Refining α-synuclein seed amplification assays to distinguish Parkinson’s disease from multiple system atrophy

Abstract Background Parkinson’s disease (PD) and multiple system atrophy (MSA) are two distinct α-synucleinopathies traditionally differentiated through clinical symptoms. Early diagnosis of MSA is problematic, and seed amplification assays (SAAs), such as real-time quaking-induced conversion (RT-Qu...

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Main Authors: James A. Wiseman, Clinton P. Turner, Richard L. M. Faull, Glenda M. Halliday, Birger Victor Dieriks
Format: Article
Language:English
Published: BMC 2025-02-01
Series:Translational Neurodegeneration
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Online Access:https://doi.org/10.1186/s40035-025-00469-6
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author James A. Wiseman
Clinton P. Turner
Richard L. M. Faull
Glenda M. Halliday
Birger Victor Dieriks
author_facet James A. Wiseman
Clinton P. Turner
Richard L. M. Faull
Glenda M. Halliday
Birger Victor Dieriks
author_sort James A. Wiseman
collection DOAJ
description Abstract Background Parkinson’s disease (PD) and multiple system atrophy (MSA) are two distinct α-synucleinopathies traditionally differentiated through clinical symptoms. Early diagnosis of MSA is problematic, and seed amplification assays (SAAs), such as real-time quaking-induced conversion (RT-QuIC), offer the potential to distinguish these diseases through their underlying α-synuclein (α-Syn) pathology and proteoforms. Currently, SAAs provide a binary result, signifying either the presence or absence of α-Syn seeds. To enhance the diagnostic potential and biological relevance of these assays, there is a pressing need to incorporate quantification and stratification of α-Syn proteoform-specific aggregation kinetics into current SAA pipelines. Methods Optimal RT-QuIC assay conditions for α-Syn seeds extracted from PD and MSA patient brains were determined, and assay kinetics were assessed for α-Syn seeds from different pathologically relevant brain regions (medulla, substantia nigra, hippocampus, middle temporal gyrus, and cerebellum). The conformational profiles of disease- and region-specific α-Syn proteoforms were determined by subjecting the amplified reaction products to concentration-dependent proteolytic digestion with proteinase K. Results Using our protocol, PD and MSA could be accurately delineated using proteoform-specific aggregation kinetics, including α-Syn aggregation rate, maximum relative fluorescence, the gradient of amplification, and core protofilament size. MSA cases yielded significantly higher values than PD cases across all four kinetic parameters in brain tissues, with the MSA-cerebellar phenotype having higher maximum relative fluorescence than the MSA-Parkinsonian phenotype. Statistical significance was maintained when the data were analysed regionally and when all regions were grouped. Conclusions Our RT-QuIC protocol and analysis pipeline can distinguish between PD and MSA, and between MSA phenotypes. MSA α-Syn seeds induce faster propagation and exhibit higher aggregation kinetics than PD α-Syn, mirroring the biological differences observed in brain tissue. With further validation of these quantitative parameters, we propose that SAAs could advance from a yes/no diagnostic to a theranostic biomarker that could be utilised in developing therapeutics.
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spelling doaj-art-2a93c7805d8448549110597f39eb19cb2025-02-09T12:52:05ZengBMCTranslational Neurodegeneration2047-91582025-02-0114111810.1186/s40035-025-00469-6Refining α-synuclein seed amplification assays to distinguish Parkinson’s disease from multiple system atrophyJames A. Wiseman0Clinton P. Turner1Richard L. M. Faull2Glenda M. Halliday3Birger Victor Dieriks4Department of Anatomy and Medical Imaging, University of AucklandLabPlus, Department of Anatomical PathologyDepartment of Anatomy and Medical Imaging, University of AucklandBrain and Mind Centre & Faculty of Medicine and Health School of Medical Sciences, The University of SydneyDepartment of Anatomy and Medical Imaging, University of AucklandAbstract Background Parkinson’s disease (PD) and multiple system atrophy (MSA) are two distinct α-synucleinopathies traditionally differentiated through clinical symptoms. Early diagnosis of MSA is problematic, and seed amplification assays (SAAs), such as real-time quaking-induced conversion (RT-QuIC), offer the potential to distinguish these diseases through their underlying α-synuclein (α-Syn) pathology and proteoforms. Currently, SAAs provide a binary result, signifying either the presence or absence of α-Syn seeds. To enhance the diagnostic potential and biological relevance of these assays, there is a pressing need to incorporate quantification and stratification of α-Syn proteoform-specific aggregation kinetics into current SAA pipelines. Methods Optimal RT-QuIC assay conditions for α-Syn seeds extracted from PD and MSA patient brains were determined, and assay kinetics were assessed for α-Syn seeds from different pathologically relevant brain regions (medulla, substantia nigra, hippocampus, middle temporal gyrus, and cerebellum). The conformational profiles of disease- and region-specific α-Syn proteoforms were determined by subjecting the amplified reaction products to concentration-dependent proteolytic digestion with proteinase K. Results Using our protocol, PD and MSA could be accurately delineated using proteoform-specific aggregation kinetics, including α-Syn aggregation rate, maximum relative fluorescence, the gradient of amplification, and core protofilament size. MSA cases yielded significantly higher values than PD cases across all four kinetic parameters in brain tissues, with the MSA-cerebellar phenotype having higher maximum relative fluorescence than the MSA-Parkinsonian phenotype. Statistical significance was maintained when the data were analysed regionally and when all regions were grouped. Conclusions Our RT-QuIC protocol and analysis pipeline can distinguish between PD and MSA, and between MSA phenotypes. MSA α-Syn seeds induce faster propagation and exhibit higher aggregation kinetics than PD α-Syn, mirroring the biological differences observed in brain tissue. With further validation of these quantitative parameters, we propose that SAAs could advance from a yes/no diagnostic to a theranostic biomarker that could be utilised in developing therapeutics.https://doi.org/10.1186/s40035-025-00469-6α-SynucleinParkinson’s diseaseMultiple system atrophySeed amplification assaysα-Synuclein strainsConformational variability
spellingShingle James A. Wiseman
Clinton P. Turner
Richard L. M. Faull
Glenda M. Halliday
Birger Victor Dieriks
Refining α-synuclein seed amplification assays to distinguish Parkinson’s disease from multiple system atrophy
Translational Neurodegeneration
α-Synuclein
Parkinson’s disease
Multiple system atrophy
Seed amplification assays
α-Synuclein strains
Conformational variability
title Refining α-synuclein seed amplification assays to distinguish Parkinson’s disease from multiple system atrophy
title_full Refining α-synuclein seed amplification assays to distinguish Parkinson’s disease from multiple system atrophy
title_fullStr Refining α-synuclein seed amplification assays to distinguish Parkinson’s disease from multiple system atrophy
title_full_unstemmed Refining α-synuclein seed amplification assays to distinguish Parkinson’s disease from multiple system atrophy
title_short Refining α-synuclein seed amplification assays to distinguish Parkinson’s disease from multiple system atrophy
title_sort refining α synuclein seed amplification assays to distinguish parkinson s disease from multiple system atrophy
topic α-Synuclein
Parkinson’s disease
Multiple system atrophy
Seed amplification assays
α-Synuclein strains
Conformational variability
url https://doi.org/10.1186/s40035-025-00469-6
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