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Stanley B. Prusiner

Regarding a recommendation for better screening assays for BSE, Dr. Chesebro noted the stiff competition among several companies to have their assays used for this purpose. Finally, the recommendations to improve the research infrastructure are particularly important, according to Dr. Among the difficulties of the research are the facts that prions are infectious and the incubation periods lengthy. Therefore, TSE laboratories must not only have biological safety level 2 or 3 isolation facilities, but also they must wait years to get results, so the financial cost is high.

I have great respect for people who work with these agents. Furthermore, the experiments take a long time. When you transmit the disease to animals, sometimes they don't get sick very fast. And you may have to wait for the results of some experiments before you can do others. NIH needs to understand that because most of the grants are cut to 4 years. The report did a great job of pointing this out. But the government needs to pay attention. The first, or interim, report was published in January , before research funds were distributed.

The second, or final, report was not formally published until March. The Institute of Medicine IOM report, Advancing Prion Science , highlighted these nine recommendations as priorities for improving prion disease detection and surveillance. Colleague's E-mail is Invalid. Your message has been successfully sent to your colleague.

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For more information, please refer to our Privacy Policy. Subscribe to eTOC. Maybe we should have more than one CJD surveillance center funded by the government. Johnson, who chaired the member committee that prepared the Institute of Medicine report, said it is still not clear what prions are made of. Your Name: optional. Your Email:. Colleague's Email:. All previously known pathogens, such as bacteria and viruses, contain nucleic acids, which enable them to reproduce.


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The prion hypothesis explained why the mysterious infectious agent is resistant to ultraviolet radiation, which breaks down nucleic acids, but is susceptible to substances that disrupt proteins. Some scientists hypothesized that the distorted protein could bind to other proteins of the same type and induce them to change their conformation as well, producing a chain reaction that propagates the disease and generates new infectious material. Since then, the gene for this protein has been successfully cloned, and studies using transgenic mice have bolstered the prion hypothesis.

The evidence in support of the hypothesis is now very strong, though not incontrovertible. First, the mounting experimental evidence has generated great interest in what appears to be a totally new kind of mechanism of disease. Second, the demonstration that prions are responsible for 'mad cow' disease bovine spongiform encephalopathy , which has infected large numbers of cattle in Great Britain and panicked the public, has lent new urgency to the quest for a cure--especially since the discovery that infected cows might be responsible for several new cases of CJD in humans.

Finally, I and my colleagues have recently determined that a phenomenon much like prion infection exists in yeast. These genetic traits had been known for many years, but their baffling patterns of inheritance for example, they can be passed along through a cell's cytoplasm, rather than the nucleus where the DNA resides had eluded explanation. We now know that the genetic trait is transmitted by proteins that are encoded in the nucleus but that can change their conformation in the cytoplasm.

Once this change has occurred, the reconfigured proteins induce other newly made proteins of the same type to change their conformation, too. Molecular genetic research on yeast should speed up the resolution of fundamental questions about the workings of protein-folding chain reactions.

And more important, it suggests that the prion mechanism is ubiquitous among living things and may be responsible for many phenomena other than neurodegenerative diseases like CJD. Mark Rogers in the department of zoology and the Biotechnology Centre at University College, Dublin, adds some further information:. Prusiner of the University of California School of Medicine at San Francisco in to distinguish the infectious agent that causes scrapie in sheep, Creutzfeldt-Jakob disease CJD in humans and bovine spongiform encephalopathy BSE in cattle from other, more typical infectious agents.

The prion hypothesis postulates that these diseases are caused not by a conventional virus or bacterium but by a protein that has adopted an abnormal form. Recently scientists have developed a molecular model of both variants and have published papers describing the structure of prion proteins as manufactured by E. Further work using magnetic resonance imaging and x-ray crystallography should help us understand the key structural elements that allow the prion to co-opt the normal cellular form into the disease-producing variant.

It is likely that other cellular components assist in this process, so work on understanding the cell biology of both forms of the protein is also vital. Shaun Heaphy in the department of microbiology and immunology at Leicester University provides this overview:.

Introduction

Prions also cause disease in a wide variety of other animals, including scrapie in sheep and bovine spongiform encephalopathy BSE in cows. Collectively these diseases are known as transmissible spongiform encephalopathies. B Corticostriatal slice cultures were prepared from day-old Tga 20 pups in similar fashion as cerebellar slice cultures and maintained for 42 dpi before testing on RT-QuIC assay.

