Comprehensive Insights into Prion Diseases: Classification, Mechanisms of Action, Detection Methods, and Preventive Strategies

The normal cellular prion protein (PrPc) plays a dual role in transmissible spongiform encephalopathies (TSE), which is a group of lethal disease affecting humans and a variety of animal species. First PrPC is hypothesized to be the source of the causative agent “the prion” in TSE, as the pathologic missfolded scrapie isoform of the prion protein (PrPSC) catalyses its own conversion from the prion protein. Second, there is substantial evidence that PrPC is the receptor mediating neurotoxicity and disease progression in TSE. In support of this theory, some antiprion antibodies have been found to induce neurotoxicity, an important discovery that may provide a new model system to investigate the pathologic interaction between PrPSC and PrPC. In the first part of the thesis I investigated antiprion mediated toxicity in vivo. Towards this goal, I established two read outs based on magnetic resonance imaging (MRI) volumetry allowing the measurment of antiprion induced neurotoxicity in intact mice over time. In the beginning I worked with Manganese Enhanced MRI (MEMRI) to visualize cerebellar lesion induction. As this was less well suited to the measurement of the neurotoxic induction in the hippocampus, I additionally established a diffusion weighted imaging (DWI) scan protocol. Diffusion restriction can be found within an hour after the injection of high concentration of monovalent fragments of the neurotoxic antiprion antibody POM1. As previously found in cerebellar organotypic slice cultures (COCS) antiprion mediated neurotoxicity was found to be target and eptiopic specific. Neuronal expression of PrPC is sufficient for lesion induction and lesion induction is independent from cross-linking. Further I investigated important signalling pathways downstream of PrPC. Reactive oxygen species (ROS) are a known mediator of neurodegenerative disease and I could demonstrate that they are important in the pathologic cascade of antiprion mediated neurotoxicitiy in vivo. As a major source of ROS, I identified NADPH oxidase 2 (NOX2). As in bona fide prion infection, I could detect fodrin cleavage as a marker for calpain activation in homogenates from antiprion injected brain tissue. These findings are indicative that similar pathways are activated in both pathological conditions. Further, my data shows that the NCX3 antiporter is a possible source of pathologic Ca2+ currents in the antiprion antibody model. In the second part of this thesis, I focused on the risk characterization of neurotoxic antiprion antibodies. Despite reports about the neurotoxic side effects of antiprion antibodies, passive immunotherapy with these ligands is still a therapeutic strategy under investigation in the treatment of TSE. Using my established MRI based quantification system and basic histological methods, I assessed the neurotoxic potential of the antiprion antibody ICSM18, which is under evaluation as a therapeutic agent for TSE in humans. Unless further investigations can confirm a safe therapeutic window for the use of these antibodies and others, my findings suggest that utmost caution is indicated. Here I show that Magnetic Resonance Imaging is a valuable tool in the assessment of anti prion mediated neurotoxicity in vivo. This technique can be used for the risk characterization of antiption antibodies. In addition, our work in vivo and in cerebellar slice cultures provides new evidence that neurotoxic antiprion antibodies model the pathologic interaction of PrPSC with PrPC. Thus, the established tool can be used in further studies to investigate prionmediated neurotoxicity, in a much shorter time frame and within biosafety level one.

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.

Background: Transmissible spongiform encephalopathies are a collection of rare neurodegenerative disorders characterized by loss of neuronal cells, astrocytosis, and plaque formation. The causative agent of these diseases is thought to be abnormally folded prions and is characterized by a conformational change from normal, cellular prion protein (PrPc) to the abnormal form (PrPTSE). Although, there is evidence that normal prion protein can contribute to these disorders. The unfolded protein response, a conserved series of pathways involved in resolving stress associated with unfolded protein accumulation in the Endoplasmic Reticulum (ER), has been shown to play a role in regulating the development of prion diseases. Methods: This review chose papers based on their relevance to current studies involved in prion protein synthesis and transformation, identifies various links between prion diseases and ER stress, and reports on current and potential treatments as they relate to ER stress and prion diseases. Conclusion: For the advancement of prion disease treatment, it is important to understand the mechanisms involved in prion formation, and ER stress is implicated in prion disease progression. Therefore, targeting the ER or pathways involved in response to stress in the ER may help us treat prion diseases.

