Cerebrospinal Fluid and Plasma Small Extracellular Vesicles and miRNAs as Biomarkers for Prion Diseases

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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Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion

mBio ◽

10.1128/mbio.00078-11 ◽

2011 ◽

Vol 2 (3) ◽

Cited By ~ 78

Author(s):

Christina D. Orrú ◽

Jason M. Wilham ◽

Lynne D. Raymond ◽

Franziska Kuhn ◽

Björn Schroeder ◽

...

Keyword(s):

Real Time ◽

Blood Test ◽

Prion Diseases ◽

Proteinase K ◽

Transmissible Spongiform Encephalopathies ◽

Serum Samples ◽

Spongiform Encephalopathies ◽

Jakob Disease

ABSTRACT A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 1014-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call “enhanced QuIC” (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples. IMPORTANCE Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals.

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Creutzfeldt-Jakob’s disease: case report with emphasis on the electroencephalographic features

Journal of Epilepsy and Clinical Neurophysiology ◽

10.1590/s1676-26492005000400007 ◽

2005 ◽

Vol 11 (4) ◽

pp. 189-192

Author(s):

Coral Patrícia ◽

Francisco M. B. Germiniani ◽

Carlos E. Silvado

Keyword(s):

Neurodegenerative Disorder ◽

Brain Mri ◽

Infectious Complications ◽

Transmissible Spongiform Encephalopathies ◽

Progressive Dementia ◽

Spongiform Encephalopathies ◽

Disease Case ◽

One Year ◽

In Vivo Diagnosis

Creuzfeldt-Jakob’s Disease is a rare neurodegenerative disorder that is included among the transmissible spongiform encephalopathies. The clinical features are those of a rapid progressive dementia with mioclonic jerks, which progresses to death in less than one year. We report the case of a 79 years old woman, with initial complaints of vertigo, visual and gait compromise, with an initial brain MRI, EEG and CSF that had no specific features of CJD. After 12 days she was again admitted to the hospital with a worsening of her symptoms, along with global spasticity, inability to walk and a further deterioration of speech and swallowing. A new EEG disclosed periodic triphasic complexes, typical of the disease. CSF had a positive 14-3-3 protein. She later evolved with mioclonic jerks, coma, multiple infectious complications and, ultimately, death 45 days after her second admission. We focus on the role of the EEG as an adjuctive eletrophysiological tool for the presumptive in vivo diagnosis of the disease.

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Gene-Based Antibody Strategies for Prion Diseases

International Journal of Cell Biology ◽

10.1155/2013/710406 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 6

Author(s):

Alessio Cardinale ◽

Silvia Biocca

Keyword(s):

Therapeutic Potential ◽

Prion Diseases ◽

Recombinant Antibody ◽

Cellular Protein ◽

Transmissible Spongiform Encephalopathies ◽

Diagnostic Tools ◽

Spongiform Encephalopathies ◽

Interaction Sites

Prion diseases or transmissible spongiform encephalopathies (TSE) are a group of neurodegenerative and infectious disorders characterized by the conversion of a normal cellular protein PrPCinto a pathological abnormally folded form, termed PrPSc. There are neither available therapies nor diagnostic tools for an early identification of individuals affected by these diseases. New gene-based antibody strategies are emerging as valuable therapeutic tools. Among these, intrabodies are chimeric molecules composed by recombinant antibody fragments fused to intracellular trafficking sequences, aimed at inhibiting,in vivo, the function of specific therapeutic targets. The advantage of intrabodies is that they can be selected against a precise epitope of target proteins, including protein-protein interaction sites and cytotoxic conformers (i.e., oligomeric and fibrillar assemblies). Herein, we address and discussin vitroandin vivoapplications of intrabodies in prion diseases, focussing on their therapeutic potential.

