This study presents a semi-automated 3D volumetric immunofluorescence quantification method for intraneural S129 phospho-α-synuclein in skin biopsies that discriminates synucleinopathies (PD/MSA) from PSP with high AUC, sensitivity and specificity in a small cohort.

An objective, burden-sensitive peripheral biomarker for α-synuclein pathology can improve diagnostic accuracy, patient stratification, and target engagement/outcome measures in Parkinson's therapeutic development, though larger neuropathologically confirmed validation is required.

BACKGROUND: Parkinson's disease (PD) and multiple system atrophy (MSA) are synucleinopathies marked by α-synuclein aggregation, while progressive supranuclear palsy (PSP) is a tauopathy. Clinical overlap between these diseases complicates diagnosis. Detection of intraneural S129 phospho-α-synuclein (pαSyn) via immunofluorescence staining (IF) in skin biopsies shows diagnostic promise. However, prior studies rarely addressed differentiation between synucleinopathies and tauopathies and lacked assessment of varying pαSyn burden-particularly relevant for this aim. METHODS: In this cross-sectional study, we analyzed skin biopsies from 29 PD, 5 MSA, and 4 PSP patients. Samples obtained at C7 and Th12 were double-immunostained with pαSyn and PGP9.5, a pan-axonal neurite marker. RESULTS: Our novel method digitizes biopsy sections semi-automatically and performs computer-assisted 3D signal reconstruction. The resulting full volumetric quantification of intraneural pαSyn load enables burden-dependent test results based on ROC-derived cut-offs. Applied as a differential diagnostic test, it showed excellent discrimination of synucleinopathies from tauopathies, achieving AUCs of 0.912 (C7) and 0.934 (Th12), with 88.2% sensitivity and 100% specificity. Intraneural pαSyn load was significantly higher in PD and MSA compared to PSP (C7 p = 0.004; Th12 p = 0.002), with no difference between PD and MSA. CONCLUSIONS: This novel technique refines IF by increasing objectivity and allowing gradual pαSyn-burden assessment, offering potential as a confirmatory differential biomarker. Validation in larger, neuropathologically confirmed cohorts of these preliminary small-group results is warranted to fully evaluate the diagnostic and prognostic potential.