Diagnosis of transmissible spongiform encephalopathies (TSEs), or prion diseases, is based on the detection of proteinase K (PK)-resistant PrP^Sc in post-mortem tissues as indication of infection and disease. Since PrP^Sc detection is not considered a reliable method for in vivo diagnosis in most TSEs, it is of crucial importance to identify an alternative source of biomarkers to provide useful alternatives for current diagnostic methodology. Ovine scrapie is the prototype of TSEs and has been known for a long time. Using this natural model of TSE, we investigated the presence of PrP^Sc in exosomes derived from plasma and cerebrospinal fluid (CSF) by protein misfolding cyclic amplification (PMCA) and the levels of candidate microRNAs (miRNAs) by quantitative PCR (qPCR). Significant scrapie-associated increase was found for miR-21-5p in plasma-derived but not in CSF-derived exosomes. However, miR-342-3p, miR-146a-5p, miR-128-3p and miR-21-5p displayed higher levels in total CSF from scrapie-infected sheep. The analysis of overexpressed miRNAs in this biofluid, together with plasma exosomal miR-21-5p, could help in scrapie diagnosis once the presence of the disease is suspected. In addition, we found the presence of PrP^Sc in most CSF-derived exosomes from clinically affected sheep, which may facilitate in vivo diagnosis of prion diseases, at least during the clinical stage.

Prion diseases are transmissible spongiform encephalopathies (TSEs) caused by a conformational conversion of the native cellular prion protein (PrP^C) to an abnormal, infectious isoform called PrP^Sc. Amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s, and Huntington’s diseases are also known as prion-like diseases because they share common features with prion diseases, including protein misfolding and aggregation, as well as the spread of these misfolded proteins into different brain regions. Increasing evidence proposes the involvement of epigenetic mechanisms, namely DNA methylation, post-translational modifications of histones, and microRNA-mediated post-transcriptional gene regulation in the pathogenesis of prion-like diseases. Little is known about the role of epigenetic modifications in prion diseases, but recent findings also point to a potential regulatory role of epigenetic mechanisms in the pathology of these diseases. This review highlights recent findings on epigenetic modifications in TSEs and prion-like diseases and discusses the potential role of such mechanisms in disease pathology and their use as potential biomarkers.

Scrapie is a prion disease affecting sheep and goats and it is considered a prototype of transmissible spongiform encephalopathies (TSEs). Mesenchymal stem cells (MSCs) have been proposed as candidates for developing in vitro models of prion diseases. Murine MSCs are able to propagate prions after previous mouse-adaptation of prion strains and, although ovine MSCs express the cellular prion protein (PrP^C), their susceptibility to prion infection has never been investigated. Here, we analyze the potential of ovine bone marrow-derived MSCs (oBM-MSCs), in growth and neurogenic conditions, to be infected by natural scrapie and propagate prion particles (PrP^Sc) in vitro, as well as the effect of this infection on cell viability and proliferation. Cultures were kept for 48–72 h in contact with homogenates of central nervous system (CNS) samples from scrapie or control sheep. In growth conditions, oBM-MSCs initially maintained detectable levels of PrP^Sc post-inoculation, as determined by Western blotting and ELISA. However, the PrP^Sc signal weakened and was lost over time. oBM-MSCs infected with scrapie displayed lower cell doubling and higher doubling times than those infected with control inocula. On the other hand, in neurogenic conditions, oBM-MSCs not only maintained detectable levels of PrP^Sc post-inoculation, as determined by ELISA, but this PrP^Sc signal also increased progressively over time. Finally, inoculation with CNS extracts seems to induce the proliferation of oBM-MSCs in both growth and neurogenic conditions. Our results suggest that oBM-MSCs respond to prion infection by decreasing their proliferation capacity and thus might not be permissive to prion replication, whereas ovine MSC-derived neuron-like cells seem to maintain and replicate PrP^Sc.

