Prion Propagation Research Articles

A brief overview of the Swi1 prion-[SWI+

Prion and prion-like phenomena are involved in the pathology of numerous human neurodegenerative diseases. The budding yeast, Saccharomyces cerevisiae, has a number of endogenous yeast prions-epigenetic elements that are transmitted as altered protein conformations and often manifested as heritable phenotypic traits. One such yeast prion, [SWI+], was discovered and characterized by our laboratory. The protein determinant of [SWI+], Swi1 was found to contain an amino-terminal, asparagine-rich prion domain. Normally, Swi1 functions as part of the SWI/SNF chromatin remodeling complex, thus, acting as a global transcriptional regulator. Consequently, prionization of Swi1 leads to a variety of phenotypes including poor growth on non-glucose carbon sources and abolishment of multicellular features-with implications on characterization of [SWI+] as being detrimental or beneficial to yeast. The study of [SWI+] has revealed important knowledge regarding the chaperone systems supporting prion propagation as well as prion-prion interactions with [PSI+] and [RNQ+]. Additionally, an intricate regulatory network involving [SWI+] and other prion elements governing multicellular features in yeast has begun to be revealed. In this review, we discuss the current understanding of [SWI+] in addition to some possibilities for future study.

Low doses of bioherbicide favour prion aggregation and propagation in vivo

Public concerns over the use of synthetic pesticides are growing since many studies have shown their impact on human health. A new environmental movement in occidental countries promoting an organic agriculture favours the rebirth of botanical pesticides. These products confer an effective alternative to chemical pesticides such as glyphosate. Among the biopesticides, the α-terthienyls found in the roots of Tagetes species, are powerful broad-spectrum pesticides. We found that an α-terthienyl analogue with herbicidal properties, called A6, triggers resistant SDS oligomers of the pathogenic prion protein PrPSc (rSDS-PrPSc) in cells. Our main question is to determine if we can induce those rSDS-PrPSc oligomers in vitro and in vivo, and their impact on prion aggregation and propagation. Using wild-type mice challenged with prions, we showed that A6 accelerates or slows down prion disease depending on the concentration used. At 5 mg/kg, A6 is worsening the pathology with a faster accumulation of PrPSc, reminiscent to soluble toxic rSDS-PrPSc oligomers. In contrast, at 10 and 20 mg/kg of A6, prion disease occurred later, with less PrPSc deposits and with rSDS-PrPSc oligomers in the brain reminiscent to non-toxic aggregates. Our results are bringing new openings regarding the impact of biopesticides in prion and prion-like diseases.

Dimerization of the cellular prion protein inhibits propagation of scrapie prions

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.

Prion infectivity is encoded exclusively within the structure of proteinase K-resistant fragments of synthetically generated recombinant PrPSc

Transmissible spongiform encephalopathies, also known as prion diseases, are a group of fatal neurodegenerative disorders affecting both humans and animals. The central pathogenic event in prion disease is the misfolding of normal prion protein (PrPC) into the pathogenic conformer, PrPSc, which self-replicates by converting PrPC to more of itself. The biochemical hallmark of PrPSc is its C-terminal resistance to proteinase K (PK) digestion, which has been historically used to define PrPSc and is still the most widely used characteristic for prion detection. We used PK-resistance as a biochemical measure for the generation of recombinant prion from bacterially expressed recombinant PrP. However, the existence of both PK- resistant and -sensitive PrPSc forms in animal and human prion disease led to the question of whether the in vitro-generated recombinant prion infectivity is due to the PK-resistant or -sensitive recombinant PrP forms. In this study, we compared undigested and PK-digested recombinant prions for their infectivity using both the classical rodent bioassay and the cell-based prion infectivity assay. Similar levels of infectivity were detected in PK-digested and -undigested samples by both assays. A time course study of recombinant prion propagation showed that the increased capability to seed the conversion of endogenous PrP in cultured cells coincided with an increase of the PK-resistant form of recombinant PrP. Moreover, prion infectivity diminished when recombinant prion was subjected to an extremely harsh PK digestion. These results demonstrated that the infectivity of recombinant prion is encoded within the structure of the PK-resistant PrP fragments. This characteristic of recombinant prion, that a simple PK digestion is able to eliminate all PK-sensitive (non-infectious) PrP species, makes possible a more homogenous material that will be ideal for dissecting the molecular basis of prion infectivity.

