Protease-Sensitive Conformers in Broad Spectrum of Distinct PrPSc Structures in Sporadic Creutzfeldt-Jakob Disease Are Indicator of Progression Rate

The origin, range, and structure of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD), are largely unknown. To investigate the molecular mechanism responsible for the broad phenotypic variability of sCJD, we analyzed the conformational characteristics of protease-sensitive and protease-resistant fractions of the pathogenic prion protein (PrPSc) using novel conformational methods derived from a conformation-dependent immunoassay (CDI). In 46 brains of patients homozygous for polymorphisms in the PRNP gene and exhibiting either Type 1 or Type 2 western blot pattern of the PrPSc, we identified an extensive array of PrPSc structures that differ in protease sensitivity, display of critical domains, and conformational stability. Surprisingly, in sCJD cases homozygous for methionine or valine at codon 129 of the PRNP gene, the concentration and stability of protease-sensitive conformers of PrPSc correlated with progression rate of the disease. These data indicate that sCJD brains exhibit a wide spectrum of PrPSc structural states, and accordingly argue for a broad spectrum of prion strains coding for different phenotypes. The link between disease duration, levels, and stability of protease-sensitive conformers of PrPSc suggests that these conformers play an important role in the pathogenesis of sCJD

Identification of novel risk loci and causal insights for sporadic Creutzfeldt-Jakob disease: a genome-wide association study

Background: Human prion diseases are rare and usually rapidly fatal neurodegenerative disorders, the most common being sporadic Creutzfeldt-Jakob disease (sCJD). Variants in the PRNP gene that encodes prion protein are strong risk factors for sCJD but, although the condition has similar heritability to other neurodegenerative disorders, no other genetic risk loci have been confirmed. We aimed to discover new genetic risk factors for sCJD, and their causal mechanisms. Methods: We did a genome-wide association study of sCJD in European ancestry populations (patients diagnosed with probable or definite sCJD identified at national CJD referral centres) with a two-stage study design using genotyping arrays and exome sequencing. Conditional, transcriptional, and histological analyses of implicated genes and proteins in brain tissues, and tests of the effects of risk variants on clinical phenotypes, were done using deep longitudinal clinical cohort data. Control data from healthy individuals were obtained from publicly available datasets matched for country. Findings: Samples from 5208 cases were obtained between 1990 and 2014. We found 41 genome-wide significant single nucleotide polymorphisms (SNPs) and independently replicated findings at three loci associated with sCJD risk; within PRNP (rs1799990; additive model odds ratio [OR] 1·23 [95% CI 1·17-1·30], p=2·68 × 10-15; heterozygous model p=1·01 × 10-135), STX6 (rs3747957; OR 1·16 [1·10-1·22], p=9·74 × 10-9), and GAL3ST1 (rs2267161; OR 1·18 [1·12-1·25], p=8·60 × 10-10). Follow-up analyses showed that associations at PRNP and GAL3ST1 are likely to be caused by common variants that alter the protein sequence, whereas risk variants in STX6 are associated with increased expression of the major transcripts in disease-relevant brain regions. Interpretation: We present, to our knowledge, the first evidence of statistically robust genetic associations in sporadic human prion disease that implicate intracellular trafficking and sphingolipid metabolism as molecular causal mechanisms. Risk SNPs in STX6 are shared with progressive supranuclear palsy, a neurodegenerative disease associated with misfolding of protein tau, indicating that sCJD might share the same causal mechanisms as prion-like disorders. Funding: Medical Research Council and the UK National Institute of Health Research in part through the Biomedical Research Centre at University College London Hospitals National Health Service Foundation Trust

Small Protease Sensitive Oligomers of PrP^Sc in Distinct Human Prions Determine Conversion Rate of PrP^C

<div><p>The mammalian prions replicate by converting cellular prion protein (PrP^C) into pathogenic conformational isoform (PrP^Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP^Sc on conversion of PrP^C in vitro using PrP^Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP^Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP^Sc. The tight correlation between conversion potency of small oligomers of human sPrP^Sc observed in vitro and duration of the disease suggests that sPrP^Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.</p> </div

Conversion potency of sCJD PrP^Sc inversely mirrors duration of the sCJD.

<p>Amplification index obtained with (<b>A</b>) sPMCA or with (<b>B</b>) QuIC for MM1 (n = 10) and MM2 (n = 10) sCJD cases. The amplification index is the ratio between the concentration of PrP^Sc before and after sPMCA or QuIC measured with CDI. The data points and bars are averages ± SEM obtained from three independent conversion experiments, each measured in triplicate with CDI.</p

Decreased conversion potency of PrP^Sc after protease treatment.

<p>The sCJD brain homogenates were either (blue bars) untreated or (red bars) treated with 100 µg/ml of PK for 1 hr at 37°C. The PK was blocked with 0.5 mM PMSF, and aprotinin and leupeptin at 5 ug/ml, respectively. Impact of protease treatment of PrP^Sc on amplification was monitored in QuIC with CDI either in MM1 (n = 3) or MM2 (n = 3) sCJD cases. The bars represent average ± SEM from three QuIC experiments, each measured in duplicate with CDI.</p

Relationship between conversion potency and the conformational stability of PrP^Sc, rPrP^Sc, and sPrP^Sc in MM1 (n = 10) and MM2 (n = 10) sCJD cases.

<p>The (<b>A</b>) conformational stability of MM1 PrP^Sc before (red diamonds) and after (red circles) PK digestion; the (<b>B</b>) conformational stability of MM2 PrP^Sc before (blue triangles) and after (blue squares) PK digestion; and the (<b>C</b>) fractional change in stability of PrP^Sc conformers induced by PK in individual sCJD samples (filled red circles) Type 1 PrP^Sc(129M), and (filled blue squares) Type 2 PrP^Sc(129M). The (<b>D</b>) inverse relationship between stability of total PrP^Sc and amplification index; (<b>E</b>) no correlation between stability of rPrP^Sc and amplification index; and (<b>F</b>) direct correlation between PK-induced change in the stability of PrP^Sc (Δ Fapp) and amplification index. The stability of prion and conversion potency of PrP^Sc was determined by CDI and expressed as Gdn HCl_1/2 or stability change (Δ Fapp) induced by PK. Each symbol represents an average of triplicate experiment followed by triplicate measurement ± SEM with CDI.</p

The inverse relationship between proportion of small oligomers of PrPSc and duration of sCJD.

<p>The levels of small oligomers of PrPSc with the highest seeding potency that eluted at 2.4–3.2 ml of sucrose gradient (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002835#ppat-1002835-g005" target="_blank"><b>Fig. 5D and Fig. 5E</b></a>) were expressed as a percentage of total PrPSc in (circle) MM1 (n = 6) and (square) MM2 (n = 6) sCJD cases. Each symbol represents an average ± SEM.</p