Introduction
Parkinson’s disease (PD), a multicentric neurodegenerative disorder that’s progression extends to decades [
31], is traditionally characterized by a loss of dopaminergic neurons in the central nervous system (CNS), mainly the brainstem, thus leading to characteristic cardinal motor symptoms such as bradykinesia, tremor, rigidity, and postural instability [
43]. Degeneration of dopaminergic neurons and the presence of misfolded alpha-synuclein (α-syn) containing aggregates, namely Lewy bodies (LBs) and Lewy neurites (LNs), are primary pathological hallmarks of PD in the CNS. Within these aggregates phosphorylation at serin
129 is the most dominant pathological α-syn form [
3]. Recently, numerous studies have indicated that besides the CNS also the autonomic nervous system (ANS) is affected [
36]. A wide spectrum of non-motor manifestations involving the urogenital and gastrointestinal (GI) system [
32,
49] has been observed leading to impaired urinary function [
5] and constipation [
21] in patients with PD preceding the motor symptoms.
One main division of the ANS is represented by the enteric nervous system (ENS), a complex network integrated within the gut wall and organized in mainly two ganglionated nerve plexus, the myenteric and submucosal plexus [
70]. Although the ENS works autonomously from the CNS [
18], many morphological and functional properties of the ENS resemble the CNS rather than peripheral autonomic ganglia. Thus, the ENS is considered as “the little brain within the gut” [
71], where it controls GI motility, mucosal blood flow, ion and water transport and resorption [
24,
25]. Similar to the CNS, the functional integrity of the ENS depends on intact synaptic transmission and plasticity involving the synthesis, release and trafficking of a broad range of inhibitory and excitatory enteric neurotransmitters [
23,
58].
Impairment of enteric neurotransmission is associated with a wide spectrum of functional GI diseases characterized by severe disturbances of GI motility [
38] including constipation [
21]. Constipation is also one of the leading premotor symptoms in patients with PD arguing for lesions within the ENS accompanying the progression of the disease [
49]. Moreover, the ENS has been intensively discussed as a putative entry route of neurotoxins in PD suggesting that neuropathological processes spread via anatomically connected structures from the ENS to the substantia nigra and then further into other regions of the CNS [
10,
47]. Thus, assessment of ENS pathology associated with PD might offer a twofold option: (1) to use the ENS as “diagnostic window into the CNS” [
41] possibly allowing early
in vivo diagnosis of PD and neuroprotective therapy, (2) to elucidate the pathogenetic mechanisms underlying the high prevalence of GI symptoms, in particular chronic constipation, in patients with PD.
Biopsies of the GI tract are the most promising peripheral tissue source to investigate the intestinal pathology linked to PD, as the tissue can be easily obtained by routine endoscopy and offers optimal conditions to study the ENS in living patients [
40]. However, the specifity and usefulness of phosphorylated (p-α-syn) and native α-syn detection in colonic biopsies as a reliable biomarker of PD remains unclear due to highly variable results [
2,
14,
17,
59] While some studies have regarded α-syn/p-α-syn in LB or LN as a discriminator between PD and controls [
29,
39,
51,
52,
57], others have shown that expression of enteric α-syn/p-α-syn can also be observed in healthy subjects [
2,
52,
61,
68]. In line with these findings, we could show previously that α-syn appears to be abundantly expressed in the ENS of healthy subjects and p-α-syn is not only detectable in patients with PD but also in controls in an age-dependent fashion [
9].
Since p-α-syn is considered as the marker of choice to delineate pathological aggregates from physiological α-syn deposits, we aimed to further refine the assessment of p-α-syn in the ENS by morphometric quantification of phospho-serin
129-α-synuclein (p-S
129-α-syn) positive aggregates both in neuronal somata and nerve fibers in rectal biopsies of patients with PD and controls. Moreover, endoscopically retrieved biopsies allow to monitor gene expression profiles associated with intestinal pathological processes [
16]. As PD is also associated with altered neurotransmitter systems [
35,
54], we further analyzed the mRNA expression of α-syn and functionally relevant enteric neurotransmitter systems.
