Evaluation of an antibody panel for alpha-synuclein detection in FFPE rectal biopsies in Parkinson’s disease

Abstract

Background

Conflicting results have been published regarding the accumulation of alpha-synuclein (aSyn) pathology, in intestinal tissues of patients with Parkinson’s disease (PD). This study investigates the use of a panel of aSyn antibodies for detecting pathological aSyn in rectal biopsy samples from PD patients and healthy individuals.

Materials and methods

A panel of antibodies targeting native, phosphorylated and conformation-specific forms of aSyn was used to characterize aSyn distribution in the substantia nigra and colonic tissues of controls and individuals with Lewy pathology. Distribution of aSyn was further analyzed in formalin-fixed and paraffin-embedded (FFPE) submucosal rectal biopsies of 24 PD patients and 20 healthy controls.

Results

All tested antibodies valuably detected aSyn pathology in the PD substantia nigra. Native aSyn was observed at high levels in colonic tissue. The immunoreactive patterns of native aSyn, conformation-specific or phosphorylated aSyn in rectal biopsies did not show notable differences between PD patients and healthy subjects.

Conclusion

The utility of immunohistochemical detection with currently commercially available antibodies for native and pathological forms of aSyn in rectal tissues appears limited. The findings advocate for the development of alternative methods to detect pathological aSyn conformers in rectal FFPE biopsies.

Introduction

A large majority of patients with Parkinson’s disease (PD) experience gastrointestinal (GI) disturbances, such as constipation or gastroparesis, at some point during the disease course (Ivan et al., 2021). The pathological aggregation of alpha-synuclein (aSyn) into Lewy bodies is the histological hallmark of PD. In line with the clinical presentation of PD, the expression of aSyn is not confined to the central nervous system (CNS), but is also observed in peripheral tissues, including the enteric nervous system (ENS) (Barrenschee et al., 2017, Böttner et al., 2012, Tanei et al., 2020).

Whereas aSyn is associated with the synaptic apparatus in both the CNS and ENS, phosphorylation of aSyn at serine 129 (P-aSyn) has been proposed as a key pathological event in PD. Indeed, this post-translationally modified form represents the predominant species found in Lewy bodies (Fujiwara et al., 2002).

Pathological aSyn deposits have been demonstrated in intestinal tissues from PD patients (Beach et al., 2010, Schaeffer et al., 2020). Based on this observation, a significant number of studies have explored the use of enteric aSyn expression as a potential diagnostic biomarker for PD (Beach et al., 2010, Bu et al., 2019, Schaeffer et al., 2020, Schneider et al., 2016, Shin et al., 2024).

However, P-aSyn is also physiologically expressed in the ENS, complicating its interpretation as a disease-specific marker (Böttner et al., 2012, Cossais et al., 2025, Pinard et al., 2025). The distribution of pathological aSyn in the gut of PD patients has been proposed to follow a rostro-caudal gradient, with Lewy pathology and P-aSyn deposits becoming progressively less detectable toward the almost distal segments of the gastrointestinal tract, including the rectum. Consequently, distinguishing between physiological and pathological forms of aSyn in gastrointestinal tissue has proven challenging (Barrenschee et al., 2017, Beach et al., 2010, Chahine et al., 2020, Ruffmann et al., 2018, Schneider et al., 2016).

The inconsistencies observed across studies are largely attributed to variations in antibody specificity, tissue processing protocols, and criteria used to interpret staining results. Multiple research groups and consortia have implemented diverse approaches to visualize aSyn pathology in GI tissues. Whole-mount staining of P-aSyn and protocols using antibodies against the native form of the protein combined with enzymatic digestion have shown promising results and reproducibility (Beach et al., 2018, Beach et al., 2016). However, such techniques require specialized expertise, limiting their feasibility in routine clinical pathology.

