Table of Contents

Volume 6, Issue 1

  January/February/March 2012

  Pages 1 - 93

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PERSPECTIVE

Harnessing prions as test agents for the development of broad-range disinfectants

Katja Wagenführ and Michael Beekes
Pages 1 - 6
http://dx.doi.org/10.4161/pri.6.1.18556
Abstract | Full Text | PDF

The development of disinfectants with broad-range efficacy against bacteria, viruses, fungi, protozoa and prions constitutes an ongoing challenge. Prions, the causative agents of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease (CJD) or its variant (vCJD) rank among the pathogens with the highest resistance to disinfection. Pilot studies have shown that different procedures devised for prion disinfection were also highly effective against microbial pathogens. This fueled the idea to systematically exploit prions as test pathogens for the identification of new potential broad-range disinfectants. Prions essentially consist of misfolded, aggregated prion protein (PrP) and putatively replicate by nucleation-dependent, or seeded PrP polymerization. Recently, we have been able to establish PrP seeding activity as a quantitative in vitro indicator for the disinfection of 263K scrapie prions on steel wires used as surrogates for medical instruments. The seeding activity on wires re-processed in different disinfectants could be (1) biochemically determined by quantitative protein misfolding cyclic amplification (qPMCA), (2) biologically detected after qPMCA in a cell assay and (3) correctly translated into residual titers of scrapie infectivity. Our approach will substantially facilitate the identification of disinfectants with efficacy against prions as promising candidates for a further microbiological validation of broad-range activity.




MEETING REPORT

Global Alzheimer Research Summit: Basic and clinical research: Present and future Alzheimer research

Danielle Gonsalves, Katarina Jovanovic, Bianca Da Costa Dias and Stefan F.T. Weiss
Pages 7 - 10
http://dx.doi.org/10.4161/pri.6.1.18854
Abstract | Full Text | PDF

We report here on the proceedings of the Global Alzheimer Summit that took place September 22–23, 2011 in Madrid, Spain. As Alzheimer disease (AD) is the leading cause of neurodegeneration in elderly individuals and as yet has no effective therapeutic option, it continues to stimulate global research interests. At the conference, leaders in the field of AD research provided insights into current developments in various areas of research, namely molecular mechanisms, genetics, novel aspects of AD research and translational research. Emphasis was also placed on the importance of biomarkers in the diagnosis of AD and development of current therapeutic strategies.




COMMENTARIES & VIEWS

Open Access Article

Lichens: Unexpected anti-prion agents?

Cynthia M. Rodriguez, James P. Bennett and Christopher J. Johnson
Pages 11 - 16
http://dx.doi.org/10.4161/pri.6.1.17414
Abstract | PDF

The prion diseases sheep scrapie and cervid chronic wasting disease are transmitted, in part, via an environmental reservoir of infectivity; prions released from infected animals persist in the environment and can cause disease years later. Central to controlling disease transmission is the identification of methods capable of inactivating these agents on the landscape. We have found that certain lichens, common, ubiquitous, symbiotic organisms, possess a serine protease capable of degrading prion protein (PrP) from prion-infected animals. The protease functions against a range of prion strains from various hosts and reduces levels of abnormal PrP by at least two logs. We have now tested more than 20 lichen species from several geographical locations and from various taxa and found that approximately half of these species degrade PrP. Critical next steps include examining the effect of lichens on prion infectivity and cloning the protease responsible for PrP degradation. The impact of lichens on prions in the environment remains unknown. We speculate that lichens could have the potential to degrade prions when they are shed from infected animals onto lichens or into environments where lichens are abundant. In addition, lichens are frequently consumed by cervids and many other animals and the effect of dietary lichens on prion disease transmission should also be considered.

