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 immunoreactive 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.
- Table 1 Summary of data from the SNPR and SCB for individuals with or without an appendectomy.
Prevalence of PD in the general
populationNo PD diagnosis, n PD diagnosis, n Regression
coefficientOdds ratio
(95% CI)P All Appendectomy 551,003 644 −0.18 ± 0.05 0.831 (0.756–0.907) 8.1 × 10−5 Controls 1,144,745 1608 Rural Appendectomy 342,209 373 −0.26 ± 0.06 0.769 (0.681–0.867) 1.5 × 10−5 Controls 703,075 997 Urban Appendectomy 208,794 271 −0.06 ± 0.07 0.939 (0.817–1.082) 0.383 Controls 441,670 611 Age at PD diagnosis Diagnosis age
(years)*Hazard ratio
(95% CI)Appendectomy >0 years before PD All Appendectomy 479 74.93 ± 0.51 0.990 (0.890–1.101) 0.851 Controls 1176 74.90 ± 0.31 Rural Appendectomy 277 75.09 ± 0.60 0.989 (0.860–1.138) 0.877 Controls 686 74.53 ± 0.41 Urban Appendectomy 202 74.71 ± 0.88 0.965 (0.818–1.137) 0.666 Controls 490 75.42 ± 0.47 Appendectomy ≥20 years before PD All Appendectomy 101 74.85 ± 0.81 0.793 (0.642–0.980) 0.027 Controls 658 73.23 ± 0.33 Rural Appendectomy 63 74.09 ± 1.09 0.753 (0.575–0.988) 0.034 Controls 417 72.12 ± 0.44 Urban Appendectomy 38 76.12 ± 1.15 0.802 (0.567–1.135) 0.202 Controls 241 75.14 ± 0.45 *Means ± SEM.
Supplementary Materials
www.sciencetranslationalmedicine.org/cgi/content/full/10/465/eaar5280/DC1
Materials and Methods
Fig. S1. Age of PD onset in individuals with and without a family history of PD in the PPMI data.
Fig. S2. An appendectomy delays the age of PD onset in individuals with a family history of PD that is not explained by genetic risk factors of PD.
Fig. S3. Validation of proteinase K digestion protocol in the human appendix used to assess aggregated α-synuclein.
Fig. S4. Evaluation of α-synuclein proteolysis under different tissue processing conditions.
Fig. S5. Extraction of detergent-soluble and -insoluble α-synuclein from appendix and brain tissue.
Fig. S6. Active cleavage of α-synuclein in the in vitro shaking assay with appendix tissue lysates.
Fig. S7. Effect of protease inhibition on α-synuclein cleavage and aggregation induced by appendix lysates.
Fig. S8. Identification of α-synuclein using TD-MS.
Fig. S9. Schematic of proposed model for the contributions of the vermiform appendix to PD.
Table S1. Characteristics of study samples from the SNPR.
Table S2. Appendectomy in relation to age of PD onset for patients with PD in the SNPR study.
Table S3. Incidence of PD in males and females living in rural and urban areas, SNPR study.
Table S4. Demographic and clinical information of patients with PD in the PPMI.
Table S5. Appendectomy in relation to age of PD onset for patients with PD in PPMI.
Table S6. PD age of onset is delayed in individuals with an appendectomy but not in individuals with non-GI immune conditions or other surgeries.
Table S7. Patients who had an appendectomy 30 or more years before PD do not show changes in PD symptom severity, as measured by the Hoehn and Yahr scale and UPDRS.
Table S8. α-Synuclein aggregates are detected in the appendix of both young and older adult individuals and are present in normal and inflamed appendix.
Table S9. Demographic and clinical information of sample sets used in PPMI study.
Table S10. Appendectomy delays age of PD onset in patients with de novo PD of PPMI.
Data file S1. Source data for biochemical assays.
References (92–96)
Additional Files
The PDF file includes:
- Materials and Methods
- Fig. S1. Age of PD onset in individuals with and without a family history of PD in the PPMI data.
- Fig. S2. An appendectomy delays the age of PD onset in individuals with a family history of PD that is not explained by genetic risk factors of PD.
- Fig. S3. Validation of proteinase K digestion protocol in the human appendix used to assess aggregated α-synuclein.
- Fig. S4. Evaluation of α-synuclein proteolysis under different tissue processing conditions.
- Fig. S5. Extraction of detergent-soluble and -insoluble α-synuclein from appendix and brain tissue.
- Fig. S6. Active cleavage of α-synuclein in the in vitro shaking assay with appendix tissue lysates.
- Fig. S7. Effect of protease inhibition on α-synuclein cleavage and aggregation induced by appendix lysates.
- Fig. S8. Identification of α-synuclein using TD-MS.
- Fig. S9. Schematic of proposed model for the contributions of the vermiform appendix to PD.
- Table S1. Characteristics of study samples from the SNPR.
- Table S2. Appendectomy in relation to age of PD onset for patients with PD in the SNPR study.
- Table S3. Incidence of PD in males and females living in rural and urban areas, SNPR study.
- Table S4. Demographic and clinical information of patients with PD in the PPMI.
- Table S5. Appendectomy in relation to age of PD onset for patients with PD in PPMI.
- Table S6. PD age of onset is delayed in individuals with an appendectomy but not in individuals with non-GI immune conditions or other surgeries.
- Table S7. Patients who had an appendectomy 30 or more years before PD do not show changes in PD symptom severity, as measured by the Hoehn and Yahr scale and UPDRS.
- Table S8. α-Synuclein aggregates are detected in the appendix of both young and older adult individuals and are present in normal and inflamed appendix.
- Table S9. Demographic and clinical information of sample sets used in PPMI study.
- Table S10. Appendectomy delays age of PD onset in patients with de novo PD of PPMI.
- References (92–96)
Other Supplementary Material for this manuscript includes the following:
- Data file S1 (Microsoft Excel format). Source data for biochemical assays.