Subcellular Localization of Disease-Associated Prion Protein in the Human Brain

Prion particles (prion) are proteinaceous infectious particles and it has been proved that there are two different structures exist for prion proteins. The normal cellular prion protein, denoted as PrPC, may convert into an abnormal scrapie cellular prion, denoted as PrPSc, through a process whereby a portion of its α-helical structure is refolded into β-sheet. The presence of PrPSc will catalyze the transformation of PrPC to PrPSc by the misfolding process. When PrPSc forms aggregate through hydrogen bonds and Van der Waals interactions, the amyloid fibril will be formed and cause the neurodegenerative disease. The transmissible spongiform encephalopathies (TSE) is one of the neurodegenerative diseases that caused by the amyloid fibril depositions. Different protein sequences may have different pathogenicities and distinctive amyloid fibril structures. Therefore, understanding structural conversion of amyloid fibril is very important. Owing to the low solublity and non-crystalline characteristics of amyloid fibrils, it is difficult to use conventional experimental techniques such as solution-state NMR and XRD to analyze the structure of amyloid fibrils. Hence, solid-state NMR is still the most suitable method to characterize structures of this kind. In this study, we mutated the 117 position of Syrian hamster prion protein 109-122 fragment (SHaPrP109-122, A117I, Ac-MKHMAGAAIAGAVV-NH2) from Ala to Ile. The idea is to compare the structural difference induced by mutation on the aligned position of β-sheet in the native SHaPrP109-122. We have incubated the amyloid fibril formed by SHaPrP109-122, A117I successfully. We report the ThT fluorescence, TEM, AFM experiments to observe the formation of amyloid fibrils. From the TEM and AFM images, we have measured the matured fibrils about 24 nm in width, 1.1 nm in height and 380 nm in length. From the isotope-edited FTIR study, the fibril has an anti-parallel β-strand secondary structure and showed an obvious alignment at I117. The chemical shift and linewidth data obtained from ssNMR showed that β-sheet structure exists in the core region. The distance about 4.9±0.2A between the two β layers is determined by fpRFDR-CT experiment. From the experimental data it has been inferred that the fibrils formed by A117I SHaPrP109-122 has the steric zipper structure. Finally, a molecular model for the fibrils was constructed by molecular dynamics simulations incorporated with structural constraints obtained from ssNMR measurements. The results point out that the fibrils still maintain the steric zipper structure after mutating alanine at residue 117 to isoleucine, which has a very bulky side chain. In addition, we have also investigated the amyloid fibrils formed by the residues 127-147 of the human prion protein (HuPrP127-147, Ac-GYMLGSAMSRPIIHFGSDYED-NH2) by TEM to trace the initial stages of amyloid fibrils formation and observe the morphology of the fibril and spherical aggregates. The fibrils are about 6 nm in width from the sample incubated for 5 minutes only. The distance between the β-strands was determined by ssNMR to be 5±0.1 A at the proline residue. It demonstrates that the side chain structure of P137 does not disrupt the β-sheet structure.

Prion diseases such as Creutzfeldt-Jakob disease (CJD) and Kuru in humans, scrapie in sheep and bovine spongiform encephalopathy (BSE) in cattle are a group of neurodegenerative diseases that invariably lead to death. The current hypothesis states that the cellular prion protein (PrPC) gets converted into a misfolded form PrPSc, characterized by a high β-sheet content (Aguzzi and Calella, 2009). The misfolded PrPSc oligomerizes and grows into fibrils. Broken fibrils can then serve as a seed and lead to further conversion and oligomerization, and therefore be used as a surrogate for infectivity (Knowles et al., 2009). The in vivo conformation of prion fibrils is not defined; hence, developing specific inhibitors remains challenging. Other therapies targeting both prion replication and the intracellular signalling pathways that mediate neurotoxicity have not been successful. Consequently, to date no effective prion therapy exists. Prion disease represents one if not the best-studied protein aggregation disease. An in vitro model for prion-induced pathology has been established in our laboratory. When cerebellar organotypic cultured slices (COCS) are infected with prions, they exhibit all the characteristic features as prion replication, astro- and microgliosis, vacuolation and neurotoxicity (Falsig et al., 2008; Falsig et al., 2012). Luminescent conjugated polythiophenes (LCP) are polymeric fluorescent molecules that preferentially bind to protein aggregates with regular cross-β-sheet structures, including those formed by PrPSc, and can be used to stain many different amyloids in tissues (Klingstedt and Nilsson, 2012). Recently, our laboratory found that treatment of prion-infected brain homogenates and prion-infected COCS with LCPs reduced infectivity. Interestingly, the prionostatic effect seems to rely on hyperstabilization, rather than dissociation, of PrP aggregates (Margalith et al., 2012). More recent findings from our lab have shown that full length, monovalent antibodies or single chain antibodies that target the globular domain (termed globular domain ligands; GDL) of prion protein (PrPC) lead to dramatic neuronal cell loss when applied in cultured organotypic cerebellar slices or stereotactically injected in the cerebellum of mice (Sonati et al., 2013). It was also found that neurotoxicity involves the production of reactive oxygen species and activation of calpains. This thesis focuses on the evaluation of LCPs as a therapy in a mouse model of prion diseases, the comparison of the pathogenetic mechanisms underlying neurotoxicity elicited by GDL or prions and signaling mechanisms involved in prion induced neuronal cell death.

