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The properties of infectious prions and the pathology of the diseases they cause are dependent upon the unique conformation of each prion strain. How the pathology of prion disease correlates with different strains and genetic backgrounds has been investigated via in vivo assays, but how interactions between specific prion strains and cell types contribute to the pathology of prion disease has been dissected more effectively using in vitro cell lines. Observations made through in vivo and in vitro assays have informed each other with regard to not only how genetic variation influences prion properties, but also how infectious prions are taken up by cells, modified by cellular processes and propagated, and the cellular components they rely on for persistent infection. These studies suggest that persistent cellular infection results from a balance between prion propagation and degradation. This balance may be shifted depending upon how different cell lines process infectious prions, potentially altering prion stability, and how fast they can be transported to the lysosome. Thus, in vitro studies have given us a deeper understanding of the interactions between different prions and cell types and how they may influence prion disease phenotypes in vivo.

Accumulation of insoluble aggregates of infectious, partially protease-resistant prion protein (PrPD) generated via the misfolding of protease sensitive prion protein (PrPC) into the same infectious conformer, is a hallmark of prion diseases. Aggregated PrPD is taken up and degraded by cells, a process likely involving changes in aggregate structure that can be monitored by accessibility of the N-terminus of full-length PrPD to cellular proteases. We therefore tracked the protease sensitivity of full-length PrPD before and after cellular uptake for two murine prion strains, 22L and 87V. For both strains, PrPD aggregates were less stable following cellular uptake with increased accessibility of the N-terminus to cellular proteases across most aggregate sizes. However, a limited size range of aggregates was able to better protect the N-termini of full-length PrPD, with the N-terminus of 22L-derived PrPD more protected than that of 87V. Interestingly, changes in aggregate structure were associated with minimal changes to the protease-resistant core of PrPD. Our data show that cells destabilize the aggregate quaternary structure protecting PrPD from proteases in a strain-dependent manner, with structural changes exposing protease sensitive PrPD having little effect on the protease-resistant core, and thus conformation, of aggregated PrPD.

Prion diseases arise when PrPSc, an aggregated, infectious, and insoluble conformer of the normally soluble mammalian prion protein, PrPC, catalyzes the conversion of PrPC into more PrPSc, which then accumulates in the brain leading to disease. PrPSc is the primary, if not sole, component of the infectious prion. Despite the stability and protease insensitivity of PrPSc aggregates, they can be degraded after cellular uptake. However, how cells disassemble and degrade PrPSc is poorly understood. In this work, we analyzed how the protease sensitivity and size distribution of PrPSc aggregates from two different mouse-adapted prion strains, 22L, that can persistently infect cells and 87V, that cannot, changed during cellular uptake. We show that within the first 4 h following uptake large PrPSc aggregates from both prion strains become less resistant to digestion by proteinase K (PK) through a mechanism that is dependent upon the acidic environment of endocytic vesicles. We further show that during disassembly, PrPSc aggregates from both strains become more resistant to PK digestion through the apparent removal of protease-sensitive PrPSc, with PrPSc from the 87V strain disassembled more readily than PrPSc from the 22L strain. Taken together, our data demonstrate that the sizes and stabilities of PrPSc from different prion strains change during cellular uptake and degradation, thereby potentially impacting the ability of prions to infect cells.

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In Escherichia coli, Braun's lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We used a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

In the human prion disease Creutzfeldt-Jakob disease (CJD), different CJD neuropathological subtypes are defined by the presence in normal prion protein (PrPC) of a methionine or valine at residue 129, by the molecular mass of the infectious prion protein PrPSc, by the pattern of PrPSc deposition, and by the distribution of spongiform change in the brain. Heterozygous cases of CJD potentially add another layer of complexity to defining CJD subtypes since PrPSc can have either a methionine (PrPSc-M129) or valine (PrPSc-V129) at residue 129. We have recently demonstrated that the relative amount of PrPSc-M129 versus PrPSc-V129, i.e. the PrPSc allotype ratio, varies between heterozygous CJD cases. In order to determine if differences in PrPSc allotype correlated with different disease phenotypes, we have inoculated 10 cases of heterozygous CJD (7 sporadic and 3 iatrogenic) into two transgenic mouse lines overexpressing PrPC with a methionine at codon 129. In one case, brain-region specific differences in PrPSc allotype appeared to correlate with differences in prion disease transmission and phenotype. In the other 9 cases inoculated, the presence of PrPSc-V129 was associated with plaque formation but differences in PrPSc allotype did not consistently correlate with disease incubation time or neuropathology. Thus, while the PrPSc allotype ratio may contribute to diverse prion phenotypes within a single brain, it does not appear to be a primary determinative factor of disease phenotype.

