Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid

Abstract

Misfolded alpha-synuclein (αSyn) aggregates are a hallmark event in Parkinson's disease (PD) and other synucleinopathies. Recently, αSyn seed amplification assays (αSyn-SAAs) have shown promise as a test for biochemical diagnosis of synucleinopathies. αSyn-SAAs use the intrinsic self-replicative nature of misfolded αSyn aggregates (seeds) to multiply them in vitro. In these assays, αSyn seeds circulating in biological fluids are amplified by a cyclical process that includes aggregate fragmentation into smaller self-propagating seeds, followed by elongation at the expense of recombinant αSyn (rec-αSyn). Amplification of the seeds allows detection by fluorescent dyes specific for amyloids, such as thioflavin T. Several αSyn-SAA reports have been published in the past under the names 'protein misfolding cyclic amplification' (αSyn-PMCA) and 'real-time quaking-induced conversion'. Here, we describe a protocol for αSyn-SAA, originally reported as αSyn-PMCA, which allows detection of αSyn aggregates in cerebrospinal fluid samples from patients affected by PD, dementia with Lewy bodies or multiple-system atrophy (MSA). Moreover, this αSyn-SAA can differentiate αSyn aggregates from patients with PD versus those from patients with MSA, even in retrospective samples from patients with pure autonomic failure who later developed PD or MSA. We also describe modifications to the original protocol introduced to develop an optimized version of the assay. The optimized version shortens the assay length, decreases the amount of rec-αSyn required and reduces the number of inconclusive results. The protocol has a hands-on time of ~2 h per 96-well plate and can be performed by personnel trained to perform basic experiments with specimens of human origin.

Fig. 1 |. Seeding nucleation mechanism and the principle behind the amplification of misfolded αSyn aggregates.

a, Schematic representation of the seeding/nucleation model of protein misfolding and aggregation. The seeding nucleation mechanism identifies three phases for formation of highly organized protein aggregates: the lag or nucleation phase, the growth or elongation phase and the stationary phase. During the lag phase, there is no observable aggregation, and the early misfolding events leading to formation of nuclei take place. Once enough nuclei have been formed, there is a rapid increase in the amount of observable aggregates, resulting in the formation of protofibrils and fibrils. In the stationary phase, the consumption of substrate slows down the aggregation, and the solution enters an equilibrium in which there is no further change in aggregation. If a seed is added to the aggregation process, the lag phase is decreased significantly, because the reaction bypasses the formation of nuclei. This figure aims only to be a schematic representation of the process and not to indicate the exact sizes of seeds, which are still unknown. b, Scheme for the αSyn-SAA reaction. Accelerated aggregation in the absence of self-aggregation is achieved by means of fragmentation and elongation of the endogenous αSyn seeds present in biological samples. Fragmentation effectively increases the number of active seeds, which is followed by elongation induced by quiescent incubation at 37 °C. During elongation the recombinant protein is converted into more aggregates, which are then fragmented again cyclically to amplify the misfolded protein biomarker. By the end of the reaction, the in vitro–generated aggregates represent the vast majority of the aggregates present in the solution, and most of the recombinant protein is consumed and incorporated into aggregates. The cyclic amplification of the biomarker allows its detection by conventional methods, such as thioflavin T fluorescence.

Fig. 2 |. Flowchart of the protocol.

The protocol depicted here consists of three main phases: preparation of basic assay reagents and consumables (green), acquisition or production of rec-αSyn substrate (blue) and implementation of the laboratory equipment used for the assay (yellow). Once those procedures and equipment are implemented, the protocol for the assay is very straightforward (orange). BSC, biosafety cabinet; DLS, dynamic light scattering; IMAC, immobilized metal affinity chromatography; QC, quality control; RT, room temperature; SEC, size-exclusion chromatography.

Fig. 3 |. Performance of the original αSyn-SAA for the detection of αSyn aggregates in CSF samples from patients with PD or MSA.

CSF samples (40 μl) from 10 donors with MSA, 10 donors with PD and 10 HC donors were analyzed in triplicate in a 96-well plate. The seed amplification assay was started by adding rec-αSyn monomers (1 mg/ml) and ThT (5 μm) in 100 mM PIPES pH 6.5 containing 500 mM NaCl. The plate was incubated at 37 °C with intermittent shaking at 500 rpm for 1 min every 30 min. The extent of aggregation was monitored by the increase in ThT fluorescence (excitation of 435 nm and emission of 485 nm). a, The graph illustrates the kinetics of aggregation measured by ThT fluorescence for a typical αSynSAA reaction in the presence of seeds coming from PD or MSA CSF, as well as HC samples. Data are displayed as the mean ± SEM for 10 patients. b, $F_{\text{max}}$measured at the plateau of aggregation. This parameter provides information about the amount of aggregates at the end of the assay as well as about structural differences between them, because different aggregates may have different modes of interaction with ThT, resulting in distinct fluorescence values. c, $T_{50}$corresponds to the time to reach 50% of maximum fluorescence. This parameter reflects the speed of the aggregation reaction. In a seeded assay, $T_{50}$provides information about the number of seeds present in the biological sample. Differences were analyzed by Student’s t test (****, P < 0.0001).

Fig. 4 |. Performance of optimized αSyn-SAA for the detection of αSyn aggregates in CSF samples of patients with PD.

CSF samples (40 μl) from three donors with PD and three HC donors were analyzed in triplicate in a 96-well plate. The seed amplification assay was performed as described in the text. The extent of aggregation was monitored by the increase in ThT fluorescence. Data are displayed as the mean ± SEM for three patients.