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.

Transmissible spongiform encephalopathies (TSE) are sub-acute neurodegenerative disorders which include scrapie in sheep, bovine spongiform encephalopathy in cows, and Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler syndrome, and fatal familial insomnia in humans. The essential pathogenic component of TSE is an abnormal isoform of the prion protein designated PrPSc (for reviews and discussions see 1.) The naturally occurring, cellular prion protein, called PrPC, encoded by the Prnp gene, is expressed in neurons (2) and glia (3). PrPC is glycosilated and anchored to the plasma membrane, but little is known about its cellular function (see references in 1.) During the disease process, PrPC is converted into PrPSc in both neurons and astrocytes. The “converted” PrPSc is partly resistant to proteinase K digestion and has an altered conformational state whereby the amount α-helical structure decreases and the content of β-sheet increases (for review see 1). Prion-induced encephalopathies are characterized by intracerebral accumulation of PrPSc and deposition of PrP amyloid (for review see 1,4). The observation that Prnp knockout mice do not develop spontaneous scrapie (5) supports the view that the accumulation of PrPSc and PrP amyloid in the brain mediate neuronal toxicity in the central nervous system.

The cellular prion protein (PrPC) is a glycoprotein with unknown function constitutively expressed in mammalian neurons. PrPC converts to a pathogenic misfolded isomer (PrPSc) through a poorly understood process, resulting in a group of fatal neurodegenerative diseases collectively known as transmissible spongiform encephalopathy or prion disease. Elucidating the molecular mechanisms behind prion conversion requires production of PrPC in recombinant systems. This study was designed to generate transgenic tobacco plants expressing recombinant mouse prion protein (mPrP). Using a synthetic gene encoding the mouse prion protein, plant expression vectors were constructed for constitutive mPrP expression in the apoplast (pGreen35SmPrP-Apo), cytosol (pGreen35SmPrP-Cyto) and endoplasmic reticulum (pGreen35SmPrP-ER). Putative transgenic plants transformed with either pGreen35SmPrP-Cyto or pGreen35SmPrP-ER were analysed by PCR, ELISA and immunoblot for transgene integration and expression. However, no viable plants were obtained from the pGreen35SmPrP-Apo transformants. ELISA analysis showed that recombinant mPrP accumulated up to 0.0024% of total soluble leaf protein in transgenic tobacco leaves transformed with the pGreen35SmPrP-Cyto construct and 0.0016% of total soluble leaf protein in plants designed to sequester recombinant mPrP to the ER. Furthermore, immunoblot analysis showed that ER-targeted recombinant mPrP was mainly unglycosylated, although a glycosylated mPrP isoform was observed indicating that transgenic tobacco plants process ER-targeted recombinant mPrP in a manner analogous to mammalian systems. The nutrient composition of several transgenic plants were analysed to determine the phenotypic effect of expressing recombinant mPrP in tobacco plants. The analysis revealed that transgenic lines expressing cytosolic-mPrP had elevated average levels of Mn2+ and Fe2+. In addition, kanamycin-treated transgenic plants expressing cytosolic-mPrP developed a non-rooting phenotype. Conversely, the average Cu2+ level was increased in analysed transgenic plants designed to sequester recombinant mPrP in the ER. Furthermore, the plants developed no visible phenotype upon kanamycin treatment. This result support studies that suggest that the PrPC has functional role in metal homeostasis and loss of its thermodynamic structure leads to metal dyshomeostasis which in turn has been linked to prion disease associated neurotoxicity. Finally, the recombinant mPrP was purified via affinity chromatography facilitated by the presence of a C-terminal polyhistidine tag on the synthetic gene constructs.

