Pathogenesis Of Transmissible Spongiform Encephalopathies Research Articles

Disease-associated F198S Mutation Increases the Propensity of the Recombinant Prion Protein for Conformational Conversion to Scrapie-like Form

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.

Grand ideas floating freely. Conference on the new prion biology: basic science, diagnosis and therapy.

In the exquisite gothic Palazzo Franchetti on the bank of the Grand Canal in Venice, the International Symposium on ‘New Prion Biology’ took place on April 12–13, 2002. The fabulous setting and excellent organization by the Scientific Committee together with the promotion and sponsorship of the Istituto Veneto di Scienze, Lettere ed Arti resulted in a stimulating and informative gathering for participants from a wide variety of countries. ![][1] Prion protein (PrP) is critically involved in the transmission and pathogenesis of the transmissible spongiform encephalopathies (TSEs), which include scrapie in sheep, variant and sporadic Creutzfeldt–Jakob disease (CJD) in humans and bovine spongiform encephalopathy (BSE) in cattle. The normal, cellular PrPc protein encoded by the Prnp gene is required for susceptibility to TSE. The interconversion of this normal form into the highly protease‐resistant PrPSc conformation leads to the formation of PrP aggregates and amyloid fibrils in the brain and ultimately to neurodegeneration in the affected animal. It has been proposed that this abnormal form of PrP is itself the infectious agent of transmission; however, this matter remains unsettled for a variety of reasons (Chesebro, 1998, 1999). The topics covered during this meeting included the structure and function of PrP, TSE pathogenesis, new efforts towards diagnostic tests, and the normal and abnormal roles of the newly described ‘doppel’ protein in gametogenesis and neurodegeneration. ### Doppel talk In the past several years, various laboratories have used different strategies to generate knock‐out mice in which normal PrP expression is eliminated. Although the first two of these strains had no obvious neurological problems, the two that were generated subsequently developed cerebellar degeneration in the first months of life. These findings led to the discovery of a gene located downstream of Prnp , known as Prnd . This gene encodes a protein called Doppel (Dpl) (Moore et … [1]: /embed/graphic-1.gif

Variant Creutzfeldt-Jakob disease: an unfolding epidemic of misfolded proteins.

Variant Creutzfeldt-Jakob disease (vCJD) is an emerging infectious disease believed to be the human manifestation of bovine spongiform encephalopathy (BSE). Variant CJD belongs to a family of human and animal diseases called transmissible spongiform encephalopathies (TSE). The pathogenesis of TSE is not fully understood, but a modified form of a normal cellular protein plays a central role. Current measures to control vCJD aim to prevent transmission of the infectious agent from animals to humans through food or pharmaceutical products and to prevent transmission from person to person via medical interventions. The anticipated development of preclinical diagnostic tests and treatments for vCJD will create new control options and difficult choices.

Filipin Prevents Pathological Prion Protein Accumulation by Reducing Endocytosis and Inducing Cellular PrP Release

Conversion of the normal membrane-bound prion protein (PrP-sen) to its pathological isoform (PrP-res) is a key event in the pathogenesis of transmissible spongiform encephalopathies. Although the subcellular sites of conversion are poorly characterized, several lines of evidence have suggested the involvement of membrane lipid rafts in the conversion process. Here we report that copper stimulates the endocytosis of PrP-sen via a caveolin-dependent pathway in both microglia and neuroblastoma cells. We show that the polyene antibiotic filipin both limits endocytosis of PrP-sen and dramatically reduces the amount of membrane-bound PrP-sen. This reduction results from a rapid and massive release of full matured PrP-sen into the culture medium. Finally, we demonstrate that filipin is a potent inhibitor of PrP-res formation into chronically infected neuroblastoma cells. Our results reinforce the role of rafts in PrP trafficking and raise the possibility that the release of PrP-sen from the plasma membrane decreases the amount of available substrate PrP-sen at the conversion sites.

BSE: a consequence of cattle feeding with glycated molecules host-unknown?

Although there is much evidence supporting a prion contribution in the pathogenesis of transmissible spongiform encephalopathies, a novel point of view as to the induction of the diseases can be proposed. It is hypothesized that neurodegenerative diseases, such as scrapie in sheep and goats and bovine spongiform encephalopathy in cattle (BSE), originate from the consumption of glycated proteins contained in their feed. These components are obtained during a high-temperature glycation process.

Involvement of macrophages in the pathogenesis of transmissible spongiform encephalopathies.