Modeling Multiple System Atrophy Prion Propagation in Astrocytes From Transgenic Mice

Background/AimsThere is a growing body of evidence that astrocytes contain α‐synuclein deposits in Parkinson's disease (PD), multiple system atrophy (MSA), and other α‐synucleinopathies at advanced stages of disease. The native protein isoform of α‐synuclein misfolds into a prion conformation that aggregates and spreads throughout the brain, resulting in neurodegeneration in each of these conditions. We aim to use a new in vitro model of α‐synuclein prion propagation to clarify the role of astrocytes in disease progression of MSA. This system may also prove useful for elucidating the molecular mechanisms of neurotoxicity.MethodsWe isolated primary cell culture astrocytes from the brains of transgenic mice expressing either the wild‐type (wt) form of human α‐synuclein or with the A53T mutation, which is associated with familial PD. Astrocytes were exposed to brain homogenates from MSA patients, mouse‐passaged MSA, recombinant wt α‐synuclein fibrils, and cell lysate from MSA‐infected astrocyte cultures. Following exposure, cells were thoroughly washed and cultured in fresh medium. We used confocal microscopy and high content analysis to examine α‐synuclein intracellular morphology and phosphorylation at multiple time points.ResultsWhile exogenously added α‐synuclein fibrils persisted unphosphorylated, they efficiently induced aggregation and phosphorylation of endogenously expressed α‐synuclein in exposed astrocytes. We observed rapid, progressive, and dose‐dependent α‐synuclein aggregate formation in astrocytes expressing either wt or A53T human α‐synuclein exposed to fibrils or MSA homogenates. Moreover, confocal microscopy revealed two morphologically distinct types of α‐synuclein assemblies – filamentous and granular. Both types progressively formed glial cytoplasmic inclusions, were hyperphosphorylated, thioflavin S positive, ubiquitinated, and co‐localized with the p62 marker, as is also seen in the brains of human MSA patients.ConclusionsOur studies investigate the formation of α‐synuclein aggregates in astrocytes and report these cells as potential candidates in which to examine various pathological forms of α‐synuclein prions. This cell culture model may allow studies on mechanisms of neurotoxicity and might provide a novel approach for drug discovery against MSA and other α‐synucleinopathies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glial alterations in human prion diseases: A correlative study of astroglia, reactive microglia, protein deposition, and neuropathological lesions.

Background:Neuroinflammation has recently been proposed to be a major component of neurodegenerative diseases. The aim of this study was to determine how the interaction between microglia and astroglia, which are the primary immune cell populations in the brain, and pathological prion protein (PrPsc) could influence the development and propagation of this neurodegenerative disease. Because a relevant role for glial response in prion disease has been clearly demonstrated in our previous studies using the natural animal model, a similar approach has been taken here using the natural human model.Methods:A morphological approach has been developed to analyze cerebellar samples from patients with Creutzfeldt-Jakob disease (CJD) in comparison with healthy control cases. Histopathological lesions were assessed, and PrPsc, glial fibrillary acidic protein (GFAP) and reactive microglia were immunolabelled by specific antibodies. Furthermore, co-location studies using confocal microscopy were performed to determine the possible relationships between both types of glial cells in all samples.Results:The results presented in this study support the involvement of both types of glial cells in CJD. Evidence of increased astrocyte and microglia reactivity can be observed in all CJD cases, and a close relationship between the types of glia is demonstrated by co-location studies.Conclusion:Proteinopathies such as Alzheimer, Parkinson, and Huntington diseases, where aberrant proteins spread throughout the brain during disease progression, may share a molecular basis and mechanisms of propagation. Therefore, studies elucidating the interaction between gliosis and prion propagation may be relevant to these other neurodegenerative diseases and may provide new targets for therapeutic intervention.

