Novel amplification mechanism of prions through disrupting sortilin-mediated trafficking

Conformational conversion of the cellular prion protein, PrP^C, into the abnormally folded isoform of prion protein, PrP^Sc, which leads to marked accumulation of PrP^Sc in brains, is a key pathogenic event in prion diseases, a group of fatal neurodegenerative disorders caused by prions. However, the exact mechanism of PrP^Sc accumulation in prion-infected neurons remains unknown. We recently reported a novel cellular mechanism to support PrP^Sc accumulation in prion-infected neurons, in which PrP^Sc itself promotes its accumulation by evading the cellular inhibitory mechanism, which is newly identified in our recent study. We showed that the VPS10P sorting receptor sortilin negatively regulates PrP^Sc accumulation in prion-infected neurons, by interacting with PrP^C and PrP^Sc and trafficking them to lysosomes for degradation. However, PrP^Sc stimulated lysosomal degradation of sortilin, disrupting the sortilin-mediated degradation of PrP^C and PrP^Sc and eventually evoking further accumulation of PrP^Sc in prion-infected neurons. These findings suggest a positive feedback amplification mechanism for PrP^Sc accumulation in prion-infected neurons.     

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.     

Cellular factors implicated in prion replication

Prions are the unconventional infectious agents responsible for prion diseases, which are composed mainly by the misfolded prion protein (PrP^Sc) that replicates by converting the host associated cellular prion protein (PrP^C). Several lines of evidence suggest that other cellular components participate in prion conversion, however, the identity or even the chemical nature of such factors are entirely unknown. In this article we study the conversion factor activity by complementation of a PMCA procedure employing purified PrP^C and PrP^Sc. Our results show that the conversion factor is present in all major organs of diverse mammalian species, and is predominantly located in the lipid raft fraction of the cytoplasmic membrane. On the other hand, it is not present in the lower organisms tested (yeast, bacteria and flies). Surprisingly, treatments that eliminate the major classes of chemical molecules do not affect conversion activity, suggesting that various different compounds may act as conversion factor in vitro. This conclusion is further supported by experiments showing that addition of various classes of molecules have a small, but detectable effect on enhancing prion replication in vitro. More research is needed to elucidate the identity of these factors, their detailed mechanism of action and whether or not they are essential component of the infectious particle.     

Prion Protein—Antibody Complexes Characterized by Chromatography-Coupled Small-Angle X-Ray Scattering

Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly α-helical cellular prion protein (PrP^C) into a β-sheet-rich disease-causing isoform (PrP^Sc) is the key molecular event in the formation of PrP^Sc aggregates. The molecular mechanisms underlying the PrP^C-to-PrP^Sc conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP^Sc in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23–230)] and N-terminally truncated recPrP(89–230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP^C into PrP^Sc.     

Sequestration of free cholesterol in cell membranes by prions correlates with cytoplasmic phospholipase A<Subscript>2</Subscript>activation

Background  

The transmissible spongiform encephalopathies (TSEs), otherwise known as the prion diseases, occur following the conversion of the normal cellular prion protein (PrP^C) to an alternatively folded isoform (PrP^Sc). The accumulation of PrP^Sc within the brain leads to neurodegeneration through an unidentified mechanism. Since many neurodegenerative disorders including prion, Parkinson's and Alzheimer's diseases may be modified by cholesterol synthesis inhibitors, the effects of prion infection on the cholesterol balance within neuronal cells were examined.     

Prion Infection Impairs Cholesterol Metabolism in Neuronal Cells

Conversion of prion protein (PrP^C) into a pathological isoform (PrP^Sc) during prion infection occurs in lipid rafts and is dependent on cholesterol. Here, we show that prion infection increases the abundance of cholesterol transporter, ATP-binding cassette transporter type A1 (ATP-binding cassette transporter type A1), but reduces cholesterol efflux from neuronal cells leading to the accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was confirmed in prion-infected mice. Mechanistically, conversion of PrP^C to the pathological isoform led to PrP^Sc accumulation in rafts, displacement of ABCA1 from rafts and the cell surface, and enhanced internalization of ABCA1. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression with liver X receptor agonist or overexpression of heterologous ABCA1 reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, which plays an important role in the pathogenesis of prion infection in neuronal cells.     

Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrP^Sc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrP^Sc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrP^Sc degradation were evaluated. The accumulation of PrP^Sc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann–Sträussler–Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrP^Sc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrP^Sc from either the 22L or the Chandler strain, indicating that the degradation of PrP^Sc in host cells might be strain-dependent.     

Prion infection: Seeded fibrillization or more?

The prion infection is a conversion of host encoded prion protein (PrP) from its cellular isoform PrP^C into the pathological and infectious isoform PrP^Sc; the conversion process was investigated by in vitro studies using recombinant and cellular PrP and natural PrP^Sc. We present a brief summary of the results determined with our in vitro conversion system and the derived mechanistic models. We describe well characterized intermediates and precursor states during the conversion process, kinetic studies of spontaneous and seeded fibrillogenesis and the impact of the membrane environment.Key words: prion protein conversion, seeding, fibril, dimer, precursor state, kinetics, membrane     

Cellular uptake of the prion protein fragment PrP106-126 in vitro

The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrP^c). This pathological isoform (PrP^Sc) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrP^Sc in affected tissue. To investigate the possible uptake mechanisms that may be required for PrP^Sc-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrP^c expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrP^Sc in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.     

Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC

The mammalian prions replicate by converting cellular prion protein (PrP^C) into pathogenic conformational isoform (PrP^Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP^Sc on conversion of PrP^C in vitro using PrP^Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP^Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP^Sc. The tight correlation between conversion potency of small oligomers of human sPrP^Sc observed in vitro and duration of the disease suggests that sPrP^Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.     

Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrP^C) to the infectious scrapie form (PrP^Sc). However, the mechanism that exogenous PrP^Sc infects cells and where pathologic conversion of PrP^C to the PrP^Sc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrP^C to the pathologic PrP^Sc form in N2a cells exposed to strain RML PrP^Sc infected brain homogenates, suggesting that a critical determinant of PrP^C conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP^Sc infects cells by lipid raft dependent, macropinocytosis.     

Insight into early events in the aggregation of the prion protein on lipid membranes

The key molecular event underlying prion diseases is the conversion of the monomeric and α-helical cellular form of the prion protein (PrP^C) to the disease-associated state, which is aggregated and rich in β-sheet (PrP^Sc). The molecular details associated with the conversion of PrP^C into PrP^Sc are not fully understood. The prion protein is attached to the cell membrane via a GPI lipid anchor and evidence suggests that the lipid environment plays an important role in prion conversion and propagation. We have previously shown that the interaction of the prion protein with anionic lipid membranes induces β-sheet structure and promotes prion aggregation, whereas zwitterionic membranes stabilize the α-helical form of the protein. Here, we report on the interaction of recombinant sheep prion protein with planar lipid membranes in real-time, using dual polarization interferometry (DPI). Using this technique, the simultaneous evaluation of multiple physical properties of PrP layers on membranes was achieved. The deposition of prion on membranes of POPC and POPC/POPS mixtures was studied. The properties of the resulting protein layers were found to depend on the lipid composition of the membranes. Denser and thicker protein deposits formed on lipid membranes containing POPS compared to those formed on POPC. DPI thus provides a further insight on the organization of PrP at the surface of lipid membranes.     

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.     

Lipopolysaccharide induced conversion of recombinant prion protein

The conformational conversion of the cellular prion protein (PrP^C) to the β-rich infectious isoform PrP^Sc is considered a critical and central feature in prion pathology. Although PrP^Sc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrP^C to proteinase K resistant PrP^Sc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrP^C conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrP^C to PrP^Sc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232).     

Pressure-assisted dissociation and degradation of “proteinase K-resistant” fibrils prepared by seeding with scrapie-infected hamster prion protein

The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP^C, into a fibrous pathogenic form, PrP^Sc, which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP^Sc into PrP^C is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP^Sc from PrP^C eventually in vivo. Here we show that, by applying pressures at kbar range, the “proteinase K-resistant” fibrils (rHaPrP^res) prepared from hamster prion protein (rHaPrP [23–231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP^res fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP^Sc from PrP^C is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP^Sc from various systems of pathogenic concern.     

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.     

Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure

Prions are molecular pathogens, able to convert a normal cellular prion protein (PrP^C) into a prion (PrP^Sc). The information necessary for this conversion is contained in the conformation of PrP^Sc. Mass spectrometry (MS) and small-molecule covalent reactions have been used to study prions. Mass spectrometry has been used to detect and quantitate prions in the attomole range (10^?18 mole). MS-based analysis showed that both possess identical amino acid sequences, one disulfide bond, a GPI anchor, asparagine-linked sugar antennae, and unoxidized methionines. Mass spectrometry has been used to define elements of the secondary and tertiary structure of wild-type PrP^Sc and GPI-anchorless PrP^Sc. It has also been used to study the quaternary structure of the PrP^Sc multimer. Small molecule reagents react differently with the same lysine in the PrP^C conformation than in the PrP^Sc conformation. Such differences can be detected by Western blot using mAbs with lysine-containing epitopes, such as 3F4 and 6D11. This permits the detection of PrP^Sc without the need for proteinase K pretreatment and can be used to distinguish among prion strains. These results illustrate how two important chemical tools, mass spectrometry and covalent modification by small molecules, are being applied to the detection and structural study of prions. Furthermore these tools are or can be applied to the study of the other protein misfolding diseases such as Alzheimer Disease, Parkinson Disease, or ALS.     

The Strain-Encoded Relationship between PrPSc Replication,Stability and Processing in Neurons is Predictive of the Incubation Period of Disease

Prion strains are characterized by differences in the outcome of disease, most notably incubation period and neuropathological features. While it is established that the disease specific isoform of the prion protein, PrP^Sc, is an essential component of the infectious agent, the strain-specific relationship between PrP^Sc properties and the biological features of the resulting disease is not clear. To investigate this relationship, we examined the amplification efficiency and conformational stability of PrP^Sc from eight hamster-adapted prion strains and compared it to the resulting incubation period of disease and processing of PrP^Sc in neurons and glia. We found that short incubation period strains were characterized by more efficient PrP^Sc amplification and higher PrP^Sc conformational stabilities compared to long incubation period strains. In the CNS, the short incubation period strains were characterized by the accumulation of N-terminally truncated PrP^Sc in the soma of neurons, astrocytes and microglia in contrast to long incubation period strains where PrP^Sc did not accumulate to detectable levels in the soma of neurons but was detected in glia similar to short incubation period strains. These results are inconsistent with the hypothesis that a decrease in conformational stability results in a corresponding increase in replication efficiency and suggest that glia mediated neurodegeneration results in longer survival times compared to direct replication of PrP^Sc in neurons.     

Interactome Analyses Identify Ties of PrPC and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones

The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP^C) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrP^C interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrP^C paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrP^C and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrP^C with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrP^Sc. A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrP^C organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.