PrP Sc Aggregates Research Articles

Αnti-prion effects of anthocyanins

Prion diseases, also known as Transmissible Spongiform Encephalopathies (TSEs), are protein-based neurodegenerative disorders (NDs) affecting humans and animals. They are characterized by the conformational conversion of the normal cellular prion protein, PrPC, into the pathogenic isoform, PrPSc. Prion diseases are invariably fatal and despite ongoing research, no effective prophylactic or therapeutic avenues are currently available. Anthocyanins (ACNs) are unique flavonoid compounds and interest in their use as potential neuroprotective and/or therapeutic agents against NDs, has increased significantly in recent years. Therefore, we investigated the potential anti-oxidant and anti-prion effects of Oenin and Myrtillin, two of the most common anthocyanins, using the most accepted in the field overexpressing PrPScin vitro model and a cell free protein aggregation model. Our results, indicate both anthocyanins as strong anti-oxidant compounds, upregulating the expression of genes involved in the anti-oxidant response, and reducing the levels of Reactive Oxygen Species (ROS), produced due to pathogenic prion infection, through the activation of the Keap1-Nrf2 pathway. Importantly, they showcased remarkable anti-prion potential, as they not only caused the clearance of pathogenic PrPSc aggregates, but also completely inhibited the formation of PrPSc fibrils in the Cerebrospinal Fluid (CSF) of patients with Creutzfeldt–Jakob disease (CJD). Therefore, Oenin and Myrtillin possess pleiotropic effects, suggesting their potential use as promising preventive and/or therapeutic agents in prion diseases and possibly in the spectrum of neurodegenerative proteinopathies.

Propagation of PrPSc in mice reveals impact of aggregate composition on prion disease pathogenesis

Infectious prions consist of PrPSc, a misfolded, aggregation-prone isoform of the host’s prion protein. PrPSc assemblies encode distinct biochemical and biological properties. They harbor a specific profile of PrPSc species, from small oligomers to fibrils in different ratios, where the highest infectivity aligns with oligomeric particles. To investigate the impact of PrPSc aggregate complexity on prion propagation, biochemical properties, and disease pathogenesis, we fractionated elk prions by sedimentation velocity centrifugation, followed by sub-passages of individual fractions in cervidized mice. Upon first passage, different fractions generated PrPSc with distinct biochemical, biophysical, and neuropathological profiles. Notably, low or high molecular weight PrPSc aggregates caused different clinical signs of hyperexcitability or lethargy, respectively, which were retained over passage, whereas other properties converged. Our findings suggest that PrPSc quaternary structure determines an initial selection of a specific replication environment, resulting in transmissible features that are independent of PrPSc biochemical and biophysical properties.

Quantitative analysis of prion disease using an AI-powered digital pathology framework

Prion disease is a fatal neurodegenerative disorder characterized by accumulation of an abnormal prion protein (PrPSc) in the central nervous system. To identify PrPSc aggregates for diagnostic purposes, pathologists use immunohistochemical staining of prion protein antibodies on tissue samples. With digital pathology, artificial intelligence can now analyze stained slides. In this study, we developed an automated pipeline for the identification of PrPSc aggregates in tissue samples from the cerebellar and occipital cortex. To the best of our knowledge, this is the first framework to evaluate PrPSc deposition in digital images. We used two strategies: a deep learning segmentation approach using a vision transformer, and a machine learning classification approach with traditional classifiers. Our method was developed and tested on 64 whole slide images from 41 patients definitively diagnosed with prion disease. The results of our study demonstrated that our proposed framework can accurately classify WSIs from a blind test set. Moreover, it can quantify PrPSc distribution and localization throughout the brain. This could potentially be extended to evaluate protein expression in other neurodegenerative diseases like Alzheimer's and Parkinson's. Overall, our pipeline highlights the potential of AI-assisted pathology to provide valuable insights, leading to improved diagnostic accuracy and efficiency.