The seeding activity from RT-QuIC traces demonstrates that the competence of seeding activity is highly dynamic and similar to seeds obtained from organotypic cerebellar slices. It is imperative to develop such integrated models for drug development against chronic neurodegenerative conditions like prions and related protein misfolding diseases that can remain asymptomatic for several years.

However, the caveat for both approaches is that they require adequate technical expertise to prepare and long-term maintenance of the cultures. Nevertheless, we demonstrate the efficacy of prion-infected organotypic slice cultures coupled with seeding assays like RT-QuIC to quantify seeding prions generated in the cultures in a more cost-effective way. Among various in vitro culture models, the major advantage of organotypic slice culture is that it preserves morphologic architecture and physiologic interconnectivity between various cell types like neurons and neuroglia.

In addition, chronic animal distress associated with in vivo prion infection would be circumvented while permitting continuous access to brain tissue for experimental procedures throughout the course of infection. The slices were maintained in culture for several weeks with good viability. Slices infected with scrapie accumulated PK-resistant prions in culture while demonstrating neurotoxicity Fig. We found the RT-QuIC assay amplifies prions from minute quantities of protein extracted from slice cultures in both a seed concentration- and culture time-dependent manner Fig. Importantly, prion knockout slices infected with RML scrapie did not show any seeding activity across a wide range of seed dilutions Fig.

The level of seeding activity sustained in our WT slice cultures increased with the duration of the culture period and is comparable to published values Indeed, the seeding activity that we detected as early as 7 dpi was unexpected, suggesting that the OSCAR model is more sensitive than the prevailing methods of detecting infectivity such as Western blots. This rapid amplification is relevant to recent discoveries that the replication of PrP Sc was observed in vasculature very early after microinjecting prions into mouse brains Further more, the inoculum used was not detectable in 1-dpi slices possibly due to 1 the washing steps involved in culturing the slices and 2 the homogenized slices were further diluted before testing.

We demonstrated the sensitivity of detecting prions with our OSCAR model of ex vivo prion infection and characterized the kinetics of prion amplification at various time points in culture Fig. The time course of prion amplification in slice cultures is analogous to the kinetic pattern of in vivo bioassays as PK-resistant prions rapidly reach maximum titer towards the terminal stage of infection, which becomes accelerated in the ex vivo preparation.

Based on multiple endpoint titrations for RML scrapie prions in slice culture, seeding activity shows a progressive and time-dependent amplification. Detection of prions by traditional methods depends on various factors such as PK concentration, incubation time, and choice of antibody that could create discrepancies in the results obtained between samples. However, it remains to be tested whether this model can be used successfully for other strains and species.

As the detection strategies continue to improve for a broad range of prions and strains 28 , 29 , 30 , 31 , we believe that our OSCAR model could be useful in developing procedures that disrupt prion propagation. Distinct kinetic traces from an OSCAR assay could help us to understand more characteristics of prion strains. Finally, using recombinant bank vole Myodes glareolus PrP as a substrate, a variety of prion strains could be rapidly amplified in an RT-QuIC assay to better understand mechanisms and alternative therapeutic approaches This OSCAR model offers a high-throughput platform for extending the utility of integrated prion models to support rapid drug discovery efforts and antemortem diagnostics.

As proof-of-principle, we used the OSCAR system to test a small panel of anti-prion compounds that were characterized in animal models of prion diseases 11 , 32 , The mechanism of action of this polyanion is its high-binding affinity to amyloids that hyperstabilizes the aggregates 34 , thus interfering with the formation of toxic oligomers.

Although Congo red is neuroprotective in prion-infected cerebellar slices 35 , a paradoxical increase in PK-resistant prions, as shown with stronger immunoblotted band intensity, has also been reported Next, we evaluated the small molecule quinacrine. Previous reports suggest treatment with quinacrine results in a strong anti-prion effect in cell culture and it is routinely used in screening libraries 36 , However, efforts to translate this compound in human trials failed As further studies determined that quinacrine exhibits poor CNS bioavailability, attempts were made to increase its bioavailability using mice deficient in the P-glycoprotein multi-drug resistance MDR transporter.

However, this study showed only a transient reduction in both PrP Sc and its conformational stability while encouraging drug resistance In slice cultures, chronic treatment with quinacrine showed an intermediate decrease in seeding activity when compared to control slices infected with RML scrapie Fig.

This anti-prion effect in cultured slices could be attributed to direct interaction of the drug with the brain slices and early initiation of the treatment regimen. Astemizole crosses the blood-brain barrier and reduced the PK-resistant PrP in the persistently infected cell culture model. Although its exact anti-prion mechanism of action is not known, it has been shown to stimulate autophagy. Conversely, in our study, astemizole did not show any reduction in seeding activity and the seeding kinetics remained similar to that of cerebellar slice cultures infected with RML scrapie Fig.