Although transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders, the immune system is also involved, at least in the early stages of their pathogenesis. Extensive studies have focused on cells targeted by the TSE agent for its replication but few on the possible involvement of macrophages in its clearance, as in more conventional diseases. This review summarises some of the experiments aimed at demonstrating a role for macrophages in TSE and presents the application to TSE of the macrophage "suicide" technique, which has been used to clarify the implication of these cells in the early steps of TSE pathogenesis.

Glycosylation influences cross-species formation of protease-resistant prion protein.

A key event in the transmissible spongiform encephalopathies (TSEs) is the formation of aggregated and protease-resistant prion protein, PrP-res, from a normally soluble, protease-sensitive and glycosylated precursor, PrP-sen. While amino acid sequence similarity between PrP-sen and PrP-res influences both PrP-res formation and cross-species transmission of infectivity, the influence of co- or post-translational modifications to PrP-sen is unknown. Here we report that, if PrP-sen and PrP-res are derived from different species, PrP-sen glycosylation can significantly affect PrP-res formation. Glycosylation affected PrP-res formation by influencing the amount of PrP-sen bound to PrP-res, while the amino acid sequence of PrP-sen influenced the amount of PrP-res generated in the post-binding conversion step. Our results show that in addition to amino acid sequence, co- or post-translational modifications to PrP-sen influence PrP-res formation in vitro. In vivo, these modifications might contribute to the resistance to infection associated with transmission of TSE infectivity across species barriers.

Deletion of beta-strand and alpha-helix secondary structure in normal prion protein inhibits formation of its protease-resistant isoform.

A fundamental event in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conversion of a normal, proteinase K-sensitive, host-encoded protein, PrP-sen, into its protease-resistant isoform, PrP-res. During the formation of PrP-res, PrP-sen undergoes conformational changes that involve an increase of beta-sheet secondary structure. While previous studies in which PrP-sen deletion mutants were expressed in transgenic mice or scrapie-infected cell cultures have identified regions in PrP-sen that are important in the formation of PrP-res, the exact role of PrP-sen secondary structures in the conformational transition of PrP-sen to PrP-res has not yet been defined. We constructed PrP-sen mutants with deletions of the first beta-strand, the second beta-strand, or the first alpha-helix and tested whether these mutants could be converted to PrP-res in both scrapie-infected neuroblastoma cells (Sc(+)-MNB cells) and a cell-free conversion assay. Removal of the second beta-strand or the first alpha-helix significantly altered both processing and the cellular localization of PrP-sen, while deletion of the first beta-strand had no effect on these events. However, all of the mutants significantly inhibited the formation of PrP-res in Sc(+)-MNB cells and had a greatly reduced ability to form protease-resistant PrP in a cell-free assay system. Thus, our results demonstrate that deletion of the beta-strands and the first alpha-helix of PrP-sen can fundamentally affect PrP-res formation and/or PrP-sen processing.

Does an ultra violet photooxidation of the manganese-loaded/copper-depleted prion protein in the retina initiate the pathogenesis of TSE?

Ecosystems supporting clusters of sporadic transmissible spongiform encephalopathy (TSE) are characterized by common properties of high-manganese/low-copper, zinc, selenium mineral status, and high-altitude/snow-covered/pre-cambrian mountain terrain where above-average intensities of ultra violet/ozone oxidants are prevalent. Cell culture trials have confirmed the hypothesis that manganese (Mn) substitutes at Prion Protein’s (PrP’s) vacated copper (Cu) domain, whereupon PrP loses its Cu-mediated antioxidant function, transforming into a protease-resistant misfolded isoform that aggregates into fibril ‘tombstone’ structures – the key hallmark distinguishing TSE central nervous system (CNS) pathology. The cellular localisation of PrP suggests PrP serves a ‘front line’ contributory role in neutralizing radicals generated by incoming environmental oxidants, whilst an intensive expression of PrP messenger ribonucleic acid (mRNA) in the retina, melanocytes, epidermis, etc., suggests PrP performs a key antioxidant role as a ‘photooxidative shock absorber’; binding of porphyrin IX, Congo red and other photosensitisers to PrPc suggests PrPc serves as an integral associate of the porphyrin/melanin chromophore electron transfer chain; thereby serving as a quencher of singlet O2/superoxide generated by photoenergised chromophores/xeno photosensitisers. It is proposed that sporadic TSE pathogenesis is initiated in the retina of environmentally/genetically predisposed individuals via a two-stage chronic toxic process – Mn substitution at PrP’s Cu domain forming a stable Mn2+-PrP complex, followed by an ultra violet in situ photo-oxidization of the Mn2+ component; whereby the latent ‘Jekyll and Hyde’ capacity of the Mn2+-PrP conjugate is activated into the fully fledged, ‘infectious’ lethal auto-oxidizing, Mn3+-PrP ‘prion’ agent. Thus, PrPc’s Cu-mediated antioxidant function is replaced by a Mn3+-mediated autooxidant dysfunction. Could the UK’s increased loading of a cocktail of environmental oxidants that penetrated the CNS of the UK bovine (ultra violet microwaves/ozone/systemic cu-chelating insecticides) account for a more virulent Mn4+ mediated acceleration of the TSE degenerative process in Mn-contaminated/genetically predisposed individuals, manifesting as the widespread emergence of new-variant bovine spongiform encephalopathy (BSE)/variant Creutzfeldt–Jacob disease (VCJD)/FSE in younger mammals?

