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Keywords:

  • enteric nervous system;
  • α-synuclein;
  • Parkinson's disease;
  • mucosal plexus;
  • vermiform appendix

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Roles
  8. Financial Disclosures
  9. References
  10. Supporting Information

Parkinson's disease is characterized by the pathological aggregation of Alpha-synuclein. The dual-hit hypothesis proposed by Braak implicates the enteric nervous system as an initial site of α-synuclein aggregation with subsequent spread to the central nervous system. Regional variations in the spatial pattern or levels of α-synuclein along the enteric nervous system could have implications for identifying sites of onset of this pathogenic cascade. We performed immunohistochemical staining for α-synuclein on gastrointestinal tissue from patients with no history of neurological disease using the established LB509 antibody and a new clone, MJFR1, characterized for immunohistochemistry here. We demonstrate that the vermiform appendix is particularly enriched in α-synuclein–containing axonal varicosities, concentrated in its mucosal plexus rather than the classical submucosal and myenteric plexuses. Unexpectedly, intralysosomal accumulations of α-synuclein were detected within mucosal macrophages of the appendix. The abundance and accumulation of α-synuclein in the vermiform appendix implicate it as a candidate anatomical locus for the initiation of enteric α-synuclein aggregation and permits the generation of testable hypotheses for Parkinson's disease pathogenesis. © 2013 International Parkinson and Movement Disorder Society

Aberrant aggregation of the synaptic protein Alpha-Synuclein (α-syn) in neurons is central to the pathogenesis of Parkinson's disease (PD),[1] and α-syn represents the principal protein constituent of Lewy bodies and Lewy neurites, the histopathological hallmarks of PD.[2, 3]

Clinical and experimental evidence supports a “prion-like” mechanism mediating the intracerebral spread of α-syn aggregation.[4-8] Braak has described a temporal sequence of α-syn aggregation in the central nervous system (CNS) in PD beginning in the olfactory bulb and dorsal motor nucleus of the vagus nerve (dmX) in the medulla.[9-13] This dual-hit hypothesis suggests that α-syn aggregation in the dmX may originate in the enteric nervous system (ENS),[14, 15] followed by retrograde transport from postganglionic vasoactive intestinal peptide (VIP)-ergic neurons in the ENS to preganglionic cholinergic neurons of the dmX.[12, 16]

To provide a normative anatomical basis for this hypothesis, we have compared the density as well as the intramural pattern of α-syn immunoreactivity along the gastrointestinal (GI) tract of neurologically intact humans, focusing on the stomach, appendix and surrounding regions known from rodent studies to receive particularly dense innervation from the dmX.[17-20]

Materials and Methods

  1. Top of page
  2. ABSTRACT
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Roles
  8. Financial Disclosures
  9. References
  10. Supporting Information

Specimen Acquisition

Formalin-fixed, paraffin-embedded blocks were retrieved from archived surgical specimens in the Department of Pathology and Laboratory Medicine of The Ottawa Hospital. These specimens were accrued from patients with no diagnosed synucleinopathy who underwent right hemicolectomy (n = 10; average age = 73 years) or subtotal gastrectomy (n = 10; average age = 63 years). Pertinent clinical and demographic information is presented in Supporting Table 1.

Immunohistochemical Staining

Sections of 5 μm thickness were cut from formalin-fixed, paraffin-embedded tissue blocks. Staining was performed using the automated standard methods as described, along with the immunofluorescence (IF) procedure and the characterization of MJFR1 (Supporting Figure 1), in the Supporting Methods; antibodies used are listed in Supporting Table 2.

Results

  1. Top of page
  2. ABSTRACT
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Roles
  8. Financial Disclosures
  9. References
  10. Supporting Information

α-syn–positive Nerve Fibers Are Abundant in the Appendiceal Lamina Propria

In marked contrast to the other areas, the vermiform appendix demonstrated abundant punctate α-syn immunoreactivity arranged along filamentous structures in the lamina propria (Fig. 1A-D). These α-syn immunoreactive fibers formed a prominent lattice-like network consistent with the mucosal plexus.[21-24] Staining was characterized by an apicobasal gradient of increasing fiber density.

image

Figure 1. α-syn immunoreactivity in the mucosal plexus of the vermiform appendix. A: Low magnification view of α-syn staining with (b) and (c) indicating locations of subsequent panels. BD: Higher magnification images showing the fine reticular pattern of α-syn staining in the inner proprial subplexus (B), with denser staining making individual fibers difficult to distinguish in the external propria (C) and pericryptal (D) mucosal subplexuses. The lamina propia (lp) occupies the space between the muscularis mucosae (mm) and the surface epithelium, the single cell layer structure beneath the lumen (lum). Scale bars: 50 μm. cr, crypt.

