Quaternary Structure Changes for PrPSc Predate PrPC Downregulation and Neuronal Death During Progression of Experimental Scrapie Disease

Ghazaleh Eskandari-Sedighi,Klinton Shmeit,Valerie Sim,Nathalie Daude,Jing Yang,David Westaway,Leonardo M Cortez

doi:10.1007/s12035-020-02112-z

Ghazaleh Eskandari-Sedighi, Klinton Shmeit + Show 5 more

Open Access

https://doi.org/10.1007/s12035-020-02112-z

Copy DOI

Journal: Molecular Neurobiology	Publication Date: Sep 21, 2020
Citations: 9	License type: open-access

Affiliation: University of Alberta

Abstract

Prion diseases are fatal neurodegenerative diseases in mammals with the unique characteristics of misfolding and aggregation of the cellular prion protein (PrPC) to the scrapie prion (PrPSc). Although neuroinflammation and neuronal loss feature within the disease process, the details of PrPC/PrPSc molecular transition to generate different aggregated species, and the correlation between each species and sequence of cellular events in disease pathogenesis are not fully understood. In this study, using mice inoculated with the RML isolate of mouse-adapted scrapie as a model, we applied asymmetric flow field-flow fractionation to monitor PrPC and PrPSc particle sizes and we also measured seeding activity and resistance to proteases. For cellular analysis in brain tissue, we measured inflammatory markers and synaptic damage, and used the isotropic fractionator to measure neuronal loss; these techniques were applied at different timepoints in a cross-sectional study of disease progression. Our analyses align with previous reports defining significant decreases in PrPC levels at pre-clinical stages of the disease and demonstrate that these decreases become significant before neuronal loss. We also identified the earliest PrPSc assemblies at a timepoint equivalent to 40% elapsed time for the disease incubation period; we propose that these assemblies, mostly composed of proteinase K (PK)–sensitive species, play an important role in triggering disease pathogenesis. Lastly, we show that the PK-resistant assemblies of PrPSc that appear at timepoints close to the terminal stage have similar biophysical characteristics, and hence that preparative use of PK-digestion selects for this specific subpopulation. In sum, our data argue that qualitative, as well as quantitative, changes in PrP conformers occur at the midpoint of subclinical phase; these changes affect quaternary structure and may occur at the threshold where adaptive responses become inadequate to deal with pathogenic processes.

Highlights

Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Prion diseases are fatal neurodegenerative diseases in humans and animals with the common feature of misfolding and aggregation of the cellular prion glycoprotein (PrPC) to proteaseresistant “Scrapie” prion protein (PrPSc) [1,2,3]
With regard to determining the disease-related endpoint measure of cell loss, we used the isotropic fractionation technique; this is a non-stereological method to quantify different cell populations in the brain based on counting isolated nuclei following detergent lysis of cell membranes and its accuracy has been verified by cross-referencing to other techniques [27,28,29,30,31]; we took advantage of this method to determine the number of cells in the brain at different timepoints in RML inoculated animals, as well as in matched controls
In parallel to these measurements, we observed a significant decrease in body weight of the RML-infected mice; the wet weight of brains showed no significant changes in any of the cohorts excluding this parameter as a confound for data interpretation (Fig. 1d, e)

Summary

Introduction

Prion diseases are fatal neurodegenerative diseases in humans and animals with the common feature of misfolding and aggregation of the cellular prion glycoprotein (PrPC) to proteaseresistant “Scrapie” prion protein (PrPSc) [1,2,3]. The pathogenesis of prion diseases is associated with a progressive accumulation of PrPSc molecules. Prion diseases with different incubation periods share a relatively long subclinical stage during which levels of infectious titer do not grow exponentially, but instead reach a maximum [4]. The molecular mechanism of this plateau effect, as well as the process through which the subsequent exit to overt clinical disease occurs, is debated, previous work suggests that a decrease in PrPC protein levels at pre-clinical stages of the disease could be of importance in controlling pathogenesis [5, 6]

Results

Discussion

Conclusion