doi:10.1093/brain/awv138
BRAIN 2015: 138; 2310–2321
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Phosphorylated a-synuclein in skin nerve fibres
differentiates Parkinson’s disease from multiple
system atrophy
Leonora Zange,1 Cornelia Noack,1 Katrin Hahn,1 Werner Stenzel2, and Axel Lipp1,
See Tolosa and Vilas (doi:10.1093/brain/awv164) for a scientific commentary on this article.
*These authors contributed equally to this work.
Deposition of phosphorylated SNCA (also known as a-synuclein) in cutaneous nerve fibres has been shown pre- and post-mortem
in Parkinson’s disease. Thus far, no pre-mortem studies investigating the presence of phosphorylated SNCA in skin sympathetic
nerve fibres of multiple system atrophy, another synucleinopathy, have been conducted. In this in vivo study, skin from the ventral
forearm of 10 patients with multiple system atrophy and 10 with Parkinson’s disease, together with six control subjects with
essential tremor, were examined by immunohistochemistry. Phosphorylated SNCA deposits in skin sympathetic nerve fibres and
dermal nerve fibre density were assessed. All patients with Parkinson’s disease expressed phosphorylated SNCA in sympathetic skin
nerve fibres, correlating with an age-independent denervation of autonomic skin elements. In contrast, no phosphorylated SNCA
was found in autonomic skin nerve fibres of patients with multiple system atrophy and essential tremor control subjects. These
findings support that phosphorylated SNCA deposition is causative for nerve fibre degeneration in Parkinson’s disease. Moreover,
pre-mortem investigation of phosphorylated SNCA in cutaneous nerve fibres may prove a relevant and easily conductible diagnostic procedure to differentiate Parkinson’s disease from multiple system atrophy.
1 Department of Neurology, Charité – University Medicine Berlin, Augustenburger Platz 1, 13355 Berlin, Germany
2 Department of Neuropathology, Charité – University Medicine Berlin, Charitéplatz 1, 10117 Berlin, Germany
Correspondence to: Werner Stenzel,
Department of Neuropathology,
Charité – University Medicine Berlin,
Charitéplatz 1,
10117 Berlin,
Germany
E-mail: werner.stenzel@charité.de
Keywords: synucleinopathy; neurodegeneration biomarkers; Parkinson’s disease; Parkinson’s disease cellular mechanisms; multiple
system atrophy
Abbrevations: ART = arterial blood vessels; CASS = Composite Autonomic Scoring Scale; H/M-ratio = heart to mediastinum
ratio; MAP = arrector pili muscles; MIBG = metaiodobenzylguanidine; pSNCA = phosphorylated SNCA (a-synuclein); SGD =
sweat glands dermal duct; SGS = sweat gland secretory portion; SRcontra = specific binding ratio contralateral to clinically affected
side; UMSARS = Unified Multiple System Atrophy rating scale; UPDRS = Unified Parkinson’s Disease Rating Scale
Received January 25, 2015. Revised March 15, 2015. Accepted March 23, 2015. Advance Access publication May 27, 2015
ß The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
Phosphorylated SNCA in PD and MSA
Introduction
Synucleinopathies such as idiopathic Parkinson’s disease
and multiple system atrophy are sporadic, adult-onset neurodegenerative disorders, characterized by accumulation of
modified SNCA that present clinically with a ‘Parkinson’
phenotype (Jellinger, 2003). Movement disorders (rigidity,
hypokinesia) are the clinical hallmark of Parkinson’s disease and multiple system atrophy, whereas non-motor
symptoms, foremost autonomic dysfunction as orthostatic
hypotension, bladder dysfunction and constipation
are more severe in multiple system atrophy (Barone et al.,
2009). Thus, clinical differentiation between Parkinson’s
disease and multiple system atrophy is challenging, especially in the early stage of the disease. In a clinicopathological study of 100 patients with Parkinson’s disease,
sensitivity of clinical diagnostic criteria for Parkinson’s disease was limited to 90–91.1%, whereas the positive predictive value ranged between 92–98.6% (Hughes et al.,
2001, 2002).
Serine 129 phosphorylated SNCA (pSNCA) is a major
component of neuronal Lewy bodies in Lewy body disorders such as Parkinson’s disease and of glial cytoplasmatic inclusions in multiple system atrophy (Fujiwara
et al., 2002). In Parkinson’s disease, neuronal populations
most vulnerable to Lewy body-related pathology include
the substantia nigra, locus coeruleus, raphe nuclei, pedunculopontine nucleus, basal nucleus of Meynert and dorsal
motor nucleus of the vagus (Jellinger, 2003). In addition,
neurodegeneration in Parkinson’s disease leads to pathological SNCA deposits within peripheral autonomic nerve
fibres innervating the heart, abdominopelvic organs, salivary glands, sympathetic ganglia as well as skin, which may
contribute to autonomic dysfunction in Parkinson’s disease
(Iwanaga et al., 1999; Minguez-Castellanos et al., 2007;
Orimo et al., 2007, 2008; Beach et al., 2010, 2013; Del
Tredici et al., 2010; Lebouvier et al., 2010; Cersosimo
et al., 2011; Pouclet et al., 2012a; Folgoas et al., 2013;
Hilton et al., 2014; Ito et al., 2014). In contrast, neurodegeneration in multiple system atrophy is largely limited to
the CNS including autonomic centres of the brainstem
(catecholaminergic neurons of the rostral ventrolateral medulla, nucleus of the solitary tract) causing predominant
preganglionic autonomic failure (Sung et al., 1979;
Wenning et al., 1996; Benarroch et al., 1998). Thus, detection of pSNCA in sympathetic postganglionic nerve fibres
seems promising to differentiate Parkinson’s disease from
multiple system atrophy. Recently, Donadio et al. (2014)
demonstrated pSNCA in skin sympathetic nerve fibres of
patients with clinically diagnosed Parkinson’s disease, but
not in other tau-related parkinsonian syndromes such as
progressive supranuclear palsy and corticobasal degeneration. To date, no comparative pre-mortem study assessed
the differential involvement of skin nerve fibres with respect
to pSNCA pathology in synucleinopathies such as
Parkinson’s disease and multiple system atrophy. In the
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present study we tested the hypothesis that pSNCA can
be detected in skin sympathetic nerve fibres in
Parkinson’s disease but not multiple system atrophy.