The development of multiple cell culture models of prion infection over the last two decades has led to a significant increase in our understanding of how prions infect cells. In particular, new techniques to distinguish exogenous from endogenous prions have allowed us for the first time to look in depth at the earliest stages of prion infection through to the establishment of persistent infection. These studies have shown that prions can infect multiple cell types, both neuronal and nonneuronal. Once in contact with the cell, they are rapidly taken up via multiple endocytic pathways. After uptake, the initial replication of prions occurs almost immediately on the plasma membrane and within multiple endocytic compartments. Following this acute stage of prion replication, persistent prion infection may or may not be established. Establishment of a persistent prion infection in cells appears to depend upon the achievement of a delicate balance between the rate of prion replication and degradation, the rate of cell division, and the efficiency of prion spread from cell to cell. Overall, cell culture models have shown that prion infection of the cell is a complex and variable process which can involve multiple cellular pathways and compartments even within a single cell.

Prions represent a class of universally fatal and transmissible neurodegenerative disorders that affect humans and other mammals. The prion agent contains a pathologically aggregated form of the host prion protein that can transmit infectivity without any bacterial or viral component and is thus difficult to inactivate using disinfection protocols designed for infectious microorganisms. Methods for prion inactivation include treatment with acids, bases, detergents, bleach, prolonged autoclaving and incineration. During these procedures, the sample is often either destroyed or damaged such that further analysis for research purposes is compromised. In this study we show that a straightforward denaturation and in-gel protease digestion protocol used to prepare prion-infected samples for mass spectroscopy leads to the loss of at least 7 logs of prion infectivity, yielding a final product that fails to transmit prion disease in vivo. We further show that the resultant sample remains suitable for mass spectrometry-based protein identifications. Thus, the procedure described can be used to prepare prion-infected samples for mass spectrometry analysis with greatly reduced biosafety concerns.

The development of multiple cell culture models of prion infection over the last two decades has led to a significant increase in our understanding of how prions infect cells. In particular, new techniques to distinguish exogenous from endogenous prions have allowed us for the first time to look in depth at the earliest stages of prion infection through to the establishment of persistent infection. These studies have shown that prions can infect multiple cell types, both neuronal and nonneuronal. Once in contact with the cell, they are rapidly taken up via multiple endocytic pathways. After uptake, the initial replication of prions occurs almost immediately on the plasma membrane and within multiple endocytic compartments. Following this acute stage of prion replication, persistent prion infection may or may not be established. Establishment of a persistent prion infection in cells appears to depend upon the achievement of a delicate balance between the rate of prion replication and degradation, the rate of cell division, and the efficiency of prion spread from cell to cell. Overall, cell culture models have shown that prion infection of the cell is a complex and variable process which can involve multiple cellular pathways and compartments even within a single cell.

Prions, characterized by self-propagating protease-resistant prion protein (PrP) conformations, are agents causing prion disease. Recent studies generated several such self-propagating protease-resistant recombinant PrP (rPrP-res) conformers. While some cause prion disease, others fail to induce any pathology. Here we showed that although distinctly different, the pathogenic and non-pathogenic rPrP-res conformers were similarly recognized by a group of conformational antibodies against prions and shared a similar guanidine hydrochloride denaturation profile, suggesting a similar overall architecture. Interestingly, two independently generated non-pathogenic rPrP-res were almost identical, indicating that the particular rPrP-res resulted from cofactor-guided PrP misfolding, rather than stochastic PrP aggregation. Consistent with the notion that cofactors influence rPrP-res conformation, the propagation of all rPrP-res formed with phosphatidylglycerol/RNA was cofactor-dependent, which is different from rPrP-res generated with a single cofactor, phosphatidylethanolamine. Unexpectedly, despite the dramatic difference in disease-causing capability, RT-QuIC assays detected large increases in seeding activity in both pathogenic and non-pathogenic rPrP-res inoculated mice, indicating that the non-pathogenic rPrP-res is not completely inert in vivo. Together, our study supported a role of cofactors in guiding PrP misfolding, indicated that relatively small structural features determine rPrP-res’ pathogenicity, and revealed that the in vivo seeding ability of rPrP-res does not necessarily result in pathogenicity., Author summary Many neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease and Prion disease, are caused by misfolded proteins that can self-propagate in vivo and in vitro. Misfolded self-replicating recombinant prion protein (PrP) conformers have been generated in vitro with defined cofactors, some of which are highly infectious and cause bona fide prion diseases, while others completely fail to induce any pathology. Here we compare these misfolded recombinant PrP conformers and show that the non-pathogenic misfolded recombinant PrP is not completely inert in vivo. We also found that the pathogenic and non-pathogenic recombinant PrP conformers share a similar overall architecture. Importantly, our study clearly shows that in vivo seeded spread of misfolded conformation does not necessarily lead to pathogenic change or cause disease. These findings not only are important for understanding the molecular basis for prion infectivity, but also may have important implications for the “prion-like” spread of misfolded proteins in other neurodegenerative diseases.

