Prion aggregates transfer through tunneling nanotubes in endocytic vesicles

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the misfolding of the cellular prion protein to an infectious form PrP^Sc. The intercellular transfer of PrP^Sc is a question of immediate interest as the cell-to-cell movement of the infectious particle causes the inexorable propagation of disease. We have previously identified tunneling nanotubes (TNTs) as one mechanism by which PrP^Sc can move between cells. Here we investigate further the details of this mechanism and show that PrP^Sc travels within TNTs in endolysosomal vesicles. Additionally we show that prion infection of CAD cells increases both the number of TNTs and intercellular transfer of membranous vesicles, thereby possibly playing an active role in its own intercellular transfer via TNTs.     

Stimulating the Release of Exosomes Increases the Intercellular Transfer of Prions

Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP^C, into the disease-associated isoform, PrP^Sc. The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP^C, leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions.     

De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype

Prions are the proteinaceous infectious agents responsible for Transmissible Spongiform Encephalopathies. Compelling evidence supports the hypothesis that prions are composed exclusively of a misfolded version of the prion protein (PrP^Sc) that replicates in the body in the absence of nucleic acids by inducing the misfolding of the cellular prion protein (PrP^C). The most common form of human prion disease is sporadic, which appears to have its origin in a low frequency event of spontaneous misfolding to generate the first PrP^Sc particle that then propagates as in the infectious form of the disease. The main goal of this study was to mimic an early event in the etiology of sporadic disease by attempting de novo generation of infectious PrP^Sc in vitro. For this purpose we analyzed in detail the possibility of spontaneous generation of PrP^Sc by the protein misfolding cyclic amplification (PMCA) procedure. Under standard PMCA conditions, and taking precautions to avoid cross-contamination, de novo generation of PrP^Sc was never observed, supporting the use of the technology for diagnostic applications. However, we report that PMCA can be modified to generate PrP^Sc in the absence of pre-existing PrP^Sc in different animal species at a low and variable rate. De novo generated PrP^Sc was infectious when inoculated into wild type hamsters, producing a new disease phenotype with unique clinical, neuropathological and biochemical features. Our results represent additional evidence in support of the prion hypothesis and provide a simple model to study the mechanism of sporadic prion disease. The findings also suggest that prion diversity is not restricted to those currently known, and that likely new forms of infectious protein foldings may be produced, resulting in novel disease phenotypes.     

Tunnelling nanotubes: A highway for prion spreading?

