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Chronic wasting disease (CWD) is caused by an unknown spectrum of prions and has become enzootic in populations of cervid species that express cellular prion protein (PrPC) molecules varying in amino acid composition. These PrPC polymorphisms can affect prion transmission, disease progression, neuropathology, and emergence of new prion strains, but the mechanistic steps in prion evolution are not understood. Here, using conformation-dependent immunoassay, conformation stability assay, and protein-misfolding cyclic amplification, we monitored the conformational and phenotypic characteristics of CWD prions passaged through deer and transgenic mice expressing different cervid PrPC polymorphisms. We observed that transmission through hosts with distinct PrPC sequences diversifies the PrPCWD conformations and causes a shift toward oligomers with defined structural organization, replication rate, and host range. When passaged in host environments that restrict prion replication, distinct co-existing PrPCWD conformers underwent competitive selection, stabilizing a new prion strain. Nonadaptive conformers exhibited unstable replication and accumulated only to low levels. These results suggest a continuously evolving diversity of CWD conformers and imply a critical interplay between CWD prion plasticity and PrPC polymorphisms during prion strain evolution.

Tau protein accumulation is a common denominator of major dementias, but this process is inhomogeneous, even when triggered by the same germline mutation. We considered stochastic misfolding of human tau conformers followed by templated conversion of native monomers as an underlying mechanism and derived sensitive conformational assays to test this concept. Assessments of brains from aged TgTauP301L transgenic mice revealed a prodromal state and three distinct signatures for misfolded tau. Frontotemporal lobar degeneration (FTLD)-MAPT-P301L patients with different clinical phenotypes also displayed three signatures, two resembling those found in TgTauP301L mice. As physicochemical and cell bioassays confirmed diverse tau strains in the mouse and human brain series, we conclude that evolution of diverse tau conformers is intrinsic to the pathogenesis of this uni-allelic form of tauopathy. In turn, effective therapeutic interventions in FTLD will need to address evolving repertoires of misfolded tau species rather than singular, static molecular targets. Electronic supplementary material The online version of this article (10.1007/s00401-020-02148-4) contains supplementary material, which is available to authorized users.

There is a limited understanding of structural attributes that encode the iatrogenic transmissibility and various phenotypes of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Here we report the detailed structural differences between major sCJD MM1, MM2, and VV2 prions determined with two complementary synchrotron hydroxyl radical footprinting techniques—mass spectrometry (MS) and conformation dependent immunoassay (CDI) with a panel of Europium-labeled antibodies. Both approaches clearly demonstrate that the phenotypically distant prions differ in a major way with regard to their structural organization, and synchrotron-generated hydroxyl radicals progressively inhibit their seeding potency in a strain and structure-specific manner. Moreover, the seeding rate of sCJD prions is primarily determined by strain-specific structural organization of solvent-exposed external domains of human prion particles that control the seeding activity. Structural characteristics of human prion strains suggest that subtle changes in the organization of surface domains play a critical role as a determinant of human prion infectivity, propagation rate, and targeting of specific brain structures., Author summary Sporadic human prion diseases are conceivably the most heterogenous neurodegenerative disorders and a growing body of research indicates that they are caused by distinct strains of prions. By parallel monitoring their replication potency and progressive hydroxyl radical modification of amino acid side chains during synchrotron irradiation, we identified major differences in the structural organization that correlate with distinct inactivation susceptibility of a given human prion strain. Furthermore, our data demonstrated, for the first time, that seeding activity of different strains of infectious brain-derived human prions is primarily function of distinct solvent-exposed structural domains, and implicate them in the initial binding of cellular isoform of prion protein (PrPC) as a critical step in human prion replication and infectivity.