Discussion
Positive p-α-syn staining in patients with PD and controls
Several
in vivo studies of patients with PD using α-syn and/or p-α-syn immunohistochemistry applied to GI tract biopsies reported different results in positive staining rates (for reviews see [
2,
59,
64]) with a range from 5 to 100% for PD and from 0 to 100% for controls. The high variability of these staining rates might be explained by the different GI tract sites studied and technical work-up of biopsies (e.g. tissue handling, fixation, antigen retrieval, primary antibodies). In most of the studies p-α-syn immunoreactivity was not observed in healthy controls suggesting p-α-syn detection as an useful and specific tool for identifying patients with PD [
29,
52,
57].
However, a few studies [
9,
52,
68] reported the presence of p-α-syn in colonic biopsies also in healthy controls. By using conventional immunohistochemistry and the highly specific PET-blot technique
Visanji et al. revealed p-α-syn positive aggregates in mucosal biopsies of all healthy individuals investigated [
68]. The authors concluded that enteric p-α-syn immunoreactivity is not an appropiate discriminator between healthy controls and patients with PD. In line with these findings, we could also observe positive p-α-syn staining not only in patients with PD but also in all biopsies obtained from controls. By using fluorescence p-α-syn immunohistochemistry applied to rectal wholemount biopsies, Pouclet
et al. found mild somatic labelling of submucosal neurons both in patients with PD and controls and described dot-like pathological inclusions in p-α-syn positive structures of patients with PD compared to controls. Most of these structures are immunoreactive for the neuronal marker neurofilament 200 kDa, but some of the inclusions do not display neurofilament 200 kDa immunoreactivity, even though the authors find, that their morphology is highly suggestive of Lewy neurites [
52]. Although our findings of p-α-syn positive neuronal and non-neuronal submucosal structures (somatic labeling and dot-like pathological inclusions in p-α-syn positive nerve fibers, see Fig.
2) matched that of Pouclet and colleagues, we did not find a significant difference in p-α-syn staining rates in patients with PD, as p-a-syn staining rates for nerve fibers in the present study were 25% for patients with PD and 36% for controls (Fig.
3b). Since both studies used the same antibody, the discrepancies might be related to technical aspects (e.g. unsectioned wholemount tissue vs. tissue sections, different antibody dilutions) or due to different biopsy sampling sites in the GIT (colon vs. rectum).
In summary, given that p-α-syn was readily detectable within the submucosal plexus likewise in patients with PD and controls, the present data suggest that the mere presence of p-α-syn cannot be regarded as specific for PD. For that reason we have carried out a refined quantitative morphometric analysis of the amount and pattern of p-α-syn aggregates aiming at better discriminating between patients with PD and healthy subjects.
Increased number and area of p-α-syn positive aggregates in patients with PD
Morphological heterogeneity of LB-like structures has been already observed in the CNS appearing as either diffuse granular pleomorphic structures of variable shape and size or intraneuritic dot-like structures [
7,
20]. In the ENS
Gold et al. [
27], have found also different staining pattern ranging from light, diffuse or punctate α-syn positive labeling limited to nerve terminals to large clustering of rings and puncta in some but not all myenteric somata. Consistent with these findings we could also describe both homogeneous and granular p-α-syn labeling in myenteric and submucosal ganglia [
9].
Although additional parameters to characterize p-α-syn aggregates in more detail (e.g. distributional pattern) appear to be required, so far this quantitative morphometric approach has not been carried out previously. In fact, the assessment of the total number and area of p-α-syn positive structures per neuron revealed that patients with PD showed a significant increase of both parameters compared to controls. The overlap of values between both groups was lower for the number of p-α-syn positive structures per neuron suggesting that this parameter discriminates even better between patients with PD and controls than the area of p-α-syn positive structures.
Distinct distributional pattern of p-α-syn aggregates according to size in patients with PD
Further subdivision of neuronal p-α-syn aggregates according to size revealed a significant increase of small and large sized p-α-syn aggregates in patients with PD compared to controls, while medium sized p-α-syn aggregates were equally distributed in both groups. The best discriminator was the number of small sized p-α-syn aggregates - only one third of patients with PD fell within the range of controls, whereas two thirds showed values well above the control group.
