INVESTIGATING THE ROLE OF PRION PROTEIN POLYMORPHISMS ON PRION PATHOGENESIS

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are lethal and infectious neurodegenerative diseases of humans and animals. The misfolding of the normal, or cellular isoform of the prion protein (PrPC) into the abnormal disease-associated isoform of PrP (PrPSc) could change the properties of PrP, consequently, PrPSc has lethal infectivity to transmit diseases. The proteinaceous infectious particle consisting mainly of PrPSc is called prion. Transmissibility of prions is strongly influenced by multiple factors including PrP polymorphisms, species barriers (PrP sequence specificity) and prion strains (conformational specificity) by unknown mechanisms. Even though the ability of prions to cross a species barrier has been recognized, the precise mechanisms of interspecies prion transmission remain unclear. This dissertation research was conducted in order to learn more about the molecular mechanisms of conversion, propagation and transmission of PrPSc; about determinants of genetic susceptibility to infection in prion diseases; and about understanding those mechanisms, which might govern the zoonotic potential of prion diseases. First, we investigated the transmissibility risk of multiple strains of Chronic Wasting Disease, which is a cervid TSE, with humanized transgenic mice and showed that the transmission barriers between cervid and the humanized mice are high. Next, the structural factors underlying the species barrier of prion diseases were studied using cell culture systems by systematically introducing amino acid substitutions in the regions of PrP, where the most divergences of different PrP species are recognized. Thirdly, we investigated the effects of the genetic susceptibility to prions as well as conversion kinetics and properties of PrPSc using Tg mice expressing ovine PrP polymorphism (OvPrP) at codon 136 either alanine (A) or valine (V). The templating characteristics of OvPrPSc-V136 were dominant over OvPrPSc-A136 under co-expressions of OvPrPC-A136 and OvPrPC-V136. Finally, the function of PrP was studied in relation to the pathogenesis of Alzheimer’s disease. These studies demonstrated that the conformational compatibility between PrPC and PrPSc contributed to the conversion kinetics and species barrier. We concluded that the conformational compatibility of PrPC to PrPSc is controlled not only by the PrP sequence specificity but also by the tertiary structure of PrPC

Million-fold sensitivity enhancement in proteopathic seed amplification assays for biospecimens by Hofmeister ion comparisons

Recent work with prion diseases and synucleinopathies indicates that accurate diagnostic methods for protein-folding diseases can be based on the ultrasensitive, amplified measurement of pathological aggregates in biospecimens. A better understanding of the physicochemical factors that control the seeded polymerization of such aggregates, and their amplification in vitro, should allow improvements in existing assay platforms, as well as the development of new assays for other proteopathic aggregates. Here, we systematically investigated the effects of the ionic environment on the polymerization of tau, α-synuclein, and the prion protein (PrP) induced by aggregates in biospecimens. We screened salts of the Hofmeister series, a relative ordering of strongly and weakly hydrated salts that tend to precipitate or solubilize proteins. We found that sensitivities of tau-based assays for Alzheimer’s seeds and PrP-based assays for prions were best in weakly hydrated anions. In contrast, we saw an inverse trend with different tau-based assays, improving detection sensitivity for progressive supranuclear palsy seeds by ≈106. Hofmeister analysis also improved detection of sporadic Creutzfeldt–Jakob disease prions in human nasal brushings and chronic wasting disease prions in deer-ear homogenates. Our results demonstrate strong and divergent influences of ionic environments on the amplification and detection of proteopathic seeds as biomarkers for protein-folding diseases

A single ultrasensitive assay for detection and discrimination of tau aggregates of Alzheimer and Pick diseases

Abstract: Multiple neurodegenerative diseases are characterized by aggregation of tau molecules. Adult humans express six isoforms of tau that contain either 3 or 4 microtubule binding repeats (3R or 4R tau). Different diseases involve preferential aggregation of 3R (e.g Pick disease), 4R (e.g. progressive supranuclear palsy), or both 3R and 4R tau molecules [e.g. Alzheimer disease and chronic traumatic encephalopathy]. Three ultrasensitive cell-free seed amplification assays [called tau real-time quaking induced conversion (tau RT-QuIC) assays] have been developed that preferentially detect 3R, 4R, or 3R/4R tau aggregates in biospecimens. In these reactions, low-fg amounts of a given self-propagating protein aggregate (the seed) are incubated with a vast excess of recombinant tau monomers (the substrate) in multi-well plates. Over time, the seeds incorporate the substrate to grow into amyloids that can then be detected using thioflavin T fluorescence. Here we describe a tau RT-QuIC assay (K12 RT-QuIC) that, using a C-terminally extended recombinant 3R tau substrate (K12CFh), enables sensitive detection of Pick disease, Alzheimer disease, and chronic traumatic encephalopathy seeds in brain homogenates. The discrimination of Pick disease from Alzheimer disease and chronic traumatic encephalopathy cases is then achieved through the quantitative differences in K12 RT-QuIC assay thioflavin T responses, which correlate with structural properties of the reaction products. In particular, Fourier transform infrared spectroscopy analysis of the respective K12CFh amyloids showed distinct β-sheet conformations, suggesting at least partial propagation of the original seed conformations in vitro. Thus, K12 RT-QuIC provides a single assay for ultrasensitive detection and discrimination of tau aggregates comprised mainly of 3R, or both 3R and 4R, tau isoforms

