Toward alpha-synuclein seed amplification assay in clinical practice

Abstract

Introduction: Seed amplification assays (SAAs) demonstrate remarkable diagnostic performance in alpha-synucleinopathies. However, existing protocols lack accessibility in routine laboratories, mainly due to the requirement for in-house production of recombinant alpha-synuclein (aSyn). This study proposes a cerebrospinal fluid (CSF) aSyn-SAA protocol using solely commercial reagents to facilitate its clinical implementation.

Methods: Routine clinical care CSF samples from 126 patients, comprising 47 with Lewy body diseases (LBD) (41 with dementia with Lewy bodies, six with Parkinson's disease), 37 without alpha-synucleinopathy, and 42 with Alzheimer's disease (AD), underwent assessment for aSyn-SAA activity.

Results: CSF aSyn-SAA showed a sensitivity of 72.3% and a specificity of 100% when distinguishing clinically diagnosed LBD patients from those without alpha-synucleinopathy. In AD patients, 14.3% were tested positive for aSyn.

Discussion: The commercial-only CSF aSyn-SAA protocol exhibited excellent specificity when applied to a real-life cohort, signaling progress toward the accessibility of an aSyn biomarker in clinical settings.

Highlights: Diagnosis of LBD through aSyn-SAA lacks accessibility.This commercial-only aSyn-SAA has satisfactory performance in a real-life cohort.A negative aSyn-SAA does not completely exclude a synucleinopathy.Some technical points must be considered when developing aSyn-SAA.aSyn-SAA must be confined to expert laboratories due to prion-like risk management.

Keywords: Lewy body disease (LBD); Parkinson's disease (PD); aSyn; alpha‐synuclein (α‐synuclein); biomarker; brain homogenate (BH); cerebrospinal fluid (CSF); dementia with Lewy bodies (DLB); neurodegenerative diseases; proteinopathies; real‐life cohort; real‐time quaking‐induced conversion (RT‐QuIC); seed amplification assay (SAA); synucleinopathies.

FIGURE 1

Alpha‐synuclein‐SAA's principle. The sample (here BH or CSF) containing a small amount of the misfolded aSyn protein is added to a buffer in an excess of recombinant aSyn protein and Thioflavin T (that intercalates into the newly formed aggregates and then emits fluorescence during the experiment). Several cycles of shaking and rest at a controlled temperature are performed to allow elongation and fragmentation of the newly formed aggregates. Fluorescence is regularly measured to follow the reaction. Quantitative parameters are calculated from the kinetics and include the lag phase (time to reach cutoff), the ThT Max (maximum fluorescence reached), and AUC (area under the curve). BH: brain homogenate; CSF, cerebrospinal fluid.

FIGURE 2

Alpha‐synuclein SAA kinetics curves. (A) Overall aSyn seeding activity observed in all Lewy body diseases cases (ALL SYN, N = 34/47 aSyn+ patients, 72.3% sensitivity) versus non‐ASYN patients (N = 0/37 aSyn+ patients, 100% specificity). (B) Seeding activity in the DLB cases (N = 28/41 aSyn+ patients, 68.3% sensitivity). (C) Seeding activity in PD cases (N = 6/6 aSyn+ patients, 100% sensitivity). (D) Seeding activity in the AD group overall (N = 42 cases) including positive (N = 6) and negative (N = 36) patients (see Figure S2 for details on the aSyn‐SAA amplification kinetics in AD aSyn‐ and AD aSyn+ subgroups). AD, Alzheimer's disease; aSyn, alpha‐synuclein; SAA, seed amplification assays.

FIGURE 3

Parameters characterizing kinetics of aSyn aggregation in aSyn‐SAA assay. (A) Comparative analysis of ThT Max values across each experimental group. (B) Evaluation of AUC for each diagnostic group examined. (C) Assessments of lag‐phase duration within aSyn‐SAA positive groups. The colors of the box plots correspond to those depicted in the kinetics curves illustrated in Figure 2. Each box plot displays the median and the 5th to 95th percentiles of the data distributions. AUC, area under the curve; aSyn, alpha‐synuclein; SAA, seed amplification assays.