Prion protein quantification in cerebrospinal fluid as a tool for prion disease drug development (original) (raw)
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Proceedings of the National Academy of Sciences of the United States of America, 2019
Reduction of native prion protein (PrP) levels in the brain is an attractive and genetically validated strategy for the treatment or prevention of human prion diseases. Development of any PrP-reducing therapeutic will require an appropriate pharmacodynamic biomarker: a practical and robust method for quantifying PrP, and reliably demonstrating its reduction, in the CNS of a living patient. Here we evaluate the potential of enzyme-linked immunosorbent assay (ELISA)based quantification of human PrP in human cerebrospinal fluid (CSF) to serve as a biomarker for PrP-reducing therapeutics. We show that CSF PrP is highly sensitive to plastic adsorption during handling and storage, but its loss can be minimized by addition of detergent. We find that blood contamination does not affect CSF PrP levels, and CSF PrP and hemoglobin are uncorrelated, together suggesting that CSF PrP is CNS-derived, supporting its relevance for monitoring the tissue of interest and in keeping with high PrP abundance in brain relative to blood. In a cohort with controlled sample handling, CSF PrP exhibits good test-retest reliability (mean coefficient of variation 13% over 8-11 weeks), suggesting that therapeutically meaningful reductions in brain PrP could be readily detected in CSF. Together, these findings supply a method for monitoring the effect of a PrP-reducing drug in the CNS, which will be critical to the development of any prion therapeutic with this mechanism of action.
Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry
Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have reproducibly shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. To date it has been unclear whether the reduced abundance of PrP in CSF results from its incorporation into plaques, retention in intracellular compartments, downregulation as a function of the disease process, or other factors. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a targeted mass spectrometry...
Diagnostic and Prognostic Value of Human Prion Detection in Cerebrospinal Fluid
Annals of neurology, 2016
Objective-Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second-generation real-time quaking-induced conversion (RT-QuIC). The objective of this study was to investigate the diagnostic performance of the RT-QuIC prion test in the broad phenotypic spectrum of prion diseases. Methods- We performed CSF RT-QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases which were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and Type 1 or Type 2 of human prions on diagnostic performance. Results-The 98.5% diagnostic specificity and 92% sensitivity of CSF RT-QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT-QuIC differentiated 94% of cases of sporadic Creutzfeldt-Jak...
Biochemical and Biophysical Research Communications, 1992
The cellular prion protein (PrP c) is a 33-35 kDa sialoglycoprotein anchored to the external surface of neural and non-neural cells by a glycosyl phosphatidylinositol moiety. In addition, a secretory form of PrP c has been found in cell-free translation systems and in cell cultures. On this basis, we investigated human cerebrospinal fluid for the presence of soluble PrP and identified a protein whose molecular weight, antigenic determinants, N-terminal amino acid sequence and sensitivity to protease digestion corresponded to those of PrP C. In prion-related encephalopathies of humans and animals, the secretory form of PrP c might be converted into the abnormal isoform PrP sc and play a role in the dissemination of the disease process and amyloid formation. The cellular prion protein (PrP c) is a 33-35 kDa sialoglycoprotein encoded by a gene that in humans is located on chromosome 20 (1-8). The PrP gene is expressed in neural and non-neural tissues, the highest concentration of mRNA being in neurons (9-13). The translation product consists of 253 amino acids (14, 15) and undergoes several post-translational modifications. In hamsters, a signal peptide of 22 amino acids is cleaved at the N-terminus, 23 amino acids are removed from the C-terminus on addition of a glycosyl phosphatidylinositol (GPI) anchor, and asparagine-linked oligosaccharides are attached to residues 181 and 197 in a loop formed by a disulfide bond (8, 16-20). In prion-related encephalopathies, such as Creutzfeldt-Jakob disease and Gerstmann-Strtiussler-Scheinker disease of humans, scrapie of sheep and goats, and spongiform encephalopathy of
Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion
Nature Medicine, 2011
The development of technologies for the in vitro amplification of the abnormal conformers of prion protein (PrP Sc ) has generated the potential for a novel diagnostic assay for prion disease. Previously, we developed a new PrP Sc amplification assay designated quaking-induced conversion (QUIC), which involves intermittent, automated shaking of the substrate, soluble recombinant PrP. We further improved the rapidity and practicality of this method by combining it with thioflavin T fluorescence to monitor the amyloid fibril formation. This assay, termed "real-time QUIC (RT-QUIC)", allows within 48 h, the detection of ≥1 fg of PrP Sc in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects, and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the ante-mortem evaluation of suspected CJD. 4 Transmissible spongiform encephalopathies or prion diseases are characteristically associated with the accumulation of PrP Sc in the central nervous system through auto-catalytic conversion of normal cellular PrP (PrP c ) into replicate misfolded isomers 1,2 . Despite occasional reported exceptions 3,4 , PrP Sc remains the best characterized and most reliable marker of prion disease.