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In addition, in all cases, α-syn–positive ganglion cells were noted in the appendiceal mucosa proximate to the muscularis mucosae (Fig. 2A, also Figs. 1D and 3D-F). Almost all perikarya in appendiceal submucosal ganglia (Fig. 2B) and the great majority of those in myenteric ganglia (Fig. 2C,D) displayed cytoplasmic α-syn immunoreactivity. In addition, intensely α-syn immunostained axon terminals appeared as punctae closely apposed to cell bodies.

image

Figure 2. α-syn–immunoreactive ganglia of the enteric nervous system. A: Mucosal ganglion surrounded by α-syn–positive fibers of the mucosal plexus in close proximity to muscularis mucosae. B: Submucosal ganglion with fibers extending toward the mucosa. C, D: Ganglion in the circular muscle layer (C) and 2 myenteric ganglia (D) with scant large α-syn–positive nerve fiber bundles as well as abundant small nerve fiber bundles and rare single varicose axons coursing between smooth muscle cells of the muscularis. Scale bars: 50 μm. cr, crypt; cm, circular muscle layer; lm, longitudinal muscle layer; mm, muscularis mucosae.

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image

Figure 3. Double-labeling IF demonstrating: colocalization of (A–C) α-syn (A) and SYP (B), (DF) α-syn (D) and UCH-L1 (E), and (GI) α-syn (G) and VIP (H) in mucosal nerve fibers; and (JL) lack of colocalization of αsyn (J) and CD34 (K) in vascular channels of the appendiceal lamina propria. C,F,I,L: Merged images. Note Schwann cell nuclei apposing nerve fibers (arrowheads) and a fine punctate pattern of SYP staining in the muscularis mucosae (mm in B and C). A mucosal ganglion is also readily visible in DF (arrow). Scale bars: (AC) 20 μm; (DF) 60 μm; (GI) 67 μm; and (JL) 50 μm. mm, muscularis mucosae; IF, immunofluorescence; VIP, vasoactive intestinal peptide.

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α-syn immunoreactive fibers colocalized with synaptophysin (SYP; Fig. 3A-C), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a reliable neural marker (Fig. 3D-F), and VIP-immunoreactive fibers found throughout the lamina propria of the appendix (Fig. 3G-I). In contrast, there was a complete absence of α-syn-positive punctae along CD34-immunoreactive vascular channels (Fig. 3J-L). Thus, the α-syn-immunoreactive fibers represent nerve bundles, not vascular channels.

α-syn Fibers Are Less Prominent Elsewhere in the GI Tract

On examination of gastric corpus mucosa, there were only scattered stained punctae, rarely filamentous, presumably corresponding to α-syn–immunoreactive axon terminals (Fig. 4B). Only rare ganglionic perikarya were α-syn–positive.

image

Figure 4. α-syn immunoreactivity in the mucosa of the gastric corpus (A,B), terminal ileum (C,D), and ascending colon (E,F). Arrows in (B) and (D) indicate fine nerve fibers. Arrowhead in (F) shows a α-syn–immunoreactive colonic macrophage. Scale bars: 50 μm. cr, crypt; lp, lamina propria; lum, lumen; sm, submucosa.

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Within the lamina propria of the terminal ileum, sparse α-syn stained fibers were encountered, particularly circumscribing the cryptal epithelium (Fig. 4C,D).

α-syn immunostaining in the lamina propria of the ascending colon consisted mainly of diffuse cytoplasmic staining of cells concentrated adluminally (Fig. 4E,F), identified as macrophages on CD68 double-IF studies (Fig. 5I-K). α-syn–immunoreactive varicosities were sparse.

image

Figure 5. Macrophages in the intestinal lamina propria. A: α-syn–immunoreactive structures in appendiceal mucosa. B: Membrane-bound α-syn (arrowheads in C) is found within CD68-positive lysosomes (arrows in D) by IF (merge in E). F–H: α-Syn (F, green) is not found within CD68-positive macrophages (G, red) of Peyer's patches of the terminal ileum. I–K: α-syn (I, green) is diffusely positive in CD68-positive colonic macrophages (J, red), a pattern distinct from that in the appendix. Scale bars: (A,B) 50 μm; (CK) 10 μm. IF, immunofluorescence.