Chronic wasting disease: Fingerprinting the culprit in risk assessments

Martin L. Daus and Michael Beekes
Pages 17 - 22
http://dx.doi.org/10.4161/pri.6.1.17776
Abstract | Full Text | PDF

Transmissible spongiform encephalopathies (prion diseases) in animals may be associated with a zoonotic risk potential for humans as shown by the occurrence of variant Creutzfeldt-Jakob disease in the wake of the bovine spongiform encephalopathy epidemic. Thus, the increasing exposure of humans in North America to cervid prions of chronic wasting disease (CWD) in elk and deer has prompted comprehensive risk assessments. The susceptibility of humans to CWD infections is currently under investigation in different studies using macaques as primate models. The necessity for such studies was recently reinforced when disease-associated prion protein and its seeding activity were detected in muscles of clinically inconspicuous CWD-infected white-tailed deer (WTD). Increasing evidence points to the existence of different CWD strains, and CWD prions may also change or newly emerge over time. Therefore, CWD isolates examined in macaques should be characterized as precisely as possible for their molecular identity. On this basis other CWD field samples collected in the past, present or future could be systematically compared with macaque-tested inocula in order to assess whether they are covered by the ongoing risk assessments in primates. CWD typing by Fourier transform-infrared spectroscopy of pathological prion protein may provide a method of choice for this purpose.

Two-steps control of cellular prion physiology by the Extracellular Regulated Kinase-1 (ERK1)

Frédéric Checler
Pages 23 - 25
http://dx.doi.org/10.4161/pri.6.1.18004
Abstract | Full Text | PDF

Cellular prion (PrP^c) undergoes a regulated α-secretase-like cleavage by the disintegrin ADAM17 similar to the one taking place on β-Amyloid Precursor Protein (βAPP). Because these cleavages give rise to biologically active fragments, understanding their regulation could be of importance. We have established that the Extracellular Regulated Kinase-1 (ERK1) controls PrP^c processing by modulating ADAM17 phosphorylation in a protein kinase C-dependent manner. Strikingly, we also demonstrated that ERK1 acts upstream to increase PrP^c promoter transactivation in an AP-1 dependent manner. Therefore, ERK1 exerts a dual control of both PrP^c metabolism and expression. Interestingly, α-secretase cleavage of βAPP appears to be independent of ERK1. I describe here similarities and differences in α-secretase-mediated PrP^c and βAPP processing pathways and discuss putative physiopathological implications.

Insights into the disparate action of osmolytes and macromolecular crowders on amyloid formation

Shahar Sukenik and Daniel Harries
Pages 26 - 31
http://dx.doi.org/10.4161/pri.6.1.18132
Abstract | Full Text | PDF

It is widely recognized that amyloid formation sensitively responds to conditions set by myriad cellular solutes. These cosolutes include two important classes: macromolecular crowders and compatible osmolytes. We have recently found that addition of macromolecular PEG only slightly affects fibril formation of a model peptide in vitro. Polyol osmolytes, in contrast, lengthen the lag time for aggregation, and lead to larger fibril mass at equilibrium. To further hypothesize on the molecular underpinnings of the disparate effect of the two cosolute classes, we have further analyzed the experiments using an available kinetic mechanism describing fibril aggregation. Model calculations suggest that all cosolutes similarly lengthen the time required for nucleation, possibly due to their excluded volume effect. However, PEGs may in addition promote fibril fragmentation, leading to lag times that are overall almost unvaried. Moreover, polyols effectively slow the monomer-fibril detachment rates, thereby favoring additional fibril formation. Our analysis provides first hints that cosolutes act not only by changing association or dissociation rates, but potentially also by directing the formation of fibrils of varied morphologies with different mechanical properties. Although additional experiments are needed to unambiguously resolve the action of excluded cosolutes on amyloid formation, it is becoming clear that these compounds are important to consider in the search for ways to modulate fibril formation.

Misfolded PrP and a novel mechanism of proteasome inhibition

Ralph Andre and Sarah J. Tabrizi
Pages 32 - 36
http://dx.doi.org/10.4161/pri.6.1.18272
Abstract | Full Text | PDF

