Detection of Prions

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

1) Alpha-synuclein RT-QuIC seed amplification assay (SAA) positivity has been shown to have value as a binary diagnostic biomarker for Parkinson’s disease. However, studies of the prognostic value of this biomarker have been limited to small, single-center studies, with short follow up. We aimed to assess the diagnostic and prognostic value of quantitative CSF a-syn RT-QuIC kinetic measures in Parkinson’s disease. In a longitudinal cohort study, we collected and analyzed data from participants with Parkinson’s disease, progressive supranuclear palsy, and healthy controls enrolled in three cohort studies: the UK parkinsonism cohort; the Parkinson’s Progression Markers Initiative (PPMI) international observational study; and the Tuebingen Parkinson’s disease cohort. Baseline CSF a-syn RT-QUIC data and longitudinal clinical data were collected between Jan 1, 2005, and Nov 1, 2023. The following seeding kinetic measures were calculated from the a-syn RT-QUIC curve for each RT-QUIC positive sample: time to threshold (TTT) for a positive RT-QUIC result; the maximum fluorescence during the reaction time (MaxThT); and the area under the fluorescence curve during the reaction time (AUC). We compared seeding kinetic measures between sporadic Parkinson’s disease and progressive supranuclear palsy, and between sporadic Parkinson’s disease and monogenic Parkinson’s disease. We used time-to-event analyses to assess the ability of a-syn RT-QUIC kinetic measures to predict unfavourable outcome in Parkinson’s disease, adjusting for sex, age and disease duration at RT-QUIC testing. We analysed data from 1631 participants: newly generated data from the UK parkinsonism cohort (Parkinson’s disease, n=66; progressive supranuclear palsy, n=52; controls n=9); and previously generated data from the PPMI (Parkinson’s disease, n=1,036; controls, n=239) and Tuebingen (Parkinson’s disease, n=229) cohorts. In the UK parkinsonism cohort, a-syn RT-QUIC was positive in 63/66 (96%) Parkinson’s disease and 8/52 (15%) progressive supranuclear palsy samples, with positive PSP samples having distinct ‘low and slow’ seeding kinetics (low MaxThT and high TTT) as a marker of Lewy body co-pathology. TTT was faster in GBA-Parkinson’s disease compared with sporadic PD in both the PPMI (p=0.04) and Tuebingen (p=0.01) cohorts. Unfavourable outcome was observed in 593/810 (73%) a-syn RT-QUIC positive sporadic and monogenic Parkinson’s disease participants in the PPMI cohort during a median follow-up period of 4.5 years (IQR 7 years). TTT at baseline predicted only cognitive decline (MoCA ≤21) as a component of unfavourable outcome in Parkinson’s disease in both the PPMI (n=824, HR 2.36, 95% CI 1.60-3.46, p=0.001) and Tuebingen (n=135, HR 2.17, 95% CI 1.07-4.41, p=0.03) cohorts. TTT also predicted cognitive decline in a sub-cohort of PPMI Parkinson’s disease participants who were Alzheimer’s disease biomarker negative (n=355, HR 1.80, 95% CI 1.03-3.18, p=0.04). Overall, these results suggest that assessing a-syn RT-QuIC kinetic measures might aid in the diagnostic differentiation of Parkinson’s disease from progressive supranuclear palsy with Lewy body co-pathology. Furthermore, faster seeding kinetics are found in GBA-Parkinson’s disease and predict cognitive decline in Parkinson’s disease independently of Alzheimer’s disease co-pathology. 2) Disease 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 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. 3) Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and other synucleinopathies are characterized by the accumulation of abnormal, self-propagating aggregates of α-synuclein. RT-QuIC or seed amplification assays are currently showing unprecedented diagnostic sensitivities and specificities for synucleinopathies even in prodromal phases years in advance of the onset of parkinsonian signs or dementia. However, commonly used α-synuclein seed amplification assays take ≥48 h to perform as applied to patient’s diagnostic biospecimens. We have now developed a still faster α-synuclein RT-QuIC assay that is as analytically sensitive as prior assays of this type, but can be completed in ≤12 h for brain, skin and intestinal mucosa, with positive signals often arising in <5 h. CSF assays took a few hours longer. Our same-day α-synuclein RT-QuIC (sdRT-QuIC) assay should increase the practicality, cost-effectiveness, and throughput of measurements of pathological forms of α-synuclein for fundamental research, clinical diagnosis, and therapeutics development. 4) According to the “body-first” concept, pathogenetic processes eventuating in Parkinson disease (PD) can begin in the autonomic nervous system. Whether body-first progression occurs in the related central Lewy body disease dementia with Lewy bodies (DLB) has been unknown. Testing this notion requires long-term clinical and laboratory assessments and post-mortem examinations. We helped David Goldstein (NINDS) and Guillaume Lamotte (Univ. of Utah) and their colleagues to analyze two individuals developed DLB after participating in the prospective, longitudinal PDRisk study, which assessed whether in individuals at risk for PD based on genetics, olfaction, dream enactment behavior, and orthostatic hypotension biomarkers of catecholamine deficiency in the brain or heart predict a central Lewy body disease. Both patients initially