Introduction
In recent years, non-motor symptoms in Parkinson’s disease (PD) have received much attention, and PD is now considered a multi-systemic disorder of the nervous system. Deposits within neurons and neurites mainly consisting of phosphorylated alpha-synuclein (p-alpha-synuclein) are the neuropathological hallmark of the disease and are supposed to initially occur within brain regions, such as the vagal, glossopharyngeal or olfactory nucleus, receiving innervation from mucosal surface areas. From there, alpha-synuclein deposits spread in a characteristic stage-dependent pattern [
3]. Several studies have detected alpha-synuclein deposits within structures of the peripheral nervous system (PNS) like dermal nerve fibers, pharyngeal nerves, nerves of the submandibular and minor salivary glands and nerves and ganglia of the enteric nervous system [
1,
2,
8,
15,
20,
24,
31,
38‐
40]. PD patients also have a reduced density of small intraepidermal nerve fibers and diminished innervation of sweat gland and erector pili muscles [
8,
40,
58]. Wang et al. [
58] recently reported an association between dermal alpha-synuclein deposition and reduced autonomic innervation, using an alpha-synuclein-antibody that is not specific to the phosphorylated form. The finding of alpha-synuclein deposits in skin nerve fibers raises the possibility of a disease-related peripheral neurodegeneration in PD. However, others found an association of the peripheral neuropathy with cumulative levodopa intake and reduced levels of vitamin B12 and B6, suggesting a pharmacotoxic cause: either due to interactions of levodopa metabolism with the methylation pathways of vitamin B12 or the interference of intestinal levodopa and vitamin uptake [
5,
25,
37,
43,
46,
53].
Here we hypothesized that loss of peripheral nerve fibers was intrinsic to PD and that peripheral pathology might reflect known features of neurodegeneration within the central nervous system, which could potentially render skin biopsy a useful tool for studying the pathogenesis of PD and establishing a reliable pre-mortem histopathological diagnosis of the disease.
Discussion
In the current study, we combined, for the first time, detection of p-alpha-synuclein, quantification of small and large nerve fibers in skin biopsies and assessment of vitamin B12 and B6 levels in PD patients. We found p-alpha-synuclein deposits in cutaneous nerves of 16/31 PD patients, a moderate length-dependent reduction of small and large nerve fibers and severe non-length-dependent reduction of SP-positive fibers. In our cohort, low levels of B vitamins were uncommon and we found no correlation between cumulative levodopa intake and loss of nerve fibers, suggesting that dermal nerve fiber loss, reduction of SNAP and alpha-synuclein deposits were rather disease related than caused by a toxic effect of drug therapy. These findings are surprising with respect to recent studies describing a high prevalence of peripheral neuropathy in PD (up to 55 %) due to levodopa-related hypovitaminosis [
5,
37,
43,
53,
54]. However, we did not preselect patients based on clinical symptoms or electrophysiological signs of peripheral nerve involvement unlike some previous studies, our cohort did not include patients with clinical symptoms of peripheral neuropathy, and even QST as a test for impaired small fiber function was normal. Therefore, comparability concerning vitamin levels and SNAP between our and former studies is limited. However, the absence of patients with relevant hypovitaminosis or other alternative causes of peripheral neuropathy in our study is important as it allows us to assume that involvement of peripheral nerves is intrinsic to PD in our patients, which is a prerequisite for analyzing the distinct features of PD related peripheral neuropathy.
To address the question whether pathology of the PNS reflects central nervous system (CNS) pathology and might be useful for the study of PD pathogenesis, we compared our findings with known features of brain pathology (Table
3). Morphological appearance of p-alpha-synuclein deposits in the skin did not differ from Lewy neurites found in sections of the substantia nigra that served as positive controls (Fig.
3). As described in the literature they appeared thread-like and sometimes presented with irregular varicosities [
3]. Regarding the type of nerve fibers involved in p-alpha-synuclein pathology, deposits were restricted to unmyelinated fibers as reported in studies of the CNS [
4]. They were found in VIP-, TH-, SP- and CGRP-positive fibers but were most frequent in TH-immunoreactive fibers. Alpha-synuclein deposits within SP-and TH-immunoreactive neurons/fibers have been reported in the CNS [
7,
16,
28], and VIP- and TH-positive fibers containing alpha-synuclein aggregates were found in the enteric nervous system and in sympathetic ganglia [
57]. In the brain, alpha-synuclein deposition is prevalent in the autonomic vagal nucleus early in the course of disease [
3]. Correspondingly, alpha-synuclein deposits were mostly found in autonomic fibers in our study and by others [
38,
58]. However, in contrast to previous studies, we also show p-alpha-synuclein depositions in somatosensory fibers of the subepidermal plexus and intraepidermal fibers, giving evidence that alpha-synuclein pathology in the peripheral nervous system is not restricted to autonomic fibers. All four patients with somatosensory fiber involvement were in an advanced stage of disease. This corresponds to observations of the CNS, where alpha-synuclein spreads to sensory association areas at later stages of disease finally also including nociceptive as well as sympathetic and parasympathetic neurons of the spinal cord [
3,
10]. The observation that alpha-synuclein deposition within somatosensory nerve fibers was more often found in patients with tremor-dominant PD needs to be addressed in further studies.