Eco-evolutionary trade-offs in the dynamics of prion strain competition.

Prion and prion-like molecules are a type of self-replicating aggregate protein that have been implicated in a variety of neurodegenerative diseases. Over recent decades, the molecular dynamics of prions have been characterized both empirically and through mathematical models, providing insights into the epidemiology of prion diseases and the impact of prions on the evolution of cellular processes. At the same time, a variety of evidence indicates that prions are themselves capable of a form of evolution, in which changes to their structure that impact their rate of growth or fragmentation are replicated, making such changes subject to natural selection. Here we study the role of such selection in shaping the characteristics of prions under the nucleated polymerization model (NPM). We show that fragmentation rates evolve to an evolutionary stable value which balances rapid reproduction of PrPSc aggregates with the need to produce stable polymers. We further show that this evolved fragmentation rate differs in general from the rate that optimizes transmission between cells. We find that under the NPM, prions that are both evolutionary stable and optimized for transmission have a characteristic length of three times the critical length below which they become unstable. Finally, we study the dynamics of inter-cellular competition between strains, and show that the eco-evolutionary trade-off between intra- and inter-cellular competition favours coexistence.

Synthesis and anti-prion aggregation activity of acylthiosemicarbazide analogues

Prions are infectious protein particles known to cause prion diseases. The biochemical entity of the pathogen is the misfolded prion protein (PrPSc) that forms insoluble amyloids to impair brain function. PrPSc interacts with the non-pathogenic, cellular prion protein (PrPC) and facilitates conversion into a nascent misfolded isoform. Several small molecules have been reported to inhibit the aggregation of PrPSc but no pharmacological intervention was well established thus far. We, here, report that acylthiosemicarbazides inhibit the prion aggregation. Compounds 7x and 7y showed almost perfect inhibition (EC50 = 5 µM) in prion aggregation formation assay. The activity was further confirmed by atomic force microscopy, semi-denaturing detergent agarose gel electrophoresis and real-time quaking induced conversion assay (EC50 = 0.9 and 2.8 µM, respectively). These compounds also disaggregated pre-existing aggregates in vitro and one of them decreased the level of PrPSc in cultured cells with permanent prion infection, suggesting their potential as a treatment platform. In conclusion, hydroxy-2-naphthoylthiosemicarbazides can be an excellent scaffold for the discovery of anti-prion therapeutics.

18F]PM-PBB3-PET Reveals Clinical and [18F]FDG-PET Mimics of 4-Repeat Tauopathy Caused by Creutzfeld-Jakob Disease.

This image depicts [18F] FDG and [18F]PM-PBB3 uptake surface projections of two male patients with complex movement disturbances. They were referred for PET imaging with the suspected diagnosis of a 4-repeat (4R)-tauopathy Figure 1. Patient #1 (79 years) presented with an aggressive, non-levodopa responsive hypokinetic-rigid syndrome with early falls and a vertical supranuclear gaze palsy. Patient #2 (68 years) presented with unusually rapidly progressive dystonia, right-sided rigor, bradykinesia, alien limb phenomenon and action myoclonus of the right arm, likewise without response to levodopa. Disease duration was 11 and 5 months, respectively. CSF levels of tau and amyloid in either patient were not indicative for Alzheimer's disease. In both, [18F] FDG-PET revealed a corticobasal degeneration-like bilateral reduction of cerebral glucose metabolism in frontoparietal cortical areas (arrowheads) and in the thalamus.1 A clear asymmetry to the detriment of the right hemisphere was observed in #1, whereas hypometabolism of the left hemisphere was only slightly pronounced in #2. Subsequent imaging with tau ligand [18F]PM-PBB32 showed elevated binding (arrowheads, asterisks indicate unspecific uptake of the choroid plexus) suggestive for pathological tau aggregates in frontal and parietal areas of #1, supporting the diagnosis of a 4R-tauopathy.2 In contrast, the lack of specific [18F]PM-PBB3 binding in patient #2 questioned the diagnosis of a 4R-tau related corticobasal syndrome (CBS; eg, progressive supranuclear palsy—CBS). Diagnosis of Creutzfeld-Jakob disease in this patient was made via 14–3-3 and PrPSc aggregation assays. In conclusion, [18F]PM-PBB3-PET reveals clinical and [18F] FDG-PET mimics of 4R-tau CBS caused by Creutzfeld-Jakob disease. (1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical analysis: A. Design, B. Execution, C. Review and critique; (3) Manuscript: A. Writing of the first draft, B. Review and critique. N.S.: 1A, B, 1C, 2C, 3A. G.B.: 1C, 2B, 2C, 3B. P.T.M.: 1B, 2A, 2C, 3B. M.R.: 1B, 2C, 3B. J.B.: 1C, 2A, 2C, 3A. Materials required for the synthesis of [18F] APN-1607 were generously provided by Aprinoia Therapeutics Limited. Open Access funding enabled and organized by Projekt DEAL. Ethical Compliance Statement: The authors confirm that the approval of an institutional review board was not required for this work. Both patients gave written informed consent for the scientific use of imaging and clinical data. Funding Sources and Conflicts of Interest: Nils Schröter, Berta-Ottenstein-Programme for Clinician Scientists, Faculty of Medicine, University of Freiburg. The authors have no potential conflict of interest to report. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. Financial Disclosures for the Previous 12 Months: The authors have nothing to disclose.

Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

Creutzfeldt-Jakob disease after COVID-19: infection-induced prion protein misfolding? A case report

ABSTRACT Creutzfeldt-Jakob disease (CJD) is a rare, fatal disease presenting with rapidly progressive neurological deficits caused by the accumulation of a misfolded form (PrPSc) of prion protein (PrPc). Coronavirus disease 2019 (COVID-19) is a primarily respiratory syndrome caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); many diverse neurological complications have been observed after COVID-19. We describe a young patient developing CJD two months after mild COVID-19. Presenting symptoms were visuospatial deficits and ataxia, evolving into a bedridden state with preserved consciousness and diffuse myoclonus. Diagnostic work-up was suggestive of CJD. The early age of onset and the short interval between respiratory and neurological symptoms might suggest a causal relationship: a COVID-19-related neuroinflammatory state may have induced the misfolding and subsequent aggregation of PrPSc. The present case emphasizes the link between neuroinflammation and protein misfolding. Further studies are needed to establish the role of SARS-CoV-2 as an initiator of neurodegeneration.

Crystal structure of the prion protein regulatory domain in complex with the Alzheimer's peptide

The endogenous prion protein (PrP) has been shown to play a key role in both Alzheimer's disease (AD) and prion diseases. On a fundamental level, these two neurodegenerative disorders share a common cause, the misfolding of a protein or peptide and its subsequent aggregation. In prion diseases, the healthy, properly folded cellular PrP (PrPC) can be converted into the toxic conformation, scrapie PrP (PrPSc), via a poorly understood mechanism. Aggregation of PrPSc leads to cell death. AD is most commonly associated with a peptide called amyloid beta 1-42 (Aβ(1-42)) that aggregates extracellularly and eventually forms amyloid plaques, causing neuronal degeneration in concert with intracellular neurofibrillary tangles.

Astrocyte in prion disease: a double-edged sword.