We performed skin punch biopsies and assessed the appearance of intra-axonal pSNCA in patients diagnosed as
Parkinson’s disease and multiple system atrophy based on
clinical consensus criteria, autonomic involvement and nuclear imaging.
Materials and methods
Patients
The study cohort was composed of 10 patients with
Parkinson’s disease (three females; age 69.6 8.77 years; disease duration 5.8 4.34 years) and 10 patients with multiple
system atrophy (three females, age 60.5 10.96 years; disease
duration 4.1 2.10 years). Six subjects without a neurodegenerative disorder but essential tremor served as controls (two
females, age 62.0 10.96 years; disease duration 14.7 11.8
years). Recruitment was limited to mild and moderate cases
[Hoehn and Yahr stage 1–3, mean standard deviation (SD):
1.95 0.9 points]. Patients were excluded, if they had a
history of heart disease, dementia (Mini-Mental State
Examination Score 5 24 points), polyneuropathy, diabetes
mellitus or alcoholism. Patients were enrolled from the movement disorders outpatient clinic of our hospital, Charité –
University Medicine Berlin. The study was approved by our
local ethical review board (approval number EA 2/133/09) and
all participants gave their written informed consent before
entering the study.
Diagnostic procedure
To increase diagnostic certainty, the clinical diagnosis of multiple system atrophy and Parkinson’s disease was performed
according to current diagnostic criteria [Parkinson’s disease:
UK Parkinson’s Disease Society Brain Bank criteria (Gibb
and Lees, 1988; Hughes et al., 1992, 2001); multiple system
atrophy: Gilman criteria (Gilman et al., 2008)] and validated
by a clinical follow-up. Clinical classification of multiple
system atrophy and Parkinson’s disease were further supported
by nuclear imaging and autonomic reflex screen.
Clinical testing
All participants underwent a detailed neurological examination
by two movement disorder specialists (C.N., A.L.). Motor impairment was evaluated by disease specific scoring scales
[Parkinson’s disease: Unified Parkinson’s Disease Rating
Scale, part III (UPDRS); multiple system atrophy: Unified
Multiple System Atrophy rating scale, part II (UMSARS)].
All patients (multiple system atrophy and Parkinson’s disease)
exhibited parkinsonism, which was unresponsive or only minimally responsive to levodopa treatment in those cases with
multiple system atrophy (UMSARS 5 20%; off-state versus
response to 200 mg levodopa/benserazide). A clinical follow-up
exam was performed in all patients to gain information on
disease progression and to validate the phenotype of the
underlying disease.
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Autonomic function testing
Autonomic reflex screening was performed by continuous
heart rate recording (3-lead ECG-monitoring) and non-invasive
blood pressure measurement based on beat-to-beat finger
Õ
photoplethysmography (Finometer midi, FMS) verified on
the contralateral arm by automatic sphygmomanometry
Õ
(Dinamap Pro 100, GE Healthcare). Data were digitalized
by an Analog-to-Digital converter (DI 720 series and
DI-205-C, DATAQ Instruments, Inc.) with a sample rate of
500 Hz and 14-bit resolution using data acquisition software
Õ
(WINDAQ , DATAQ Instruments, Inc.). All vasoactive medications including antiarrhythmic and dopaminergic agents
were discontinued for at least five half-lives before testing.
Patients also had to refrain from alcohol, caffeine and nicotine
for 24 h before the study. Autonomic reflex screening was performed in a supine position in a temperature-controlled room
at least 3 h after a slight meal. Parasympathetic (heart rate
response to deep breathing and Valsalva manoeuvre) and sympathetic (blood-pressure response to passive head up tilt and
Valsalva manoeuvre) functions were graded by an age- and
gender-adjusted Composite Autonomic Scoring Scale (CASS),
an objective scale ranging from 0 (no deficit) to 7 (maximal
deficit; without assessment of sudomotor function). Subjects
with a summed score of 43 on the CASS have mild autonomic failure; those with a summed score of 56 have severe
autonomic failure.