Mitochondria are crucial to proper neuronal function and overall brain health. Mitochondrial dysfunction within the brain has been observed in many neurodegenerative diseases, including prion disease. Several markers of decreased mitochondrial activity during prion infection have been reported, yet the bioenergetic respiratory status of mitochondria from prion-infected animals is unknown. Here we show that clinically ill transgenic mice overexpressing hamster prion protein (Tg7) infected with the hamster prion strain 263K suffer from a severe deficit in mitochondrial oxygen consumption in response to the respiratory complex II substrate succinate. Characterization of the mitochondrial proteome of purified brain mitochondria from infected and uninfected Tg7 mice showed significant differences in the relative abundance of key mitochondrial electron transport proteins in 263K-infected animals relative to that in controls. Our results suggest that at clinical stages of prion infection, dysregulation of respiratory chain proteins may lead to impairment of mitochondrial respiration in the brain. IMPORTANCE Mitochondrial dysfunction is present in most major neurodegenerative diseases, and some studies have suggested that mitochondrial processes may be altered during prion disease. Here we show that hamster prion-infected transgenic mice overexpressing the hamster prion protein (Tg7 mice) suffer from mitochondrial respiratory deficits. Tg7 mice infected with the 263K hamster prion strain have little or no signs of mitochondrial dysfunction at the disease midpoint but suffer from a severe deficit in mitochondrial respiration at the clinical phase of disease. A proteomic analysis of the isolated brain mitochondria from clinically affected animals showed that several proteins involved in electron transport, mitochondrial dynamics, and mitochondrial protein synthesis were dysregulated. These results suggest that mitochondrial dysfunction, possibly exacerbated by prion protein overexpression, occurs at late stages during 263K prion disease and that this dysfunction may be the result of dysregulation of mitochondrial proteins.

Cellular prion protein (PrPC) is a mammalian glycoprotein which is usually found anchored to the plasma membrane via a glycophosphatidylinositol (GPI) anchor. PrPC misfolds to a pathogenic isoform PrPSc, the causative agent of neurodegenerative prion diseases. The precise function of PrPC remains elusive but may depend upon its cellular localization. Here we show that PrPC is present in brain mitochondria from 6–12 week old wild-type and transgenic mice in the absence of disease. Mitochondrial PrPC was fully processed with mature N-linked glycans and did not require the GPI anchor for localization. Protease treatment of purified mitochondria suggested that mitochondrial PrPC exists as a transmembrane isoform with the C-terminus facing the mitochondrial matrix and the N-terminus facing the intermembrane space. Taken together, our data suggest that PrPC can be found in mitochondria in the absence of disease, old age, mutation, or overexpression and that PrPC may affect mitochondrial function.

Prion diseases are a heterogeneous group of neurodegenerative disorders affecting various mammals including humans. Prion diseases are characterized by a misfolding of the host-encoded prion protein (PrP(C)) into a pathological isoform termed PrP(Sc). In wild-type mice, PrP(C) is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP(Sc) typically accumulates in diffuse nonamyloid deposits with gray matter spongiosis. By contrast, when mice lacking the GPI anchor are infected with the same prion inoculum, PrP(Sc) accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. In order to evaluate whether different host biochemical pathways were implicated in these two phenotypically distinct prion disease models, we utilized a proteomics approach. In both models, infected mice displayed evidence of a neuroinflammatory response and complement activation. Proteins involved in cell death and calcium homeostasis were also identified in both phenotypes. However, mitochondrial pathways of apoptosis were implicated only in the nonamyloid form, whereas metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. Thus, following infection with a single prion strain, PrP(C) anchoring to the plasma membrane correlated not only with the type of PrP(Sc) deposition but also with unique biochemical pathways associated with pathogenesis.