The discovery of tunnelling nanotubes (TNTs) and their proposed role in long intercellular transport of organelles, bacteria and viruses have led us to examine their potential role during prion spreading. We have recently shown that these membrane bridges can form between neuronal cells, as well as between dendritic cells and primary neurons and that both endogenous and exogenous PrP^Sc appear to traffic through these structures between infected and non-infected cells. Furthermore, prion infection can be efficiently transmitted from infected dendritic cells to primary neurons only in co-culture conditions permissive for TNT formation. Therefore, we propose a role for TNTs during prion spreading from the periphery to the central nervous system (CNS). Here, we discuss some of the key steps where TNTs might play a role during prion neuroinvasion.Key words: tunnelling nanotubes, TNTs, prion, PrP^Sc, prion spreading, dendritic cells, neuroinvasionPrion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that have been found in a number of species, including scrapie in sheep, bovine spongiform encephalopathy in cattle (BSE), Chronic wasting disease in deer and Creutzfeldt-Jacob, the Gerstmann-Straüssler-Scheinker syndrome, fatal familial insomnia and kuru in humans (reviewed in ref. ¹). Human TSEs can be sporadic, genetic or acquired by infection. A new variant of Creutzfeldt-Jakob disease (termed vCJD) was reported from the UK in 1996.² The majority of vCJD cases diagnosed to date resulted from a peripheral exposure via the consumption of BSE-contaminated food. Pathological features of TSE diseases can include gliosis, neuronal cell loss and spongiform changes, but the common feature of all members of this group of diseases is the build-up of an aberrant form of the host cellular protein PrP^C, named PrP^Sc (from scrapie). The normal cellular isoform, PrP^C, is an endogenous glycosylphosphatidyl inositol (GPI)-anchored protein present in numerous tissues in mammals, including neurons and lymphoid cells. While the exact function of PrP^C remains unclear, evidence suggest putative roles in neuroprotection, cell adhesion and signal transduction (reviewed in refs. ³ and ⁴). According to the ‘protein-only hypothesis,’ the causative agents of prion diseases are proteinaceous infectious particles (‘prions’), which are composed essentially of misfolded PrP^C, or PrP^Sc.^5,6 Prions replicate through a molecular mechanism in which abnormally folded PrP^Sc acts as a catalyst and serves as a template to convert normal PrP^C molecules into PrP^Sc.^5,6 PrP^Sc differs from PrP^C in the conformation of its polypeptide chain, which is enriched in β-sheets and is protease resistant. Although the conversion process is believed to have a predominant role in the pathogenesis of prion diseases, the cellular and molecular basis for the pathogenic conversion of PrP are still unknown.Another important question is how PrP^Sc spreads to and within the brain. After oral exposure, PrP^Sc accumulates into lymphoid tissues, such as the spleen, lymph nodes or Peyer''s patches, prior to neuroinvasion.^7–9 The exact mechanisms and specific cells involved in the spreading from the gastrointestinal track to the lymphoid system and to the peripheral nervous system (PNS), leading to neuroinvasion of the CNS remain to be elucidated. However, a range of evidence suggests that the accumulation of PrP^Sc within lymphoid tissues is necessary for efficient neuroinvasion.^9–11 In particular it has been shown that PrP^Sc accumulates first within follicular dendritic cells (FDCs)¹² and macrophages.¹³ FDCs are stromal-differentiated cells in the germinal centres of activated lymphoid follicles. A number of studies have demonstrated that FDCs play a critical role during spreading of infection since their absence greatly impaires the neuroinvasion process.^8,11,14,15 However, because FDCs are immobile cells, it is not clear how they acquire PrP^Sc and how it spreads from the FDCs to the PNS. FDCs and nerve synapses occupy different anatomical sites^16,17 and therefore the lack of physical contact between the gut and FDCs and between FDCs and the nerve periphery imply the presence of intermediate mechanisms for the transport of PrP^Sc. Dendritic cells (DCs) have been proposed to play a critical role in the transport of PrP^Sc from the gut to FDCs.¹⁸ DCs function as sentinels for incoming pathogens. Bone-marrow dendritic cells (BMDCs) are migratory cells that are able to transport proteins within Peyer''s patches and into mesenteric lymph nodes.¹⁹ Interestingly, mucosal dendritic cells which play a role in the transport of intestinal antigen for presentation to Peyer''s patches and to mesenteric lymph nodes, can also extend trans-epithelial dendrites to directly sample bacteria in the gut.^20,21 However, the transport of PrP^Sc from FDCs to the PNS remains controversial and evidence for a direct role of DCs during this process has been debated.