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Lookup NU author(s): Dr Jon Marles-WrightORCiD, Dr David Koss, Professor Alan ThomasORCiD, Professor Tiago OuteiroORCiD, Dr Ahmad Khundakar
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 2025 The Authors. Published by American Chemical Society.The aggregation of α-synuclein is crucial to the development of Lewy body diseases, including Parkinson’s disease and dementia with Lewy bodies. The aggregation pathway of α-synuclein typically involves a defined sequence of nucleation, elongation, and secondary nucleation, exhibiting prion-like spreading. This study employed Raman spectroscopy and machine learning analysis, alongside complementary techniques, to characterize the biomolecular changes during the fibrillation of purified recombinant wild-type α-synuclein protein. Monomeric α-synuclein was produced, purified, and subjected to a 7-day fibrillation assay to generate preformed fibrils. Stages of α-synuclein fibrillation were analyzed using Raman spectroscopy, with aggregation confirmed through negative staining transmission electron microscopy, mass spectrometry, and light scattering analyses. A machine learning pipeline incorporating principal component analysis and uniform manifold approximation and projection was used to analyze the Raman spectral data and identify significant peaks, resulting in differentiation between sample groups. Notable spectral shifts in α-synuclein were found in various stages of aggregation. Early changes (D1) included increases in α-helical structures (1303, 1330 cm-1) and β-sheet formation (1045 cm-1), with reductions in COO- and CH2 bond regions (1406, 1445 cm-1). By D4, these structural shifts persist with additional β-sheet features. At D7, a decrease in β-sheet H-bonding (1625 cm-1) and tyrosine ring breathing (830 cm-1) indicates further structural stabilization, suggesting a shift from initial helical structures to stabilized β-sheets and aggregated fibrils. Additionally, alterations in peaks related to tyrosine, alanine, proline, and glutamic acid were identified, emphasizing the role of these amino acids in intramolecular interactions during the transition from α-helical to β-sheet conformational states in α-synuclein fibrillation. This approach offers insight into α-synuclein aggregation, enhancing the understanding of its role in Lewy body disease pathophysiology and potential diagnostic relevance.
Author(s): Coles NP, Elsheikh S, Quesnel A, Butler L, Jennings C, Tarzi C, Achadu OJ, Islam M, Kalesh K, Occhipinti A, Angione C, Marles-Wright J, Koss DJ, Thomas AJ, Outeiro TF, Filippou PS, Khundakar AA
Publication type: Article
Publication status: Published
Journal: ACS Chemical Neuroscience
Year: 2025
Pages: epub ahead of print
Online publication date: 28/01/2025
Acceptance date: 21/01/2025
Date deposited: 17/02/2025
ISSN (electronic): 1948-7193
Publisher: American Chemical Society
URL: https://doi.org/10.1021/acschemneuro.4c00726
DOI: 10.1021/acschemneuro.4c00726
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