The vermiform appendix impacts the risk of developing Parkinson's disease

Abstract

The pathogenesis of Parkinson's disease (PD) involves the accumulation of aggregated α-synuclein, which has been suggested to begin in the gastrointestinal tract. Here, we determined the capacity of the appendix to modify PD risk and influence pathogenesis. In two independent epidemiological datasets, involving more than 1.6 million individuals and over 91 million person-years, we observed that removal of the appendix decades before PD onset was associated with a lower risk for PD, particularly for individuals living in rural areas, and delayed the age of PD onset. We also found that the healthy human appendix contained intraneuronal α-synuclein aggregates and an abundance of PD pathology-associated α-synuclein truncation products that are known to accumulate in Lewy bodies, the pathological hallmark of PD. Lysates of human appendix tissue induced the rapid cleavage and oligomerization of full-length recombinant α-synuclein. Together, we propose that the normal human appendix contains pathogenic forms of α-synuclein that affect the risk of developing PD.

Fig. 1.. An appendectomy reduces the risk of PD in a general population.

Data from the SNPR involving 1,698,000 individuals. (A) Cumulative incidence plot showing the rate of PD diagnosis in individuals who previously had an appendectomy and matched nonappendectomized controls. Incidence rate (PD cases per 100,000 person-years) in appendectomized individuals (n = 551,003 without PD, n = 644 with PD) was 1.60 (95% CI, 1.46 to 1.75); in the general population (n = 1,144,745 without PD, n = 1608 with PD), the PD incidence rate was 1.98 (95% CI, 1.87 to 2.10). (B) Age at PD diagnosis in individuals who had an appendectomy 20 or more years prior relative to nonappendectomized controls. n = 101 patients with PD with an appendectomy and n = 658 patients with PD without this surgery; hazard ratio, 0.793 (95% CI, 0.642 to 0.980). (C) Cumulative incidence plot showing the rate of PD diagnosis in appendectomized and nonappendectomized individuals living in rural or urban regions of Sweden. Incidence rate (PD cases per 100,000 person-years) in appendectomized individuals in the rural population: 1.49 (95% CI, 1.31 to 1.68); in appendectomized individuals in the urban population: 1.77 (95% CI, 1.55 to 2.02); in the general rural population: 2.00 (95% CI, 1.87 to 2.15); in the general urban population: 1.97 (95% CI, 1.79 to 2.16).

Fig. 2.. Epidemiological analysis of PPMI data shows that an appendectomy delays the age of PD onset.

(A) Age of PD onset in patients who had an appendectomy 30 or more years prior relative to that of nonappendectomized patients. n = 39 patients with PD with an appendectomy (mean PD age of onset, 62.6; 95% CI, 60.1 to 65.0) and n = 780 PD controls without this procedure (mean age of PD onset, 59.0; 95% CI, 58.2 to 59.7). (B and C) PD age of onset in patients with PD with an immune condition that does not involve the GI tract (B) or a surgery that is not appendectomy (C). Patients with PD were matched on the basis of age of condition/surgery (i.e., age of appendectomy, non-GI immune condition diagnosis, and age of other surgery), sex, ethnicity, number of education years, family history, and mutation status. The effect of appendectomy, non-GI immune conditions, or other surgery occurring 30 or more years before PD was examined. Top panels show the age of PD onset in non-GI immune conditions or other surgeries compared to patients with PD without such conditions/other surgeries. Bottom panels show age of PD onset in patients who had an appendectomy relative to patients with non-GI immune conditions or other surgeries. Mean age of PD onset for the appendectomy group (n = 39), the non–GI tract immune condition group (n = 39), the other surgery group (n = 22), and the no-condition/surgery group (n = 366) is 62.6 (95% CI, 60.1 to 65.0), 58.6 (95% CI, 55.7 to 61.5), 57.7 (95% CI, 55.4 to 59.9), and 58.0 (95% CI, 56.8 to 59.2), respectively. n.s., not significant.

Fig. 3.. α-Synuclein pathology is prevalent in the healthy human appendix.