Prion diseases are infectious protein misfolding disorders of the central nervous system that result from misfolding of the cellular prion protein (PrPC) into the pathologic isoform PrPSc. Pathologic hallmarks of prion disease are depositions of pathological prion protein PrPSc, neuronal loss, spongiform degeneration and astrogliosis in the brain. Prion diseases affect human and animals, there is no effective therapy, and they invariably remain fatal. For a long time, neuronal loss was considered the sole reason for neurodegeneration in prion pathogenesis, and the contribution of non-neuronal cells like microglia and astrocytes was considered less important. Recent evidence suggests that neurodegeneration during prion pathogenesis is a consequence of a complex interplay between neuronal and non-neuronal cells in the brain, but the exact role of these non-neuronal cells during prion pathology is still elusive. Astrocytes are non-neuronal cells that regulate brain homeostasis under physiological conditions. However, astrocytes can deposit PrPSc aggregates and propagate prions in prion-infected brains. Additionally, sub-populations of reactive astrocytes that include neurotrophic and neurotoxic species have been identified, differentially expressed in the brain during prion infection. Revealing the exact role of astrocytes in prion disease is hampered by the lack of in vitro models of prion-infected astrocytes. Recently, we established a murine astrocyte cell line persistently infected with mouse-adapted prions, and showed how such astrocytes differentially process various prion strains. Considering the complexity of the role of astrocytes in prion pathogenesis, we need more in vitro and in vivo models for exploring the contribution of sub-populations of reactive astrocytes, their differential regulation of signaling cascades, and the interaction with neurons and microglia during prion pathogenesis. This will help to establish novel in vivo models and define new therapeutic targets against prion diseases. In this review, we will discuss the complex role of astrocytes in prion disease, the existing experimental resources, the challenges to analyze the contribution of astrocytes in prion disease pathogenesis, and future strategies to improve the understanding of their role in prion disease.

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

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

The Latest Research on RT-QuIC Assays—A Literature Review

The misfolding of proteins such as the prion protein, α-synuclein, and tau represents a key initiating event for pathogenesis of most common neurodegenerative disorders, and its presence correlates with infectivity. To date, the diagnosis of these disorders mainly relied on the recognition of clinical symptoms when neurodegeneration was already at an advanced phase. In recent years, several efforts have been made to develop new diagnostic tools for the early diagnosis of prion diseases. The real-time quaking-induced conversion (RT–QuIC) assay, an in vitro assay that can indirectly detect very low amounts of PrPSc aggregates, has provided a very promising tool to improve the early diagnosis of human prion diseases. Over the decade since RT–QuIC was introduced, the diagnosis of not only prion diseases but also synucleinopathies and tauopathies has greatly improved. Therefore, in our study, we summarize the current trends and knowledge of RT–QuIC assays, as well as discuss the diagnosis of neurodegenerative diseases using RT–QuIC assays, which have been updated in recent years.

Extracellular Protein Aggregates Colocalization and Neuronal Dystrophy in Comorbid Alzheimer's and Creutzfeldt-Jakob Disease: A Micromorphological Pilot Study on 20 Brains.

Alzheimer’s disease (AD) and sporadic Creutzfeldt–Jakob disease (sCJD) are both characterized by extracellular pathologically conformed aggregates of amyloid proteins—amyloid β-protein (Aβ) and prion protein (PrPSc), respectively. To investigate the potential morphological colocalization of Aβ and PrPSc aggregates, we examined the hippocampal regions (archicortex and neocortex) of 20 subjects with confirmed comorbid AD and sCJD using neurohistopathological analyses, immunohistochemical methods, and confocal fluorescent microscopy. Our data showed that extracellular Aβ and PrPSc aggregates tended to be, in most cases, located separately, and “compound” plaques were relatively rare. We observed PrPSc plaque-like structures in the periphery of the non-compact parts of Aβ plaques, as well as in tau protein-positive dystrophic structures. The AD ABC score according to the NIA-Alzheimer’s association guidelines, and prion protein subtype with codon 129 methionine–valine (M/V) polymorphisms in sCJD, while representing key characteristics of these diseases, did not correlate with the morphology of the Aβ/PrPSc co-aggregates. However, our data showed that PrPSc aggregation could dominate during co-aggregation with non-compact Aβ in the periphery of Aβ plaques.

The Size and Stability of Infectious Prion Aggregates Fluctuate Dynamically during Cellular Uptake and Disaggregation.

