Dataset Information

Prion MRM assay development and clinical study

ABSTRACT: 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 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. Because misfolding or proteolytic cleavage could potentially render PrP undetectable by ELISA, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified by stable isotope dilution analysis. Analytical validation experiments showed intra-day and inter-day assay reproducibility coefficients of variation less than 15% (below the best practices threshold), 3 orders of dynamic linear range that encompass the entire expected range of PrP concentration in CSF, lower limits of detection near 10 ng/mL and a similar recovery response from both CSF and brain homogenate matrices. Critical for preclinical assay development, comparable assay performance was found for peptides unique to 4 species. In N=55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to individuals with non-prion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in pre-symptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.

ORGANISM(S): Homo Sapiens Rattus Norvegicus Mus Musculus Macaca Fascicularis

SUBMITTER: Eric Kuhn

PROVIDER: PXD014781 | panorama | Tue Nov 19 00:00:00 GMT 2019

REPOSITORIES: PanoramaPublic

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Publications

Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.

Minikel Eric Vallabh EV Kuhn Eric E Cocco Alexandra R AR Vallabh Sonia M SM Hartigan Christina R CR Reidenbach Andrew G AG Safar Jiri G JG Raymond Gregory J GJ McCarthy Michael D MD O'Keefe Rhonda R Llorens Franc F Zerr Inga I Capellari Sabina S Parchi Piero P Schreiber Stuart L SL Carr Steven A SA

Molecular & cellular proteomics : MCP 20190926 12

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 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. Because misfold ...[more]

PMID: 31558565

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Project description:s synapse degeneration is an early event in Alzheimer’s disease (AD) pathophysiology, a biofluid marker of synapse loss in living patients could be a surrogate marker of disease prognosis and therefore would be an excellent addition to the AD biomarker arsenal. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins that could be potential biomarkers of underlying synapse degeneration. The aim of this study was to identify, verify and evaluate a set of synaptic proteins as surrogate CSF markers of underlying synapse loss in AD patients. In the Discovery Stage, we combined high-throughput shotgun proteomics of the CSF with an exhaustive search of the literature and public databases for proteomic studies of the CSF and synapse. A thorough characterization of the synaptic component of the CSF identified 210 synaptic proteins that are detectable in human CSF. We selected an initial panel of 22 candidate biomarkers for evaluation. In the Verification Stage, 12 proteins were discarded due to poor detection by targeted mass spectrometry (Selected Reaction Monitoring, SRM). We confirmed the expression of the remaining 10 proteins either directly at (Calsynytenin-1, GluR2, GluR4, Neurexins 2A and 3A, Neuroligin-2, Syntaxin-1B, Thy-1 and Vamp-2), or surrounding (Tenascin-R), the human synapse using Array Tomography microscopy and biochemical fractionation methods. We quantified the synaptic panel by SRM in CSF samples from 2 independent clinical cohorts of cognitively normal controls and all clinical stages of AD (n=140). A set of the panel proteins demonstrated a non-linear profile distinct to that of existing biomarkers whereby the CSF levels were decreased at the earliest preclinical stage of AD, reflecting reduced synaptic density in these asymptomatic individuals and elevated at later symptomatic stages when neurodegeneration is widespread. In conclusion, we have identified a set of novel synapse-specific proteins that could have clinical value for assessing disease progression in individuals at-risk for AD and potentially in other neurological disorders characterized by early synapse loss.

Dataset Information

Prion MRM assay development and clinical study

Publications

Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.

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