Accelerated high fidelity prion amplification within and across prion species barriers

Experimental obstacles have impeded our ability to study prion transmission within and, more particularly, between species. Here, we used cervid prion protein expressed in brain extracts of transgenic mice, referred to as Tg(CerPrP), as a substrate for in vitro generation of chronic wasting disease (CWD) prions by protein misfolding cyclic amplification (PMCA). Characterization of this infectivity in Tg(CerPrP) mice demonstrated that serial PMCA resulted in the high fidelity amplification of CWD prions with apparently unaltered properties. Using similar methods to amplify mouse RML prions and characterize the resulting novel cervid prions, we show that serial PMCA abrogated a transmission barrier that required several hundred days of adaptation and subsequent stabilization in Tg(CerPrP) mice. While both approaches produced cervid prions with characteristics distinct from CWD, the subtly different properties of the resulting individual prion isolates indicated that adaptation of mouse RML prions generated multiple strains following inter-species transmission. Our studies demonstrate that combined transgenic mouse and PMCA approaches not only expedite intra- and inter-species prion transmission, but also provide a facile means of generating and characterizing novel prion strains.     

In vitro methods in the study of viral and prion permeability across the blood-brain barrier

(1) Infectious agents capable of entering the central nervous system (CNS) produce some of the most dreaded diseases known to man. The infectious agent within the CNS is often protected by the blood-brain barrier (BBB), shielded from endogenous and exogenous anti-infectious agents. (2) The use of in vitro methods offers many advantages to the study of how infectious agents interact with the BBB. Two such agents which negotiate the BBB early in the course of disease before damage to the BBB are the autoimmune deficiency syndrome virus, or human immunodeficiency virus 1, and scrapie prion. Our laboratories have used in vitro methods to study these agents. (3) Here, we review some of the results form our laboratories and those of others.     

Passage of murine scrapie prion protein across the mouse vascular blood-brain barrier

Prions are the infectious agents associated with transmissible spongiform encephalopathies and are composed mainly of a misfolded form of the endogenous prion protein. Prion protein must enter the brain to produce disease. Previous work has emphasized various mechanisms which partially bypass the blood-brain barrier (BBB). Here, we used the brain perfusion method to directly assess the ability of mouse scrapie protein (PrP(SC)) to cross the mouse BBB independent of the influences of neural pathways or circulating immune cells. We found that PrP(SC) oligomers rapidly crossed the BBB without disrupting it with a unidirectional influx rate of about 4.4microl/g-min. HPLC and capillary depletion confirmed that PrP(SC) crossed the entire width of the capillary wall to enter brain parenchyma. PrP(SC) also entered the cerebrospinal fluid (CSF) compartment. These results show that a prion protein can cross the intact BBB to enter both the parenchymal and CSF compartments of the brain.     

Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type

The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrPSc) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two-dimensional immunoblot profiles of detergent insoluble and proteinase K-resistant PrP species in a type of sporadic Creutzfeldt-Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full-length and truncated PrP forms present in the insoluble fractions were also separately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrPSc, but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent-insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two-dimensional blots of the proteinase K-resistant PrP fraction suggesting that different PrP species are selected for conversion to proteinase K-resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N-linked glycans. We also show that the over-representation of the highly glycosylated forms distinctive of the proteinase K-resistant PrPSc of vCJD in one-dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases.     

Common structural traits across pathogenic mutants of the human prion protein and their implications for familial prion diseases

Human (Hu) familial prion diseases are associated with about 40 point mutations of the gene coding for the prion protein (PrP). Most of the variants associated with these mutations are located in the globular domain of the protein. We performed 50 ns of molecular dynamics for each of these mutants to investigate their structure in aqueous solution. Overall, 1.6 μs of molecular dynamics data is presented. The calculations are based on the AMBER(parm99) force field, which has been shown to reproduce very accurately the structural features of the HuPrP wild type and a few variants for which experimental structural information is available. The variants present structural determinants different from those of wild-type HuPrP and the protective mutation HuPrP(E219K-129M). These include the loss of salt bridges in α₂-α₃ regions and the loss of π-stacking interactions in the β₂-α₂ loop. In addition, in the majority of the mutants, the α₃ helix is more flexible and Y169 is more solvent exposed. The presence of similar traits in this large spectrum of mutations hints to a role of these fingerprints in their known disease-causing properties. Overall, the regions most affected by disease-linked mutations in terms of structure and/or flexibility are those involved in the pathogenic conversion to the scrapie form of the protein and in the interaction with cellular partners. These regions thus emerge as optimal targets for antibody- and ligand-binding studies.     