Alternative approaches using P-aSyn immunohistochemistry and conformation-specific antibodies, including clone 5G4 (Kumar et al., 2020), have shown encouraging specificity and sensitivity in distinguishing PD from control samples, particularly in duodenal tissues (Emmi et al., 2023, Kovacs et al., 2012, Skorvanek et al., 2018). Still, antibodies directed against pathological aSyn forms have not yet been systematically evaluated for use on rectal biopsy specimens.

Given the practical advantages of rectal biopsies in clinical settings, there remains a strong interest in validating simple and reliable immunohistochemical methods for detecting pathological aSyn in this tissue type. The present study aims to reappraise the utility of a panel of antibodies—including phosphorylation- and conformation-specific clones—for the detection of pathological aSyn deposits in routinely processed rectal biopsies.

Material and methods

Patients

Twenty-four patients with PD, diagnosed according to the Movement Disorder Society (MDS) Clinical Diagnostic Criteria for Parkinson’s Disease (Postuma et al., 2015), and 20 healthy individuals (controls) were included in the study (Table 1). All individuals underwent colonoscopy for colorectal cancer screening at the Interdisciplinary Endoscopy, Department of Internal Medicine, University Hospital Schleswig-Holstein, Campus Kiel.

Table 1.

Clinical data and results.

	Controls (n = 20)	PD (n = 24)	Sig. PD vs controls (p)
Sex m/f	9/11	20/4	0.008 *b
Age years, mean ± SD	66.2 ± 8.1	65.3 ± 8.3	0.417a
Disease duration years, mean ± SD	-	7.0 ± 6.0	-
LEDD mean ± SD	-	410.2 ± 367.1	-
MDS-UPDRS part III total score, mean ± SD, (range)	0.6 ± 1.3(0−5)	25.2 ± 13.5(2−54)	< 0.001 *a
Wexner´s constipation score total score, mean ± SD, (range)	3.2 ± 3.7 (0−10)	4.5 ± 4.5 (0−15)	0.154a
IHC-IR positive (%)
syn211
neuronal	5 (25)	8 (33)	0.546
- neurites	4 (20)	6 (25)	0.974
- ganglia	2 (10)	6 (25)	0.117
non-neuronal (mucosal)	4 (20)	4 (17)	0.775
N-term aSyn
neuronal	5 (25)	5 (21)	0.743
- neurites	3 (15)	4 (17)	0.880
- ganglia	2 (10)	2 (8)	0.848
non-neuronal (mucosal)	20 (100)	24 (100)	n.a.
P-S129 aSyn (D1R1R)
neuronal	0 (0)	4 (16.6)	0.114
- neurites	0 (0)	0 (0)	n.a.
- ganglia	0 (0)	4 (16.6)	0.114
non-neuronal (mucosal)	0 (0)	0 (0)	n.a.
P-S129 aSyn (EP1536Y)
neuronal	0 (0)	0 (0)	n.a.
- neurites	0 (0)	0 (0)	n.a.
- ganglia	0 (0)	0 (0)	n.a.
non-neuronal (mucosal)	0 (0)	0 (0)
P-S129 aSyn (MJF-R13)
neuronal	16 (80)	20 (83)	0.498
- neurites	16 (80)	20 (83)	0.498
- ganglia	8 (40)	3 (12)	0.036 *
non-neuronal (mucosal)	19 (95)	24 (100)	0.268
P-S129 aSyn (pSyn#64)
neuronal	7 (35)	18 (75)	0.001 *
- neurites	7 (35)	18 (75)	0.001 *
- ganglia	4 (20)	14 (58)	0.010 *
non-neuronal (mucosal)	18 (90)	24 (100)	0.113
P-Y39 aSyn
neuronal	3 (15)	1 (4)	0.213
- neurites	0 (0)	0 (0)	n.a.
- ganglia	3 (15)	1 (4)	0.213
non-neuronal (mucosal)	18 (90)	19 (79)	0.328
5G4
neuronal	0 (0)	1 (4)	0.356
- neurites	0 (0)	1 (4)	0.356
- ganglia	0 (0)	0 (0)	n.a.
non-neuronal (mucosal)	15 (75)	12 (50)	0.039 *
MJF-R14
neuronal	20 (100)	20 (83)	0.056
- neurites	20 (100)	20 (83)	0.056
- ganglia	7 (35)	4 (17)	0.162
non-neuronal (mucosal)	20 (100)	24 (100)	n.a.