Table 3
Comparison of characteristic findings of the CNS and PNS in Parkinson’s disease
p-Alpha-synuclein deposition | Restricted to unmyelinated fibers | Restricted to unmyelinated fibers [ 4] |
Autonomic >> somatosensory small fibers | Autonomic structures at early stages, sensory association areas at later stages [ 3] |
TH- >CGRP-, SP-, VIP-positive fibers | Colocalization of Lewy bodies with TH- and SP-immunoreactivity [ 12, 16, 28] |
Loss of neurons/nerve fibers | Marked loss of SP-positive fibers | Loss of SP-levels and SP-positive neurons [ 14, 16] |
Length-dependent loss of intraepidermal fibers | Evidence of impaired axonal transport [ 7, 42] |
Potential pathogenetic mechanisms | Antegrade neurodegeneration: non-length-dependent loss of SP- and CGRP-positive fibers => damage to sensory neuron | Neuronal loss in substantia nigra, vagal/glossopharyngeal nucleus, reticular formation, coeruleus complex [ 3, 13, 27, 44] |
Retrograde neurodegeneration: length-dependent loss of intraepidermal nerve fibers => impaired axonal transport | Evidence of impaired axonal transport [ 7] and primary degeneration of “long-range” projection neurons [ 4] |
Length-dependent nerve fiber loss is generally assumed to be the effect of a dying-back neuropathy due to axonal damage, whereas non-length-dependent loss is supposed to be caused by loss of sensory neurons in the dorsal root ganglia (e.g. in ganglionitis) [
18]. Predominant loss of SP-immunoreactive intraepidermal nerve fibers in our patients in a non-length-dependent pattern suggests anterograde degeneration of SP-positive neurons in the dorsal root ganglia. This finding is paralleled by studies reporting loss of SP-containing neurons and reduction of SP levels in the CNS [
14,
16,
19,
52]. Furthermore, SP-immunoreactive neurons and nerve fibers have been found to be the major site of alpha-synuclein deposition in colon biopsies and in the olfactory system early in the course of PD [
49,
55].
In spite of extensive research, the exact pathomechanism leading to neurodegeneration in PD is still unknown. While detailed knowledge of the involvement of different regions of the brain in the course of disease has been gained in recent years [
3], the question where the neurodegenerative process begins on the cellular level is still unanswered [
6]. Neuronal loss due to alpha-synuclein deposition, impaired axonal transport and mitochondrial dysfunction are discussed. Based on alpha-synuclein deposits in skin biopsies as well as quantification of nerve fibers, two patterns of peripheral involvement could be differentiated in our study: IENFD and myelinated dermal fibers were reduced in a length-dependent pattern, whereas SP- and CGRP-immunoreactive intraepidermal fibers showed a non-length-dependent reduction. P-alpha-synuclein deposition was more frequently found at more proximal sites as already suggested in former studies [
24,
38], reflecting non-length-dependent distribution as well. Those two patterns might correspond to two distinct neurodegenerative processes currently discussed in PD brain pathology: (1) programmed cell death mediating soma destruction and (2) axonal degeneration [
6]. The ongoing debate has been fostered by the observed discrepancy between nigral cell loss and striatal dopaminergic denervation in the early stages of the disease.
Neuronal death is a characteristic feature of PD in the CNS and death of peripheral neurons of the dorsal root ganglia might be a correlate in the PNS resulting in non-length-dependent peripheral nerve degeneration. So far, the question whether p-alpha-synuclein directly leads to neuronal death is still unresolved [
9]. Alpha-synuclein deposits in SP-containing neurons in the dorsal root ganglia or in proximal neurites and their subsequent degeneration might explain non-length-dependent loss of SP-positive intraepidermal nerve fibers independent of duration of disease. This notion might be worth to be further investigated in post-mortem studies.