Spiroplasma sp. 16S rDNA in Creutzfeldt-Jakob disease and scrapie as shown by PCR and DNA sequence analysis.

The pathogenesis of the transmissible spongiform encephalopathies (TSE), which include Creutzfeldt-Jakob disease (CJD) in humans and scrapie in sheep, remains an enigma. In this paper we present evidence for the association of Spiroplasma sp., a wall-less prokaryote, with TSE. We have shown PCR amplification of Spiroplasma 16S rDNA in TSE-infected brain tissues (13 of 13 CJD cases and 5 of 9 scrapie cases) and not in control brains (0 of 50). Direct sequencing of the amplified PCR products has confirmed the presence of Spiroplasma-like DNA in all 5 of the TSE brains tested. Our evidence is not necessarily in conflict with involvement of a PrPres--a protease-resistant host-derived protein referred to as the prion--in the pathogenesis of TSE, since there is evidence that another factor is involved. We propose a bacterium, namely Spiroplasma, as this associated factor although the role of Spiroplasma in TSE cannot be determined from these experiments. The presence of the nucleic acid sequence of this microbe in all cases of TSE in our laboratory and not in controls provides direct evidence of the association of Spiroplasma sp. with TSE.

On the mechanism of alpha-helix to beta-sheet transition in the recombinant prion protein.

It is believed that the critical event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of the prion protein from an alpha-helical form, PrP(C), to a beta-sheet-rich conformer, PrP(Sc). Recently, we have shown that incubation of the recombinant prion protein under mildly acidic conditions (pH 5 or below) in the presence of low concentrations of guanidine hydrochloride results in a transition to PrP(Sc)-like beta-sheet-rich oligomers that show fibrillar morphology and an increased resistance to proteinase K digestion [Swietnicki, W., Morillas, M, Chen, S., Gambetti, P., and Surewicz, W. K. (2000) Biochemistry 39, 424-431]. To gain insight into the mechanism of this transition, in the present study we have characterized the biophysical properties of the recombinant human prion protein (huPrP) at acidic pH in the presence of urea and salt. Urea alone induces unfolding of the protein but does not result in protein self-association or a conversion to beta-sheet structure. However, a time-dependent transition to beta-sheet structure occurs upon addition of both urea and NaCl to huPrP, even at a sodium chloride concentration as low as 50 mM. This transition occurs concomitantly with oligomerization of the protein. At a given protein and sodium chloride concentration, the rate of monomeric alpha-helix to oligomeric beta-sheet transition is strongly dependent on the concentration of urea. Low and medium concentrations of the denaturant accelerate the reaction, whereas strongly unfolding conditions are not conducive to the conversion of huPrP into an oligomeric beta-sheet-rich structure. The present data strongly suggest that partially unfolded intermediates may be involved in the transition of the monomeric recombinant prion protein into the oligomeric scrapie-like form.

