Detection of prion seeding activity in the olfactory mucosa of patients with Fatal Familial Insomnia

Veronica Redaelli,Michele Di Bari,Martina Rossi,Umberto Agrimi,Giuseppe Legname,Giulia Salzano,Gianluigi Forloni,Fabrizio Tagliavini,Ignazio Roiter,Gianluigi Zanusso,Chiara Maria Giulia De Luca,Fabio Moda,Edoardo Bistaffa,Sara Maria Portaleone,Luca Sacchetto

doi:10.1038/srep46269

Abstract

Fatal Familial Insomnia (FFI) is a genetic prion disease caused by a point mutation in the prion protein gene (PRNP) characterized by prominent thalamic atrophy, diffuse astrogliosis and moderate deposition of PrPSc in the brain. Here, for the first time, we demonstrate that the olfactory mucosa (OM) of patients with FFI contains trace amount of PrPSc detectable by PMCA and RT-QuIC. Quantitative PMCA analysis estimated a PrPSc concentration of about 1 × 10−14 g/ml. In contrast, PrPSc was not detected in OM samples from healthy controls and patients affected by other neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease and frontotemporal dementia. These results indicate that the detection limit of these assays is in the order of a single PrPSc oligomer/molecule with a specificity of 100%.

Highlights

Transmissible Spongiform Encephalopathies (TSEs) are a group of fatal and incurable neurodegenerative diseases that might present as sporadic, acquired or genetic conditions
To assess whether PrPSc associated to Fatal Familial Insomnia (FFI) (FFI-PrPSc) was able to amplify by means of Protein Misfolding Cyclic Amplification (PMCA), we have performed spiking experiment diluting FFI brain homogenate into 10% healthy bank vole brain homogenates (BvBH) carrying methionine at position 109 of the prion protein (M109)
Real Time Quaking-Induced Conversion (RT-QuIC) assay on olfactory mucosa (OM) samples obtained by nasal swabbing has been largely tested in patients with sporadic or genetic forms of Creutzfeldt-Jakob disease (CJD), showing a specificity of 100% and sensitivity of 97 and 75%, respectively, but never in patients with FFI or at risk of developing FFI

Summary

Introduction

Transmissible Spongiform Encephalopathies (TSEs) are a group of fatal and incurable neurodegenerative diseases that might present as sporadic, acquired or genetic conditions. PMCA12 consists of cycles of incubation and sonication of samples containing small amount of PrPSc in the presence of an excess of PrPC This enables the exponential amplification of PrPSc, and can begin the reaction with the equivalent of a single molecule of PrPSc, which after amplification can give rise to billions of PrPSc molecules[13]. RT-QuIC18 alternates phases of incubation to phases of vigorous shaking where soluble recombinant prion proteins (recPrP) are used as substrate for detecting femtomolar amounts of PrPSc. The reaction is monitored by Thioflavin-T (ThT) fluorescence and enabled detection of PrP seeds in the cerebrospinal fluid (CSF) of patients with sporadic CJD (sCJD) or genetic forms of prion disease (e.g. GSS and FFI) either in the symptomatic[19] or pre-symptomatic stage of the disease[20,21]. These findings suggest that RT-QuIC and PMCA have a huge potential to detect trace-amount of PrPSc (≥1 femtogram) in peripheral tissues

Methods

Results

Conclusion