Prion diseases arise when PrPSc, an aggregated, infectious, and insoluble conformer of the normally soluble mammalian prion protein, PrPC, catalyzes the conversion of PrPC into more PrPSc, which then accumulates in the brain leading to disease. PrPSc is the primary, if not sole, component of the infectious prion. Despite the stability and protease insensitivity of PrPSc aggregates, they can be degraded after cellular uptake. However, how cells disassemble and degrade PrPSc is poorly understood. In this work, we analyzed how the protease sensitivity and size distribution of PrPSc aggregates from two different mouse-adapted prion strains, 22L, that can persistently infect cells and 87V, that cannot, changed during cellular uptake. We show that within the first 4 h following uptake large PrPSc aggregates from both prion strains become less resistant to digestion by proteinase K (PK) through a mechanism that is dependent upon the acidic environment of endocytic vesicles. We further show that during disassembly, PrPSc aggregates from both strains become more resistant to PK digestion through the apparent removal of protease-sensitive PrPSc, with PrPSc from the 87V strain disassembled more readily than PrPSc from the 22L strain. Taken together, our data demonstrate that the sizes and stabilities of PrPSc from different prion strains change during cellular uptake and degradation, thereby potentially impacting the ability of prions to infect cells.

Creutzfeldt-Jakob Disease: An Overview and a Preliminary Study on Its Impacts on the Beef and Cattle Production Industry in the United Kingdom and the United States

Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal neurodegenerative disease, that affects 1 in 1,000,000 people, with there being 350 new cases of the pathology every year. In specific, it is a prion disease that is caused by a misfolded prion protein, termed PrPSc, which is the infectious form of the prion protein PrPC. Rather than being recycled by the body, the PrPSc aggregates in the brain as plaques, leading to neurodegeneration of surrounding cells and the spongiform characteristics of the pathology. This paper gives an overview of the current understanding of CJD, by looking at the pathophysiology genetic features of CJD, the different forms of CJD, before exploring the societal impact of CJD. In specific, this societal impact is inspected by investing the correlation between the beef and cattle production industry and the number of CJD cases in the United Kingdom and the United States, through the lens of the 1996 outbreak of variant Creutzfeldt-Jakob disease (vCJD), a form of CJD, in the United Kingdom (UK). This outbreak was caused by an infection of bovine spongiform encephalopathy in the UK’s cattle, which was then transmitted via cattle products. In its wake and seeing that it is always fatal, the outbreak forced many societal changes in an effort to prevent further infection and spread, with an emphasis being placed on the beef and cattle production industries. Hence, it is important to understand the correlation between the beef and cattle production industry and CJD cases. To do this, I looked at the correlation between the United States and United Kingdom’s cattle and beef production industries and CJD cases (vCJD cases for the UK), over the course of the past 25 years. This was done by carrying out a Pearson Correlation test between the number of CJD cases in each country and the rate of cattle production during this period of time and running a linear regression on the dataset. In doing so, it was discovered that the linear regression model fit the dataset with very low accuracy (R2 ≈ 0.5664). Moreover, it was revealed that there was only a moderative negative correlation between the number of CJD cases in the United States and the rate of production of the United States’ beef and cattle production industry (r = -0.7418), however, it was also found that there was a strong upward correlation between the number of vCJD cases in the UK and the rate of production of the UK’s beef and cattle production industry (r = 0.7632), indicating that vCJD does have a strong impact on the UK’s beef and cattle production industry.

Structural and dynamical mechanisms of a naturally occurring variant of the human prion protein in preventing prion conversion**Project supported by the Key Program of the National Key Research and Development Program of China (Grant No. 2016YFA0501702) and the National Natural Science Foundation of China (Grant No. 11674065).