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Quantitative analysis (see Supplementary Methods) confirmed that α-syn-immunoreactive fibers are much more abundant in the mucosa of the vermiform appendix than in the stomach, ileum or colon (Supporting Figure 2).

Other α-syn–Positive Structures in the Appendiceal Lamina Propria Are Located in Macrophages Within CD68-Positive Vesicles

The appendiceal mucosa displayed large α-syn–immunoreactive structures apparently confined to cell bodies (Fig. 5A), many with a botryoid or “bunch-of-grapes” appearance. They failed to stain for ubiquitin or phospho-S129 α-syn and did not bind thioflavin T or thiazine red (not shown). These cells were CD68-immunoreactive macrophages, as confirmed in single (Fig. 5B) and double-labeling studies (Fig. 5C-E).The α-syn signal within macrophages colocalized with a gold autofluorescence visible with a 4′,6-diamidino-2-phenylindole (DAPI) filter that was somewhat diminished by CuSO4, consistent with lipofuscin.[25]

In the terminal ileum, CD68-positive cells found in the lamina propria and Peyer's patches were uniformly α-syn–negative (Fig. 5F-H). Many of the CD68-immunoreactive cells in Peyer's patches demonstrated the multilocular pattern found in the appendix but these lacked staining for α-syn (Fig. 5F-H), consistent with intestinal pigment cells.[26]

Discussion

  1. Top of page
  2. ABSTRACT
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Roles
  8. Financial Disclosures
  9. References
  10. Supporting Information

Despite the perikaryal localization of Lewy bodies, α-syn aggregation begins in axon terminals.[1, 27] If synaptic α-syn in the ENS is targeted at the initial stages of PD,[16] then a higher density of α-syn innervation in any particular ENS region would render it attractive as a candidate locus for initiation of the PD pathogenetic cascade.

In our cohort of neurologically intact subjects, we detected α-syn immunostaining in all subjects in all GI regions, contrasting with studies by Shannon and colleagues[28] and Gold and colleagues,[29] who detected α-synimmunoreactivity in only 8% and 52% of colons from neurologically normal subjects, respectively. This discrepancy may be related to differences in the sensitivity of the techniques employed (ie, staining with the MJFR1 antibody characterized in Supporting Figure 1) or our use of surgical specimens (used by Shannon and colleagues[28] but not by Gold and colleagues[29]), obviating issues of postmortem alterations in α-syn immunoreactivity.

This study revealed the relative abundance of α-syn–immunoreactive nerve terminals in the lamina propria of the vermiform appendix, consistently the densest mucosal α-syn positivity among the sites sampled. This contrasts with reports of decreasing rostrocaudal gradients of Lewy body number[30] and phosphorylated α-syn aggregates[31] within the GI tract of synucleinopathy patients, studies omitting the appendix.

The localization of punctate α-syn staining along the length of appendiceal nerve fibers labeled with SYP, UCH-L1, and VIP suggests that it is present in axonal varicosities en passant. The presence of VIP-positive, α-syn–negative fibers indicates that α-syn is expressed in a subpopulation of mucosal axons.

The architecture of the ENS is complex (reviewed in Brehmer and colleagues[32]) but the mucosal plexus has been described in detail,[21, 23, 33] including in the appendix.[34] It is a ganglionic plexus composed of unmyelinated nerve fibers subdivided into several subplexuses.[21-23] The relative abundance of α-syn renders the appendiceal mucosa an attractive candidate as an initial focus of environmentally-induced α-syn aggregation. According to the dual-hit hypothesis, an unidentified neurotropic pathogen in the GI lumen triggers α-syn aggregation after achieving access to the CNS through the myenteric plexus of the stomach, via postganglionic VIPergic neurons,[16] but this localization can be disputed based on our findings. First, α-syn–immunoreactive neurons are rare (3%) in the gastric myenteric plexus,[35] and our study confirms this and extends it to fibers in the mucosa. Second, the stomach was implicated as a site for initiation of α-syn aggregation based largely on its significant dmX-derived innervation,[12] but the rat cecum (analogous to the human appendix) also receives substantial vagal efferents.[17-20] The third issue is the requirement for the putative luminal agent to breach the brush border and the mucosal barrier in order to access enteric neurons.[12] The dense α-syn–positive innervation that we have demonstrated in the mucosal plexus of the appendix places it in close proximity to any luminal trigger. In addition, this pattern provides an opportunity to propose that blood-borne agents could also easily access mucosal α-syn due to the lack of a blood-ENS barrier.[36] The GI tract, and specifically the appendix, would then be especially vulnerable to the α-syn aggregation process through its rich endowment of α-syn combined with its lack of a blood-tissue barrier.

