Search

Mammalian prion or PrP

The transformation of astrocytes into reactive states constitutes a biological response of the central nervous system under a variety of pathological insults. Astrocytes display diverse homeostatic identities, which are developmentally predetermined and regionally specified. Upon transformation into reactive states associated with neurodegenerative diseases and other neurological disorders, astrocytes acquire diverse reactive phenotypes. However, it is not clear whether their reactive phenotypes are dictated by regionspecific homeostatic identity or, alternatively, by the nature of an insult. To address this question, regionspecific gene expression profiling was performed for four brain regions (cortex, hippocampus, thalamus and hypothalamus) in mice using a custom Nanostring panel consisting of selected sets of genes that report on astrocyte functions and their reactivity for five conditions: prion disease, traumatic brain injury, brain ischemia, 5XFAD Alzheimer’s disease model and normal aging. Upon transformation into reactive states, genes that are associated predominantly with astrocytes were found to preserve region-specific signatures suggesting that they respond to insults in a region-specific manner. A common gene set was found to be involved in astrocyte remodeling across insults and normal aging. Regardless of the nature of an insult or insult-specificity of astrocyte response, strong correlations between the degree of astrocyte reactivity and perturbations in their homeostasis-associated genes were observed within each individual brain region. The insult-specific populations did not separate well from each other and instead partially overlapped, forming continuums of phenotypes. The current study demonstrates that astrocytes acquire their reactive phenotypes according to their region-specific homeostatic identities. Within these region-specified identities, reactive phenotypes show continuums of states, partially overlapping between individual insults.

Prion diseases are a group of fatal neurodegenerative diseases caused by misfolding of the normal cellular form of the prion protein or PrPC, into a disease-associated self-replicating state or PrPSc. PrPC and PrPSc are posttranslationally modified with N-linked glycans, in which the terminal positions occupied by sialic acids residues are attached to galactose predominantly via α2-6 linkages. The sialylation status of PrPSc is an important determinant of prion disease pathogenesis, as it dictates the rate of prion replication and controls the fate of prions in an organism. The current study tests whether a knockout of ST6Gal1, one of the two mammalian sialyltransferases that catalyze the sialylation of glycans via α2-6 linkages, reduces the sialylation status of PrPSc and alters prion disease pathogenesis. We found that a global knockout of ST6Gal1 in mice significantly reduces the α2-6 sialylation of the brain parenchyma, as determined by staining with Sambucus Nigra agglutinin. However, the sialylation of PrPSc remained stable and the incubation time to disease increased only modestly in ST6Gal1 knockout mice (ST6Gal1-KO). A lack of significant changes in the PrPSc sialylation status and prion pathogenesis is attributed to the redundancy in sialylation and, in particular, the plausible involvement of a second member of the sialyltransferase family that sialylate via α2-6 linkages, ST6Gal2.

The possibility that the etiology of late onset Alzheimer's disease is linked to viral infections of the CNS has been actively debated in recent years. According to the antiviral protection hypothesis, viral pathogens trigger aggregation of Aβ peptides that are produced as a defense mechanism in response to infection to entrap and neutralize pathogens. To test the causative relationship between viral infection and Aβ aggregation, the current study examined whether Aβ plaques protect the mouse brain against Herpes Simplex Virus 1 (HSV-1) infection introduced via a physiological route and whether HSV-1 infection triggers formation of Aβ plaques in a mouse model of late-onset AD that does not develop Aβ pathology spontaneously. In aged 5XFAD mice infected via eye scarification, high density of Aβ aggregates did not improve survival time or rate when compared with wild type controls. In 5XFADs, viral replication sites were found in brain areas with a high density of extracellular Aβ deposits, however, no association between HSV-1 and Aβ aggregates could be found. To test whether HSV-1 triggers Aβ aggregation in a mouse model that lacks spontaneous Aβ pathology, 13-month-old hAβ/APOE4/Trem2*R47H mice were infected with HSV-1 via eye scarification with the McKrae HSV-1 strain, intracranial inoculation with McKrae, intracranial inoculation after priming with LPS for 6 weeks, or intracranial inoculation with high doses of McKrae or 17syn + strains that represent different degrees of neurovirulence. No signs of Aβ aggregation were found in any of the experimental groups. Instead, extensive infiltration of peripheral leukocytes was observed during the acute stage of HSV-1 infection, and phagocytic activity of myeloid cells was identified as the primary defense mechanism against HSV-1. The current results argue against a direct causative relationship between HSV-1 infection and Aβ pathology.

Posttranslational modifications are a common feature of proteins associated with neurodegenerative diseases including prion protein (PrP(C)), tau, and α-synuclein. Alternative self-propagating protein states or strains give rise to different disease phenotypes and display strain-specific subsets of posttranslational modifications. The relationships between strain-specific structure, posttranslational modifications, and disease phenotype are poorly understood. We previously reported that among hundreds of PrP(C) sialoglycoforms expressed by a cell, individual prion strains recruited PrP(C) molecules selectively, according to the sialylation status of their N-linked glycans. Here we report that transmission of a prion strain to a new host is accompanied by a dramatic shift in the selectivity of recruitment of PrP(C) sialoglycoforms, giving rise to a self-propagating scrapie isoform (PrP(Sc)) with a unique sialoglycoform signature and disease phenotype. The newly emerged strain has the shortest incubation time to disease and is characterized by colocalization of PrP(Sc) with microglia and a very profound proinflammatory response, features that are linked to a unique sialoglycoform composition of PrP(Sc). The current work provides experimental support for the hypothesis that strain-specific patterns of PrP(Sc) sialoglycoforms formed as a result of selective recruitment dictate strain-specific disease phenotypes. This work suggests a causative relationship between a strain-specific structure, posttranslational modifications, and disease phenotype.