Amyloid beta (A beta) deposition in the neocortex is a major hallmark of Alzheimers disease (AD), but the extent of deposition does not readily explain phenotypic diversity and rate of disease progression. The prion strain-like model of disease heterogeneity suggests the existence of different conformers of A beta. We explored this paradigm using conformation-dependent immunoassay (CDI) for A beta and conformation-sensitive luminescent conjugated oligothiophenes (LCOs) in AD cases with variable progression rates. Mapping the A beta conformations in the frontal, occipital, and temporal regions in 20 AD patients with CDI revealed extensive interindividual and anatomical diversity in the structural organization of A beta with the most significant differences in the temporal cortex of rapidly progressive AD. The fluorescence emission spectra collected in situ from A beta plaques in the same regions demonstrated considerable diversity of spectral characteristics of two LCOs-quatroformylthiophene acetic acid and heptaformylthiophene acetic acid. Heptaformylthiophene acetic acid detected a wider range of A beta deposits, and both LCOs revealed distinct spectral attributes of diffuse and cored plaques in the temporal cortex of rapidly and slowly progressive AD and less frequent and discernible differences in the frontal and occipital cortex. These and CDI findings indicate a major conformational diversity of A beta accumulating in the neocortex, with the most notable differences in temporal cortex of cases with shorter disease duration, and implicate distinct A beta conformers (strains) in the rapid progression of AD. Funding Agencies|Alberta Innovates Bio Solutions [FP00209618]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [1RF1AG061797, AG066512, AG060882]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2019-04405, 2016-00748]; Swedish Alzheimer Foundation; Swedish Brain Foundation; Torsten Soderberg Foundation

Rapidly progressive Alzheimer’s disease (rpAD) is a particularly aggressive form of Alzheimer’s disease, with a median survival time of 7–10 months after diagnosis. Why these patients have such a rapid progression of Alzheimer’s disease is currently unknown. To further understand pathological differences between rpAD and typical sporadic Alzheimer’s disease (sAD) we used localized proteomics to analyze the protein differences in amyloid plaques in rpAD and sAD. Label-free quantitative LC–MS/MS was performed on amyloid plaques microdissected from rpAD and sAD patients (n = 22 for each patient group) and protein expression differences were quantified. On average, 913 ± 30 (mean ± SEM) proteins were quantified in plaques from each patient and 279 of these proteins were consistently found in plaques from every patient. We found significant differences in protein composition between rpAD and sAD plaques. We found that rpAD plaques contained significantly higher levels of neuronal proteins (p = 0.0017) and significantly lower levels of astrocytic proteins (p = 1.08 × 10−6). Unexpectedly, cumulative protein differences in rpAD plaques did not suggest accelerated typical sAD. Plaques from patients with rpAD were particularly abundant in synaptic proteins, especially those involved in synaptic vesicle release, highlighting the potential importance of synaptic dysfunction in the accelerated development of plaque pathology in rpAD. Combined, our data provide new direct evidence that amyloid plaques do not all have the same protein composition and that the proteomic differences in plaques could provide important insight into the factors that contribute to plaque development. The cumulative protein differences in rpAD plaques suggest rpAD may be a novel subtype of Alzheimer’s disease.

The molecular mechanism that determines under physiological conditions transmissibility of the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD) is unknown. We report the synthesis of new human prion from the recombinant human prion protein expressed in bacteria in reaction seeded with sCJD MM1 prions and cofactor, ganglioside GM1. These synthetic human prions were infectious to transgenic mice expressing non-glycosylated human prion protein, causing neurologic dysfunction after 459 and 224 days in the first and second passage, respectively. The neuropathology, replication potency, and biophysical profiling suggest that a novel, particularly neurotoxic human prion strain was created. Distinct biological and structural characteristics of our synthetic human prions suggest that subtle changes in the structural organization of critical domains, some linked to posttranslational modifications of the pathogenic prion protein (PrPSc), play a crucial role as a determinant of human prion infectivity, host range, and targetting of specific brain structures in mice models., Synthetic prions have previously been generated from recombinant rodent PrP. Here the authors generate synthetic human prions, by seeding human PrP with CJD prions, and characterize its infectivity in mice.