^22,23 Several mechanisms have been proposed for the intercellular transfer of PrP^Sc, including cell-cell contact, transfer via exosomes or by GPI-painting.^24–26 For example, similar to other types of pathogens such as HIV-1, which was proposed to follow the “exosomal” pathway to be released from the cells,²⁷ it has been shown that the supernatant of prion infected cells contain large amount of PrP^Sc in membranous vesicles known as exosomes.^25,28 Thus, it was suggested that exosomes might be a way to spread prion infection in vivo.^25,28 Recently, a different type of vesicles known as plasma membrane-derived microvesicles, were also described as a potential spreading mechanism during neuroinvasion.²⁹In 2004, Rustom and colleagues discovered a new mechanism of long distance intercellular communication in mammalian cells, called tunnelling nanotubes (TNTs).³⁰ TNTs are transient, long, actin-rich projections that allow for long-distance intercellular communication (reviewed in refs. ^31–33). TNT-like structures have been described to form in vitro between numerous cell types, including neuronal and immune cells.^30,34,35 These studies demonstrated that TNT-like structures formed bridges or channels between distant cells that can be used to transfer material between cells, including Lysotracker positive or endosomal vesicles, calcium fluxes, bacteria or viruses through their cytoplasms or along the surface of the nanotubes.^31–33 Interestingly, a model GPI-anchored protein, GFP-GPI, was found to move at the surface of these tubes³⁴ and while studying the neuritic transport of prions in neuronal cells, Magalhães and colleagues noticed a strong correlation between internalized PrP-res and Lysotracker positive vesicles in neurites,³⁶ suggesting that PrP-res might also be able to transfer through TNTs during prion cell-cell spreading.The results from the studies mentioned above and random observations of TNT-like structures in neuronal model cell cultures first led us to study whether these structures could in fact provide an efficient mechanism for prion cell-cell spreading.³⁷ We initially characterized TNT-like structures in the mouse catecholaminergic neuronal cell line, Cath.a-Differentiated cells (CAD cells) a well-recognized neuronal cell model for prion infection.³⁸ Under our culturing conditions, over 40% of the CAD cells could efficiently form actin-rich TNT-like structures between differentially labelled cell populations. In CAD cells, these nanotubes were very heterogeneous, both in length and in diameters. Indeed, TNT-like structures had lengths ranging from 10 to 80 µm and while over 70% of the nanotubes had diameters smaller than 200 nm, the remaining TNT-like structures had larger diameters (200 to 800 nm). We demonstrated that vesicles of lysosomal origins, a fluorescent form of PrP (GFP-PrP), infectious Alexa-PrP^Sc, as well as both endogenous and exogenous PrP^Sc could traffic within TNTs between neuronal cells (Fig. 1). The lysosomal and GFP-PrP vesicles observed to move through TNTs had a directed movement with a speed in the range of actin-mediated motors,³⁷ consistent with previous studies suggesting the involvement of an actomyosin-dependent transport.³⁹ Interestingly, active transfer of endogenous PrP^Sc, lysosomal or GFP-PrP vesicles occurred through TNTs with larger diameters, suggesting distinct roles for the different TNT-like structures observed.³⁷ These results do not seem to be specific to CAD cells since the transfer of GFP-PrP throught TNTs was observed in different types of transfected cells, including HEK293 cells (unpublished data). Furthermore, these results were in agreement with previous observations by Onfelt and colleagues showing the presence of a fluorescent GPI model protein (GFP-GPI) in TNTs formed between EBV-transformed human B cells³⁴ suggesting that different GPI-anchored proteins can be transferred along the surface and inside vesicles within TNTs. In order to determine the relevance of this type of intercellular communication in the case of prion diseases, it was necessary to evaluate the trafficking of the pathological form of PrP (PrP^Sc) within TNTs, by analyzing the transfer of endogenous PrP^Sc between chronically infected ScCAD cells and non-infected CAD cells. By immunofluorescence after guanidium treatment, endogenous PrP^Sc was found inside TNTs and in the cytoplasm of recipient non-infected CAD cells. Similar to exogenous PrP^Sc, endogenous PrP^Sc particles were not present in non-infected CAD cells not in contact with ScCAD cells after overnight co-cultures, thus excluding exosomal transfer or protein shedding.