Novel dominant-negative prion protein mutants identified from a randomized library

Prion diseases are untreatable neurodegenerative disorders characterized by accumulation of PrP(Sc), an aggregated isoform of the cellular prion protein (PrP(C)). We generated a library of PrP variants with random mutations in the helix-3 domain and screened for dominant-negative mutants (DNMs) that would inhibit replication of prions (the Rocky Mountain Laboratory strain) in infected N2a cells. Two of the identified PrP mutants, Q167R and Q218K, were already known to counteract prion replication, thereby validating the effectiveness of this approach. In addition, novel DNMs were found efficiently to antagonize PrP(Sc) propagation in cells. In contrast to Q167R and Q218K, the newly identified DNMs S221P and Y217C resided on the cell surface at a substantially lower level, suggesting that robust cell surface display of DNM might not be a general prerequisite for efficient prion antagonism. The newly identified DNMs point to useful target-selective therapeutic tools for the treatment of prion diseases.     

Prion-protein-specific aptamer reduces PrPSc formation

The critical initial event in the pathophysiology of transmissible spongiform encephalopathies (TSEs) appears to be the conversion of the cellular prion protein (PrP(C)) into the abnormal isoform PrP(Sc). This isoform forms high-molecular-weight protease K (PK) resistant aggregates that accumulate in the central nervous system of affected individuals. We have selected nuclease-resistant 2'-amino-2'-deoxypyrimidine-modified RNA aptamers which recognize a peptide comprising amino acid residues 90-129 of the human prion protein with high specificity. This domain of prion proteins is thought to be functionally important for the conversion of PrP(C) into its pathogenic isoform PrP(Sc) and is highly homologous among prion proteins of various species including mouse, hamster, and man. Consequently, aptamer DP7 binds to the full-length human, mouse, and hamster prion protein. At low concentrations in the growth medium of persistently prion-infected neuroblastoma cells, aptamer DP7 significantly reduced the relative proportion of de novo synthesized PK-resistant PrP(Sc) within only 16 h. These findings may open the door towards a rational development of a new class of drugs for the therapy or prophylaxis of prion diseases.     

Disruption of the X-loop turn of the prion protein linked to scrapie resistance

The prion diseases are a class of neurodegenerative diseases caused by the misfolding and aggregation of the prion protein (PrP(C)) into toxic and infectious oligomers (PrP(Sc)). These oligomers are critical to understanding and combating these diseases. Differences in the sequence of PrP affect disease susceptibility, likely by shifting the tolerance of the protein for adaptation to PrP(Sc) conformations and/or the recognition event between PrP(Sc) and PrP(C) prior to conversion of the PrP(C). We selected two sets of PrP(Sc)-resistant mutant sequences for solvated atomistic molecular dynamics simulation to investigate the structural basis of resistance. The first group involved mutation in the X-loop (residues 164-171) resulting from selective breeding of sheep. The second group included eight mutants in mice identified by random mutagenesis targeting helix C followed by screening in cell cultures. Multiple simulations were performed of 14 different mutant and control constructs under different pH conditions for a total of 3.6 μs of simulation time. The X-loop formed a stable turn at neutral pH in wild-type PrP from both species. PrP(Sc)-resistant mutations disrupted this turn even though only one of the mutants is in the X-loop. The X-loop is compact and buried in our previously described spiral models of PrP(Sc)-like oligomers. On the basis of the findings presented here and in the context of the spiral oligomer model, we propose that expansion of the X-loop disrupts protofibril packing, providing a structural basis for resistance.     