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Detection of Pathognomonic Biomarker PrPSc and the Contribution of Cell Free-Amplification Techniques to the Diagnosis of Prion Diseases

Biomolecules ◽

10.3390/biom10030469 ◽

2020 ◽

Vol 10 (3) ◽

pp. 469

Author(s):

Hasier Eraña ◽

Jorge M. Charco ◽

Ezequiel González-Miranda ◽

Sandra García-Martínez ◽

Rafael López-Moreno ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Protein Misfolding ◽

Prion Diseases ◽

Immunohistochemical Analysis ◽

Body Fluids ◽

Detection Methods ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathies ◽

Prion Propagation

Transmissible spongiform encephalopathies or prion diseases are rapidly progressive neurodegenerative diseases, the clinical manifestation of which can resemble other promptly evolving neurological maladies. Therefore, the unequivocal ante-mortem diagnosis is highly challenging and was only possible by histopathological and immunohistochemical analysis of the brain at necropsy. Although surrogate biomarkers of neurological damage have become invaluable to complement clinical data and provide more accurate diagnostics at early stages, other neurodegenerative diseases show similar alterations hindering the differential diagnosis. To solve that, the detection of the pathognomonic biomarker of disease, PrPSc, the aberrantly folded isoform of the prion protein, could be used. However, the amounts in easily accessible tissues or body fluids at pre-clinical or early clinical stages are extremely low for the standard detection methods. The solution comes from the recent development of in vitro prion propagation techniques, such as Protein Misfolding Cyclic Amplification (PMCA) and Real Time-Quaking Induced Conversion (RT-QuIC), which have been already applied to detect minute amounts of PrPSc in different matrixes and make early diagnosis of prion diseases feasible in a near future. Herein, the most relevant tissues and body fluids in which PrPSc has been detected in animals and humans are being reviewed, especially those in which cell-free prion propagation systems have been used with diagnostic purposes.

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Complex proteinopathies and neurodegeneration: insights from the study of transmissible spongiform encephalopathies

Arquivos de Neuro-Psiquiatria ◽

10.1590/0004-282x20180111 ◽

2018 ◽

Vol 76 (10) ◽

pp. 705-712

Author(s):

Pedro Piccardo ◽

David M. Asher

Keyword(s):

Protein Misfolding ◽

Wide Spectrum ◽

Prion Diseases ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathies ◽

Duration Of Illness ◽

Secondary Result ◽

Different Strains ◽

New Treatments ◽

Misfolding Diseases

ABSTRACT Protein misfolding diseases are usually associated with deposits of single “key” proteins that somehow drive the pathology; β-amyloid and hyperphosphorylated tau accumulate in Alzheimer's disease, α-synuclein in Parkinson's disease, or abnormal prion protein (PrPTSE) in transmissible spongiform encephalopathies (TSEs or prion diseases). However, in some diseases more than two proteins accumulate in the same brain. These diseases might be considered “complex” proteinopathies. We have studied models of TSEs (to explore deposits of PrPTSE and of “secondary proteins”) infecting different strains and doses of TSE agent, factors that control incubation period, duration of illness and histopathology. Model TSEs allowed us to investigate whether different features of histopathology are independent of PrPTSE or appear as a secondary result of PrPTSE. Better understanding the complex proteinopathies may help to explain the wide spectrum of degenerative diseases and why some overlap clinically and histopathologically. These studies might also improve diagnosis and eventually even suggest new treatments for human neurodegenerative diseases.

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Toxicological Evaluation of Anti-Scrapie Trimethoxychalcones and Oxadiazoles

Anais da Academia Brasileira de Ciências ◽

10.1590/0001-3765201520140712 ◽

2015 ◽

Vol 87 (2 suppl) ◽

pp. 1421-1434 ◽

Cited By ~ 1

Author(s):

CLAUDIA P. FIGUEIREDO ◽

NATALIA C. FERREIRA ◽

GISELLE F. PASSOS ◽

ROBSON DA COSTA ◽

FERNANDA S. NEVES ◽

...

