Cervical skin denervation associates with alpha-synuclein aggregates in Parkinson disease

Abstract

Objective: Autonomic nervous system is involved at the onset of Parkinson disease (PD), and alpha-synuclein (α-Syn) and its phosphorylated form (p-αSyn) have been detected in dermal autonomic nerve fibers of PD. We assessed disease specific conformation variant of α-Syn immunoreactivity in cutaneous nerves and characterized skin denervation patterns in PD and atypical parkinsonism (AP).

Methods: We enrolled 49 subjects, 19 with PD, 17 age-matched healthy controls, and 13 with AP. The manifestations of disease were rated on clinical scales. Skin biopsies from ankle, thigh, and neck were analyzed by immunofluorescence for p-αSyn, 5G4 as a conformation specific antibody to pathogenic α-Syn and PGP9.5 as axonal marker. Intraepidermal nerve fiber density was measured in all anatomical sites as marker of neurodegeneration. Thirteen of the 19 PD underwent a 1 year follow-up visit plus skin biopsies.

Results: PD subjects displayed more severe cervical skin denervation (P < 0.03), which correlated to disease duration and worsened between initial and follow-up examination (P < 0.001). p-αSyn and 5G4 were equally sensitive and specific for the diagnosis of PD (area under the ROC was 0.839 for p-αSyn and 0.886 for 5G4). PD and AP with possible alpha-synucleinopathies share the features of marked cervical denervation and the presence of 5G4. In contrast AP with possible tauopathies were normal.

Interpretation: Conformational specific forms of α-Syn are detectable in skin biopsy by immunofluorescence in PD, with a promising diagnostic efficiency similar to p-αSyn. Cervical cutaneous denervation correlates with disease duration and increases over time standing out as a potential biomarker of PD progression.

Figure 1

Phosphorylated alpha synuclein deposits are more expressed in PD. Confocal images of immunofluorescence with PGP9.5 (green) and p‐αSyn (red) of dermal nerves around sweat glands in PD T0 (A–B), in AP‐SYN (D–E), AP‐TAU (G–H), and healthy subject (J–L). In yellow colocalization of p‐αSyn and PGP 9.5 (C,F,I,M) along axons. Scale bar 50 μm. White arrows indicate positive structures, asterisks indicate unspecific staining in non‐neuronal structures. The percentage of p‐αSyn is higher in PD T0 patients compared to other groups (P = 0.028) (N), but is not different among localizations (O). A major colocalization area p‐αSyn/PGP9.5 was measured in PD with a proximal to distal gradient (P). PD, Parkinson disease.

Figure 2

5G4 deposits are more expressed in PD and AP‐SYN patients. Confocal images of immunofluorescence with PGP9.5 (green) and 5G4 (red) of dermal nerves around sweat glands in PD T0 (A–B), AP‐SYN (D–E), AP‐TAU (G–H), and healthy subject (J–L). In yellow colocalization of 5G4 and PGP 9.5 (C,F,I,M) along axons. Scale bar 50 μm. White arrows indicate positive structures, asterisks indicate unspecific staining in non‐neuronal structures. The percentage of 5G4 is higher in PD T0 compared to HC and AP‐TAU (P _FDR  < 0.012), but not compared to AP‐SYN, and in AP‐SYN more than in AP‐TAU (P _FDR  = 0.04) (N); no significant differences among localizations but a tendency of higher 5G4 at cervical site in PD (O). A major colocalization area 5G4/PGP9.5 was measured in PD with a proximal to distal gradient (P).

Figure 3

ROC analysis for p‐αSyn and 5G4 in PD. Sensitivity and specificity tests with each antibody in each anatomical site. N° PD T0 and N° HC are the number of subjects for which each test is available (A). Comparison of the diagnostic yield of test performed with p‐αSyn (blue), 5G4 (red), both markers (green) and 5G4 in cervical area only (yellow) (B). Statistical power of the ROC curves was: p‐αSyn (0.946), 5G4 (0.987), both markers (0.987) and 5G4 in cervical area only (0.998).

Figure 4

Skin innervation: intraepidermal nerve fiber density (IENFD). Immunofluorescence staining with anti‐PGP9.5 (green) and DAPI (blue) of cervical skin in an healthy subject (A) and PD (C); in A the dotted white line shows the border between epidermis and dermis: IENFD is calculated as the number of nerve fibers (arrows) crossing the border per mm. Dermal nerve fibers around sweat glands are shown in an healthy subject (B) and PD (D). The pictures shows skin denervation in PD more evident at level of autonomic nerve fibers of sweat gland. Scale bar 50 μm.

Figure 5

Cervical skin denervation correlates to disease duration and significantly increases at 12 months follow‐up. IENFD is lower in PD T0 patients compare to AP‐TAU and HC subjects (P < 0.03), but not compared with AP‐SYN (A). Total IENFD is significantly reduced in PD females versus PD males (P = 0.012), in PD females versus HC females (P = 0.002) and versus AP‐TAU (P < 0.001) (B). In PD after 12 months IENFD is significantly lower at the thigh (P = 0.002) and at the cervical site (P = 0.001) (C). Western blot of skin lysates (an example) (D). Densitometry of PGP9.5 and β‐actin bands measured by LICOR software (n = 12 PD, n = 8 HC) PGP9.5 values normalized to β‐actin, show a major denervation at cervical site (P < 0.05) (E). PGP9.5 quantification negatively correlates to disease duration at cervical site in PD at T0 (F).

Figure 6

Cervical skin denervation in PD differs according to disease gravity. PD patients were divided in two groups based on H&Y severity. The group with H&Y ≥ 3 is significant older (P = 0.005) (A), with a lower disease duration (P = 0.036) (B), and no difference in LEDD (C). After 12 months IENFD denervation is greater in more disabled patients at cervical site (P = 0.019) (D).