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

Abstract

Prion diseases are fatal neurodegenerative diseases in mammals with the unique characteristics of misfolding and aggregation of the cellular prion protein (PrP^C) to the scrapie prion (PrP^Sc). Although neuroinflammation and neuronal loss feature within the disease process, the details of PrP^C/PrP^Sc 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 PrP^C and PrP^Sc 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 PrP^C levels at pre-clinical stages of the disease and demonstrate that these decreases become significant before neuronal loss. We also identified the earliest PrP^Sc 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 PrP^Sc 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.

Keywords: Asymmetric flow field-flow fractionation; Neuronal loss; PrP assemblies; PrPC downregulation; Prion disease; RML.

Fig. 1

Quantification of brain cell populations by isotropic fractionation. a Quantification of total brain cells indicates a significantly higher number of cells at 120 DPI and a significant decrease in the number of cells at 150 DPI in RML-inoculated animals. b Quantification of neuronal cells in the brain indicates a significant decrease in the number of neurons at 150 DPI in RML-inoculated animals. c Quantification of non-neuronal cell populations suggests a trend towards increase at 90 DPI, and a significantly higher number of cells at 120 DPI in RML-inoculated animals. d Monitoring weight changes of NBH and RML-infected animals reveals a significant decrease in animal weight at 120 and 150 DPI in RML-inoculated mice. e No significant difference was detected in brain wet weight between NBH and RML inoculated animals. Data obtained from 5 animals were averaged per group in each timepoint. Error bars represent SD. (p values * ≤ 0.05, ** ≤ 0.01, and *** ≤ 0.001)

Fig. 2

H&E staining of brain sections from RML and NBH inoculated animals at different timepoints during disease progression. a A section of the brain consisting of the hippocampus and part of the cerebral cortex are presented. Vacuoles are first noticeable at 90 DPI. b Quantification of H&E staining on half-brain sagittal sections by Weka image segmentation and ImageJ. The values obtained at terminal-stage were adjusted to 1 and the rest of the timepoints were normalized relative to that. * indicates p value ≤ 0.05 and *** indicates p value ≤ 0.001. n.s indicates not significant

Fig. 3

PrP^Sc immunostaining of brain sections from RML and NBH inoculated animals at different timepoints. a A section of the brain consisting of the hippocampus and part of the cerebral cortex are presented. PK-resistant PrP is first noticeable at 90 DPI. b Quantification of PrP immunostaining on half-brain sagittal sections by ImageJ. The values obtained at terminal-stage were adjusted to 1 and the rest of the timepoints were normalized relative to that. * indicates p value ≤ 0.05, ** indicates p value ≤ 0.01, and *** indicates p value ≤ 0.001. n.s indicates not significant

Fig. 4

GFAP immunostaining of brain slices from RML and NBH inoculated animals. a A section of the brain consisting of the hippocampus and part of the cerebral cortex are presented. Increased GFAP immunoreactivity is first noticeable at 90 DPI. b Quantification of GFAP immunostaining on half-brain sagittal sections by ImageJ. * indicates p value ≤ 0.05

Fig. 5

Assessment of inflammatory and synaptic health biomarkers at different timepoints during disease progression. a Immunoblotting of TREM2 protein in solubilized brain homogenate of RML-inoculated animals. Antibody (anti-TREM2 1/5000). b Immunoblotting of Iba1 protein in solubilized brain homogenate of RML-inoculated animals. Antibody (anti-Iba1 1/5000). c Immunoblotting of synaptophysin protein in solubilized brain homogenate of RML-inoculated animals. A 150-DPI sample from an NBH-inoculated animal was used as a control for panels a–c. Antibody (anti-synaptophysin 1/2000). Quantification of the blots was done by ImageJ. Data represents an average of 3 repeats. Error bars represent SD. * indicates p value ≤ 0.05. n.s indicates not significant

Fig. 6

Total and PK-resistant PrP in solubilized brain homogenates. a Immunoblotting of total PrP in solubilized brain homogenate of RML-inoculated animals. A NBH-inoculated brain homogenate at 150 DPI is used as the control. b Immunoblotting of PK-resistant PrP^Sc in solubilized brain homogenate of RML-inoculated animals at different timepoints during disease progression. A NBH-inoculated brain homogenate at 150 DPI was used as a control as per Fig. 5. Antibody Sha 31 (1/30,000). Quantification of the blots was done by ImageJ. Data represents an average of 3 repeats. * indicates p value ≤ 0.05, ** indicates p value ≤ 0.01, and *** indicates p value ≤ 0.001. n.s indicates not significant

Fig. 7

AF4 analysis of solubilized brain homogenate of RML-infected animals at different timepoints during disease progression. The Y-axis represents the intensity of total PrP in arbitrary units (see supplementary Fig. 1). a A representative graph from averaged fractionated brain of NBH-inoculated animals at 150 DPI, b fractionated brain of RML-inoculated mice sacrificed at 30 DPI, c fractionated brain of RML-inoculated mice sacrificed at 60 DPI, d fractionated brain of RML-inoculated mice sacrificed at 90 DPI, e fractionated brain of RML-inoculated mice sacrificed at 120 DPI, and f fractionated brain of RML-inoculated mice sacrificed at terminal stage (150 DPI). Data represent an average from 2 biological replicates (i.e., 2 animals). Error bars represent SD

Fig. 8

PrP^C levels in brain homogenate of RML-inoculated animals at different timepoints. Protein levels were quantified by integrating the values from fractions corresponding to PrP^C obtained from fractionation of brains from NBH-inoculated mice (green trace, see Fig. 7b–f) and expressing the integrated values from infected animals as a percentage of this reference value. There is a trend towards decrease at 90 DPI, and a significant reduction at 120 and 150 DPI (29% and 38% respectively). Biological replicates are as per Fig. 7. Error bars represent SD. ** indicates p value ≤ 0.01. n.s indicates not significant

Fig. 9

PK-resistant PrP^Sc in fractionated brain of RML-inoculated animals at different timepoints. The Y-axis represents the intensity of Pk-digested PrP in arbitrary units (see supplementary Fig. 2). a A representative graph from PK-digested fractionated brain of a NBH-inoculated animal at 150 DPI, b PK-digested fractionated brain of RML-inoculated mice sacrificed at 30 DPI, c PK-digested fractionated brain of RML-inoculated mice sacrificed at 60 DPI, d PK-digested fractionated brain of RML-inoculated mice sacrificed at 90 DPI, e PK-digested fractionated brain of RML-inoculated mice sacrificed at 120 DPI, and f PK-digested fractionated brain of RML-inoculated mice sacrificed at terminal stage (150 DPI). Biological replicates are as per Fig. 7. Error bars represent SD

Fig. 10

Seeding activity of total brain homogenate of RML-inoculated animals at different timepoints. Two microliters of 10% brain homogenate was used to assess the seeding potential of PrP species present in each timepoint. Data represents an average of 5 repeats. Error bars represent SEM