Single-photon emission computed tomography
123
I-flouropropyl (FP)-CITSPECT reflects nigrostriatal transmission by visualizing presynaptic dopamine transporters in
the striatum. SPECT imaging and data analysis was performed
as described before (Plotkin et al., 2005). Briefly, 4 h after administration of 200 MBq 123I-FP-CIT the cerebral distribution
of the tracer was assessed by a triple head camera system
(Mulitspect 3, Siemens Medical Systems). The ratio of the specific binding within the putamen contralateral to the clinically
more affected side in terms of movement impairment (SRcontra)
versus an occipital reference region was used for further
analysis.
The integrity of postganglionic sympathetic innervation of
the myocardium was assessed by 123I-metaiodobenzylguanidine (123I-MIBG) myocardial scintigraphy after thyroid blockade with sodium perchlorate. Planar images were recorded 4 h
post-injection by a double-headed gamma camera with a low
energy high resolution (LEHR) collimator (either Millenium
VG5 Hawkeye with VPC-45 K collimator, GE Medical
Systems-EU; or Symbia TruePoint SPECT-CT, Siemens) at a
photopeak on 159 keV of a 15% window. SPECT imaging
was performed on a 128 128 matrix as control. The heart
to mediastinum ratio (H/M-ratio) of the 123I-MIBG uptake at
4 h was analysed, a cut-off value of 1.7 was defined.
Skin biopsy and
immunohistochemistry
Skin biopsy, specimen preparation and staining
All subjects underwent 3 mm punch biopsy from the uninjured
skin of the volar forearm of the clinically more affected
side under 2% lidocaine local anaesthesia using sterile technique according to current guidelines (Lauria et al., 2010).
L. Zange et al.
The obtained biopsy consisted of a 3-mm core of skin (an
‘excision’) including epidermis and subpapillary dermis, to a
depth of 3 mm. The resulting lesion was allowed to heal by
granulation with re-epithelialization. Biopsy specimens were immediately immersion-fixed with formaldehyde (formaldehyde
4% buffered, Herbeta Arzneimittel) for 24 h to 2 weeks,
embedded in paraffin and sectioned in 3-mm thick sections
by a microtome (HM355, MICROM Internation). One section
per patient was stained with haematoxylin and eosin for
morphological orientation within the sample. For immunohistochemistry, serial sections were stained by an autoimmunostainer (Benchmark or Benchmark XT, Ventana
Medical Systems) with primary antibodies as listed in Table
1. Primary antibodies were detected by application of the
TM
iVIEW DAB detection Kit (Ventana) based on a labelled
streptavidin biotin method. Diaminobenzidine (Ventana)
served as chromogen of the peroxidase reaction and counterstaining was performed with Blueing-haematoxylin (Ventana).
Primary antibodies were omitted as negative controls. To
validate the intra-axonal localization of pSNCA deposits we
performed double-labelling immunofluorescence using antipSNCA and a neuronal marker [anti-protein gene product 9.5
(PGP 9.5)] in selected individuals. As secondary antibodies Cy3
Õ
and Alexa Fluor were used. The fluorescence-labelled sections
were mounted aqueously. Photomicrographs were taken with
the Zeiss Observer.Z1 Microscope (Immunofluorescence).
Morphological analysis and
assessment
Sections were examined with an Olympus BX 50 Microscope,
and photomicrographic images were taken with the digital
camera DP25 (Olympus) and cell^D software (Olympus Soft
Imaging Solutions). A representative sample of each antibody
was independently evaluated in a non-blinded manner by two
neuropathologists (W.S., L.Z.). Skin sympathetic nerve fibres
around sweat glands (dermal duct, SGD; secretory portion,
SGS), arrector pili muscles (MAP) and arterial blood vessels
(ART) were analysed within at least two histological levels.
Antibody staining was assessed both qualitatively and semiquantitatively. Detection rate (qualitative measure) was defined
as the percentage of antibody positive skin elements (SGD,
SGS, MAP, ART) related to all detected skin elements.
Total-antibody-staining based on an antibody specific-semiquantitative scoring scale. Phosphorylated SNCA antibody
staining was graded as: 0 = absence of staining; 0.5 = slightly
reduced staining pattern; 1 = strong staining pattern. The
innervation of autonomic structures visualized by UCHL1antibody and anti-TH were scored: 0 = absence of staining;
1 = severely reduced staining; 2 = slightly reduced staining;
3 = strong staining pattern (Supplementary Fig. 1).
