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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.

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.

Under normal conditions, astrocytes perform a number of important physiological functions centered around neuronal support and synapse maintenance. In neurodegenerative diseases including Alzheimer’s, Parkinson’s and prion diseases, astrocytes acquire reactive phenotypes, which are sustained throughout the disease progression. It is not known whether in the reactive states associated with prion diseases, astrocytes lose their ability to perform physiological functions and whether the reactive states are neurotoxic or, on the contrary, neuroprotective. The current work addresses these questions by testing the effects of reactive astrocytes isolated from prion-infected C57BL/6J mice on primary neuronal cultures. We found that astrocytes isolated at the clinical stage of the disease exhibited reactive, pro-inflammatory phenotype, which also showed downregulation of genes involved in neurogenic and synaptogenic functions. In astrocyte-neuron co-cultures, astrocytes from prion-infected animals impaired neuronal growth, dendritic spine development and synapse maturation. Toward examining the role of factors secreted by reactive astrocytes, astrocyte-conditioned media was found to have detrimental effects on neuronal viability and synaptogenic functions via impairing synapse integrity, and by reducing spine size and density. Reactive microglia isolated from prion-infected animals were found to induce phenotypic changes in primary astrocytes reminiscent to those observed in prion-infected mice. In particular, astrocytes cultured with reactive microglia-conditioned media displayed hypertrophic morphology and a downregulation of genes involved in neurogenic and synaptogenic functions. In summary, the current study provided experimental support toward the non-cell autonomous mechanisms behind neurotoxicity in prion diseases and demonstrated that the astrocyte reactive phenotype associated with prion diseases is synaptotoxic.

Phagocytosis is one of the most important physiological functions of the glia directed at maintaining a healthy, homeostatic environment in the brain. Under a homeostatic environment, the phagocytic activities of astrocytes and microglia are tightly coordinated in time and space. In neurodegenerative diseases, both microglia and astrocytes contribute to neuroinflammation and disease pathogenesis, however, whether their phagocytic activities are up- or downregulated in reactive states is not known. To address this question, this current study isolated microglia and astrocytes from C57BL/6J mice infected with prions and tested their phagocytic activities in live-cell imaging assays that used synaptosomes and myelin debris as substrates. The phagocytic uptake by the reactive microglia was found to be significantly upregulated, whereas that of the reactive astrocytes was strongly downregulated. The up- and downregulation of phagocytosis by the two cell types were observed irrespective of whether disease-associated synaptosomes, normal synaptosomes, or myelin debris were used in the assays, indicating that dysregulations are dictated by cell reactive states, not substrates. Analysis of gene expression confirmed dysregulation of phagocytic functions in both cell types. Immunostaining of animal brains infected with prions revealed that at the terminal stage of disease, neuronal cell bodies were subject to engulfment by reactive microglia. This study suggests that imbalance in the phagocytic activities of the reactive microglia and astrocytes, which are dysregulated in opposite directions, is likely to lead to excessive microglia-mediated neuronal death on the one hand, and the inability of astrocytes to clear cell debris on the other hand, contributing to the neurotoxic effects of glia as a whole.

Alzheimer’s disease (AD) is a devastating fatal neurodegenerative disease. An alternative to the amyloid cascade hypothesis is that a viral infection is key to the etiology of late-onset AD, with β-amyloid (Aβ) peptides playing a protective role. In the current study, young 5XFAD mice that overexpress mutant human amyloid precursor protein with the Swedish, Florida, and London familial AD mutations were infected with one of two strains of herpes simplex virus 1 (HSV-1), 17syn+ and McKrae, at three different doses. Contrary to previous work, 5XFAD genotype failed to protect mice against HSV-1 infection. The region- and cell-specific tropisms of HSV-1 were not affected by the 5XFAD genotype, indicating that host–pathogen interactions were not altered. Seven- to ten-month-old 5XFAD animals in which extracellular Aβ aggregates were abundant showed slightly better survival rate relative to their wild-type (WT) littermates, although the difference was not statistically significant. In these 5XFAD mice, HSV-1 replication centers were partially excluded from the brain areas with high densities of Aβ aggregates. Aβ aggregates were free of HSV-1 viral particles, and the limited viral invasion to areas with a high density of Aβ aggregates was attributed to phagocytic activity of reactive microglia. In the oldest mice (12–15 months old), the survival rate did not differ between 5XFAD and WT littermates. While the current study questions the antiviral role of Aβ, it neither supports nor refutes the viral etiology hypothesis of late-onset AD.