³⁷ Similarly, no transfer was observed between cells in direct contact with one another or upon treatment with latrunculin, which inhibits TNT formation. Strikingly, the transfer of endogenous PrP^Sc was visible only when TNTs were present, demonstrating that in vitro, PrP^Sc can efficiently exploit TNTs to spread between cells of neuronal origin. These data suggested that TNTs could be a mechanism for prion spreading within the cells of the CNS.Open in a separate windowFigure 1Endogenous PrP^Sc transfer from ScCAD cells to CAD cells via TNTs. Endogenous PrP^Sc is found in punctate structures inside TNTs and in the cytoplasms of recipient cells. CAD cells were transfected with Cherry-PLAP (red) and co-cultured with ScCAD for 24 h. Cells were fixed, treated with Gnd and immunostained for PrP using SAF32 Ab (green). (A) Merge projection of Z-stacks obtained with a confocal Andor spinning-disk microscope. (B) Three-dimensional reconstruction of (A) using OsiriX software. (C) Zoom in on TNT-like structures. PrP^Sc is found in vesicular structures inside TNTs and in the cytoplasm of the recipient non-infected CAD cells (see blue arrow heads). Scale bar represents 10 µm.Interestingly, DCs were shown to form networks of TNTs both in vitro⁴⁰ and in vivo.⁴¹ In an elegant study, Watkins and Salter demonstrated that DCs could propagate calcium flux upon cell stimulation to other cells hundreds of microns away through TNTs, both between DCs and between DCs and THP-1 monocytes.⁴⁰ These data suggested the possibility that DCs could form tubular connections with neuronal cells in order to transport PrP^Sc to the PNS via TNTs. Using BMDCs in co-cultures with both cerebellar granular neurons (CGNs) and primary hippocampal neurons, we showed that BMDCs could form networks of TNTs with both types of neurons. Furthermore, these TNTs appeared to be functional, allowing for the transport of Lysotracker positive vesicles and infectious Alexa-PrP^Sc between loaded BMDCs and primary neurons, suggesting that DCs could transfer the infectious prion agent to primary neuronal cultures through TNTs. By using filters and conditions unfavorable for other mechanisms of transport, we found that moRK13 cells,²⁸ as well as CGNs (unpublished data), could be infected by co-cultures with BMDCs loaded with infectious brain homogenate.³⁷ Overall, these data indicate that TNTs could be an efficient mechanism of prion transmission between immune cells and neuronal cells, as well as between neuronal cultures. Since DCs can interact with peripheral neurons,⁴² we propose that TNTs could be involved in the process of neuroinvasion at multiple stages, from the peripheral site of entry to the PNS by neuroimmune interactions with DCs, allowing neurons to retrogradely transport prions to the CNS, and within the CNS (Fig. 2).Open in a separate windowFigure 2Transport of PrP^Sc via TNTs, an alternative spreading mechanism during neuroinvasion. Studies in our laboratory suggest that TNTs allow for the intracellular transport of PrP^Sc between dendritic cells and neurons and between neurons (see inset). The exact mechanism of transport remains to be determined. For instance, it is still not clear, whether PrP^Sc is strictly transported within endocytic vesicles, or whether it can slide along the surface or be transported as aggregosomes within the tubes. Similarly, the types of motors used, as well as the possible gated mechanisms to enter the recipient cells are not known. Because of the high propensity of DCs to form TNTs with different cell types, we propose that TNTs could play important roles in delivering PrP^Sc to the proper cell types along the neuroinvasion route. For instance, DCs could deliver PrP^Sc from the peripheral entry sites to FDCs in the secondary lymphoid tissues (2) or in a less efficient manner, they might occasionally directly transport PrP^Sc to the PNS (1). They could also bridge the immobile FDC networks and the PNS (3), since we have shown that DCs can form TNTs with nerve cells. Finally, once PrP^Sc has reached its final destination within the CNS, TNTs might play a final role in the spreading of PrP^Sc within the brain between neurons and possibly between neuronal cells and astrocytes (4).Recently, it was demonstrated that the distance between FDCs and the neighbouring PNS was critical for prion neuroinvasion.⁴³ Indeed, in the spleen of CD19^−/− mice, FDC networks were found to be 50% closer to the nerve fibers compared to wild-type mice.⁴³ The authors suggested that the increase in prion spreading efficiency in these mice was directly dependent on the reduction in the distance between the FDC networks and the PNS in these mice. These results would be consistent with a mechanism of transfer such as exosomes release. However, shortening the distance between FDCs and the PNS would also reduce the route of transport that mobile cells would have to travel and increase the chances for transfer of prions to the PNS, resulting in an increase in prion spreading efficiency. While the importance of FDCs in prion replication during the spreading to the CNS seems to be clear,^11,14,15 their specific role in the transfer of prions and their possible interactions with other mobile cells are much more debated.