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

Misfolded alpha-synuclein (alpha Syn) aggregates are a hallmark event in Parkinson's disease (PD) and other synucleinopathies. Recently, alpha Syn seed amplification assays (alpha Syn-SAAs) have shown promise as a test for biochemical diagnosis of synucleinopathies. alpha Syn-SAAs use the intrinsic self-replicative nature of misfolded alpha Syn aggregates (seeds) to multiply them in vitro. In these assays, alpha Syn 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 alpha Syn (rec-alpha Syn). Amplification of the seeds allows detection by fluorescent dyes specific for amyloids, such as thioflavin T. Several alpha Syn-SAA reports have been published in the past under the names 'protein misfolding cyclic amplification' (alpha Syn-PMCA) and 'real-time quaking-induced conversion'. Here, we describe a protocol for alpha Syn-SAA, originally reported as alpha Syn-PMCA, which allows detection of alpha Syn aggregates in cerebrospinal fluid samples from patients affected by PD, dementia with Lewy bodies or multiple-system atrophy (MSA). Moreover, this alpha Syn-SAA can differentiate alpha Syn 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-alpha Syn required and reduces the number of inconclusive results. The protocol has a hands-on time of similar to 2 h per 96-well plate and can be performed by personnel trained to perform basic experiments with specimens of human origin.

Automated summary

Misfolded alpha-synuclein aggregates can be amplified in vitro using alpha Syn seed amplification assays, which have shown promise as a biochemical test for synucleinopathies. This assay can detect alpha Syn aggregates in cerebrospinal fluid samples from patients with Parkinson's disease, dementia with Lewy bodies, or multiple-system atrophy, and distinguish between samples from patients with Parkinson's disease and those with multiple-system atrophy.