Toward the quantification of α-synuclein aggregates with digital seed amplification assays

• 成果类型: Article
• 署名作者: Gilboa, Tal; Swank, Zoe; Thakur, Rohan; Gould, Russell A.; Ooi, Kean Hean; Norman, Maia; Flynn, Elizabeth A.; Deveney, Brendan T.; Chen, Anqi; Borberg, Ella; Kuzkina, Anastasia; Ndayisaba, Alain; Khurana, Vikram; Weitz, David A.; Walt, David R.
• 署名单位: Harvard University; Harvard University Medical Affiliates; Brigham & Women's Hospital; Harvard University; Harvard University; Harvard Medical School; Harvard University; Harvard University; Harvard University; Harvard Medical School; Harvard University; Harvard University Medical Affiliates; Brigham & Women's Hospital; Harvard University; Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; Harvard University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-9844
• DOI: 10.1073/pnas.2312031121
• 发表日期: 2024-01-16
• 关键词: multiple system atrophy; generation; antibodies; proteins; disease

摘要

The quantification and characterization of aggregated alpha-synuclein in clinical samples offer immense potential toward diagnosing, treating, and better understanding neuro-degenerative synucleinopathies. Here, we developed digital seed amplification assays to detect single alpha-synuclein aggregates by partitioning the reaction into microcompartments. Using pre-formed alpha-synuclein fibrils as reaction seeds, we measured aggregate concentrations as low as 4 pg/mL. To improve our sensitivity, we captured aggregates on antibody-coated magnetic beads before running the amplification reaction. By first characterizing the pre-formed fibrils with transmission electron microscopy and size exclusion chromatography, we determined the specific aggregates targeted by each assay platform. Using brain tissue and cerebrospinal fluid samples collected from patients with Parkinson's Disease and multiple system atrophy, we demonstrated that the assay can detect endogenous pathological alpha-synuclein aggregates. Furthermore, as another application for these assays, we studied the inhibition of alpha-synuclein aggregation in the presence of small-molecule inhibitors and used a custom image analysis pipeline to quantify changes in aggregate growth and filament morphology.