Protective Efficacy of Neutralizing Monoclonal Antibodies in a Nonhuman Primate Model of Ebola Hemorrhagic Fever

Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF

シロリムス ヨウシュツ ステント リュウチ 7ネンゴ ニ ハジメテ ゾウエイザイ ステント シュウイ シミダシゾウ オ ミトメタ イチレイ

A 74-year-old man who had a history of percutaneous coronary intervention ［left anterior descending coronary artery ＃6‐7, sirolimus eluting stent （SES） （Cypher stent,3.0×18mm）, left circumflex coronary artery ＃13, SES （Cypher stent, 2.5×23mm）］ for angina pectoris experienced chest pain on effort after seven years from the coronary intervention. He was introduced to our hospital and coronary angiography revealed late acquired peri-stent contrast staining （PSS）, which is defined as an angiographical finding of contrast medium stain outside the stent being ＞20％ of the stent diameter, in the SES of the left anterior descending artery. Drug-eluting stent （DES） significantly inhibits neointimal proliferation, thereby significantly reducing in-stent restenosis. However, the risk of very late stent thrombosis has become a major problem after the DES implantation against the bare-metal stent implantation. PSS has been reported that PSS after SES implantation could predict late stent thrombosis and incomplete stent apposition of the lesion with PSS. In this case, PSS was pointed out for the first time in seven years after SES implantation nevertheless it did not be pointed out in three years. The mechanism and prognosis of PSS is unclear. But, we found the increase in local coagulation at the coronary artery in this case and the degree of prothrombin fragment F1＋2, one of the coagulation marker, was greater in seven years after SES implantation than in three years. We thought these findings might reflect that PSS after SES implantation was associated with very late stent thrombosis. So we started the dual antiplatelet therapy for the prevention of stent thrombosis. Careful long-term observation might be recommended in patients with late acquired PSS and elevated local coagulation response following SES implantation

Automated Workflow for Preparation of cDNA for Cap Analysis of Gene Expression on a Single Molecule Sequencer

Background: Cap analysis of gene expression (CAGE) is a 59 sequence tag technology to globally determine transcriptional starting sites in the genome and their expression levels and has most recently been adapted to the HeliScope single molecule sequencer. Despite significant simplifications in the CAGE protocol, it has until now been a labour intensive protocol. Methodology: In this study we set out to adapt the protocol to a robotic workflow, which would increase throughput and reduce handling. The automated CAGE cDNA preparation system we present here can prepare 96 ‘HeliScope ready ’ CAGE cDNA libraries in 8 days, as opposed to 6 weeks by a manual operator.We compare the results obtained using the same RNA in manual libraries and across multiple automation batches to assess reproducibility. Conclusions: We show that the sequencing was highly reproducible and comparable to manual libraries with an 8 fold increase in productivity. The automated CAGE cDNA preparation system can prepare 96 CAGE sequencing samples simultaneously. Finally we discuss how the system could be used for CAGE on Illumina/SOLiD platforms, RNA-seq and fulllengt

Epigenetic dominance of prion conformers

Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer's and Parkinson's diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg) mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A) at (OvPrP-A136) infected with SSBP/1 scrapie prions propagated a relatively stable (S) prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V) at 136 (OvPrP-V136) infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U), diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb) PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to the otherwise unfavorable U conformer. This epigenetic mechanism thus expands the range of selectable conformations that can be adopted by PrP, and therefore the variety of options for strain propagation

A single ultrasensitive assay for detection and discrimination of tau aggregates of Alzheimer and Pick diseases.

Multiple neurodegenerative diseases are characterized by aggregation of tau molecules. Adult humans express six isoforms of tau that contain either 3 or 4 microtubule binding repeats (3R or 4R tau). Different diseases involve preferential aggregation of 3R (e.g Pick disease), 4R (e.g. progressive supranuclear palsy), or both 3R and 4R tau molecules [e.g. Alzheimer disease and chronic traumatic encephalopathy]. Three ultrasensitive cell-free seed amplification assays [called tau real-time quaking induced conversion (tau RT-QuIC) assays] have been developed that preferentially detect 3R, 4R, or 3R/4R tau aggregates in biospecimens. In these reactions, low-fg amounts of a given self-propagating protein aggregate (the seed) are incubated with a vast excess of recombinant tau monomers (the substrate) in multi-well plates. Over time, the seeds incorporate the substrate to grow into amyloids that can then be detected using thioflavin T fluorescence. Here we describe a tau RT-QuIC assay (K12 RT-QuIC) that, using a C-terminally extended recombinant 3R tau substrate (K12CFh), enables sensitive detection of Pick disease, Alzheimer disease, and chronic traumatic encephalopathy seeds in brain homogenates. The discrimination of Pick disease from Alzheimer disease and chronic traumatic encephalopathy cases is then achieved through the quantitative differences in K12 RT-QuIC assay thioflavin T responses, which correlate with structural properties of the reaction products. In particular, Fourier transform infrared spectroscopy analysis of the respective K12CFh amyloids showed distinct β-sheet conformations, suggesting at least partial propagation of the original seed conformations in vitro. Thus, K12 RT-QuIC provides a single assay for ultrasensitive detection and discrimination of tau aggregates comprised mainly of 3R, or both 3R and 4R, tau isoforms