Alteration of the chronic wasting disease species barrier by in vitro prion amplification

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids now detected in 19 states of the United States, three Canadian provinces, and South Korea. Whether noncervid species can be infected by CWD and thereby serve as reservoirs for the infection is not known. To investigate this issue, we previously used serial protein misfolding cyclic amplification (sPMCA) to demonstrate that CWD prions can amplify in brain homogenates from several species sympatric with cervids, including prairie voles (Microtus ochrogaster) and field mice (Peromyscus spp.). Here, we show that prairie voles are susceptible to mule deer CWD prions in vivo and that sPMCA amplification of CWD prions in vole brain enhances the infectivity of CWD for this species. Prairie voles inoculated with sPMCA products developed clinical signs of TSE disease approximately 300 days prior to, and more consistently than, those inoculated with CWD prions from deer brain. Moreover, the deposition patterns and biochemical properties of protease-resistant form of PrP (PrP(RES)) in the brains of affected voles differed from those in cervidized transgenic (CerPrP) mice infected with CWD. In addition, voles inoculated orally with sPMCA products developed clinical signs of TSE and were positive for PrP(RES) deposition, whereas those inoculated orally with deer-origin CWD prions did not. These results demonstrate that transspecies sPMCA of CWD prions can enhance the infectivity and adapt the host range of CWD prions and thereby may be useful to assess determinants of prion species barriers.     

Presymptomatic detection or exclusion of prion protein gene defects in families with inherited prion diseases.

The identification of defects in the prion protein (PrP) gene in families with inherited Creutzfeldt-Jakob disease or Gerstmann-Straussler syndrome allows presymptomatic diagnosis or exclusion of these disorders in subjects at risk. After counseling, PrP gene analysis was performed in three such individuals: two from families with a 144-bp insert and one with a point mutation at codon 102 in the PrP gene. The presence of a PrP gene defect was confirmed in one and excluded in two. Despite the potential problems of using PrP gene analysis in genetic prediction - specifically, uncertainty about penetrance and, generally, problems of presymptomatic testing in any inherited late-onset neurodegenerative disorder - we conclude that it has a role to play in improved genetic counseling for families with inherited prion diseases.     

Therapeutics in Alzheimer's and prion diseases

There is increasing recognition that numerous neurodegenerative conditions have the same underlying pathogenetic mechanism, namely a change in protein conformation, where the beta-sheet content is increased. In Alzheimer's disease, amyloid deposition in the form of neuritic plaques and congophilic angiopathy is driven by the conversion of normal soluble amyloid-beta peptide (sA beta) to A beta plaques; while in the prionoses the critical event is the conversion of normal prion protein, PrP(C), to the disease-associated form, PrP(Sc). This common theme in the pathogenesis of these disorders and the extracellular localization of the accumulating abnormal protein make them highly amenable to therapeutic approaches based on experimental manipulation of protein conformation and clearance. A number of different approaches under current development include drugs which affect the processing of the precursor proteins drugs the clearance of the amyloidogenic protein, and which inhibit or prevent the conformation change and immunological approaches. Particularly interesting are compounds termed 'beta-sheet breakers' that directly target the abnormal conformational change both for A beta- and PrP(Sc)-related deposits. In addition, immune system activation can serve as beta-sheet breakers and/or to increase the clearance of the disease-associated proteins. These conformation-based approaches appear to hold the best promise for therapies for this devastating group of disorders.     