Prion diseases: from molecular biology to intervention strategies

Prion diseases are fatal neurodegenerative infectious disorders for which no therapeutic or prophylactic regimens exist. Understanding the molecular process of conformational conversion of the cellular prion protein (PrP(c)) into its pathological isoform (PrP(Sc)) will be necessary to devise effective antiprion strategies. In recent years, new findings in the cell biology of PrP(c), in the molecular pathogenesis of PrP(Sc), and in the cellular quality control mechanisms involved in these scenarios have accumulated. A function of the prion protein in signalling, the possible impact of the proteasome, and aggresomes as intracellular waste deposits have been described. Here, important pathogenetic similarities with the more frequent neurodegenerative disorders are evident. The need for therapeutic, postexposure, and prophylactic possibilities was drastically illustrated by the emergence of variant Creutzfeldt-Jakob disease (vCJD), a new human prion disease caused by bovine spongiform encephalopathy (BSE) derived prions. Although prion infectivity in humans is usually restricted to the central nervous system, in vCJD patients prions are present in the lympho-reticular system, posing a theoretical risk of accidental human-to-human transmission. A variety of chemical antiprion substances have been reported in in vitro and cell culture based assays or in animal studies. Occasionally, they have also made their way into the first human trials. In addition, various promising interference strategies have been devised in transgenic models, although they are usually hard to transfer into nontransgenic in vivo situations. New findings in the fields of peripheral prion pathogenesis and immune system involvement fuelled the search for antiprion strategies formerly considered to be entirely impossible. This opened the door towards classical immunological interference techniques. Remarkably, passive and even active vaccination approaches now seem to be realistic goals.     

Prions and transmissible spongiform encephalopathy (TSE) chemotherapeutics: A common mechanism for anti-TSE compounds?

No validated treatments exist for transmissible spongiform encephalopathies (TSEs or prion diseases) in humans or livestock. The search for TSE therapeutics is complicated by persistent uncertainties about the nature of mammalian prions and their pathogenic mechanisms. In pursuit of anti-TSE drugs, we and others have focused primarily on blocking conversion of normal prion protein, PrP(C), to the TSE-associated isoform, PrP(Sc). Recently developed high-throughput screens have hastened the identification of new inhibitors with strong in vivo anti-TSE activities such as porphyrins, phthalocyanines, and phosphorthioated oligonucleotides. New routes of administration have enhanced beneficial effects against established brain infections. Several different classes of TSE inhibitors share structural similarities, compete for the same site(s) on PrP(C), and induce the clustering and internalization of PrP(C) from the cell surface. These activities may represent a common mechanism of action for these anti-TSE compounds.     

How an Infection of Sheep Revealed Prion Mechanisms in Alzheimer’s Disease and Other Neurodegenerative Disorders

Although it is not yet universally accepted that all neurodegenerative diseases (NDs) are prion disorders, there is little disagreement that Alzheimer’s disease (AD), Parkinson’s disease, frontotemporal dementia (FTD), and other NDs are a consequence of protein misfolding, aggregation, and spread. This widely accepted perspective arose from the prion hypothesis, which resulted from investigations on scrapie, a common transmissible disease of sheep and goats. The prion hypothesis argued that the causative infectious agent of scrapie was a novel proteinaceous pathogen devoid of functional nucleic acids and distinct from viruses, viroids, and bacteria. At the time, it seemed impossible that an infectious agent like the one causing scrapie could replicate and exist as diverse microbiological strains without nucleic acids. However, aggregates of a misfolded host-encoded protein, designated the prion protein (PrP), were shown to be the cause of scrapie as well as Creutzfeldt–Jakob disease (CJD) and Gerstmann–Sträussler–Scheinker syndrome (GSS), which are similar NDs in humans. This review discusses historical research on diseases caused by PrP misfolding, emphasizing principles of pathogenesis that were later found to be core features of other NDs. For example, the discovery that familial prion diseases can be caused by mutations in PrP was important for understanding prion replication and disease susceptibility not only for rare PrP diseases but also for far more common NDs involving other proteins. We compare diseases caused by misfolding and aggregation of APP-derived Aβ peptides, tau, and α-synuclein with PrP prion disorders and argue for the classification of NDs caused by misfolding of these proteins as prion diseases. Deciphering the molecular pathogenesis of NDs as prion-mediated has provided new approaches for finding therapies for these intractable, invariably fatal disorders and has revolutionized the field.     

