and progenitor cells. StemCells2017;35:754–765 Model for PrPCaction on Notch and stemness in neural progenitors: PrPCpositively regulates the expression of Notch ligands and that of the Notch receptors and its ablation compromises Notch signaling;proper Notch signaling supports N‐cadherin‐mediated cell‐cell contacts as well as the expression of NSC markers and exerts a positive feedback action on PrPC;downstream from PrPC, Notch signaling additionally upregulates the expression of EGFR, which, when activated, negatively regulates both PrPCand Notch. Thus, PrPC, the Notch pathway and EGFR belong to a gene regulatory network, whose imbalance may shift toward “more stemness” (higher Notch activity induces higher PrPCexpression and vice versa) or “less stemness” (EGFR activation dampens Notch signaling and PrPCexpression).

ABSTRACTMapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrPCstructure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrPCnor for the formation of a stable prion structure.

Although the pivotal implication of the host-encoded Prion protein, PrP, in the neuropathology of transmissible spongiform encephalopathy is known for decades, its biological role remains mostly elusive. Genetic inactivation is one way to assess such issue but, so far, PrP-knockout mice did not help much. However, recent reports involving (1) further studies of these mice during embryogenesis, (2) knockdown experiments in Zebrafish and (3) knockdown of Shadoo, a protein with PrP-like functional domains, in PrP-knockout mice, all suggested a role of the Prion protein family in early embryogenesis. This view is challenged by the recent report that PrP/Shadoo knockout mice are healthy and fertile. Although puzzling, these apparently contradictory data may on the contrary help at deciphering the Prion protein family role through focusing scientific attention outside the central nervous system and by helping the identification of other loci involved in the genetic robustness associated with PrP.

Upon prion infection, abnormal prion protein (PrPSc) self-perpetuate by conformational conversion of α-helix-rich PrPCinto β sheet enriched form, leading to formation and deposition of PrPScaggregates in affected brains. However the process remains poorly understood at the molecular level and the regions of PrP critical for conversion are still debated. Minimal amino acid substitutions can impair prion replication at many places in PrP. Conversely, we recently showed that bona fide prions could be generated after introduction of eight and up to 16 additional amino acids in the H2-H3 inter-helix loop of PrP. Prion replication also accommodated the insertions of an octapeptide at different places in the last turns of H2. This reverse genetic approach reveals an unexpected tolerance of prions to substantial sequence changes in the protease-resistant part which is associated with infectivity. It also demonstrates that conversion does not require the presence of a specific sequence in the middle of the H2-H3 area. We discuss the implications of our findings according to different structural models proposed for PrPScand questioned the postulated existence of an N- or C-terminal prion domain in the protease-resistant region.

A sensitive blood diagnostic assay can identify patients with variant Creutzfeldt-Jakob disease before symptoms appear and during the clinical phase.

ABSTRACTMeasurements of the presence of prions in biological tissues or fluids rely more and more on cell-free assays. Although protein misfolding cyclic amplification (PMCA) has emerged as a valuable, sensitive tool, it is currently hampered by its lack of robustness and rapidity for high-throughput purposes. Here, we made a number of improvements making it possible to amplify the maximum levels of scrapie prions in a single 48-h round and in a microplate format. The amplification rates and the infectious titer of the PMCA-formed prions appeared similar to those derived from the in vivolaboratory bioassays. This enhanced technique also amplified efficiently prions from different species, including those responsible for human variant Creutzfeldt-Jakob disease. This new format should help in developing ultrasensitive, high-throughput prion assays for cognitive, diagnostic, and therapeutic applications.IMPORTANCEThe method developed here allows large-scale, fast, and reliable cell-free amplification of subinfectious levels of prions from different species. The sensitivity and rapidity achieved approach or equal those of other recently developed prion-seeded conversion assays. Our simplified assay may be amenable to high-throughput, automated purposes and serve in a complementary manner with other recently developed assays for urgently needed antemortem diagnostic tests, by using bodily fluids containing small amounts of prion infectivity. Such a combination of assays is of paramount importance to reduce the transfusion risk in the human population and to identify asymptomatic carriers of variant Creutzfeldt-Jakob disease.