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Lethal neurodegenerative prion diseases result from the continuous accumulation of infectious and variably protease-resistant prion protein aggregates (PrP D ) which are misfolded forms of the normally detergent soluble and protease-sensitive cellular prion protein. Molecular chaperones like Grp78 have been found to reduce the accumulation of PrP D , but how different cellular environments and other chaperones influence the ability of Grp78 to modify PrP D is poorly understood. In this work, we investigated how pH and protease-mediated structural changes in PrP D from two mouse-adapted scrapie prion strains, 22L and 87V, influenced processing by Grp78 in the presence or absence of chaperones Hsp90, DnaJC1, and Stip1. We developed a cell-free in vitro system to monitor chaperone-mediated structural changes to, and disaggregation of, PrP D . For both strains, Grp78 was most effective at structurally altering PrP D at low pH, especially when additional chaperones were present. While Grp78, DnaJC1, Stip1, and Hsp90 were unable to disaggregate the majority of PrP D from either strain, pretreatment of PrP D with proteases increased disaggregation of 22L PrP D compared to 87V, indicating strain-specific differences in aggregate structure were impacting chaperone activity. Hsp90 also induced structural changes in 87V PrP D as indicated by an increase in the susceptibility of its n-terminus to proteases. Our data suggest that, while chaperones like Grp78, DnaJC1, Stip1, and Hsp90 disaggregate only a small fraction of PrP D , they may still facilitate its clearance by altering aggregate structure and sensitizing PrP D to proteases in a strain and pH-dependent manner., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The PINK1/Parkin pathway of mitophagy has been implicated in the pathogenesis of Parkinson's disease. In prion diseases, a transmissible neurodegenerative disease caused by the misfolded and infectious prion protein (PrPSc), expression of both PINK1 and Parkin are elevated, suggesting that PINK1/Parkin mediated mitophagy may also play a role in prion pathogenesis. Using mice in which expression of either PINK1 (PINK1KO) or Parkin (ParkinKO) has been ablated, we analyzed the potential role of PINK1 and Parkin in prion pathogenesis. Prion infected PINK1KO and ParkinKO mice succumbed to disease more rapidly (153 and 150 days, respectively) than wild-type control C57Bl/6 mice (161 days). Faster incubation times in PINK1KO and ParkinKO mice did not correlate with altered prion pathology in the brain, altered expression of proteins associated with mitochondrial dynamics, or prion-related changes in mitochondrial respiration. However, the expression level of mitochondrial respiration Complex I, a major site for the formation of reactive oxygen species (ROS), was higher in prion infected PINK1KO and ParkinKO mice when compared to prion infected control mice. Our results demonstrate a protective role for PINK1/Parkin mitophagy during prion disease, likely by helping to minimize ROS formation via Complex I, leading to slower prion disease progression., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

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., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Prion diseases are a group of fatal, transmissible neurodegenerative diseases of mammals. In the brain, axonal loss and neuronal death are prominent in prion infection, but the mechanisms remain poorly understood. Sterile alpha and heat/Armadillo motif 1 (SARM1) is a protein expressed in neurons of the brain that plays a critical role in axonal degeneration. Following damage to axons, it acquires an NADase activity that helps to regulate mitochondrial health by breaking down NAD+, a molecule critical for mitochondrial respiration. SARM1 has been proposed to have a protective effect in prion disease, and we hypothesized that it its role in regulating mitochondrial energetics may be involved. We therefore analyzed mitochondrial respiration in SARM1 knockout mice (SARM1KO) and wild-type mice inoculated either with prions or normal brain homogenate. Pathologically, disease was similar in both strains of mice, suggesting that SARM1 mediated axonal degradation is not the sole mechanism of axonal loss during prion disease. However, mitochondrial respiration was significantly increased and disease incubation time accelerated in prion infected SARM1KO mice when compared to wild-type mice. Increased levels of mitochondrial complexes II and IV and decreased levels of NRF2, a potent regulator of reactive oxygen species, were also apparent in the brains of SARM1KO mice when compared to wild-type mice. Our data suggest that SARM1 slows prion disease progression, likely by regulating mitochondrial respiration, which may help to mitigate oxidative stress via NRF2., Competing Interests: The authors have declared that no competing interests exist.