Assessment of antibody staining was limited to those nerve
fibres that surrounded autonomic skin structures (SGD, SGS,
MAP, ART). Staining of isolated, potentially sensory nerve
fibres was ignored. Total antibody-staining (in per cent) was
defined as the ratio between the actual detected staining and
the maximum possible staining of an antibody summarized
overall detected skin elements. As such, total pSNCAPin sympathetic skin nerve fibres was assessed as: 100% Score
pSNCA (SGD + SGS + MAP + ART) / (maximum pSNCA
score [1] number of detected skin elements). Total UCHL1
Phosphorylated SNCA in PD and MSA
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Table 1 Characteristics of primary antibodies used for immunohistochemistry
Target
Clone, animal
Source
Dilution ratio
Positive control
Anti-pSNCA
Anti-UCHL1
anti-TH
Serin 129 phosphorylated SNCA
Monoclonal, mouse
Wako
1:1000
Brain tissue of a clinically and neuropathologically
confirmed patient with multiple system atrophy
Nerve fibres
Polyclonal, rabbit
Millipore
1:100
Nerve fascicles as internal control
Adrenergic (sympathetic) nerve fibres
Monoclonal, mouse
Sigma-Aldrich
1:100
Adrenal gland
Table 2 Relevant clinical and paraclinical data of patients with Parkinson’s disease, multiple system atrophy and
control subjects (essential tremor)
Parkinson’s disease
Epidemographic data
Age, years
Body mass index, kg/m2
Disease duration, years
Follow-up time, months
Autonomic function data
CASSv, points
CASSa, points
SBP, mHg
Nuclear imaging data
SRcontra
H/M–ratio
Multiple system atrophy
69.60 8.77
24.74 4.43
5.85 4.34
26.44 13.14
60.50 10.96
24.13 4.45
4.10 2.10
22.80 15.46
0 (0–1.0)
1.0 (1.0–1.0)z
3.50 13.51z
0 (0–2.0)
2.0 (1.0–3.0)†
29.30 18.49†
0.69 0.27†
1.54 0.43†
0.56 029†
1.65 0.32†
Controls
62.00 10.78
25.89 4.50
14.67 11.83
35.33 4.04
P-value
0.252a
0.748b
0.436c
0.396b
0 (0–0)
1.0 (0.75–1.0)
6.83 14.93
0.262a
0.001a
50.001b
2.27 0.32
2.33 0.45
50.001b
0.002b
SBP = Change of systolic blood pressure during head-up tilt; CASSa/v = adrenergic/vagal subscore of the CASS; HY = Hoehn and Yahr Scale; SRcontra = specific binding ratiocontralateral to the clinically more affected side, n.a. = not available; n.d. = not detectable.
Post hoc between group analysis: (pairwise post hoc Dunn’s test and Pairwise post hoc Tukey’s test) †P 5 0.05 versus ET; zP 5 0.05 versus multiple system atrophy.
a
Kruskal-Wallis test.
b
One-way ANOVA.
c
Mann-Whitney U-test (multiple system atrophy versus Parkinson’s disease).
P
and total TH were calculated as: 100% Score
(SGD + SGS + MAP + ART) / (maximum score [3] number
of detected skin elements).
Statistical analysis
Because of small sample size, normal distribution for quantitative data was assumed if skewness was 1 5 0 4 1 (Miles
and Shevlin, 2001). For parametric data (BMI, SBP; H/Mratio; SRcontra), between group analysis was performed by oneway ANOVA and Tukey’s multiple comparison test for post
hoc analysis. Non-parametric data [age, disease duration,
CASSv (vagal subscore); CASSa (adrenergic subscore); immunhistochemical scores] were analysed by Kruskal-Wallis test,
and post hoc analysis consisted of Dunn’s multiple comparisons. Mann-Whitney U-test was performed to compare nonparametric data between two groups (disease duration of
Parkinson’s disease and multiple system atrophy). Wilcoxon
rank-sum test compared data of two correlated samples
(sweat gland innervations SGD and SGS within one patient).
Fisher’s exact test tested the relationship between disease and
presence of pSNCA in sympathetic skin nerve fibres.
Spearman’s rho (r) was used to express the relation between
anti-pSNCA and anti- UCHL1, UPDRS, H/M-ratio, SRcontra,
SBP and CASSv. Regression analysis estimated the relation
between age and pSNCA in patients with Parkinson’s
disease. Quantitative data were expressed as mean SD.
Semiquantitative and ordinal data were expressed as median
[interquartile range, (IQR)]. A P-value 4 0.05 was considered
statistically
significant,
P-value
adjustment
strategy
(Bonferroni) for multiple comparisons were performed when
Õ
Õ
appropriate. Data were analysed using IBM SPSS Statistics
software, Version 21.00.0.0 (IBM Corporation) and Prism 5
software for Windows, Version 5.02 (GraphPad Software,
Inc.).
Results
Clinical data
The study cohort comprised 26 patients diagnosed as
Parkinson’s disease (n = 10), multiple system atrophy
(n = 10) and essential tremor (n = 6, controls). Clinical
phenotypes of multiple system atrophy were cerebellar in
three cases and parkinsonian in seven cases. Clinical characteristics and results of autonomic function tests and
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nuclear imaging are summarized in Supplementary Table 1
and Table 2. The initial diagnostic classification was confirmed by a clinical follow-up in all cases (mean follow-up
period: 26 13.7 month) except for one patient with multiple system atrophy and three controls that were lost to
follow-up. Autonomic impairment, foremost adrenergic
CASS score and orthostatic hypotension, was significantly
higher in multiple system atrophy (P = 0.001) compared to
Parkinson’s disease and control subjects, indicating the
more severe and widespread autonomic involvement
in the former. However, orthostatic hypotension of
520 mmHg occurred in 6 of 10 patients with Parkinson’s
disease. Twenty-four patients underwent 123I-FP-CIT
SPECT. Striatal dopaminergic function was impaired in
all cases (multiple system atrophy, Parkinson’s disease)
but preserved in controls (essential tremor) as indicated
by significant reduction of SR (P 5 0.001) contralateral to
the clinically more affected side. Sympathetic cardiac innervation was significantly affected in both Parkinson’s disease and multiple system atrophy patients (H/M-ratio
multiple system atrophy: 1.65 0.32; Parkinson’s disease:
1.54 0.43) but normal in controls (H/M-ratio controls:
2.33 0.45). However, myocardial 123I-MIBG SPECT was
not able to differentiate between a pre- and postganglionic
autonomic lesion (H/M-ration multiple system atrophy
versus Parkinson’s disease: P 4 0.5; Tukey’s post hoc
test), and predictability of individual cases was less distinct
(unexpected 123I-MIBG reduction: 1/6 controls, 4/10 multiple system atrophy; unexpected normal 123I-MIBG: 3/10
patients with Parkinson’s disease).