In lethal prion neurodegenerative diseases, misfolded prion proteins (PrP Sc ) replicate by redirecting the folding of the cellular prion glycoprotein (PrP C ). Infections of different durations can have a subclinical phase with constant levels of infectious particles, but the mechanisms underlying this plateau and a subsequent exit to overt clinical disease are unknown. Using tandem biophysical techniques, we show that attenuated accumulation of infectious particles in presymptomatic disease is preceded by a progressive fall in PrP C level, which constricts replication rate and thereby causes the plateau effect. Furthermore, disease symptoms occurred at the threshold associated with increasing levels of small, relatively less protease-resistant oligomeric prion particles (oPrP Sc ). Although a hypothetical lethal isoform of PrP cannot be excluded, our data argue that diminishing residual PrP C levels and continuously increasing levels of oPrP Sc are crucial determinants in the transition from presymptomatic to symptomatic prion disease. IMPORTANCE Prions are infectious agents that cause lethal brain diseases; they arise from misfolding of a cell surface protein, PrP C to a form called PrP Sc . Prion infections can have long latencies even though there is no protective immune response. Accumulation of infectious prion particles has been suggested to always reach the same plateau in the brain during latent periods, with clinical disease only occurring when hypothetical toxic forms (called PrP L or TPrP) begin to accumulate. We show here that infectivity plateaus arise because PrP C precursor levels become downregulated and that the duration of latent periods can be accounted for by the level of residual PrP C , which transduces a toxic effect, along with the amount of oligomeric forms of PrP Sc .

The infectious pathogen responsible for prion diseases is the misfolded, aggregated form of the prion protein, PrPSc. In contrast to recent progress in studies of laboratory rodent-adapted prions, current understanding of the molecular basis of human prion diseases and, especially, their vast phenotypic diversity is very limited. Here, we have purified proteinase resistant PrPSc aggregates from two major phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD), determined their conformational stability and replication tempo in vitro, as well as characterized structural organization using recently emerged approaches based on hydrogen/deuterium (H/D) exchange coupled with mass spectrometry. Our data clearly demonstrate that these phenotypically distant prions differ in a major way with regard to their structural organization, both at the level of the polypeptide backbone (as indicated by backbone amide H/D exchange data) as well as the quaternary packing arrangements (as indicated by H/D exchange kinetics for histidine side chains). Furthermore, these data indicate that, in contrast to previous observations on yeast and some murine prion strains, the replication rate of sCJD prions is primarily determined not by conformational stability but by specific structural features that control the growth rate of prion protein aggregates., Author Summary Sporadic Creutzfeldt-Jakob disease (sCJD) represents ~90% of all human prion diseases worldwide. This neurodegenerative disease, which is transmissible and invariably fatal, is characterized by variable progression rates and remarkable diversity of clinical and pathological traits. The infectious sCJD prions propagating the pathology mainly in the brain are assemblies of abnormally folded isoform (PrPSc) of a host-encoded prion protein (PrPC). The structure and replication mechanisms of human prions are unknown, and whether the PrPSc subtypes identified by proteolytic fragmentation represent distinct strains of sCJD prions has been debated. Here, we isolated sCJD prions from patients with two very distant phenotypes of the disease, compared their structural organization using recently developed biophysical techniques, and investigated their replication in vitro. Our data indicate that these sCJD prions are characterized by different secondary structure organization and quaternary packing arrangements, and that these structural differences are responsible for distinct prion replication rates and unique phenotypic characteristics of the disease. Furthermore, our analysis reveals that, contrary to previous observations for yeast prions, the replication tempo of sCJD prions is determined not so much by their conformational stability but by specific structural features that control the growth speed of prion particles.