Evolutionary Conservation of Variant‐dependent Prion‐promoting Hsp40 Functions in Plants

The J‐protein (Hsp40) Sis1 is required for the propagation of yeast prions and for the efficient curing of the prion [PSI+] by overexpression of the disaggregase Hsp104. Interestingly, some of the prion‐supporting functions of Sis1 are conserved in homologs from higher organisms: Sis1 homologs from fruit flies and humans have been shown to substitute for Sis1 in yeast in the propagation of some, but not all, yeast prions. The number and diversity of J‐proteins is greatest in the cytosol and the number of J‐protein genes in the eukaryotic genome generally scales with the overall biochemical complexity of the organism and genome size. As such, yeast has 23 J‐proteins, humans have 41, and we recently reported that the model plant Arabidopsis thaliana has 106. Not surprisingly, at least 6 functional homologs of Sis1 have been identified in A. thaliana. Here we present the results of our ongoing investigation, using yeast complementation assays and multiple prions, to examine which prion‐maintaining functions of Sis1 are conserved in various plant Sis1 homologs. Most notably, preliminary results indicate that these homologs give rise to distinct patterns of prion maintenance, indicating that the diversification of Sis1‐homolog structure has created alternative combinations of functions that affect prion‐propagation. In addition to providing information about J‐protein evolution, our investigation is expected to lead to a greater understanding of how the amino acid sequences of various J‐proteins allow for prion‐specific interactions in yeast.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The autophagy inducers AR‐12 and AR‐14 control prion infection

Prion diseases are fatal infectious neurodegenerative disorders that affect both man and animals. The autocatalytic conversion of the cellular prion protein (PrPC) into the pathologic isoform PrPSc is a key feature in prion pathogenesis. Loss of neurons, astrogliosis and mild microglia activation are the main pathological features of prion diseases. This results in a progressive spongiform degeneration of the central nervous system, leading to ataxia, behavioral changes and, in humans, highly progressive loss of intellectual abilities. In the last two decades, great efforts have been made to establish treatment options for prion diseases. These included testing existing drugs for anti‐prion activity in experimental models with only a few agents progressing to human studies of patients with prion diseases. Investigations to date have not resulted in a recognized/proven treatment for prion diseases. AR‐12 is an IND‐approved derivative of celecoxib that demonstrated preclinical activity against several microbial diseases. Recently, AR‐12 has been shown to enhance clearance of misfolded proteins through autophagy stimulation. The latter proposes AR‐12 to be a potential therapeutic agent for neurodegenerative disorders. In the present study, we evaluated the role of AR‐12 and its derivatives in controlling prion infection. We tested AR‐12 in prion infected neuronal and non‐neuronal cell lines. Immunoblotting and confocal microscopy results showed that AR‐12 and its analogue AR‐14 reduced PrPSc levels after only 72 hours of treatment. Furthermore, infected cells were cured of PrPSc after exposure of AR‐12 or AR‐14 for only two weeks. We partially attribute the influence of the AR compounds on prion propagation to autophagy stimulation. Currently, we are investigating the effect of AR‐compounds in vivo. We are also evaluating the effect of combining AR‐ compounds with other anti‐prion agents to investigate the effect on PrPSc and prion seeding activity. Taken together, this study demonstrates that AR‐12 and the AR‐14 analogue are potential new therapeutic agents for prion diseases and possibly protein misfolding disorders involving prion‐like mechanisms.Support or Funding InformationThis work was supported by grants from the Alberta Prion Research Institute (201200002), the National Institute of Health (R01 NS076853‐01A1), the Canada Foundation for Innovation (Leaders Opportunity Fund project #32212) and was performed within the framework of the Calgary Prion Research Unit (CPRU; APRI grant 201600010). B.A.A. is an Alberta Innovates Health Solutions postgraduate fellow, D. A. is an Eyes High postgraduate fellow. S. T. is an Eyes High and Killam PhD student. S.G. is supported by the Canada Research Chair program. There are no conflicts of interest. There was no financial support from ARNO Therapeutics for performing this study. U.S. Patent Application entitled “COMPOSITIONS AND METHODS FOR REDUCING PRION LEVELS” filed February 28, 2017 as 15/445,964; VLP Ref: 2242‐00‐018U01.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Complex Effects of J‐protein Alterations on Hsp104‐mediated Curing of Prion [PSI+

The amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded protein, passed to daughter cells following fragmentation by a set of molecular chaperones which includes the J‐protein Sis1, Hsp70, and Hsp104. Overexpression of Hsp104 efficiently cures the prion [PSI+], a phenomenon which has promoted the exploration of Hsp104 as a potential therapeutic agent for neurodegenerative diseases. However, the mechanism of [PSI+] elimination by Hsp104 overexpression has been the subject of significant debate for the past two decades and has garnered significant interest in the recent literature as multiple conflicting models have been proposed. Yeast prion propagation is inexorably reliant on the function of molecular chaperones of the Hsp100, Hsp70, and Hsp40 classes. Specifically, four Hsp40s (also called J‐proteins) have been implicated in various aspects of yeast prion biology: Sis1, Ydj1, Apj1, and Swa2. We found that overexpression of Sis1 or Apj1 accelerates strong [PSI+] elimination by Hsp104 overexpression, yet Ydj1 overexpression has a profound and opposing effect, completely blocking Hsp104‐mediated curing, indicating that Apj1 and Sis1 likely have similar and partially overlapping roles in this process. Interestingly results for weak variants of [PSI+] indicated potentially no role for J‐proteins in curing as no J‐protein alteration whatsoever affected the ability of Hsp104 to cure these variants. Additional experiments to determine the specific J‐protein domains responsible for various effects, as well as J‐protein requirements in cell backgrounds harboring both [PSI+] and [RNQ+] are underway. Overall our data support the hypothesis that Hsp104‐mediated curing may occur by biochemically distinct, variant‐specific mechanisms, only some of which involve J‐proteins.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tuning Hsp70 Function: Investigating the Ability of Hsp40/Hsp70 Extragenic Suppressors to Promote Prion Propagation in Yeast

Hsp70s are a versatile class of molecular chaperones, essential for a variety of cellular functions ranging from protein folding and transport to iron‐sulfur cluster biogenesis. Hsp70s function with obligate Hsp40 co‐chaperones, also called J‐proteins, which stimulate Hsp70 ATPase activity via their characteristic J‐domains. In Saccharomyces cerevisiae, the J‐protein Sis1 is essential for cell viability and the propagation of prions. However, the means by which Sis1 drives specific functions of Hsp70 are largely unknown. We recently reported the discovery of gain‐of‐function substitutions in both the J‐protein Ydj1 and the Hsp70 Ssa1 which allow cell survival without Sis1. Here we present additional data that support the ability of the suppressor mutants to substitute for Sis1‐dependent roles, specifically the maintenance of yeast prions. Specifically, we tested the ability of the two most robust suppressor mutations in both Ydj1 and Ssa1 to maintain two prions, [PSI‐+] and [RNQ+], and included both phenotypically strong and weak variants of [PSI‐+] in our analysis. Interestingly, we found similar results for all mutants: both Ydj1 mutants could substitute for the propagation of strong [PSI‐+], but not weak [PSI‐+] nor [RNQ+]. Similarly, we found that the Ssa1 mutants could not support the maintenance of weak [PSI‐+] nor [RNQ+]. Unfortunately, due to the severe [PSI‐+] toxicity in the compromised strain bearing strong [PSI‐+], we could not confidently determine the ability of the Ssa1 mutants to support strong [PSI‐+] propagation. Further investigation of additional Ydj1 and Ssa1 mutants may provide greater resolution regarding the specific functions required of J‐proteins by distinct prions for propagation.Support or Funding InformationThis work was supported by National Institutes of Health (https://www.nih.gov/) grants GM31107 and GM27870 (EAC) and R15GM110606 (JKH). This work was also supported by the Lafayette College Chemistry Department, the EXCEL research scholarship program, and the John F and Dorothy M Dorflinger Summer Research Endowment Fund (JKH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction of Peptide Aptamers with Prion Protein Central Domain Promotes \u03b1-Cleavage of PrPC

Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis or treatment exists. Prions are composed of the misfolded isoform PrPSc of the cellular prion protein PrPC. Expression of PrPC is a prerequisite for prion infection, and conformational conversion of PrPC is induced upon its direct interaction with PrPSc. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrPC as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrPSc replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrPC α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrPC at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aβ oligomers characteristic for Alzheimer’s disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrPC and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.