One of the general pathophysiological mechanisms resulting in length-dependent damage of peripheral nerves (as for example found in diabetic neuropathy) is impaired axonal transport. Indeed, a number of studies provide evidence of impaired axonal and mitochondrial transport in PD in the CNS and PNS [
7,
35,
50]. Altered axonal transport proteins have been reported in the CNS of PD patients [
7]. Alpha-synuclein oligomers have been found to disturb axonal transport, and more severe Wallerian degeneration after nerve transection has been detected in axons with alpha-synuclein deposition in transgenic mice over-expressing alpha-synuclein, providing a possible link between these two suspected pathogenetic mechanisms [
32,
42,
50]. Distal alpha-synuclein deposition might induce retrograde axonal degeneration, a notion which is supported by the observation that alpha-synuclein aggregates in cardiac autonomic nerves are more abundant in the distal part of the nerve and precede aggregation in the corresponding sympathetic ganglia [
41]. Correlation between reduced distal IENFD and duration of disease in our patients is well in line with impaired axonal transport and retrograde axonal degeneration as it implies a slowly progressive degeneration of nerve fibers with advancing disease.
P-alpha-synuclein depositions were equally found in patients with early and late stages of disease indicating an early involvement of cutaneous nerve fibers in PD. The observation that alpha-synuclein aggregates in cardiac autonomic nerves are more abundant in the distal part of the nerve [
41] provides evidence that skin as the most distal site of the peripheral nervous system is not only easily accessible but might also provide an opportunity for early detection of alpha-synuclein pathology [
20]. Here we found p-alpha-synuclein deposition in 16/31 PD patients, but not in any normal control. The number of positive cases is higher compared to former studies also using antibodies specific against p-alpha-synuclein, probably because biopsies of different sites were analyzed in our study and our cryosections were thicker compared to other studies that used paraffin-embedded material [
24,
38]. Wang et al. recently published a study analyzing alpha-synuclein deposition within dermal nerve fibers using an alpha-synuclein antibody that is not specific to the phosphorylated form. They found an increase of alpha-synuclein in PD patients compared to controls and an association between loss of autonomic innervation and alpha-synuclein deposition. This study is of great interest as it implies that the impact of alpha-synuclein pathology in peripheral nerve fibers is larger than so far suspected. However, the use of an antibody that is not specific for p-alpha-synuclein requires complex immunohistochemical procedures with threshold dilution of antibodies to detect a difference in the amount of immunoreactivity between patients and controls and the cut-off value to distinguish between physiological and pathological conditions is not clear. Therefore, this method does not seem suitable for application as a biomarker in routine diagnostic work-up. Using conventional immunohistochemical staining procedures with an antibody specifically recognizing p-alpha-synuclein, we found immunoreactivity in 16 patients but not in any normal control, allowing good specificity but low sensitivity for diagnostic purposes. The high specificity compared to normal controls makes it worthwhile to discuss a diagnostic value especially at early stages when a clinical diagnosis may still be flawed by large uncertainties.
Our study has some limitations and further studies are needed to determine the exact sensitivity and specificity of skin biopsy as a diagnostic tool and to potentially improve it using higher numbers of samples or by targeted sampling, which would include hair follicles and sweat glands in each biopsy. The present study provides data of a small but well-characterized cohort of patients with a clinical diagnosis of PD. When evaluating the frequency of cutaneous p-alpha-synuclein deposition, we need to take into account that diagnosis of PD in our patients is based on clinical symptoms and sensitivity of clinical diagnosis ranges between 70 and 90 % [
22,
23,
34,
48]. Detection of alpha-synuclein in skin biopsies very much depends on whether autonomic structures like sweat glands, vessels or erector pili muscles are included in the skin section. Furthermore, the inclusion of patients with atypical Parkinsonism would be of interest.
In summary, our data provide evidence for peripheral neurodegeneration as an intrinsic feature of PD. P-alpha-synuclein deposition was found in about half of all patients and may be of diagnostic value, especially in early stages of disease. We demonstrate a strong analogy between CNS and PNS pathology concerning alpha-synuclein deposition, pattern of nerve fiber loss and evidence of impaired axonal transport, implying that skin biopsy might be an interesting “in vivo” method of studying the pathogenesis of PD. In addition to better accessibility compared to autopsy material, skin biopsies could be taken longitudinally to assess individual disease progression, which is a principal shortcoming of any post-mortem study reflecting the terminal stage of a disease. Based on our findings we speculate that two independent mechanisms lead to peripheral neuropathy in PD, retrograde axonal degeneration due to impaired axonal transport and anterograde degeneration due to sensory neuronal cell death. Detection of phosphorylated alpha-synuclein in dermal nerve fibers might be a useful diagnostic test for PD with high specificity but low sensitivity.