BackgroundChronic neuroinflammation is recognized as a major neuropathological hallmark in a broad spectrum of neurodegenerative diseases including Alzheimer’s, Parkinson’s, Frontal Temporal Dementia, Amyotrophic Lateral Sclerosis, and prion diseases. Both microglia and astrocytes exhibit region-specific homeostatic transcriptional identities, which under chronic neurodegeneration, transform into reactive phenotypes in a region- and disease-specific manner. Little is known about region-specific identity of glia in prion diseases. The current study was designed to determine whether the region-specific homeostatic signature of glia changes with the progression of prion diseases, and whether these changes occur in a region-dependent or universal manner. Also of interest was whether different prion strains give rise to different reactive phenotypes.MethodsTo answer these questions, we analyzed gene expression in thalamus, cortex, hypothalamus and hippocampus of mice infected with 22L and ME7 prion strains using Nanostring Neuroinflammation panel at subclinical, early clinical and advanced stages of the disease.ResultsWe found that at the preclinical stage of the disease, region-specific homeostatic identities were preserved. However, with the appearance of clinical signs, region-specific signatures were partially lost and replaced with a neuroinflammation signature. While the same sets of genes were activated by both prion strains, the timing of neuroinflammation and the degree of activation in different brain regions was strain-specific. Changes in astrocyte function scored at the top of activated pathways. Moreover, clustering analysis suggested that the astrocyte function pathway responded to prion infection prior to activated microglia or neuron and neurotransmission pathways.ConclusionsThe current work established neuroinflammation gene expression signature associated with prion diseases. Our results illustrate that with the disease progression, the region-specific homeostatic transcriptome signatures are replaced by region-independent neuroinflammation signature, which was common for prion strains with different cell tropism. The prion-associated neuroinflammation signature identified in the current study overlapped only partially with the microglia degenerative phenotype and the disease-associated microglia phenotype reported for animal models of other neurodegenerative diseases.

Chronic neuroinflammation involves reactive microgliosis and astrogliosis, and is regarded as a common pathological hallmark of neurodegenerative diseases including Alzheimer’s, Parkinson’s, ALS and prion diseases. Reactive astrogliosis, routinely observed immunohistochemically as an increase in glial fibrillary acidic protein (GFAP) signal, is a well-documented feature of chronic neuroinflammation associated with neurodegenerative diseases. Recent studies on single-cell transcriptional profiling of a mouse brain revealed that, under normal conditions, several distinct subtypes of astrocytes with regionally specialized distribution exist. However, it remains unclear whether astrocytic response to pro-inflammatory pathological conditions is uniform across whole brain or is region-specific. The current study compares the response of microglia and astrocytes to prions in mice infected with 22L mouse-adapted prion strain. While the intensity of reactive microgliosis correlated well with the extent of PrPSc deposition, reactive astrogliosis displayed a different, region-specific pattern. In particular, the thalamus and stratum oriens of hippocampus, which are both affected by 22L prions, displayed strikingly different response of astrocytes to PrPSc. Astrocytes in stratum oriens of hippocampus responded to accumulation of PrPSc with visible hypertrophy and increased GFAP, while in the thalamus, despite stronger PrPSc signal, the increase of GFAP was milder than in hippocampus, and the change in astrocyte morphology was less pronounced. The current study suggests that astrocyte response to prion infection is heterogeneous and, in part, defined by brain region. Moreover, the current work emphasizes the needs for elucidating region-specific changes in functional states of astrocytes and exploring the impact of these changes to chronic neurodegeneration.

Previous studies established that transmissible prion diseases could be induced by in vitro -produced recombinant prion protein (PrP) fibrils with structures that are fundamentally different from that of authentic PrP scrapie isoform (PrP Sc ). To explain evolution of synthetic prions, a new mechanism referred to as deformed templating was introduced. Here, we asked whether an increase in expression level of the cellular form of PrP (PrP C ) speeds up the evolution of synthetic strains in vivo . We found that in transgenic mice that overexpress hamster PrP C , PrP C overexpression accelerated recombinant PrP fibril-induced conversion of PrP C to the abnormal proteinase K-resistant state, referred to as atypical PrPres, which was the first product of PrP C misfolding in vivo . However, overexpression of PrP C did not facilitate the second step of synthetic strain evolution-transition from atypical PrPres to PrP Sc , which is attributed to the stochastic nature of rare deformed templating events. In addition, the potential of atypical PrPres to interfere with replication of a short-incubation time prion strain was investigated. Atypical PrPres was found to interfere strongly with replication of 263K in vitro ; however, it did not delay prion disease in animals. The rate of deformed templating does not depend on the concentration of substrate and is hence more likely to be controlled by the intrinsic rate of conformational errors in templating alternative self-propagating states.

Figure 6 of the original publication [1] contained an error in the Wavenumber in panels B and C. The wavenumbers 1616 (Cm-1) in panels B and C should have been 1516 (cm-1). The updated figure has been published in this correction article; the original article has been updated.

Last decade witnessed an enormous progress in generating authentic infectious prions or PrPSc in vitro using recombinant prion protein (rPrP). Previous work established that rPrP that lacks posttranslational modification is able to support replication of highly infectious PrPSc with assistance of cofactors of polyanionic nature and/or lipids. Unexpectedly, previous studies also revealed that seeding of rPrP by brain-derived PrPSc gave rise to new prion strains with new disease phenotypes documenting loss of a strain identity upon replication in rPrP substrate. Up to now, it remains unclear whether prion strain identity can be preserved upon replication in rPrP. The current study reports that faithful replication of hamster strain SSLOW could be achieved in vitro using rPrP as a substrate. We found that a mixture of phosphatidylethanolamine (PE) and synthetic nucleic acid polyA was sufficient for stable replication of hamster brain-derived SSLOW PrPSc in serial Protein Misfolding Cyclic Amplification (sPMCA) that uses hamster rPrP as a substrate. The disease phenotype generated in hamsters upon transmission of recombinant PrPSc produced in vitro was strikingly similar to the original SSLOW diseases phenotype with respect to the incubation time to disease, as well as clinical, neuropathological and biochemical features. Infrared microspectroscopy (IR-MSP) indicated that PrPSc produced in animals upon transmission of recombinant PrPSc is structurally similar if not identical to the original SSLOW PrPSc. The current study is the first to demonstrate that rPrP can support replication of brain-derived PrPSc while preserving its strain identity. In addition, the current work is the first to document that successful propagation of a hamster strain could be achieved in vitro using hamster rPrP. Electronic supplementary material The online version of this article (10.1186/s40478-018-0597-y) contains supplementary material, which is available to authorized users.