Prion diseases comprise a family of fatal neurodegenerative disorders caused by the conformational re-arrangement of a normal host-encoded protein, PrP^C, to an abnormal infectious isoform termed PrP^Sc. Currently, the precise cellular mechanism(s) underlying prion disease pathogenesis remain unclear. Evidence suggests a role for the ubiquitin proteasome system (UPS), a protein degradation pathway that is critical for maintaining cellular proteostasis. Dysfunction of the UPS has been implicated in various neurodegenerative diseases. However, the mechanisms of this impairment remain unknown in many cases, and evidence that disease-associated misfolded proteins are able to directly inhibit the function of the proteasome has been lacking. Recently, we have shown data describing a mechanism of proteasome impairment by the direct interaction of β-sheet-rich PrP to reduce gate opening and inhibit substrate entry. This novel mechanism may provide a model for how other misfolded, disease-associated proteins might interact with the proteasome to disrupt its function. Targeting the UPS to restore proteostasis in neurodegenerative disorders in which misfolded proteins accumulate offers a possible target for therapeutic intervention.

Open Access Article

Early structural features in mammalian prion conformation conversion

Giuseppe Legname
Pages 37 - 39
http://dx.doi.org/10.4161/pri.6.1.18425
Abstract | Full Text | PDF

The conversion to a disease-associated conformer (PrP^Sc) of the cellular prion protein (PrP^C) is the central event in prion diseases. Wild-type PrP^C converts to PrP^Sc in the sporadic forms of the disorders through an unknown mechanism. These forms account for up to 85% of all human (Hu) occurrences; the infectious types contribute for less than 1%, while genetic incidence of the disease is about 15%. Familial Hu prion diseases are associated with about 40 point mutations of the gene coding for the PrP denominated PRNP. Most of the variants associated with these mutations are located in the globular domain of the protein. In a recent work in collaboration with the German Research School for Simulation Science, in Jülich, Germany, we performed molecular dynamics simulations for each of these mutants to investigate their structure in aqueous solution. Structural analysis of the various point mutations present in the globular domain unveiled common folding traits that may allow to a better understanding of the early conformational changes leading to the formation of monomeric PrP^Sc. Recent experimental data support these findings, thus opening novel approaches to determine initial structural determinants of prion formation.

Ion channels induced by the prion protein: Mediators of neurotoxicity

Isaac H. Solomon, Emiliano Biasini and David A. Harris
Pages 40 - 45
http://dx.doi.org/10.4161/pri.6.1.18627
Abstract | Full Text | PDF

Prion diseases comprise a group of rapidly progressive and invariably fatal neurodegenerative disorders for which there are no effective treatments. While conversion of the cellular prion protein (PrP^C) to a β-sheet rich isoform (PrP^Sc) is known to be a critical event in propagation of infectious prions, the identity of the neurotoxic form of PrP and its mechanism of action remain unclear. Insights into this mechanism have been provided by studying PrP molecules harboring deletions and point mutations in the conserved central region, encompassing residues 105–125. When expressed in transgenic mice, PrP deleted for these residues (Δ105–125) causes a spontaneous neurodegenerative illness that is reversed by co-expression of wild-type PrP. In cultured cells, Δ105–125 PrP confers hypersensitivity to certain cationic antibiotics and induces spontaneous ion channel activity that can be recorded by electrophysiological techniques. We have utilized these drug-hypersensitization and current-inducing activities to identify which PrP domains and subcellular locations are required for toxicity. We present an ion channel model for the toxicity of Δ105–125 PrP and related mutants and speculate how a similar mechanism could mediate PrP^Sc-associated toxicity. Therapeutic regimens designed to inhibit prion-induced toxicity, as well as formation of PrP^Sc, may prove to be the most clinically beneficial.




RESEARCH PAPERS

Comparative peptidome analyses of the profiles of the peptides ranging 
from 1­–10 KD in CSF samples pooled from probable sporadic CJD 
and non-CJD patients

Cao Chen, Di Xiao, Wei Zhou, Yong-Chan Zhang, Qi Shi, Chan Tian, Jin Zhang, Chun-Xi Zhou, Jian-Zhong Zhang and Xiao-Ping Dong
Pages 46 - 51
http://dx.doi.org/10.4161/pri.6.1.18082
Abstract | Full Text | PDF