PD: Parkinson’s disease; IHC-IR: immunohistochemistry immunoreactivity; LEDD: L-Dopa equivalent daily dose; Sig.: statistical significance, ^a Mann-Whitney U test, ^b Chi-square test. * statistically significant result. n.a.: not applicable

The MDS Unified Parkinson's Disease Rating Scale (MDS UPDRS-III) and Wexner’s constipation score were obtained for both groups. The levodopa-equivalent daily dose (LEDD) was determined for PD patients. The study was approved by the local ethics committee of the Faculty of Medicine, Christian-Albrechts University of Kiel, Germany (D452/19).

Retrieval and processing of rectal biopsies

From each individual, we obtained two deep submucosal biopsies from the rectum during screening colonoscopy (Jumbo Radial Jaw 3.2 mm biopsy forceps; Boston Scientific, Malborough, MA, USA). The samples were fixed in 4 % paraformaldehyde in phosphate-buffered saline (PBS) prior to paraffin embedding. Paraffin-embedded tissue blocks were cut in sections of 6 µm thickness, transferred onto glass slides, and stored at room temperature until further processing. Immunohistochemical analyses were performed as detailed below.

Retrieval and processing of substantia nigra and colon specimens

Substantia nigra tissue was obtained post-mortem from three body donors with diagnosed PD, as well as from two control individuals without known neurological manifestation (two females, three males, age range: 74–92 years), who were recruited from the body donation program of the Institute of Anatomy, Kiel University, in compliance with the Local Ethics Committee of the Faculty of Medicine, Kiel University, Germany (D512/24).

Retrieval of surgical specimens of descending colon was performed as previously described (Cossais et al., 2025). In particular, control colon samples were obtained from three patients who underwent partial colectomy for nonobstructive colorectal carcinoma. In addition, a surgical specimen from one individual with diverticulitis and additional anorectal outlet obstruction, presenting with incidental enteric Lewy pathology, was further included in the study. The study was approved by the Local Ethics Committee of the Faculty of Medicine, Kiel University, Germany (B299/07).

All samples were fixed in 4 % paraformaldehyde in PBS until they were embedded in paraffin. The paraffin embedded tissue-blocks were then cut into 6 µm thick sections that were transferred onto glass slides and stored at room temperature until further processing.

Immunohistochemical staining

Immunohistochemical staining was performed in accordance with a standard protocol (Cossais et al., 2021). Briefly, sections were pretreated with xylol for deparaffinization, alcohol with descending concentrations for rehydration, and citrate buffer (pH=6.0) to unmask antigens. For staining with clone 5G4 (Kovacs et al., 2012) as well as for P-Y39 aSyn, the sections were additionally incubated for 1 min in 98 % formic acid (Sigma). Sections were rinsed with distilled water and PBS buffer (pH=7.4) and incubated overnight with the primary antibodies (supplementary Table 1, supplementary Fig. 1) in antibody diluent (Thermo Fisher Scientific, Carlsbad, USA). After washing with PBS, the sections were incubated for one hour with the secondary antibodies. For immunohistochemical investigation we used the BrightVision 1 step detection system with anti-mouse horseradish peroxidase (HRP) and anti-rabbit HRP (ImmunoLogic). ImmPACT DAB EqV was used as substrate for HRP (Vector Laboratories). Nuclei were counterstained with hematoxylin. Images were acquired using a Leica Aperio CS2 slide scanner or a Keyence BZ-x800e inverted microscope. Blank controls were performed for all tissue types by omitting primary antibodies. supplementary methods Immunoreactive signal (IR) and tissue area was quantified using Aperio ImageScope software (v12.4.6.5003, Leica) running the “positive pixel count 2004–08–11” algorithm. Mean IR-positive area of five different mucosal fields for each biopsy was measured. Results are represented as percentage of the total measured area. Samples were not analyzed in a blinded fashion.