It has become evident that the prion protein (PrP) can form a diverse range of self-replicating structures in addition to bona fide PrP(Sc) or strain-specific PrP(Sc) variants. Some self-replicating states can be only produced in vitro, whereas others can be formed in vivo and in vitro. While transmissible, not all states that replicate in vivo are truly pathogenic. Some of them can replicate silently without causing symptoms or clinical diseases. In the current article we discuss the data on PK-digestion patterns of different self-replicating PrP states in connection with other structural data available to date and assess possible relationships between different self-replicating states. Even though different self-replicating PrP states appear to have significantly different global folding patterns, it seems that the C-terminal region exhibits a cross-β-sheet structure in all self-replicating states, as this region acquires the proteolytically most stable conformation. We also discuss the possibility of the transformation of self-replicating states and triggering of PrP(Sc) formation within the frame of the deformed templating model. The spread of silent self-replicating states is of a particular concern because they can lead to transmissible prion disease. Moreover, examples on how different replication requirements favor different states are discussed. This knowledge can help in designing conditions for selective amplification of a particular PrP state in vitro.

The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Successful cross-species transmission is often accompanied by strain adaptation and result in stable changes of strain-specific disease phenotype. Amino acid sequences of host PrPC and donor PrPSc as well as strain-specific structure of PrPSc are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult if possible at all. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster strain 263K was propagated under normal or RNA-depleted conditions using serial Protein Misfolding Cyclic Amplification (PMCA) conducted first in mouse and then hamster substrates. We found that 263K propagated under normal conditions in mouse and then hamster substrates induced the disease phenotype similar to the original 263K. Surprisingly, 263K that propagated first in RNA-depleted mouse substrate and then normal hamster substrate produced a new disease phenotype upon serial transmission. Moreover, 263K that propagated in RNA-depleted mouse and then RNA-depleted hamster substrates failed to induce clinical diseases for three serial passages despite a gradual increase of PrPSc in animals. To summarize, depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging of PMCA-derived materials in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation., Author summary The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Amino acid sequences of host prion protein and donor prions are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster prion strain was propagated under normal or RNA-depleted conditions in vitro first using mouse and then hamster substrates. This work demonstrated that depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation.

Protein misfolding cyclic amplification (PMCA) amplifies infectious prions in vitro. Over the past decade, PMCA has become an essential tool in prion research. The current chapter describes in detail the PMCA format with beads (PMCAb) and several methods that rely on PMCAb for assessing strain-specific prion amplification rates, for selective amplification of subtypes of PrP(Sc) from a mixture, and a PMCAb approach that can replace animal titration of scrapie material. Development of PMCAb-based methodology is important for addressing a number of research topics including prion strain evolution, selection and adaptation, strain-typing, prion detection, and biochemical requirements for prion replication.

Aims Naturally occurring transmissible spongiform encephalopathies (TSEs) accumulate disease-specific forms of prion protein on cell membranes in association with pathognomonic lesions. We wished to determine whether synthetic prion protein disorders recapitulated these and other subcellular TSE-specific changes. Methods SSLOW is a TSE initiated with refolded synthetic prion protein. Five terminally sick hamsters previously intracerebrally inoculated with third passage SSLOW were examined using light and immunogold electron microscopy. Results SSLOW-affected hamsters showed widespread abnormal prion protein (PrPSSLOW) and amyloid plaques. PrPSSLOW accumulated on plasma lemmas of neurites and glia without pathological changes. PrPSSLOW also colocalized with increased coated vesicles and pits, coated spiral membrane invaginations and membrane microfolding. PrPSSLOW was additionally observed in lysosomes of microglial cells but not of neurones or astrocytes. Conclusions PrPSSLOW is propagated by cell membrane conversion of normal PrP and lethal disease may be linked to the progressive growth of amyloid plaques. Cell membrane changes present in SSLOW are indistinguishable from those of naturally occurring TSEs. However, some lesions found in SSLOW are absent in natural animal TSEs and vice versa. SSLOW may not entirely recapitulate neuropathological features previously described for natural disease. End-stage neuropathology in SSLOW, particularly the nature and distribution of amyloid plaques may be significantly influenced by the early redistribution of seeds within the inoculum and its recirculation following interstitial, perivascular and other drainage pathways. The way in which seeds are distributed and aggregate into plaques in SSLOW has significant overlap with murine APP overexpressing mice challenged with Aβ.

A number of disease-associated PrP forms characterized by abnormally short proteinase K-resistant fragments (atypical PrPres) were recently described in prion diseases. The relationship between atypical PrPres and PrP(Sc), and their role in etiology of prion diseases, remains unknown. We examined the relationship between PrP(Sc) and atypical PrPres, a form characterized by short C-terminal proteinase K-resistant fragments, in a prion strain of synthetic origin. We found that the two forms exhibit distinct neuronal tropism, deposition patterns, and degree of pathological lesions. Immunostaining of brain regions demonstrated a partial overlap in anatomic involvement of the two forms and revealed the sites of their selective deposition. The experiments on amplification in vitro suggested that distinct neuronal tropism is attributed to differences in replication requirements, such as preferences for different cellular cofactors and PrP(C) glycoforms. Remarkably, deposition of atypical PrPres alone was not associated with notable pathological lesions, suggesting that it was not neurotoxic, but yet transmissible. Unlike PrP(Sc), atypical PrPres did not show significant perineuronal, vascular, or perivascular immunoreactivity. However, both forms showed substantial synaptic immunoreactivity. Considering that atypical PrPres is not associated with substantial lesions, this result suggests that not all synaptic disease-related PrP states are neurotoxic. The current work provides important new insight into our understanding of the structure-pathogenicity relationships of transmissible PrP states.

The innate immune system provides the first line of defense against pathogens. To recognize pathogens, this system detects a number of molecular features that discriminate pathogens from host cells, including terminal sialylation of cell surface glycans. Mammalian cell surfaces, but generally not microbial cell surfaces, have sialylated glycans. Prions or PrPSc are proteinaceous pathogens that lack coding nucleic acids but do possess sialylated glycans. We proposed that sialylation of PrPSc is essential for evading innate immunity and infecting a host. In this study, the sialylation status of PrPSc was reduced by replicating PrPSc in serial Protein Misfolding Cyclic Amplification using sialidase-treated PrPC substrate and then restored to original levels by replication using non-treated substrate. Upon intracerebral administration, all animals that received PrPSc with original or restored sialylation levels were infected, whereas none of the animals that received PrPSc with reduced sialylation were infected. Moreover, brains and spleens of animals from the latter group were completely cleared of prions. The current work established that the ability of prions to infect the host via intracerebral administration depends on PrPSc sialylation status. Remarkably, PrPSc infectivity could be switched off and on in a reversible manner by first removing and then restoring PrPSc sialylation.