The shotgun strategy applying tandem mass spectrometry has been widely used to identify the proteins that are differentially distributed among diseases for its high reliability and efficiency. To find out the potential difference of protein profiles in cerebrospinal fluids (CSF) between Creutzfeldt-Jakob disease (CJD) and non-CJD patients, especially in the fraction ranging from 1–10 KD, the CSF samples of 40 probable sporadic CJD (sCJD) patients, 32 non-CJD cases with dementia and 17 non-CJD cases without dementia were separately pooled and enriched by the magnetic beads based weak cation exchange chromatography (MB-WCX). After trypsin digestion, each enriched CSF was separated and identified by RP-HPLC-ESI-QTOF MS/MS. In total, 42, 53 and 47 signals of proteins were identified in the pooled CSF fraction less than 10 KD of probable sCJD, non-CJD with dementia and non-CJD without dementia, respectively. Compared with that of probable sCJD, the similarity of CSF protein profiles of non-CJD with dementia (76.2%) were higher than that of non-CJD without dementia (57.1%). Nine CSF proteins were found to be specially observed in probable sCJD group. Those data may help to select the potential biomarkers for diagnosis of CJD. Additionally, further studies of the small segments of cellular proteins in CSF of CJD patients may also provide scientific clues for understanding the neuropathogenesis of TSEs.

Salivary prions in sheep and deer

Gültekin Tamgüney, Jürgen A. Richt, Amir N. Hamir, Justin J. Greenlee, Michael W. Miller, Lisa L. Wolfe, Tracey M. Sirochman, Alan J. Young, David V. Glidden, Natrina L. Johnson, Kurt Giles, Stephen J. DeArmond and Stanley B. Prusiner
Pages 52 - 61
http://dx.doi.org/10.4161/pri.6.1.16984
Abstract | Full Text | PDF

Scrapie of sheep and chronic wasting disease (CWD) of cervids are transmissible prion diseases. Milk and placenta have been identified as sources of scrapie prions but do not explain horizontal transmission. In contrast, CWD prions have been reported in saliva, urine and feces, which are thought to be responsible for horizontal transmission. While the titers of CWD prions have been measured in feces, levels in saliva or urine are unknown. Because sheep produce ~17 L/day of saliva, and scrapie prions are present in tongue and salivary glands of infected sheep, we asked if scrapie prions are shed in saliva. We inoculated transgenic (Tg) mice expressing ovine prion protein, Tg(OvPrP) mice, with saliva from seven Cheviot sheep with scrapie. Six of seven samples transmitted prions to Tg(OvPrP) mice with titers of -0.5 to 1.7 log ID₅₀ U/ml. Similarly, inoculation of saliva samples from two mule deer with CWD transmitted prions to Tg(ElkPrP) mice with titers of -1.1 to -0.4 log ID₅₀ U/ml. Assuming similar shedding kinetics for salivary prions as those for fecal prions of deer, we estimated the secreted salivary prion dose over a 10-mo period to be as high as 8.4 log ID₅₀ units for sheep and 7.0 log ID₅₀ units for deer. These estimates are similar to 7.9 log ID₅₀ units of fecal CWD prions for deer. Because saliva is mostly swallowed, salivary prions may reinfect tissues of the gastrointestinal tract and contribute to fecal prion shedding. Salivary prions shed into the environment provide an additional mechanism for horizontal prion transmission.

Therapeutic effect of CHF5074, a new γ-secretase modulator, in a mouse model of scrapie

Giorgio Poli, Erica Corda, Barbara Lucchini, Maria Puricelli, Piera Anna Martino, Paola Dall’Ara, Gino Villetti, Silvio R. Bareggi, Cristiano Corona, Elena Vallino Costassa, Paola Gazzuola, Barbara Iulini, Maria Mazza, Pierluigi Acutis, Paolo Mantegazza, Cristina Casalone and Bruno P. Imbimbo
Pages 62 - 72
http://dx.doi.org/10.4161/pri.6.1.18317
Abstract | Full Text | PDF

In transmissible spongiform encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrP^c is a mediator of a synaptic dysfunction induced by Aβ oligomers. We tested a novel γ-secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 d longer than vehicle-treated mice (p < 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrP^Sc in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared with the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrP^Sc toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.