Statistical analyses

Prism GraphPad version 9.0 and SPSS version 29 were used for statistical analysis. Statistical differences between two groups were evaluated by the t-test and for three or more groups using ANOVA followed by Tukey’s post-hoc tests. Differences between PD and control groups for categorical variables were assessed with the Chi-square test. Test results with a p-value < 0.05 were considered statistically significant. Spearman’s rank correlation (two-sided) was applied for correlation analyses.

Results

Validation of anti-αSyn antibodies in substantia nigra and colon samples

Comparative α-synuclein antibody immunoreactivity in the substantia nigra of parkinson’s disease and control cases

Immunoreactive pattern of a panel of aSyn and P-aSyn antibodies was first exemplarily evaluated on postmortem nigral tissues of three PD body donors and two body donors without known neurological disorders (Fig. 1). The tested antibodies showed various degrees of immunoreactivity in nigral neurons and neuropile under control conditions (Table 2). Nonetheless, all antibodies clearly revealed Lewy bodies (LB) and Lewy neurites (LN) in the PD substantia nigra (Fig. 1a-i). The D1R1R antibody was more prone to stain LN than alternative P-S129 aSyn antibodies (Fig. 1c).

Fig. 1.

Localization pattern of aSyn in the substantia nigra of PD patients and healthy control individual. Immunohistochemistry showing the presence of Lewy bodies (arrows) and Lewy neurites (arrowheads), stained with aSyn antibodies Syn211 (a), N-term aSyn (b), D1R1R (c), EP1536Y (d), MJF-R13 (e), pSyn#64 (f), P-Y39 aSyn (g), 5G4 (h) and MJFR-14 (i) in the substantia nigra of a PD individual. Physiological presence of neuromelanin is also observed in nigral neurons in the control and PD specimens. Scale bar = 60 µm.

Table 2.

Immunoreactivity of aSyn antibodies in the substantia nigra of PD and controls donors and in colon specimen of an individual with incidental enteric aSyn pathology and control individuals. LB: Lewy bodies, LN: Lewy neurites, IM fibers: intramuscular nerve fibers, IR: immunoreactivity. Staining pattern: strong ++, moderate +, weak ±, absent -.

	Substantia nigra			Colon			Unspecific IR
Antibodies	neuronal (controls)	LB (PD)	LN (PD)	ganglia (controls)	IM fibers (controls)	aSyn deposits
Syn211	+	++	+	++	++	+	mucosa, immune cells
N-term aSyn	+	++	+	++	++	+	mucosa, immune cells, cell nuclei
P-aSyn - D1R1R	-	++	++	±	-	++	very low
P-aSyn - EP1536Y	-	++	±	-	-	+	very low
P-aSyn - MJF-R13	-	++	++	++	+	+	mucosa, immune cells, cell nuclei
P-aSyn - pSyn#64	-	++	++	+	-	++	mucosa (faint)
P-Y39 aSyn	-	++	++	±	-	++	immune cells (faint)
5G4	±	++	±	±	-	++	mucosa, immune cells
MJFR−14	+	++	+	++	++	+	mucosa, immune cells

Comparative α-synuclein antibody immunoreactivity in colon of individuals with synuclein pathology and control cases

Antibodies immunoreactivity was further exemplary assessed in colonic tissue of one individual with incidental enteric Lewy (aSyn) pathology (iLP) and two control individuals. Immunoreactivity of total aSyn was observed at high level in the enteric nervous system, including myenteric ganglia, intramuscular nerve fiber strands and submucosal ganglia (Fig. 2 and data not shown). Immunoreactivity for the syn211 and N-term antibodies was also observed at various extents in the intestinal epithelium and in unidentified cells within the lamina propria, which was considered as unspecific signal. Immunoreactivity was further observed in pathological aSyn aggregates within myenteric ganglia of an individual with iLP. Nonetheless, because of the intensity of the DAB staining in enteric neurons, aSyn pathological deposits were difficult to distinguish from the global neuronal staining (Fig. 2a-b, Table 2).

