Carr - CSF_Brain_Prion_TSQ_directMRM

PrP concentration in the central nervous system: regional variability, genotypic effects, and pharmacodynamic impact
Data License: CC BY 4.0 | ProteomeXchange: PXD031432 | doi:
  • Organism: Homo sapiens, Rattus norvegicus
  • Instrument: TSQ Quantiva
  • SpikeIn: Yes
  • Keywords: cerebrospinal fluid, LC-MRM-MS, TSQ Quantiva, Skyline, antisense oligonucleotide, prion protein
  • Lab head: Steve Carr Submitter: Eric Kuhn
Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across six species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varies dramatically between different brain regions in mice, cynomolgus macaques, and humans. PrP expression does not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP is lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55% and of D178N just 31% the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA is reflected in brain parenchyma PrP, and in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs, and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.
Experiment Description
Samples of dried digested human or rat cerebrospinal fluid were reconstituted in 12 µL 3% acetonitrile/5% acetic acid, vortexed for 5 min at RT, centrifuged 12,000 x g for 5 min and 10 µL were transferred into an HPLC vial (Waters 186000273). Samples were analyzed on a TSQ Quantiva triple quadrupole mass spectrometer equipped with a nanoLC 1200 and a custom built nanospray source (James A. Hill Instrument Services). Ion source was set to positive ion mode with capillary temperature of 300°C, spray voltage of 2,000 and sweep gas set to 0. Samples were injected (2 µL, 20% of digested sample) onto a 75 um ID PicoFrit (New Objective) column pulled to a 10 µm emitter and custom-packed to 20 cm with 1.9 µm 200Å C18-AQ Reprosil beads (Dr. Maisch). Mobile phases consisted of 3% acetonitrile/0.1% formic acid as solvent A, 90% acetonitrile/0.1% formic acid as solvent B. The LC gradient was 0% B to 30% B for 55 min, 30% B to 60% B in 5 min, 60% B to 90 % B in 1 min using a flow rate of 200 nL/min.
Sample Description
CSF from asymptomatic PRNP mutation carriers and controls was collected through the Massachusetts General Hospital prion disease biomarker study as previously and include samples previously described (11). Participants were recruited through, Rally (Mass General Brigham), Prion Alliance, CJD Foundation. Participants analyzed here had no mutation (N=21), E200K (N=12), D178N (N=6), P102L (N=4), or other PRNP mutation (N=4) and had each made 1-5 study visits (mean: 2.3) spanning a time period of up to ~3.5 years. CSF was collected from Sprague-Dawley males (age study) and females (pharmacodynamic study) rats under terminal anesthesia.
Created on 2/3/22, 7:26 PM