^22,23 In order to bridge the gap between FDCs and the PNS, a role for DCs as possible carriers of PrP^Sc has been postulated. Aucouturier and colleagues have previously shown, using RAG-1^−/− mice, which are deficient in FDCs, that CD11c⁺ DCs infected with 139A were able to carry prion infection to the CNS, without accumulation and replication in lymphoid organs,²² thus suggesting that DCs are able to transport prions from the periphery to nerve cells. Recently, however, another study using TNFR1^−/− mice, deficient for FDCs, suggested that DCs were unlikely candidates in the transport of prion to the PNS.²³ In this study, the authors showed that in TNFR1^−/− mice, ME7 or 139A infected DCs were inefficient in transferring infection to the PNS. The authors suggested that the differences between the results obtained with RAG-1^−/− mice and TNFR1^−/− mice could be due to the differences in the levels of innervation of the spleen in RAG-1^−/−mice compared to TNFR1^−/− mice. They suggested that in RAG-1^−/− mice, DCs could spread prion infection because their spleens are highly innervated, compared to TNFR1^−/− or wild-type mice, therefore increasing the propensity of DCs to encounter nerve cells and tranfer the prion agents. Because of the reduction in the levels of innervation in the spleens of wild-type mice, the authors concluded that DCs are unlikely candidate for the transport of prions directly to the PNS [see (1) in Fig. 2]. However, since these studies are using mice deficient for FDCs, it remains unclear what type of interactions might occur between FDCs and DCs, and how DCs might be able to transport prions from FDCs to the PNS in wild-type mice [see (2) in Fig. 2]. Indeed, both studies show that under the right circumstances, DCs can interact with nerve cells, similar to what was recently shown in infected mice⁴² and in agreement with our findings that DCs can form TNTs with neurons.³⁷ Within this scenario, it is clear that to determine the specific role of DCs during the spreading of prions from the gut to the PNS, the transfer mechanisms between DCs and other cell types, especially FDCs and peripheral neurons, need to be better characterized.Overall, these in vitro data strongly point toward TNTs as one possible mechanism of prion spreading. The next step will be to identify these structures in vivo and to determine whether prion spreading in vivo is the result of passive mechanisms, such as exosome release, active intercellular transport along and within TNTs or whether prions will use any means available to reach their targets. Recently, TNT-like structures were imaged in a mouse cornea,⁴¹ suggesting that while challenging, the visualization of in vivo trafficking of prions in lymphoid tissues such as in lymph nodes or in the spleen as well as in brain organotypic cultures might be possible and could be used to reveal the presence of TNTs.The discovery of the existence of nanotubular membrane bridges in vitro has opened-up a new field of research. Channels, called plasmodesmata,⁴⁴ connecting plant cells have long been known to play crucial roles in the transport of nutrients, molecules and signals during development and some of their functions were recently compared with some of the recently proposed functions of TNTs.⁴⁵ Furthermore, in vivo long, actin-rich filopodia like structures were found to be crucial during development.^46–49 For example, these structures exist in developing sea urchin embryos and were proposed to play a role in signalling and patterning during gastrulation.⁴⁷ Similar roles were proposed for thin filopodia-like structures observed during dorsal closure in drosophila.⁴⁹ In addition, TNT-like structures were observed in the mouse cornea between DCs and were shown to increase under inflammatory conditions.⁴¹ The authors postulated that these TNT-like structures could play a role in Ag-specific signalling, especially as a response to eye inflammation. Therefore, the possibility that TNTs might play numerous roles during cell development, in the immune system and as conduits for the spreading of pathogens could lead to major changes in the way we view animal cell interactions. Specifically, understanding how pathogens usurp these cellular connections to spread could allow for the screening and the identification of new therapeutic inhibitors. To this aim, characterizing the basic mechanism of TNT formation within cell model systems will be necessary to improve the knowledge of TNTs in general, to analyze the transfer of pathogens more specifically, and to identify key molecules during this process. In the case of prions, whether they hijack nanotubes to spread between cells or whether prions increase the formation of filopodia and TNT-like structures similar to some viruses^33,50 and/or the efficiency of transfer remain to be determined. Overall, in this specific field, the constant improvement of cell imaging techniques and the emergence of imaging tools to study prion spreading^{36,37,51–53} could lead to exciting new insights both in the physiology of these intercellular connections and in the pathology of these devastating diseases.     

Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor

Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrP^C molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrP^C is pre-incubated with poly(A) RNA and PrP^Sc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrP^Sc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrP^Sc molecules, most likely through an epitope containing residue 172.     

Activities of curcumin-related compounds in two cell lines persistently infected with different prion strains

BackgroundCultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrP^Sc). In this study, two types of cells persistently infected with prion were treated with curcumin-related compounds. We found that the compounds behave differently in neuroblastoma neuro-2a (N2a) cells infected with different prion strains.MethodsCurcumin and related compounds were applied to the two types of persistently prion infected cells to analyze the different activities of the compounds.ResultsIn ScN2a cells, which were infected with the Rocky Mountain Laboratory prion strain, two of the six compounds significantly reduced the PrP^Sc level in a dose-dependent manner. On the other hand, in N167 cells, effective suppression of the total amount of PrP^Sc was not observed; instead, two other compounds promoted the formation of covalently linked PrP^Sc dimers.ConclusionsChemometric analysis was used to determine the factors that contributed to the different effects of the six compounds. It showed that the ability to form hydrogen bonds, such as phenolic hydroxyl groups, and hydrophobic molecular properties predominantly contributed to the reduction of the PrP^Sc level in the ScN2a cells and the dimer formation of PrP^Sc in the N167 cells, respectively.General significanceThe extracted information can be used to delineate the differences among prion strains and to design compounds that are directed toward their respective activities.     

Anti-LRP/LR Antibody W3 Hampers Peripheral PrPSc Propagation in Scrapie Infected Mice

We identified the 37kDa/67kDa laminin receptor (LRP/LR) as a cell surface receptor for the cellular prion protein (PrP^c) and the infectious prion protein (PrP^Sc). Recently, we showed that anti-LRP/LR antibody W3 cured scrapie infected N2a cells. Here, we demonstrate that W3 delivered by passive immunotransfer into C57BL/6 mice reduced the PrP^Sc content in the spleen significantly by 66%, demonstrating an impairment of the peripheral PrP^Sc propagation. In addition, we observed a 1.8-fold increase in survival of anti-LRP/LR antibody W3 treated mice (mean survival of 31 days) compared to preimmune serum treated control animals (mean survival of 17 days). We conclude that the significant effect of anti-LRP/LR antibody W3 on the reduction of peripheral PrP^Sc propagation might be due to the blockage of the prion receptor LRP/LR which is required, as previously shown in vitro, for PrP^Sc propagation in vivo.Key Words: 37kDa/67kDa laminin receptor, LRP/LR, prion, PrP, TSE-therapy     

Chemically Induced Accumulation of GAGs Delays PrPSc Clearance but Prolongs Prion Disease Incubation Time

Prion diseases are a group of fatal neurodegenerative diseases affecting humans and animals. The only identified component of the infectious prion is PrP^Sc, an aberrantly folded isoform of PrP^C. Glycosaminoglycans, which constitute the main receptor for prions on cells, play a complex role in the pathogenesis of prion diseases. For example, while agents inducing aberrant lysosomal accumulation of GAGs such as Tilorone and Quinacrine significantly reduced PrP^Sc content in scrapie-infected cells, administration of Quinacrine to prion-infected subjects did not improve their clinical status. In this study, we investigated the association of PrP^Sc with cells cultured with Tilorone. We found that while the initial incorporation of PrP^Sc was similar in the treated and untreated cells, clearance of PrP^Sc from the Tilorone-treated cells was significantly impaired. Interestingly, prolonged administration of Tilorone to mice prior to prion infection resulted in a significant delay in disease onset, concomitantly with in vivo accumulation of lysosomal GAGs. We hypothesize that GAGs may complex with newly incorporated PrP^Sc in lysosomes and further stabilize the prion protein conformation. Over-stabilized PrP^Sc molecules have been shown to comprise reduced converting activity.     