Fig. 2.

Localization pattern of aSyn in colonic myenteric ganglia of control individuals and one individual with incidental enteric Lewy pathology (LP). Immunohistochemistry showing variable diffuse aSyn immunoreactivity in myenteric ganglia (dotted line) stained with aSyn antibodies Syn211 (a), N-term aSyn (b), D1R1R (c), EP1536Y (d), MJF-R13 (e), pSyn#64 (f), P-Y39 aSyn (g), 5G4 (h) and MJFR-14 (i). Presence of aSyn aggregates was observed in LP ganglia (arrows). Scale bar = 60 µm.

Immunoreactivity of P-aSyn was further assessed in colonic tissues. High staining variability was observed when using a panel of four different antibodies against P-aSyn (Fig. 2c-f, Table 2). Although varying degrees of diffuse immunoreactivity were observed in enteric neurons, pathological aSyn deposits were clearly observed within enteric ganglia using the D1R1R and EP1536Y antibodies, whereas no to very limited staining was observed along intramuscular nerve fibers (Fig. 2c-d, Table 2). Similar results were obtained using the P-aSyn#64 (Fig. 2f). In contrast, widespread immunoreactivity was observed within enteric ganglia using the MJF-R13 antibody, although this antibody also stained pathological aSyn aggregates (Fig. 2e, Table 2). Whereas P-Y39 aSyn immunoreactivity was barely detectable within control enteric ganglia, P-Y39 aSyn was found localized in pathological enteric aSyn aggregates (Fig. 2g).

Finally, the immunoreactivity of MJFR-14 and 5G4 antibodies was evaluated in colonic specimens (Fig. 2h-i). Immunoreactivity for 5G4 was observed within enteric neurons in control individuals. Signal was further observed within the intestinal epithelium , which was considered unspecific. Immunoreactivity of 5G4 was further found localized within pathological enteric aSyn aggregates (Fig. 2h). In contrast, MJFR-14-IR was widely present in enteric ganglia, as well as in intramuscular and interganglionic nerve fiber strands of the myenteric and submucosal plexus (Fig. 2i, Table 2).

Immunoreactive pattern of αSyn in FFPE rectal biopsies of PD patients and healthy controls

Clinical characterization of the study group

The healthy control and PD group were comparable regarding age (mean age controls: 66, SD 8 years; PD: 65, SD 8 years; p = 0.42; Table 1), while the PD group comprised more men (male sex controls: 45 %, PD: 83 %; p = 0.008; Table 1). No group difference was seen for the Wexner’s constipation score (Table 1).

Distribution pattern of total aSyn in FFPE rectal biopsies of PD patients and healthy controls

The distribution pattern of total aSyn was then evaluated by immunohistochemistry in FFPE deep submucosal rectal biopsies of PD patients and healthy control individuals, which contained mucosal tissue as well as a part of the muscularis mucosae and submucosal connective tissue including the submucosal ganglia. The presence of neuronal fibers within the biopsies was first confirmed by staining for beta-III tubulin (TUBB3, supplementary Fig. 2). Immunoreactivity for aSyn was detectable in enteric neurites and ganglia in about 25 % controls and PD specimens, using either syn211 or N-term-aSyn antibodies (Fig. 3, Table 1). Both antibodies also displayed immunoreactivity in non-neuronal cells within the mucosa in almost all specimens which was largely considered unspecific (Table 2). Nonetheless, because recent reports have shown that mucosal aSyn measurement may correlate with PD diagnosis, the mucosal immunoreactive area was measured. The positive area covered after staining with syn211 or N-term-aSyn antibodies did not differ significantly between PD and control biopsies (Fig. 3c-d).

Fig. 3.

