Recent studies have proposed that nucleic acids act as potential cofactors for protein aggregation and prionogenesis. By means of sedimentation, transmission electron microscopy, circular dichroism, static and dynamic light scattering, we have studied how RNA can influence the aggregation of the murine recombinant prion protein (rPrP). We find that RNA, independent of its sequence, source and size, modulates rPrP aggregation in a bimodal fashion, affecting both the extent and the rate of rPrP aggregation in a concentration dependent manner. Analogous to RNA-induced liquid-liquid phase transitions observed for other proteins implicated in neurodegenerative diseases, high protein to RNA ratios stimulate rPrP aggregation, while low ratios suppress it. However, the latter scenario also promotes formation of soluble oligomeric aggregates capable of seeding de novo rPrP aggregation. Furthermore, RNA co-aggregates with rPrP and thereby gains partial protection from RNase digestion. Our results also indicate that rPrP interacts with the RNAs with its N-terminus. In summary, this study elucidates the proposed adjuvant role of RNA in prion protein aggregation and propagation, and thus advocates an auxiliary role of the nucleic acids in protein aggregation in general.

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Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, arise from the structural conversion of the monomeric, cellular prion protein (PrP C ) into its multimeric scrapie form (PrP Sc ). These pathologies comprise a group of intractable, rapidly evolving neurodegenerative diseases. Currently, a definitive diagnosis of TSE relies on the detection of PrP Sc and/or the identification of pathognomonic histological features in brain tissue samples, which are usually obtained postmortem or, in rare cases, by brain biopsy (antemortem). Over the past two decades, several paraclinical tests for antemortem diagnosis have been developed to preclude the need for brain samples. Some of these alternative methods have been validated and can provide a probable diagnosis when combined with clinical evaluation. Paraclinical tests include in vitro cell-free conversion techniques, such as the real-time quakinginduced conversion (RT-QuIC), as well as immunoassays, electroencephalography (EEG), and brain bioimaging methods, such as magnetic resonance imaging (MRI), whose importance has increased over the years. PrP Sc is the main biomarker in TSEs, and the RT-QuIC assay stands out for its ability to detect PrP Sc in cerebrospinal fluid (CSF), olfactory mucosa, and dermatome skin samples with high sensitivity and specificity. Other biochemical biomarkers are the proteins 14-3-3, tau, neuronspecific enolase (NSE), astroglial protein S100B, α-synuclein, and neurofilament light chain protein (NFL), but they are not specific for TSEs. This paper reviews the techniques employed for definite diagnosis, as well as the clinical and paraclinical methods for possible and probable diagnosis, both those in use currently and those no longer employed. We also discuss current criteria, challenges, and perspectives for TSE diagnosis. An early and accurate diagnosis may allow earlier implementation of strategies to delay or stop disease progression.

The search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrP) conversion into protease-resistant forms (protease-resistant PrP [PrP] or the scrapie form of PrP [PrP]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identified to be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrP mRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP) as the substrate and PrP seeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrP globular domain. J8 also reduced the fibrillation of mouse rPrP seeded with -produced fibrils. Furthermore, most of the compounds decreased the amount of PrP on the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrP content on the cell surface.

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