Transcriptional Activation of Prion Protein Gene in Growth-Arrested and Differentiated Mouse Erythroleukemia and Human Neoplastic Cells

The prion protein (PrP) is a GPI-anchored sialoglycoprotein that has attracted worldwide attention over the years due to its involvement in the pathogenesis of transmissible spongiform encephalopathies in sheep (scrapie), cattle (BSE), and humans (CJD). To understand the precise role of the Prn-p gene in cell growth and differentiation we investigated the expression pattern of the Prn-p gene in proliferating cells and in cells arrested in growth either by confluency or by induction of terminal differentiation. Viral-transformed mouse spleen hematopoietic cells named murine erythroleukemia (MEL) and other types of inducible cells (human neuroectodermal RD/TE-671, myoid RD cells) were employed. Cells grown exponentially, at confluency, or irreversibly arrested in growth at terminal differentiation state were analyzed by fluorescence cell sorting and Northern blot hybridization to estimate the steady-state level of PrP mRNA at different phases of the cell cycle. MEL cells that failed to differentiate from treatment with N6-methyladenosine (N6mAdo), an inhibitor of differentiation, were also analyzed for PrP mRNA level. Our results indicate the following: (a) growth arrest of cells at G1 phase by confluency or by induction of terminal differentiation led to increased accumulation of PrP mRNA transcripts, an event observed also in differentiated MEL, RD/TE-671, and RD cells independent of the inducer used; (b) treatment of MEL cells with N6mAdo prevented early activation of the Prn-p gene in cells treated with the inducer; and (c) cell-free nuclear run-off studies showed enhanced expression of the Prn-p gene due to transcriptional activation. These findings indicate, for the first time, that the Prn-p gene, which is thought to be a housekeeping gene, is transcriptionally activated in G1 phase in confluent and terminally differentiated cells. This information may be valuable in understanding the overaccumulation of PrP in some differentiated tissues and may let us repress Prn-p gene activation by novel agents.

Prion Protein Affects Ca2+-Activated K+ Currents in Cerebellar Purkinje Cells

The prion protein (PrPC) has a primary role in the pathogenesis of transmissible spongiform encephalopathies. Its physiological function is not known yet. Altered late afterhyperpolarization has been observed in hippocampal CA1 pyramidal cells of prion protein-deficient mice (Prnp0/0 mice) presumably caused by a disruption of Ca2+-activated K+ currents. An alteration of these currents has been recently described in scrapie-infected animals, and loss of function of PrPC has been put forward as one possible pathophysiological mechanism in prion diseases. This work focuses on patch-clamp studies of Ca2+-activated K+ currents in cerebellar Purkinje cells in the slice preparation of Prnp0/0 mice as well as of transgenic mice. A significant correlation between PrPC expression in Purkinje cells and the maximal amplitude of TEA-insensitive Ca2+-activated K+ currents was observed, with reduced current amplitudes in Prnp0/0 mice and a rescue of the phenotype in transgenic mice where PrPC had been reintroduced. Further studies of the intracellular free calcium concentration revealed an alteration of the maximal increase of intracellular calcium concentration with depolarization in the Prnp0/0 mouse Purkinje cells. These data provide strong evidence that Ca2+-activated K+ currents in Prnp0/0 mice are reduced due to an alteration of intracellular calcium homeostasis.

A novel erythroid-specific marker of transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSE) are a group of invariably fatal neurodegenerative diseases and include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in deer and elk, and Kuru disease, Creutzfeldt-Jakob disease (CJD) and variant CJD in humans. The pathological effects of disease occur predominantly in the CNS (central nervous system), where common hallmarks include vacuolation, gliosis, accumulation of a protease-resistant, abnormally folded isoform of the prion protein (PrPSc) and neuronal cell death. Lack of understanding of the molecular mechanisms underlying disease pathogenesis, particularly in non-CNS tissues, means that there are currently no effective strategies for early diagnosis or therapeutic intervention of TSEs. Here we report the first identification of a molecular marker that is easily detectable in readily accessible tissues. We demonstrate that a dramatic decrease in expression of a transcript specific to erythroid lineage cells is a common feature of TSEs. Our findings indicate a previously unrecognized role for involvement of the erythroid lineage in the etiology of TSE pathogenesis and should provide a new focus for research into diagnostic and therapeutic strategies.

The pathogenesis of transmissible spongiform encephalopathy: routes to the brain and the erection of therapeutic barricades.

Classical and modern studies of the pathogenesis of transmissible spongiform encephalopathy are reviewed, with particular attention paid to recent investigations of the routes of neuroinvasion. In various experimental models, a heirarchy of paths to the brain includes direct neural transit from the site of infection, replication in the spleen and neural entry through the spinal cord, and hematogenous spread. Possible modes and sites of therapeutic intervention are suggested.