The emerging principles of mammalian prion propagation and transmissibility barriers: Insight from studies in vitro

Self-perpetuating conformational conversion of the cellular prion protein PrP(C) into the beta-sheet-rich "scrapie" conformer (PrP(Sc)) is believed to be the central molecular event in pathogenesis of a group of diseases known as transmissible spongiform encephalopathies. Recent advances provide growing support for the notion that a misfolded protein alone might act as an infectious agent. Furthermore, findings regarding the mechanism of prion protein structural rearrangement, the role of folding intermediates in conformational conversion, and "conformational adaptability" in the propagation of prion amyloids in vitro yield molecular-level insight into such phenomena as inherited prion diseases, prion transmission barriers, and prion strains.     

Propagation and Dissemination Strategies of Transmissible Spongiform Encephalopathy Agents in Mammalian Cells

Transmissible spongiform encephalopathies or prion disorders are fatal infectious diseases that cause characteristic spongiform degeneration in the central nervous system. The causative agent, the so-called prion, is an unconventional infectious agent that propagates by converting the host-encoded cellular prion protein PrP into ordered protein aggregates with infectious properties. Prions are devoid of coding nucleic acid and thus rely on the host cell machinery for propagation. While it is now established that, in addition to PrP, other cellular factors or processes determine the susceptibility of cell lines to prion infection, exact factors and cellular processes remain broadly obscure. Still, cellular models have uncovered important aspects of prion propagation and revealed intercellular dissemination strategies shared with other intracellular pathogens. Here, we summarize what we learned about the processes of prion invasion, intracellular replication and subsequent dissemination from ex vivo cell models.     

Predicted secondary structure and membrane topology of the scrapie prion protein

The integral membrane sialoglycoprotein PrP^Sc is the only identifiablecomponent of the scrapie prion. Scrapie in animals and Creutzfeldt-Jakobdisease in humans are transmissible, degenerative neurologicaldiseases caused by prions. Standard predictive strategies havebeen used to analyze the secondary structure of the prion proteinin conjunction with Fourier analysis of the primary sequencehydrophobicities to detect potential amphipathic regions. Severalhydrophobic segments, a proline- and glycine-rich repeat regionand putative glycosylation sites are incorporated into a modelfor the integral membrane topology of PrP. The complete aminoacid sequences of the hamster, human and mouse prion proteinsare compared and the effects of residue substitutions upon thepredicted conformation of the polypeptide chain are discussed.While PrP has a unique primary structure, its predicted secondarystructure shares some interesting features with the serum amyloidA proteins. These proteins undergo a post-translational modificationto yield amyloid A, molecules that share with PrP the abilityto polymerize into birefringent filaments. Our analyses mayexplain some experimental observations on PrP, and suggest furtherstudies on the properties of the scrapie and cellular PrP isoforms.     

Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system

Syrian hamster prion protein (PrPC) and a truncated Syrian hamster prion protein lacking the glycosylphosphatidylinositol (GPI) anchor C- terminal signal sequence (GPI-) were expressed in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. The CHO cell clones expressing the GPI- PrP secreted the majority of the protein into the media, whereas most of the PrP produced by clones expressing the full-length protein with the GPI anchor was located on the cell surface, as demonstrated by its release upon treatment with phosphatidylinositol-specific phospholipase C (PIPLC). A cell clone that expressed the highest levels of full length PrP was subcloned to obtain clone 30C3-1. PrP from clone 30C3-1 was shown to be sensitive to proteolysis by proteinase K and to react with monoclonal and polyclonal antibodies that recognize native PrPC. The recombinant PrP migrated as a diffuse band of 19-40 kDa but removal of the N-linked oligosaccharides with peptide N-glycosidase F (PNGase F) revealed three protein species of 19, 17 and 15 kDa. The 19 kDa band corresponding to deglycosylated full-length PrP was quantified and found to be expressed at a level approximately 14-fold higher than that of PrPC found in Syrian hamster brain.      

