Increased intestinal permeability correlates with sigmoid mucosa alpha-synuclein staining and endotoxin exposure markers in early Parkinson's disease

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. The pathological hallmark of PD is neuronal inclusions termed Lewy bodies whose main component is alpha-synuclein protein. The finding of these Lewy bodies in the intestinal enteric nerves led to the hypothesis that the intestine might be an early site of PD disease in response to an environmental toxin or pathogen. One potential mechanism for environmental toxin(s) and proinflammatory luminal products to gain access to mucosal neuronal tissue and promote oxidative stress is compromised intestinal barrier integrity. However, the role of intestinal permeability in PD has never been tested. We hypothesized that PD subjects might exhibit increased intestinal permeability to proinflammatory bacterial products in the intestine. To test our hypothesis we evaluated intestinal permeability in subjects newly diagnosed with PD and compared their values to healthy subjects. In addition, we obtained intestinal biopsies from both groups and used immunohistochemistry to assess bacterial translocation, nitrotyrosine (oxidative stress), and alpha-synuclein. We also evaluated serum markers of endotoxin exposure including LPS binding protein (LBP). Our data show that our PD subjects exhibit significantly greater intestinal permeability (gut leakiness) than controls. In addition, this intestinal hyperpermeability significantly correlated with increased intestinal mucosa staining for E. coli bacteria, nitrotyrosine, and alpha-synuclein as well as serum LBP levels in PD subjects. These data represent not only the first demonstration of abnormal intestinal permeability in PD subjects but also the first correlation of increased intestinal permeability in PD with intestinal alpha-synuclein (the hallmark of PD), as well as staining for gram negative bacteria and tissue oxidative stress. Our study may thus shed new light on PD pathogenesis as well as provide a new method for earlier diagnosis of PD and suggests potential therapeutic targets in PD subjects.

Trial registration: Clinicaltrials.gov NCT01155492.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Consort 2010 flow diagram of this study.

12 research subjects were invited to participate in this study. Two were excluded because they failed to meet inclusion criteria (prohibited medications). One study subject incorrectly performed the 24-hour urine collection for intestinal permeability testing and his data were excluded from analysis. Nine study subjects completed all study assessments.

Figure 2. Intestinal permeability to lactulose and mannitol is similar in control and PD subjects.

PD subjects and healthy controls were assessed for intestinal permeability using an oral sugar solution containing sucrose, lactulose, mannitol, and sucralose and GC analysis of 24 hour urine samples as described in Materials and Methods. Urinary lactulose and mannitol as well as the L/M ratio (all primarily measures of small intestinal permeability) were not significantly different in PD patients compared to controls. Data are presented as mean % urinary excretion of the oral dose in 24 hours ± SE. * p

Figure 3. Intestinal permeability to sucralose is significantly greater in PD patients.

PD subjects and healthy controls were assessed for intestinal permeability using an oral sugar solution containing sucrose, lactulose, mannitol, and sucralose and GC analysis of 24 hour urine samples as described in Materials and Methods. Urinary sucralose, a measure of whole intestine permeability, was significantly greater in PD subjects vs. healthy age matched controls. Data are presented as mean % urinary excretion of the oral dose in 24 hours ± SE. * p

Figure 4. Plasma LBP is significantly lower in PD patients.

Plasma levels of LPS binding protein (LBP), an indirect measure of systemic endotoxin exposure, were determined for PD subjects and healthy controls as described in Materials and Methods. Values for plasma LBP in PD subjects were significantly lower than in healthy controls. Data are presented as means (ng/ml) ± SE. *p

Figure 5. Immunohistochemical staining of intestinal biopsies for E. coli, α-synuclein, and 3-NT is significantly greater in PD subjects.

Intestinal biopsies from PD subjects and healthy controls were formalin fixed, paraffin embedded, and then cut to 5 µm and processed as described in Materials and Methods for each respective antibody to either E.coli gram negative bacteria (A,D,G), α-synuclein (B,E,H) or 3-nitrotyrosine (3-NT)(C,F,I). Representative images are shown from a PD subject at 15× (A–C) and 40× (D–F) magnification using the same field of view and from a healthy control subject (G–I) at 40×. Staining data were analyzed by blinded observers and PD subjects were found to stain significantly greater for E. coli, α-synuclein, and 3-NT. Scale bars = A–C 50 µm, D–I 100 µm.