Skin α-Synuclein Aggregation Seeding Activity as a Novel Biomarker for Parkinson Disease

Key Points Question Does the pathological α-synuclein (αSynP) detected by immunohistochemistry in the skin of individuals with Parkinson disease (PD) have aggregation seeding activity, and is skin αSynP seeding activity a potential biomarker for diagnosis of PD and other synucleinopathies? Findings In this diagnostic study including skin samples from 160 autopsies and 41 biopsies, a statistically significant increase in αSynP seeding activity was observed in individuals with PD and synucleinopathies compared with controls with tauopathies and nonneurodegenerative diseases. Meaning Skin αSynP has aggregation seeding activity in patients with PD and non-PD synucleinopathies and may be a biomarker for antemortem diagnosis of PD and other synucleinopathies.

set up the RT-QuIC and PMCA assay conditions and then blindly tested scalp skin samples from the same 20 PD while they were re-coded by the sample provider along with 10 additional coded non-PD samples. We subsequently validated our findings with an increased number of PD and control cases including other synucleinopathies and tauopathies. Finally, we tested our assays with all biopsy samples available.

RT-QuIC Analysis
The skin homogenates were spun at 2,000 g for 2 min at 4°C prior to making serial dilutions.
The 96-well plate was then sealed with a plate sealer film (Nalgene Nunc International) and incubated at 42°C in a BMG FLUOstar Omega plate reader with cycles of 1 min shaking (400 rpm double orbital) and 1 min rest throughout the indicated incubation time. ThT fluorescence measurements (450 +/-10 nm excitation and 480 +/-10 nm emission; bottom read) were taken every 45 minutes. Four replicate reactions were seeded with the same dilution of an individual sample. The average fluorescence values per sample were calculated using fluorescence values from all four replicate wells regardless of whether these values crossed the threshold described below. To compensate for minor differences in baselines between fluorescent plate readers and across multiple experiments, some data sets were normalized to a percentage of the maximal fluorescence response (260,000 rfu) of the plate readers as described, 27,28 and plotted versus different reaction times. Reactions were classified as RT-QuIC positive based on criteria similar to those previously described. 27,28 A ThT fluorescence threshold for a reaction to be considered positive was based on the mean ThT value of all negative control samples at 60 hours, plus 3 standard deviations yielding an ~27% threshold. At least 2 of 4 replicate wells must cross this threshold and lag phase less than 50 hours for a sample to be considered positive.

PMCA Analysis
At various time points, a 2 µL of sample aliquot was removed and incubated with 198 µL ThT solution (20 µM ThT, 50 mM glycine, pH 8.5) in a 96-well plate (Greiner Bio-One) for 5 minutes at room temperature. ThT readings were performed on a BioTek plate reader with an optimized gain of 100 (excitation at 440 nm and emission at 480 nm).

Immunohistochemistry and Immunofluorescence staining
Immunohistochemistry and immunofluorescence staining were conducted as previously described. 41 Antigen retrieval with pressure cooking using decloaking buffer (Biocare) or with 80% formic acid for 30 min was performed prior to blocking with 10% normal goat serum (NGS). Primary antibodies (PGP9.5, Genetex; and alpha synuclein Ser 129, BioLegend) were incubated overnight. For immunohistochemical staining, goat-anti-mouse or goat-anti-rabbit secondary antibodies (Millipore) were incubated for 30 minutes followed by a 60 min incubation in either mouse or rabbit peroxidase-anti-peroxidase (Jackson ImmunoResearch). After rinsing in Tis buffer (50 mM Tris, pH = 7.6) sections were developed with DAB reagent (Biocare), dehydrated and cover-slipped. For double label immunofluorescence, sections were rehydrated and primary antibodies were incubated together overnight. After rinsing in PBS, Alexafluor 488 and 568 species specific conjugated secondary antibodies (Invitrogen) were incubated at RT for 1 hr. After 3 rinses in PBS, sections were subjected to autofluorescence quenching step (Vector TrueVIEW kit), and cover slipped with Vectashield with DAPI mounting media. Images were obtained on a Zeiss Axiovert.