Bistability explains threshold phenomena in protein aggregation both in vitro and in vivo

Neurodegenerative disease can originate from the misfolding and aggregation of proteins, such as Amyloid-beta, SOD1, or Huntingtin. Fortunately, all cells possess protein quality control machinery that sequesters misfolded proteins, either refolding or degrading them, before they can self-associate into proteotoxic oligomers and aggregates. This activity is largely performed by the stress response chaperones (i.e., Hsp70). However, the expression level of molecular chaperones varies widely among cell types. To understand the potential consequence of this variation, we studied the process of protein aggregation in the presence of molecular chaperones using mathematical modeling. We demonstrate that protein aggregation, in the presence of molecular chaperones, is a bistable process. Bistability in protein aggregation offers an explanation for threshold transitions to high aggregate concentration, which are observed both in vitro and in vivo. Additionally, we show that slight variations in chaperone concentration, due to natural fluctuations, have important consequences in a bistable system for the onset of protein aggregation. Therefore, our results offer a possible theoretical explanation for neuronal vulnerability observed in vivo and the onset of neurodegenerative phenotypes in neurons lacking an effective heat-shock response.     

Prions and prion diseases: fundamentals and mechanistic details

Prion diseases, often called transmissible spongiform encephalopathies (TSEs), are infectious diseases that accompany neurological dysfunctions in many mammalian hosts. Prion diseases include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE, "mad cow disease") in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elks. The cause of these fatal diseases is a proteinaceous pathogen termed prion that lacks functional nucleic acids. As demonstrated in the BSE outbreak and its transmission to humans, the onset of disease is not limited to a certain species but can be transmissible from one host species to another. Such a striking nature ofprions has generated huge concerns in public health and attracted serious attention in the scientific communities. To date, the potential transmission ofprions to humans via foodbome infectiorn and iatrogenic routes has not been alleviated. Rather, the possible transmission of human to human or cervids to human aggravates the terrifying situation across the globe. In this review, basic features about prion diseases including clinical and pathological characteristics, etiology, and transmission of diseases are described. Based on recently accumulated evidences, the molecular and biochemical aspects of prions, with an emphasis on the molecular interactions involved in prion conversion that is critical during prion replication and pathogenesis, are also addressed.     

Wide but not impermeable: Testing the riverine barrier hypothesis for an Amazonian plant species

Wallace's riverine barrier hypothesis postulates that large rivers, such as the Amazon and its tributaries, reduce or prevent gene flow between populations on opposite banks, leading to allopatry and areas of species endemism occupying interfluvial regions. Several studies have shown that two major tributaries, Rio Branco and Rio Negro, are important barriers to gene flow for birds, amphibians and primates. No botanical studies have considered the potential role of the Rio Branco as a barrier, while a single botanical study has evaluated the Rio Negro as a barrier. We studied an Amazon shrub, Amphirrhox longifolia (A. St.‐Hil.) Spreng (Violaceae), as a model to test the riverine barrier hypothesis. Twenty‐six populations of A. longifolia were sampled on both banks of the Rio Branco and Rio Negro in the core Amazon Basin. Double‐digest RADseq was used to identify 8,010 unlinked SNP markers from the nuclear genome of 156 individuals. Data relating to population structure support the hypothesis that the Rio Negro acted as a significant genetic barrier for A. longifolia. On the other hand, no genetic differentiation was detected among populations spanning the narrower Rio Branco, which is a tributary of the Rio Negro. This study shows that the strength of riverine barriers for Amazon plants is dependent on the width of the river separating populations and species‐specific dispersal traits. Future studies of plants with contrasting life history traits will further improve our understanding of the landscape genetics and allopatric speciation history of Amazon plant diversity.     

Diagnosing prion diseases: needs, challenges and hopes

Prion diseases are among the most intriguing infectious diseases and are associated with unconventional proteinaceous infectious agents known as prions. Prions seem to lack nucleic acid and propagate by transmission of protein misfolding. The nature of prions and their unique mode of transmission present challenges for early diagnosis of prion diseases. In this article, state-of-the-art prion diagnostic techniques, together with the new strategies that are being used to develop sensitive, early and non-invasive diagnoses for these diseases are reviewed.     