During the course of prion infection, the normally soluble and protease-sensitive mammalian prion protein (PrPC) is refolded into an insoluble, partially protease-resistant, and infectious form called PrPSc. The conformational conversion of PrPC to PrPSc is a critical event during prion infection and is essential for the production of prion infectivity. This chapter briefly summarizes the ways in which cell biological approaches have enhanced our understanding of how PrP contributes to different aspects of prion pathogenesis.

Aggregated and protease-resistant mammalian prion protein (PrPSc) is the primary protein component of infectious prions. Enriched PrPSc preparations are often used to study the mechanisms underlying prion disease. However, most enrichment procedures are relatively nonspecific and tend to yield significant amounts of non-PrPSc components, including various proteins that could confound functional and structural studies. It is thus important to identifythese proteins and assess their potential relevance to prion pathogenesis. Following proteinase K treatment and phosphotungstic acid precipitation of brain homogenate, we have used mass spectrometry to analyze the protein content of PrPSc isolated from prion-infected mice, multiple cases of sporadic Creutzfeldt-Jakob disease (sCJD), and human growth hormone associated casesof iatrogenic CJD (iCJD). Creatine kinase was the primary protein contaminant in all PrPSc samples while many of the other proteins identified were also found in non-CJD controls, suggesting that they are not CJD specific. Interestingly, the Alzheimer’s disease associated peptide amyloid β 1-42 (Aβ1-42) was identified in the majority of the sCJD cases as well as non-CJD age-matched controls while apoliprotein E was found in greater abundance in the sCJD cases. By contrast, while some of the iCJD cases showed evidence of higher molecular weight Aβ oligomers, monomeric Aβ1-42 peptide was not detected by immunoblot and only one case had significant levels of apolipoprotein E. Our data are consistent with the age-associated deposition of Aβ1-42 in older sporadic CJD and non-CJD patients and suggest that both apolipoprotein E and Aβ1-42 abundance can differ depending upon the type of CJD.

Prions are unconventional agents composed of misfolded prion protein that cause fatal neurodegenerative diseases in mammals. Prion strains induce specific neuropathological changes in selected brain areas. The mechanism of strain-specific cell tropism is unknown. We hypothesised that prion strains rely on different endocytic routes to invade and replicate within their target cells. Using prion permissive cells, we determined how impairment of endocytosis affects productive infection by prion strains 22L and RML. We demonstrate that early and late stages of prion infection are differentially sensitive to perturbation of clathrin- and caveolin-mediated endocytosis. Manipulation of canonical endocytic pathways only slightly influenced prion uptake. However, blocking the same routes had drastic strain-specific consequences on the establishment of infection. Our data argue that prion strains use different endocytic pathways for infection and suggest that cell type-dependent differences in prion uptake could contribute to host cell tropism.

The ability of prions to infect some species and not others is determined by the transmission barrier. This unexplained phenomenon has led to the belief that certain species were not susceptible to transmissible spongiform encephalopathies (TSEs) and therefore represented negligible risk to human health if consumed. Using the protein misfolding cyclic amplification (PMCA) technique, we were able to overcome the species barrier in rabbits, which have been classified as TSE resistant for four decades. Rabbit brain homogenate, either unseeded or seeded in vitro with disease-related prions obtained from different species, was subjected to serial rounds of PMCA. De novo rabbit prions produced in vitro from unseeded material were tested for infectivity in rabbits, with one of three intracerebrally challenged animals succumbing to disease at 766 d and displaying all of the characteristics of a TSE, thereby demonstrating that leporids are not resistant to prion infection. Material from the brain of the clinically affected rabbit containing abnormal prion protein resulted in a 100% attack rate after its inoculation in transgenic mice overexpressing rabbit PrP. Transmissibility to rabbits (>470 d) has been confirmed in 2 of 10 rabbits after intracerebral challenge. Despite rabbits no longer being able to be classified as resistant to TSEs, an outbreak of “mad rabbit disease” is unlikely.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are characterized by the accumulation of an aggregated isoform of the prion protein (PrP). This pathological isoform, termed PrPSc, appears to be the primary component of the TSE infectious agent or prion. However, it is not clear to what extent other protein co-factors may be involved in TSE pathogenesis or whether there are PrPSc-associated proteins which help to determine TSE strain-specific disease phenotypes. We enriched PrPSc from the brains of mice infected with either 22L or Chandler TSE strains and examined the protein content of these samples using nanospray liquid chromatography-tandem mass spectrometry. These samples were compared to “mock” PrPSc preparations from uninfected brains. Prion protein was the major component of the infected samples and ferritin was the most abundant impurity. By contrast, mock enrichments contained no detectable prion protein but did contain a significant amount of ferritin. Of the total proteins identified, 32% were found in both mock and infected samples. The similarities between PrPSc samples from 22L and Chandler TSE strains suggest that the non-PrPSc protein components found in standard enrichment protocols are not strain-specific.