PRND 3′UTR polymorphism may be associated with behavioral disturbances 
in Alzheimer disease

Marcin Flirski, Monika Sieruta, Ewa Golańska, Iwona Kłoszewska, Paweł P. Liberski and Tomasz Sobów
Pages 73 - 80
http://dx.doi.org/10.4161/pri.6.1.18428
Abstract | Full Text | PDF

The etiology of behavioral and psychological symptoms of dementia (BPSD) is complex, including putative biological, psychological, social and environmental factors. Recent years have witnessed accumulation of data on the association between genetic factors and behavioral abnormalities in Alzheimer disease (AD). In this research paper, our aim is to evaluate the association between the APOE, CYP46, PRNP and PRND genes and the profile of neuropsychiatric symptoms in Polish subjects with AD and mild cognitive impairment (MCI). We studied 99 patients with AD and 48 subjects with MCI. The presence and profile of BPSD were evaluated at baseline and prospectively with the Neuropsychiatric Inventory (NPI). Patients were dichotomized into those having ever experienced a particular symptom and those who did not over the whole disease period. Genotyping was performed using previously described standard protocols. The prevalence of comorbid behavioral symptoms and the overall level of behavioral burden were significantly greater in AD compared with the MCI group. In AD patients, carrier status of the T allele of the 3′UTR (untranslated region) PRND polymorphism was associated with an increased cumulative behavioral load and an elevated risk for delusions, anxiety, agitation/aggression, apathy and irritability/emotional ability. Among MCI subjects, APOE ε4 carriers demonstrated a reduced risk for nighttime behavior change. No other statistically significant genotype-phenotype correlations were observed, including the APOE, CYP46 and PRNP genes. A precise estimation of the exact significance of particular polymorphisms in BPSD etiology requires future studies on large populations.

Y145Stop is sufficient to induce de novo generation prions using protein misfolding cyclic amplification

Ahmed Abdallah, Ping Wang, Juergen A. Richt and Srinand Sreevatsan
Pages 81 - 88
http://dx.doi.org/10.4161/pri.6.1.18493
Abstract | Full Text | PDF

A point mutation in Prnp that converts tyrosine (Y) at position 145 into a stop codon leading to a truncated prion molecule as found in an inherited transmissible spongiform encephalopathy (TSE), Gertsmann-Sträussler-Scheincker syndrome, suggests that the N-terminus of the molecule (spanning amino acids 23–144) likely plays a critical role in prion misfolding as well as in protein-protein interactions. We hypothesized that Y145Stop molecule represents an unstable part of the prion protein that is prone to spontaneous misfolding. Utilizing protein misfolding cyclic amplification (PMCA) we show that the recombinant polypeptide corresponding to the Y145Stop of sheep and deer PRNP can be in vitro converted to PK-resistant PrP^Sc in presence or absence of preexisting prions. In contrast, recombinant protein full-length PrP^C did not show a propensity for spontaneous conformational conversion to protease resistant isoforms. Further, we show that seeded or spontaneously misfolded Y145Stop molecules can efficiently convert purified mammalian PrP^C into protease resistant isoforms. These results establish that the N-terminus of PrP^C molecule corresponding to residues 23–144 plays a role in seeding and misfolding of mammalian prions.




SHORT COMMUNICATION

Comparative analysis of Japanese and foreign L-type BSE prions

Kentaro Masujin, Ritsuko Miwa, Hiroyuki Okada, Shirou Mohri and Takashi Yokoyama
Pages 89 - 93
http://dx.doi.org/10.4161/pri.6.1.18429
Abstract | Full Text | PDF

L-type bovine spongiform encephalopathy (BSE) is an atypical form of BSE. To characterize the Japanese L-type BSE prion, we conducted a comparative study of the Japanese and foreign L-type BSE isolates. The L-type BSE isolates of Japan, Germany, France and Canada were intracerebrally inoculated into bovinized prion protein-overexpressing transgenic mice (TgBoPrP). All the examined L-type BSE isolates were transmitted to TgBoPrP mice, and no clear differences were observed in their biological and biochemical properties. Here, we present evidence that the Japanese and Canadian L-type BSE prions are identical to those from the European cases.