Streamlined alpha-synuclein RT-QuIC assay for various biospecimens in Parkinson’s disease and dementia with Lewy bodies

Bargar, Connor; Wang, Wen; Gunzler, Steven A.; LeFevre, Alexandra; Wang, Zerui; Lerner, Alan J.; Singh, Neena; Tatsuoka, Curtis; Appleby, Brian S.; Zhu, Xiongwei; Xu, Rong; Haroutunian, Vahram; Zou, Wen-Quan; Ma, Jiyan; Chen, Shu G.

doi:10.1186/s40478-021-01175-w

Cited by 88 publications

(94 citation statements)

References 39 publications

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“…The in vitro generated αSyn fibrils are rich in β-sheets, enabling detection by the amyloid-specific dye Thioflavin T (ThT). αSyn-SAAs are capable of detecting and amplifying αSyn seeds from multiple biospecimens, including brain [11][12][13], cerebrospinal fluid (CSF) [6][7][8][9][10][13][14][15][16], skin [17][18][19], olfactory mucosa [20,21], submandibular gland [22], and gut [23]. Moreover, they have demonstrated accurate detection of αSyn seeds in clinically and pathologically validated cohorts of PD patients [6,7,9,10,13,17,18,24].…”

Section: Introductionmentioning

confidence: 99%

High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson’s disease

Russo

Orrú

Concha‐Marambio

et al. 2021

acta neuropathol commun

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Alpha-synuclein seed amplification assays (αSyn-SAAs) are promising diagnostic tools for Parkinson’s disease (PD) and related synucleinopathies. They enable detection of seeding-competent alpha-synuclein aggregates in living patients and have shown high diagnostic accuracy in several PD and other synucleinopathy patient cohorts. However, there has been confusion about αSyn-SAAs for their methodology, nomenclature, and relative accuracies when performed by various laboratories. We compared αSyn-SAA results obtained from three independent laboratories to evaluate reproducibility across methodological variations. We utilized the Parkinson’s Progression Markers Initiative (PPMI) cohort, with DATSCAN data available for comparison, since clinical diagnosis of early de novo PD is critical for neuroprotective trials, which often use dopamine transporter imaging to enrich their cohorts. Blinded cerebrospinal fluid (CSF) samples for a randomly selected subset of PPMI subjects (30 PD, 30 HC, and 20 SWEDD), from both baseline and year 3 collections for the PD and HC groups (140 total CSF samples) were analyzed in parallel by each lab according to their own established and optimized αSyn-SAA protocols. The αSyn-SAA results were remarkably similar across laboratories, displaying high diagnostic performance (sensitivity ranging from 86 to 96% and specificity from 93 to 100%). The assays were also concordant for samples with results that differed from clinical diagnosis, including 2 PD patients determined to be clinically inconsistent with PD at later time points. All three assays also detected 2 SWEDD subjects as αSyn-SAA positive who later developed PD with abnormal DAT-SPECT. These multi-laboratory results confirm the reproducibility and value of αSyn-SAA as diagnostic tools, illustrate reproducibility of the assay in expert hands, and suggest that αSyn-SAA has potential to provide earlier diagnosis with comparable or superior accuracy to existing methods.

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Section: Introductionmentioning

confidence: 99%

High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson’s disease

Russo

Orrú

Concha‐Marambio

et al. 2021

acta neuropathol commun

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“…A quantitative mapping of the temporal–spatial course of the distribution of αSyn PD seeding activity in humans using highly sensitive real-time quaking induced conversion (RT-QuIC) [ 112 , 113 , 114 , 115 ] or protein misfolding cyclic amplification (PMCA) [ 116 ] assays could also provide important contributions to the elucidation of neuroanatomical spreading pathways of αSyn pathology in PD. αSyn PD seeding activity is assumed to constitute the molecular driver underlying the propagation and spatial spread of pathological αSyn aggregation in PD.…”

Section: Discussionmentioning

confidence: 99%

“…This would possibly also help to settle the controversy on reports claiming the detection of pathological αSyn PD deposits in colonic and other gastro-intestinal biopsies years before the onset of PD motor symptoms [ 27 ]. That detection of αSyn PD seeding activity by RT-QuIC assay in GI tissue from the stomach or colon of PD patients is feasible has been recently demonstrated in two independent studies [ 99 , 115 ]. Thus, using the relatively new and powerful analytical tools of RT-QuIC or PMCA for the quantitative detection of αSyn PD -associated seeding activity in human postmortem tissues and bioptic samples may open new perspectives on both the neural gut–brain axis of pathological protein aggregation and the ultrasensitive diagnostic detection of αSyn PD seeds in PD.…”