Innervation of autonomic skin structures
UCHL1 immunoreactivity appeared as dotted and filamentous staining pattern in dermal nerve fibres of all patients
and controls (detection rate 100%, Fig. 3A–I). UCHL1
staining in general was strong or slightly reduced (grading
2–3) with a predominance in SGS compared to SGD that
was significant in Parkinson’s disease only [Parkinson’s disease P = 0.006; Wilcoxon rank-sum test (adjusted significance level P 5 0.008)]. Total UCHL1 staining was
significantly reduced in Parkinson’s disease when compared
to multiple system atrophy and controls [UCHL1 total
score: Parkinson’s disease 43% (38–56%), multiple
system atrophy 67% (48–77%), controls 72.5% (64.3–
83.5%); P = 0.004, Kruskal-Wallis test (adjusted significance level P 5 0.008)] (Fig. 4B). In Parkinson’s disease,
loss of UCHL1-positive nerve fibres was pronounced
around SGD [Parkinson’s disease 1.0 (interquartile range,
IQR 1.0–1.0); P = 0.007] and MAP [Parkinson’s disease 0.5
(IQR 0.5–0.6); P = 0.003]. UCHL1 staining in multiple
system atrophy was slightly reduced around SGS [3.0
(IQR 2.0–3.0)] but pronounced around SGD [2.0 (IQR
2.0–3.0)] when compared to controls [SGS: 3.0 (IQR
3.0–3.0); SGD: 1.0 (IQR 1.0–1.0)]. Mann-Whitney U-test
revealed a significant reduction of pilomotor nerve fibres in
multiple system atrophy [1.0 (1.0–2.75)] compared to those
of Parkinson’s disease [0.5 (0.5–0.63); U-test = 2.5;
L. Zange et al.
P = 0.003 (adjusted significance level P 5 0.008)]. Because
MAP was detected in only one essential tremor sample, the
control group was excluded from statistical analysis.
Anti-TH staining resulted in a variable, less distinct
dotted and filamentous pattern of nerve fibres surrounding
autonomic skin structures. TH immunoreactivity was least
frequent in controls (detection rate: controls 53%, multiple
system atrophy 78%, Parkinson’s disease 65%). However,
total-TH-staining did not differ significantly between
groups [multiple system atrophy 37.5%, Parkinson’s disease 31%, controls 20%; P = 0.11, Kruskal-Wallis test
(adjusted significance level P 5 0.008)].
Phosphorylated SNCA in skin
sympathetic nerve fibres
Neuropathological data are summarized in Supplementary
Table 2. Phosphorylated SNCA is visualized as finegranular staining pattern in small nerve fibres surrounding
autonomic skin structures. In patients with Parkinson’s disease, positive staining for pSNCA was observed in 7 of 10
SGS [0.5 (IQR 0–1.0); Fig. 1A and B], 8 of 10 ART [0.75
(IQR 0.38–1.0); Fig. 2A] and in three of three samples with
MAP [0.5 (IQR 0.5–n.a.); Fig. 2B]. Phosphorylated SNCA
staining was not discernible in nerve fibres supplying SGD
of patients with Parkinson’s disease. The co-localization of
anti-pSNCA and the neuronal marker UCHL1 as shown in
Fig. 6 proves the intra-axonal localization of pSNCA deposits and distinguishes from unspecific background staining. In contrast to Parkinson’s disease, dermal autonomic
nerves fibres of multiple system atrophy patients (Fig. 1C,
Fig. 2C and D) and control subjects (Fig. 1D, Fig. 2E and
F) were devoid of pSNCA [Fisher’s exact test 2 = 27;
P 5 0.001 (adjusted significance P 5 0.0125)].
Correlation of phosphorylated SNCA
deposition and autonomic
denervation
The parameter ‘total antibody-staining’ is a normalized
semiquantitative measure of skin autonomic innervation
(see ‘Materials and methods’ section) that allows
between-group comparison independent from the number
of detected dermal autonomic structures. Total pSNCA in
patients with Parkinson’s disease ranged between 17–75%
[33% (31–67%)] and was absent in multiple system atrophy patients and controls (Fig. 4A). There was a negative
correlation between total pSNCA and the degree of dermal
denervation assessed by anti-UCHL1 staining (r = 0.57;
P = 0.002).