Chronic 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. To answer these questions, we analyzed gene expression in the thalamus, cortex, hypothalamus and hippocampus of mice infected with 22L and ME7 prion strains using a Nanostring Neuroinflammation panel at the subclinical, early clinical and advanced stages of the disease. We found that at the preclinical stage of the disease, the region-specific homeostatic identities were preserved. However, with the appearance of clinical signs, the 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 the activated pathways. Moreover, clustering analysis suggested that the astrocyte function pathway responded to prion infection prior to the Activated Microglia or Neuron and Neurotransmission pathways. The 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 the region-independent neuroinflammation signature, which is 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.

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.

Mammalian prions are unconventional infectious agents that invade and replicate in an organism by recruiting a normal form of a prion protein (PrPC) and converting it into misfolded, disease-associated state referred to as PrPSc. PrPC is posttranslationally modified with two N-linked glycans. Prion strains replicate by selecting substrates from a large pool of PrPC sialoglycoforms expressed by a host. Brain regions have different vulnerability to prion infection, however, molecular mechanisms underlying selective vulnerability is not well understood. Toward addressing this question, the current study looked into a possibility that sialylation of PrPSc might be involved in defining selective vulnerability of brain regions. The current work found that in 22L -infected animals, PrPSc is indeed sialylated in a region dependent manner. PrPSc in hippocampus and cortex was more sialylated than PrPSc from thalamus and stem. Similar trends were also observed in brain materials from RML- and ME7-infected animals. The current study established that PrPSc sialylation status is indeed region-specific. Together with previous studies demonstrating that low sialylation status accelerates prion replication, this work suggests that high vulnerability of certain brain region to prion infection could be attributed to their low sialylation status.

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.

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.

Prion diseases display multiple disease phenotypes characterized by diverse clinical symptoms, different brain regions affected by the disease, distinct cell tropism and diverse PrPSc deposition patterns. The diversity of disease phenotypes within the same host is attributed to the ability of PrPC to acquire multiple, alternative, conformationally distinct, self-replicating PrPSc states referred to as prion strains or subtypes. Structural diversity of PrPSc strains has been well documented, yet the question of how different PrPSc structures elicit multiple disease phenotypes remains poorly understood. The current article reviews emerging evidence suggesting that carbohydrates in the form of sialylated N-linked glycans, which are a constitutive part of PrPSc, are important players in defining strain-specific structures and disease phenotypes. This article introduces a new hypothesis, according to which individual strain-specific PrPSc structures govern selection of PrPC sialoglycoforms that form strain-specific patterns of carbohydrate epitopes on PrPSc surface and contribute to defining the disease phenotype and outcomes.