The Role of Disulfide Bridge in the Folding and Stability of the Recombinant Human Prion Protein

It is believed that the critical step in the pathogenesis of transmissible spongiform encephalopathies is a transition of prion protein (PrP) from an alpha-helical conformation, PrP(C), to a beta-sheet-rich form, PrP(Sc). Native prion protein contains a single disulfide bond linking Cys residues at positions 179 and 214. To elucidate the role of this bridge in the stability and folding of the protein, we studied the reduced form of the recombinant human PrP as well as the variant of PrP in which cysteines were replaced with alanine residues. At neutral pH, the reduced prion protein and the Cys-free mutant were insoluble and formed amorphous aggregates. However, the proteins could be refolded in a monomeric form under the conditions of mildly acidic pH. Spectroscopic experiments indicate that the monomeric Cys-free and reduced PrP have molten globule-like properties, i.e. they are characterized by compromised tertiary interactions, an increased exposure of hydrophobic surfaces, lack of cooperative unfolding transition in urea, and partial loss of native (alpha-helical) secondary structure. In the presence of sodium chloride, these partially unfolded proteins undergo a transition to a beta-sheet-rich structure. However, this transition is invariably associated with protein oligomerization. The present data argue against the notion that reduced prion protein can exist in a stable monomeric form that is rich in beta-sheet structure.

Spongiform encephalopathies: Insights from transgenic models

Publisher Summary This chapter summarizes some of the transgenic mouse models that contributed to the current understanding of the pathogenesis of transmissible spongiform encephalopathies. Prion diseases, or transmissible spongiform encephalopathies, are neurological disorders caused by transmissible pathogens termed prions. Human prion diseases are characterized by extended incubation periods ranging from several months to decades that are followed by a progressive clinical phase presenting with severe dementia and ataxia. Clinical disease is always lethal: death can occur within as short a period as a few weeks but occasionally in a period of up to a few years. Peripheral prion pathogenesis, and ultimately neuroinvasion are dependent on the components of the host-immune system. Collectively, these processes require either B cells or their products. At least one B cell-dependent event is the acquisition of a functional follicular dendritic cell network within the germinal centers of peripheral lymphoid tissue. These cells are the major sites of extraneuronal prion protein (PrP c ) expression and probably the principal sites of PrP Sc accumulation.

Follicular dendritic cells in TSE pathogenesis

The pathogenesis of transmissible spongiform encephalopathies (TSEs) often includes a replication phase in lymphoid tissues before infection spreads to the central nervous system. Recent studies show that the follicular dendritic cells of the germinal centres are critical for this replication. These cells are therefore potential targets for therapy or prophylaxis in natural TSEs, such as variant Creutzfeldt-Jakob disease.

Interactions between heterologous forms of prion protein: Binding, inhibition of conversion, and species barriers

The self-induced formation of the disease-associated, protease-resistant prion protein (PrP-res) from the normal protease-sensitive isoform (PrP-sen) appears to be a key event in the pathogenesis of transmissible spongiform encephalopathies. The amino acid sequence specificity of PrP-res formation correlates with, and may account for, the species specificity in transmission of transmissible spongiform encephalopathy agents in vivo. To analyze the mechanism controlling the sequence specificity of PrP-res formation, we compared the binding of PrP-sen to PrP-res with its subsequent acquisition of protease resistance by using cell-free systems consisting of heterologous versus homologous mouse and hamster PrP isoforms. Our studies showed that heterologous PrP-sen can bind to PrP-res with little conversion to the protease-resistant state and, in doing so, can interfere with the conversion of homologous PrP-sen. The interference occurred with molar ratios of homologous to heterologous PrP-sen molecules as low as 1:1. The interference was due primarily to the inhibition of conversion, but not the binding, of the homologous PrP-sen to PrP-res. The results provide evidence that the sequence specificity of PrP-res formation in this model is determined more by the conversion to protease resistance than by the initial binding step. These findings also imply that after the initial binding, further intermolecular interactions between PrP-sen and PrP-res are required to complete the process of conversion to the protease-resistant state.

Porphyrin and Phthalocyanine Antiscrapie Compounds

The transmissible spongiform encephalopathies (TSEs) are fatal, neurodegenerative diseases for which no effective treatments are available. The likelihood that a bovine form of TSE has crossed species barriers and infected humans underscores the urgent need to identify anti-TSE drugs. Certain cyclic tetrapyrroles (porphyrins and phthalocyanines) have recently been shown to inhibit the in vitro formation of PrP-res, a protease-resistant protein critical for TSE pathogenesis. We now report that treatment of TSE-infected animals with three such compounds increased survival time from 50 to 300%. The significant inhibition of TSE disease by structurally dissimilar tetrapyrroles identifies these compounds as anti-TSE drugs.