Prion diseases arise when PrP Sc , an aggregated, infectious, and insoluble conformer of the normally soluble mammalian prion protein, PrP C , catalyzes the conversion of PrP C into more PrP Sc , which then accumulates in the brain leading to disease. PrP Sc is the primary, if not sole, component of the infectious prion. Despite the stability and protease insensitivity of PrP Sc aggregates, they can be degraded after cellular uptake. However, how cells disassemble and degrade PrP Sc is poorly understood. In this work, we analyzed how the protease sensitivity and size distribution of PrP Sc 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 PrP Sc 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, PrP Sc aggregates from both strains become more resistant to PK digestion through the apparent removal of protease-sensitive PrP Sc , with PrP Sc from the 87V strain disassembled more readily than PrP Sc from the 22L strain. Taken together, our data demonstrate that the sizes and stabilities of PrP Sc 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 proteins 2,3 . In Escherichia coli, Braun's lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability 4 ; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present 5 . 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 (PrP C ) of a methionine or valine at residue 129, by the molecular mass of the infectious prion protein PrP Sc , by the pattern of PrP Sc 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 PrP Sc can have either a methionine (PrP Sc -M129) or valine (PrP Sc -V129) at residue 129. We have recently demonstrated that the relative amount of PrP Sc -M129 versus PrP Sc -V129, i.e. the PrP Sc allotype ratio, varies between heterozygous CJD cases. In order to determine if differences in PrP Sc 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 PrP C with a methionine at codon 129. In one case, brain-region specific differences in PrP Sc allotype appeared to correlate with differences in prion disease transmission and phenotype. In the other 9 cases inoculated, the presence of PrP Sc -V129 was associated with plaque formation but differences in PrP Sc allotype did not consistently correlate with disease incubation time or neuropathology. Thus, while the PrP Sc 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.

Prion protein (PrPC) is a protease-sensitive and soluble cell surface glycoprotein expressed in almost all mammalian cell types. PrPSc, a protease-resistant and insoluble form of PrPC, is the causative agent of prion diseases, fatal and transmissible neurogenerative diseases of mammals. Prion infection is initiated via either ingestion or inoculation of PrPSc or when host PrPC stochastically refolds into PrPSc. In either instance, the early events that occur during prion infection remain poorly understood. We have used transgenic mice expressing mouse PrPC tagged with a unique antibody epitope to monitor the response of host PrPC to prion inoculation. Following intracranial inoculation of either prion-infected or uninfected brain homogenate, we show that host PrPC can accumulate both intra-axonally and within the myelin membrane of axons suggesting that it may play a role in axonal loss following brain injury. Moreover, in response to the inoculation host PrPC exhibits an increased insolubility and protease resistance similar to that of PrPSc, even in the absence of infectious prions. Thus, our results raise the possibility that damage to the brain may be one trigger by which PrPC stochastically refolds into pathogenic PrPSc leading to productive prion infection., Competing Interests: The authors have declared that no competing interests exist.

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., (Published by Elsevier B.V.)

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., (2018 Published by Elsevier B.V.)

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., (Copyright © 2017 American Society for Microbiology.)

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.

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.

Cellular prion protein (PrP C ) is a mammalian glycoprotein which is usually found anchored to the plasma membrane via a glycophosphatidylinositol (GPI) anchor. PrP C misfolds to a pathogenic isoform PrP Sc , the causative agent of neurodegenerative prion diseases. The precise function of PrP C remains elusive but may depend upon its cellular localization. Here we show that PrP C is present in brain mitochondria from 6-12 week old wild-type and transgenic mice in the absence of disease. Mitochondrial PrP C 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 PrP C 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 PrP C can be found in mitochondria in the absence of disease, old age, mutation, or overexpression and that PrP C may affect mitochondrial function., Competing Interests: The authors declare no competing financial interests.

Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP C ) influences PrP C misfolding into the disease-associated isoform, PrP res , as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP C away from rafts. Previous studies showed that nonraft transmembrane PrP C variants resist conversion to PrP res when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP C and GPI-anchored PrP res in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP C might convert to PrP res if seeded by an exogenous source of PrP res not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP C To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP C supported persistent PrP res propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP C is required for persistent PrP res propagation, implicating raft microdomains as a location for conversion., Importance: Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP C ) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrP C or transmembrane-anchored PrP C , which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrP res We show that only GPI-anchored PrP C was able to convert to PrP res and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrP C to rafts are crucial to the ability of PrP C to propagate as a prion., (Copyright © 2017 American Society for Microbiology.)