Anti-Prion Systems in Yeast and Inositol Polyphosphates.

The amyloid-based yeast prions are folded in-register parallel β-sheet polymers. Each prion can exist in a wide array of variants, with different biological properties resulting from different self-propagating amyloid conformations. Yeast has several anti-prion systems, acting in normal cells (without protein overexpression or deficiency). Some anti-prion proteins partially block prion formation (Ssb1,2p, ribosome-associated Hsp70s); others cure a large portion of prion variants that arise [Btn2p, Cur1p, Hsp104 (a disaggregase), Siw14p, and Upf1,2,3p, nonsense-mediated decay proteins], and others prevent prion-induced pathology (Sis1p, essential cytoplasmic Hsp40). Study of the anti-prion activity of Siw14p, a pyrophosphatase specific for 5-diphosphoinositol pentakisphosphate (5PP-IP5), led to the discovery that inositol polyphosphates, signal transduction molecules, are involved in [PSI+] prion propagation. Either inositol hexakisphosphate or 5PP-IP4 (or 5PP-IP5) can supply a function that is needed by nearly all [PSI+] variants. Because yeast prions are informative models for mammalian prion diseases and other amyloidoses, detailed examination of the anti-prion systems, some of which have close mammalian homologues, will be important for the development of therapeutic measures.

Dissecting the Structural Mechanism of a Naturally Occuring Variant of the Prion Protein in Preventing Prion Disease

Prion diseases are a group of fatal neurodegenerative disorders associated with the misfolding and aggregation of mostly alpha-helical cellular prion protein (PrPC) into the beta-rich infectious (scrapie) form of the prion protein (PrPSc). A recent experimental study reported that the naturally occurring G127V variant of human PrPC is intrinsically resistant to prion conversion and completely prevents prion disease. However, the structural basis of the protective effect of V127 variant remains mostly unknown. Herein we performed multiple microsecond-scale molecular dynamics simulations on both wildtype and V127 variant of human prion protein at neutral pH with the aim to understand why V127 variant can prevent prion conversion. Our simulations show that the G127V mutation not only increases the rigidity of the loop between strand-2 (S2) and helix-2 (H2) but also enhances the stability of H2 through the formation of hydrophobic interactions between V127 and P165 and that of the salt bridge between R164 and D178. The increased rigidity of S2-H2 loop and the enhanced H2 stability in V127 variant may inhibit the prion conversion of human PrPC, thus prevents prion disease. Our hypothesis is supported by the fact that prions are poorly transmissible to animals with their PrPC carrying a rigid loop and a recent finding showing that stabilization of H2 of prion protein prevents its misfolding and oligomerization. In addition, our MD simulations at acidic pH demonstrate that G127V mutation facilitates the helix-to-sheet transition of the C-terminal region of H2 that is critical to the misfolding of wildtype PrPC. Our findings offer structural basis for understanding the role of human 127V variant in preventing prion conversion and may provide key mechanistic insights into prion propagation and the development of rational therapeutics.