Spongiform encephalopathies, also called "prion diseases", are fatal degenerative diseases of the central nervous system which can occur in animals (such as the "mad cow disease" in cattle) and also in humans. This paper presents a novel medical theory concerning the pathogenic mechanisms for various human and animal spongiform encephalopathies. It is hypothesized that various forms of prion diseases are essentially autoimmune diseases, resulting from chronic autoimmune attack of the central nervous system. A key step in the pathogenic process leading towards the development of spongiform encephalopathies involves the production of specific autoimmune antibodies against the disease-causing prion protein (PrPsc) and possibly other immunogenic macromolecules present in the brain. As precisely explained in this paper, the autoimmune antibodies produced against PrPsc are responsible for the conversion of the normal cellular prion protein (PrPc) to PrPsc, for the accumulation of PrPsc in the brain and other peripheral tissues, and also for the initiation of an antibody-mediated chronic autoimmune attack of the central nervous system neurons, which would contribute to the development of characteristic pathological changes and clinical symptoms associated with spongiform encephalopathies. The validity and correctness of the proposed theory is supported by an overwhelming body of experimental observations that are scattered in the biomedical literature. In addition, the theory also offers practical new strategies for early diagnosis, treatment, and prevention of various human and animal prion diseases.

Deadly Conformations—Protein Misfolding in Prion Disease

Transmissible spongiform encephalopathies (TSEs), better known as prion diseases, are a group of progressive neurodegenerative diseases that affect both animals and humans. The most well‐known example, bovine spongiform encephalopathy (BSE), better known as “mad cow disease,” is a progressive neurological disorder of cattle that is created as a result of a mutation in the cellular prion protein (PrPc) into a toxic isoform called PrP scrapie (PrPSc). This disease first gained public attention when a British outbreak affected about 180,000 cattle between 1986 and 2001, devastating numerous agricultural communities. The Independent School BioClub SMART Team used 3‐D modeling and printing technology to examine PrPc and PrPSc molecular structures, focusing specifically on the protein’s metal‐binding properties. Copper (II) ion (Cu2+), PrPc’s highest affinity metal ion, plays a significant role in the interdomain interaction between the globular C‐terminal domain, consisting of three □‐helices and one short □‐sheet, and the flexible N‐terminal domain. Cu2+ ion binds to the PrPc N‐terminal octarepeat domain (OR), which is considered fundamental due to its series of four (or more) tandem repeats. Each tandem repeat can bind one Cu2+ ion, which directly interacts with a specific highly conserved, negatively‐charged region of the globular domain defined by the exposed surface of helices 2 and 3. The N‐terminal domain of PrPc causes severe neurotoxicity unless the globular domain properly regulates it. In humans, this prion protein mutation leads to Creutzfeldt‐Jakob disease (CJD), which has the same neurodegenerative symptoms as BSE. A negatively‐charged region on the surface of helix 3 occupied by the Cu2+ ion also overlaps with the epitopes of many PrPc antibodies associated with prion diseases. A better understanding of these interactions could potentially lead to discovering a cure for neurodegenerative diseases caused by PrPc’s misfolded form, such as CJD and Alzheimer’s.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are progressive neurodegenerative disorders of the central nervous system that affect humans and animals as sporadic, inherited, and infectious forms. Similarly to Alzheimer's disease and other neurodegenerative disorders, any attempt to reduce TSEs' lethality or increase the life expectancy of affected individuals has been unsuccessful. Typically, the onset of symptoms anticipates the fatal outcome of less than 1 year, although it is believed to be the consequence of a decades-long process of neuronal death. The duration of the symptoms-free period represents by itself a major obstacle to carry out effective neuroprotective therapies. Prions, the infectious entities of TSEs, are composed of a protease-resistant protein named prion protein scrapie (PrPSc) from the prototypical TSE form that afflicts ovines. PrPSc misfolding from its physiological counterpart, cellular prion protein (PrPC), is the unifying pathogenic trait of all TSEs. PrPSc is resistant to intracellular turnover and undergoes amyloid-like fibrillation passing through the formation of soluble dimers and oligomers, which are likely the effective neurotoxic entities. The failure of PrPSc removal is a key pathogenic event that defines TSEs as proteopathies, likewise other neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, characterized by alteration of proteostasis. Under physiological conditions, protein quality control, led by the ubiquitin-proteasome system, and macroautophagy clears cytoplasm from improperly folded, redundant, or aggregation-prone proteins. There is evidence that both of these crucial homeostatic pathways are impaired during the development of TSEs, although it is still unclear whether proteostasis alteration facilitates prion protein misfolding or, rather, PrPSc protease resistance hampers cytoplasmic protein quality control. This review is aimed to critically analyze the most recent advancements in the cause-effect correlation between PrPC misfolding and proteostasis alterations and to discuss the possibility that pharmacological restoring of ubiquitin-proteasomal competence and stimulation of autophagy could reduce the intracellular burden of PrPSc and ameliorate the severity of prion-associated neurodegeneration.