Keyword(s):

Prion Protein ◽

Aromatic Compounds ◽

Prion Diseases ◽

Intraperitoneal Administration ◽

Neuroblastoma Cells ◽

Cellular Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Toxicological Evaluation ◽

Spongiform Encephalopathies

An altered form of the cellular prion protein, the PrPScor PrPRes, is implicated in the occurrence of the still untreatable transmissible spongiform encephalopathies. We have previously synthesized and characterized aromatic compounds that inhibit protease-resistant prion protein (PrPRes) accumulation in scrapie-infected cells. These compounds belong to different chemical classes, including acylhydrazones, chalcones and oxadiazoles. Some of the active compounds were non-toxic to neuroblastoma cells in culture and seem to possess drugable properties, since they are in agreement with the Lipinski´s rule of 5 and present desirable pharmacokinetic profiles as predicted in silico. Before the evaluation of the in vivo efficacy of the aromatic compounds in scrapie-infected mice, safety assessment in healthy mice is needed. Here we used Swiss mice to evaluate the acute toxicity profile of the six most promising anti-prionic compounds, the 2,4,5-trimethoxychalcones (J1, J8, J20 and J35) and the 1,3,4-oxadiazoles (Y13 and Y17). One single oral administration (300 mg/kg) of J1, J8, J20, J35, Y13 and Y17 or repeated intraperitoneal administration (10 mg/kg, 3 times a week, for 4 weeks) of J1, J8 and J35, did not elicit toxicity in mice. We strongly believe that the investigated trimethoxychalcones and oxadiazoles are interesting compounds to be further analyzed in vivo against prion diseases.

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Development of antibody fragments for immunotherapy of prion diseases

Biochemical Journal ◽

10.1042/bj20081541 ◽

2009 ◽

Vol 418 (3) ◽

pp. 507-515 ◽

Cited By ~ 26

Author(s):

Vincenza Campana ◽

Lorena Zentilin ◽

Ilaria Mirabile ◽

Agata Kranjc ◽

Philippe Casanova ◽

...

Keyword(s):

Structural Model ◽

Prion Diseases ◽

Neuronal Cell ◽

Cellular Protein ◽

Proteinase K ◽

Active Immunotherapy ◽

Transmissible Spongiform Encephalopathies ◽

Single Chain ◽

Infected Cells

Prions are infectious proteins responsible for a group of fatal neurodegenerative diseases called TSEs (transmissible spongiform encephalopathies) or prion diseases. In mammals, prions reproduce themselves by recruiting the normal cellular protein PrPC and inducing its conversion into the disease-causing isoform denominated PrPSc. Recently, anti-prion antibodies have been shown to permanently cure prion-infected cells. However, the inability of full-length antibodies and proteins to cross the BBB (blood-brain barrier) hampers their use in the therapy of TSEs in vivo. Alternatively, brain delivery of prion-specific scFv (single-chain variable fragment) by AAV (adeno-associated virus) transfer delays the onset of the disease in infected mice, although protection is not complete. We investigated the anti-prion effects of a recombinant anti-PrP (D18) scFv by direct addition to scrapie-infected cell cultures or by infection with both lentivirus and AAV-transducing vectors. We show that recombinant anti-PrP scFv is able to reduce proteinase K-resistant PrP content in infected cells. In addition, we demonstrate that lentiviruses are more efficient than AAV in gene transfer of the anti-PrP scFv gene and in reducing PrPSc content in infected neuronal cell lines. Finally, we have used a bioinformatic approach to construct a structural model of the D18scFv–PrPC complex. Interestingly, according to the docking results, ArgPrP151 (Arg151 from prion protein) is the key residue for the interactions with D18scFv, anchoring the PrPC to the cavity of the antibody. Taken together, these results indicate that combined passive and active immunotherapy targeting PrP might be promising strategies for therapeutic intervention in prion diseases.