During the course of prion infection, the normally soluble and protease-sensitive mammalian prion protein (PrP C ) is refolded into an insoluble, partially protease-resistant, and infectious form called PrP Sc . The conformational conversion of PrP C to PrP Sc 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 (PrP Sc ) is the primary protein component of infectious prions. Enriched PrP Sc preparations are often used to study the mechanisms that underly prion disease. However, most enrichment procedures are relatively nonspecific and tend to yield significant amounts of non-PrP Sc components including various proteins that could confound functional and structural studies. It is thus important to identify these 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 PrP Sc isolated from prion-infected mice, multiple cases of sporadic Creutzfeldt-Jakob disease (sCJD), and human growth hormone associated cases of iatrogenic CJD (iCJD). Creatine kinase was the primary protein contaminant in all PrP Sc samples, while many of the other proteins identified were also found in non-CJD controls, which suggests 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.

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrP(Sc)). The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrP(C)) into infectious PrP(Sc). By contrast, human growth hormone-associated cases of iatrogenic CJD (iCJD) in the United Kingdom (UK) are associated with exposure to an exogenous source of PrP(Sc). In both forms of CJD, heterozygosity at residue 129 for methionine (M) or valine (V) in the prion protein gene may affect disease phenotype, onset and progression. However, the relative contribution of each PrP(C) allotype to PrP(Sc) in heterozygous cases of CJD is unknown. Using mass spectrometry, we determined that the relative abundance of PrP(Sc) with M or V at residue 129 in brain specimens from MV cases of sCJD was highly variable. This result is consistent with PrP(C) containing an M or V at residue 129 having a similar propensity to misfold into PrP(Sc) thus causing sCJD. By contrast, PrP(Sc) with V at residue 129 predominated in the majority of the UK human growth hormone associated iCJD cases, consistent with exposure to infectious PrP(Sc) containing V at residue 129. In both types of CJD, the PrP(Sc) allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Therefore, factors other than PrP(Sc) allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD.

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.

Prion diseases are a heterogeneous class of fatal neurodegenerative disorders associated with misfolding of host cellular prion protein (PrP(C)) into a pathological isoform, termed PrP(Sc). Prion diseases affect various mammals, including humans, and effective treatments are not available. Prion diseases are distinguished from other protein misfolding disorders - such as Alzheimer's or Parkinson's disease - in that they are infectious. Prion diseases occur sporadically without any known exposure to infected material, and hereditary cases resulting from rare mutations in the prion protein have also been documented. The mechanistic underpinnings of prion and other neurodegenerative disorders remain poorly understood. Various proteomics techniques have been instrumental in early PrP(Sc) detection, biomarker discovery, elucidation of PrP(Sc) structure and mapping of biochemical pathways affected by pathogenesis. Moving forward, proteomics approaches will likely become more integrated into the clinical and research settings for the rapid diagnosis and characterization of prion pathogenesis.

Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrP(Sc)) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrP(Sc) is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrP(Sc)). Although evidence suggests that sPrP(Sc) may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte's ability to degrade PrP(Sc). Furthermore, the cell does not appear to distinguish between sPrP(Sc) and protease-resistant PrP(Sc), suggesting that sPrP(Sc) could contribute to prion infection., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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.

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.

During prion infection, the normal, protease-sensitive conformation of prion protein (PrP(C)) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP(Sc)). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP(Sc) in prion-infected brain homogenates as an initiating seed to convert PrP(C) and trigger the self-propagation of PrP(Sc) over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP(Sc). More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP(Sc) seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP(Sc). However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.

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.

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.

Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrP(Sc) ) varies between prion strains and these unique conformations may determine strain-specific disease phenotypes. However, the relative amounts of α-helix, β-sheet and other secondary structures have not always been consistent between studies, suggesting that other proteins might be confounding the analysis of PrP(Sc) secondary structure. We have used FTIR and LC-MS/MS to analyze enriched PrP(Sc) from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co-purify with PrP(Sc) . Our data show that non-PrP proteins do contribute to absorbances that have been associated with α-helical, loop, turn and β-sheet structures attributed to PrP(Sc) . The major contaminant, the α-helical protein ferritin, absorbs strongly at 1652 cm(-1) in the FTIR spectrum associated with PrP(Sc) . However, even the removal of more than 99% of the ferritin from PrP(Sc) did not completely abolish absorbance at 1652 cm(-1) . Our results show that contaminating proteins alter the FTIR spectrum attributed to PrP(Sc) and suggest that the α-helical, loop/turn and β-sheet secondary structure that remains following their removal are derived from PrP(Sc) itself., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 PrP(Sc), appears to be the primary component of the TSE infectious agent or prion. However, it is not clear to what extent other protein cofactors may be involved in TSE pathogenesis or whether there are PrP(Sc)-associated proteins which help to determine TSE strain-specific disease phenotypes. We enriched PrP(Sc) from the brains of mice infected with either 22L or Chandler TSE strains and examined the protein content of these samples using nanospray LC-MS/MS. These samples were compared with "mock" PrP(Sc) preparations from uninfected brains. PrP was the major component of the infected samples and ferritin was the most abundant impurity. Mock enrichments contained no detectable PrP 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 PrP(Sc) samples from 22L and Chandler TSE strains suggest that the non-PrP(Sc) protein components found in standard enrichment protocols are not strain specific.

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.

The hallmark of transmissible spongiform encephalopathies (TSEs or prion diseases) is the accumulation of an abnormally folded, partially protease-resistant form (PrP-res) of the normal protease-sensitive prion protein (PrP-sen). PrP-sen is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. In vitro, the anchor and the local membrane environment are important for the conversion of PrP-sen to PrP-res. In vivo, however, the anchor is not necessary because transgenic mice expressing anchorless PrP-sen accumulate PrP-res and replicate infectivity. To clarify the role of the GPI anchor in TSE infection, cells expressing GPI-anchored PrP-sen, anchorless PrP-sen, or both forms of PrP-sen were exposed to the mouse scrapie strain 22L. Cells expressing anchored PrP-sen produced PrP-res after exposure to 22L. Surprisingly, while cells expressing anchorless PrP-sen made anchorless PrP-res in the first 96 h postinfection, no PrP-res was detected at later passes. In contrast, when cells expressing both forms of PrP-sen were exposed to 22L, both anchored and anchorless PrP-res were detected over multiple passes. Consistent with the in vitro data, scrapie-infected cells expressing anchored PrP-sen transmitted disease to mice whereas cells expressing anchorless PrP-sen alone did not. These results demonstrate that the GPI anchor on PrP-sen is important for the persistent infection of cells in vitro. Our data suggest that cells expressing anchorless PrP-sen are not directly infected with scrapie. Thus, PrP-res formation in transgenic mice expressing anchorless PrP-sen may be occurring extracellularly.

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 (PrP). They are unique among protein misfolding disorders in that they are transmissible and have different strains of infectious agents 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 PrP within the context of what is known about the biology of prion disease.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease, bovine spongiform encephalopathy and sheep scrapie. Although one of the earliest events during TSE infection is the cellular uptake of protease resistant prion protein (PrP-res), this process is poorly understood due to the difficulty of clearly distinguishing input PrP-res from either PrP-res or protease-sensitive PrP (PrP-sen) made by the cell. Using PrP-res tagged with a unique antibody epitope, we examined PrP-res uptake in neuronal and fibroblast cells exposed to three different mouse scrapie strains. PrP-res uptake was rapid and independent of scrapie strain, cell type, or cellular PrP expression, but occurred in only a subset of cells and was influenced by PrP-res preparation and aggregate size. Our results suggest that PrP-res aggregate size, the PrP-res microenvironment, and/or host cell-specific factors can all influence whether or not a cell takes up PrP-res following exposure to TSE infectivity.

The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrP(C)) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrP(C) trafficking and tested whether this process controls PrP(C)-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrP(C), induced PrP(C) endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrP(C); however, heterologous expression of PrP(C) reconstituted both PKA and ERK1/2 activation. In contrast, a PrP(C) mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrP(C) endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.