Localization pattern of aSyn in rectal biopsies of PD patients and healthy controls. Immunohistochemistry showing the distribution pattern of aSyn, clone syn211 (a) and N-term aSyn (c) in rectal biopsies. Immunoreactivity was observed in neural structures as well as in some unidentified cells within the lamina propria. No difference was observed by morphometrical quantification of the percentage of syn211 (b) and N-term aSyn (d) immunoreactive (IR) signal to total area between PD and control (n = 24 and n = 20 respectively, Mann-Whitney U-test). Arrows indicate IR-positive ganglionic structures and arrowheads indicate IR-positive unidentified cells in the mucosa. Scale bar = 60 µm.

Distribution pattern of phosphorylated-aSyn in FFPE rectal biopsies of PD patients and healthy controls

The immunoreactive pattern of P-S129 aSyn was similarly assessed in FFPE rectal biopsies from PD patients and healthy controls. The signal intensity for D1R1R and EP1536Y was generally at the limit of visual detection or was observed as faint diffuse staining within submucosal ganglia in nearly all analyzed biopsies. Given the very low levels observed, the immunoreactive-positive area for D1R1R and EP1536Y did not differ significantly between the PD and control groups when assessed computationally. In contrast, both MJF-R13 and pSyn#64 were observed in submucosal ganglia and nerve fibers within mucosal and submucosal tissue of PD and control individuals (Fig. 4, Table 1). In particular, neuronal distribution of MJF-R13 was observed in 80 % of controls and 83 % of PD patients (p = 0.498, Table 1). Neuronal immunoreactivity for pSyn#64 was significantly more frequently observed in PD patients in comparison to control individuals (75 % versus 35 %, respectively, p < 0.001). Nonetheless, mucosal immunoreactive area covered by MJF-R13 or pSyn#64 did not differ significantly between PD biopsies and controls (Fig. 4f, h).

Fig. 4.

Localization pattern of phosphorylated aSyn in rectal biopsies of PD patients and healthy controls. Immunohistochemistry showing the distribution pattern of phosphorylated aSyn using the D1R1R (a), EP1536Y (c), MJF-R13 (e) and pSyn#64 (g) and P-Y39 (i) antibodies. No difference was observed by morphometrical quantification of the percentage of D1R1R (b), EP1536Y (d), MJF-R13 (f), pSyn#64 (h) or P-Y39 (j) IR signal between PD and control (n = 24 and 20 respectively, Mann-Whitney U-test). Arrows indicate IR-positive ganglionic structures and arrowheads indicate unspecific staining in the mucosa. Scale bar = 60 µm.

Immunoreactive pattern of conformation-specific antibodies in FFPE rectal biopsies of PD patients and healthy controls

The immunoreactive signal for the 5G4 antibody was seen as disperse spots in the mucosa and submucosa in 12 of 24 PD patients and 15 of 20 healthy controls (Fig. 5a, Table 1). The staining pattern obtained for the 5G4 antibody was mainly observed in cells within the lamina propria, which may correspond to intestinal immune cells. The area covered by the 5G4 staining was not significantly changed in biopsies of PD patients in comparison to controls (Fig. 5b).

Fig. 5.

Localization pattern of conformation-specific aSyn in rectal biopsies of PD patients and healthy controls. Immunohistochemistry showing the distribution pattern of aSyn using the conformation-specific antibodies 5G4 (a) and MJFR-14 (c) in rectal biopsies. No difference was observed by quantification of the percentage of 5G4 IR-positive signal (b) or MJFR-14 IR-signal (d) between PD and controls (n = 24 and n = 20 respectively, Mann-Whitney U-test). Arrows indicate IR-positive ganglionic structures and arrowheads indicate IR-positive unidentified cells in the mucosa. Scale bar = 60 µm.

MJFR-14 immunoreactivity was detected in nerve fibers and ganglia in 22 of 24 PD patients, as well as in 20 of 20 of control subjects (Fig. 5c-d, Table 1). In most cases, MJFR-14 staining was also seen in unidentified cells in the lamina propria, most likely corresponding to immune cell populations (Fig. 5c, Table 1). The area covered by the MJFR-14 staining was not significantly changed in biopsies of PD patients in comparison to controls (Fig. 5d).

