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

Abstract

Importance: Deposition of the pathological α-synuclein (αSynP) in the brain is the hallmark of synucleinopathies, including Parkinson disease (PD), Lewy body dementia (LBD), and multiple system atrophy (MSA). Whether real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays can sensitively detect skin biomarkers for PD and non-PD synucleinopathies remains unknown.

Objective: To develop sensitive and specific skin biomarkers for antemortem diagnosis of PD and other synucleinopathies.

Design, setting, and participants: This retrospective and prospective diagnostic study evaluated autopsy and biopsy skin samples from neuropathologically and clinically diagnosed patients with PD and controls without PD. Autopsy skin samples were obtained at 3 medical centers from August 2016 to September 2019, and biopsy samples were collected from 3 institutions from August 2018 to November 2019. Based on neuropathological and clinical diagnoses, 57 cadavers with synucleinopathies and 73 cadavers with nonsynucleinopathies as well as 20 living patients with PD and 21 living controls without PD were included. Specifically, cadavers and participants had PD, LBD, MSA, Alzheimer disease, progressive supranuclear palsy, or corticobasal degeneration or were nonneurodegenerative controls (NNCs). A total of 8 approached biopsy participants either refused to participate in or were excluded from this study due to uncertain clinical diagnosis. Data were analyzed from September 2019 to April 2020.

Main outcomes and measures: Skin αSynP seeding activity was analyzed by RT-QuIC and PMCA assays.

Results: A total of 160 autopsied skin specimens from 140 cadavers (85 male cadavers [60.7%]; mean [SD] age at death, 76.8 [10.1] years) and 41 antemortem skin biopsies (27 male participants [66%]; mean [SD] age at time of biopsy, 65.3 [9.2] years) were analyzed. RT-QuIC analysis of αSynP seeding activity in autopsy abdominal skin samples from 47 PD cadavers and 43 NNCs revealed 94% sensitivity (95% CI, 85-99) and 98% specificity (95% CI, 89-100). As groups, RT-QuIC also yielded 93% sensitivity (95% CI, 85-97) and 93% specificity (95% CI, 83-97) among 57 cadavers with synucleinopathies (PD, LBD, and MSA) and 73 cadavers without synucleinopathies (Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, and NNCs). PMCA showed 82% sensitivity (95% CI, 76-88) and 96% specificity (95% CI, 85-100) with autopsy abdominal skin samples from PD cadavers. From posterior cervical and leg skin biopsy tissues from patients with PD and controls without PD, the sensitivity and specificity were 95% (95% CI, 77-100) and 100% (95% CI, 84-100), respectively, for RT-QuIC and 80% (95% CI, 49-96) and 90% (95% CI, 60-100) for PMCA.

Conclusions and relevance: This study provides proof-of-concept that skin αSynP seeding activity may serve as a novel biomarker for antemortem diagnoses of PD and other synucleinopathies.

Figure 1.. Immunohistochemistry (IHC) and Immunofluorescence (IF) Microscopy of Pathological α-Synuclein (αSyn^P)

A, IHC of skin tissue section from a Parkinson disease (PD) cadaver stained with the pS129-Syn antibody for phosphorylated αSyn^P. B, IHC of skin tissue section from a non-PD control cadaver stained with the pS129-Syn antibody for phosphorylated αSyn^P. C, IHC of brain tissue section from a PD cadaver stained with the pS129-Syn antibody for phosphorylated αSyn^P. D, IF microscopy of skin tissue section from a patient with PD stained with the pS129-Syn antibody. E, IF microscopy of skin tissue section from a patient with PD stained with the PGP9.5 antibody directed against an axonal marker PGP9.5 protein. F, Merged image of IF microscopy of skin tissue section from a patient with PD stained with the pS129-Syn antibody and PGP9.5 antibody. G, IF microscopy of skin tissue section from a control without PD stained with the ps129-Syn antibody. H, IF microscopy of skin tissue section from a control without PD stained with the PGP9.5 protein. I, Merged image of IF microscopy of skin tissue section from a control without PD stained with the pS129-Syn antibody and PGP9.5 antibody.