THERPA: A small molecule database related to prion protein regulation and prion diseases progression

Prion diseases are fatal neurodegenerative disorders that affect humans and animals. Although various small molecules have been evaluated for application in the treatment of prion diseases, none have been shown to be efficacious. Expanding our knowledge of these molecules is important for understanding of the complex mechanisms of prion diseases. To improve access to the scattered information on small molecules related to prion diseases, we built a database of therapeutic molecules associated with prion diseases (THERPA, therpa.pythonanywhere.com). THERPA includes 119 small molecules and their 283 relationships with prion diseases. THERPA is an interactive visual database and useful for improving search efficiency which can help researchers identify intrinsic small molecules that can be used for developing therapeutics for prion diseases.     

Peptidylarginine deiminase and protein citrullination in prion diseases

The post-translational citrullination (deimination) process is mediated by peptidylarginine deiminases (PADs), which convert peptidylarginine into peptidylcitrulline in the presence of high calcium concentrations. Over the past decade, PADs and protein citrullination have been commonly implicated as abnormal pathological features in neurodegeneration and inflammatory responses associated with diseases such as multiple sclerosis, Alzheimer disease and rheumatoid arthritis. Based on this evidence, we investigated the roles of PADs and citrullination in the pathogenesis of prion diseases. Prion diseases (also known as transmissible spongiform encephalopathies) are fatal neurodegenerative diseases that are pathologically well characterized as the accumulation of disease-associated misfolded prion proteins, spongiform changes, glial cell activation and neuronal loss. We previously demonstrated that the upregulation of PAD2, mainly found in reactive astrocytes of infected brains, leads to excessive citrullination, which is correlated with disease progression. Further, we demonstrated that various cytoskeletal and energy metabolism-associated proteins are particularly vulnerable to citrullination. Our recent in vivo and in vitro studies elicited altered functions of enolase as the result of citrullination; these altered functions included reduced enzyme activity, increased protease sensitivity and enhanced plasminogen-binding affinity. These findings suggest that PAD2 and citrullinated proteins may play a key role in the brain pathology of prion diseases. By extension, we believe that abnormal increases in protein citrullination may be strong evidence of neurodegeneration.     

Breaking the species barrier: use of SCID mouse-human chimeras for the study of human infectious diseases

Mouse-human chimeras have become a novel way to model the interactions between microbial pathogens and human cells, tissues or organs. Diseases studied with human xenografts in severe combined immunodeficient (SCID) mice include Pseudomonas aeruginosa infection in cystic fibrosis, group A streptococci and impetigo, bacillary and amoebic dysentery, and AIDS. In many cases, disease in the human xenograft appears to accurately reproduce the disease in humans, providing a powerful model for identifying virulence factors, host responses to infection and the effects of specific interventions on disease. In this review, we summarize recent studies that have used mouse-human chimeras to understand the pathophysiology of specific bacterial and protozoan infections.     

Dominant-negative effects in prion diseases: insights from molecular dynamics simulations on mouse prion protein chimeras

Mutations in the prion protein (PrP) can cause spontaneous prion diseases in humans (Hu) and animals. In transgenic mice, mutations can determine the susceptibility to the infection of different prion strains. Some of these mutations also show a dominant-negative effect, thus halting the replication process by which wild type mouse (Mo) PrP is converted into Mo scrapie. Using all-atom molecular dynamics (MD) simulations, here we studied the structure of HuPrP, MoPrP, 10?Hu/MoPrP chimeras, and 1 Mo/sheepPrP chimera in explicit solvent. Overall, ～2?μs of MD were collected. Our findings suggest that the interactions between α1 helix and N-terminal of α3 helix are critical in prion propagation, whereas the β2–α2 loop conformation plays a role in the dominant-negative effect.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:4.     

Pathogenesis of prion diseases: current status and future outlook

The prion, a conformational variant of a host protein, is the infectious particle responsible for transmissible spongiform encephalopathy (TSE), a fatal neurodegenerative disease of humans and animals. The principal target of prion pathology is the brain, yet most TSEs also display prion replication at extra-cerebral locations, including secondary lymphoid organs and sites of chronic inflammation. Despite significant progress in our understanding of this infectious agent, many fundamental questions relating to the nature of the prion, including the mechanism of replication and the molecular events underlying brain damage, remain unanswered. Here we focus on the unresolved issues pertaining to prion pathogenesis, particularly on the role played by the immune system.