Dermal phospho-alpha-synuclein deposits confirm REM sleep behaviour disorder as prodromal Parkinson's disease

Abstract

Phosphorylated alpha-synuclein (p-alpha-syn) deposits, one of the neuropathological hallmarks of Parkinson's disease (PD), have recently been detected in dermal nerve fibres in PD patients with good specificity and sensitivity. Here, we studied whether p-alpha-syn may serve as a biomarker in patients with a high risk of developing PD, such as those with REM sleep behaviour disorder (RBD). We compared the presence and distribution of p-alpha-syn deposits in dermal nerve fibres in 18 patients with RBD, 25 patients with early PD and 20 normal controls. Skin biopsy was taken at C7, Th10, and the upper and lower leg. Presynaptic dopamine transporter imaging using FP-CIT-SPECT was performed in all patients with RBD and in 11 patients with PD. All RBD patients underwent olfactory function testing. The likelihood ratio (LR) for prodromal PD was calculated for each patient based on published research criteria. Skin serial sections were assessed by double-immunofluorescence labelling with antibodies to pSer129-alpha-syn under blinded conditions. P-alpha-syn was visualized in 10/18 patients with RBD (sensitivity of 55.6%) and in 20/25 early PD patients (sensitivity of 80%) but in none of the controls (specificity of 100%). The percentage of dermal structures innervated by p-alpha-syn-positive fibres was negatively correlated with dopamine transporter binding in the FP-CIT-SPECT (ρ = -0.377, p = 0.048), with olfactory function (ρ = -0.668, p = 0.002), and positively correlated with the total LR for RBD to present prodromal PD (ρ = 0.531, p = 0.023). Dermal p-alpha-syn can be considered a peripheral histopathological marker of synucleinopathy and can be detected in a subgroup of RBD patients presumably representing prodromal PD. Dermal p-alpha-syn is detectable in RBD patients without PD motor symptoms, thereby stratifying a patient group that is of great interest for clinical trials testing disease-modifying drugs.

Keywords: Alpha-synuclein; FP-CIT-SPECT; Parkinson’s disease; REM sleep behaviour disorder; Skin biopsy.

Fig. 1

Immunofluorescence staining with anti-p-alpha-syn (red) and anti-PGP9.5 (green, c). Dermal p-alpha-syn deposition was found within axons of somatosensory or autonomic nerve fibres (b) and resembled Lewy neurites that are typically found in the CNS (a). Lewy bodies as seen in CNS tissue (a, arrow) are not detectable in skin biopsies as the cell bodies of somatosensory neurons are located in the dorsal root ganglia and sympathetic ganglia. P-alpha-syn immunoreactivity forms a punctuated line within the nerve fibres. c illustrates the localization of p-alpha-syn deposition within a nerve fibre of a dermal nerve bundle. Scale bar 10 µm p-alpha-syn phosphorylated alpha-synuclein

Fig. 2

Confocal photomicrographs of double-immunofluorescence with anti-p-alpha-syn (red) and anti-PGP 9.5 (axonal marker, green). P-alpha-syn -immunoreactive nerve fibres can be found in vasomotor nerve fibres around vessels (a, b), in somatosensory nerve fibres of the subepidermal plexus (c, d), in pilomotor nerve fibres of erector pilorum muscles (e, f) and in sudomotor fibres around sweat glands (g, h). Scale bar 20 µm. p-alpha-syn phosphorylated alpha-synuclein

Fig. 3

Box plots illustrating skin biopsy, FP-CIT-SPECT, LR and TDI data. The box plots of the upper panels a–c illustrate the median percentage of p-alpha-syn-positive dermal nerve structures per patient (y-axis) in patients with abnormal/normal FP-CIT-SPECT (a), normosmic (TDI > 26)/hyposmic (TDI ≤ 26) patients (b) and patients fulfilling/not fulfilling the research MDS criteria of prodromal PD (x-axis) (c). In a, upper panel, PD patients are included, all other panels only represent the RBD group. The black line marks the median, the box represents the quartiles, the whiskers mark the range. Individual patient values are represented by circles. The percentage of p-alpha-syn-positive dermal structures shows a trend towards higher numbers in PD, declining from PD to RBD with abnormal, FP-CIT-SPECT (i.e. reduced specific to non-specific binding ratio) and further to RBD with normal FP-CIT-SPECT (a). P-alpha-syn deposition is not found in normosmic patients, but is high in hyposmic patients (b) and is much more abundant in patients fulfilling the MDS criteria of prodromal PD (c). The box plots of the lower images a–c compare the lowest putaminal FP-CIT-SPECT values, TDI values and the probability of prodromal PD (y-axis) in patients without (negative) and with (positive) p-alpha-syn deposition in the skin biopsy (x-axis). Putaminal FP-CIT-SPECT values tend to be lower in patients with at least one p-alpha-syn deposition (positive) vs in patients with no deposition (negative) (a). In a the punctate line indicates the lower normal value (2.0) of the FP-CIT-SPECT value. TDI values (b) are significantly lower in patients with p-alpha-syn deposits in the skin biopsy and the probability of prodromal PD is higher in patients with p-alpha-syn deposition (c). In a, lower panel, only 17 individual patient values are represented by circles as the FP-CIT-SPECT of one patient was not included into the quantitative analysis due to lack of raw data. FP-CIT-SPECT ¹²³I-2beta-carbomethoxy-3beta-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane single photon emission computed tomography, p-alpha-syn phosphorylated alpha-synuclein, PD Parkinson’s disease, RBD REM sleep behaviour disorder, TDI olfactory threshold, discrimination and identification score