Figure 2.. Detection of Pathological α-Synuclein (αSyn^P) Seeding Activity in Autopsy Skin Samples From Parkinson Disease (PD) Cadavers by Real-Time Quaking-Induced Conversion (RT-QuIC) or Protein Misfolding Cyclic Amplification (PMCA) Assay

A, Dose-dependent αSyn^P seeding activity in the abdominal skin homogenate from a PD cadaver by RT-QuIC assay. Abdominal skin homogenate from a control was used as a negative control. The skin homogenates were diluted to 10⁻³ through 10⁻⁶ dilution. Commercially purchased recombinant human αSyn (rPeptide) was used. B, RT-QuIC detection of αSyn^P seeding activity in the abdominal skin homogenates from 20 patients with PD and 4 controls with PD. Synthesized in-house recombinant human αSyn was used. C, RT-QuIC detection of αSyn^P seeding activity in skin homogenates from the scalp skin tissues of 20 patients with PD and 10 additional controls without PD, all of which were coded for the blinded test. Commercially purchased recombinant human αSyn (rPeptide) was used. D, PMCA detection of αSyn^P seeding activity in the abdominal skin homogenates from 23 patients with PD and 7 controls without PD. Synthesized in-house recombinant human αSyn was used. All data were normalized to percentages of the maximal fluorescence response.

Figure 3.. Comparison of Pathological α-Synuclein (αSyn^P) Seeding Activity in Autopsy Skin Samples From Cadavers With Synucleinopathies (SOPs), Tauopathies, or Nonneurodegenerative Controls (NNCs) by Real-Time Quaking-Induced Conversion (RT-QuIC) and Protein Misfolding Cyclic Amplification (PMCA) Assays

A, Scatter graph of RT-QuIC thioflavin T (ThT) fluorescence intensity at 60 hours of αSyn^P seeding activity of abdominal skin samples from cadavers with SOPs, including 47 Parkinson disease (PD) cadavers, 3 multiple system atrophy (MSA) cadavers, and 7 Lewy body dementia (LBD) cadavers, as well as non-SOPs, including 17 Alzheimer disease (AD) cadavers, 8 progressive supranuclear palsy (PSP) cadavers, 5 corticobasal degeneration (CBD) cadavers, and 43 nonneurodegenerative controls (NNCs). B and C, Receiver operating characteristic (ROC) curves and derived area under the curve (AUC) calculations for αSyn^P comparisons between PD and NNC cadavers and between SOP and non-SOP cadavers. D, Scatter graph of PMCA ThT fluorescence intensity at 100 hours of αSyn^P seeding activity of abdominal skin samples from cadavers with SOPs, including 24 PD cadavers, 3 MSA cadavers, and 5 LBD cadavers, as well as non-SOPs, including 5 PSP cadavers, 5 CBD cadavers, and 8 NNC cadavers. E and F, ROC curve and AUC calculation for comparisons of αSyn^P seeding activity between PD and NNC cadavers and between SOP and non-SOP cadavers. The dotted lines indicate the threshold defining the positive and negative cases. Commercially purchased recombinant human αSyn was used. ^aP < .001.

Figure 4.. Detection of Pathological α-Synuclein (αSyn^P) Seeding Activity in Biopsy Skin Samples From Living Patients With Parkinson Disease (PD) and Controls Without PD by Real-Time Quaking-Induced Conversion (RT-QuIC) and Protein Misfolding Cyclic Amplification (PMCA) Assays

A, Representative αSyn^P RT-QuIC spectra of biopsy skin samples from 2 posterior neck areas (cervical samples 1 and 2) and 2 leg areas (leg samples 1 and 2) of a living patient with PD. Positive autopsy skin controls from PD cadavers (positive samples 1 and 2) and negative autopsy controls from non-PD cadavers (negative samples 1, 2, and 3) were included. Data were normalized to percentage of the maximal fluorescence response. B, Scatter graph of maximal thioflavin T (ThT) fluorescence percentage at 60 hours of the αSyn^P seeding activity of the posterior neck or leg biopsy skin samples from 20 living patients with PD and 21 living controls without PD by RT-QuIC assay. C, ROC curve and AUC calculation for comparison of αSyn^P seeding activity between patients with PD and controls without PD. D, Scatter graph of maximal ThT fluorescence percentage at 100 hours of αSyn^P seeding activity of the posterior neck or leg biopsy skin samples from 10 living patients with PD and 10 living controls without PD by PMCA. E, ROC curve and AUC calculation between PD and non-PD controls. The dotted lines represent the thresholds defining the samples with positive vs negative findings. Commercially purchased recombinant human αSyn was used. ^aP < .001.