(A) Proteinase K–resistant α-synuclein (red) in the appendix of healthy individuals. Hematoxylin was used as a nuclear counterstain (purple). Tissue distribution of proteinase K–resistant α-synuclein aggregates in a representative cross section of human appendix (i and ii), in the muscularis externa (iii), and mucosa of the appendix (iv). Scale bars, 2 mm (i), 250 µm (ii), and 100 µm (iii and iv). (B) Phosphorylated α-synuclein in the human appendix. Sections were probed for α-synuclein phosphorylated at serine 129 (pSer129) using antibody AB51253 and detected with peroxidase-conjugated antibodies. Top panels depict plexus containing pSer129 puncta (arrows). Bottom panels depict pSer129 staining in the cingulate cortex in PD patient brain tissue. Scale bars, 50 µm (left) and 10 µm (right). (C) Cellular localization of human α-synuclein aggregates. Proteinase K–digested tissue sections were probed with antibodies against peripherin (top panels, green) or synaptophysin (bottom panels, green). Sections were also probed for proteinase K–resistant α-synuclein (red) and 4′,6-diamidino-2-phenylindole (DAPI) stain (blue). Sections were imaged by confocal microscopy. Depicted are orthogonal projections for each emission channel individually and with all channels merged in the last panel. Fluorescent signal in the z axis is depicted for the area of interest (crosshairs). All images are representative of staining done on n ≥ 9 individuals. Scale bars, 25 µm.

Fig. 4.. The appendix contains an abundance of truncated α-synuclein proteoforms.

(A) Representative blot showing immunoprecipitation of α-synuclein from the Triton X-100–soluble fraction of substantia nigra (SN) and appendix of control individuals (C) and patients with PD (PD). α-Synuclein immunoprecipitation (IP), 20 µg of protein of the Triton X-100–soluble fraction (input), and 20 µg of protein of the remaining sample after immunoprecipitation (FT) were resolved on SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the anti–α-synuclein antibody SYN1 (clone 42/α-synuclein). Low and high exposure included to show all immuno-reactive proteoforms. HMW, high molecular weight. (B) Densitometric analysis showing the ratio of α-synuclein cleavage product to monomer in the Triton X-100–soluble fraction. n = 8 healthy control appendix, n = 5 PD appendix, n = 4 healthy control substantia nigra, n = 4 PD substantia nigra. (C) Blot showing Triton X-100–insoluble fractions from appendix tissues. Triton X-100–insoluble fractions were extracted with 8 M urea and blotted using an antibody against α-synuclein (MJFR1). The relative abundance of (D) monomeric and (E) cleaved α-synuclein was determined by densitometric analysis. α-Synuclein immunoreactivity was normalized to in-gel protein abundance as measured by Coomassie blue staining. Data are representative of n = 8 healthy controls and n = 6 patients with PD. Red arrows highlight the position of cleavage product. *P < 0.05, **P < 0.01 by one-way or two-way analysis of variance (ANOVA).

Fig. 5.. Rapid cleavage and aggregation of α-synuclein in the human appendix.

Triton X-100–soluble appendix lysates were combined with purified, full-length recombinant human α-synuclein and then vigorously shaken for up to 48 hours. α-Synuclein proteoforms were detected on SDS-PAGE by immunoblotting with MJFR1 antibody. (A to C) The formation of α-synuclein cleavage products and oligomers over a 48-hour time course. Representative blot (A) and densitometric analysis of cleavage products (B) and oligomers (C), n = 4 healthy appendix samples. (D to F) Time course using tissue lysate from appendix (AP) and substantia nigra (SN) from healthy individuals (C) and patients with PD. (D) Representative blots of shaking assay performed with purified α-synuclein combined with appendix or substantia nigra lysates. Left blot probed with anti-α-synuclein antibody (MJFR1); right blot probed with α-synuclein aggregate-preferring antibody (5G4). Time scale is 0, 24, and 48 hours. Densitometric analysis of cleavage products (E) and oligomers (F), n = 3 to 4 samples per group. Repeated-measures ANOVA for cleavage products showed a main effect of time [F(2,36) = 5.0, P < 0.05] and tissue [F(1,36) = 31.6, P < 10⁻⁵]; repeated-measures ANOVA for oligomers showed a main effect of time [F(2,30) = 30.0, P < 10⁻⁷] and tissue [F(1,30) = 9.3, P < 0.005]. *P < 0.05, **P < 0.01 by post hoc Tukey test.

Fig. 6.. Identification of α-synuclein truncation products in the human appendix using TD-MS.

(A) Blot showing α-synuclein immunoprecipitated from the healthy human appendix. (B) Graphical representation of the α-synuclein proteoforms identified by TD-MS. Analysis of the TD-MS fragmentation data by TDPortal identified 11 α-synuclein proteoforms present in the human appendix (n = 1 individual), corresponding to full-length α-synuclein and 10 α-synuclein truncation products. α-Synuclein proteoforms in the appendix are depicted with the amino acid start and end position marked. (C) MS spectrum shows full-length α-synuclein (marked with “*”) in addition to truncation products. m/z, mass/charge ratio. (D) Deconvoluted spectrum showing relative abundance of the α-synuclein proteoforms in the human appendix and their respective protein masses.