Recent studies demonstrated that the efficiency, rate, and yield of prion amplification in vitro could be substantially improved by supplementing protein misfolding cyclic amplification (PMCA) with Teflon beads [Gonzalez-Montalban et al. (2011) PLoS Pathog. 7, e1001277]. Here we employed the new PMCA format with beads (PMCAb) to gain insight into the mechanism of prion amplification. Using a panel of six hamster prion strains, the effect of beads on amplification was found to be strain-specific, with the largest improvements in efficiency observed for strains with the highest conformational stability. This result suggests a link between PrP(Sc) conformational stability and its fragmentation rate and that beads improved amplification by assisting fragmentation. Furthermore, while exploring the PrP(Sc)-independent bead effect mechanism, a synergy between the effects of RNA and beads on amplification was observed. Consistent with previous studies, amplification of all six hamster strains tested here was found to be RNA-dependent. Under sonication conditions used for PMCA, large RNA molecules were found to degrade into smaller fragments of a size that was previously shown to be the most effective in facilitating prion conversion. We speculate that sonication-induced changes in RNA size distribution could be one of the rate-limiting steps in prion amplification.

Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrPSc). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP that had been converted into the cross-β-sheet amyloid form and subjected to annealing. Serial transmission gave rise to a disease phenotype with highly unique clinical and neuropathological features. Among them were the deposition of large PrPSc plaques in subpial and subependymal areas in brain and spinal cord, very minor lesioning of the hippocampus and cerebellum, and a very slow progression of disease after onset of clinical signs despite the accumulation of large amounts of PrPSc in the brain. The length of the clinical duration is more typical of human and large animal prion diseases, than those of rodents. Our studies establish that transmissible prion disease can be induced in wild-type animals by inoculation of rPrP and introduce a valuable new model of prion diseases. Electronic supplementary material The online version of this article (doi:10.1007/s00401-009-0633-x) contains supplementary material, which is available to authorized users.

Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-β-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions—perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including αB-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.

The primary structure of the prion protein (PrP) is believed to be the key factor in regulating the species barrier of prion transmission. Because the strength of the species barrier was found to be affected by the prion strain, the extent to which the barrier can indeed be attributed to differences in the PrP primary structures of either donor and acceptor species remains unclear. In this study, we exploited the intrinsic property of PrP to polymerize spontaneously into disease-related amyloid conformations in the absence of a strain-specified template and analyzed polymerization of mouse and hamster full-length recombinant PrPs. Unexpectedly, we found no evidence of species specificity in cross-seeding polymerization assays. Even when both recombinant PrP variants were present in mixtures, preformed mouse or hamster fibrils displayed no selectivity in elongation reactions and consumed equally well both homologous and heterologous substrates. Analysis of individual fibrils revealed that fibrils can elongate in a bidirectional or unidirectional manner. Our work revealed that, in the absence of a cellular environment, post-translational modifications, or strain-specified conformational constraints, PrP fibrils are intrinsically promiscuous and capable of utilizing heterologous PrP variants as a substrate in a highly efficient manner. This study suggests that amyloid structures are capable of accommodating local perturbations arising because of a mismatch in amino acid sequences and highlights the promiscuous nature of the self-propagating activity of amyloid fibrils.

Defects in axonal transport and synaptic dysfunctions are associated with early stages of several neurodegenerative diseases including Alzheimer's, Huntington's, Parkinson's, and prion diseases. Here, we tested the effect of full-length mammalian prion protein (rPrP) converted into three conformationally different isoforms to induce pathological changes regarded as early subcellular hallmarks of prion disease. We employed human embryonal teratocarcinoma NTERA2 cells (NT2) that were terminally differentiated into neuronal and glial cells and co-cultured together. We found that rPrP fibrils but not alpha-rPrP or soluble beta-sheet rich oligomers caused degeneration of neuronal processes. Degeneration of processes was accompanied by a collapse of microtubules and aggregation of cytoskeletal proteins, formation of neuritic beads, and a dramatic change in localization of synaptophysin. Our studies demonstrated the utility of NT2 cells as valuable human model system for elucidating subcellular events of prion pathogenesis, and supported the emerging hypothesis that defects in neuronal transport and synaptic abnormalities are early pathological hallmarks associated with prion diseases.

Introduction Previous studies established that prion disease with unique strain-specific phenotypes could be induced by in vitro-formed recombinant PrP (rPrP) fibrils with structures different from that of authentic prions, or PrPSc. To explain the etiology of prion diseases, new mechanism proposed that in animals the transition from rPrP fibrils to PrPSc consists of two main steps: the first involves fibril-induced formation of atypical PrPres, a self-replicating but clinically silent state, and the second consists of atypical PrPres-dependent formation of PrPSc via rare deformed templating events. Results In the current study, atypical PrPres with characteristics similar to those of brain-derived atypical PrPres was generated in vitro. Upon inoculation into animals, in vitro-generated atypical PrPres gave rise to PrPSc and prion disease with a phenotype similar to those induced by rPrP fibrils. Significant differences in the sialylation pattern between atypical PrPres and PrPSc suggested that only a small sub-fraction of the PrPC that is acceptable as a substrate for PrPSc could be also recruited by atypical PrPres. This can explain why atypical PrPres replicates slower than PrPSc and why PrPSc outcompetes atypical PrPres. Conclusions This study illustrates that transmissible prion diseases with very similar disease phenotypes could be produced via two alternative procedures: direct inoculation of recombinant PrP amyloid fibrils or in vitro-produced atypical PrPres. Moreover, this work showed that preparations of atypical PrPres free of PrPSc can give rise to transmissible diseases in wild type animals and that atypical PrPres generated in vitro is an adequate model for brain-derived atypical PrPres. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0248-5) contains supplementary material, which is available to authorized users.

Prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded and aggregated form of a sialoglycoprotein called prion protein or PrPC. PrPC has two sialylated N-linked carbohydrates. In PrPSc, the glycans are directed outward, with the terminal sialic acid residues creating a negative charge on the surface of prion particles. The current study proposes a new hypothesis that electrostatic repulsion between sialic residues creates structural constraints that control prion replication and PrPSc glycoform ratio. In support of this hypothesis, here we show that diglycosylated PrPC molecules that have more sialic groups per molecule than monoglycosylated PrPC were preferentially excluded from conversion. However, when partially desialylated PrPC was used as a substrate, recruitment of three glycoforms into PrPSc was found to be proportional to their respective populations in the substrate. In addition, hypersialylated molecules were also excluded from conversion in the strains with the strongest structural constraints, a strategy that helped reduce electrostatic repulsion. Moreover, as predicted by the hypothesis, partial desialylation of PrPC significantly increased the replication rate. This study illustrates that sialylation of N-linked glycans creates a prion replication barrier that controls replication rate and glycoform ratios and has broad implications.

Almost all proteins contain charged residues, and their chain distribution is tailored to fulfill essential ionic interactions for folding, binding and catalysis. Among proteins, the hinged two-domain chain of the cellular prion protein (PrP C) exhibits a peculiar charge structure with unclear consequences in its structural malleability. To decipher the charge design role, we generated charge-reverted mutants for each domain and analyzed their effect on conformational and metabolic features. We found that charges contain the information for interdomain interactions. Use of dynamic light scattering and thermal denaturation experiments delineates the compaction of the α-fold by an electrostatic compensation between the polybasic 23-30 region and the α3 electronegative surface. This interaction increases stability and disfavors fibrillation. Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency. In the fibrillar state, use of circular dichroism, atomic-force and fluorescence microscopies reveal that the N-terminal positive clusters and the α3 electronegative surface dictate the secondary structure, the assembly hierarchy and the growth length of the fibril state. These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

The central molecular event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC), which is a sialoglycoprotein, into the disease-associated, transmissible form denoted PrPSc. Recent studies revealed a correlation between the sialylation status of PrPSc and incubation time to disease and introduced a new hypothesis that progression of prion diseases could be controlled or reversed by altering the sialylation level of PrPC. Of the four known mammalian sialidases, the enzymes that cleave off sialic acid residues, only NEU1, NEU3 and NEU4 are expressed in the brain. To test whether cellular sialidases control the steady-state sialylation level of PrPC and to identify the putative sialidase responsible for desialylating PrPC, we analyzed brain-derived PrPC from knockout mice deficient in Neu1, Neu3, Neu4, or from Neu3/Neu4 double knockouts. Surprisingly, no differences in the sialylation of PrPC or its proteolytic product C1 were noticed in any of the knockout mice tested as compared to the age-matched controls. However, significantly higher amounts of the C1 fragment relative to full-length PrPC were detected in the brains of Neu1 knockout mice as compared to WT mice or to the other knockout mice. Additional experiments revealed that in neuroblastoma cell line the sialylation pattern of C1 could be changed by an inhibitor of sialylatransferases. In summary, this study suggests that targeting cellular sialidases is apparently not the correct strategy for altering the sialylation levels of PrPC, whereas modulating the activity of sialylatransferases might offer a more promising approach. Our findings also suggest that catabolism of PrPC involves its α-cleavage followed by desialylation of the resulting C1 fragments by NEU1 and consequent fast degradation of the desialylated products.

In contrast to most amyloidogenic proteins or peptides that do not contain any significant post-translational modifications, the prion protein (PrP) is modified with either one or two polysaccharides and a GPI anchor which attaches PrP to the plasma membrane. Like other amyloidogenic proteins, however, PrP adopts a fibrillar shape when converted to a disease-specific conformation. Therefore, PrP polymerization offers a unique opportunity to examine the effects of biologically relevant non-peptidic modifications on conversion to the amyloid conformation. To test the extent to which a long hydrophobic chain at the C-terminus affects the intrinsic amyloidogenic propensity of PrP, we modified recombinant PrP with a N-myristoylamido-maleimidyl group, which can serve as a membrane anchor. We show that while this modification increases the affinity of PrP for the cell membrane, it does not alter the structure of the protein. Myristoylation of PrP affected amyloid formation in two ways: (i) it substantially decreased the extent of fibrillation, presumably due to off-pathway aggregation, and (ii) it prohibited assembly of filaments into higher-order fibrils by preventing their lateral association. The negative effect on lateral association was abolished if the myristoylated moiety at the C-terminus was replaced by a polar group of similar size or by a hydrophobic group of smaller size. When preformed PrP fibrils were provided as seeds, myristoylated PrP supported fibril elongation and formation of higher-order fibrils composed of several filaments. Our studies illustrate that, despite a bulky hydrophobic moiety at C-terminus, myristoylated PrP can still incorporate into fibrillar structure, and that the C-terminal hydrophobic substitution does not affect the size of the proteinase K resistant core, but controls the mode of lateral assembly of filaments into higher-order fibrils.

Despite possessing a common cross-beta core, amyloid fibrils are known to exhibit great variations in their morphologies. To date, the mechanism responsible for the polymorphism in amyloid fibrils is poorly understood. Here we report that two variants of mammalian full-length prion protein (PrP), hamster (Ha) and mouse (Mo) PrPs, produced morphologically distinguishable subsets of mature fibrils under identical solvent conditions. To gain insight into the origin of this morphological diversity we analyzed the early stages of polymerization. Unexpectedly, we found that despite a highly conserved amyloidogenic region (94% identity within the residues 90-230), Ha and Mo PrPs followed two distinct pathways for lateral assembly of protofibrils into mature, higher order fibrils. The protofibrils of Ha PrP first formed irregular bundles characterized by a peculiar palm-type shape, which ultimately condensed into mature fibrils. The protofibrils of Mo PrP, on the other hand, associated in pairs in a pattern resembling dichotomous coalescence. These pathways are referred to here as the palm-type and dichotomous mechanisms. Two distinct mechanisms for lateral assembly explain striking differences in morphology of mature fibrils produced from closely related Mo and Ha PrPs. Remarkable similarities between subtypes of amyloid fibrils generated from different proteins and peptides suggest that the two mechanisms of lateral assembly may not be limited to prion proteins but may be a common characteristic of polymerization of amyloidogenic proteins and peptides in general.