In neurodegenerative diseases, including prion diseases, cellular in vitro models appear as fundamental tools for the study of pathogenic mechanisms and potential therapeutic compounds. Two-dimensional (2D) monolayer cell culture systems are the most used cell-based assays, but these platforms are not able to reproduce the microenvironment of in vivo cells. This limitation can be surpassed using three-dimensional (3D) culture systems such as spheroids that more effectively mimic in vivo cell interactions. Herein, we evaluated the effect of scrapie prion infection in monolayer-cultured ovine bone marrow-derived mesenchymal stem cells (oBM-MSCs) and oBM-MSC-derived spheroids in growth and neurogenic conditions, analyzing their cell viability and their ability to maintain prion infection. An MTT assay was performed in oBM-MSCs and spheroids subjected to three conditions: inoculated with brain homogenate from scrapie-infected sheep, inoculated with brain homogenate from healthy sheep, and non-inoculated controls. The 3D conditions improved the cell viability in most cases, although in scrapie-infected spheroids in growth conditions, a decrease in cell viability was observed. The levels of pathological prion protein (PrP^Sc) in scrapie-infected oBM-MSCs and spheroids were measured by ELISA. In neurogenic conditions, monolayer cells and spheroids maintained the levels of PrP^Sc over time. In growth conditions, however, oBM-MSCs showed decreasing levels of PrP^Sc throughout time, whereas spheroids were able to maintain stable PrP^Sc levels. The presence of PrP^Sc in spheroids was also confirmed by immunocytochemistry. Altogether, these results show that a 3D culture microenvironment improves the permissiveness of oBM-MSCs to scrapie infection in growth conditions and maintains the infection ability in neurogenic conditions, making this model of potential use for prion studies.

Epilepsy is one of the most prevalent complex neurological diseases in both the canine and human species, with the idiopathic form as its most common diagnosis. MicroRNAs (miRNAs) are small, noncoding RNA molecules that play a role in gene regulation processes and appear to be a promising biological target for convulsion control. These molecules have been reported as constituents of the internal content of exosomes, which are small extracellular vesicles released by cells. In this study, exosome samples were isolated from the plasma of 23 dogs, including 9 dogs with epilepsy responsive to treatment, 6 dogs with drug-resistant epilepsy, and 8 control dogs. Plasma exosomes were then characterized by electron transmission microscopy, nanoparticle tracking analysis, and dot blotting. Afterwards, the microRNA-enriched RNA content of exosomes was isolated, and miRNA quantification was performed by quantitative real-time PCR. Seven circulating miRNAs that have been previously described in the literature as potential diagnostic or prognostic biomarkers for epilepsy were evaluated. We observed significant differences in miR-16 (p < 0.001), miR-93-5p (p < 0.001), miR-142 (p < 0.001), miR-574 (p < 0.01), and miR-27 (p < 0.05) levels in dogs with refractory epilepsy compared to the control group. In drug-sensitive epileptic dogs, miR-142 (p < 0.01) showed significant differences compared to healthy dogs. Moreover, distinct levels of miR-16 (p < 0.05), miR-93-5p (p < 0.01), miR-132 (p < 0.05), and miR-574 (p < 0.05) were also found between drug-sensitive and drug-resistant epileptic dogs. Our results present plasma-circulating exosomes as an advantageous source of epileptic biomarkers, highlighting the potential of miRNAs as prognostic and diagnostic biomarkers of canine idiopathic epilepsy.

Prion diseases affect both animals and humans. Research in the natural animal model of the disease could help in the understanding of neuropathological mechanisms and in the development of biomarkers for human pathologies. For this purpose, we studied the expression of 10 genes involved in prion propagation in vitro in the central nervous system of scrapie-infected sheep. Dysregulated genes (BAMBI and CHGA) were further analysed in a transgenic murine model (Tg338) of scrapie, and their protein distribution was determined using immunohistochemistry and Western blot. Their potential as biomarkers was finally assessed using enzyme-linked immunosorbent assay (ELISA) in cerebrospinal fluid (CSF) of scrapie sheep and Creutzfeldt-Jakob disease (CJD) patients. Protein BAMBI was upregulated in highly affected brain areas and CHGA was overexpressed along the brain in both models. Moreover, BAMBI and CHGA immunostaining scores strongly correlated with spongiosis and microgliosis in mice. Finally, levels of BAMBI were significantly higher in the CSF of clinical sheep and CJD patients. In addition to their potential as biomarkers, our work confirms the role of BAMBI and CHGA in prion neuropathology in vivo, but besides prion replication, they seem to be involved in the characteristic neuroinflammatory response associated to prion infection.