Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC

Abstract

The diagnosis and treatment of synucleinopathies such as Parkinson disease and dementia with Lewy bodies would be aided by the availability of assays for the pathogenic disease-associated forms of α-synuclein (αSynD) that are sufficiently sensitive, specific, and practical for analysis of accessible diagnostic specimens. Two recent αSynD seed amplification tests have provided the first prototypes for ultrasensitive and specific detection of αSynD in patients’ cerebrospinal fluid. These prototypic assays require 5–13 days to perform. Here, we describe an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay that has similar sensitivity and specificity to the prior assays, but can be performed in 1–2 days with quantitation. Blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases [12 Parkinson’s and 17 dementia with Lewy bodies] and 31 non-synucleinopathy controls, including 16 Alzheimer’s cases, yielded 93% diagnostic sensitivity and 100% specificity for this test so far. End-point dilution analyses allowed quantitation of relative amounts of αSynD seeding activity in cerebrospinal fluid samples, and detection in as little as 0.2 μL. These results confirm that αSynD seeding activity is present in cerebrospinal fluid. We also demonstrate that it can be rapidly detected, and quantitated, even in early symptomatic stages of synucleinopathy.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0508-2) contains supplementary material, which is available to authorized users.

Seeding variability of different alpha synuclein strains in synucleinopathies

“…Therefore, the observed increase in the β-sheets content mirrors the RT-QuIC results, providing an unbiased validation of the formation of aggregates during the course of the reaction. Remarkably, Grovemann et al observed in CSF-seeded RT-QuIC reactions containing K23Q/ α-synuclein substrate that PD α-synuclein seeds exhibit lower signal responses in RT-QuIC when compared to DLB, 49 which confirms our findings. 43 Fibril compactness and size appeared increased in FCx-seeded reactions compared to reactions containing an SNc seed.…”

“…Importantly, because ThT affects the Raman signal, 48 all RT-QuIC reactions were prepared without adding ThT. Indeed, it has been recently demonstrated by different groups 43,49,50 that PD-derived α-synuclein possesses the ability to amplify recombinant α-synuclein when tested by RT-QuIC. Moreover, morphological studies conducted by AFM and TEM showed that DLB-seeded reactions may generate rod-shaped amyloid-fibrils of different sizes differing from other α-synuclein aggregates and exhibiting a more circular and compact appearance.…”

α‐Synuclein real‐time quaking‐induced conversion in the cerebrospinal fluid of uncertain cases of parkinsonism

“…[1][2][3][4] However, in these studies, selected CSF samples or brain homogenates of clinically straightforward or neuropathologically confirmed cases were used. The high specificity and positive predictive values indicate that the vast majority of patients with an undefined diagnosis of parkinsonism and a positive α-syn RT-QuIC will have an underlying α-synucleinopathy.…”

“…[1][2][3][4] However, this test has thus far only been evaluated in clear-cut clinical cases and/or neuropathologically confirmed cases, whereas clinicians would rather use the test in ambiguous cases. [1][2][3][4] However, this test has thus far only been evaluated in clear-cut clinical cases and/or neuropathologically confirmed cases, whereas clinicians would rather use the test in ambiguous cases.…”

A proteomic signature for dementia with Lewy bodies

“…With this initial screening approach, appropriate referrals can be made for subsequent specialty examinations and confirmatory diagnostic biomarkers (imaging, CSF), following the multistage models used for diagnosing cancer [31]. For example, Groveman et al [20] recently demonstrated the accuracy of a rapid and ultrasensitive seed amplification technique for detection of α‐synuclein. In the present proposed context, a blood‐based screening tool can be utilized to rule out the vast majority of patients who do not need to undergo lumbar puncture for biomarker confirmatory diagnostics.…”