A species barrier may protect humans from this disease., Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy, or prion disease, that affects deer, elk, and moose. Human susceptibility to CWD remains unproven despite likely exposure to CWD-infected cervids. We used 2 nonhuman primate species, cynomolgus macaques and squirrel monkeys, as human models for CWD susceptibility. CWD was inoculated into these 2 species by intracerebral and oral routes. After intracerebral inoculation of squirrel monkeys, 7 of 8 CWD isolates induced a clinical wasting syndrome within 33–53 months. The monkeys’ brains showed spongiform encephalopathy and protease-resistant prion protein (PrPres) diagnostic of prion disease. After oral exposure, 2 squirrel monkeys had PrPres in brain, spleen, and lymph nodes at 69 months postinfection. In contrast, cynomolgus macaques have not shown evidence of clinical disease as of 70 months postinfection. Thus, these 2 species differed in susceptibility to CWD. Because humans are evolutionarily closer to macaques than to squirrel monkeys, they may also be resistant to CWD.

Normal cellular and abnormal disease-associated forms of prion protein (PrP) contain a C-terminal glycophosphatidyl-inositol (GPI) membrane anchor. The importance of the GPI membrane anchor in prion diseases is unclear but there are data to suggest that it both is and is not required for abnormal prion protein formation and prion infection. Utilizing an in vitro model of prion infection we have recently demonstrated that, while the GPI anchor is not essential for the formation of abnormal prion protein in a cell, it is necessary for the establishment of persistent prion infection. In combination with previously published data, our results suggest that GPI anchored PrP is important in the amplification and spread of prion infectivity from cell to cell.

Transmissible spongiform encephalopathies (TSEs or prion diseases) are a rare group of invariably fatal neurodegenerative disorders that affect humans and other mammals. TSEs are protein misfolding diseases that involve the accumulation of an abnormally aggregated form of the normal host prion protein. They are unique among protein misfolding disorders in that they are transmissible and have different strains of infectious agent that are associated with unique phenotypes in vivo. A wealth of biological and biophysical evidence now suggests that the molecular basis for prion diseases may be encoded by protein conformation. The purpose of this review is to provide an overview of the existing structural information for prion protein within the context of what is known about the biology of prion disease.

Amyloid fibrils have been classically defined as linear, nonbranched polymeric proteins with a cross beta-sheet structure and the ability to alter the optical properties of the amyloid-specific dye Congo Red. Mounting evidence suggests that soluble oligomeric peptide assemblies approximately 2-20 nm in diameter are critical intermediates in amyloid formation. Using a pathogenic prion protein peptide comprised of residues 23-144, we demonstrate that, under quiescent but not agitated conditions, much larger globular assemblies up to 1 mum in diameter are made. These globules precede fibril formation and directly interact with growing fibril bundles. Fibrils made via these large spherical peptide assemblies displayed a remarkable diversity of ultrastructural features. Fibrillization of the Abeta1-40 peptide under similar conditions yielded similar results, suggesting a mechanism of general amyloid formation that can proceed through intermediates much larger than those previously described. Our data suggest that simply changing the physical microenvironment can profoundly influence the mechanism of amyloid formation and yield fibrils with novel ultrastructural properties.

In prion and Alzheimer's diseases, the roles played by amyloid versus nonamyloid deposits in brain damage remain unresolved. In scrapie-infected transgenic mice expressing prion protein (PrP) lacking the glycosylphosphatidylinositol (GPI) membrane anchor, abnormal protease-resistant PrPres was deposited as amyloid plaques, rather than the usual nonamyloid form of PrPres. Although PrPres amyloid plaques induced brain damage reminiscent of Alzheimer's disease, clinical manifestations were minimal. In contrast, combined expression of anchorless and wild-type PrP produced accelerated clinical scrapie. Thus, the PrP GPI anchor may play a role in the pathogenesis of prion diseases.

