A BS TRACT: Background: Identification of a peripheral biomarker is a major roadblock in the diagnosis of PD. Immunohistological identification of p-serine 129 αsynuclein in the submandibular gland tissues of PD patients has been recently reported. Objective: We report on a proof-of-principle study for using an ultra-sensitive and specific, real-time quakinginduced conversion assay to detect pathological α-synuclein in the submandibular gland tissues of PD patients. Methods: The α-synuclein real-time quaking-induced conversion assay was used to detect and quantify pathological α-synuclein levels in PD, incidental Lewy body disease, and control submandibular gland tissues as well as in formalin-fixed paraffin-embedded sections. Results: We determined the quantitative seeding kinetics of pathological α-synuclein present in submandibular gland tissues from autopsied subjects using the α-synuclein real-time quaking-induced conversion assay. A total of 32 cases comprising 13 PD, 3 incidental Lewy body disease, and 16 controls showed 100% sensitivity and 94% specificity. Interestingly, both PD and incidental Lewy body disease tissues showed 100% concordance for elevated levels of pathological α-synuclein seeding activity compared to control tissues. End-point dilution kinetic analyses revealed that the submandibular gland had a wide dynamic range of pathological α-synuclein seeding activity. Conclusions: Our results are the first to demonstrate the utility of using the real-time quaking-induced conversion assay on peripherally accessible submandibular gland tissues and formalin-fixed paraffin-embedded tissue sections to detect PD-related pathological changes with high sensitivity and specificity. Additionally, the detection of seeding activity from incidental Lewy body disease cases containing immunohistochemically undetected pathological α-synuclein demonstrates the α-synuclein real-time quaking-induced conversion assay's potential utility for identifying prodromal PD in submandibular gland tissues.

A BS TRACT: Background: An unmet clinical need in Parkinson's disease (PD) is to identify biomarkers for diagnosis, preferably in peripherally accessible tissues such as skin. Immunohistochemical studies have detected pathological α-synuclein (αSyn) in skin biopsies from PD patients albeit sensitivity needs to be improved. Objective: Our study provides the ultrasensitive detection of pathological αSyn present in the skin of PD patients, and thus, pathological αSyn in skin could be a potential biomarker for PD. Methods: The real-time quaking-induced conversion assay was used to detect pathological αSyn present in human skin tissues. Further, we optimized this ultra-sensitive and specific assay for both frozen and formalin-fixed paraffinembedded sections of skin tissues. We determined the seeding kinetics of the αSyn present in the skin from autopsied subjects consisting of frozen skin tissues from 25 PD and 25 controls and formalin-fixed paraffinembedded skin sections from 12 PD and 12 controls. Results: In a blinded study of skin tissues from autopsied subjects, we correctly identified 24/25 PD and 24/25 controls using frozen skin tissues (96% sensitivity and 96% specificity) compared to 9/12 PD and 10/12 controls using formalin-fixed paraffinembedded skin sections (75% sensitivity and 83% specificity). Conclusions: Our blinded study results clearly demonstrate the feasibility of using skin tissues for clinical diagnosis of PD by detecting pathological αSyn. Moreover, this peripheral biomarker discovery study may have broader translational value in detecting misfolded proteins in skin samples as a longitudinal progression marker.

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