Genetic and environmental factors that increase the risk of late-onset Alzheimer disease are now well recognized but the cause of variable progression rates and phenotypes of sporadic Alzheimer's disease is largely unknown. We aimed to investigate the relationship between diverse structural assemblies of amyloid-β and rates of clinical decline in Alzheimer's disease. Using novel biophysical methods, we analysed levels, particle size, and conformational characteristics of amyloid-β in the posterior cingulate cortex, hippocampus and cerebellum of 48 cases of Alzheimer's disease with distinctly different disease durations, and correlated the data with APOE gene polymorphism. In both hippocampus and posterior cingulate cortex we identified an extensive array of distinct amyloid-β42 particles that differ in size, display of N-terminal and C-terminal domains, and conformational stability. In contrast, amyloid-β40 present at low levels did not form a major particle with discernible size, and both N-terminal and C- terminal domains were largely exposed. Rapidly progressive Alzheimer's disease that is associated with a low frequency of APOE e4 allele demonstrates considerably expanded conformational heterogeneity of amyloid-β42, with higher levels of distinctly structured amyloid-β42 particles composed of 30-100 monomers, and fewer particles composed of < 30 monomers. The link between rapid clinical decline and levels of amyloid-β42 with distinct structural characteristics suggests that different conformers may play an important role in the pathogenesis of distinct Alzheimer's disease phenotypes. These findings indicate that Alzheimer's disease exhibits a wide spectrum of amyloid-β42 structural states and imply the existence of prion-like conformational strains.

Background:While the genetic and environmental factors that increase the risk of late onset Alzheimer disease (LOAD) are well recognized, the cause of variable progression rates and phenotypes of sporadic LOAD is largely unknown. We aimed to investigate the relationship between diverse conformational structures of amyloid beta (Ab) and variable rates of clinical decline in LOAD.Methods:Using novel biophysical methods, we analyzed conformational structural characteristics of Ab in the posterior cingulate cortex, hippocampus, and cerebellum of 49 cases of LOAD with distinctly different disease durations, and correlated the data with clinical profiles and APOE gene polymorphism. Results: In both hippocampus and posterior cingulate cortex we identified an extensive array of distinct Ab42 particles that differ in conformational structure, size, and display of critical domains. In contrast, Ab40 present at low levels did not form a major particle with discernible size, and both N-terminal and Cterminal domains were largely exposed. Rapidly progressive cases demonstrated distinctly structured Ab42 forming unique spectrum of oligomeric particles. Conclusions: The link between rapid clinical decline and levels of Ab42 with distinct structural characteristics suggests that differently structured conformers play an important role in the pathogenesis od LOAD. Specifically, we assert that the variable propagation tempo and phenotypic characteristics of the disease may be encoded in the distinct structures of Ab42 by prion-like mechanism.

The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrPSc), which is derived from its cellular precursor (PrPC), as well as downregulation of the PrP-like Shadoo (Sho) glycoprotein. Given the overlapping cellular environments for PrPC and Sho, we inferred that PrPC levels might also be altered as part of a host response during prion infection. Using rodent models, we found that, in addition to changes in PrPC glycosylation and proteolytic processing, net reductions in PrPC occur in a wide range of prion diseases, including sheep scrapie, human Creutzfeldt-Jakob disease, and cervid chronic wasting disease. The reduction in PrPC results in decreased prion replication, as measured by the protein misfolding cyclic amplification technique for generating PrPSc in vitro. While PrPC downregulation is not discernible in animals with unusually short incubation periods and high PrPC expression, slowly evolving prion infections exhibit downregulation of the PrPC substrate required for new PrPSc synthesis and as a receptor for pathogenic signaling. Our data reveal PrPC downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains.

The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion proteins (PrP(Sc)). The molecular mechanism responsible for the adaptation, mutation, and evolution of prions observed in cloned cells and upon crossing the species barrier remains unsolved. Using biophysical techniques and conformation-dependent immunoassays in tandem, we isolated two distinct populations of PrP(Sc) particles with different conformational stabilities and aggregate sizes, which frequently co-exist in the most common human prion disease, sporadic Creutzfeldt-Jakob disease. The protein misfolding cyclic amplification replicates each of the PrP(Sc) particle types independently and leads to the competitive selection of those with lower initial conformational stability. In serial propagation with a nonglycosylated mutant PrP(C) substrate, the dominant PrP(Sc) conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to its lowest stability. Cumulatively, the data show that sporadic Creutzfeldt-Jakob disease PrP(Sc) is not a single conformational entity but a dynamic collection of two distinct populations of particles. This implies the co-existence of different prions, whose adaptation and evolution are governed by the selection of progressively less stable, faster replicating PrP(Sc) conformers.