had normal cognition, no parkinsonism, normal putamen 18F-DOPA-derived radioactivity, and low cardiac 18F-dopamine-derived radioactivity, consistent with the body-first concept; however, both also initially had anosmia, low cerebrospinal fluid (CSF) levels of 3,4-dihydroxyphenylacetic acid (a biomarker of central dopamine deficiency), and elevated CSF alpha-synuclein seeding activity. Post-mortem analyses documented intra-neuronal alpha-synuclein deposition in the sympathetic ganglionic chain and profound myocardial and putamen catecholamine deficiency. We concluded that although some clinical laboratory abnormalities in our patients fit with the body-first sequence, others noted upon initial evaluation raise the possibility of multicentric, approximately simultaneous disease progression in the brain and periphery in DLB. 5) We also helped the Goldstein group (NINDS) assess the efficiency of cerebrospinal fluid (CSF) alpha-synuclein seeding activity (SSA) via a seed amplification assay for predicting central Lewy body diseases (LBDs), in a prospective, longitudinal, observational study. Participants self-reported risk factors (genetics, olfactory dysfunction, dream enactment behavior, orthostatic intolerance or hypotension); those with ≥3 confirmed risk factors had CSF sampling and were followed for up to 7.5 years. Those developing a central LBD (LBD+) were compared to those who did not (LBD-). Quadruplicate SSA areas under the curve (AUCs) were averaged. Among 27 at-risk individuals, of 9 with average AUCs above 500,000 units 6 (67%) developed a central LBD during follow-up compared to 0 of 18 (0%) with AUCs below the cutoff value (p=0.0003). Conversely, all LBD+ participants had elevated initial AUCs. These result suggest that increased CSF SSA predicts central LBDs, and individuals who develop a central LBD during long-term follow-up have elevated initial SSA AUCs. 6) Neuronal hyperexcitability precedes synapse loss in certain neurodegenerative diseases, yet the synaptic membrane interactions and downstream signaling events remain unclear. The disordered amino terminus of the prion protein (PrPC) has been implicated in aberrant signaling in prion and Alzheimer’s disease. To disrupt neuronal interactions and signaling linked to the amino terminus, the group of Christina Sigurdson (UCSD) generated CRISPR-engineered a knock-in mouse expressing mutant PrPC (G92N), generating an N-linked glycosylation site between two functional motifs. Mice developed seizures and necrosis of hippocampal pyramidal neurons, similar to prion-infected mice and consistent with excitotoxicity. However, our group showed that they had no RT-QuIC seeding activity that would be indicative of infectious prions. Phosphoproteomics revealed phosphorylated glutamate receptors and calcium-sensitive kinases, including protein kinase C (PKC). Additionally, 92N-PrPC-expressing neurons show persistent calcium influx as well as dendritic beading, which was rescued by an NMDA receptor antagonist. Finally, survival of Prnp92N mice was prolonged by blocking active NMDA receptor channels. We proposed dysregulated PrPC – NMDA receptor - induced signaling can trigger excitatory – inhibitory imbalance, spongiform degeneration, and neurotoxicity, and that calcium dysregulation is central to PrPC-linked neurodegeneration. 7) The assembly of tau into amyloid filaments is a defining characteristic of Alzheimer’s disease (AD) and other tauopathies. Cryo-electron microscopy (cryo-EM) showed that specific tau folds characterise different diseases, and that in vitro models often yield filaments with folds that do not replicate those that form in disease. Working with the groups of Michele Vendruscolo (Cambridge U.) and Schors Scheres (MRC LMB) we investigated the aggregation of full-length recombinant 0N3R tau, using wild-type, or mutations C322A or C322S and a real-time quaking-induced conversion (RT-QuIC) assay with brain homogenate seeding. The assembly of C322A 0N3R tau resulted in filaments with a structure resembling the AD fold in paired helical filaments (PHFs), with differences attributed to the C322A mutation. C322S 0N3R tau formed structurally more distinct filaments with respect to PHFs, with an ordered carboxy-terminal region. Both mutant filaments retained the ability to seed a second round of aggregation. Meanwhile, wild-type 0N3R tau exhibited poor reproducibility and formed predominantly unfolded aggregates. Our findings emphasise the need for optimised assembly conditions to obtain disease-relevant filament folds. Refining these methodologies could enhance our understanding of the molecular origins of tauopathies and facilitate the development of targeted therapeutic strategies for these conditions. 8) I co-chaired a Prion biology and disease diagnostics working group organized by the University of Minnesota’s CIDRAP program aimed at summarizing the state-of the art in diagnosing CWD in cervid species and identifying current and future challenges in detecting and addressing potential transmissions of CWD into humans or livestock species that we eat. Our collective efforts (with other working groups) led to a publicly available document: Chronic Wasting Disease Spillover Preparedness and Response: Charting an Uncertain Future. Center for Infectious Disease Research and Policy, University of Minnesota 2025 Jan 8. https://www.cidrap.umn.edu/chronic-wasting-disease/cwd-spillover-report-2025.