We described α-syn–positive structures in enlarged lysosomes within macrophages located in the appendiceal lamina propria, but not in those of the ascending colon or terminal ileum. CD68 decorates lysosomal membranes[37, 38] and colocalizes with macrophage-specific markers[39] in the normal intestinal mucosa. Phillips and Powley[40] demonstrated that macrophages within the muscularis propria of the rat GI tract maintain ENS synaptic architecture, with direct contacts between macrophages and α-syn–positive axonal varicosities or aggregates in the muscularis. They postulate that intestinal macrophages may ingest pathological α-syn aggregates.

The accumulation of α-syn within lysosomes in neurologically intact patients is a novel finding because hitherto this association has been confined to human synucleinopathies[41] and α-syn–overexpression models in mice.[42, 43] Furthermore, we find it intriguing that 3 human PD-linked gene products can be associated with macrophage function in vivo; ie, α-syn (this report and Phillips and Powley[40]), and leucine-rich-repeat kinase 2 (LRRK2) and glucocerebrosidase (GBA1) (in Hakimi and colleagues[44]). Given that appendectomy is a common medical procedure[45] and the age of removal is generally known, it is possible to test hypotheses with respect to the role of the appendix in PD. It will be important to determine whether α-syn in appendiceal lysosomes reflects the PD pathogenetic process at its inception.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Roles
  8. Financial Disclosures
  9. References
  10. Supporting Information

We thank Dr. Robert Nussbaum for the kind gift of α-syn transgenic mice and Dr. David Grynspan, Mansoureh Hakimi, and Dr. Julianna J. Tomlinson for helpful discussions. M.T.G. is funded by studentships from the Canadian Institutes of Health Research (CIHR) and the Parkinson's Research Consortium of Ottawa (PRC). J.M.W. is funded by the PRC, Parkinson's Society Canada, and a CIHR grant held jointly with D.A.G. M.G.S. holds the Bhargava Research Chair in Neurodegeneration and the Canada Research Chair in Parkinson's Disease and Translational Neuroscience (CIHR) and his research is supported by CIHR and the Canadian Foundation for Innovation.

Author Roles

  1. Top of page
  2. ABSTRACT
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Roles
  8. Financial Disclosures
  9. References
  10. Supporting Information

MTG: Research project: A. Conception, B. Organization, C. Execution Manuscript: A. Writing of the first draft, B. Review and Critique. DGM: Research project: A. Conception, B. Organization Manuscript: B. Review and Critique. DAG: Research project: A. Conception, B. Organization, Manuscript: B. Review and Critique. MGS: Research project: A. Conception, B. Organization, Manuscript: B. Review and Critique. JMW: Research project: A. Conception, B. Organization, C. Execution Manuscript: A. Writing of the first draft, B. Review and Critique.

Financial Disclosures

  1. Top of page
  2. ABSTRACT
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Roles
  8. Financial Disclosures
  9. References
  10. Supporting Information

MTG: Studentships from Canadian Institute of Health Research and the Parkinson's Research Consortium. DGM: Honoraria as Speaker from Novartis and Janssen. DAG: Grants from the Canadian Institutes of Health Research, the Cancer Research Society Inc., and the Canadian Cancer Society Research Institute. Employee of the Ottawa Hospital. Member of the scientific advisory board of the Cancer Research Society Inc. MGS: Intellectual Property Rights: MGS is a co-inventor of USPTO Notification #3875410; Consultancies: MGS has been paid for consulting services by Bristol Meyers and BiogenIdec; Honoraria: Teva Neurosciences. JMW: Research Grants from the Parkinson Society of Canada, The Canadian Institute of Health Research, and the University of Ottawa Department of Pathology and Laboratory Medicine. Dr. Woulfe was employed as Staff Neuropathologist at, and received a salary for said services from, The Ottawa Hospital.

References

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  2. ABSTRACT
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Roles
  8. Financial Disclosures
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. ABSTRACT
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Roles
  8. Financial Disclosures
  9. References
  10. Supporting Information

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