An abnormal isoform, PrP(Sc), of the normal cellular prion protein (PrP(C)) is the major component of the causative agent of prion diseases. Both isoforms were found to possess the same covalent structures, including a C-terminal glycosylphosphatidylinositol anchor, but different secondary and tertiary structures. In this study, a variant of full-length PrP with an unpaired cysteine at the C terminus was recombinantly produced in Escherichia coli, covalently coupled to a thiol-reactive phospholipid, and incorporated into liposomes to serve as a model for studying possible changes in structure and stability of recombinant PrP upon membrane attachment. Covalent coupling of PrP to liposomes did not result in significant structural changes observable by far-UV circular dichroism. Moreover, limited proteolysis experiments failed to detect changes in the stability of liposome-bound PrP relative to soluble PrP. These data suggest that the requirement of raft localization for the PrP(C) to PrP(Sc) conversion, observed previously in cell culture models, is not because of a direct influence of raft lipids on the structure and stability of membranebound PrP(C) but caused by other factors, e.g. increased local PrP concentrations or high effective concentrations of membrane-associated conversion factors. The availability of recombinant PrP covalently attached to liposomes provides the basis for systematic in vitro conversion assays with recombinant PrP on the surface of membranes. In addition, our results indicate that the three-dimensional structure of mammalian PrP(C) in membranes is identical to that of recombinant PrP in solution.

Prion diseases, also known as transmissible spongiform encephalopathies, are rapidly progressive, uniformly fatal brain diseases that can infect humans and animals, including cattle, sheep, goats, mink, deer, elk, cats, and zoo ungulates. In humans, prion diseases can occur as a sporadic or inherited disease, or as a result of iatrogenic transmission. Prion diseases generated great public concern after an outbreak of bovine spongiform encephalopathy occurred in many European countries and scientific evidence indicated its transmission to humans. Research in prion diseases is hampered by certain unconventional properties of the presumed etiologic agent and the long incubation period associated with these diseases. Most conventional laboratory methods used to study viruses and bacteria may not be applicable. In the past, the etiologic agent of transmissible spongiform encephalopathies was believed to be a slow virus, primarily because of its transmissibility, ability to retain infectivity after filtration, and long incubation period. The successful transmission of scrapie, a centuries-old prion disease of sheep, to mice in 1961 greatly facilitated identification and characterization of the scrapie agent. Several characteristics of the scrapie agent suggest that the agent is not a virus but is likely composed primarily of a protein. The agent's characteristics include the absence of disease-specific nucleic acids; resistance to radiation, nucleases, and standard sterilization and disinfection methods; and inactivation by protein-modifying procedures. These observations and purification of the scrapie prion in the early 1980s led to widespread acceptance of the prion hypothesis. Since the 1980s, both the scope and nature of prion disease research has progressed rapidly. The economic and human cost associated with the bovine spongiform encephalopathy outbreak fueled the need to better understand the etiologic agent of prion diseases and their basic transmission mechanism. Prions and Prion Diseases: Current Perspectives summarizes the advances in prion disease research. It expands on a previous volume edited by David Harris that was published in 1999 under the title Prions: Molecular and Cellular Biology. The book's 10 chapters describe the biochemical and molecular features of prions and the normal prion protein, various laboratory methods for studying prions, and advances in the pathogenesis and immunology of prion diseases. Chapters 2 through 6 detail laboratory methods developed to study the unconventional agent of prion diseases. Chapter 2 describes a cell-free conversion reaction system to study how pathogenic prions associated with different interact with host cellular prion protein. Such systems have been used to study the biochemical mechanisms of prion diseases and can potentially be used to screen new therapies for their effectiveness against prion diseases. Chapter 3 describes the mechanisms underlying the biosynthesis and cell biology of the cellular prion protein by using cell culture systems. Understanding the detailed biochemical properties of the cellular prion protein will help show the molecular basis of its interaction with, and conversion to, the pathogenic prions. Subsequent chapters in the book describe other laboratory methods, including transgenic mouse models, which can be used to investigate the transmissibility of prions among different species, the extent and degree of the species barrier, the mechanism of prion propagation, and prion disease pathogenesis. Overall, the book provides a wealth of information on the progress made in understanding the molecular, immunologic, and genetic aspects of prion diseases and the laboratory methods used to study them. This book will be valuable to prion disease researchers, to scientists who want to gain more knowledge about the progress made in understanding the mechanisms of prion propagation, and to persons just beginning to study these unconventional, fatal brain diseases.