To further ensure the specificity of the MJFR14 staining, colocalization experiments with the neuronal marker PGP9.5 were performed on a subcohort of patients, confirming that MJFR14 immunoreactivity corresponded to neuronal structures in both PD patients and healthy controls. Colocalization measurements for MJFR14 and PGP9.5 did not statistically differ between the control and patient groups (supplementary Fig. 3).

Discussion

Considerable efforts have been made to assess the diagnostic value of misfolded aSyn in GI tissues from people with PD. Nonetheless, immunohistochemical characterization of aSyn in mucosal biopsies remains challenging, particularly when using rectal tissue (supplementary Table 2).

In our study, using a panel of antibodies, we confirmed the presence of aSyn in its native and phosphorylated forms in rectal tissues of PD patients and healthy controls. However, antibodies targeting pathological aSyn strains still appear to have limited diagnostic utility in rectal biopsies using routine immunohistochemistry.

The detection of pathological aSyn in intestinal tissues has been the subject of numerous studies (supplementary Table 2). Efforts by dedicated consortia have achieved good sensitivity and specificity in identifying pathological aSyn deposits, particularly through whole-mount staining of P-aSyn (Lebouvier et al., 2008). However, the developed protocols require specialized staining techniques and expert pathological evaluation and interpretation, making them less suitable for routine diagnostic use in standard pathology laboratories.

The performance of aSyn detection is influenced by various technical factors, including fixation protocols and antigen retrieval techniques. Furthermore, antibody specificity depends on complex post-translational modifications and truncations that aSyn may undergo in the gut environment (Lashuel et al., 2022). In our analysis, aSyn-specific antibodies — despite validation for labeling Lewy bodies and Lewy neurites in the substantia nigra—did not yield consistent or diagnostically useful staining in rectal tissue. This finding is particularly notable given that some of these antibodies have demonstrated reliability in other GI regions. For instance, quantitation of 5G4 immunoreactivity in duodenal mucosa of PD patients has shown high sensitivity and specificity (Emmi et al., 2023). However, we failed to reproduce similar results in the rectal mucosa using a similar methodology.

Although immunoreactivity for the EP1536Y and D1R1R antibodies remained below detection level in most rectal mucosal biopsies, our previous and present data indicate that these antibodies are suitable to detect both physiological and pathological P-aSyn deposits in human colonic tissues (Cossais et al., 2025). These results are in line with observation made by an independent research group using the EP1536Y antibody (Shin et al., 2024, Shin et al., 2017). In their study, Shin and colleagues reported that the sensitivity of P-aSyn detection decreased from 64.3 % to 23.8 % when the assessment was restricted to the mucosal layer, compared with full-thickness intestinal specimens (Shin et al., 2024). Although the number of full-thickness intestinal specimens included in our study was too low to perform a similar assessment, we observed that P-aSyn deposits were predominantly confined to the myenteric and submucosal ganglia, with only rare isolated immunoreactive spots within the mucosa (data not shown). This distribution pattern may further reduce the likelihood of detecting such immunoreactive spots in histological sections of intestinal mucosal biopsies.

In PD, pathological aSyn deposits—whether as Lewy bodies or diffuse deposits—have been observed throughout the whole GI tract (supplementary Table 2). However, only five studies have specifically addressed pathological aSyn distribution in rectal biopsies of PD patients, reporting the lowest detection rate in comparison to upper parts of the GI tract (Supplementary Table 2). Indeed, aSyn distribution appears to follow a rostrocaudal gradient corresponding to the vagus nerve's innervation territory, with higher detection rates in the stomach and duodenum and lowest detection in the rectum (Beach et al., 2010, Borghammer, 2021, Borghammer, 2018, Shin et al., 2024). Nonetheless, the underlying mechanisms for this gradient remain unclear. It is noteworthy that aSyn pathology has been observed in the sacral spinal cord and the pelvic autonomic outflow that innervates the rectum (Del Tredici and Braak, 2012, Raunio et al., 2022, Tamura et al., 2012, Wakabayashi, 2020), suggesting a complex pattern of distribution of aSyn pathology in the almost distal neuroanatomical structures of the gut-brain axis. Whether the rectal ENS presents tissue-specific aSyn strains and post-translational modifications which may contribute to this observed discrepancy remains to be determined.