The mammalian prions replicate by converting cellular prion protein (PrPC) into pathogenic conformational isoform (PrPSc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrPSc on conversion of PrPC in vitro using PrPSc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrPSc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrPSc. The tight correlation between conversion potency of small oligomers of human sPrPSc observed in vitro and duration of the disease suggests that sPrPSc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease., Author Summary Mammalian prion diseases were originally characterized by accumulation of protease-resistant prion protein (PrPSc), often forming large amyloid deposits and fibrils. However, the apparent absence of protease-resistant PrPSc or amyloid fibrils in growing number of prion diseases raised several fundamental questions; specifically, whether presumably protease-sensitive forms of PrPSc exist as distinct conformers; and whether they comprise the initial steps in prion replication or are related to the alternative misfolding pathway generating noninfectious aggregates. We investigated the conformational characteristics of protease sensitive conformers of PrPSc and their role in the pathogenesis of sporadic Creutzfeldt-Jakob disease (sCJD). Using two different in vitro prion protein (PrPC) conversion techniques in tandem with biophysical methods, we identified small oligomers of protease sensitive PrPSc present in sCJD brains as the most potent initiators of PrPC conversion. Their concentration and conformational stability determine the distinctly different replication potency of PrPSc in individual isolates of sCJD and each of these characteristics correlates tightly with duration of the disease. These features argue for a broad range of distinct prion strains causing the sCJD and imply that small oligomers of protease sensitive conformers of pathogenic prion protein are encoding incubation time and progression rate of the disease.

The origin, range, and structure of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD), are largely unknown. To investigate the molecular mechanism responsible for the broad phenotypic variability of sCJD, we analyzed the conformational characteristics of protease-sensitive and protease-resistant fractions of the pathogenic prion protein (PrPSc) using novel conformational methods derived from a conformation-dependent immunoassay (CDI). In 46 brains of patients homozygous for polymorphisms in the PRNP gene and exhibiting either Type 1 or Type 2 western blot pattern of the PrPSc, we identified an extensive array of PrPSc structures that differ in protease sensitivity, display of critical domains, and conformational stability. Surprisingly, in sCJD cases homozygous for methionine or valine at codon 129 of the PRNP gene, the concentration and stability of protease-sensitive conformers of PrPSc correlated with progression rate of the disease. These data indicate that sCJD brains exhibit a wide spectrum of PrPSc structural states, and accordingly argue for a broad spectrum of prion strains coding for different phenotypes. The link between disease duration, levels, and stability of protease-sensitive conformers of PrPSc suggests that these conformers play an important role in the pathogenesis of sCJD., Author Summary Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common human prion disease worldwide. This neurodegenerative disease, which is transmissible and invariably fatal, is characterized by the accumulation of an abnormally folded isoform (PrPSc) of a host-encoded protein (PrPC), predominantly in the brain. Most researchers believe that PrPSc is the infectious agent and five or six subtypes of sCJD have been identified. Whether or not these subtypes represent distinct strains of sCJD prions is debated in the context of the extraordinary variability of sCJD phenotypes, frequent co-occurrence of different PrPSc fragments in the same brain, and the fact that up to 90% of protease-sensitive PrPSc eludes the conventional analysis because it is destroyed by protease treatment. Using novel conformational methods, we identified within each clinical and pathological category an array of PrPSc structures that differ in protease-sensitivity, display of critical domains, and conformational stability. Each of these features offers evidence of a distinct conformation. The link between the rate at which the disease progresses, on the one hand, and the concentration and stability of protease-sensitive conformers of PrPSc on the other, suggests that these conformers play an important role in how the disease originates and progresses.