Neuroinflammation is recognized as one of the obligatory pathogenic features of neurodegenerative diseases including Alzheimer’s, Parkinson’s or prion diseases. In prion diseases, space and time correlations between deposition of disease-associated, pathogenic form of the prion protein or PrPSc and microglial-mediated neuroinflammation has been established. Yet, it remains unclear whether activation of microglia is triggered directly by a contact with PrPSc, and what molecular features of PrPSc microglia sense and respond to that drive microglia to inflammatory states. The current study asked the questions whether PrPSc can directly trigger activation of microglia and whether the degree of microglia response depends on the nature of terminal carbohydrate groups on the surface of PrPSc particles. PrPSc was purified from brains of mice infected with mouse-adapted prion strain 22L or neuroblastoma N2a cells stably infected with 22L. BV2 microglial cells or primary microglia were cultured in the presence of purified 22L. We found that exposure of BV2 cells or primary microglia to purified PrPSc triggered proinflammatory responses characterized by an increase in the levels of TNFα, IL6, nitric oxide (NO) and expression of inducible Nitric Oxide Synthase (iNOS). Very similar patterns of inflammatory response were induced by PrPSc purified from mouse brains and neuroblastoma cells arguing that microglia response is independent of the source of PrPSc. To test whether the microglial response is mediated by carbohydrate epitopes on PrPSc surface, the levels of sialylation of PrPSc N-linked glycans was altered by treatment of purified PrPSc with neuraminidase. Partial cleavage of sialic acid residues was found to boost the inflammatory response of microglia to PrPSc. Moreover, transient degradation of Iκβα observed upon treatment with partially desialylated PrPSc suggests that canonical NFκB activation pathway is involved in inflammatory response. The current study is the first to demonstrate that PrPSc can directly trigger inflammatory response in microglia. In addition, this work provides direct evidence that the chemical nature of the carbohydrate groups on PrPSc surface is important for microglial activation.

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.

The aim of this paper is to estimate the Hurst parameter of Fractional Gaussian Noise (FGN) using Bayesian inference. We propose an estimation technique that takes into account the full correlation structure of this process. Instead of using the integrated time series and then applying an estimator for its Hurst exponent, we propose to use the noise signal directly. As an application we analyze the time series of the Nile River, where we find a posterior distribution which is compatible with previous findings. In addition, our technique provides natural error bars for the Hurst exponent.

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.

Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases including Alzheimer’s, Parkinson’s, Huntington’s, prion and other diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features that were once thought to be unique to only one neurodegenerative disorder. However, the origin for the overlap remains unclear. One possibility is that diseases with mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by aggregated states of unrelated proteins. In the current study we examined whether prion replication can be induced by cross-seeding by α-synuclein or Aβ peptide. We found that α-synuclein aggregates formed in cultured cells or in vitro display cross-seeding activity and trigger misfolding of the prion protein (PrPC) in serial Protein Misfolding Cyclic Amplification reactions, producing self-replicating PrP states characterized by a short C-terminal proteinase K (PK)-resistant region referred to as PrPres. Non-fibrillar α-synuclein or fibrillar Aβ failed to cross-seed misfolding of PrPC. Remarkably, PrPres triggered by aggregated α-synuclein in vitro propagated in animals and, upon serial transmission, produced PrPSc and clinical prion disease characterized by spongiosis and astrocytic gliosis. The current study demonstrates that aggregated α-synuclein is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-replicating states that can lead to transmissible prion diseases upon serial passaging in wild type animals. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies., Author summary Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features specific to two or more neurodegenerative diseases in individual patients. However, the origin for the overlap remains unclear. One possibility is that disease that have mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by fibrillar states of unrelated proteins. The current study examined whether prion replication can be induced by cross-seeding by α-synuclein or Aβ peptide in their aggregated states. We found that α-synuclein aggregates display cross-seeding activity and trigger misfolding of the prion protein (PrPC) in vitro, producing self-replicating PrP states. Non-fibrillar α-synuclein or fibrillar Aβ failed to cross-seed misfolding of PrPC. Remarkably, misfolded PrP triggered by fibrillar α-synuclein in vitro propagated in animals and, upon serial transmission, produced clinical prion diseases. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies.

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.

Prions are composed of the misfolded prion protein (PrPSc) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrPSc aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrPSc aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrPSc aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrPSc aggregates and the incubation periods for the strains studied. The relative presence of PrPSc in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrPSc aggregates in prion-induced neurodegeneration.

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.