The development of aSyn pathology remains poorly understood in the brain and, even more so, in peripheral tissues. Evidence indicates that aSyn pathology is site-specific, with distinct conformers identified in different brain regions (Wiseman et al., 2024). Although P-S129 antibodies appear effective for detecting aSyn pathology in intestinal tissue, we noted high variability in the observed immunoreactive patterns for different P-aSyn antibodies. Additional investigations are needed to clarify the temporal dynamics of aSyn pathology in the gut and to define the range of pathological aSyn species present in the intestine and their recognition by specific antibodies.

Finally, our study cohort showed an imbalance in gender distribution between PD and control groups. Although no conclusive evidence currently links gender with differences in enteric aSyn pathology, future studies should consider gender matching to minimize potential confounding effects.

Despite recent advances, our findings suggest that routine immunohistochemical characterization of aSyn deposits in mucosal rectal tissue using currently commercially available antibodies remains of limited diagnostic value in PD, supporting the conclusions of previous studies (Antunes et al., 2016, Barrenschee et al., 2017, Desmet et al., 2017, Ruffmann et al., 2018, Shin et al., 2024). Nevertheless, in rare cases, characterization of enteric aSyn pathology in full-thickness colo-rectal specimens may offer insights into associated GI dysfunctions (Cossais et al., 2025, Kupsky et al., 1987).

This data further highlights the urgent need for alternative biochemical approaches to improve pathological aSyn detection accuracy in intestinal tissue. Development of sensitive biochemical detection assays and complementary strategies — such as transcriptomic profiling, assessment of intestinal barrier integrity, immune cell population and microbiome analysis — may help advance biomarker discovery and deepen our understanding of the role of gut pathology in PD (Derkinderen et al., 2025, Derkinderen et al., 2022, Zacharias et al., 2022).

CRediT authorship contribution statement

Daniela Berg: Writing – review & editing, Resources. Annika Kluge: Writing – review & editing, Methodology, Investigation, Data curation, Conceptualization. Sebastian Heinzel: Writing – review & editing, Methodology. Martina Böttner: Writing – review & editing, Resources, Methodology, Conceptualization. Thilo Wedel: Writing – review & editing, Resources. Manuela Pendziwiat: Writing – review & editing, Resources, Methodology. Ralph Lucius: Writing – review & editing, Resources. Mark Ellrichmann: Writing – review & editing, Resources, Methodology. Stephan Schoch: Writing – review & editing, Resources, Data curation. Katja Schröder: Writing – review & editing, Validation, Methodology, Investigation, Formal analysis, Data curation. François Cossais: Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Kristína Kulcsárová: Writing – review & editing, Supervision, Formal analysis, Conceptualization. Eva Schaeffer: Writing – review & editing, Supervision, Project administration, Methodology, Investigation, Conceptualization. Carmen Kintrup: Writing – review & editing, Visualization, Methodology, Investigation, Formal analysis, Data curation.

Ethical compliance statement

The study was approved by the Local Ethics Committee of the Faculty of Medicine, Kiel University, Germany (B299/07), (D512/24) and (D452/19). All participants and body donors have given written informed consent prior to the study. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.

Funding

This project was supported by a grant of the Faculty of Medicine, Kiel University (K126422) to FC and ES. Open Access funding enabled and organized by Projekt DEAL. Funders and supporting institutions had no influence on the design, conduct, or analysis of the study.

Declaration of Competing Interest

The authors declare that there are no conflicts of interest relevant to this work.

Appendix A. Supplementary material

Supplementary material

Supplementary material

Availability of data and material

The datasets analysed during the current study are available from the corresponding authors on reasonable request.