Browsing by Author "Caughey, Byron"
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Item Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains(Public Library of Science, 2015-06) Orrú, Christina D.; Groveman, Bradley R.; Raymond, Lynne D.; Hughson, Andrew G.; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron; Department of Pathology and Laboratory Medicine, IU School of MedicinePrions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.Item Discovery of potent inhibitors of α-synuclein aggregation using structure-based iterative learning(Springer Nature, 2024) Horne, Robert I.; Andrzejewska, Ewa A.; Alam, Parvez; Brotzakis, Z. Faidon; Srivastava, Ankit; Aubert, Alice; Nowinska, Magdalena; Gregory, Rebecca C.; Staats, Roxine; Possenti, Andrea; Chia, Sean; Sormanni, Pietro; Ghetti, Bernardino; Caughey, Byron; Knowles, Tuomas P. J.; Vendruscolo, Michele; Pathology and Laboratory Medicine, School of MedicineMachine learning methods hold the promise to reduce the costs and the failure rates of conventional drug discovery pipelines. This issue is especially pressing for neurodegenerative diseases, where the development of disease-modifying drugs has been particularly challenging. To address this problem, we describe here a machine learning approach to identify small molecule inhibitors of α-synuclein aggregation, a process implicated in Parkinson's disease and other synucleinopathies. Because the proliferation of α-synuclein aggregates takes place through autocatalytic secondary nucleation, we aim to identify compounds that bind the catalytic sites on the surface of the aggregates. To achieve this goal, we use structure-based machine learning in an iterative manner to first identify and then progressively optimize secondary nucleation inhibitors. Our results demonstrate that this approach leads to the facile identification of compounds two orders of magnitude more potent than previously reported ones.Item Enhanced quantitation of pathological α-synuclein in patient biospecimens by RT-QuIC seed amplification assays(Public Library of Science, 2024-09-20) Srivastava, Ankit; Wang, Qinlu; Orrù, Christina D.; Fernandez, Manel; Compta, Yaroslau; Ghetti, Bernardino; Zanusso, Gianluigi; Zou, Wen-Quan; Caughey, Byron; Beauchemin, Catherine A. A.; Pathology and Laboratory Medicine, School of MedicineDisease associated pathological aggregates of alpha-synuclein (αSynD) exhibit prion-like spreading in synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Seed amplification assays (SAAs) such as real-time quaking-induced conversion (RT-QuIC) have shown high diagnostic sensitivity and specificity for detecting proteopathic αSynD seeds in a variety of biospecimens from PD and DLB patients. However, the extent to which relative proteopathic seed concentrations are useful as indices of a patient's disease stage or prognosis remains unresolved. One feature of current SAAs that complicates attempts to correlate SAA results with patients' clinical and other laboratory findings is their quantitative imprecision, which has typically been limited to discriminating large differences (e.g. 5-10 fold) in seed concentration. We used end-point dilution (ED) RT-QuIC assays to determine αSynD seed concentrations in patient biospecimens and tested the influence of various assay variables such as serial dilution factor, replicate number and data processing methods. The use of 2-fold versus 10-fold dilution factors and 12 versus 4 replicate reactions per dilution reduced ED-RT-QuIC assay error by as much as 70%. This enhanced assay format discriminated as little as 2-fold differences in αSynD seed concentration besides detecting ~2-16-fold seed reductions caused by inactivation treatments. In some scenarios, analysis of the data using Poisson and midSIN algorithms provided more consistent and statistically significant discrimination of different seed concentrations. We applied our improved assay strategies to multiple diagnostically relevant PD and DLB antemortem patient biospecimens, including cerebrospinal fluid, skin, and brushings of the olfactory mucosa. Using ED αSyn RT-QuIC as a model SAA, we show how to markedly improve the inter-assay reproducibility and quantitative accuracy. Enhanced quantitative SAA accuracy should facilitate assessments of pathological seeding activities as biomarkers in proteinopathy diagnostics and prognostics, as well as in patient cohort selection and assessments of pharmacodynamics and target engagement in drug trials.Item Million-fold sensitivity enhancement in proteopathic seed amplification assays for biospecimens by Hofmeister ion comparisons(National Academy of Sciences, 2019-11-12) Metrick, Michael A., II; do Carmo Ferreira, Natalia; Saijo, Eri; Hughson, Andrew G.; Kraus, Allison; Orrú, Christina; Miller, Michael W.; Zanusso, Gianluigi; Ghetti, Bernardino; Vendruscolo, Michele; Caughey, Byron; Pathology and Laboratory Medicine, School of MedicineRecent 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.Item A single ultrasensitive assay for detection and discrimination of tau aggregates of Alzheimer and Pick diseases(BMC, 2020-02) Metrick, Michael A., II; do Carmo Ferreira, Natália; Saijo, Eri; Kraus, Allison; Newell, Kathy; Zanusso, Gianluigi; Vendruscolo, Michele; Ghetti, Bernardino; Caughey, Byron; Pathology and Laboratory Medicine, School of MedicineMultiple 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.