Prion diseases are associated with the misfolding of the normal helical cellular form of prion protein (PrPC) into the β-sheet-rich scrapie form (PrPSc) and the subsequent aggregation of PrPSc into amyloid fibrils. Recent studies demonstrated that a naturally occurring variant V127 of human PrPC is intrinsically resistant to prion conversion and aggregation, and can completely prevent prion diseases. However, the underlying molecular mechanism remains elusive. Herein we perform multiple microsecond molecular dynamics simulations on both wildtype (WT) and V127 variant of human PrPC to understand at atomic level the protective effect of V127 variant. Our simulations show that G127V mutation not only increases the rigidity of the S2–H2 loop between strand-2 (S2) and helix-2 (H2), but also allosterically enhances the stability of the H2 C-terminal region. Interestingly, previous studies reported that animals with rigid S2–H2 loop usually do not develop prion diseases, and the increase in H2 C-terminal stability can prevent misfolding and oligomerization of prion protein. The allosteric paths from G/V127 to H2 C-terminal region are identified using dynamical network analyses. Moreover, community network analyses illustrate that G127V mutation enhances the global correlations and intra-molecular interactions of PrP, thus stabilizing the overall PrPC structure and inhibiting its conversion into PrPSc. This study provides mechanistic understanding of human V127 variant in preventing prion conversion which may be helpful for the rational design of potent anti-prion compounds.

Global analysis of protein degradation in prion infected cells

Prion diseases are rare, neurological disorders caused by the misfolding of the cellular prion protein (PrPC) into cytotoxic fibrils (PrPSc). Intracellular PrPSc aggregates primarily accumulate within late endosomes and lysosomes, organelles that participate in the degradation and turnover of a large subset of the proteome. Thus, intracellular accumulation of PrPSc aggregates has the potential to globally influence protein degradation kinetics within an infected cell. We analyzed the proteome-wide effect of prion infection on protein degradation rates in N2a neuroblastoma cells by dynamic stable isotopic labeling with amino acids in cell culture (dSILAC) and bottom-up proteomics. The analysis quantified the degradation rates of more than 4,700 proteins in prion infected and uninfected cells. As expected, the degradation rate of the prion protein is significantly decreased upon aggregation in infected cells. In contrast, the degradation kinetics of the remainder of the N2a proteome generally increases upon prion infection. This effect occurs concurrently with increases in the cellular activities of autophagy and some lysosomal hydrolases. The resulting enhancement in proteome flux may play a role in the survival of N2a cells upon prion infection.

Isolation of infectious, non-fibrillar and oligomeric prions from a genetic prion disease.

Prions are transmissible agents causing lethal neurodegenerative diseases that are composed of aggregates of misfolded cellular prion protein (PrPSc). Despite non-fibrillar oligomers having been proposed as the most infectious prion particles, prions purified from diseased brains usually consist of large and fibrillar PrPSc aggregates, whose protease-resistant core (PrPres) encompasses the whole C-terminus of PrP. In contrast, PrPSc from Gerstmann-Sträussler-Scheinker disease associated with alanine to valine substitution at position 117 (GSS-A117V) is characterized by a small protease-resistant core, which is devoid of the C-terminus. We thus aimed to investigate the role of this unusual PrPSc in terms of infectivity, strain characteristics, and structural features. We found, by titration in bank voles, that the infectivity of GSS-A117V is extremely high (109.3 ID50 U/g) and is resistant to treatment with proteinase K (109.0 ID50 U/g). We then purified the proteinase K-resistant GSS-A117V prions and determined the amount of infectivity and PrPres in the different fractions, alongside the morphological characteristics of purified PrPres aggregates by electron microscopy. Purified pellet fractions from GSS-A117V contained the expected N- and C-terminally cleaved 7 kDa PrPres, although the yield of PrPres was low. We found that this low yield depended on the low density/small size of GSS-A117V PrPres, as it was mainly retained in the last supernatant fraction. All fractions were highly infectious, thus confirming the infectious nature of the 7 kDa PrPres, with infectivity levels that directly correlated with the PrPres amount detected. Finally, electron microscopy analysis of these fractions showed no presence of amyloid fibrils, but only very small and indistinct, non-fibrillar PrPresparticles were detected and confirmed to contain PrP via immunogold labelling. Our study demonstrates that purified aggregates of 7 kDa PrPres, spanning residues ∼90-150, are highly infectious oligomers that encode the biochemical and biological strain features of the original sample. Overall, the autocatalytic behaviour of the prion oligomers reveals their role in the propagation of neurodegeneration in patients with Gerstmann-Sträussler-Scheinker disease and implies that the C-terminus of PrPSc is dispensable for infectivity and strain features for this prion strain, uncovering the central PrP domain as the minimal molecular component able to encode infectious prions. These findings are consistent with the hypothesis that non-fibrillar prion particles are highly efficient propagators of disease and provide new molecular and morphological constraints on the structure of infectious prions.

