Objective: Assess seed amplification assay (SAA) and immunohistochemistry (IHC) in peripheral gut mucosal biospecimens as PD diagnostics.

Background: SAA detects misfolded alpha-synuclein (α-syn) aggregates across biospecimens (CSF, skin, blood)^1-5. Despite its sensitivity, the biological source of seeds and their relation to Lewy body pathology (LBP) remain unclear.

Method: We applied in vitro SAA, in situ (iSAA), and IHC to peripheral and central nervous system tissues from age/race/gender-matched healthy controls (HC), PD and multiple systems atrophy (MSA). PD patients were stratified by severity: mild (non-medicated), moderate (medicated), both Hoehn and Yahr (H&Y) I–II; and severe (medicated) H&Y ≥3^6,7. IHC for proteinase K-resistant phosphorylated α-syn (PS129) was conducted on sigmoid, duodenum, and midbrain (HC, PD). iSAA and IHC were applied to PD midbrain and olfactory bulb (OB) to explore α-syn seed origins. A blinded rater evaluated IHC images.

Results: SAA sensitivity for PD was moderate: ileum 49% (18/37) [Figure1], sigmoid 64% (36/56) [Figure2], and duodenum 62.5% (5/8); 71.4% (5/7) MSA sigmoid. Specificity was high: 2.5% (1/40) HC sigmoid positive. Ileum SAA rates were stable across PD severity (mild 50%, moderate 48%, severe 50%) [Figure1], while sigmoid rates increased with PD severity (mild 11%, moderate 71%, severe 81%) [Figure2]. Sigmoid IHC showed low PD sensitivity (26%, n=38) but high specificity (0% HC), with rates increasing modestly with PD severity (mild 0%, moderate 23%, severe 43%) [Figure3], but remaining lower than SAA. To increase sampling rate, IHC was performed on full 4 mm sigmoid biopsies (regular endoscopy biopsy forceps) in select SAA-positive cases, but sensitivity did not improve. In PD brains, iSAA detected seeding throughout the midbrain, and OB linked to neuromelanin pigments and possibly lipofuscin, as well as some Lewy bodies/Lewy neurites [Figure4]. PS129-positive Lewy pathology was more abundant overall.

Conclusion: Despite the assumption that LBP is the primary source of α-syn seeds, SAA and LBP markers are discordant. Gut mucosa SAA shows promise as a PD biomarker, while IHC has limited sensitivity, likely due to low sampling and sparse PS129-positive aggregates. Future studies should examine the spatial overlap between α-syn seeds and aggregate markers in the peripheral and central nervous systems.

α-Syn Seed Assay in PD Ileal Biopsies

α-Syn Seed Assay in PD Sigmoid Biopsies

PK-Resistant pS129 in PD Sigmoid Biopsies

iSAA & PK-Resistant pS129 in PD & MSA Brains

References: 1 Gibbons, C. H. et al. Skin Biopsy Detection of Phosphorylated α-Synuclein in Patients With Synucleinopathies. Jama 331, 1298–1306 (2024). https://doi.org/10.1001/jama.2024.0792
2 Wang, Z. et al. Skin α-Synuclein Aggregation Seeding Activity as a Novel Biomarker for Parkinson Disease. JAMA Neurol 78, 1–11 (2020). https://doi.org/10.1001/jamaneurol.2020.3311
3 Zheng, Y. et al. Comparison of biospecimens for α-synuclein seed amplification assays in Parkinson’s disease: A systematic review and network meta-analysis. Eur J Neurol 30, 3949–3967 (2023). https://doi.org/10.1111/ene.16041
4 Soto, C. α-Synuclein seed amplification technology for Parkinson’s disease and related synucleinopathies. Trends Biotechnol 42, 829–841 (2024). https://doi.org/10.1016/j.tibtech.2024.01.007
5 Otero-Jimenez, M. et al. Novel in situ seeding immunodetection assay uncovers neuronal-driven alpha-synuclein seeding in Parkinson’s disease. npj Parkinson’s Disease 11, 259 (2025). https://doi.org/10.1038/s41531-025-01111-y
6 Goetz, C. G. et al. Movement Disorder Society Task Force report on the Hoehn and Yahr staging scale: status and recommendations. Mov Disord 19, 1020–1028 (2004). https://doi.org/10.1002/mds.20213
7 Skorvanek, M. et al. Differences in MDS-UPDRS Scores Based on Hoehn and Yahr Stage and Disease Duration. Mov Disord Clin Pract 4, 536–544 (2017). https://doi.org/10.1002/mdc3.12476

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MDS Abstracts - https://www.mdsabstracts.org/abstract/seed-amplification-assay-is-more-sensitive-to-detecting-%ce%b1-synuclein-in-gut-biopsies-than-immunohistochemistry/