Amyloid fibrils were produced from the full-length mouse prion protein (PrP) under solvent conditions similar to those used for the generation of synthetic prions from PrP 89-230. Analysis of the ultrastructure by atomic force microscopy revealed extremely broad polymorphism in fibrils formed under a single growth condition. Fibrils varied with respect to the number of constitutive filaments and the manner in which the filaments were assembled. PrP polymerization was found to show several peculiar features: (i) the higher-order fibrils/ribbons were formed through a highly hierarchical mechanism of assembly of lower-order fibrils/ribbons; (ii) the lateral assembly proceeded stepwise; at each step, a semi-stable fibrillar species were generated, which were then able to enter the next level of assembly; (iii) the assembly of lower into higher-order fibrils occurred predominantly in a vertical dimension via stacking of ribbons on top of each other; (iv) alternative modes of lateral association co-existed under a single growth condition; (iv) the fibrillar morphology changed even within individual fibrils, illustrating that alternative modes of filament assembly are inter-convertible and thermodynamically equivalent. The most predominant fibrillar types were classified into five groups according to their height, each of which was divided in up to three subgroups according to their width. Detailed analysis of ultrastructure revealed that the fibrils of the major subtype (height 3.61(+/-0.28)nm, width 31.1(+/-2.0)nm) were composed of two ribbons, each of which was composed of two filaments. The molecular volume calculations indicated that a single PrP molecule occupied a distance of approximately 1.2 nm within a single filament. High polymorphism in fibrils generated in vitro is reminiscent of high morphological diversity of scrapie-associated fibrils isolated from scrapie brains, suggesting that polymorphism is peculiar for polymerization of PrP regardless of whether fibrils are formed in vitro or under pathological conditions in vivo.

We report the generation of a previously unknown class of water-soluble organic-inorganic hybrid nanoclusters composed of silver and thioflavin T with remarkable fluorescent properties. These hybrid nanoclusters display an extra bright fluorescence in aqueous solutions without any detectable photobleaching. Furthermore, the fluorescent nanoclusters can be generated in situ by sensitized photoreduction of Ag+. Organic-inorganic nanoclusters have remarkable advantages over the known classes of fluorescent probes for the development of ultrasensitive biological assays, cell imaging, or studies of single molecules. As an example of a practical biological application, imaging of amyloid fibrils produced from recombinant mammalian prion proteins and nonprion proteins using hybrid nanoclusters is presented.

The Kalman filter is an established tool for the analysis of dynamic systems with normally distributed noise, and it has been successfully applied in numerous application areas. It provides sequentially calculated estimates of the system states along with a corresponding covariance matrix. For nonlinear systems, the extended Kalman filter is often used which is derived from the Kalman filter by linearization around the current estimate. A key issue in metrology is the evaluation of the uncertainty associated with the Kalman filter state estimates. The "Guide to the Expression of Uncertainty in Measurements" (GUM) and its supplements serve as the de facto standard for uncertainty evaluation in metrology. We explore the relationship between the covariance matrix produced by the Kalman filter and a GUM-compliant uncertainty analysis. In addition, also the results of a Bayesian analysis are considered. For the case of linear systems with known system matrices, we show that all three approaches are compatible. When the system matrices are not precisely known, however, or when the system is nonlinear, this equivalence breaks down and different results can be reached then. Though for precisely known nonlinear systems the result of the extended Kalman filter still corresponds to the linearized uncertainty propagation of GUM. The extended Kalman filter can suffer from linearization and convergence errors. These disadvantages can be avoided to some extent by applying Monte Carlo procedures, and we propose such a method which is GUM-compliant and can also be applied online during the estimation. We illustrate all procedures in terms of a two-dimensional dynamic system and compare the results with those obtained by particle filtering, which has been proposed for the approximate calculation of a Bayesian solution. Finally, we give some recommendations based on our findings., 27 pages, 5 figures

The molecular mechanisms underlying structural diversity of amyloid fibrils or prion strains formed within the same primary structure is considered to be one of the most enigmatic questions in prion biology. We report here on the direct characterization of amyloid structures using a novel spectroscopic technique, hydrogen-deuterium exchange ultraviolet Raman spectroscopy. This method enables us to assess the structural differences within highly ordered cross-β-cores of two amyloid states produced within the same amino acid sequence of full-length mammalian prion protein. We found that while both amyloid states consisted of β-structures, their cross-β-cores exhibited hydrogen bonding of different strengths. Moreover, Raman spectroscopy revealed that both amyloid states displayed equally narrow crystalline-like bands, suggesting uniform structures of cross-β-cores within each state. Taken together, these data suggest that highly polymorphous fibrils can display highly uniform structures of their cross-β-core and belong to the same prion strain.

Prion replication occurs via a template-assisted mechanism, which postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a template. The concept of prion-like template-assisted propagation of an abnormal protein conformation has been expanded to amyloidogenic proteins associated with Alzheimer, Parkinson, Huntington diseases, amyotrophic lateral sclerosis and others. Recent studies demonstrated that authentic PrP (Sc) and transmissible prion disease could be generated in wild type animals by inoculation of recombinant prion protein amyloid fibrils, which are structurally different from PrP (Sc) and lack any detectable PrP (Sc) particles. Here we discuss a new replication mechanism designated as "deformed templating," according to which fibrils with one cross-β folding pattern can seed formation of fibrils or particles with a fundamentally different cross-β folding pattern. Transformation of cross-β folding pattern via deformed templating provides a mechanistic explanation behind genesis of transmissible protein states induced by amyloid fibrils that are considered to be non-infectious. We postulate that deformed templating is responsible for generating conformationally diverse amyloid populations, from which conformers that are fit to replicate in a particular cellular environment are selected. We propose that deformed templating represents an essential step in the evolution of transmissible protein states.

Misfolding and aggregation of prion protein are related to several neurodegenerative diseases in humans such as Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. A growing number of applications in the prion field including assays for detection of PrPSc and methods for production of PrPSc de novo require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, oxidative modifications, or truncation, which is typically generated as a result of spontaneous oxidation or degradation. We also describe methods for preparation of amyloid fibrils from recombinant PrP in vitro. Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of PrPSc for development of prion diagnostics including generation of PrPSc-specific antibody.

