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

Nature Protocols volume 18, pages 1179–1196 (2023)Cite this article

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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.

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Fig. 1: Seeding nucleation mechanism and the principle behind the amplification of misfolded αSyn aggregates.
Fig. 2: Flowchart of the protocol.
Fig. 3: Performance of the original αSyn-SAA for the detection of αSyn aggregates in CSF samples from patients with PD or MSA.
Fig. 4: Performance of optimized αSyn-SAA for the detection of αSyn aggregates in CSF samples of patients with PD.

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Data availability

The authors declare that the main data discussed in this protocol are available in the supporting primary research papers16,21. The raw datasets are available for research purposes from the corresponding authors upon reasonable request.

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Acknowledgements

We thank various members of the laboratory who contributed to the identification and testing of the experimental conditions described here. This work was partially supported by grants from the NIH (R01AG055053, R01AG061069 and R01AG059321) to C.S., R21NS114884 to M.S., and R01 NS119689 to M.S. and S.P., as well as grants from the Michael J. Fox Foundation for Parkinson’s disease to C.S. and S.P., and a grant from the American Parkinson Disease Association to M.S.

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Authors and Affiliations

  1. R&D Unit, Amprion Inc., San Diego, CA, USA

    Luis Concha-Marambio, Carly M. Farris & Claudio Soto

  2. Mitchell Center for Alzheimer’s Disease and Related Brain Disorders, University of Texas McGovern Medical School, Houston, TX, USA

    Sandra Pritzkow, Mohammad Shahnawaz & Claudio Soto

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  1. Luis Concha-Marambio
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Contributions

L.C.-M., C.M.F, S.P. and M.S. contributed to the development of the protocols. With the help of S.P., C.M.F. and M.S., L.C.-M. wrote the first draft of the article. C.S. is the principal investigator who provided funding and supervision for this research. C.S. produced the final version of the article.

Corresponding author

Correspondence to Claudio Soto.

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Competing interests

L.C.-M. and C.M.F. are employees of Amprion Inc., a biotechnology company that focuses on the commercial use of SAAs (PMCA and RT-QuIC) for high-sensitivity detection of misfolded protein aggregates. C.S. is a Founder, Chief Scientific Officer and Member of the Board of Directors of Amprion Inc. The University of Texas Health Science Center at Houston has licensed patents and patent applications to Amprion. The other authors declare no competing interests.

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Key references using this protocol

Shahnawaz, M. et al. Nature 578, 273–277 (2020): https://doi.org/10.1038/s41586-020-1984-7

Shahnawaz, M. et al. JAMA Neurol. 74, 163–172 (2017): https://doi.org/10.1001/jamaneurol.2016.4547

Kang, U. J. et al. Mov. Disord. 34, 536–544 (2019): https://doi.org/10.1002/mds.27646

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Concha-Marambio, L., Pritzkow, S., Shahnawaz, M. et al. Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid. Nat Protoc 18, 1179–1196 (2023). https://doi.org/10.1038/s41596-022-00787-3

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