Novel Biomarkers and the Diagnosis of Prion Diseases
Journal of Molecular Biomarkers & Diagnosis, 2016
In fatal neurodegenerative diseases, prion diseases have high risk of transmission ability and no cure and effective treatment. This abnormal folded protein disease in brain pose a serious threat to public health and the development of early diagnostic markers and new therapeutic approaches is in pronounced plea. Prion disease show infectious and incurable irrepressible nature and long period of silent incubations and nature of prion diseases, development of early diagnostic markers is in great demand to prevent a potential spread of the disease and for early diagnosis of the disease given the long incubation periods of disease. Moreover discovery of novel biomarkers can lead to development of new therapeutic targets and better understanding of the underlying pathogenesis of the Prion diseases.
Pathogens, 2021
The possibilities for diagnosing prion diseases have shifted significantly over the last 10 years. The RT-QuIC assay option has been added for neuropsychiatric symptoms, supporting biomarkers and final post-mortem confirmation. Samples of brain homogenates used for final diagnosis, archived for many years, provide the possibility for retrospective studies. We used a second-generation RT-QuIC assay to detect seeding activity in different types of sporadic and genetic prion diseases in archival brain homogenates and post-mortem CSF samples that were 2 to 15 years old. Together, we tested 92 archival brain homogenates: 39 with definite prion disease, 28 with definite other neurological disease, and 25 with no signs of neurological disorders. The sensitivity and specificity of the assay were 97.4% and 100%, respectively. Differences were observed in gCJD E200K, compared to the sporadic CJD group. In 52 post-mortem CSF samples—24 with definite prion disease and 28 controls—we detected th...
Rapid communications in mass spectrometry : RCM, 2007
More sensitive detection of prions in brain is important because it would allow early detection of disease in young animals and assure a safer food supply. We have quantitated the amount of proteinase K-resistant prion protein (PrP 27-30) by use of nano-scale liquid chromatography coupled to tandem mass spectrometry using the multiple reaction monitoring mode of operation. We used a method based on the detection of VVEQMCTTQYQK (residues 209-220) obtained by reduction, alkylation and digestion with trypsin. Quantitation of the amount of PrP 27-30 in the brains of Syrian hamsters was possible as early as 24 h after inoculation. Our results show sensitive detection of 180 fmol of PrP 27-30 per g brain (wet weight) as early as 24 h after inoculation. Clinical symptoms are not observed until 9 weeks after inoculation.
A versatile prion replication assay in organotypic brain slices
Nature Neuroscience, 2008
A versatile prion replication assay in organotypic brain slices Abstract Methods enabling prion replication ex vivo are important for advancing prion studies. However, few such technologies exist, and many prion strains are not amenable to them. Here we describe a prion organotypic slice culture assay (POSCA) that allows prion amplification and titration ex vivo under conditions that closely resemble intracerebral infection. Thirty-five days after contact with prions, mouse cerebellar slices had amplified the abnormal isoform of prion protein, PrP(Sc), >10(5)-fold. This is quantitatively similar to amplification in vivo, but fivefold faster. PrP(Sc) accumulated predominantly in the molecular layer, as in infected mice. The POSCA detected replication of prion strains from disparate sources, including bovines and ovines, with variable detection efficiency. Pharmacogenetic ablation of microglia from POSCA slices led to a 15-fold increase in prion titers and PrP(Sc) concentrations over those in microglia-containing slices, as well as an increase in susceptibility to infection. This suggests that the extensive microglial activation accompanying prion diseases represents an efficacious defensive reaction. Running title: Microglia-mediated prion removal. 2 Methods enabling prion replication ex vivo are important for advancing prion science. However, few such technologies exist and many prion strains are intractable with them. Here we describe a prion organotypic slice culture assay (POSCA) which allows for prion amplification and titration ex vivo under conditions that closely resemble intracerebral infection. Thirty-five days after contact with prions, cerebellar slices from wild-type mice had amplified PrP Sc >10 5 -fold. This is quantitatively similar to amplification in vivo, but ca. 5-fold faster. PrP Sc accumulated predominantly in the molecular layer, similarly to infected mice. The POSCA detected replication of prion strains from disparate sources, including bovines and ovines. Pharmacogenetic ablation of microglia from POSCA slices led to a 15-fold increase in prion titers and PrP Sc concentration, as well as a 10-fold increase in susceptibility of slices to infection. This suggests that the extensive microglial activation and proliferation accompanying prion diseases represent a defensive reaction that significantly decelerates prion replication.