The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrPSc), which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrPSc template. Here we report that authentic PrPSc and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrPSc and lack any detectable PrPSc particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres) before authentic PrPSc evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrPSc and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as “deformed templating” postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrPSc can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases., Author Summary The transmissible agent of prion disease consists of a prion protein in its abnormal conformation (PrPSc), which replicates itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrPSc. The current study reports that infectious prions and transmissible prion disease can be triggered in wild type animals by amyloid fibrils produced from recombinant prion prtotein, which are structurally different from PrPSc and lacks any detectable PrPSc particles. This work introduces a new hypothesis that transmissible prion diseases can be induced by prion protein structures different from that of authentic PrPSc and suggests that a new mechanism for triggering PrPSc formation different from the classical templating exists. The current work provides important new insight into the mechanisms underlying genesis and evolution of the transmissible states of the prion protein and has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.

Bioassay by end-point dilution has been used for decades for routine determination of prion infectivity titre. Here we show that the new protein misfolding cyclic amplification with beads (PMCAb) technique can be used to estimate titres of the infection-specific forms of the prion protein with a higher level of precision and in 3-6 days as opposed to 2 years, when compared with the bioassay. For two hamster strains, 263 K and SSLOW, the median reactive doses determined by PCMAb (PMCAb(50)) were found to be 10(12.8) and 10(12.2) per gram of brain tissue, which are 160- and 4,000-fold higher than the corresponding median infectious dose (ID(50)) values measured by bioassay. The 10(2)- to 10(3)-fold differences between ID(50) and PMCAb(50) values could be due to a large excess of PMCAb-reactive prion protein seeds with little or no infectivity. Alternatively, the differences between ID(50) and PMCAb(50) could be due to higher rate of clearance of infection-specific prion protein seeds in animals versus PMCAb reactions. A well-calibrated PMCAb reaction can be an efficient and cost-effective method for the estimation of infection-specific prion protein titre.

BACKGROUND According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells. METHODOLOGY/PRINCIPAL FINDINGS For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein (PrP(C)) at high levels confirming that cytotoxicity was in part PrP(C)-dependent. Silencing of PrP(C) expression by small hairpin RNAs designed to silence expression of human PrP(C) (shRNA-PrP(C)) diminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrP(C)-mediated and PrP(C)-independent mechanisms depends on the structure of the aggregates. CONCLUSIONS/SIGNIFICANCE This work provides a direct illustration that the relationship between an amyloid's physical dimension and its toxic potential is not unidirectional but is controlled by the molecular structure of prion protein (PrP) molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrP(C) expression can be exploited to reduce their deleterious effects.

In this study we propose a Bayesian approach to the estimation of the Hurst exponent in terms of linear mixed models. Even for unevenly sampled signals and signals with gaps, our method is applicable. We test our method by using artificial fractional Brownian motion of different length and compare it with the detrended fluctuation analysis technique. The estimation of the Hurst exponent of a Rosenblatt process is shown as an example of an H-self-similar process with non-Gaussian dimensional distribution. Additionally, we perform an analysis with real data, the Dow-Jones Industrial Average closing values, and analyze its temporal variation of the Hurst exponent.

We report the results of solid state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the full-length prion protein PrP (residues 23−231, Syrian hamster sequence). Measurements of intermolecular 13C−13C dipole−dipole couplings in selectively carbonyl-labeled samples indicate that β-sheets in these fibrils have an in-register parallel structure, as previously observed in amyloid fibrils associated with Alzheimer’s disease and type 2 diabetes and in yeast prion fibrils. Two-dimensional 13C−13C and 15N−13C solid state NMR spectra of a uniformly 15N- and 13C-labeled sample indicate that a relatively small fraction of the full sequence, localized to the C-terminal end, forms the structurally ordered, immobilized core. Although unique site-specific assignments of the solid state NMR signals cannot be obtained from these spectra, analysis with a Monte Carlo/simulated annealing algorithm suggests that the core is comprised primarily of residues in the 173−224 range. These results are consistent with earlier electron paramagnetic resonance studies of fibrils formed by residues 90−231 of the human PrP sequence, formed under somewhat different conditions [Cobb, N. J., Sonnichsen, F. D., McHaourab, H., and Surewicz, W. K. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 18946−18951], suggesting that an in-register parallel β-sheet structure formed by the C-terminal end may be a general feature of PrP fibrils prepared in vitro.

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro., Author Summary Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro. While PMCA has become an important tool in prion research, its application is limited because of low yield, poor efficiency and, sometimes, stochastic behavior. The current study introduces a new PMCA format that dramatically improves the efficiency, yield, and robustness of prion conversion in vitro and reduces the time of the reaction. These improvements have numerous implications. The method opens new opportunities for improving prion detection and for generating large amounts of PrPSc in vitro. Furthermore, the results demonstrate that in vitro conversion is not limited by lack of convertible PrPC nor by concentrations of cellular cofactors required for prion conversion.

Misfolding and aggregation of prion protein (PrP) is related to several neurodegenerative diseases in humans such as Creutzfeldt-Jacob disease, fatal familial insomnia, and Gerstmann-Straussler-Sheinker disease. Amyloid fibrils prepared from recombinant PrP in vitro share many features of the infectious prions. These fibrils can be used as a synthetic surrogate of PrP(Sc) for development of prion diagnostics, including generation of PrP(Sc)-specific antibody, for screening of antiprion drugs, or for development of antiprion decontamination procedures. Here, we describe the methods of preparation of prion protein fibrils in vitro and biochemical assays for assessing physical properties and the quality of fibrils.

Certain applications in the prion field require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, which are normally generated as a result of spontaneous oxidation or degradation.

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native alpha-helical forms and the conformational stability of alpha-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in alpha-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.

The question of whether distinct self-propagating structures could be formed within the same amino acid sequence in the absence of external cofactors or templates has important implications for a number of issues, including the origin of prion strains and the engineering of smart, self-assembling peptide-based biomaterials. In the current study, we showed that chemically identical prion protein can give rise to conformationally distinct, self-propagating amyloid structures in the absence of cellular cofactors, post-translational modification, or PrPSc-specified templates. Even more surprising, two self-replicating states were produced under identical solvent conditions, but under different shaking modes. Individual prion conformations were inherited by daughter fibrils in seeding experiments conducted under alternative shaking modes, illustrating the high fidelity of fibrillation reactions. Our study showed that the ability to acquire conformationally different self-propagating structures is an intrinsic ability of protein fibrillation and strongly supports the hypothesis that conformational variation in self-propagating protein states underlies prion strain diversity.