Nonsense-mediated mRNA decay factors cure most [PSI+] prion variants

The yeast prion [PSI+] is a self-propagating amyloid of Sup35p with a folded in-register parallel β-sheet architecture. In a genetic screen for antiprion genes, using the yeast knockout collection, UPF1/NAM7 and UPF3, encoding nonsense-mediated mRNA decay (NMD) factors, were frequently detected. Almost all [PSI+] variants arising in the absence of Upf proteins were eliminated by restored normal levels of these proteins, and [PSI+] arises more frequently in upf mutants. Upf1p, complexed with Upf2p and Upf3p, is a multifunctional protein with helicase, ATP-binding, and RNA-binding activities promoting efficient translation termination and degradation of mRNAs with premature nonsense codons. We find that the curing ability of Upf proteins is uncorrelated with these previously reported functions but does depend on their interaction with Sup35p and formation of the Upf1p-Upf2p-Upf3p complex (i.e., the Upf complex). Indeed, Sup35p amyloid formation in vitro is inhibited by substoichiometric Upf1p. Inhibition of [PSI+] prion generation and propagation by Upf proteins may be due to the monomeric Upf proteins and the Upf complex competing with Sup35p amyloid fibers for available Sup35p monomers. Alternatively, the association of the Upf complex with amyloid filaments may block the addition of new monomers. Our results suggest that maintenance of normal protein-protein interactions prevents prion formation and can even reverse the process.

Prion propagation and inositol polyphosphates.

The [PSI+] prion is a folded in-register parallel β-sheet amyloid (filamentous polymer) of Sup35p, a subunit of the translation termination factor. Our searches for anti-prion systems led to our finding that certain soluble inositol polyphosphates (IPs) are important for the propagation of the [PSI+] prion. The IPs affect a wide range of processes, including mRNA export, telomere length, phosphate and polyphosphate metabolism, energy regulation, transcription and translation. We found that 5-diphosphoinositol tetra(or penta)kisphosphate or inositol hexakisphosphate could support [PSI+] prion propagation, and 1-diphosphoinositol pentakisphosphate appears to inhibit the process.

Recombinant PrP and Its Contribution to Research on Transmissible Spongiform Encephalopathies.

The misfolding of the cellular prion protein (PrPC) into the disease-associated isoform (PrPSc) and its accumulation as amyloid fibrils in the central nervous system is one of the central events in transmissible spongiform encephalopathies (TSEs). Due to the proteinaceous nature of the causal agent the molecular mechanisms of misfolding, interspecies transmission, neurotoxicity and strain phenomenon remain mostly ill-defined or unknown. Significant advances were made using in vivo and in cellula models, but the limitations of these, primarily due to their inherent complexity and the small amounts of PrPSc that can be obtained, gave rise to the necessity of new model systems. The production of recombinant PrP using E. coli and subsequent induction of misfolding to the aberrant isoform using different techniques paved the way for the development of cell-free systems that complement the previous models. The generation of the first infectious recombinant prion proteins with identical properties of brain-derived PrPSc increased the value of cell-free systems for research on TSEs. The versatility and ease of implementation of these models have made them invaluable for the study of the molecular mechanisms of prion formation and propagation, and have enabled improvements in diagnosis, high-throughput screening of putative anti-prion compounds and the design of novel therapeutic strategies. Here, we provide an overview of the resultant advances in the prion field due to the development of recombinant PrP and its use in cell-free systems.

Generation and propagation of yeast prion [URE3] are elevated under electromagnetic field.

In this study, we studied the effect of 2.0GHz radio frequency electromagnetic field (RF-EMF) and 50Hz extremely low frequency electromagnetic field (ELF-EMF) exposure on prion generation and propagation using two budding yeast strains, NT64C and SB34, as model organisms. Under exposure to RF-EMF or ELF-EMF, the de novo generation and propagation of yeast prions [URE3] were elevated in both strains. The elevation increased over time, and the effects of ELF-EMF occurred in a dose-dependent manner. The transcription and expression levels of the molecular chaperones Hsp104, Hsp70-Ssa1/2, and Hsp40-Ydj1 were not statistically significantly changed after exposure. Furthermore, the levels of ROS, as well as the activities of superoxide dismutase (SOD) and catalase (CAT), were significantly elevated after short-term, but not long-term exposure. This work demonstrated for the first time that EMF exposure could elevate the de novo generation and propagation of yeast prions and supports the hypothesis that ROS may play a role in the effects of EMF on protein misfolding. The effects of EMF on protein folding and ROS levels may mediate the broad effects of EMF on cell function.