In Parkinson’s disease, total pSNCA did not correlate
with clinical or nuclear imaging measures: disease duration
(r = 0.57; P = 0.086), UPDRS (r = 0.38; P = 0.82),
Hoehn and Yahr (r = 0.25; P = 0.492), H/M-ratio
(r = 0.29; P = 0.423), SRcontra (r = 0.18; P = 0.62), SBP
(r = 0.4; P = 0.25), CASSv (r = 0.2; P = 0.638). Innervation
Phosphorylated SNCA in PD and MSA
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Figure 1 Immunohistochemistry with phosphorylated SNCA in sweat glands of patients with Parkinson’s disease, multiple
system atrophy and essential tremor (controls). In Parkinson’s disease (A and B), pSNCA immunostaining appeared as punctate staining
pattern (brown-purple) in sudomotor nerve fibres of the secretory portion (SGS), but was absent in those of the sweat gland dermal duct
(D). Absence of pSNCA immunoreactivity in sudomotor nerve fibres of multiple system atrophy (C) and essential tremor patients
(D). The arrowheads indicate pSNCA deposition. Counterstain = haematoxylin. Scale bar = 50 mm.
of autonomic skin structures (total UCHL1 staining) was
significantly reduced in Parkinson’s disease (P = 0.004, see
above). This loss of UCHL1 immunoreactivity was independent from patients age (r = 0.12; P = 0.73; Fig. 5)
and disease duration (r = 0.35; P = 0.31).
Discussion
Our study addressed the detection and distribution of
pathological SNCA in dermal sympathetic nerve fibres
in vivo in two clinically similar but neuropathologically
distinct neurodegenerative conditions, multiple system atrophy and Parkinson’s disease. The main results of our study
are as follows: (i) pSNCA deposits were detected in nerve
fibres innervating skin autonomic structures (sweat gland,
arrector pili muscle and arterial blood vessels) in SNCA (asynuclein) dependent neurodegenerative disorders; (ii)
pSNCA deposition was limited to Parkinson’s disease
whereas dermal nerve fibres of multiple system atrophy patients were devoid of pSNCA; and (iii) pSNCA deposition
correlated with dermal denervation (UCHL1 staining).
Phosphorylated SNCA is considered the pathognomonic
protein underlying Parkinson’s disease and multiple system
atrophy. The neuropathological diagnosis of multiple
system atrophy and Parkinson’s disease is based on specific
regional distribution of pSNCA in glial cells and neurons of
the CNS, respectively. The involvement of peripheral postganglionic autonomic neurons in Parkinson’s disease only
led to the particular diagnostic value of autonomic function
tests [thermoregulatory sweat test and quantitative sudomotor axon reflex test (QSART)] to separate Parkinson’s
disease and multiple system atrophy (Kimpinski et al.,
2012; Orimo et al., 2012). Nevertheless, sensitivity and
specificity of clinical diagnostic tools remain limited
(Kimpinski et al., 2012; Orimo et al., 2012).
Wang et al. (2013) reported a higher incidence of SNCA
in sympathetic skin nerve fibres in patients with Parkinson’s
disease compared to controls, and thus proposed SNCA
deposits in skin nerve fibres as a diagnostic marker of
Parkinson’s disease (Wang et al., 2013). However, the
used antibody stained physiological soluble SNCA, a ubiquitous predominately presynaptic protein, and was not
specific for the underlying pathogenic protein—pSNCA.
Our finding of peripheral pSNCA deposits within skin
nerve fibres is supported by post-mortem studies in neuropathologically confirmed Parkinson’s disease, demonstrating pSNCA in skin wedge samples of the upper arm,
abdomen and scalp (Ikemura et al., 2008; Beach et al.,
2010). In a recent study, Donadio et al. (2014) reported
pSNCA in sympathetic nerve fibres of intravital cervical
skin samples in 21/21 patients with Parkinson’s disease.
Phosphorylated SNCA staining was pronounced in dermal
nerve bundles and nerves of arterioles (22%, respectively)
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L. Zange et al.
Figure 2 Immunohistochemistry with phosphorylated SNCA in arterioles (ART) and arrector pilorum muscles (MAP) of
patients with Parkinson’s disease, multiple system atrophy and controls (essential tremor). In Parkinson’s disease, pSNCA was
present in vasomotor (A) and pilomotor (B) sympathetic nerve fibres. In multiple system atrophy (C and D) and essential tremor (E and F) no
pSNCA immunoreactivity was detectable in sympathetic nerve fibres of arterioles (C and E) and arrector pili muscles (D and F). The arrowheads
indicate pSNCA deposition. The arrow marks an unspecific staining, presumably a macrophage. Counterstain = haematoxylin. Scale bar = 50 mm.
and thus differed slightly from our findings (MAP 100%,
ART 80% and SGS 70%).
In our present study, all patients with Parkinson’s disease
presented pSNCA depositions within at least one dermal
nerve fibre innervating autonomic skin structures (sensitivity of 100%). In contrast, evidence of dermal pSNCA deposits in previous histopathological studies ranged from
70% in skin samples of the upper arm and abdomen
(Ikemura et al., 2008) to even 0% immunoreactivity in
scalp and abdominal skin samples (Beach et al., 2010).