Misfolding and aggregation of prion protein (PrP) is related to several neurodegenerative diseases in humans such as Creutzfeldt-Jacob disease, fatal familial insomnia, and Gerstmann-Straussler-Sheinker disease. Amyloid fibrils prepared from recombinant PrP in vitro share many features of the infectious prions. These fibrils can be used as a synthetic surrogate of PrP(Sc) for development of prion diagnostics, including generation of PrP(Sc)-specific antibody, for screening of antiprion drugs, or for development of antiprion decontamination procedures. Here, we describe the methods of preparation of prion protein fibrils in vitro and biochemical assays for assessing physical properties and the quality of fibrils.

Certain applications in the prion field require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, which are normally generated as a result of spontaneous oxidation or degradation.

Defects in axonal transport and synaptic dysfunctions are associated with early stages of several neurodegenerative diseases including Alzheimer's, Huntington's, Parkinson's, and prion diseases. Here, we tested the effect of full-length mammalian prion protein (rPrP) converted into three conformationally different isoforms to induce pathological changes regarded as early subcellular hallmarks of prion disease. We employed human embryonal teratocarcinoma NTERA2 cells (NT2) that were terminally differentiated into neuronal and glial cells and co-cultured together. We found that rPrP fibrils but not alpha-rPrP or soluble beta-sheet rich oligomers caused degeneration of neuronal processes. Degeneration of processes was accompanied by a collapse of microtubules and aggregation of cytoskeletal proteins, formation of neuritic beads, and a dramatic change in localization of synaptophysin. Our studies demonstrated the utility of NT2 cells as valuable human model system for elucidating subcellular events of prion pathogenesis, and supported the emerging hypothesis that defects in neuronal transport and synaptic abnormalities are early pathological hallmarks associated with prion diseases.

The coexistence of multiple strains or subtypes of the disease-related isoform of prion protein (PrP) in natural isolates, together with the observed conformational heterogeneity of PrP amyloid fibrils generated in vitro, indicates the importance of probing the conformation of single particles within heterogeneous samples. Using an array of PrP-specific antibodies, we report the development of a novel immunoconformational assay. Uniquely, application of this new technology allows the conformation of multimeric PrP within a single fibril or particle to be probed without pretreatment of the sample with proteinase K. Using amyloid fibrils prepared from full-length recombinant PrP, we demonstrated the utility of this assay to define (i) PrP regions that are surface-exposed or buried, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of PrP fibrils as measured for either the entire fibrillar population or for individual fibrils. Specifically, PrP regions 159-174 and 224-230 were shown to be buried and were the most resistant to denaturation. The 132-156 segment of PrP was found to be cryptic under native conditions and solvent-exposed under partially denaturing conditions, whereas the region 95-105 was solvent-accessible regardless of the solvent conditions. Remarkably, a subfraction of fibrils showed immunoreactivity to PrPSc-specific antibodies designated as IgGs 89-112 and 136-158. The immunoreactivity of the conformational epitopes was reduced upon exposure to partially denaturing conditions. Unexpectedly, PrPSc -specific antibodies revealed conformational polymorphisms even within individual fibrils. Our studies provide valuable new insight into fibrillar substructure and offer a new tool for probing the conformation of single PrP fibrils.

In recent studies, we developed a protocol for in vitro conversion of full-length mouse recombinant PrP (Mo rPrP23-230) into amyloid fibrils [Bocharova et al. (2005) J. Mol. Biol. 346, 645-659]. Because amyloid fibrils produced from recombinant Mo PrP89-230 display infectivity [Legname et al. (2004) Science 305, 673-676], polymerizatiom of rPrPs in vitro represents a valuable model for elucidating the mechanism of prion conversion. Unexpectedly, when the same conversion protocol was used for hamster (Ha) rPrP23-231, we experienced substantial difficulties in forming fibrils. While searching for potential reasons of our failure to produce fibrils, we probed the effect of methionine oxidation in rPrP. We found that oxidation of methionines interferes with the formation of rPrP fibrils and that this effect is more profound for Ha than for Mo rPrP. To minimize the level of spontaneous oxidation, we developed a new protocol for rPrP purification, in which highly amyloidogenic Ha rPrP with minimal levels of oxidized residues was produced. Furthermore, our studies revealed that oxidation of methionines in preformed fibrils inhibited subsequent maturation of fibrils into proteinase K-resistant PrP(Sc)-like conformation (PrP-res). Our data are consistent with the proposition that conformational changes within the central region of the protein (residues 90-140) are essential for adopting PrP-res conformation and demonstrate that methionine oxidation interferes with this process. These studies provide new insight into the mechanism of prion polymerization, solve a long-standing practical problem in producing PrP-res fibrils from full-length PrP, and may help in identifying new genetic and environmental factors that modulate prion disease.

The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC) into the disease-associated, transmissible form (PrPSc). PrPC is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrPC and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrPC glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrPSc glycoform ratio. The current study demonstrates that undersialylated PrPC is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrPC. As a result, PMCAb-derived PrPSc was less sialylated than brain-derived PrPSc. A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrPC using sialidase was found to increase the rate of PrPSc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrPC reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrPC was also found to control PrPSc glycoform ratio. A decrease in PrPC sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrPSc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrPC differed from that of spleen-derived PrPC. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrPC, suggesting that Neu1 is not responsible for desialylation of PrPC. The current work highlights previously unappreciated role of PrPC sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches., Author Summary The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC) into disease-associated, transmissible form (PrPSc). The amino acid sequence of PrPC and strain-specific structure of PrPSc are among the key parameters that control prion replication and transmission. The current study showed that PrPC posttranslational modification, specifically sialylation of N-linked glycans, plays a key role in regulating prion replication rate, infectivity, cross-species barrier and PrPSc glycoform ratio. A decrease in PrPC sialylation level increased the rate of prion replication in a strain-specific manner and reduced or eliminated a species barrier when prion replication was seeded by heterologous seeds. At the same time, a decrease in sialylation correlated with a drop in infectivity of PrPSc material produced in vitro. The current study also demonstrated that the PrPSc glycoform ratio, which is an important feature used for strain typing, is not only controlled by prion strain or host but also the sialylation status of PrPC. This study opens multiple new directions in prion research, including development of new therapeutic approaches.