The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently observed upon experimental transmission to rodent and swine models. This study aimed to assess ...whether the inoculation of atypical scrapie could induce BSE-like disease in cattle. Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease and tested negative for PrPSc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prion seeding activity was detected during in vitro propagation of brain samples from the inoculated animals. These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle.
Prion diseases are diagnosed in the symptomatic stage, when the neuronal damage is spread throughout the central nervous system (CNS). The assessment of biological features that allow the detection ...of asymptomatic cases is needed, and, in this context, scrapie, where pre-symptomatic infected animals can be detected through rectal biopsy, becomes a good study model. Neurogranin (Ng) and neurofilament light chain (NfL) are proteins that reflect synaptic and axonal damage and have been studied as cerebrospinal fluid (CSF) biomarkers in different neurodegenerative disorders. In this study, we evaluated Ng and NfL both at the protein and transcript levels in the CNS of preclinical and clinical scrapie-affected sheep compared with healthy controls and assessed their levels in ovine CSF. The correlation between these proteins and the main neuropathological events in prion diseases, PrP
deposition and spongiosis, was also assessed. The results show a decrease in Ng and NfL at the protein and gene expression levels as the disease progresses, and significant changes between the control and preclinical animals. On the contrary, the CSF levels of NfL increased throughout the progression of the disease. Negative correlations between neuropathological markers of prion disease and the concentration of the studied proteins were also found. Although further research is needed, these results suggest that Ng and NfL could act as biomarkers for neurodegeneration onset and intensity in preclinical cases of scrapie.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Scrapie is a neurodegenerative disorder belonging to the group of transmissible spongiform encephalopathies or prion diseases, which are caused by an infectious isoform of the innocuous cellular ...prion protein (PrP
) known as PrP
. DNA methylation, one of the most studied epigenetic mechanisms, is essential for the proper functioning of the central nervous system. Recent findings point to possible involvement of DNA methylation in the pathogenesis of prion diseases, but there is still a lack of knowledge about the behavior of this epigenetic mechanism in such neurodegenerative disorders. Here, we evaluated by immunohistochemistry the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels in sheep and mouse brain tissues infected with scrapie. Expression analysis of different gene coding for epigenetic regulatory enzymes (
,
,
,
,
,
, and
) was also carried out. A decrease in 5mC levels was observed in scrapie-affected sheep and mice compared to healthy animals, whereas 5hmC displayed opposite patterns between the two models, demonstrating a decrease in 5hmC in scrapie-infected sheep and an increase in preclinical mice. 5mC correlated with prion-related lesions in mice and sheep, but 5hmC was associated with prion lesions only in sheep. Differences in the expression changes of epigenetic regulatory genes were found between both disease models, being differentially expressed
,
, and
in mice and
in sheep. Our results support the evidence that DNA methylation in both forms, 5mC and 5hmC, and its associated epigenetic enzymes, take part in the neurodegenerative course of prion diseases.
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Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrP
to its abnormal and misfolded isoform PrP
...is the main event in the pathogenesis of prion diseases of all origins. In spontaneous prion diseases, the mechanisms that trigger the formation of PrP
in the central nervous system remain unknown. Several reports have demonstrated that the accumulation of PrP
can induce endoplasmic reticulum (ER) stress and proteasome impairment from the early stages of the prion disease. Both mechanisms lead to an increment of PrP aggregates in the secretory pathway, which could explain the pathogenesis of spontaneous prion diseases. Here, we investigate the role of ER stress and proteasome impairment during prion disorders in a murine model of spontaneous prion disease (TgVole) co-expressing the Ub
-GFP reporter, which allows measuring the proteasome activity in vivo. Spontaneously prion-affected mice showed a significantly higher accumulation of the PKR-like ER kinase (PERK), the ER chaperone binding immunoglobulin protein (BiP/Grp78), the ER protein disulfide isomerase (PDI) and the Ub
-GFP reporter than age-matched controls in certain brain areas. The upregulation of PERK, BiP, PDI and ubiquitin was detected from the preclinical stage of the disease, indicating that ER stress and proteasome impairment begin at early stages of the spontaneous disease. Strong correlations were found between the deposition of these markers and neuropathological markers of prion disease in both preclinical and clinical mice. Our results suggest that both ER stress and proteasome impairment occur during the pathogenesis of spontaneous prion diseases.
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Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrP
), spongiform changes, neuronal loss, and gliosis. Growing ...evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion diseases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed the gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated the possible correlations between gene expression and the pathological hallmarks of prion disease. We observed significant changes in TLR expression in scrapie-infected sheep that correlate with the degree of spongiosis, PrP
deposition, and gliosis in each of the regions studied. Remarkably,
was the only gene upregulated in all regions, regardless of the severity of neuropathology. In the hippocampus, we observed milder neuropathology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, a comparison of the findings in naturallyinfected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLRgene expression.