Pre-mortem studies of patients with clinically diagnosed
Parkinson’s disease present with likewise heterogeneous results; pSNCA was detected in skin samples of the distal and
proximal leg, back and finger in 16/35 (45.71%) patients
(Doppler et al., 2014); of the chest wall in 2/20 (10%)
patients (Miki et al., 2010) but was absent in biopsies of
the ventral forearm (Kawada et al., 2009) and distal leg
(Navarro-Otano et al., 2014). The variable detection of
pSNCA deposits in Parkinson’s disease skin biopsies may
be a result of different skin sample size, tissue fixation,
antibody pretreatment and biopsy site. The selection of
the biopsy site should consider the physiological distribution of SNCA. Physiological SNCA is predominately
located in presynaptic nerve terminals and implicated in
synaptic plasticity (Watson et al., 2009), learning (George
et al., 1995), neurotransmitter release (Burre et al., 2010),
and synaptic vesicle pool maintenance (Dikiy and Eliezer,
2012). Nerve terminals are located at sympathetic effector
Phosphorylated SNCA in PD and MSA
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| 2317
Figure 3 UCHL1 Immunostaining of a dermal sweat gland duct (SGD) and secretory portion (SGS), arterioles (ART) and
arrector pili muscles (MAP) in Parkinson’s disease (A–C), multiple system atrophy (multiple system atrophy, D–F) and controls
(essential tremor, G–I). In controls, UCHL1 sympathetic skin nerve fibres were well preserved in sweat glands secretory portion (G),
cutaneous arterioles (H) and arrector pili muscle (I). In contrast, patients with Parkinson’s disease had an obviously reduced innervation pattern in
sweat gland secretory portion (A), and pilomotor nerve fibres (C) compared to controls (G–I). Sympathetic nerve fibres of sweat gland dermal
duct and arrector pili muscle were more abundant in multiple system atrophy (D–F) than in Parkinson’s disease (G–I). Positive UCHL1 staining
was verified by staining of nerve fascicles (N). T = sebaceous gland; Counterstain = haematoxylin. Scale bar = 50 mm.
organs, e.g. sweat glands. Sweat gland density is higher at
the ventral forearm (159/cm2) compared to the proximal
sites abdomen (81–141/cm2), back (106–132/cm2), neck
(126/cm2), upper arm (94–102/cm2) and distal sites such
as leg (31–132/cm2), except finger (126–350/cm2) and
scalp (70–195/cm2) (Taylor and Machado-Moreira,
2013). Previous studies proposed a proximo-distal gradient
of pSNCA appearance with the highest incidence cervically
(Donadio et al., 2014), and decreasing levels at the back
(Doppler et al., 2014), abdomen, leg, upper arm or finger
(Ikemura et al., 2008). Variable sweat gland density may
contribute to this effect. High variability of pSNCA expression at different localizations requires a consensus with respect to biopsy location and sample preparation. Our data
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| BRAIN 2015: 138; 2310–2321
L. Zange et al.
Figure 4 Comparison of total pSNCA and total UCHL1 in sympathetic skin nerve fibres between controls (essential tremor),
multiple system atrophy and Parkinson’s disease. Total antibody is the ratio of the summed scores of the present innervation for each
detected skin element to the sum of the greatest possible score of detected skin elements. PSNCA was expressed and quantified in Parkinson’s
disease (PD), but absent in essential tremor (ET) and multiple system atrophy (MSA) (A). Total UCHL1 was significantly reduced in Parkinson’s
disease compared to multiple system atrophy and essential tremor (B). The data are presented as median IQR. *Tukey post hoc test P 5 0.05.
Figure 5 Linear regression of total UCHL1 versus age.
Patients with Parkinson’s disease presented a loss of UCHL1 positive nerve fibres independent of age [Parkinson’s disease (PD)
r2 = 0.01; essential tremor (ET) r2 = 0.00; multiple system atrophy
(MSA) r2 = 0.23]. Regression line is plotted for patients with
Parkinson’s disease.
promote the ventral forearm as preferred biopsy site for
pSNCA detection in sympathetic skin nerve fibres.
The pathological hallmark of multiple system atrophy are
cytoplasmatic pSNCA deposits in oligodendrocytes (glial
cytoplasmatic inclusions) and to a lesser extend in nuclei
of oligodendrocytes as well in the nuclei and cytoplasm of
neurons (Ahmed et al., 2012). The pathological process
affects central and brainstem autonomic centres as well as
preganglionic sympathetic neurons in the intermediolateral
cell column (Wenning et al., 2004). Our present study is
the first investigating pSNCA in dermal nerve fibres of multiple system atrophy patients in vivo. We demonstrate that
despite the severe autonomic impairment, nerve fibres surrounding autonomic skin structures in multiple system atrophy are devoid of pSNCA deposits. Our finding is
consistent with the results of Ikemura et al. (2008) demonstrating absence of pSNCA staining in skin samples of
abdomen and upper arm of three patients with neuropathologically confirmed multiple system atrophy. In contrast,
autopsy studies on peripheral sympathetic ganglia in multiple system atrophy patients revealed neuronal SNCA
inclusions in 25% (Nishie et al., 2004) and Lewy bodies
in late stage multiple system atrophy in 42.3% (mean disease duration 12.8 5.3 years) (Sone et al., 2005).
