Parkinson's disease is not associated with gastrointestinal myenteric ganglion neuron loss

Abstract

Gastrointestinal dysfunction is a prominent non-motor feature of Parkinson's disease (PD) that contributes directly to the morbidity of patients, complicates management of motor symptoms, and may herald incipient PD in patients without motor disability. Although PD has traditionally been considered a disease of dopaminergic neurons in the substantia nigra, analyses of gastrointestinal samples from PD patients have consistently revealed pathology in the enteric nervous system. The relationship of PD pathology to GI dysmotility is poorly understood, and this lack of understanding has led to limited success in developing treatments for PD-related GI symptoms. We have quantitatively compared myenteric neuron density and relative abundance of NO, VIP, and catecholamine neurons between patients with PD and control individuals along the length of the GI tract. In addition, we have examined the frequency of GI α-synuclein neuritic pathology and its co-localization with the same neuronal markers. We have included a comparison with a small population of patients with incidental Lewy bodies found at autopsy. These data indicate that there is no neuronal loss in the myenteric plexus in PD. Lewy body pathology parallels parasympathetic autonomic input from the dorsal motor nucleus of the vagus, not the distribution of extrinsic sympathetic input or intrinsic enteric neurons, and is only rarely co-localized with tyrosine hydroxylase. These data provide a critical background to which further analyses of the effect of PD on the GI tract may be compared and suggest that neuropathology in myenteric neurons is unlikely to be a causative factor in PD-related GI dysmotility.

Fig. 1. Quantitative assessment of myenteric neurons

Absolute numbers (neurons/mm²) and relative proportions of myenteric neuron phenotypes were assessed using double-label immunofluorescence. a. Myenteric ganglion from colon demonstrating double-label immunofluorescence for NOS (green) and HuC/D (red). Double-labeled NOS-positive neurons are yellow/orange. NOS-negative neurons (HuC/D only) are red. NOS-positive processes are green (arrows). b. Myenteric ganglion from stomach demonstrating double-label immunofluorescence for VIP (green) and HuC/D (red). Double-labeled VIP-positive neurons are yellow/orange. VIP-negative neurons (HuC/D only) are red. VIP-positive processes are green (arrows). c. Myenteric ganglion from stomach demonstrating double-label immunofluorescence for TH (green) and HuC/D (red). Double-labeled TH-positive neurons are yellow/orange. TH-negative neurons (HuC/D only) are red. TH-positive processes are green. Dotted lines outline ganglion area. LM, longitudinal muscle layer; CM, circular muscle layer. Scale bar = 100 μm

Fig. 2. Numbers of myenteric neurons (per mm² ganglion area) in stomach, duodenum, and colon

a-c. Scatter plots of total neurons (HuD-positive) counted per mm² from all cases for (a) stomach (R² = 0.84), (b) duodenum (R² = 0.77), and (c) colon (R² = 0.90). d–f. Dot plots of NOS neurons counted per mm² for (d) stomach, (e) duodenum, and (f) colon. g–i. Dot plots of VIP neurons counted per mm² for (g) stomach, (h) duodenum, and (i) colon. j–l. Dot plots of TH neurons counted per mm² for (j) stomach, (k) duodenum, and (l) colon

Fig. 3. Myenteric neuron number and ganglion area (per mm intestinal length) in PD and ILB

a. HUD neurons per mm length b. Ganglion area per mm length. Bars are mean ± SEM from 12 control individuals, 13 PD patients, and 4 ILB patients. There is no difference between controls and patients in any segment. Density of ganglia per mm length varies based on GI segment examined

Fig. 4. Relative proportions of major myenteric neuron subtypes in PD and ILB

a. NOS b. VIP c. TH. Bars are mean ± SEM from 12 control individuals, 13 PD patients, and 4 ILB patients. There is no difference in the proportion of any neurochemical phenotype between controls and patients in any segment. The relative proportions of individual neurochemical subtypes vary based on GI tract segment

Fig. 5. Examples of Lewy pathology in gut from PD and ILB patients in different layers from different segments of the gut

a. Mucosal staining in the stomach (lumen on lower left). b–d demonstrate submucosal staining in stomach (b), duodenum (c), and colon (d). Asterisks (*) indicate submucosal blood vessels. Arrows in c and d indicate neurons in submucosal ganglia. e. Synuclein staining in circular muscle (stomach). Staining in muscle layers was extremely rare with this the only observable example in the study. f–g. Myenteric ganglia staining in ileum (f, high magnification) and colon (g, lower magnification). Arrows indicate myenteric neurons in ganglia surrounded by intestinal smooth muscle. All scale bars = 50 μm

Fig. 6. Distribution of Lewy pathology by gut segment in PD and ILB patients

a. The percent of cases in which Lewy bodies were detected in any layer of any gut segment after examination of one section per segment (a total of 5 sections per case). N = 13 for PD and 4 for ILB. No Lewy bodies were detected in control samples (not shown). b. The percent of the total number of myenteric ganglia counted that contained Lewy bodies for each patient group. Total number of ganglia examined: stomach (123 PD and 24 ILB), duodenum (104 PD and 33 ILB), ileum (221 PD and 62 ILB), colon (214 PD and 64 ILB), and rectum (27 PD and 37 ILB)

Fig. 7. Distribution of Lewy pathology by gut layer in PD patients

Distribution of Lewy pathology in PD patients by layer across all gut segments. MP, myenteric plexus; SM, submucosa. Submucosal (SM) staining is broken out into 3 categories: submucosal blood vessels only (white), submucosal plexus only (shaded), and staining in both structures (hatched)

Fig. 8. Lewy bodies in gut from PD patients were not observed to co-localize with NOS or VIP, and only very rarely with TH

a. LB509 (red) and NOS (green). b. LB509 (red) and VIP (green). c. LB509 (red) and TH (green). All scale bars = 100 μm. Inset in c shows a small area of double-labeling of LB509-positive neurites with TH.