This difference may be attributed to a highly sensitive readout using RT-QuIC assay to detect seeding prions. These initial studies demonstrate the translational potential for the OSCAR model to further test novel compounds and may have utility in future treatment paradigms involving a wide range of prion strains.

No drugs are available for halting or reversing prion diseases or any other prion-like neurodegenerative disorders Additionally, drugs demonstrating excellent effects in cell culture and in vivo models of scrapie paradoxically fail to show any effect on CJD prions 15 or in clinical trials Also, mouse cell lines persistently infected with prions were developed to establish in vitro models of scrapie 41 , 42 for drug testing, but compounds identified through a high-throughput drug screening 43 had little or no effect against CJD prions.

Furthermore, no cell culture model can propagate the CJD prions in culture. In summary, we have established a highly sensitive integrated technique for the efficient detection of seeding prions Fig. Our OSCAR model using an organotypic slice culture assay coupled to the RT-QuIC assay may be able to accelerate translational research by testing a large number of anti-prion compounds, including small molecules, against prion infection.

In addition, using OSCAR with transgenic mouse models and genetic manipulation may provide mechanistic insights into the cell-to-cell transmission of prion-like aggregates and the progression of neurodegenerative diseases.

Advancing Prion Science Guidance for the National Prion Research Program Interim Report

The brain slice cultures were prepared in organotypic fashion obtained from neonatal mice and embedded in compressing lip and sliced. Treatments with compounds of interest began at 14 dpi in this study and slices were harvested at 31 dpi.

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The seeding activity was monitored in real time based on seeding using a thioflavin T ThT fluorescence readout and quantified. Positive wells can be clearly visualized based on the traces that cross the threshold fluorescence and by the rapid increase in intensity. The well-area scan image shows the intense amyloid deposits in the well. Louis, MO. PICM , E. Horse serum, penicillin, streptomycin, and low-melting agarose Cat No. Alexa Fluor conjugated goat anti-mouse IgG was purchased from Invitrogen. Organotypic cerebellar slices for prion assay were prepared as previously described 6 , Culture media was exchanged every other day with fresh media until the infected slices reached their pre-determined endpoints.

Lysates were prepared in 1X PBS by three freeze-thaw cycles and subjected to five sec sonication pulses each in a cup sonicator. When testing anti-prion compounds, the compounds were added from stock solutions X to slices at 15 days post-inoculation dpi , and then fresh media with compounds was exchanged every other day and harvested at 31 dpi as described above.

Viability assays in organotypic cerebellar slices were performed as previously described with modifications All the images were processed using ImageJ with constant threshold settings. Western blot analyses and limited proteolysis were performed as described previously 6 , 7 , 25 , 45 with minor modifications. After electrophoresis, proteins were transferred to a nitrocellulose membrane. For limited proteolysis, we used partial Proteinase-K digestion methods as previously described with a few modifications 6 , 12 , Secondary antibody incubations and washings were done as described above and fluorescence was captured using the Odyssey IR Imaging system.

In brief, the slice cultures were harvested at pre-determined endpoints, and homogenates were prepared and protein concentrations were determined as described above. Next, 0. After fixing the slices, antibody treatments were performed as described previously 6 , 25 , 47 with minor changes. The culture membranes were removed from the inserts and mounted directly on microscope slides, with membranes facing the slide, using Fluoromount mounting medium Sigma and imaged with a SPOT color digital camera attached to a Nikon TEU microscope.

Recombinant prion protein rPrP was expressed and purified using previously reported protocols 23 , 28 , 48 , 49 , Briefly, N-histidine-tagged prion protein-encoding plasmids of Syrian golden hamster residues for full length 23— or truncated 90— in pET vector EMD biosciences were transformed into E.

Cryo-EM and Prion Disease: How Our Proteins Turn Against Us

On-column refolding was performed using gradient reduction of GuHCl. Protein concentration was typically in the range of 0. Each batch was routinely tested for quality and activity using Western blot and RT-QuIC assay respectively, before using on test samples. RT-QuIC assay was performed using standard protocols from published reports 23 , 30 , 51 with slight modifications.

All samples were run at least in triplicates, and samples were judged to be positive as reported previously when samples were run in quadruplicates 30 , Whenever triplicates were run, we averaged their fluorescence readings, and additionally we selected 10xSD standard deviation of negative controls as the criteria for determining the threshold. R8 or Gen 5 version 2. How to cite this article: Kondru, N. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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