Furthermore, pSNCA has rarely been shown in the enteric
and myocardial nervous system in multiple system atrophy
(Orimo et al., 2008; Pouclet et al., 2012b). These findings
might be limited to advanced stages of the disease as they
were detected in post-mortem studies only, or in cases of
concurrent Lewy body disorders. Our present study does
not support the notion of an involvement of postganglionic
sympathetic skin nerve fibres in multiple system atrophy
with a clinical duration up to 7 years. Thus, testing sympathetic skin nerve fibres for pSNCA deposits appears
worthy to differentiate Parkinson’s disease from multiple
system atrophy. Additional studies with patients at different
stages of the diseases are necessary to verify the involvement of skin sympathetic nerve fibres in multiple system
atrophy.
Loss of dermal nerve fibres and reduced epidermal
nerve fibre density in Parkinson’s disease has been repeatedly demonstrated (Dabby et al., 2006; Nolano et al.,
2008; Novak et al., 2009; Navarro-Otano et al., 2014).
However, quantification of autonomic dermal nerve fibres
is complex due to insufficiency of sectional images depicting sterical organization of 3D sympathetic nerve fibre
plexus. Accurate quantification requires immunofluorescence staining, confocal microscopy and is time consuming
(Donadio et al., 2014). Our present study was not intended
to access dermal nerve fibre density but detection of dermal
Phosphorylated SNCA in PD and MSA
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Figure 6 Evidence of intraaxonal localization of anti-pSNCA depositions. Double-labelling immunofluorescence of anti-pSNCA (A)
and anti-protein gene product (B), UCHL1. Phosphorylated SNCA is strongly co-staining with UCHL1 within axonal structures surrounding
sweat glands in a patient with idiopathic Parkinson’s disease indicating intra-axonal localization of pSNCA deposition (C). Scale bar = 50 mm.
intra-axonal pSNCA depositions, which is why we deviated
from the EFNS guidelines on the use of skin biopsy (Lauria
et al., 2010). Nevertheless, our data support the finding of
dermal denervation in Parkinson’s disease by a significant
reduction of total-UCHL1 staining that occurred independent from patient’s age and correlated with the extent of
intra-axonal pSNCA deposition. This is in line with
recent studies proposing a length-independent loss of
intraepidermal nerve fibres associated with appearance of
pSNCA at proximal sites (Doppler et al., 2014) and sweat
gland denervation correlating with pSNCA distribution
(Donadio et al., 2014). To avoid any confounding effect
of potential small-fibre neuropathy, we excluded patients
suffering from diabetes mellitus or alcoholism.
The present study has several limitations. First, diagnostic
classification of patients relied on clinical criteria and putative neuropathological abnormalities are not definite (no
neuropathological confirmation available). To increase
diagnostic reliability, additional diagnostic procedures (nuclear imaging, autonomic testing) and clinical follow-up
supported the clinical diagnosis. Second, specificity of nuclear imaging, especially of 123I-MIBG-SPECT is limited as
indicated by 8 of 26 conflicting 123I-MIBG results in our
study. Overlapping results of myocardial 123I-MIBG
SPECT in multiple system atrophy and Parkinson’s disease
have been reported before (Kimpinski et al., 2012; Orimo
et al., 2012). Although we excluded patients suffering from
diabetes mellitus and alcoholism, inapparent peripheral
neuropathy is highly prevalent in the age of Parkinson’s
disease and potentially decreases nerve fibre density (totalUCHL1 staining). Anti-TH staining, applied to distinguish
sympathetic from sensory dermal nerve fibres, resulted in
highly variable patterns of immunoreactivity unsuitable for
double-immunofluorescence staining (anti-UCHL1 and
anti-TH). We therefore limited assessment of antibody
staining to dermal nerve fibres surrounding autonomic
skin structures (SGD, SGS, MAP, ART) ignoring isolated,
potentially sensory nerve fibres. Whether those dermal
nerve fibres are affected as well by pSNCA depositions is
uncertain. Finally, semiquantitative scoring of staining patterns underlie subjectivity and thus reduce reliability.
In summary, we present the first in vivo comparison of
pSNCA deposits in dermal sympathetic nerve fibres in
Parkinson’s disease, multiple system atrophy and controls
of similar age and gender. We were able to separate multiple system atrophy from Parkinson’s disease on the basis
of peripheral pSNCA pathology that was limited to the
latter. pSNCA deposits may contribute to cutaneous nerve
fibre loss and be related to autonomic symptoms in
Parkinson’s disease. Immunhistochemical analysis of
pSNCA in skin biopsies appears sensitive, feasible, safe
and inexpensive when compared to current diagnostic
methods such as 123FP-CIT-SPECT and myocardial
123
MIBG. Further studies with larger sample sizes and at
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| BRAIN 2015: 138; 2310–2321
different disease stages are needed to prove whether detection of peripheral pSNCA pathology in skin biopsies will
increase diagnostic reliability of synucleinopathies.
Acknowledgements
We thank Mrs Petra Matylewski for outstanding technical
laboratory assistance. We also gratefully acknowledge Dr
Michail Plotkin for support in analysing nuclear imaging
data.
Funding
This work was supported in part by the German Research
Foundation (DFG 1301/2-1 to A.L.).
Supplementary material
Supplementary material is available at Brain online.
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