Transmissible spongiform encephalopathies are accompanied by the accumulation of a pathologic isoform of a host-encoded protein, termed prion protein (PrP). Despite the widespread distribution of the cellular isoform of PrP (protease-sensitive PrP; PrP-sen), the disease-associated isoform (protease-resistant PrP; PrP-res) appears to be primarily restricted to cells of the nervous and lymphoreticular systems. In order to study why scrapie infection appears to be restricted to certain cells, we followed acute and persistent PrP-res formation upon exposure of cells to different scrapie agents. We found that, independent of the cell type and scrapie strain, initial PrP-res formation occurred rapidly in cells. However, sustained generation of PrP-res and persistent infection did not necessarily follow acute PrP-res formation. Persistent PrP-res formation and scrapie infection was restricted to one cell line inoculated with the mouse scrapie strain 22L. In contrast to cells that did not become scrapie-infected, the level of PrP-res in the 22L-infected cells rapidly increased in the absence of a concomitant increase in the number of PrP-res-producing cells. Furthermore, the protein banding pattern of PrP-res in these cells changed over time as the cells became chronically infected. Thus, our results suggest that the events leading to the initial formation of PrP-res may differ from those required for sustained PrP-res formation and infection. This may, at least in part, explain the observation that not all PrP-sen-expressing cells appear to support transmissible spongiform encephalopathy agent replication.

The risk of contamination of tissue culture cells with transmissible spongiform encephalopathy (TSE) agents as a result of the use of animal products as medium components has been considered to be low, in part, because only a few brain-derived cell lines have been reported to be susceptible to TSE infection. In the present study, we demonstrate that the common laboratory fibroblast cell lines NIH/3T3 and L929, which express low levels of cellular mouse prion protein, are susceptible to infection with mouse-adapted scrapie. Our results show that the susceptibility of a cell line to TSE infection cannot be predicted on the basis of its tissue origin or its level of expression of the cellular prion protein, and they suggest that any cell line expressing normal host prion protein could have the potential to support propagation of TSE agents. Thus, testing of cells for TSE susceptibility might be necessary for all cell lines that are routinely used in vaccine production and in other medical applications.

Transmissible spongiform encephalopathies (TSEs) are neurological diseases that are associated with the conversion of the normal host-encoded prion protein (PrP-sen) to an abnormal protease-resistant form, PrP-res. Transmission of the TSE agent from one species to another is usually inefficient and accompanied by a prolonged incubation time. Species barriers to infection by the TSE agent are of particular importance given the apparent transmission of bovine spongiform encephalopathy to humans. Among the few animal species that appear to be resistant to infection by the TSE agent are rabbits. They survive challenge with the human kuru and Creutzfeldt-Jakob agents as well as with scrapie agent isolated from sheep or mice. Species barriers to the TSE agent are strongly influenced by the PrP amino acid sequence of both the donor and recipient animals. Here we show that rabbit PrP-sen does not form PrP-res in murine tissue culture cells persistently infected with the mouse-adapted scrapie agent. Unlike other TSE species barriers that have been studied, critical amino acid residues that inhibit PrP-res formation are located throughout the rabbit PrP sequence. Our results suggest that the resistance of rabbits to infection by the TSE agent is due to multiple rabbit PrP-specific amino acid residues that result in a PrP structure that is unable to refold to the abnormal isoform associated with disease.

Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are fatal neurodegenerative diseases for which there is no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPsc or PrPres). Both in vitro (cell culture and cell free conversion assays) and in vivo (animal) studies have demonstrated the strong dependence of this conversion process on protein sequence homology between the initial prion inoculum and the host’s own cellular prion protein. The presence of non-homologous (heterologous) proteins is often inhibitory to this conversion process. We hypothesize that the presence of heterologous prion proteins from one species might therefore constitute an effective treatment for prion disease in another species. To test this hypothesis, we infected mice intracerebrally with murine adapted RML-Chandler scrapie and treated them with heterologous prion protein (purified bacterially expressed recombinant hamster prion protein) or vehicle alone. Treated animals demonstrated reduced disease associated pathology, decreased accumulation of protease-resistant disease-associated prion protein, with delayed onset of clinical symptoms and motor deficits. This was concomitant with significantly increased survival times relative to mock-treated animals. These results provide proof of principle that recombinant hamster prion proteins can effectively and safely inhibit prion disease in mice, and suggest that hamster or other non-human prion proteins may be a viable treatment for prion diseases in humans.

Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a protease-sensitive host glycoprotein, prion protein or PrP-sen, to a protease-resistant form (PrP-res). PrP-res molecules that accumulate in the brain and lymphoreticular system of the host consist of three differentially glycosylated forms. Analysis of the relative amounts of the PrP-res glycoforms has been used to discriminate TSE strains and has become increasingly important in the differential diagnosis of human TSEs. However, the molecular basis of PrP-res glycoform variation between different TSE agents is unknown. Here we report that PrP-res itself can dictate strain-specific PrP-res glycoforms. The final PrP-res glycoform pattern, however, can be influenced by the cell and significantly altered by subtle changes in the glycosylation state of PrP-sen. Thus, strain-specific PrP-res glycosylation profiles are likely the consequence of a complex interaction between PrP-res, PrP-sen, and the cell and may indicate the cellular compartment in which the strain-specific formation of PrP-res occurs.

The transmissible spongiform encephalopathy diseases are transmissible, neurodegenerative, and inevitably fatal diseases of the central nervous system. There are no sensitive diagnostic tests to detect infection early during disease and no effective treatments are available. Accumulation of an abnormally aggregated and protease resistant form of the mammalian prion protein (PrP) is a key facet of TSE pathogenesis and an obvious target for early diagnosis and therapeutic intervention. Synthetic peptides to certain regions of the PrP gene have been shown to inhibit formation of the abnormal disease-associated PrP isoform. Peptides based on the PrP sequence may be useful as anti-TSE therapeutics. Specific interactions between PrP peptides and the abnormal PrP protein could also form the basis of a specific and sensitive diagnostic test.

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 β-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 β-strand, the second β-strand, or the first α-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 β-strand or the first α-helix significantly altered both processing and the cellular localization of PrP-sen, while deletion of the first β-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 β-strands and the first α-helix of PrP-sen can fundamentally affect PrP-res formation and/or PrP-sen processing.

In the transmissible spongiform encephalopathies, disease is closely associated with the conversion of the normal proteinase K-sensitive host prion protein (PrP-sen) to the abnormal proteinase K-resistant form (PrP-res). Amino acid sequence homology between PrP-res and PrP-sen is important in the formation of new PrP-res and thus in the efficient transmission of infectivity across species barriers. It was previously shown that the generation of mouse PrP-res was strongly influenced by homology between PrP-sen and PrP-res at amino acid residue 138, a residue located in a region of loop structure common to PrP molecules from many different species. In order to determine if homology at residue 138 also affected the formation of PrP-res in a different animal species, we assayed the ability of hamster PrP-res to convert a panel of recombinant PrP-sen molecules to protease-resistant PrP in a cell-free conversion system. Homology at amino acid residue 138 was not critical for the formation of protease-resistant hamster PrP. Rather, homology between PrP-sen and hamster PrP-res at amino acid residue 155 determined the efficiency of formation of a protease-resistant product induced by hamster PrP-res. Structurally, residue 155 resides in a turn at the end of the first alpha helix in hamster PrP-sen; this feature is not present in mouse PrP-sen. Thus, our data suggest that PrP-res molecules isolated from scrapie-infected brains of different animal species have different PrP-sen structural requirements for the efficient formation of protease-resistant PrP.

Antibodies to the prion protein (PrP) have been critical to the neuropathological and biochemical characterization of PrP-related degenerative diseases in humans and animals. Although PrP is highly conserved evolutionarily, there is some sequence divergence among species; as a consequence, anti-PrP antibodies have a wide spectrum of reactivity when challenged with PrP from diverse species. We have produced an antibody [monoclonal antibody (mAb) 2-40] raised against a synthetic peptide corresponding to residues (106-126 of human PrP and have characterized it by epitope mapping, Western immunoblot analysis, and immunohistochemistry. The antibody recognizes not only human PrP isoforms but also pathological PrP from all species tested (i.e., sheep, hamsters, and mice). Together with the fact that it recognizes the whole PrP in both cellular and scrapie isoforms, mAb 2-40 may be helpful in studying conformational changes of the PrP, as well as establishing a possible connection between human and animal diseases.