Role of prion protein aggregation in neurotoxicity

In several neurodegenerative diseases, such as Parkinson, Alzheimer's, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.     

The Effect of Octapeptide Repeats on Prion Folding and Misfolding

Misfolding of prion protein (PrP) into amyloid aggregates is the central feature of prion diseases. PrP has an amyloidogenic C-terminal domain with three α-helices and a flexible tail in the N-terminal domain in which multiple octapeptide repeats are present in most mammals. The role of the octapeptides in prion diseases has previously been underestimated because the octapeptides are not located in the amyloidogenic domain. Correlation between the number of octapeptide repeats and age of onset suggests the critical role of octapeptide repeats in prion diseases. In this study, we have investigated four PrP variants without any octapeptides and with 1, 5 and 8 octapeptide repeats. From the comparison of the protein structure and the thermal stability of these proteins, as well as the characterization of amyloids converted from these PrP variants, we found that octapeptide repeats affect both folding and misfolding of PrP creating amyloid fibrils with distinct structures. Deletion of octapeptides forms fewer twisted fibrils and weakens the cytotoxicity. Insertion of octapeptides enhances the formation of typical silk-like fibrils but it does not increase the cytotoxicity. There might be some threshold effect and increasing the number of peptides beyond a certain limit has no further effect on the cell viability, though the reasons are unclear at this stage. Overall, the results of this study elucidate the molecular mechanism of octapeptides at the onset of prion diseases.     

Structural Mechanism of Barriers to Interspecies Seeding Transmissibility of Full-Length Prion Protein Amyloid

A puzzling feature of prion diseases is the cross-species barriers. The detailed molecular mechanisms underlying these interspecies barriers remain poorly understood because of a lack of high-resolution structural information on the scrapie isoform of the prion protein (PrP^Sc). In this study we identified the critical role of the residues 165/167 in the barrier to seeding mouse PrP (mPrP) fibril seeds to human cellular prion protein (PrP^C). Solid-state NMR revealed a C-terminal β-sheet core spanning residues 165–230 and the packing arrangement of mPrP fibrils. Residues 165/167 are located on one end of the fibril core. Molecular dynamics simulations demonstrated that the stabilities of the seeding-induced β-strand structures are significantly impacted by hydrogen bonds involving the side chain of residue 167 and steric resistance involving residue 165. These findings suggest that the α2–β2 loop containing residues 165/167 could be the initial site of seed–template conformational conversion.     

Prion-Associated Neurodegeneration Causes Both Endoplasmic Reticulum Stress and Proteasome Impairment in a Murine Model of Spontaneous Disease

Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrP^C to its abnormal and misfolded isoform PrP^Sc is the main event in the pathogenesis of prion diseases of all origins. In spontaneous prion diseases, the mechanisms that trigger the formation of PrP^Sc in the central nervous system remain unknown. Several reports have demonstrated that the accumulation of PrP^Sc can induce endoplasmic reticulum (ER) stress and proteasome impairment from the early stages of the prion disease. Both mechanisms lead to an increment of PrP aggregates in the secretory pathway, which could explain the pathogenesis of spontaneous prion diseases. Here, we investigate the role of ER stress and proteasome impairment during prion disorders in a murine model of spontaneous prion disease (TgVole) co-expressing the Ub^G76V-GFP reporter, which allows measuring the proteasome activity in vivo. Spontaneously prion-affected mice showed a significantly higher accumulation of the PKR-like ER kinase (PERK), the ER chaperone binding immunoglobulin protein (BiP/Grp78), the ER protein disulfide isomerase (PDI) and the Ub^G76V-GFP reporter than age-matched controls in certain brain areas. The upregulation of PERK, BiP, PDI and ubiquitin was detected from the preclinical stage of the disease, indicating that ER stress and proteasome impairment begin at early stages of the spontaneous disease. Strong correlations were found between the deposition of these markers and neuropathological markers of prion disease in both preclinical and clinical mice. Our results suggest that both ER stress and proteasome impairment occur during the pathogenesis of spontaneous prion diseases.     