Evidence for sortilin modulating regional accumulation of human tau prions in transgenic mice

Misfolding of tau proteins into prions and their propagation along neural circuits are thought to result in neurodegeneration causing Alzheimer's disease, progressive supranuclear palsy, chronic traumatic encephalopathy, and other tauopathies. Little is known about the molecular processes mediating tau prion replication and spreading in different brain regions. Using transgenic (Tg) mice with a neuronal promoter driving expression of human mutant (P301S) tau, we found that tau prion formation and histopathologic deposition is largely restricted to the hindbrain. Unexpectedly, tau mRNA and protein levels did not differ between the forebrain and hindbrain, suggesting that other factors modulating the conversion of tau into a prion exist and are region specific. Using a cell-based prion propagation assay, we discovered that tau prion replication is suppressed by forebrain-derived inhibitors, one of which is sortilin, a lysosomal sorting receptor. We also show that sortilin expression is higher in the forebrain than the hindbrain across the life span of the Tg mice, suggesting that sortilin, at least in part, inhibits forebrain tau prion replication in vivo. Our findings provide evidence for selective vulnerability in mice resulting in highly regulated levels of tau prion propagation, thus affording a model for identification of additional molecules that could mitigate the levels of tau prions in human tauopathies.

Proteolysis suppresses spontaneous prion generation in yeast

Prions are infectious proteins that cause fatal neurodegenerative disorders including Creutzfeldt-Jakob and bovine spongiform encephalopathy (mad cow) diseases. The yeast [PSI+] prion is formed by the translation-termination factor Sup35, is the best-studied prion, and provides a useful model system for studying such diseases. However, despite recent progress in the understanding of prion diseases, the cellular defense mechanism against prions has not been elucidated. Here, we report that proteolytic cleavage of Sup35 suppresses spontaneous de novo generation of the [PSI+] prion. We found that during yeast growth in glucose media, a maximum of 40% of Sup35 is cleaved at its N-terminal prion domain. This cleavage requires the vacuolar proteases PrA-PrB. Cleavage occurs in a manner dependent on translation but independently of autophagy between the glutamine/asparagine-rich (Q/N-rich) stretch critical for prion formation and the oligopeptide-repeat region required for prion maintenance, resulting in the removal of the Q/N-rich stretch from the Sup35 N terminus. The complete inhibition of Sup35 cleavage, by knocking out either PrA (pep4Δ) or PrB (prb1Δ), increased the rate of de novo formation of [PSI+] prion up to ∼5-fold, whereas the activation of Sup35 cleavage, by overproducing PrB, inhibited [PSI+] formation. On the other hand, activation of the PrB pathway neither cleaved the amyloid conformers of Sup35 in [PSI+] strains nor eliminated preexisting [PSI+]. These findings point to a mechanism antagonizing prion generation in yeast. Our results underscore the usefulness of the yeast [PSI+] prion as a model system to investigate defense mechanisms against prion diseases and other amyloidoses.

Complement Regulatory Protein Factor H Is a Soluble Prion Receptor That Potentiates Peripheral Prion Pathogenesis.

Several complement proteins exacerbate prion disease, including C3, C1q, and CD21/35. These proteins of the complement cascade likely increase uptake, trafficking, and retention of prions in the lymphoreticular system, hallmark sites of early prion propagation. Complement regulatory protein factor H (fH) binds modified host proteins and lipids to prevent C3b deposition and, thus, autoimmune cell lysis. Previous reports show that fH binds various conformations of the cellular prion protein, leading us to question the role of fH in prion disease. In this article, we report that transgenic mice lacking Cfh alleles exhibit delayed peripheral prion accumulation, replication, and pathogenesis and onset of terminal disease in a gene-dose manner. We also report a biophysical interaction between purified fH and prion rods enriched from prion-diseased brain. fH also influences prion deposition in brains of infected mice. We conclude from these data and previous findings that the interplay between complement and prions likely involves a complex balance of prion sequestration and destruction via local tissue macrophages, prion trafficking by B and dendritic cells within the lymphoreticular system, intranodal prion replication by B and follicular dendritic cells, and potential prion strain selection by CD21/35 and fH. These findings reveal a novel role for complement-regulatory proteins in prion disease.