Metal homeostasis disturbances in neurodegenerative disorders, with special emphasis on Creutzfeldt-Jakob disease - Potential pathogenetic mechanism and therapeutic implications.

Creutzfeldt-Jakob disease (CJD) is characterized by a rapidly progressive dementia often accompanied by myoclonus and other signs of brain dysfunction, relying on the conversion of the normal cellular form of the prion protein (PrPC) to a misfolded form (PrPSc). The neuropathological changes include spongiform degeneration, neuronal loss, astrogliosis, and deposition of PrPSc. It is still unclear how these pathological changes correlate with the development of CJD symptoms because few patients survive beyond 2years after diagnosis. Inasmuch as the symptoms of CJD overlap some of those observed in Alzheimer's, Parkinson's, and Huntington's diseases, there may be some underlying pathologic mechanisms associated with CJD that may contribute to the symptoms of non-prion neurodegenerative diseases as well. Data suggest that imbalance of metals, including copper, zinc, iron, and manganese, induces abnormalities in processing and degradation of prion proteins that are accompanied by self-propagation of PrPSc. These events appear to be responsible for glutamatergic synaptic dysfunctions, neuronal death, and PrPSc aggregation. Given that the prodromal symptoms of CJD such as sleep disturbances and mood disorders are associated with brain stem and limbic system dysfunction, the pathological changes may initially occur in these brain regions, then spread throughout the entire brain. Alterations in cerebrospinal fluid homeostasis, which may be linked to imbalance of these metals, seem to be more important than neuroinflammation in causing the cell death. It is proposed that metal dyshomeostasis could be responsible for the initiation and progression of the pathological changes associated with symptoms of CJD and other neurodegenerative disorders.

Recent advances in the histo-molecular pathology of human prion disease.

Prion diseases are progressive neurodegenerative disorders affecting humans and other mammalian species. The term prion, originally put forward to propose the concept that a protein could be infectious, refers to PrPSc , a misfolded isoform of the cellular prion protein (PrPC ) that represents the pathogenetic hallmark of these disorders. The discovery that other proteins characterized by misfolding and seeded aggregation can spread from cell to cell, similarly to PrPSc , has increased interest in prion diseases. Among neurodegenerative disorders, however, prion diseases distinguish themselves for the broader phenotypic spectrum, the fastest disease progression and the existence of infectious forms that can be transmitted through the exposure to diseased tissues via ingestion, injection or transplantation. The main clinicopathological phenotypes of human prion disease include Creutzfeldt-Jakob disease, by far the most common, fatal insomnia, variably protease-sensitive prionopathy, and Gerstmann-Sträussler-Scheinker disease. However, clinicopathological manifestations extend even beyond those predicted by this classification. Because of their transmissibility, the phenotypic diversity of prion diseases can also be propagated into syngenic hosts as prion strains with distinct characteristics, such as incubation period, pattern of PrPSc distribution and regional severity of histopathological changes in the brain. Increasing evidence indicates that different PrPSc conformers, forming distinct ordered aggregates, encipher the phenotypic variants related to prion strains. In this review, we summarize the most recent advances concerning the histo-molecular pathology of human prion disease focusing on the phenotypic spectrum of the disease including co-pathologies, the characterization of prion strains by experimental transmission and their correlation with the physicochemical properties of PrPSc aggregates.