Copper binding to the neurotoxic peptide PrP106-126: thermodynamic and structural studies

The human prion protein fragment PrP(106-126) is a highly fibrillogenic peptide, resistant to proteinases and toxic to neurons; it derives from the normal prion protein (PrP(C)), with which it can interact, thus inhibiting its superoxide dismutase-like activity. The same properties are also shown by the abnormal isoform of the prion protein (PrP(Sc)), and this similarity makes PrP(106-126) an interesting model for the neurotoxic action of PrP(Sc). A role for copper in PrP(106-126) aggregation and toxicity has recently been evidenced, and the interaction of terminal Lys, His and Met residues with the copper ion at neutral pH has been suggested. In order to shed more light on the complex-formation equilibria of PrP(106-126) with the copper ion, a thorough investigation has been carried out by means of several experimental techniques: potentiometry, solution calorimetry, VIS spectrophotometry, circular dichroism, EPR and NMR spectroscopy. A shorter and more soluble fragment-PrP(106-113), which lacks the hydrophobic C-terminal domain of PrP(106-126) but contains all the potential donor groups-has also been considered for the sake of comparison. The involvement of terminal amino, imidazolic and amido nitrogens in complex formation has been confirmed, while no evidence was found for the interaction of side chains of Met and Lys residues with the copper ion. Solution structures for the main complexes are suggested.     

Liberation of GPI-Anchored Prion from Phospholipids Accelerates Amyloidogenic Conversion

Prion diseases or transmissible spongiform encephalopathies are a rare group of fatal neurodegenerative illnesses in humans and animals caused by misfolding of prion protein (PrP). Prion protein is a cell-surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mostly in the central and peripheral nervous system, and this membrane-bound protein can be cleaved from the cell membranes by phosphoinositide phospholipase C. Numerous studies have investigated GPI-free recombinant PrP, but the role of GPI on misfolding of PrP is not well known. In this study, we synthesized a GPI analog that was covalently linking to a PrP S230C mutant, resulting in S230C-GPI. The structural changes in S230C-GPI upon binding to lipid vesicles composed of mixtures of the zwitterionic lipid (POPC) and the anionic lipid (POPG) were analyzed by circular dichroism spectroscopy, and the amyloid aggregation of S230C-GPI in the liberation from phospholipid vesicles was monitored by proteinase K-digestion assay. Our results indicate that S230C-GPI in the liberation of lipid vesicles has high tendency to misfold into amyloid fibrils, while the membrane-bound S230C-GPI proteins are highly stable and rarely convert into amyloid forms. In addition, the role of cholesterol in S230C-GPI was studied. The effect of GPI, cholesterol and phospholipid vesicles on misfolding of PrP is further discussed.     

Cervid Prion Protein Polymorphisms: Role in Chronic Wasting Disease Pathogenesis

Chronic wasting disease (CWD) is a prion disease found in both free-ranging and farmed cervids. Susceptibility of these animals to CWD is governed by various exogenous and endogenous factors. Past studies have demonstrated that polymorphisms within the prion protein (PrP) sequence itself affect an animal’s susceptibility to CWD. PrP polymorphisms can modulate CWD pathogenesis in two ways: the ability of the endogenous prion protein (PrP^C) to convert into infectious prions (PrP^Sc) or it can give rise to novel prion strains. In vivo studies in susceptible cervids, complemented by studies in transgenic mice expressing the corresponding cervid PrP sequence, show that each polymorphism has distinct effects on both PrP^C and PrP^Sc. It is not entirely clear how these polymorphisms are responsible for these effects, but in vitro studies suggest they play a role in modifying PrP epitopes crucial for PrP^C to PrP^Sc conversion and determining PrP^C stability. PrP polymorphisms are unique to one or two cervid species and most confer a certain degree of reduced susceptibility to CWD. However, to date, there are no reports of polymorphic cervid PrP alleles providing absolute resistance to CWD. Studies on polymorphisms have focused on those found in CWD-endemic areas, with the hope that understanding the role of an animal’s genetics in CWD can help to predict, contain, or prevent transmission of CWD.     