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

During prion infection, the normal, protease-sensitive conformation of prion protein (PrP^C) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP^Sc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP^Sc in prion-infected brain homogenates as an initiating seed to convert PrP^C and trigger the self-propagation of PrP^Sc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP^Sc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP^Sc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP^Sc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.     

Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrP^Sc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). Stable variations in PrP^Sc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrP^Sc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrP^Sc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrP^Sc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrP^Sc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrP^Sc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrP^Sc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.     

An improved method for cell-to-cell transmission of infectious prion

Prion diseases are characterized by the accumulation of a pathological form of prion protein (PrP^Sc), which behaves as an infectious agent. Here we developed an in vitro co-culture system to analyze the PrP^Sc transmission from ScN2a cell, which persistently retains PrP^Sc, to naïve N2a cell. In this cell-to-cell transmission system, PrP^Sc transmitted to recipient N2a cell was able to be detected within 5-7 days. Further characterization showed that higher cell density greatly facilitated the transmission of PrP^Sc. This improved in vitro transmission method may become a useful tool for unveiling the molecular mechanism of PrP^Sc transmission.     

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans^9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrP^C) and the infectious protein (PrP^Sc), an abnormally-folded isoform of PrP^{C 8}.One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation¹³. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrP^Sc are poorly characterized due to their unusual conformation and aggregation states.PrP^Sc can seed the conversion of PrP^C to PrP^Scin vitro¹⁴. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrP^Sc, at an accelerated rate, from a system containing excess amounts of PrP^C and minute amounts of the PrP^Sc seed¹⁹. This technique has proven to effectively recapitulate the species and strain specificity of PrP^Sc conversion from PrP^C, to emulate prion strain interference, and to amplify very low levels of PrP^Sc from infected tissues, fluids, and environmental samples^6,7,16,23 .This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.     

Differential effects of prion particle size on infectivity in vivo and in vitro

The conversion of cellular prion protein to the disease-associated isoform (PrP^Sc) has been suggested to follow a mechanism of seeded aggregation. Here, we show that fragmentation of PrP^Sc aggregates by sonication increases converting activity in cell culture in a way similar to in vitro conversion assays. In contrast, under the same conditions the infectious titer of sonicated samples in vivo was reduced. We modified the size distribution of PrP^Sc by adsorption to nitrocellulose, which resulted in a reduction of the infectious titer in non-sonicated samples and an increase in sonicated samples. Our results indicate that NC-adsorption can (i) block some active sites of PrP^Sc aggregates and (ii) reduce the rate of clearance from the brain. For large particles with low clearance the effect of NC-particles on the number of available active sites may dominate, whereas for smaller particles (i.e. sonicated samples) the effect of NC-adsorption on clearance dominates resulting in increased infectivity.     

The Distribution of Prion Protein Allotypes Differs Between Sporadic and Iatrogenic Creutzfeldt-Jakob Disease Patients

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrP^Sc). The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrP^C) into infectious PrP^Sc. By contrast, human growth hormone-associated cases of iatrogenic CJD (iCJD) in the United Kingdom (UK) are associated with exposure to an exogenous source of PrP^Sc. In both forms of CJD, heterozygosity at residue 129 for methionine (M) or valine (V) in the prion protein gene may affect disease phenotype, onset and progression. However, the relative contribution of each PrP^C allotype to PrP^Sc in heterozygous cases of CJD is unknown. Using mass spectrometry, we determined that the relative abundance of PrP^Sc with M or V at residue 129 in brain specimens from MV cases of sCJD was highly variable. This result is consistent with PrP^C containing an M or V at residue 129 having a similar propensity to misfold into PrP^Sc thus causing sCJD. By contrast, PrP^Sc with V at residue 129 predominated in the majority of the UK human growth hormone associated iCJD cases, consistent with exposure to infectious PrP^Sc containing V at residue 129. In both types of CJD, the PrP^Sc allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Therefore, factors other than PrP^Sc allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD.     

