PMCA-replicated PrPD in urine of vCJD patients maintains infectivity and strain characteristics of brain PrPD: Transmission study

Ignazio Cali,Silvio Notari,Brian Appleby,Jody Lavrich,Claudio Soto,Diane Kofskey,Pierluigi Gambetti,Fabio Moda,Satish Kumar Nemani,Fabrizio Tagliavini

doi:10.1038/s41598-019-41694-0

Abstract

The presence of abnormal, disease-related prion protein (PrPD) has recently been demonstrated by protein misfolding cyclic amplification (PMCA) in urine of patients affected with variant Creutzfeldt-Jakob disease (vCJD), a prion disease typically acquired from consumption of prion contaminated bovine meat. The complexity and multistage process of urine excretion along with the obligatory use of PMCA raise the issue of whether strain characteristics of the PrPD present in vCJD brains, such as infectivity and phenotype determination, are maintained in urine excreted PrPD and following amplification by PMCA. We inoculated transgenic mice expressing normal human PrP with amplified urine and brain homogenate achieving the same 100% attack rate, similar incubation periods (in both cases extremely long) and histopathological features as for type and severity of the lesions. Furthermore, PrPD characteristics analyzed by immunoblot and conformational stability immunoassay were indistinguishable. Inoculation of raw vCJD urine caused no disease, confirming the extremely low concentration of PrPD in vCJD urine. These findings show that strain characteristics of vCJD brain PrPD, including infectivity, are preserved in PrPD present in urine and are faithfully amplified by means of PMCA; moreover, they suggest that the PrPD urine test might allow for the diagnosis and identification of disease subtype also in sporadic CJD.

Highlights

The diversity of human prion diseases is in part due to the presence of three etiological formssporadic, inherited and acquired by infectionwhile in all other neurodegenerative diseases only the sporadic and inherited forms are currently recognized[1,2]
These results were comparable to those obtained with the transmission of variant CreutzfeldtJakob disease (vCJD) brain homogenate (BH) where attack rate again was always 100% and incubation periods varied between 574 ± 104 and 648 ± 39 dpi, according to the 10% or 1% BH concentration of the inoculum (Table 1)
We have recently shown that urine of patients affected by vCJD harbors minute amounts of PrPD estimated at 1 × 10−16 g/mL, which is approximately 12 orders of magnitude smaller than the PrPD amount in the vCJD brain tissue (1 × 10−4 g/g)[11]

Summary

Introduction

The diversity of human prion diseases is in part due to the presence of three etiological formssporadic, inherited and acquired by infectionwhile in all other neurodegenerative diseases only the sporadic and inherited forms are currently recognized[1,2]. The complex, multistage process of urine formation, raises the possibility that, while spreading to urine, PrPD undergo subtle conformational changes altering strain characteristics. This issue is further compounded by the extreme under-representation of PrPD in urine that requires extensive amplification or enrichment procedures[11,12]. Transmission to appropriate hosts has been shown to be a suitable approach to define and compare prion strain properties[13] To this aim, we inoculated transgenic (Tg) mice expressing human PrP 129 M (Tg40) with PrPD obtained from vCJD urine following PMCA and untreated vCJD brain homogenate (BH). None of the animals inoculated with untreated vCJD urine developed a prion disease

Methods

Results

Conclusion