The (patho-) physiological mechanisms leading to the formation of differently sized p-α-syn aggregates observed within the ENS remains unclear. The well-known morphological heterogeneity of LBs within the CNS has fed speculations that the spectrum of α-syn inclusions could represent different stages of LB development [
28,
55].
Gomez-Tortosa et al. hypothesized that clouds of rich α-syn containing structures could represent very early stages of LB pathology. Such lesions would progress to more compact and better-defined α-syn aggregates and some of these inclusions could be recognized as pale bodies in H&E stain considered as precursors of LBs [
69], which in turn condense to classical LBs. As p-α-syn is a subcomponent of LBs [
62], it might be assumed that the differently sized p-α-syn positive aggregates observed within enteric ganglia could represent different LB development stages ranging from small sized granula to the assembly and fusion into larger aggregates.
It is of note that although the number of small and large sized p-α-syn aggregates was significant increased in patients with PD, all three sizes of p-α-syn aggregates were also observed in controls. This suggests that p-α-syn aggregation in the gut is a physiological process, but may be dysregulated and increased in patients with PD. The following observations strengthen this hypothesis: (1) Both p-α-syn and α-syn are physiologically expressed in the human [
8,
9] and rat ENS [
50]. (2) α-syn aggregates are primarily cytoplasmic [
63] and although most of the studies observed physiological cytoplasmic α-syn as monomer [
12], some studies indicate that α-syn can assemble also physiologically as oligomers [
6,
37] and upon membrane binding as multimers [
11]. (3) Environmental toxins (e.g. rotenone) trigger PD-like progression by promoting α-syn release from enteric neurons, uptake by presynaptic neurites and retrograde transport to and accumulation in neuronal somata. [
48]. Thus, it is suggestive that increased p-α-syn positive aggregates in enteric neurons, in particular the small sized type, in PD patients reflect the inital steps of an enhanced α-syn release upon external insults.
Gene expression of α-syn
Interestingly, no difference in the SNCA mRNA expression between PD and controls was observed, even though total number and area of p-α-syn positive aggregates were significantly increased in PD patients. However, also in the CNS PD, particularly the sporadic form, is not consistently associated with increased mRNA levels of SNCA [
15,
67]. In addition, some studies showed that LB formation is also not associated with an increase in SNCA mRNA expression [
34]. In contrast to that, α-syn secretion normally is associated with an increase in SNCA mRNA expression [
44]. This strengthen the idea that our observed increase in neuronal p-α-syn positive aggregates is the product of rather a disturbed α-syn aggregation process than an increase in α-syn secretion.
Altered gene expression of neurotransmitter systems
In line with our findings a decrease of 5-HT4 receptors in the tunica muscularis has also been observed in a mouse-model of PD [
74]
. Therefore, the dysregulated 5-HT4 and 5-HT3 receptor mRNA expression observed in our study might reflect mechanisms underlying colonic dysmotility in PD. DA is known to inhibit GI motility via D1 receptors [
72] and the M3 receptor is an important subtype mediating the contraction of intestinal smooth muscle [
66]. Thus, both the significant upregulation of D1 receptor mRNA and downregulation of M3 receptor mRNA may further contribute to constipation symptoms in patients with PD. However, the upregulation of VIP mRNA does not fit into this concept, as this neuropeptide is downregulated in patients with idiopathic constipation [
46]. Generally, interpretation of enteric neurotransmitter disturbances observed within the mucosal/submucosal layer retrieved by biopsies should be made carefully in regard to its effects on GI motility, as GI motility is primary driven by the myenteric plexus. Full-thickness biopsies including all enteric nerve plexus would provide optimized conditions to study these aspects. In addition in a previous study Annerino
et al. investigated the relative abundance of NO, VIP or TH neurons between patients with PD and controls, by counting nitric oxide synthase (NOS), VIP, or TH fluorescence labelled neurons [
4]. The authors reported no difference in relative abundance of NO, VIP, or TH neurons in myenteric plexus of any GI segment of patients with PD. Thus, neuropathology in myenteric neurons as causative factor for PD-related GI dysmotility is still under discussion.