Identification of an Intracellular Site of Prion Conversion

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP^C), denoted PrP^Sc, which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrP^C into PrP^Sc and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrP^C and PrP^Sc and measured PrP^Sc levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.     

Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds,Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate,Chlorpromazine, and U18666A,in Prion-Infected Mouse Neuroblastoma Cells

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP^Sc) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP^Sc formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP^C) and PrP^Sc in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP^Sc levels without altering intracellular distribution of PrP^Sc. PPS did not change the distribution and levels of PrP^C, whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP^C to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP^Sc formation. In contrast, CPZ and U18666A initiated the redistribution of PrP^Sc from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP^C. The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP^Sc redistribution by CPZ or U18666A partly antagonized PrP^Sc degradation, suggesting that the transfer of PrP^Sc to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP^Sc degradation. This study revealed that precise analysis of the intracellular dynamics of PrP^C and PrP^Sc provides important information for understanding the mechanism of anti-prion agents.     

Early Appearance but Lagged Accumulation of Detergent-Insoluble Prion Protein in the Brains of Mice Inoculated with a Mouse-Adapted Creutzfeldt–Jakob Disease Agent

SUMMARY 1. To elucidate mechanisms for the generation of the detergent-insoluble, proteinase K-resistant prion protein (PrP^Sc) from the detergent-soluble, proteinase K-sensitive PrP (PrP^C) and the replication of the infectious agent in prion diseases, we followed the kinetics of detergent-insoluble PrP and PrP^Sc levels, infectious titers, and associated pathological changes in the brains of mice inoculated with a mouse-adapted Creutzfeldt–Jakob disease agent.2. PrP^Sc in brain homogenate and detergent-insoluble PrP enriched by two-cycle ultracentrifugation were detected by immunoblotting and their relative amounts were estimated according to a standard curve plotted between the amount of PrP and signal intensity on immunoblotting. The titer of infectivity was determined by the incubation periods of mice inoculated with the unfractionated homogenate on the basis of a standard curve plotted between the titer and incubation period.3. Detergent-insoluble PrP became detectable 4 weeks postinoculation (p.i.) well before the detection of PrP^Sc. The low level of detergent-insoluble PrP continued until dramatic accumulation occurred at 14 weeks p.i., correlating well with the accumulation of PrP^Sc and development of pathological changes. The infectious titer was undetectable at 4 weeks p.i. and its logarithmic increase occurred 10 weeks p.i. preceding the logarithmic accumulation of PrPs.4. The lag time of detergent-insoluble PrP accumulation and the discrepancy between infectious titers and PrPs observed during the early period after inoculation suggest a slow and rate-limiting step for the detergent-insoluble PrP to become the infectious agent-associated PrP^Sc.     

The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy

The structure of the infectious prion protein (PrP^Sc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrP^Sc replicates by converting the non-infectious, cellular prion protein (PrP^C) into the misfolded, infectious conformer through an unknown mechanism. PrP^Sc and its N-terminally truncated variant, PrP 27–30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27–30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 ų, predicted the height of each PrP 27–30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.     

Generation of antisera to purified prions in lipid rafts

Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrP^C into alternately folded PrP^Sc. Immunoassays toward pre-clinical detection of infectious PrP^Sc have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrP^Sc selective antibodies. We report a method to purify infectious PrP^Sc from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrP^Sc was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM >20-fold and purified DRM immunogen >40-fold) relative to 1% crude brain homogenate. Purification of PrP^Sc from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrP^Sc antigen was performed using wild-type (wt) and Prnp^0/0 mice, both on Balb/cJ background. A robust immune response against PrP^Sc was observed in all inoculated Prnp^0/0 mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrP^C and PrP^Sc, while binding to other brain-derived protein was not observed. In contrast, the PrP^Sc inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.Key words: prion, scrapie, Prnp0/0 mice, purification methodology, antibody, antisera, lipid-rafts, detergent resistant membranes, neuroscience, immunization, diagnostic