Single-Chain Fragment Variable Passive Immunotherapies for Neurodegenerative Diseases

Accumulation of misfolded proteins has been implicated in a variety of neurodegenerative diseases including prion diseases, Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD). In the past decade, single-chain fragment variable (scFv) -based immunotherapies have been developed to target abnormal proteins or various forms of protein aggregates including Aβ, SNCA, Htt, and PrP proteins. The scFvs are produced by fusing the variable regions of the antibody heavy and light chains, creating a much smaller protein with unaltered specificity. Because of its small size and relative ease of production, scFvs are promising diagnostic and therapeutic reagents for protein misfolded diseases. Studies have demonstrated the efficacy and safety of scFvs in preventing amyloid protein aggregation in preclinical models. Herein, we discuss recent developments of these immunotherapeutics. We review efforts of our group and others using scFv in neurodegenerative disease models. We illustrate the advantages of scFvs, including engineering to enhance misfolded conformer specificity and subcellular targeting to optimize therapeutic action.     

Immunization with Genetically Modified Trypanosomes Provides Protection against Transmissible Spongiform Encephalopathies

Transmissible spongiform encephalopathies are incurable neurodegenerative diseases, associated with the conversion of the physiological prion protein to its disease-associated counterpart. Even though immunization against transmissible spongiform encephalopathies has shown great potential, immune tolerance effects impede the use of active immunization protocols for successful prophylaxis. In this study, we evaluate the use of trypanosomes as biological platforms for the presentation of a prion antigenic peptide to the host immune system. Using the engineered trypanosomes in an immunization protocol without the use of adjuvants led to the development of a humoral immune response against the prion protein in wild type mice, without the appearance of adverse reactions. The immune reaction elicited with this protocol displayed in vitro therapeutic potential and was further evaluated in a bioassay where immunized mice were partially protected in a representative murine model of prion diseases. Further studies are underway to better characterize the immune reaction and optimize the immunization protocol.     

Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes

Prion proteins (PrPs) are difficult to crystallize, probably due to their inherent flexibility. Several PrPs structures have been solved by nuclear magnetic resonance (NMR) techniques; however, only three structures were solved by X-ray crystallography. Here we combined in-situ proteolysis with automated microseed matrix screening (MMS) to crystallize two different PrP(C)-nanobody (Nb) complexes. Nanobodies are single-domain antibodies derived from heavy-chain-only antibodies of camelids. Initial crystallization screening conditions using in-situ proteolysis of mouse prion (23-230) in complex with a nanobody (Nb_PrP_01) gave thin needle aggregates, which were of poor diffraction quality. Next, we used these microcrystals as nucleants for automated MMS. Good-quality crystals were obtained from mouse PrP (89-230)/Nb_PrP_01, belonged to the monoclinic space group P 1 21 1, with unit-cell parameters a = 59.13, b = 63.80, c = 69.79 ?, β = 101.96° and diffracted to 2.1 ? resolution using synchrotron radiation. Human PrP (90-231)/Nb_PrP_01 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 131.86, b = 45.78, c = 45.09 ?, β = 96.23° and diffracted to 1.5 ? resolution. This combined strategy benefits from the power of the MMS technique without suffering from the drawbacks of the in-situ proteolysis. It proved to be a successful strategy to crystallize PrP-nanobodies complexes and could be exploited for the crystallization of other difficult antigen-antibody complexes.