Consistent with our results, reduced mucosal expression of 5-HT4 receptor and increased mucosal expression of 5-HT3A receptors was also observed in a mouse model of experimental colitis [
45]. Additionally, 5-HT4 receptor activation is linked to anti-inflammatory effects in the GI system [
76], and increased mRNA expression of VIP was reported in moderately inflamed mucosal epithelium [
33]. These data suggest a link between PD and inflammatory processes at the level of the GI tract, as proposed by
Devos et al., who found striking similarity between pro-inflammatory cytokine expression patterns in bowel biopsies of PD patients and patients with inflammatory bowel diseases [
19].
It has been postulated that the GI tract might be an entry route for a still unknown agens or neurotoxin that crosses the intestinal epithelial barrier, induces α-syn aggregation in the ENS and migrates retrogradely via projecting neurons towards the CNS [
10]. A disturbed intestinal barrier function was recently observed in patients with PD [
56]. In this context it is of note that increased mRNA levels of DRD1 and DRD2 in the intestinal mucosa after traumatic brain injury correlated with an impaired intestinal mucosal barrier function [
73]. Thus, our observed upregulation of D1 receptor could point to a disturbed intestinal epithelial barrier in patients with PD. This hypothesis is in line with the impressive down-regulation of the muscarinergic M3 receptor, as M3 receptor is involved in the regulation of permeability in human jejunal epithelium and discussed as main mediator of transcellular transport of macromolecules [
13]. Of note, M3R is known as an activator of cytoplasmatic phospholipase A2 via the activation of protein kinase c (PKC) [
75] and since PKC itself has been shown to play a role in the modulation of tight junction proteins such as occludin [
65], it is possible, that M3 receptors may be involved in the modulation of epithelial barrier permeability both via modulation of tight junction proteins and transcellular permeability in patients with PD.
Correlation between p-α-syn positive aggregates and gene expression data
Correlation analysis between p-α-syn positive aggregates and gene expression data yielded a negative correlation between the expression level of the M3 receptor and the number of small sized p-α-syn positive aggregates, independent of both groups where correlation was lost when analysis was made separately. This suggested a direct link between p-α-syn assembling in submucosal neurons and transcript changing of M3R.
Positive correlation between mucosal α-syn staining and increased intestinal permeability or bacterial translocation in patients with PD was found before by
Forsyth et al. [
22]. Based on the widely accepted assumption that α-syn aggregates are a consequence of oxidative injury to neurons [
62], the authors proposed that local oxidative stress caused by the translocation of luminal bacteria products leads to α-syn missfolding, aggregation and subsequent neuronal damage in the ENS. Since M3R expression may play a role in modulating the epithelial barrier, our observed direct negative correlation between M3R expression and small sized p-α-syn aggregates may further reflect a causality of increased intestinal permeability and α-syn assembling in patients with PD.
Interestingly, M3R activation is linked to α-syn in a human dopaminergic cell line in which muscarinic receptor stimulation leads to translocation of oligomeric α-syn from the plasma membrane to a light vesicle fraction in the cytoplasm [
42]. The authors suggest, that α-syn could have a physiological role in the cell, in which its release transiently disinhibits membrane bound phospholipase LD2, freeing this lipase to function in ligand-stimulated manner to muscarinic receptor endocytosis. Our data therefore also suggest a physiological process of α-syn aggregation, which is linked to M3R receptor expression and therefore perhaps dysregulated in PD.
Conclusions
In summary, we provide evidence that although suggested in previous studies, the mere presence of p-α-syn positive aggregates in the ENS cannot be regarded as a diagnostic criterion for PD, as p-α-syn is also consistently expressed in healthy controls. However, if a refined morphometric analysis is applied, a specific pattern of p-α-syn aggregates can be identified in patients with PD distinct from controls. Moreover, altered gene expression profiles observed in biopsies from patients with PD suggest that PD is associated with inflammatory processes and intestinal barrier dysfunctions possibly enhancing p-α-syn assembling. Further studies are needed to proof whether subtle quantitative and morphometric characterization of enteric p-α-syn may be an additional approach to allow diagnosis of PD by using GI biopsies.
Acknowledgements
The authors gratefully acknowledge the excellent technical assistance of Bettina Facompré, Karin Stengel and Inka Geurink (Institute of Anatomy, Kiel University).