Cyclic tetrapyrroles are among the most potent compounds with activity against transmissible spongiform encephalopathies (TSEs; or prion diseases). Here the effects of differential sulfonation and metal binding to cyclic tetrapyrroles were investigated. Their potencies in inhibiting disease-associated protease-resistant prion protein were compared in several types of TSE-infected cell cultures. In addition, prophylactic antiscrapie activities were determined in scrapie-infected mice. The activity of phthalocyanine was relatively insensitive to the number of peripheral sulfonate groups but varied with the type of metal bound at the center of the molecule. The tendency of the various phthalocyanine sulfonates to oligomerize (i.e., stack) correlated with anti-TSE activity. Notably, aluminum(III) phthalocyanine tetrasulfonate was both the poorest anti-TSE compound and the least prone to oligomerization in aqueous media. Similar comparisons of iron- and manganese-bound porphyrin sulfonates confirmed that stacking ability correlates with anti-TSE activity among cyclic tetrapyrroles.

Prion diseases are transmissible neurodegenerative disorders characterized by the accumulation in the CNS of the protease-resistant prion protein (PrPres), a structurally misfolded isoform of its physiological counterpart PrPsen. Both neuropathogenesis and prion infectivity are related to PrPres formation. Here, we report that the nonpsychoactive cannabis constituent cannabidiol (CBD) inhibited PrPres accumulation in both mouse and sheep scrapie-infected cells, whereas other structurally related cannabinoid analogs were either weak inhibitors or noninhibitory. Moreover, after intraperitoneal infection with murine scrapie, peripheral injection of CBD limited cerebral accumulation of PrPres and significantly increased the survival time of infected mice. Mechanistically, CBD did not appear to inhibit PrPres accumulation via direct interactions with PrP, destabilization of PrPres aggregates, or alteration of the expression level or subcellular localization of PrPsen. However, CBD did inhibit the neurotoxic effects of PrPres and affected PrPres-induced microglial cell migration in a concentration-dependent manner. Our results suggest that CBD may protect neurons against the multiple molecular and cellular factors involved in the different steps of the neurodegenerative process, which takes place during prion infection. When combined with its ability to target the brain and its lack of toxic side effects, CBD may represent a promising new anti-prion drug.

The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects >or=50 ag of hamster PrPSc (approximately 0.003 lethal dose) within 2-3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-microl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.

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.

The transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of rare, fatal, and transmissible neurodegenerative diseases of mammals for which there are no known viral or bacterial etiological agents. The bovine form of these diseases, bovine spongiform encephalopathy (BSE), has crossed over into humans to cause variant Creutzfeldt-Jakob disease. As a result, BSE and the TSE diseases are now considered a significant threat to human health. Understanding the basic mechanisms of TSE pathogenesis is essential for the development of effective TSE diagnostic tests and anti-TSE therapeutic regimens. This review provides an overview of the molecular mechanisms that underlie this enigmatic group of diseases.

A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD.

DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP(C)) 23-231 by systematic evolution of ligands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 10(24) distinct nucleic acid species. Sixty nanograms of rhuPrP(C)23-231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrP(C)23-231 at 10(-6) M to 10(-8) M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP(C)90-231. Theoretical thermodynamic modeling of selected aptamer sequences identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrP(C)23-231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrP(C) expressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrP(C) specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrP(C) and mammalian PrP(C) with varying affinities and can be applied to biological samples for PrP(C) enrichment and as diagnostic tools in double ligand assay systems.

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.

Transmissible spongiform encephalopathy (TSE) diseases are characterized by the accumulation in brain of an abnormal protease-resistant form of the host-encoded prion protein (PrP), PrP-res. PrP-res conformation differs among TSE agents derived from various sources, and these conformational differences are thought to influence the biological characteristics of these agents. In this study, we introduced deletions into the flexible N-terminal region of PrP (residues 34-124) and investigated the effect of this region on the conformation of PrP-res generated in an in vitro cell-free conversion assay. PrP deleted from residues 34 to 99 generated 12-16-kDa protease-resistant bands with intact C termini but variable N termini. The variable N termini were the result of exposure of new protease cleavage sites in PrP-res between residues 130 and 157, suggesting that these new cleavage sites were caused by alterations in the conformation of the PrP-res generated. Similarly truncated 12-16-kDa PrP bands were also identified in brain homogenates from mice infected with mouse-passaged hamster scrapie as well as in the cell-free conversion assay using conditions that mimicked the hamster/mouse species barrier to infection. Thus, by its effects on PrP-res conformation, the flexible N-terminal region of PrP seemed to influence TSE pathogenesis and cross-species TSE transmission.

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.