Section: Discussionmentioning

confidence: 99%

The Neural Gut–Brain Axis of Pathological Protein Aggregation in Parkinson’s Disease and Its Counterpart in Peroral Prion Infections

Beekes

2021

Viruses

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A neuropathological hallmark of Parkinson’s disease (PD) is the cerebral deposition of abnormally aggregated α-synuclein (αSyn). PD-associated αSyn (αSynPD) aggregates are assumed to act, in a prion-like manner, as proteinaceous nuclei (“seeds”) capable of self-templated propagation. Braak and colleagues put forward the idea of a neural gut-brain axis mediating the centripetal spread of αSynPD pathology from the enteric nervous system (ENS) to the brain in PD. This has sparked great interest and initiated passionate discussions both in support of and opposing the suggested hypothesis. A precedent for the spread of protein seeds or seeding from the gastro-intestinal (GI) tract to the central nervous system (CNS) had been previously revealed for pathological prion protein in peroral prion infections. This article scrutinizes the similarities and dissimilarities between the pathophysiological spread of disease-associated protein aggregation along the neural gut–brain axis in peroral prion infections and PD. On this basis, evidence supporting the proposed neural gut–brain axis in PD is concluded to be not as robust as that established for peroral prion infections. New tools for the ultrasensitive detection of αSynPD-associated seeding activity in archived or fresh human tissue samples such as real-time quaking induced conversion (RT-QuIC) or protein misfolding cyclic amplification (PMCA) assays can possibly help to address this deficit in the future.

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“…A modified version of the α-synuclein RT-QuIC assay described by Barger et al [ 31 ] was used. Dilution of rat CSF (1:10) was performed with 1x N2 supplement (Thermo Fisher) diluted with PBS.…”

Section: Methodsmentioning

confidence: 99%

Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans

Chen

Menezes

Rodgers

et al. 2021

Mol Neurodegeneration

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Background Parkinson’s disease is a disabling neurodegenerative movement disorder characterized by dopaminergic neuron loss induced by α-synuclein oligomers. There is an urgent need for disease-modifying therapies for Parkinson’s disease, but drug discovery is challenged by lack of in vivo models that recapitulate early stages of neurodegeneration. Invertebrate organisms, such as the nematode worm Caenorhabditis elegans, provide in vivo models of human disease processes that can be instrumental for initial pharmacological studies. Methods To identify early motor impairment of animals expressing α-synuclein in dopaminergic neurons, we first used a custom-built tracking microscope that captures locomotion of single C. elegans with high spatial and temporal resolution. Next, we devised a method for semi-automated and blinded quantification of motor impairment for a population of simultaneously recorded animals with multi-worm tracking and custom image processing. We then used genetic and pharmacological methods to define the features of early motor dysfunction of α-synuclein-expressing C. elegans. Finally, we applied the C. elegans model to a drug repurposing screen by combining it with an artificial intelligence platform and cell culture system to identify small molecules that inhibit α-synuclein oligomers. Screen hits were validated using in vitro and in vivo mammalian models. Results We found a previously undescribed motor phenotype in transgenic α-synuclein C. elegans that correlates with mutant or wild-type α-synuclein protein levels and results from dopaminergic neuron dysfunction, but precedes neuronal loss. Together with artificial intelligence-driven in silico and in vitro screening, this C. elegans model identified five compounds that reduced motor dysfunction induced by α-synuclein. Three of these compounds also decreased α-synuclein oligomers in mammalian neurons, including rifabutin which has not been previously investigated for Parkinson’s disease. We found that treatment with rifabutin reduced nigrostriatal dopaminergic neurodegeneration due to α-synuclein in a rat model. Conclusions We identified a C. elegans locomotor abnormality due to dopaminergic neuron dysfunction that models early α-synuclein-mediated neurodegeneration. Our innovative approach applying this in vivo model to a multi-step drug repurposing screen, with artificial intelligence-driven in silico and in vitro methods, resulted in the discovery of at least one drug that may be repurposed as a disease-modifying therapy for Parkinson’s disease.

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Streamlined alpha-synuclein RT-QuIC assay for various biospecimens in Parkinson’s disease and dementia with Lewy bodies

Cited by 88 publications

References 39 publications

High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson’s disease

High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson’s disease

The Neural Gut–Brain Axis of Pathological Protein Aggregation in Parkinson’s Disease and Its Counterpart in Peroral Prion Infections

Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans

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