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.

Transmissible spongiform encephalopathies are characterized by spongiosis, astrocytosis and accumulation of PrPSc, an isoform of the normal host protein PrPC. The exact cell types responsible for agent propagation and pathogenesis are still uncertain. To determine the possible role of astrocytes, we generated mice devoid of murine PrP but expressing hamster PrP transgenes driven by the astrocyte-specific GFAP promoter. After inoculation with hamster scrapie, these mice accumulated infectivity and PrPSc to high levels, developed severe disease after 227 +/- 5 days and died 7 +/- 4 days later. Therefore, astrocytes could play an important role in scrapie pathogenesis, possibly by an indirect toxic effect on neurons. Interestingly, mice expressing the same transgenes but also endogenous murine PrP genes propagated infectivity without developing disease.

Prion diseases are characterized by the conversion of the soluble protease-sensitive host-encoded prion protein (PrP C ) into its aggregated, protease-resistant, and infectious isoform (PrP Sc ). One of the earliest events occurring in cells following exposure to an exogenous source of prions is the cellular uptake of PrP Sc . It is unclear how the biochemical properties of PrP Sc influence its uptake, although aggregate size is thought to be important. Here we show that for two different strains of mouse prions, one that infects cells (22L) and one that does not (87V), a fraction of PrP Sc associated with distinct sedimentation properties is preferentially taken up by the cells. However, while the fraction of PrP Sc and the kinetics of uptake were similar for both strains, PrP Sc derived from the 87V strain was disaggregated more rapidly than that derived from 22L. The increased rate of PrP Sc disaggregation did not correlate with either the conformational or aggregate stability of 87V PrP Sc , both of which were greater than those of 22L PrP Sc . Our data suggest that the kinetics of disaggregation of PrP Sc following cellular uptake is independent of PrP Sc stability but may be dependent upon some component of the PrP Sc aggregate other than PrP. Rapid disaggregation of 87V PrP Sc by the cell may contribute, at least in part, to the inability of 87V to infect cells in vitro .

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP Sc ) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP Sc in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP Sc . Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.

Neurodegeneration caused by the transmissible spongiform encephalopathies is associated with the conversion of a normal host protein, PrP-sen, into an abnormal aggregated protease-resistant form, PrP-res. In scrapie-infected mouse neuroblastoma cells, mouse PrP-sen is converted into PrP-res but recombinant hamster PrP-sen expressed in these cells is not. In the present studies, recombinant hamster/mouse PrP-sen molecules were expressed in these scrapie-infected cells to define specific PrP amino acid residues critical for the conversion to PrP-res. The results showed that homology to the region of mouse PrP-sen from amino acid residues 112 to 138 was required for conversion of recombinant PrP-sen to PrP-res in scrapie-infected mouse cells. Furthermore, a single hamster-specific PrP amino acid at residue 138 could inhibit the conversion of the recombinant PrP-sen into PrP-res. The data are consistent with studies in humans which show that specific amino acid residue changes within PrP can influence disease pathogenesis and transmission of transmissible spongiform encephalopathies across species barriers.

Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrPc) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to protease-resistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction. The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPc and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.

The transmissible spongiform encephalopathies or prion diseases include scrapie in sheep, mad cow disease, and variant Creutzfeld-Jakob disease in humans. The agent of all of these diseases is thought to be a self-replicating protein or prion. In a report of a recent meeting on prion diseases, Priola, Chesebro, and Caughey discuss the latest developments in this often contentious field of research.

Species barriers in prion diseases are defined by the difficulty that prions from one species have in triggering prion infection in a new species. The amino acid sequence of the normal host prion protein (PrPC), the available pool of exogenous abnormal and infectious prion protein (PrPSc), and the ability to establish a subclinical infection are all important determinants of prion species barriers. Mechanistically, maintenance of species barriers to prion infection is likely dependent upon the conformational diversity of the PrPSc molecules in an infectious inoculum and the potential for conformational compatibility between the exogenous PrPSc and endogenous host PrPC. However, the lack of high resolution structural information for PrPSc, the potential for host factors and posttranslational modifications to PrPC to influence species barriers, and the fact that the amino acids important in prion species barriers differ between species, makes it difficult to predict prion species barriers based on PrPC sequence alone. In vivo or in vitro experimentation in relevant models of infection remains the only way to determine species barriers to prion infection.