Disease-Specific α-Synuclein Seeding in Lewy Body Disease and Multiple System Atrophy Are Preserved in Formaldehyde-Fixed Paraffin-Embedded Human Brain

Ain Kim; Ivan Martinez-Valbuena; Jun Li; Anthony E Lang; Gabor G Kovacs

doi:10.3390/biom13060936

Disease-Specific α-Synuclein Seeding in Lewy Body Disease and Multiple System Atrophy Are Preserved in Formaldehyde-Fixed Paraffin-Embedded Human Brain

Biomolecules. 2023 Jun 2;13(6):936. doi: 10.3390/biom13060936.

Authors

Ain Kim^{1

2}, Ivan Martinez-Valbuena^{1

3}, Jun Li¹, Anthony E Lang^{1

3

4}, Gabor G Kovacs^{1

2

3

4

5}

Affiliations

¹ Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON M5T 0S8, Canada.
² Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada.
³ Krembil Brain Institute, University Health Network, Toronto, ON M5T 0S8, Canada.
⁴ Edmond J. Safra Program in Parkinson's Disease and the Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, Toronto, ON M5T 2S6, Canada.
⁵ Laboratory Medicine Program, University Health Network, Toronto, ON M5G 2C4, Canada.

Abstract

Recent studies have been able to detect α-synuclein (αSyn) seeding in formaldehyde-fixed paraffin-embedded (FFPE) tissues from patients with synucleinopathies using seed amplification assays (SAAs), but with relatively low sensitivity due to limited protein extraction efficiency. With the aim of introducing an alternative option to frozen tissues, we developed a streamlined protein extraction protocol for evaluating disease-specific seeding in FFPE human brain. We evaluated the protein extraction efficiency of different tissue preparations, deparaffinizations, and protein extraction buffers using formaldehyde-fixed and FFPE tissue of a single Lewy body disease (LBD) subject. Alternatively, we incorporated heat-induced antigen retrieval and dissociation using a commercially available kit. Our novel protein extraction protocol has been optimized to work with 10 sections of 4.5-µm-thickness or 2-mm-diameter micro-punch of FFPE tissue that can be used to seed SAAs. We demonstrated that extracted proteins from FFPE still preserve seeding potential and further show disease-specific seeding in LBD and multiple system atrophy. To the best of our knowledge, our study is the first to recapitulate disease-specific αSyn seeding behaviour in FFPE human brain. Our findings open new perspectives in re-evaluating archived human brain tissue, extending the disease-specific seeding assays to larger cohorts to facilitate molecular subtyping of synucleinopathies.

Keywords: FFPE; SAA; alpha-synuclein; protein extraction; seeding behaviour.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Brain / metabolism
Formaldehyde
Humans
Lewy Body Disease*
Multiple System Atrophy*
Paraffin
Paraffin Embedding / methods
Synucleinopathies*
alpha-Synuclein / metabolism

Substances

alpha-Synuclein
Paraffin
Formaldehyde

Grants and funding

This study was supported by the Edmond J Safra Philanthropic Foundation, the Krembil Foundation, the Maybank Foundation, and the Rossy Foundation. A.K. is supported by the Centre for Research in Neurodegenerative Disease (CRND) Graduate Student Endowment, Unilever/Lipton Graduate Fellowship Award in Neurosciences, and the Jesse Keshin Graduate Student Award. The funding bodies did not take part in the design of the study, in collection, analysis, or interpretation of data, or in writing the manuscript.