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 (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.

Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm

“…Alternately, if the smallest particles with partial resistance to PK digestion simply came from the fragmentation of larger PrP res particles in the brain or during sample preparation, the 15 nm R h PrP res particles might not reflect the point at which conformational change occurs, but rather the smallest size at which a PK-resistant conformation can be maintained. Interestingly, we recently described the evolution of PrP Sc particles in the brain of RML-infected mice over the course of disease, observing that PrP res particles were also always 15 nm R h or larger [ 29 ].…”

“…The use of inline multi-angle and dynamic light scattering (MALS and DLS) detectors allows for precise measurement of the size and shape of the eluting particles. Surprisingly, the use of AF4 technology to study neurodegenerative disease-related protein aggregates has been very limited to date [26][27][28][29]. Within the field of prion disease research, AF4-MALS-DLS was first used to determine the size of the most infectious prion particle [26].…”

“…Surprisingly, the use of AF4 technology to study neurodegenerative disease-related protein aggregates has been very limited to date [ 26 – 29 ]. Within the field of prion disease research, AF4-MALS-DLS was first used to determine the size of the most infectious prion particle [ 26 ].…”

“…In this seminal work, PrP Sc from 263K-infected Syrian hamster brains was first highly purified and digested with proteinase K, then disrupted by detergent, sonication and freeze-thaw before being fractionated and analyzed. Recently, we developed a method for using AF4 on brain homogenates without the need for initial purification or sonication, and with in-line DLS measurements, we were able to ascertain the size distribution of PrP Sc particles in the brains of RML-infected mice [ 29 ]. Now we have applied our isolation method and AF4-MALS-DLS analysis to the characterization of inter- and intra-strain differences of subpopulations of PrP Sc particles in the brains of Syrian golden hamsters infected with three different prion strains.…”

Structural and Electronic Analysis of the Octarepeat Region of Prion Protein with Four Cu(II) by Polarizable MD and QM/MM Simulations

“…[5] The formation of proteinase K-sensitive PrP Sc oligomers is associated with serious pathological changes in the brain. [6] PrP C is found in several mammal species such as mice, hamsters, monkeys, sheep, goats, minks, cattle, deer and humans. [7][8][9][10][11] PrP is a typical component of many types of tissues such as lung and kidney.…”