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TSE Diagnostics: Recent Advances in Immunoassaying Prions

Clinical and Developmental Immunology ◽

10.1155/2013/360604 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 7

Author(s):

Anja Lukan ◽

Tanja Vranac ◽

Vladka Čurin Šerbec

Keyword(s):

Cerebrospinal Fluid ◽

Neurodegenerative Diseases ◽

Brain Tissue ◽

Prion Diseases ◽

Cellular Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Post Mortem ◽

Definite Diagnosis ◽

Spongiform Encephalopathies ◽

Recent Advances

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of rare fatal neurodegenerative diseases, affecting humans and animals. They are believed to be the consequence of the conversion of the cellular prion protein to its aggregation-prone,β-sheet-rich isoform, named prion. Definite diagnosis of TSEs is determinedpost mortem. For this purpose, immunoassays for analyzing brain tissue have been developed. However, the ultimate goal of TSE diagnostics is anante mortemtest, which would be sensitive enough to detect prions in body fluids, that is, in blood, cerebrospinal fluid, or urine. Such a test would be of paramount importance also for screening of asymptomatic carriers of the disease with the aim of increasing food, drugs, and blood-derived products safety. In the present paper, we have reviewed recent advances in the development of immunoassays for the detection of prions.

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Cells Expressing Anchorless Prion Protein Are Resistant to Scrapie Infection

Journal of Virology ◽

10.1128/jvi.02412-08 ◽

2009 ◽

Vol 83 (9) ◽

pp. 4469-4475 ◽

Cited By ~ 40

Author(s):

Kristin L. McNally ◽

Anne E. Ward ◽

Suzette A. Priola

Keyword(s):

Transgenic Mice ◽

Prion Protein ◽

Prion Diseases ◽

Transmitted Disease ◽

Transmissible Spongiform Encephalopathies ◽

Gpi Anchor ◽

Spongiform Encephalopathies ◽

Infected Cells

ABSTRACT The hallmark of transmissible spongiform encephalopathies (TSEs or prion diseases) is the accumulation of an abnormally folded, partially protease-resistant form (PrP-res) of the normal protease-sensitive prion protein (PrP-sen). PrP-sen is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. In vitro, the anchor and the local membrane environment are important for the conversion of PrP-sen to PrP-res. In vivo, however, the anchor is not necessary because transgenic mice expressing anchorless PrP-sen accumulate PrP-res and replicate infectivity. To clarify the role of the GPI anchor in TSE infection, cells expressing GPI-anchored PrP-sen, anchorless PrP-sen, or both forms of PrP-sen were exposed to the mouse scrapie strain 22L. Cells expressing anchored PrP-sen produced PrP-res after exposure to 22L. Surprisingly, while cells expressing anchorless PrP-sen made anchorless PrP-res in the first 96 h postinfection, no PrP-res was detected at later passes. In contrast, when cells expressing both forms of PrP-sen were exposed to 22L, both anchored and anchorless PrP-res were detected over multiple passes. Consistent with the in vitro data, scrapie-infected cells expressing anchored PrP-sen transmitted disease to mice whereas cells expressing anchorless PrP-sen alone did not. These results demonstrate that the GPI anchor on PrP-sen is important for the persistent infection of cells in vitro. Our data suggest that cells expressing anchorless PrP-sen are not directly infected with scrapie. Thus, PrP-res formation in transgenic mice expressing anchorless PrP-sen may be occurring extracellularly.

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Enhanced Antiscrapie Effect Using Combination Drug Treatment

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00715-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3447-3449 ◽

Cited By ~ 17

Author(s):

David A. Kocisko ◽

Byron Caughey ◽

John D. Morrey ◽

Richard E. Race

Keyword(s):

Combination Therapy ◽

Protein Misfolding ◽

Transmissible Spongiform Encephalopathies ◽

Pentosan Polysulfate ◽

Survival Times ◽

Combination Drug ◽

Spongiform Encephalopathies ◽

Misfolding Diseases ◽

Combination Drug Treatment

ABSTRACT Combination treatment with pentosan polysulfate and Fe(III)meso-tetra(4-sulfonatophenyl)porphine in mice beginning 14 or 28 days after scrapie inoculation significantly increased survival times. This increase may be synergistic, implying that the compounds act cooperatively in vivo. Combination therapy may therefore be more effective for treatment of transmissible spongiform encephalopathies and other protein-misfolding diseases.

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