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The role of the glycosylation status of PrP
C
in the conversion to its pathological counterpart and on cross-species transmission of prion strains has been widely discussed. Here, we assessed the ...effect on strain characteristics of bovine spongiform encephalopathy (BSE) isolates with different transmission histories upon propagation on a model expressing a non-glycosylated human PrP
C
. Bovine, ovine and porcine-passaged BSE, and variant Creutzfeldt-Jakob disease (vCJD) isolates were used as seeds/inocula in both
in vitro
and
in vivo
propagation assays using the non-glycosylated human PrP
C
-expressing mouse model (TgNN6h). After protein misfolding cyclic amplification (PMCA), all isolates maintained the biochemical characteristics of BSE. On bioassay, all PMCA-propagated BSE prions were readily transmitted to TgNN6h mice, in agreement with our previous
in vitro
results. TgNN6h mice reproduced the characteristic neuropathological and biochemical hallmarks of BSE, suggesting that the absence of glycans did not alter the pathobiological features of BSE prions. Moreover, back-passage of TgNN6h-adapted BSE prions to BoTg110 mice recovered the full BSE phenotype, confirming that the glycosylation of human PrP
C
is not essential for the preservation of the human transmission barrier for BSE prions or for the maintenance of BSE strain properties.
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Diagnosis of transmissible spongiform encephalopathies (TSEs), or prion diseases, is based on the detection of proteinase K (PK)-resistant PrPSc in post-mortem tissues as indication of infection and ...disease. Since PrPSc 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 PrPSc 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 PrPSc in most CSF-derived exosomes from clinically affected sheep, which may facilitate in vivo diagnosis of prion diseases, at least during the clinical stage.
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Chronic accumulation of misfolded proteins such as PrP
can alter the endoplasmic reticulum homeostasis triggering the unfolded protein response (UPR). In this pathogenic event, the molecular ...chaperones play an important role. Several reports in humans and animals have suggested that neurodegeneration is related to endoplasmic reticulum stress in diseases caused by the accumulation of misfolded proteins. In this study, we investigated the expression of three endoplasmic reticulum stress markers: PERK (protein kinase R-like endoplasmic reticulum kinase), BiP (binding immunoglobulin protein), and PDI (Protein Disulfide Isomerase). In addition, we evaluated the accumulation of ubiquitin as a marker for protein degradation mediated by the proteasome. These proteins were studied in brain tissues of sheep affected by scrapie in clinical and preclinical stages of the disease. Results were compared with those observed in healthy controls. Scrapie-infected sheep showed significant higher levels of PERK, BiP/Grp78 and PDI than healthy animals. As we observed before in models of spontaneous prion disease, PDI was the most altered ER stress marker between scrapie-infected and healthy sheep. Significantly increased intraneuronal and neuropil ubiquitinated deposits were observed in certain brain areas in scrapie-affected animals compared to controls. Our results suggest that the neuropathological and neuroinflammatory phenomena that develop in prion diseases cause endoplasmic reticulum stress in brain cells triggering the UPR. In addition, the significantly higher accumulation of ubiquitin aggregates in scrapie-affected animals suggests an impairment of the ubiquitin-proteasome system in natural scrapie. Therefore, these proteins may contribute as biomarkers and/or therapeutic targets for prion diseases.
The non-toxic C-terminal fragment of the tetanus toxin (TTC) has been described as a neuroprotective molecule since it binds to Trk receptors and activates Trk-dependent signaling, activating ...neuronal survival pathways and inhibiting apoptosis. Previous in vivo studies have demonstrated the ability of this molecule to increase mice survival, inhibit apoptosis and regulate autophagy in murine models of neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. Prion diseases are fatal neurodegenerative disorders in which the main pathogenic event is the conversion of the cellular prion protein (PrP
C
) into an abnormal and misfolded isoform known as PrP
Sc
. These diseases share different pathological features with other neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson’s disease or Alzheimer’s disease. Hitherto, there are no effective therapies to treat prion diseases. Here, we present a pilot study to test the therapeutic potential of TTC to treat prion diseases. C57BL6 wild-type mice and the transgenic mice Tg338, which overexpress PrP
C
, were intracerebrally inoculated with scrapie prions and then subjected to a treatment consisting of repeated intramuscular injections of TTC. Our results indicate that TTC displays neuroprotective effects in the murine models of prion disease reducing apoptosis, regulating autophagy and therefore increasing neuronal survival, although TTC did not increase survival time in these models.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase ...receptors and the p75 neurotrophin receptor (p75
). The activation of p75
can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75
and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75
, in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75
. Infected mice showed higher abundance of p75
immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75
and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75
-mediated neurodegeneration. In contrast, p75
staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75
in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.
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